In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zargar, K.; Saville, R.; Phelan, R. M.

Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation inmore » complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extract s, (iii) both activities were irreversibly inactivated upon exposure to O 2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.« less

In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community

DOE PAGES

Zargar, K.; Saville, R.; Phelan, R. M.; ...

2016-08-10

Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation inmore » complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extract s, (iii) both activities were irreversibly inactivated upon exposure to O 2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.« less

PA-1, a Versatile Anaerobe Obtained in Pure Culture, Catabolizes Benzenoids and Other Compounds in Syntrophy with Hydrogenotrophs, and P-2 plus Wolinella sp. Degrades Benzenoids

PubMed Central

Barik, Sudhakar; Brulla, W. J.; Bryant, M. P.

1985-01-01

Methanogenic enrichments catabolizing 13 mM phenylacetate or 4 mM phenol were established at 37°C, using a 10% inoculum from a municipal anaerobic digester. By using agar roll tubes of the basal medium plus 0.1% yeast extract-25 mM fumarate, a hydrogenotrophic lawn of Wolinella succinogenes and phenol or phenylacetate, strains P-2 and PA-1, respectively, were isolated in coculture with W. succinogenes. With the lawn deleted, PA-1 was isolated in pure culture. Strain P-2 is apparently a new species of anaerobic, motile, gram-negative, spindle-shaped, small rod that as yet has been grown only in coculture with W. succinogenes. It used phenol, hydrocinnamate, benzoate, and phenylacetate as energy sources. Product recovery by the coculture, per mole of phenol and 4.4 mol of fumarate used, included 2.03, 0.12, 0.08, and 3.23 mol, respectively, of acetate, propionate, butyrate, and succinate. Carbon recovery was 75% and H recovery was 80%, although CO2 and a few other possible products were not determined. That P-2 is an obligate proton-reducing acetogen and possible pathways for its degradation of phenol are discussed. Strain PA-1 is apparently a new species of anaerobic, motile, relatively small, gram-negative rod. It utilized compounds such as phenylacetate, hydrocinnamate, benzoate, phenol, resorcinol, gallate, 4-aminophenol, 2-aminobenzoate, pyruvate, Casamino Acids, and aspartate as energy sources in coculture with W. succinogenes. Per mole of phenylacetate and 1.44 mol of fumarate used, 1.04, 0.53, and 0.78 mol of acetate, propionate, and succinate, respectively, were recovered from the coculture. Only about 50% of the carbon and H were recovered. In coculture with Methanospirillum hungatei, 0.96 mol of acetate and 0.25 mol of methane were recovered per mol of pyruvate used; 0.90 mol of acetate and 0.33 mol of methane, per mol of fumarate used; 0.93 mol of acetate and 0.54 mol of methane, per mol of aspartate used; and 1.71 mol of acetate and 0.57 mol of methane, per mol of glucose used. Carbon and H recoveries, assuming CO2 and ammonia were produced in stoichiometric amounts, were 97 and 98% for pyruvate, 72.5 and 82% for fumarate, 96.5 and 98% for aspartate, and 61.8 and 76% for glucose. No explanation such as contamination could be found for the fact that the coculture PA-1 plus Wolinella sp. did not use glucose; after growth with M. hungatei on pyruvate, however, the latter coculture used glucose. The PA-1 pure culture produced 0.86 mol of propionate per mol of succinate used during growth. PA-1 produced a small amount of H2. Strain PA-1 is the most versatile anaerobic bacterium yet known that catabolizes monobenzenoids in the absence of electron acceptors such as sulfate or nitrate. PMID:16346852

Metabolism of β-valine via a CoA-dependent ammonia lyase pathway.

PubMed

Otzen, Marleen; Crismaru, Ciprian G; Postema, Christiaan P; Wijma, Hein J; Heberling, Matthew M; Szymanski, Wiktor; de Wildeman, Stefaan; Janssen, Dick B

2015-11-01

Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding β-valinyl-coenzyme A ligase (BvaA) and two genes encoding β-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate β-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only β-valine and to a lesser extent, 3-aminobutyrate and β-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate β-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

Anaerobic Degradation of Ethylbenzene by a New Type of Marine Sulfate-Reducing Bacterium

PubMed Central

Kniemeyer, Olaf; Fischer, Thomas; Wilkes, Heinz; Glöckner, Frank Oliver; Widdel, Friedrich

2003-01-01

Anaerobic degradation of the aromatic hydrocarbon ethylbenzene was studied with sulfate as the electron acceptor. Enrichment cultures prepared with marine sediment samples from different locations showed ethylbenzene-dependent reduction of sulfate to sulfide and always contained a characteristic cell type that formed gas vesicles towards the end of growth. A pure culture of this cell type, strain EbS7, was isolated from sediment from Guaymas Basin (Gulf of California). Complete mineralization of ethylbenzene coupled to sulfate reduction was demonstrated in growth experiments with strain EbS7. Sequence analysis of the 16S rRNA gene revealed a close relationship between strain EbS7 and the previously described marine sulfate-reducing strains NaphS2 and mXyS1 (similarity values, 97.6 and 96.2%, respectively), which grow anaerobically with naphthalene and m-xylene, respectively. However, strain EbS7 did not oxidize naphthalene, m-xylene, or toluene. Other compounds utilized by strain EbS7 were phenylacetate, 3-phenylpropionate, formate, n-hexanoate, lactate, and pyruvate. 1-Phenylethanol and acetophenone, the characteristic intermediates in anaerobic ethylbenzene degradation by denitrifying bacteria, neither served as growth substrates nor were detectable as metabolites by gas chromatography-mass spectrometry in ethylbenzene-grown cultures of strain EbS7. Rather, (1-phenylethyl)succinate and 4-phenylpentanoate were detected as specific metabolites in such cultures. Formation of these intermediates can be explained by a reaction sequence involving addition of the benzyl carbon atom of ethylbenzene to fumarate, carbon skeleton rearrangement of the succinate moiety (as a thioester), and loss of one carboxyl group. Such reactions are analogous to those suggested for anaerobic n-alkane degradation and thus differ from the initial reactions in anaerobic ethylbenzene degradation by denitrifying bacteria which employ dehydrogenations. PMID:12570993

Genome sequence of two members of the chloroaromatic-degrading MT community: Pseudomonas reinekei MT1 and Achromobacter xylosoxidans MT3.

PubMed

Gutierrez-Urrutia, Izabook; Miossec, Matthieu J; Valenzuela, Sandro L; Meneses, Claudio; Dos Santos, Vitor A P Martins; Castro-Nallar, Eduardo; Poblete-Castro, Ignacio

2018-06-10

We describe the genome sequence of Pseudomonas reinekei MT1 and Achromobacter xylosoxidans MT3, the most abundant members of a bacterial community capable of degrading chloroaromatic compounds. The MT1 genome contains open reading frames encoding enzymes responsible for the catabolism of chlorosalicylate, methylsalicylate, chlorophenols, phenol, benzoate, p-coumarate, phenylalanine, and phenylacetate. On the other hand, the MT3 strain genome possesses no ORFs to metabolize chlorosalicylates; instead the bacterium is capable of metabolizing nitro-phenolic and phenolic compounds, which can be used as the only carbon and energy source by MT3. We also confirmed that MT3 displays the genetic machinery for the metabolism of chlorocathecols and chloromuconates, where the latter are toxic compounds secreted by MT1 when degrading chlorosalicylates. Altogether, this work will advance our fundamental understanding of bacterial interactions. Copyright © 2018 Elsevier B.V. All rights reserved.

Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis.

PubMed

Sass, Andrea; Marchbank, Angela; Tullis, Elizabeth; Lipuma, John J; Mahenthiralingam, Eshwar

2011-07-22

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes. Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance. Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.

Bacterial conversion of phenylalanine and aromatic carboxylic acids into dihydrodiols.

PubMed Central

Wegst, W; Tittmann, U; Eberspächer, J; Lingens, F

1981-01-01

Strain E of chloridazon-degrading bacteria, when grown on L-phenylalanine accumulates cis-2,3-dihydro-2,3-dihydroxyphenylalanine. In experiments with resting cells and during growth the bacterium converts the aromatic carboxylic acids phenylacetate, phenylpropionate, phenylbutyrate and phenyl-lactate into the corresponding cis-2,3-dihydrodiol compounds. The amino acids L-phenylalanine, N-acetyl-L-phenylalanine and t-butyloxycarbonyl-L-phenylalanine were also transformed into dihydrodiols. All seven dihydrodiols, thus obtained, were characterized both by conventional analytical techniques and by the ability to serve as substrates for a cis-dihydrodiol dehydrogenase. PMID:7306016

A Nitration Reaction Puzzle for the Organic Chemistry Laboratory

ERIC Educational Resources Information Center

Wieder, Milton J.; Barrows, Russell

2008-01-01

Treatment of phenylacetic acid with 90% HNO[subscript 3] yields a product, I, whose observed melting point is 175-179 degrees C and whose equivalent weight is approximately 226 grams. Treatment of phenylacetic acid with 70% HNO[subscript 3] yields a product, II, whose observed melting point is 106-111 degrees C and whose equivalent weight is…

[Specific odor component produced by Mycobacterium lepraemurium on Ogawa yolk medium].

PubMed

Mori, T; Aishima, T

1992-11-01

When Mycobacterium lepraemurium is grown on the 1% Ogawa yolk medium, it produces a specific odor. This odor was not observed in other easily cultivable acid-fast bacilli. Therefore, identification of the components responsible for the specific odor produced by M. lepraemurium was attempted. The odor components were extracted for overnight with sterilized and distilled water from the Ogawa yolk medium on which M. lepraemurium had been cultivated for two months. The odor components in the extract was adsorbed on refined charcoal. After washing with distilled water for three times, the charcoal was dried. Then the odor components were eluted from the charcoal with ethanol and the eluate was condensed under nitrogen gas flow at 40 degrees C. The condensate was analyzed by Gas-Chromatography-Mass-Spectrum (GC-MS). Phenylethanol and phenylacetic acid were identified as major odor components. A mixture of authentic phenylacetic acid, its methyl and ethyl esters, smelled similar to the odor of cultivated medium of M. lepraemurium. Thus, phenylacetic acid was identified as the key odor component produced by M. lepraemurium. When initial isolation culture of M. lepraemurium from murine leproma was cultivated on the Ogawa yolk medium by adding phenylacetic acid, growth inhibition was brought by the compound.

Molecular Characterization of a Novel N-Acetyltransferase from Chryseobacterium sp.

PubMed Central

Yoshida, Kenji; Tanaka, Kosei; Yoshida, Ken-ichi

2014-01-01

N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of l-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l-2-phenylglycine and its chloro- and hydroxyl-derivatives. The Km and Vmax values for l-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1 · mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1). PMID:24375143

Induction of immune-related gene expression in Ctenopharyngodon idella kidney cells by secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3.

PubMed

Wu, Z-F; Liu, G-L; Zhou, Z; Wang, G-X; Xia, L; Liu, J-L

2012-08-01

This study was undertaken to isolate active secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3 and evaluate their activities using grass carp Ctenopharyngodon idella kidney (CIK) cells. By applying chromatography techniques and successive recrystallization, three purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: (1) phenylacetic acid, (2) p-hydroxyphenylacetylamide and (3) cyclo-(Gly-(L)-Pro). CIK cells were stimulated by different concentrations (1, 10 and 100 μg/ml) of the isolated compounds, and expression of MyD88, IL-1β, TNF-α, type I-IFN and IL-8 genes at different time points (2, 8 and 24 h) post-stimulation was quantified by real-time PCR. The known immunostimulatory agent lipopolysaccharide (LPS) was used as a positive control. To analyse whether these compounds are toxic to the cells, the methyl tetrazolium assay was employed to measure changes in cell viability. The obtained results revealed that transcribing level of MyD88, an important adaptor molecule in toll-like receptor signalling pathway, was augmented remarkably by all the three isolated compounds and LPS as early as 2-h exposure. These compounds also induced gene expression of cytokines such as IL-1β, TNF-α and type I-IFN. Under the experimental conditions, none of the test compounds is toxic to the CIK cells. These findings demonstrate that the immunostimulatory properties of the three metabolites [phenylacetic acid, p-hydroxyphenylacetylamide and cyclo-(Gly-(L)-Pro)] from A. faecalis FY-3 in CIK cells and highlight the potential of using these metabolites as immunostimulants in fish aquaculture. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

Survival after treatment with phenylacetate and benzoate for urea-cycle disorders.

PubMed

Enns, Gregory M; Berry, Susan A; Berry, Gerard T; Rhead, William J; Brusilow, Saul W; Hamosh, Ada

2007-05-31

The combination of intravenous sodium phenylacetate and sodium benzoate has been shown to lower plasma ammonium levels and improve survival in small cohorts of patients with historically lethal urea-cycle enzyme defects. We report the results of a 25-year, open-label, uncontrolled study of sodium phenylacetate and sodium benzoate therapy (Ammonul, Ucyclyd Pharma) in 299 patients with urea-cycle disorders in whom there were 1181 episodes of acute hyperammonemia. Overall survival was 84% (250 of 299 patients). Ninety-six percent of the patients survived episodes of hyperammonemia (1132 of 1181 episodes). Patients over 30 days of age were more likely than neonates to survive an episode (98% vs. 73%, P<0.001). Patients 12 or more years of age (93 patients), who had 437 episodes, were more likely than all younger patients to survive (99%, P<0.001). Eighty-one percent of patients who were comatose at admission survived. Patients less than 30 days of age with a peak ammonium level above 1000 micromol per liter (1804 microg per deciliter) were least likely to survive a hyperammonemic episode (38%, P<0.001). Dialysis was also used in 56 neonates during 60% of episodes and in 80 patients 30 days of age or older during 7% of episodes. Prompt recognition of a urea-cycle disorder and treatment with both sodium phenylacetate and sodium benzoate, in conjunction with other therapies, such as intravenous arginine hydrochloride and the provision of adequate calories to prevent catabolism, effectively lower plasma ammonium levels and result in survival in the majority of patients. Hemodialysis may also be needed to control hyperammonemia, especially in neonates and older patients who do not have a response to intravenous sodium phenylacetate and sodium benzoate. Copyright 2007 Massachusetts Medical Society.

Dihydroxo-bridged dimeric Cu(II) system containing sandwiched non-coordinating phenylacetate anion: Crystal structure, spectroscopic, anti-bacterial, anti-fungal and DNA-binding studies of [(phen)(H2O)Cu(OH)2Cu(H2O)(phen)]2L.6H2O: (HL = phenylacetic acid; phen = 1,10-phenanthroline)

NASA Astrophysics Data System (ADS)

Iqbal, Muhammad; Ali, Saqib; Tahir, Muhammad Nawaz; Shah, Naseer Ali

2017-09-01

This paper reports the synthesis, X-ray crystal structure, DNA-binding, antibacterial and antifungal studies of a rare dihydroxo-bridged dinuclear copper(II) complex including 1,10-phenanthroline (Phen) ligands and phenylacetate (L) anions, [Cu2(Phen)2(OH)2(H2O)2].2L.6H2O. Structural data revealed distorted square-pyramidal geometry for each copper(II) atom with the basal plane formed by the two nitrogen atoms of the phenantroline ligand and the oxygen atoms of two bridging hydroxyl groups. The apical positions are filled by the oxygen atom from a water molecule. This forms a centrosymmetric cationic dimer where the uncoordinated phenylacetate ligands serve to balance the electrical charge. The dimers interact by means of hydrogen bonds aided by the coordinated as well as uncoordinated water molecules and phenyl-acetate moieties in the crystal lattice. The binding ability of the complex with salmon sperm DNA was determined using cyclic voltammetry and absorption spectroscopy yielding binding constants 2.426 × 104 M-1 and 1.399 × 104 M-1, respectively. The complex was screened against two Gram-positive (Micrococcus luteus and Bacillus subtilis) and one Gram-negative (Escherichia coli) bacterial strains exhibiting significant activity against all the three strains. The complex exhibited significant, moderate and no activity against fungal strains Mucor piriformis, Helminthosporium solani and Aspergillus Niger, respectively. These preliminary tests indicate the competence of the complex towards the development of a potent biological drug.

Radiation- and Depleted Uranium-Induced Carcinogenesis Studies: Characterization of the Carcinogenic Process and Development of Medical Countermeasures

DTIC Science & Technology

2005-01-01

drug development. 3.2 Candidate Agents: Phenylacetate and Epigallocatechin Gallate Phenylacetate (PA) is an attractive candidate for our studies...The polyphenol, epigallocathechin-3 gallate ( EGCG ), a major constituent of green tea, is also an attractive candidate for low-dose radiation...preventive agents. Part 1: chemopreventive potential of Epigallocatechin gallate , cimigenol, cimigenol-3,15-dione, and related compounds, Bioorganic Medical

Conformational equilibrium of phenylacetic acid and its halogenated analogues through theoretical studies, NMR and IR spectroscopy

NASA Astrophysics Data System (ADS)

Levandowski, Mariana N.; Rozada, Thiago C.; Melo, Ulisses Z.; Basso, Ernani A.; Fiorin, Barbara C.

2017-03-01

This paper presents a study on the conformational preferences of phenylacetic acid (PA) and its halogenated analogues (FPA, CPA, BPA). To clarify the effects that rule these molecules' behaviour, theoretical calculations were used, for both the isolated phase and solution, combined with nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. Most conformations of phenylacetic acid and its halogenated derivatives are stabilized through the hyperconjugative effect, which rules the conformational preference. NMR analyses showed that even with the variation in medium polarity, there was no significant change in the conformation population. Infrared spectroscopy showed similar results for all compounds under study. In most spectra, two bands were found through the carbonyl deconvolution, which is in accordance with the theoretical data. It was possible to prove that variation in the nature of the substituent in the ortho position had no significant influence on the conformational equilibrium.

Possible Phenylacetate Hepatotoxicity During 4-Phenylbutyrate Therapy of Byler Disease.

PubMed

Shneider, Benjamin L; Morris, Amy; Vockley, Jerry

2016-03-01

In vitro studies have suggested that 4-phenylbutyrate (PBA) may rescue missense mutated proteins that underlie some forms of progressive familial intrahepatic cholestasis. Encouraging preliminary responses to 4-PBA have been reported in liver disease secondary to mutations in ABCB11 and ATP8B1. A 4-year-old boy with Byler disease was treated with 4-PBA in the forms of sodium PBA (5 months) and then glycerol PBA (7 months) as part of expanded access single patient protocols. During this therapy serum total bilirubin fell and his general well-being was reported to be improved, although total serum bile acids were not reduced. Discontinuation of rifampin therapy, which had been used to treat pruritus, resulted in reversible severe acute liver injury that was potentially the result of phenylacetate toxicity. Interactions between 4-PBA and cytochrome P450 enzymes should be considered in the use of this agent with special attention to potential phenylacetate toxicity.

Urinary Phenylacetylglutamine as Dosing Biomarker for Patients with Urea Cycle Disorders

PubMed Central

Mokhtarani, M; Diaz, GA; Rhead, W; Lichter-Konecki, U; Bartley, J; Feigenbaum, A; Longo, N; Berquist, W; Berry, SA; Gallagher, R; Bartholomew, D; Harding, CO; Korson, MS; McCandless, SE; Smith, W; Vockley, J; Bart, S; Kronn, D; Zori, R; Cederbaum, S; Dorrani, N; Merritt, JL; Sreenath-Nagamani, Sandesh; Summar, M; LeMons, C; Dickinson, K; Coakley, DF; Moors, TL; Lee, B; Scharschmidt, BF

2013-01-01

We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). Study Design These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6–17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. Results Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5–31.8 g/day and of sodium phenylbutyrate ranging from 1.3–31.7 g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% –5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% -to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r=0.730, p<0.001) or as total 24-hour excretion (r=0.791 p<0.001), followed by plasma phenylacetylglutamine AUC24-hour, plasma phenylacetic acid AUC24-hour and phenylbutyric acid AUC24-hour. Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. Conclusion The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring. PMID:22958974

Urinary phenylacetylglutamine as dosing biomarker for patients with urea cycle disorders.

PubMed

Mokhtarani, M; Diaz, G A; Rhead, W; Lichter-Konecki, U; Bartley, J; Feigenbaum, A; Longo, N; Berquist, W; Berry, S A; Gallagher, R; Bartholomew, D; Harding, C O; Korson, M S; McCandless, S E; Smith, W; Vockley, J; Bart, S; Kronn, D; Zori, R; Cederbaum, S; Dorrani, N; Merritt, J L; Sreenath-Nagamani, Sandesh; Summar, M; Lemons, C; Dickinson, K; Coakley, D F; Moors, T L; Lee, B; Scharschmidt, B F

2012-11-01

We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6-17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8 g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7 g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% to 5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r = 0.730, p < 0.001) or as total 24-hour excretion (r = 0.791 p<0.001), followed by plasma phenylacetylglutamine AUC(24-hour), plasma phenylacetic acid AUC(24-hour) and phenylbutyric acid AUC(24-hour). Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring. Copyright © 2012 Elsevier Inc. All rights reserved.

Fate of microbial metabolites of hydrocarbons in a coastal plain aquifer: The role of electron acceptors

USGS Publications Warehouse

Cozzarelli, I.M.; Herman, J.S.; Baedecker, M. Jo

1995-01-01

A combined field and laboratory study was undertaken to understand the distribution and geochemical conditions that influence the prevalence of low molecular weight organic acids in groundwater of a shallow aquifer contaminated with gasoline. Aromatic hydrocarbons from gasoline were degraded by microbially mediated oxidation-reduction reactions, including reduction of nitrate, sulfate, and Fe(III). The biogeochemical reactions changed overtime in response to changes in the hydrogeochemical conditions in the aquifer. Aliphatic and aromatic organic acids were associated with hydrocarbon degradation in anoxic zones of the aquifer. Laboratory microcosms demonstrated that the biogeochemical fate of specific organic acids observed in groundwater varied with the structure of the acid and the availability of electron acceptors. Benzoic and phenylacetic acid were degraded by indigenous aquifer microorganisms when nitrate was supplied as an electron acceptor. Aromatic acids with two or more methyl substituants on the benzene ring persisted under nitrate-reducing conditions. Although iron reduction and sulfate reduction were important processes in situ and occurred in the microcosms, these reactions were not coupled to the biological oxidation of aromatic organic acids that were added to the microcosms as electron donors. ?? 1995 American Chemical Society.

Microbial degradation of [C14C]polystyrene and 1,3-diphenylbutane.

PubMed

Sielicki, M; Focht, D D; Martin, J P

1978-07-01

Microbial degradation of [beta-14C]polystyrene and 1,3-diphenylbutane, a compound structurally representing the smallest repeating unit of styrene (dimer), was investigated in soil and liquid enrichment cultures. Degradation rates in soil, as determined by 14CO2 evolution from applied [14C]polystyrene, varied from 1.5 to 3.0% for a 4-month period. Although relatively low, these percentages were 15 to 30 times greater than values previously reported. Enrichment cultures, containing 1,3-diphenylbutane as the only carbon souce, were used to determine the mechanisms of microbial oxidation of the polymer chain ends. Metabolism of 1,3-diphenylbutane appeared to involve the attack by a monooxygenease to form 2-phenyl-4-hydroxyphenylbutane followed by a further oxidation and subsequent fission of the benzene ring to yield 4-phenylvaleric acid and an unidentified 5-carbon fragment via the classic meta-fission pathway. Phenylacetic acid was probably formed from 4-phenylvaleric acid by subsequent beta-oxidation of the side chain, methyl-oxidation and decarboxylation. An initial examination of the population of microorganisms in the diphenylbutane enrichment cultures indicated that these oxidative reactions are carried out by common soil microorganism of the genera Bacillus, Pseudomonas, Micrococcus, and Nocardia.

Identification of signatory secondary metabolites during mycoparasitism of Rhizoctonia solani by Stachybotrys elegans

PubMed Central

Chamoun, Rony; Aliferis, Konstantinos A.; Jabaji, Suha

2015-01-01

Stachybotrys elegans is able to parasitize the fungal plant pathogen Rhizoctonia solani AG-3 following a complex and intimate interaction, which, among others, includes the production of cell wall-degrading enzymes, intracellular colonization, and expression of pathogenic process encoding genes. However, information on the metabolome level is non-existent during mycoparasitism. Here, we performed a direct-infusion mass spectrometry (DIMS) metabolomics analysis using an LTQ Orbitrap analyzer in order to detect changes in the profiles of induced secondary metabolites of both partners during this mycoparasitic interaction 4 and 5 days following its establishment. The diketopiperazine(s) (DKPs) cyclo(S-Pro-S-Leu)/cyclo(S-Pro-S-Ile), ethyl 2-phenylacetate, and 3-nitro-4-hydroxybenzoic acid were detected as the primary response of Rhizoctonia 4 days following dual-culturing with Stachybotrys, whereas only the latter metabolite was up-regulated 1 day later. On the other hand, trichothecenes and atranones were mycoparasite-derived metabolites identified during mycoparasitism 4 and 5 days following dual-culturing. All the above secondary metabolites are known to exhibit bioactivity, including fungitoxicity, and represent key elements that determine the outcome of the interaction being studied. Results could be further exploited in programs for the evaluation of the bioactivity of these metabolites per se or their chemical analogs, and/or genetic engineering programs to obtain more efficient mycoparasite strains with improved efficacy and toxicological profiles. PMID:25972848

Isolation and characterization of a newly identified impurity in methamphetamine synthesized via reductive amination of 1-phenyl-2-propanone (P2P) made from phenylacetic acid/lead (II) acetate.

PubMed

Toske, Steven G; McConnell, Jennifer B; Brown, Jaclyn L; Tuten, Jennifer M; Miller, Erin E; Phillips, Monica Z; Vazquez, Etienne R; Lurie, Ira S; Hays, Patrick A; Guest, Elizabeth M

2017-03-01

A trace processing impurity found in certain methamphetamine exhibits was isolated and identified as trans-N-methyl-4-methyl-5-phenyl-4-penten-2-amine hydrochloride (1). It was determined that this impurity was produced via reductive amination of trans-4-methyl-5-phenyl-4-penten-2-one (4), which was one of a cluster of related ketones generated during the synthesis of 1-phenyl-2-propanone (P2P) from phenylacetic acid and lead (II) acetate. This two-step sequence resulted in methamphetamine containing elevated levels of 1. In contrast, methamphetamine produced from P2P made by other methods produced insignificant (ultra-trace or undetectable) amounts of 1. These results confirm that 1 is a synthetic marker compound for the phenylacetic acid and lead (II) acetate method. Analytical data for 1 and 4, and a postulated mechanism for the production of 4, are presented. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Current state of knowledge of hepatic encephalopathy (part I): newer treatment strategies for hyperammonemia in liver failure.

PubMed

Kristiansen, Rune Gangsoy

2016-12-01

Alterations in interorgan metabolism of ammonia play an important role in the onset of hyperammonemia in liver failure. Glutamine synthetase (GS) in muscle is an important target for ammonia removal strategies in hyperammonemia. Ornithine Phenylacetate (OP) is hypothesized to remove ammonia by providing glutamate as a substrate for increased GS activity and hence glutamine production. The newly generated glutamine conjugates with phenylacetate forming phenylacetylglutamine which can be excreted in the urine, providing an excretion pathway for ammonia. We have also shown that OP targets glycine metabolism, providing an additional ammonia reducing effect.

The Small RNA ncS35 Regulates Growth in Burkholderia cenocepacia J2315

PubMed Central

Kiekens, Sanne; Sass, Andrea; Van Nieuwerburgh, Filip; Deforce, Dieter

2018-01-01

ABSTRACT Burkholderia cenocepacia J2315 is a member of the B. cepacia complex. It has a large genome with three replicons and one plasmid; 7,261 genes code for annotated proteins, while 113 code for functional RNAs. Small regulatory RNAs of B. cenocepacia have not yet been functionally characterized. We investigated a small regulatory RNA, designated ncS35, that was discovered by differential RNA sequencing. Its expression under various conditions was quantified, and a deletion mutant, ΔncS35, was constructed. Compared to planktonic growth in a rich medium, the expression of ncS35 was elevated when B. cenocepacia J2315 was grown in biofilms and in minimal medium. Cells of the deletion mutant showed increased aggregation, higher metabolic activity, a higher growth rate, and an increased susceptibility to tobramycin. A transcriptomic analysis revealed upregulation of the phenylacetic acid and tryptophan degradation pathways in ΔncS35. Computational target prediction indicated that ncS35 likely interacts with the first gene of the tryptophan degradation pathway. Overall, we demonstrated that small RNA ncS35 is a noncoding RNA with an attenuating effect on the metabolic rate and growth. It is possible that slower growth protects B. cenocepacia J2315 against stressors acting on fast-dividing cells and enhances survival under unfavorable conditions. IMPORTANCE Small RNAs play an important role in the survival of bacteria in diverse environments. We explored the physiological role of ncS35, a small RNA expressed in B. cenocepacia J2315, an opportunistic pathogen in cystic fibrosis patients. In cystic fibrosis patients, infections can lead to “cepacia syndrome,” a rapidly progressing and often fatal pneumonia. Infections with Burkholderia spp. are difficult to threat with antibiotics because of their high intrinsic resistance and ability to form biofilms. We show that ncS35 attenuates the growth and reduces the metabolic rate of B. cenocepacia and influences biofilm structure. This demonstrates that as-yet-uncharacterized small RNAs with regulatory function can influence physiological traits of B. cenocepacia that are relevant for infection. PMID:29359187

A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory.

PubMed

Nogales, Juan; Palsson, Bernhard Ø; Thiele, Ines

2008-09-16

Pseudomonas putida is the best studied pollutant degradative bacteria and is harnessed by industrial biotechnology to synthesize fine chemicals. Since the publication of P. putida KT2440's genome, some in silico analyses of its metabolic and biotechnology capacities have been published. However, global understanding of the capabilities of P. putida KT2440 requires the construction of a metabolic model that enables the integration of classical experimental data along with genomic and high-throughput data. The constraint-based reconstruction and analysis (COBRA) approach has been successfully used to build and analyze in silico genome-scale metabolic reconstructions. We present a genome-scale reconstruction of P. putida KT2440's metabolism, iJN746, which was constructed based on genomic, biochemical, and physiological information. This manually-curated reconstruction accounts for 746 genes, 950 reactions, and 911 metabolites. iJN746 captures biotechnologically relevant pathways, including polyhydroxyalkanoate synthesis and catabolic pathways of aromatic compounds (e.g., toluene, benzoate, phenylacetate, nicotinate), not described in other metabolic reconstructions or biochemical databases. The predictive potential of iJN746 was validated using experimental data including growth performance and gene deletion studies. Furthermore, in silico growth on toluene was found to be oxygen-limited, suggesting the existence of oxygen-efficient pathways not yet annotated in P. putida's genome. Moreover, we evaluated the production efficiency of polyhydroxyalkanoates from various carbon sources and found fatty acids as the most prominent candidates, as expected. Here we presented the first genome-scale reconstruction of P. putida, a biotechnologically interesting all-surrounder. Taken together, this work illustrates the utility of iJN746 as i) a knowledge-base, ii) a discovery tool, and iii) an engineering platform to explore P. putida's potential in bioremediation and bioplastic production.

Analysis of Ethyl Acetate Extract of Enzymatic Hydrolysate from High Purity Oleuropein and DPPH Radical Scavenging Capacity

NASA Astrophysics Data System (ADS)

Yuan, Jiaojiao; Li, Bing; Qin, Frank G. F.; Tu, Junling

2018-01-01

High purify oleuropein (81.04% OL) was hydrolyzed by hemicellulase and phenols was existed in the ethyl acetate extract of enzymatic hydrolysate (EAE). The results presented that there were hydroxytyrosol (HT), tyrosol, caffeic acid, 3,4-dihydroxybenzoic acid, 3,4-dihydroxy phenylacetic acid in EAE by HPLC, and HT content was 19.36%. Antioxidant activities (DPPH radical scavenging capacity) were all added as the samples concentration increased, and dose-effect relationships also existed. HT possessed the highest DPPH radical scavenging capacity, followed by Vc, and eugenol, OL, caffeic acid, 3,4-dihydroxy phenylacetic acid and 3,4-dihydroxybenzoic acid.

Spectroscopic studies of the reaction between bovine serum amine oxidase (copper-containing) and some hydrazides and hydrazines.

PubMed Central

Morpurgo, L; Befani, O; Sabatini, S; Mondovì, B; Artico, M; Corelli, F; Massa, S; Stefancich, G; Avigliano, L

1988-01-01

The carbonyl cofactor of bovine serum amine oxidase, recently identified as pyrroloquinoline quinone [Ameyama, Hayashi, Matsushita, Shinagawa & Adachi (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, Jongejan, Frank & Duine (1984) FEBS Lett. 170, 305-309], reacts stoichiometrically and irreversibly with hydrazides of phenylacetic acid and of benzoic acid. With the phenylacetic hydrazides a reversible intermediate step was detected by competition with substrate, carbonylic reagents or phenylhydrazine, a typical inhibitor of the enzyme. All hydrazides form an intense broad band with maximum absorbance in a narrow wavelength range (350-360 nm), irrespective of the acyl group, suggesting that the transition is located on the organic cofactor. A different situation is found with some phenylhydrazines, where extended conjugation can occur between the cofactor and the phenyl pi-electron system via the azo group, as shown by the lower energy and higher intensity of the transition. In this case the transition is sensitive to substituents in the phenyl ring. The c.d. spectrum of the adducts is influenced by the type of hydrazide (derived from phenylacetic acid or benzoic acid), by pH and by NN-diethyldithiocarbamate binding to copper, probably as a result of shifts of equilibria between hydrazone-azo tautomers. PMID:3146976

Metabolism of 2-phenylethylamine and phenylacetaldehyde by precision-cut guinea pig fresh liver slices.

PubMed

Panoutsopoulos, Georgios I; Kouretas, Demetrios; Gounaris, Elias G; Beedham, Christine

2004-01-01

2-Phenylethylamine is an endogenous constituent of human brain and is implicated in cerebral transmission. It is also found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalyzed by monoamine oxidase and the oxidation of the reactive aldehyde to its acid derivative is catalyzed mainly by aldehyde dehydrogenase and perhaps aldehyde oxidase, with xanthine oxidase having minimal transformation. The present investigation examines the metabolism of 2-phenylethylamine to phenylacetaldehyde in liver slices and compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity in the oxidation of phenylacetaldehyde with precision-cut fresh liver slices in the presence/absence of specific inhibitors of each enzyme. In liver slices, phenylacetaldehyde was rapidly converted to phenylacetic acid. Phenylacetic acid was the main metabolite of 2-phenylethylamine, via the intermediate phenylacetaldehyde. Phenylacetic acid formation was completely inhibited by disulfiram (specific inhibitor of aldehyde dehydrogenase), whereas isovanillin (specific inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (specific inhibitor of xanthine oxidase) had little or no effect. Therefore, in liver slices, phenylacetaldehyde is rapidly oxidized by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.

Simultaneous LC-MS/MS determination of phenylbutyrate, phenylacetate benzoate and their corresponding metabolites phenylacetylglutamine and hippurate in blood and urine.

PubMed

Laryea, Maurice D; Herebian, Diran; Meissner, Thomas; Mayatepek, Ertan

2010-12-01

Inborn errors of urea metabolism result in hyperammonemia. Treatment of urea cycle disorders can effectively lower plasma ammonium levels and results in survival in the majority of patients. Available medications for treating urea cycle disorders include sodium benzoate (BA), sodium phenylacetate (PAA), and sodium phenylbutyrate (PBA) and are given to provide alternate routes for disposition of waste nitrogen excretion. In this study, we develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of benzoic acid, phenylacetic acid, phenylbutyric acid, phenylacetylglutamine, and hippuric acid in plasma and urine from children with inborn errors of urea synthesis. Plasma extracts and diluted urine samples were injected on a reverse-phase column and identified and quantified by selected reaction monitoring (SRM) in negative ion mode. Deuterated analogues served as internal standards. Analysis time was 7 min. Assay precision, accuracy, and linearity and sample stability were determined using enriched samples. Quantification limits of the method were 100 ng/ml (0.3-0.8 μmol/L) for all analytes, and recoveries were >90%. Inter- and intraday relative standard deviations were <10%. Our newly developed LC-MS/MS represents a robust, sensitive, and rapid method that allows simultaneous determination of the five compounds in plasma and urine.

Penicillins and other acylamino compounds synthesized by the cell-bound penicillin acylase of Escherichia coli

PubMed Central

Cole, M.

1969-01-01

1. The penicillin acylase of Eschericha coli N.C.I.B. 8743 is a reversible enzyme. Reaction rates for the two directions have been determined. 2. Measurements of the rates of enzymic synthesis of penicillins from 6-aminopenicillanic acid and various carboxylic acids revealed that p-hydroxyphenylacetic acid was the best substrate, followed by phenylacetic, 2-thienylacetic, substituted phenylacetic, 3-hexenoic and n-hexanoic acids. 3. The rate of synthesis of penicillin improved when amides or N-acylglycines were used; α-aminobenzylpenicillin and phenoxymethylpenicillin were only synthesized when using these more energy-rich compounds. 4. Phenyl-acetylglycine was the best substrate for the synthesis of benzylpenicillin compared with other derivatives of phenylacetic acid. 5. The enzyme was specific for acyl-l-amino acids, benzylpenicillin being synthesized from phenylacetyl-l-α-aminophenylacetic acid but not from phenylacetyl-d-α-aminophenylacetic acid. 6. α-Phenoxyethylpenicillin was synthesized from 6-aminopenicillanic acid and α-phenoxypropionylthioglycollic acid non-enzymically, but the rate was faster in the presence of the enzyme. 7. The E. coli acylase catalysed the acylation of hydroxylamine by acids or amides to give hydroxamic acids, the phenylacetyl group being the most suitable acyl group. The enzyme also catalysed other acyl-group transfers. PMID:4982418

Biodegradation of alkaline lignin by Bacillus ligniniphilus L1

DOE PAGES

Zhu, Daochen; Zhang, Peipei; Xie, Changxiao; ...

2017-02-21

Lignin is the most abundant aromatic biopolymer in the biosphere and it comprises up to 30% of plant biomass. Although lignin is the most recalcitrant component of the plant cell wall, still there are microorganisms able to decompose it or degrade it. Fungi are recognized as the most widely used microbes for lignin degradation. However, bacteria have also been known to be able to utilize lignin as a carbon or energy source. Bacillus ligniniphilus L1 was selected in this study due to its capability to utilize alkaline lignin as a single carbon or energy source and its excellent ability tomore » survive in extreme environments. To investigate the aromatic metabolites of strain L1 decomposing alkaline lignin, GC–MS analysis was performed and fifteen single phenol ring aromatic compounds were identified. The dominant absorption peak included phenylacetic acid, 4-hydroxy-benzoicacid, and vanillic acid with the highest proportion of metabolites resulting in 42%. Comparison proteomic analysis was carried out for further study showed that approximately 1447 kinds of proteins were produced, 141 of which were at least twofold up-regulated with alkaline lignin as the single carbon source. The up-regulated proteins contents different categories in the biological functions of protein including lignin degradation, ABC transport system, environmental response factors, protein synthesis, assembly, etc. In conclusion, GC–MS analysis showed that alkaline lignin degradation of strain L1 produced 15 kinds of aromatic compounds. Comparison proteomic data and metabolic analysis showed that to ensure the degradation of lignin and growth of strain L1, multiple aspects of cells metabolism including transporter, environmental response factors, and protein synthesis were enhanced. Based on genome and proteomic analysis, at least four kinds of lignin degradation pathway might be present in strain L1, including a Gentisate pathway, the benzoic acid pathway and the β-ketoadipate pathway. The study provides an important basis for lignin degradation by bacteria.« less

Biodegradation of alkaline lignin by Bacillus ligniniphilus L1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Daochen; Zhang, Peipei; Xie, Changxiao

Background: Lignin is the most abundant aromatic biopolymer in the biosphere and it comprises up to 30% of plant biomass. Although lignin is the most recalcitrant component of the plant cell wall, still there are microorganisms able to decompose it or degrade it. Fungi are recognized as the most widely used microbes for lignin degradation. However, bacteria have also been known to be able to utilize lignin as a carbon or energy source. Bacillus ligniniphilus L1 was selected in this study due to its capability to utilize alkaline lignin as a single carbon or energy source and its excellent abilitymore » to survive in extreme environments. Results: To investigate the aromatic metabolites of strain L1 decomposing alkaline lignin, GC-MS analyze was performed and fifteen single phenol ring aromatic compounds were identified. The dominant absorption peak included phenylacetic acid, 4-hydroxy-benzoicacid, and vanillic acid with the highest proportion of metabolites resulting in 42%. Comparison proteomic analysis were carried out for further study showed that approximately 1447 kinds of proteins were produced, 141 of which were at least 2-fold up-regulated with alkaline lignin as the single carbon source. The up-regulated proteins contents different categories in the biological functions of protein including lignin degradation, ABC transport system, environmental response factors, protein synthesis and assembly, etc. Conclusions: GC-MS analysis showed that alkaline lignin degradation of strain L1 produced 15 kinds of aromatic compounds. Comparison proteomic data and metabolic analysis showed that to ensure the degradation of lignin and growth of strain L1, multiple aspects of cells metabolism including transporter, environmental response factors, and protein synthesis were enhanced. Based on genome and proteomic analysis, at least four kinds of lignin degradation pathway might be present in strain L1, including a Gentisate pathway, the benzoic acid pathway and the β-ketoadipate pathway. The study provides an important basis for lignin degradation by bacteria.« less

Biodegradation of alkaline lignin by Bacillus ligniniphilus L1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Daochen; Zhang, Peipei; Xie, Changxiao

Lignin is the most abundant aromatic biopolymer in the biosphere and it comprises up to 30% of plant biomass. Although lignin is the most recalcitrant component of the plant cell wall, still there are microorganisms able to decompose it or degrade it. Fungi are recognized as the most widely used microbes for lignin degradation. However, bacteria have also been known to be able to utilize lignin as a carbon or energy source. Bacillus ligniniphilus L1 was selected in this study due to its capability to utilize alkaline lignin as a single carbon or energy source and its excellent ability tomore » survive in extreme environments. To investigate the aromatic metabolites of strain L1 decomposing alkaline lignin, GC–MS analysis was performed and fifteen single phenol ring aromatic compounds were identified. The dominant absorption peak included phenylacetic acid, 4-hydroxy-benzoicacid, and vanillic acid with the highest proportion of metabolites resulting in 42%. Comparison proteomic analysis was carried out for further study showed that approximately 1447 kinds of proteins were produced, 141 of which were at least twofold up-regulated with alkaline lignin as the single carbon source. The up-regulated proteins contents different categories in the biological functions of protein including lignin degradation, ABC transport system, environmental response factors, protein synthesis, assembly, etc. In conclusion, GC–MS analysis showed that alkaline lignin degradation of strain L1 produced 15 kinds of aromatic compounds. Comparison proteomic data and metabolic analysis showed that to ensure the degradation of lignin and growth of strain L1, multiple aspects of cells metabolism including transporter, environmental response factors, and protein synthesis were enhanced. Based on genome and proteomic analysis, at least four kinds of lignin degradation pathway might be present in strain L1, including a Gentisate pathway, the benzoic acid pathway and the β-ketoadipate pathway. The study provides an important basis for lignin degradation by bacteria.« less

Photo-fenton degradation of diclofenac: identification of main intermediates and degradation pathway.

PubMed

Pérez-Estrada, Leónidas A; Malato, Sixto; Gernjak, Wolfgang; Agüera, Ana; Thurman, E Michael; Ferrer, Imma; Fernández-Alba, Amadeo R

2005-11-01

In recent years, the presence of pharmaceuticals in the aquatic environment has been of growing interest. These new contaminants are important because many of them are not degraded under the typical biological treatments applied in the wastewater treatment plants and represent a continuous input into the environment. Thus, compounds such as diclofenac are present in surface waters in all Europe and a crucial need for more enhanced technologies that can reduce its presence in the environment has become evident. In this sense, advanced oxidation processes (AOPs) represent a good choice for the treatment of hazardous nonbiodegradable pollutants. This work deals with the solar photodegradation of diclofenac, an antiinflammatory drug, in aqueous solutions by photo-Fenton reaction. A pilot-scale facility using a compound parabolic collector (CPC) reactor was used for this study. Results obtained show rapid and complete oxidation of diclofenac after 60 min, and total mineralization (disappearance of dissolved organic carbon, DOC) after 100 min of exposure to sunlight. Although diclofenac precipitates during the process at low pH, its degradation takes place in the homogeneous phase governed by a precipitation-redissolution-degradation process. Establishment of the reaction pathway was made possible by a thorough analysis of the reaction mixture identifying the main intermediate products generated. Gas chromatography-mass spectrometry (GC/ MS) and liquid chromatography coupled with time-of-flight mass spectrometry (LC/TOF-MS) were used to identify 18 intermediates, in two tentative degradation routes. The main one was based on the initial hydroxylation of the phenylacetic acid moiety in the C-4 position and subsequent formation of a quinone imine derivative that was the starting point for further multistep degradation involving hydroxylation, decarboxylation, and oxidation reactions. An alternative route was based on the transient preservation of the biphenyl amino moiety that underwent a similar oxidative process of C-N bond cleavage. The proposed degradation route differs from those previously reported involving alternative degradation processes (ozonization, UV/H2O2, or photolysis), indicating that diclofenac degradation follows different pathways, depending on the treatment applied.

Resolving the smell of wood - identification of odour-active compounds in Scots pine (Pinus sylvestris L.).

PubMed

Schreiner, Linda; Bauer, Patrick; Buettner, Andrea

2018-05-29

Being one of the most common trees in forests, Pinus sylvestris L. is a frequently used raw material for wood products. Its specific odour is, however, mostly unresolved to date. Accordingly, we investigated Scots pine wood samples grown in Germany for their main odorant composition. We employed dedicated odorant analysis techniques such as gas chromatography-olfactometry (GC-O) and aroma extract dilution analysis (AEDA) and successfully detected 44 odour-active compounds; of these, 39 substances were successfully identified by gas chromatography-mass spectrometry/olfactometry (GC-MS/O) and two-dimensional gas chromatography-mass spectrometry/olfactometry (2D-GC-MS/O). Among the main odorants found were (E,E)-nona-2,4-dienal, vanillin, phenylacetic acid, 3-phenylpropanoic acid, δ-octalactone and α-pinene, all of them having been detected with high flavour dilution factors during GC-O analyses. The majority of the identified odorants were fatty acid degradation products, plus some terpenoic substances and odorous substances resulting from the degradation of lignin. Although some of the detected substances have previously been reported as constituents of wood, 11 substances are reported here for the first time as odour-active compounds in wood, amongst them heptanoic acid, γ-octalactone, δ-nonalactone and (E,Z,Z)-trideca-2,4,7-trienal.

Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis.

PubMed

Kim, Young Hwan; Cho, Kun; Yun, Sung-Ho; Kim, Jin Young; Kwon, Kyung-Hoon; Yoo, Jong Shin; Kim, Seung Il

2006-02-01

Proteomic analysis of Pseudomonas putida KT2440 cultured in monocyclic aromatic compounds was performed using 2-DE/MS and cleavable isotope-coded affinity tag (ICAT) to determine whether proteins involved in aromatic compound degradation pathways were altered as predicted by genomic analysis (Jiménez et al., Environ Microbiol. 2002, 4, 824-841). Eighty unique proteins were identified by 2-DE/MS or MS/MS analysis from P. putida KT2440 cultured in the presence of six different organic compounds. Benzoate dioxygenase (BenA, BenD) and catechol 1,2-dioxygenase (CatA) were induced by benzoate. Protocatechuate 3,4-dixoygenase (PcaGH) was induced by p-hydroxybenzoate and vanilline. beta-Ketoadipyl CoA thiolase (PcaF) and 3-oxoadipate enol-lactone hydrolase (PcaD) were induced by benzoate, p-hydroxybenzoate and vanilline, suggesting that benzoate, p-hydroxybenzoate and vanilline were degraded by different dioxygenases and then converged in the same beta-ketoadipate degradation pathway. An additional 110 proteins, including 19 proteins from 2-DE analysis, were identified by cleavable ICAT analysis for benzoate-induced proteomes, which complemented the 2-DE results. Phenylethylamine exposure induced beta-ketoacyl CoA thiolase (PhaD) and ring-opening enzyme (PhaL), both enzymes of the phenylacetate (pha) biodegradation pathway. Phenylalanine induced 4-hydroxyphenyl-pyruvate dioxygenase (Hpd) and homogentisate 1,2-dioxygenase (HmgA), key enzymes in the homogentisate degradation pathway. Alkyl hydroperoxide reductase (AphC) was induced under all aromatic compounds conditions. These results suggest that proteome analysis complements and supports predictive information obtained by genomic sequence analysis.

Regulation of sphingomyelin phosphodiesterase acid-like 3A gene (SMPDL3A) by liver X receptors.

PubMed

Noto, Paul B; Bukhtiyarov, Yuri; Shi, Meng; McKeever, Brian M; McGeehan, Gerard M; Lala, Deepak S

2012-10-01

Liver X receptor (LXR) α and LXRβ function as physiological sensors of cholesterol metabolites (oxysterols), regulating key genes involved in cholesterol and lipid metabolism. LXRs have been extensively studied in both human and rodent cell systems, revealing their potential therapeutic value in the contexts of atherosclerosis and inflammatory diseases. The LXR genome landscape has been investigated in murine macrophages but not in human THP-1 cells, which represent one of the frequently used monocyte/macrophage cell systems to study immune responses. We used a whole-genome screen to detect direct LXR target genes in THP-1 cells treated with two widely used LXR ligands [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317) and 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy] phenylacetic acid hydrochloride (GW3965)]. This screen identified the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene as a novel LXR-regulated gene, with an LXR response element within its promoter. We investigated the regulation of SMPDL3A gene expression by LXRs across several human and mouse cell types. These studies indicate that the induction of SMPDL3A is LXR-dependent and is restricted to human blood cells with no induction observed in mouse cellular systems.

Relationship between human paraoxonase-1 activity and PON1 polymorphisms in Mexican workers exposed to organophosphate pesticides.

PubMed

López-Flores, I; Lacasaña, M; Blanco-Muñoz, J; Aguilar-Garduño, C; Sanchez-Villegas, P; Pérez-Méndez, O A; Gamboa-Avila, R

2009-07-24

Paraoxonase-1 (PON1) is a serum enzyme which catalyzes the hydrolysis of organophosphate pesticides. In this study we conducted a cross-sectional study and reported on the distribution of three common genetic polymorphisms of the PON1 gene in a population of floriculture workers from Mexico as well as the association between those polymorphisms and other predictors with serum PON1 activity on paraoxon, diazoxon and phenylacetate. The genotype frequencies at position PON1(55) were 89% (LL), 10% (LM) and 0.6% (MM), at position PON1(192) they were 16% (QQ), 47% (QR) and 37% (RR), and 26% (TT), 42% (TC) and 32% (CC) at position PON1(-108). Thus, the frequencies of alleles L, Q and T were 0.94, 0.40 and 0.47, respectively. The PON1(55) polymorphism had no significant effect on serum PON1 activity on any substrate. We found a significant association between the PON1(192) polymorphism and PON1 activity towards paraoxon and diazoxon, which increased in genotypes as follows: 192RR>192QR>192QQ for paraoxonase activity and, inversely, 192QQ>192QR>192RR for diazoxonase activity. The PON1(-108) polymorphism also had a significant effect on PON1 activity level towards paraoxon in the following order among the genotype groups: -108CC>-108TC>-108TT. Serum PON1 activity towards diazoxon was not associated with the PON1(-108) polymorphism but it was influenced by the intensity exposure to pesticides at the floriculture industry and the years of the occupational exposure to pesticides. No polymorphism significantly influenced serum PON1 activity on phenylacetate.

Comparative study on cytogenetic damage induced by homo-aza-steroidal esters in human lymphocytes.

PubMed

Mourelatos, D; Papageorgiou, A; Boutis, L; Catsoulacos, P

1995-02-01

The effect of P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of 3 beta-hydroxy-N-methyl-17 alpha-aza-D-homo-5 alpha-androstan-17-one (compound 3) and 3 beta-hydroxy-17 alpha-aza-D-homo-5 alpha-androstane (compound 2) on sister-chromatid exchange (SCE) frequencies and on human lymphocytes proliferation kinetics was studied. The results are compared with those of the P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of 3 beta-hydroxy-17 alpha-aza-D-homo-5 alpha-androstan-17-one (compound 1). All compounds were found to be active in inducing markedly increased SCE rates and cell division delays. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumour activity of these compounds was observed.

A Patient with MSUD: Acute Management with Sodium Phenylacetate/Sodium Benzoate and Sodium Phenylbutyrate.

PubMed

Köse, Melis; Canda, Ebru; Kagnici, Mehtap; Uçar, Sema Kalkan; Çoker, Mahmut

2017-01-01

In treatment of metabolic imbalances caused by maple syrup urine disease (MSUD), peritoneal dialysis, and hemofiltration, pharmacological treatments for elimination of toxic metabolites can be used in addition to basic dietary modifications. Therapy with sodium phenylacetate/benzoate or sodium phenylbutyrate (NaPB) in urea-cycle disorder cases has been associated with a reduction in branched-chain amino acid (BCAA) concentrations when the patients are on adequate dietary protein intake. Moreover, NaPB in treatment of MSUD patients is also associated with reduction of BCAA levels in a limited number of cases. However, there are not enough studies in the literature about application and efficacy of this treatment. Our case report sets an example of an alternative treatment's efficacy when extracorporeal procedures are not available due to technical difficulties during attack period of the disease.

A Patient with MSUD: Acute Management with Sodium Phenylacetate/Sodium Benzoate and Sodium Phenylbutyrate

PubMed Central

Canda, Ebru; Kagnici, Mehtap; Uçar, Sema Kalkan; Çoker, Mahmut

2017-01-01

In treatment of metabolic imbalances caused by maple syrup urine disease (MSUD), peritoneal dialysis, and hemofiltration, pharmacological treatments for elimination of toxic metabolites can be used in addition to basic dietary modifications. Therapy with sodium phenylacetate/benzoate or sodium phenylbutyrate (NaPB) in urea-cycle disorder cases has been associated with a reduction in branched-chain amino acid (BCAA) concentrations when the patients are on adequate dietary protein intake. Moreover, NaPB in treatment of MSUD patients is also associated with reduction of BCAA levels in a limited number of cases. However, there are not enough studies in the literature about application and efficacy of this treatment. Our case report sets an example of an alternative treatment's efficacy when extracorporeal procedures are not available due to technical difficulties during attack period of the disease. PMID:28589054

The effect of morphine on the biosynthesis of catecholamines in the rat brain.

PubMed

Malini, M; Kwan, T K; Perumal, R

1994-02-01

In vivo studies involved monitoring the effect of morphine administration on catecholamine biosynthesis by the brain while in vitro studies involved studying the effect of morphine on the uptake of tritiated tyrosine by synaptosomes and its subsequent incorporation into the catecholamines. The extremely low levels of these endogenous compounds required the use of High Performance Liquid Chromatography with electrochemical detection. Intra-peritoneal injection of morphine at a dosage of 10 mg/kg did not produce appreciable changes in the catecholamine levels but a dosage of 30 mg/kg morphine was found to elevate dihydroxy phenylacetic acid content. At a dosage of 60 mg/kg, dopamine levels were elevated while noradrenaline was depleted. Morphine, at a concentration of 1 x 10(-5)M increases the incorporation of tritiated tyrosine into dopamine and dihydroxy phenylacetic acid in synaptosomal preparations.

Methyl jasmonate stimulates biosynthesis of 2-phenylethylamine, phenylacetic acid and 2-phenylethanol in seedlings of common buckwheat.

PubMed

Horbowicz, Marcin; Wiczkowski, Wiesław; Sawicki, Tomasz; Szawara-Nowak, Dorota; Sytykiewicz, Hubert; Mitrus, Joanna

2015-01-01

Methyl jasmonate has a strong effect on secondary metabolizm in plants, by stimulating the biosynthesis a number of phenolic compounds and alkaloids. Common buckwheat (Fagopyrum esculentum Moench) is an important source of biologically active compounds. This research focuses on the detection and quantification of 2-phenylethylamine and its possible metabolites in the cotyledons, hypocotyl and roots of common buckwheat seedlings treated with methyl jasmonate. In cotyledons of buckwheat sprouts, only traces of 2-phenylethylamine were found, while in the hypocotyl and roots its concentration was about 150 and 1000-times higher, respectively. Treatment with methyl jasmonate resulted in a 4-fold increase of the 2-phenylethylamine level in the cotyledons of 7-day buckwheat seedlings, and an 11-fold and 5-fold increase in hypocotyl and roots, respectively. Methyl jasmonate treatment led also to about 4-fold increase of phenylacetic acid content in all examined seedling organs, but did not affect the 2-phenylethanol level in cotyledons, and slightly enhanced in hypocotyl and roots. It has been suggested that 2-phenylethylamine is a substrate for the biosynthesis of phenylacetic acid and 2-phenylethanol, as well as cinnamoyl 2-phenethylamide. In organs of buckwheat seedling treated with methyl jasmonate, higher amounts of aromatic amino acid transaminase mRNA were found. The enzyme can be involved in the synthesis of phenylpyruvic acid, but the presence of this compound could not be confirmed in any of the examined organs of common buckwheat seedling.

Phenylacetic acid co-crystals with acridine, caffeine, isonicotinamide and nicotinamide: Crystal structures, thermal analysis, FTIR spectroscopy and Hirshfeld surface analysis

NASA Astrophysics Data System (ADS)

Amombo Noa, Francoise M.; Jacobs, Ayesha

2017-07-01

Co-crystals of phenylacetic acid (PAA) with acridine (ACR), caffeine (CAF), isonicotinamide (INM) and nicotinamide (NAM) have been successfully prepared and characterised by single crystal X-ray diffraction, FTIR spectroscopy, thermal analysis and Hirshfeld surface analysis. The ACR, INM and NAM co-crystals with PAA exhibit the carboxylic acid-pyridine heterosynthon. Furthermore the amide-amide supramolecular homosynthon is observed in the PAA co-crystals with INM and NAM as well as Nsbnd H⋯O interactions between the acid and the respective base. The CAF co-crystal exhibits hydrogen bonding between the imidazole nitrogen and the COOH group of the PAA. The compounds demonstrate different stoichiometries; for PAA·ACR and PAA·INM a 1:1 ratio is displayed, a 2:1 in 2PAA·CAF and a 2:2 in the case of 2PAA·2NAM.

A comparative study on cytogenetic and antineoplastic effects induced by two modified steroidal alkylating agents.

PubMed

Papageorgiou, A; Tsavdaridis, D; Geromichalos, G D; Camoutsis, C; Karaberis, E; Mourelatos, D; Chrysogelou, E; Houvartas, S; Kotsis, A

2001-01-01

We investigated the effects of two newly synthesized steroidal derivatives of nitrogen mustard on sister chromatid exchange rates and on human lymphocyte proliferation kinetics. The compound 33-hydroxy-5alpha,22alpha-spirostan- 12-one-p-(N,N-bis(2-chloroethyl)amino)phenylacetate(1) was, on a molar basis, less effective in inducing sister chromatid exchange and suppressing cell proliferation rate indices than compound 3beta-hydroxy-12alpha-aza-C-homo-5alpha,22alpha-spirostan-12-one-p-(N,N-bis(2-chloroethyl)amino)phenylacetate(2). A correlation was observed between the magnitude of the sister chromatid exchange response and the depression of cell proliferation index. We also studied the effects of the aforementioned compounds on Lewis lung carcinoma. The order of the percent inhibition of tumor growth achieved by the compounds coincides with the order of the cytogenetic effects they induce.

Biodegradation of Aromatic Compounds by Escherichia coli

PubMed Central

Díaz, Eduardo; Ferrández, Abel; Prieto, María A.; García, José L.

2001-01-01

Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications. PMID:11729263

Evaluation and Development of Radiation Countermeasures at AFRRI

DTIC Science & Technology

2005-01-01

epigallocatechin gallate ( EGCG ), benzyl styryl sulfones, CpG oligonucleotides, a superoxide dismutase mi- metic, statins, dipeptidyl peptidase inhibitors, and...candidate (Hollis-Eden Pharmaceuticals) • Soy isoflavones • Vitamin E-related compounds (Yasoo Health) • Phenylacetate, phenylbutyrate, EGCG (late

Four one-dimensional lanthanide-phenylacetate polymers exhibiting luminescence and magnetic cooling/spin-glass behavior.

PubMed

Li, Zhong-Yi; Xu, Ya-Lan; Zhang, Xiang-Fei; Zhai, Bin; Zhang, Fu-Li; Zhang, Jian-Jun; Zhang, Chi; Li, Su-Zhi; Cao, Guang-Xiu

2017-12-21

Four isostructural lanthanide coordination polymers with a phenylacetate (PAA - ) ligand, [Ln(PAA) 3 (H 2 O)] n (Ln = Eu (1); Gd (2); Tb (3); Dy (4)), were synthesized under hydrothermal conditions. Complexes 1-4 display a one-dimensional (1D) wave chain structure bridged by the carboxylate of the PAA - ligand, which was generated via the in situ decarboxylation of phenylmalonic acid. Magnetic studies suggest the presence of ferromagnetic LnLn coupling in the 1D chain of 1-4. Meanwhile, 2 has a significant cryogenic magnetocaloric effect with the maximum -ΔS m of 26.73 at 3 K and 7 T, and 3 and 4 show interesting spin-glass behavior, which is rarely reported for Ln-containing complexes. Additionally, the solid-state photophysical properties of 1 and 3 display strong characteristic Eu 3+ and Tb 3+ photoluminescence emission in the visible region, indicating that Eu- and Tb-based luminescence are sensitized by the effective energy transfer from the ligand to the metal centers.

Gas chromatography-mass spectrometry evidence for several endogenous auxins in pea seedling organs.

PubMed

Schneider, E A; Kazakoff, C W; Wightman, F

1985-08-01

Qualitative analysis by gas chromatography-mass spectrometry (GC-MS) of the auxins present in the root, cotyledons and epicotyl of 3-dold etiolated pea (Pisum sativum L., cv. Alaska) seedlings has shown that all three organs contain phenylacetic acid (PAA), 3-indoleacetic acid (IAA) and 4-chloro-3-indoleacetic acid (4Cl-IAA). In addition, 3-indolepropionic acid (IPA) was present in the root and 3-indolebutyric acid (IBA) was detected in both root and epicotyl. Phenylacetic acid, IAA and IPA were measured quantitatively in the three organs by GC-MS-single ion monitoring, using deuterated internal standards. Levels of IAA were found to range from 13 to 115 pmol g(-1) FW, while amounts of PAA were considerably higher (347-451 pmol g(-1) FW) and the level of IPA was quite low (5 pmol g(-1) FW). On a molar basis the PAA:IAA ratio in the whole seedling was approx. 15:1.

Anti-Adhesive Activity of Cranberry Phenolic Compounds and Their Microbial-Derived Metabolites against Uropathogenic Escherichia coli in Bladder Epithelial Cell Cultures.

PubMed

de Llano, Dolores González; Esteban-Fernández, Adelaida; Sánchez-Patán, Fernando; Martínlvarez, Pedro J; Moreno-Arribas, Maria Victoria; Bartolomé, Begoña

2015-05-27

Cranberry consumption has shown prophylactic effects against urinary tract infections (UTI), although the mechanisms involved are not completely understood. In this paper, cranberry phenolic compounds and their potential microbial-derived metabolites (such as simple phenols and benzoic, phenylacetic and phenylpropionic acids) were tested for their capacity to inhibit the adherence of uropathogenic Escherichia coli (UPEC) ATCC®53503™ to T24 epithelial bladder cells. Catechol, benzoic acid, vanillic acid, phenylacetic acid and 3,4-dihydroxyphenylacetic acid showed anti-adhesive activity against UPEC in a concentration-dependent manner from 100-500 µM, whereas procyanidin A2, widely reported as an inhibitor of UPEC adherence on uroepithelium, was only statistically significant (p < 0.05) at 500 µM (51.3% inhibition). The results proved for the first time the anti-adhesive activity of some cranberry-derived phenolic metabolites against UPEC in vitro, suggesting that their presence in the urine could reduce bacterial colonization and progression of UTI.

Optimization of Lipase-Mediated Synthesis of 1-Nonene Oxide Using Phenylacetic Acid and Hydrogen Peroxide

PubMed Central

Abdulmalek, Emilia; Arumugam, Mahashanon; Basri, Mahiran; Rahman, Mohd Basyaruddin Abdul

2012-01-01

Herein, an efficient epoxidation of 1-nonene is described. In a simple epoxidation system, commercially available Novozym 435, an immobilized Candida antarctica lipase B, and hydrogen peroxide (H2O2) were utilized to facilitate the in situ oxidation of phenylacetic acid to the corresponding peroxy acid which then reacted with 1-nonene to give 1-nonene oxide with high yield and selectivity. The aliphatic terminal alkene was epoxidised efficiently in chloroform to give an excellent yield (97%–99%) under the optimum reaction conditions, including temperature (35 °C), initial H2O2 concentration (30%), H2O2 amount (4.4 mmol), H2O2 addition rate (one step), acid amount (8.8 mmol), and stirring speed (250 rpm). Interestingly, the enzyme was stable under the single-step addition of H2O2 with a catalytic activity of 190.0 Ug−1. The entire epoxidation process was carried out within 12 h using a conventional water bath shaker. PMID:23202943

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

PubMed

de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Vilchez-Vargas, Ramiro; Pieper, Dietmar H

2016-01-01

Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Phenylbutyrate reduces plasma leucine concentrations without affecting the flux of leucine

USDA-ARS?s Scientific Manuscript database

Phenylbutyrate (PB) has been used as an alternative pathway to excrete nitrogen in urea cycle disorder patients for the last 20 years. PB, after oxidation to phenylacetate, is conjugated with glutamine and excreted in the urine. A reduction in the plasma concentration of branched amino acids (BCAA) ...

21 CFR 1310.04 - Maintenance of records.

Code of Federal Regulations, 2013 CFR

2013-04-01

... kilogram. 8675 Ergonovine and its salts 10 grams. 8676 Ergotamine and its salts 20 grams. 8678 Ethylamine.... 8791 Phenylacetic acid, its esters, and its salts 1 kilogram. 2704 Piperidine and its salts 500 grams. 8750 Piperonal (also called heliotropine) 4 kilograms. 8328 Propionic anhydride 1 gram. 8323 Safrole 4...

21 CFR 1310.04 - Maintenance of records.

Code of Federal Regulations, 2012 CFR

2012-04-01

... kilogram. 8675 Ergonovine and its salts 10 grams. 8676 Ergotamine and its salts 20 grams. 8678 Ethylamine.... 8791 Phenylacetic acid, its esters, and its salts 1 kilogram. 2704 Piperidine and its salts 500 grams. 8750 Piperonal (also called heliotropine) 4 kilograms. 8328 Propionic anhydride 1 gram. 8323 Safrole 4...

21 CFR 1310.04 - Maintenance of records.

Code of Federal Regulations, 2011 CFR

2011-04-01

... kilogram. 8675 Ergonovine and its salts 10 grams. 8676 Ergotamine and its salts 20 grams. 8678 Ethylamine.... 8791 Phenylacetic acid, its esters, and its salts 1 kilogram. 2704 Piperidine and its salts 500 grams. 8750 Piperonal (also called heliotropine) 4 kilograms. 8328 Propionic anhydride 1 gram. 8323 Safrole 4...

21 CFR 1310.04 - Maintenance of records.

Code of Federal Regulations, 2014 CFR

2014-04-01

... kilogram. 8675 Ergonovine and its salts 10 grams. 8676 Ergotamine and its salts 20 grams. 8678 Ethylamine.... 8791 Phenylacetic acid, its esters, and its salts 1 kilogram. 2704 Piperidine and its salts 500 grams. 8750 Piperonal (also called heliotropine) 4 kilograms. 8328 Propionic anhydride 1 gram. 8323 Safrole 4...

Degradation of endogenous and exogenous genes of genetically modified rice with Cry1Ab during food processing.

PubMed

Zhang, Wei; Xing, Fuguo; Selvaraj, Jonathan Nimal; Liu, Yang

2014-05-01

In order to assess the degradation of endogenous and exogenous genes during food processing, genetically modified rice with Cry1Ab was used as raw material to produce 4 processed foods: steamed rice, rice noodles, rice crackers, and sweet rice wine. The results showed various processing procedures caused different degrees of degradation of both endogenous and exogenous genes. During the processing of steamed rice and rice noodles, the procedures were so mild that only genes larger than 1500 bp were degraded, and no degradation of NOS terminator and Hpt gene was detected. For rice crackers, frying was the most severe procedure, followed by microwaving, baking, boiling, 1st drying, and 2nd drying. For sweet rice wine, fermentation had more impact on degradation of genes than the other processing procedures. All procedures in this study did not lead to degradation of genes to below 200 bp, except for NOS terminator. In the case of stability of the genes studied during processing of rice crackers and sweet rice wine, SPS gene was the most, followed by the Cry1Ab gene, Hpt gene, Pubi promoter, and NOS terminator. In our study, we gained some information about the degradation of endogenous and exogenous genes during 4 foods processing, compared the different stabilities between endogenous and exogenous genes, and analyzed different effects of procedure on degradation of genes. In addition, the fragments of endogenous and exogenous genes about 200 bp could be detected in final products, except NOS terminator. As a result, we provided some base information about risk assessment of genetically modified (GM) food and appropriate length of fragment to detect GM component in processed foods. © 2014 Institute of Food Technologists®

Ammonia toxicity and its prevention in inherited defects of the urea cycle.

PubMed

Walker, V

2009-09-01

The urea cycle is the final pathway for removal of surplus nitrogen from the body, and the major route in humans for detoxification of ammonia. The full complement of enzymes is expressed only in liver. Inherited deficiencies of urea cycle enzymes lead to hyperammonaemia, which causes brain damage. Severe defects present with hyperammonaemic crises in neonates. Equally devastating episodes may occur in previously asymptomatic adults with mild defects, most often X-linked ornithine transcarbamylase (OTC) deficiency. Several mechanisms probably contribute to pathogenesis. Treatment aims to reduce plasma ammonia quickly, reduce production of waste nitrogen, dispose of waste nitrogen using alternative pathways to the urea cycle and replace arginine. These therapies have increased survival and probably improve the neurological outcome. Arginine, sodium benzoate, sodium phenylbutyrate and, less often, sodium phenylacetate are used. Long-term correction is achieved by liver transplantation. Gene therapy for OTC deficiency is effective in animals, and work is ongoing to improve persistence and safety.

Successful use of alternate waste nitrogen agents and hemodialysis in a patient with hyperammonemic coma after heart-lung transplantation.

PubMed

Berry, G T; Bridges, N D; Nathanson, K L; Kaplan, P; Clancy, R R; Lichtenstein, G R; Spray, T L

1999-04-01

Lethal hyperammonemic coma has been reported in 2 adults after lung transplantation. It was associated with a massive elevation of brain glutamine levels, while plasma glutamine levels were normal or only slightly elevated. In liver tissue, glutamine synthetase activity was markedly reduced, and the histologic findings resembled those of Reye syndrome. The adequacy of therapy commonly used for inherited disorders of the urea cycle has not been adequately evaluated in patients with this form of secondary hyperammonemia. To determine whether hemodialysis, in conjunction with intravenous sodium phenylacetate, sodium benzoate, and arginine hydrochloride therapy, would be efficacious in a patient with hyperammonemic coma after solid-organ transplantation. Case report. A children's hospital. A 41-year-old woman with congenital heart disease developed a hyperammonemic coma with brain edema 19 days after undergoing a combined heart and lung transplantation. Ammonium was measured in plasma. Amino acids were quantitated in plasma and cerebrospinal fluid by column chromatography. The effectiveness of therapy was assessed by measuring plasma ammonium levels and intracranial pressure and performing sequential neurological examinations. The patient had the anomalous combination of increased cerebrospinal fluid and decreased plasma glutamine levels. To our knowledge, she is the first patient with this complication after solid-organ transplantation to survive after combined therapy with sodium phenylacetate, sodium benzoate, arginine hydrochloride, and hemodialysis. Complications of the acute coma included focal motor seizures, which were controlled with carbamazepine, and difficulty with short-term memory. The aggressive use of hemodialysis in conjunction with intravenous sodium phenylacetate, sodium benzoate, and arginine hydrochloride therapy may allow survival in patients after solid-organ transplantation. An acute acquired derangement in extra-central nervous system glutamine metabolism may play a role in the production of hyperammonemia in this illness that resembles Reye syndrome, and, as in other hyperammonemic disorders, the duration and degree of elevation of brain glutamine levels may be the important determining factors in responsiveness to therapy.

2-Diazo-1-(4-hydroxyphenyl)ethanone: A Versatile Photochemical and Synthetic Reagenta

PubMed Central

Senadheera, Sanjeewa N.; Evans, Anthony S.; Toscano, John P.; Givens, Richard S.

2014-01-01

α-Diazo arylketones are well-known substrates for Wolff rearrangement to phenylacetic acids through a ketene intermediate by either thermal or photochemical activation. Likewise, α-substituted p-hydroxyphenacyl (pHP) esters are substrates for photo-Favorskii rerrangements to phenylacetic acids by a different pathway that purportedly involves a cyclopropanone intermediate. In this paper, we show that the photolysis of a series of α-diazo-p-hydroxyacetophenones and p-hydroxyphenacyl (pHP) α-esters both generate the identical rearranged phenylacetates as major products. Since α-diazo-p-hydroxyacetophenone (1a, pHP N2) contains all the necessary functionalities for either Wolff or Favorskii rearrangement, we were prompted to probe this intriguing mechanistic dichotomy under conditions favorable to the photo-Favorskii reangement, i.e., photolysis in hydroxylic media. An investigation of the mechanism for conversion of 1a to p-hydroxyphenyl acetic acid (4a) using time-resolved infrared (TRIR) spectroscopy clearly demonstrates the formation of a ketene intermediate that is subsequently trapped by solvent or nucleophiles. The photoreaction of 1a is quenched by oxygen and sensitized by triplet sensitizers and the quantum yields for 1a–c range from 0.19 to a robust 0.25. The lifetime of the triplet, determined by Stern-Volmer quenching, is 15 ns with a rate for appearance of 4a of k = 7,1 × 106 s−1 in aq. acetonitrile (1:1 v:v). These studies establish that the primary rearrangement pathway for 1a involves ketene formation in accordance with the photo-Wolff rearrangement. Furthermore we have also demonstrated the synthetic utility of 1a as an esterification and etherification reagent with a variety of substituted α-diazo-p-hydroxyacetophenones, using them as synthons for efficiently coupling it to acids and phenols to produce pHP protect substrates. PMID:24305682

2-Diazo-1-(4-hydroxyphenyl)ethanone: a versatile photochemical and synthetic reagent.

PubMed

Senadheera, Sanjeewa N; Evans, Anthony S; Toscano, John P; Givens, Richard S

2014-02-01

α-Diazo arylketones are well-known substrates for Wolff rearrangement to phenylacetic acids through a ketene intermediate by either thermal or photochemical activation. Likewise, α-substituted p-hydroxyphenacyl (pHP) esters are substrates for photo-Favorskii rearrangements to phenylacetic acids by a different pathway that purportedly involves a cyclopropanone intermediate. In this paper, we show that the photolysis of a series of α-diazo-p-hydroxyacetophenones and p-hydroxyphenacyl (pHP) α-esters both generate the identical rearranged phenylacetates as major products. Since α-diazo-p-hydroxyacetophenone (1a, pHP N2) contains all the necessary functionalities for either Wolff or Favorskii rearrangement, we were prompted to probe this intriguing mechanistic dichotomy under conditions favorable to the photo-Favorskii rearrangement, i.e., photolysis in hydroxylic media. An investigation of the mechanism for conversion of 1a to p-hydroxyphenyl acetic acid (4a) using time-resolved infrared (TRIR) spectroscopy clearly demonstrates the formation of a ketene intermediate that is subsequently trapped by solvent or nucleophiles. The photoreaction of 1a is quenched by oxygen and sensitized by triplet sensitizers and the quantum yields for 1a-c range from 0.19 to a robust 0.25. The lifetime of the triplet, determined by Stern-Volmer quenching, is 31 ns with a rate for appearance of 4a of k = 7.1 × 10(6) s(-1) in aq. acetonitrile (1 : 1 v : v). These studies establish that the primary rearrangement pathway for 1a involves ketene formation in accordance with the photo-Wolff rearrangement. Furthermore we have also demonstrated the synthetic utility of 1a as an esterification and etherification reagent with a variety of substituted α-diazo-p-hydroxyacetophenones, using them as synthons for efficiently coupling it to acids and phenols to produce pHP protect substrates.

2,4-Dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading gene cluster in the soybean root-nodulating bacterium Bradyrhizobium elkanii USDA94.

PubMed

Hayashi, Shohei; Sano, Tomoki; Suyama, Kousuke; Itoh, Kazuhito

2016-01-01

Herbicides 2,4-dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading Bradyrhizobium strains possess tfdAα and/or cadABC as degrading genes. It has been reported that root-nodulating bacteria belonging to Bradyrhizobium elkanii also have tfdAα and cadA like genes but lack the ability to degrade these herbicides and that the cadA genes in 2,4-D-degrading and non-degrading Bradyrhizobium are phylogenetically different. In this study, we identified cadRABCK in the genome of a type strain of soybean root-nodulating B. elkanii USDA94 and demonstrated that the strain could degrade the herbicides when cadABCK was forcibly expressed. cadABCK-cloned Escherichia coli also showed the degrading ability. Because co-spiked phenoxyacetic acid (PAA) could induce the degradation of 2,4-D in B. elkanii USDA94, the lack of degrading ability in this strain was supposed to be due to the low inducing potential of the herbicides for the degrading gene cluster. On the other hand, tfdAα from B. elkanii USDA94 showed little potential to degrade the herbicides, but it did for 4-chlorophenoxyacetic acid and PAA. The 2,4-D-degrading ability of the cad cluster and the inducing ability of PAA were confirmed by preparing cadA deletion mutant. This is the first study to demonstrate that the cad cluster in the typical root-nodulating bacterium indeed have the potential to degrade the herbicides, suggesting that degrading genes for anthropogenic compounds could be found in ordinary non-degrading bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.

Abundance and Distribution of Dimethylsulfoniopropionate Degradation Genes and the Corresponding Bacterial Community Structure at Dimethyl Sulfide Hot Spots in the Tropical and Subtropical Pacific Ocean

PubMed Central

Suzuki, Shotaro; Omori, Yuko; Wong, Shu-Kuan; Ijichi, Minoru; Kaneko, Ryo; Kameyama, Sohiko; Tanimoto, Hiroshi; Hamasaki, Koji

2015-01-01

Dimethylsulfoniopropionate (DMSP) is mainly produced by marine phytoplankton but is released into the microbial food web and degraded by marine bacteria to dimethyl sulfide (DMS) and other products. To reveal the abundance and distribution of bacterial DMSP degradation genes and the corresponding bacterial communities in relation to DMS and DMSP concentrations in seawater, we collected surface seawater samples from DMS hot spot sites during a cruise across the Pacific Ocean. We analyzed the genes encoding DMSP lyase (dddP) and DMSP demethylase (dmdA), which are responsible for the transformation of DMSP to DMS and DMSP assimilation, respectively. The averaged abundance (±standard deviation) of these DMSP degradation genes relative to that of the 16S rRNA genes was 33% ± 12%. The abundances of these genes showed large spatial variations. dddP genes showed more variation in abundances than dmdA genes. Multidimensional analysis based on the abundances of DMSP degradation genes and environmental factors revealed that the distribution pattern of these genes was influenced by chlorophyll a concentrations and temperatures. dddP genes, dmdA subclade C/2 genes, and dmdA subclade D genes exhibited significant correlations with the marine Roseobacter clade, SAR11 subgroup Ib, and SAR11 subgroup Ia, respectively. SAR11 subgroups Ia and Ib, which possessed dmdA genes, were suggested to be the main potential DMSP consumers. The Roseobacter clade members possessing dddP genes in oligotrophic subtropical regions were possible DMS producers. These results suggest that DMSP degradation genes are abundant and widely distributed in the surface seawater and that the marine bacteria possessing these genes influence the degradation of DMSP and regulate the emissions of DMS in subtropical gyres of the Pacific Ocean. PMID:25862229

Widespread promoter-mediated coordination of transcription and mRNA degradation

PubMed Central

2012-01-01

Background Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. Results We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. Conclusions Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation. PMID:23237624

Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities.

PubMed

Ferrer-Sevillano, Francisco; Fernández-Cañón, José M

2007-03-01

Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).

Novel phacB-Encoded Cytochrome P450 Monooxygenase from Aspergillus nidulans with 3-Hydroxyphenylacetate 6-Hydroxylase and 3,4-Dihydroxyphenylacetate 6-Hydroxylase Activities▿

PubMed Central

Ferrer-Sevillano, Francisco; Fernández-Cañón, José M.

2007-01-01

Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (ΔphacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B). PMID:17189487

Neuromuscular blocking properties of some bistropinium esters

PubMed Central

Haining, C. G.; Johnston, R. G.

1962-01-01

The neuromuscular blocking, anti-acetylcholine and ganglion blocking properties of two series of bistropinium esters were examined. The neuromuscular blocking activities of the mandelic acid esters of NN'-polymethylenebis(tropinium halides) were found to depend upon the number of carbon atoms (n) in the linking chain. Potency was enhanced more than 50 times as n was increased from 2 to 7. Compounds in which n equalled 7, 8, 9, 10 and 12 differed little in activity, but were generally more potent than tubocurarine in cats and rabbits. A peak of ganglion blocking action was obtained at the pentamethylene member. Esterification enhanced the feeble neuromuscular blocking properties of NN'-decamethylenebis(tropinium halide), the mandelic acid ester being more effective than the tropic, benzoic or phenylacetic acid esters in cats and rabbits. When two benzoic or mandelic acid esters of tropine were linked through their nitrogen atoms by a phenylenedimethyl grouping (-CH2.C6H4.CH2-), meta substitution was more effective than was ortho or para in producing neuromuscular block. The effectiveness of esterifying acids in m-phenylenedimethyl derivatives decreased in the following order, phenylacetic> tropic or mandelic>benzoic>acetic and diphenylacetic. PMID:13903721

Comparison of different methodologies for detailed screening of Taraxacum officinale honey volatiles.

PubMed

Jerković, Igor; Marijanović, Zvonimir; Kranjac, Marina; Radonić, Ani

2015-02-01

Headspace solid-phase microextraction (HS-SPME), ultrasonic solvent extraction (USE) and solid phase extraction (SPE), followed by GC-FID/MS were used for screening of dandelion (Taraxacum officinale Weber) honey headspace, volatiles and semi-volatiles. The obtained results constitute a breakthrough towards screening of dandelion honey since dominant compounds identified in the extracts were not previously reported for this honey type. Nitriles dominated in the headspace, particularly 3-methylpentanenitrile (up to 29.9%) and phenylacetonitrile (up to 20.9%). Lower methyl branched aliphatic acids and norisoprenoids were relevant minor constituents of the headspace. The extracts contained phenylacetic acid (up to 24.0%) and dehydrovomifoliol (up to 19.3%) as predominant compounds, while 3-methylpentanenitrile and phenylacetonitrile were detected in the extracts in minor abundance. Dehydrovomifoliol can be considered more characteristic for dandelion honey in distinction from phenylacetic acid. Low molecular aliphatic acids, benzene derivatives and an array of higher aliphatic compounds were also found in the extracts. The results of SPE/GC-FID/MS were very similar to USE/GC-FID/MS with the solvent dichloromethane. The use of all applied methodologies was relevant for the comprehensive chemical fingerprinting of dandelion honey volatiles.

Putative pathway of sex pheromone biosynthesis and degradation by expression patterns of genes identified from female pheromone gland and adult antenna of Sesamia inferens (Walker).

PubMed

Zhang, Ya-Nan; Xia, Yi-Han; Zhu, Jia-Yao; Li, Sheng-Yun; Dong, Shuang-Lin

2014-05-01

The general pathway of biosynthesis and degradation for Type-I sex pheromones in moths is well established, but some genes involved in this pathway remain to be characterized. The purple stem borer, Sesamia inferens, employs a pheromone blend containing components with three different terminal functional groups (Z11-16:OAc, Z11-16:OH, and Z11-16:Ald) of Type-I sex pheromones. Thus, it provides a good model to study the diversity of genes involved in pheromone biosynthesis and degradation pathways. By analyzing previously obtained transcriptomic data of the sex pheromone glands and antennae, we identified 73 novel genes that are possibly related to pheromone biosynthesis (46 genes) or degradation (27 genes). Gene expression patterns and phylogenetic analysis revealed that one desaturase (SinfDes4), one fatty acid reductase (SinfFAR2), and one fatty acid xtransport protein (SinfFATP1) genes were predominantly expressed in pheromone glands, and clustered with genes involved in pheromone synthesis in other moth species. Ten genes including five carboxylesterases (SinfCXE10, 13, 14, 18, and 20), three aldehyde oxidases (SinfAOX1, 2 and 3), and two alcohol dehydrogenases (SinfAD1 and 3) were expressed specifically or predominantly in antennae, and could be candidate genes involved in pheromone degradation. SinfAD1 and 3 are the first reported alcohol dehydrogenase genes with antennae-biased expression. Based on these results we propose a pathway involving these potential enzyme-encoding gene candidates in sex pheromone biosynthesis and degradation in S. inferens. This study provides robust background information for further elucidation of the genetic basis of sex pheromone biosynthesis and degradation, and ultimately provides potential targets to disrupt sexual communication in S. inferens for control purposes.

Identification of three homologous latex-clearing protein (lcp) genes from the genome of Streptomyces sp. strain CFMR 7.

PubMed

Nanthini, Jayaram; Ong, Su Yean; Sudesh, Kumar

2017-09-10

Rubber materials have greatly contributed to human civilization. However, being a polymeric material does not decompose easily, it has caused huge environmental problems. On the other hand, only few bacteria are known to degrade rubber, with studies pertaining them being intensively focusing on the mechanism involved in microbial rubber degradation. The Streptomyces sp. strain CFMR 7, which was previously confirmed to possess rubber-degrading ability, was subjected to whole genome sequencing using the single molecule sequencing technology of the PacBio® RS II system. The genome was further analyzed and compared with previously reported rubber-degrading bacteria in order to identify the potential genes involved in rubber degradation. This led to the interesting discovery of three homologues of latex-clearing protein (Lcp) on the chromosome of this strain, which are probably responsible for rubber degrading activities. Genes encoding oxidoreductase α-subunit (oxiA) and oxidoreductase β-subunit (oxiB) were also found downstream of two lcp genes which are located adjacent to each other. In silico analysis reveals genes that have been identified to be involved in the microbial degradation of rubber in the Streptomyces sp. strain CFMR 7. This is the first whole genome sequence of a clear-zone-forming natural rubber- degrading Streptomyces sp., which harbours three Lcp homologous genes with the presence of oxiA and oxiB genes compared to the previously reported Gordonia polyisoprenivorans strain VH2 (with two Lcp homologous genes) and Nocardia nova SH22a (with only one Lcp gene). Copyright © 2017 Elsevier B.V. All rights reserved.

Abundance and distribution of dimethylsulfoniopropionate degradation genes and the corresponding bacterial community structure at dimethyl sulfide hot spots in the tropical and subtropical pacific ocean.

PubMed

Cui, Yingshun; Suzuki, Shotaro; Omori, Yuko; Wong, Shu-Kuan; Ijichi, Minoru; Kaneko, Ryo; Kameyama, Sohiko; Tanimoto, Hiroshi; Hamasaki, Koji

2015-06-15

Dimethylsulfoniopropionate (DMSP) is mainly produced by marine phytoplankton but is released into the microbial food web and degraded by marine bacteria to dimethyl sulfide (DMS) and other products. To reveal the abundance and distribution of bacterial DMSP degradation genes and the corresponding bacterial communities in relation to DMS and DMSP concentrations in seawater, we collected surface seawater samples from DMS hot spot sites during a cruise across the Pacific Ocean. We analyzed the genes encoding DMSP lyase (dddP) and DMSP demethylase (dmdA), which are responsible for the transformation of DMSP to DMS and DMSP assimilation, respectively. The averaged abundance (±standard deviation) of these DMSP degradation genes relative to that of the 16S rRNA genes was 33% ± 12%. The abundances of these genes showed large spatial variations. dddP genes showed more variation in abundances than dmdA genes. Multidimensional analysis based on the abundances of DMSP degradation genes and environmental factors revealed that the distribution pattern of these genes was influenced by chlorophyll a concentrations and temperatures. dddP genes, dmdA subclade C/2 genes, and dmdA subclade D genes exhibited significant correlations with the marine Roseobacter clade, SAR11 subgroup Ib, and SAR11 subgroup Ia, respectively. SAR11 subgroups Ia and Ib, which possessed dmdA genes, were suggested to be the main potential DMSP consumers. The Roseobacter clade members possessing dddP genes in oligotrophic subtropical regions were possible DMS producers. These results suggest that DMSP degradation genes are abundant and widely distributed in the surface seawater and that the marine bacteria possessing these genes influence the degradation of DMSP and regulate the emissions of DMS in subtropical gyres of the Pacific Ocean. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Response of PAH-degrading genes to PAH bioavailability in the overlying water, suspended sediment, and deposited sediment of the Yangtze River.

PubMed

Xia, Xinghui; Xia, Na; Lai, Yunjia; Dong, Jianwei; Zhao, Pujun; Zhu, Baotong; Li, Zhihuang; Ye, Wan; Yuan, Yue; Huang, Junxiong

2015-06-01

The degrading genes of hydrophobic organic compounds (HOCs) serve as indicators of in situ HOC degradation potential, and the existing forms and bioavailability of HOCs might influence the distribution of HOC-degrading genes in natural waters. However, little research has been conducted to study the relationship between them. In the present study, nahAc and nidA genes, which act as biomarkers for naphthalene- and pyrene-degrading bacteria, were selected as model genotypes to investigate the response of polycyclic aromatic hydrocarbon (PAH)-degrading genes to PAH bioavailability in the overlying water, suspended sediment (SPS), and deposited sediment of the Yangtze River. The freely dissolved concentration, typically used to reflect HOC bioavailability, and total dissolved, as well as sorbed concentrations of PAHs were determined. Phylogenetic analysis showed that all the PAH-ring hydroxylating dioxygenase gene sequences of Gram-negative bacteria (PAH-RHD[GN]) were closely related to nahAc, nagAc, nidA, and uncultured PAH-RHD genes. The PAH-RHD[GN] gene diversity as well as nahAc and nidA gene copy numbers decreased in the following order: deposited sediment>SPS>overlying water. The nahAc and nidA gene abundance was not significantly correlated with environmental parameters but was significantly correlated with the bioavailable existing forms of naphthalene and pyrene in the three phases. The nahAc gene copy numbers in the overlying water and deposited sediment were positively correlated with freely dissolved naphthalene concentrations in the overlying and pore water phases, respectively, and so were nidA gene copy numbers. This study suggests that the distribution and abundance of HOC-degrading bacterial population depend on the HOC bioavailability in aquatic environments. Copyright © 2015 Elsevier Ltd. All rights reserved.

Independent degradation in genes of the plastid ndh gene family in species of the orchid genus Cymbidium (Orchidaceae; Epidendroideae).

PubMed

Kim, Hyoung Tae; Chase, Mark W

2017-01-01

In this paper, we compare ndh genes in the plastid genome of many Cymbidium species and three closely related taxa in Orchidaceae looking for evidence of ndh gene degradation. Among the 11 ndh genes, there were frequently large deletions in directly repeated or AT-rich regions. Variation in these degraded ndh genes occurs between individual plants, apparently at population levels in these Cymbidium species. It is likely that ndh gene transfers from the plastome to mitochondrial genome (chondriome) occurred independently in Orchidaceae and that ndh genes in the chondriome were also relatively recently transferred between distantly related species in Orchidaceae. Four variants of the ycf1-rpl32 region, which normally includes the ndhF genes in the plastome, were identified, and some Cymbidium species contained at least two copies of that region in their organellar genomes. The four ycf1-rpl32 variants seem to have a clear pattern of close relationships. Patterns of ndh degradation between closely related taxa and translocation of ndh genes to the chondriome in Cymbidium suggest that there have been multiple bidirectional intracellular gene transfers between two organellar genomes, which have produced different levels of ndh gene degradation among even closely related species.

A reference gene set for sex pheromone biosynthesis and degradation genes from the diamondback moth, Plutella xylostella, based on genome and transcriptome digital gene expression analyses.

PubMed

He, Peng; Zhang, Yun-Fei; Hong, Duan-Yang; Wang, Jun; Wang, Xing-Liang; Zuo, Ling-Hua; Tang, Xian-Fu; Xu, Wei-Ming; He, Ming

2017-03-01

Female moths synthesize species-specific sex pheromone components and release them to attract male moths, which depend on precise sex pheromone chemosensory system to locate females. Two types of genes involved in the sex pheromone biosynthesis and degradation pathways play essential roles in this important moth behavior. To understand the function of genes in the sex pheromone pathway, this study investigated the genome-wide and digital gene expression of sex pheromone biosynthesis and degradation genes in various adult tissues in the diamondback moth (DBM), Plutella xylostella, which is a notorious vegetable pest worldwide. A massive transcriptome data (at least 39.04 Gb) was generated by sequencing 6 adult tissues including male antennae, female antennae, heads, legs, abdomen and female pheromone glands from DBM by using Illumina 4000 next-generation sequencing and mapping to a published DBM genome. Bioinformatics analysis yielded a total of 89,332 unigenes among which 87 transcripts were putatively related to seven gene families in the sex pheromone biosynthesis pathway. Among these, seven [two desaturases (DES), three fatty acyl-CoA reductases (FAR) one acetyltransferase (ACT) and one alcohol dehydrogenase (AD)] were mainly expressed in the pheromone glands with likely function in the three essential sex pheromone biosynthesis steps: desaturation, reduction, and esterification. We also identified 210 odorant-degradation related genes (including sex pheromone-degradation related genes) from seven major enzyme groups. Among these genes, 100 genes are new identified and two aldehyde oxidases (AOXs), one aldehyde dehydrogenase (ALDH), five carboxyl/cholinesterases (CCEs), five UDP-glycosyltransferases (UGTs), eight cytochrome P450 (CYP) and three glutathione S-transferases (GSTs) displayed more robust expression in the antennae, and thus are proposed to participate in the degradation of sex pheromone components and plant volatiles. To date, this is the most comprehensive gene data set of sex pheromone biosynthesis and degradation enzyme related genes in DBM created by genome- and transcriptome-wide identification, characterization and expression profiling. Our findings provide a basis to better understand the function of genes with tissue enriched expression. The results also provide information on the genes involved in sex pheromone biosynthesis and degradation, and may be useful to identify potential gene targets for pest control strategies by disrupting the insect-insect communication using pheromone-based behavioral antagonists.

Metabolic and spatio-taxonomic response of uncultivated seafloor bacteria following the Deepwater Horizon oil spill

DOE Office of Scientific and Technical Information (OSTI.GOV)

Handley, K. M.; Piceno, Y. M.; Hu, P.

The release of 700 million liters of oil into the Gulf of Mexico over a few months in 2010 produced dramatic changes in the microbial ecology of the water and sediment. Here, we reconstructed the genomes of 57 widespread uncultivated bacteria from post-spill deep-sea sediments, and recovered their gene expression pattern across the seafloor. These genomes comprised a common collection of bacteria that were enriched in heavily affected sediments around the wellhead. Although rare in distal sediments, some members were still detectable at sites up to 60 km away. Many of these genomes exhibited phylogenetic clustering indicative of common traitmore » selection by the environment, and within half we identified 264 genes associated with hydrocarbon degradation. Alkane degradation ability was near ubiquitous among candidate hydrocarbon degraders, whereas just three harbored elaborate gene inventories for the degradation of alkanes and aromatic and polycyclic aromatic hydrocarbons (PAHs). Differential gene expression profiles revealed a spill-promoted microbial sulfur cycle alongside gene upregulation associated with PAH degradation. Gene expression associated with alkane degradation was widespread, although active alkane degrader identities changed along the pollution gradient. Analyses suggest that a broad metabolic capacity to respond to oil inputs exists across a large array of usually rare indigenous deep-sea bacteria.« less

Genes Involved in Degradation of para-Nitrophenol Are Differentially Arranged in Form of Non-Contiguous Gene Clusters in Burkholderia sp. strain SJ98

PubMed Central

Vikram, Surendra; Pandey, Janmejay; Kumar, Shailesh; Raghava, Gajendra Pal Singh

2013-01-01

Biodegradation of para-Nitrophenol (PNP) proceeds via two distinct pathways, having 1,2,3-benzenetriol (BT) and hydroquinone (HQ) as their respective terminal aromatic intermediates. Genes involved in these pathways have already been studied in different PNP degrading bacteria. Burkholderia sp. strain SJ98 degrades PNP via both the pathways. Earlier, we have sequenced and analyzed a ~41 kb fragment from the genomic library of strain SJ98. This DNA fragment was found to harbor all the lower pathway genes; however, genes responsible for the initial transformation of PNP could not be identified within this fragment. Now, we have sequenced and annotated the whole genome of strain SJ98 and found two ORFs (viz., pnpA and pnpB) showing maximum identity at amino acid level with p-nitrophenol 4-monooxygenase (PnpM) and p-benzoquinone reductase (BqR). Unlike the other PNP gene clusters reported earlier in different bacteria, these two ORFs in SJ98 genome are physically separated from the other genes of PNP degradation pathway. In order to ascertain the identity of ORFs pnpA and pnpB, we have performed in-vitro assays using recombinant proteins heterologously expressed and purified to homogeneity. Purified PnpA was found to be a functional PnpM and transformed PNP into benzoquinone (BQ), while PnpB was found to be a functional BqR which catalyzed the transformation of BQ into hydroquinone (HQ). Noticeably, PnpM from strain SJ98 could also transform a number of PNP analogues. Based on the above observations, we propose that the genes for PNP degradation in strain SJ98 are arranged differentially in form of non-contiguous gene clusters. This is the first report for such arrangement for gene clusters involved in PNP degradation. Therefore, we propose that PNP degradation in strain SJ98 could be an important model system for further studies on differential evolution of PNP degradation functions. PMID:24376843

Biogeographical distribution analysis of hydrocarbon degrading and biosurfactant producing genes suggests that near-equatorial biomes have higher abundance of genes with potential for bioremediation.

PubMed

Oliveira, Jorge S; Araújo, Wydemberg J; Figueiredo, Ricardo M; Silva-Portela, Rita C B; de Brito Guerra, Alaine; da Silva Araújo, Sinara Carla; Minnicelli, Carolina; Carlos, Aline Cardoso; de Vasconcelos, Ana Tereza Ribeiro; Freitas, Ana Teresa; Agnez-Lima, Lucymara F

2017-07-27

Bacterial and Archaeal communities have a complex, symbiotic role in crude oil bioremediation. Their biosurfactants and degradation enzymes have been in the spotlight, mainly due to the awareness of ecosystem pollution caused by crude oil accidents and their use. Initially, the scientific community studied the role of individual microbial species by characterizing and optimizing their biosurfactant and oil degradation genes, studying their individual distribution. However, with the advances in genomics, in particular with the use of New-Generation-Sequencing and Metagenomics, it is now possible to have a macro view of the complex pathways related to the symbiotic degradation of hydrocarbons and surfactant production. It is now possible, although more challenging, to obtain the DNA information of an entire microbial community before automatically characterizing it. By characterizing and understanding the interconnected role of microorganisms and the role of degradation and biosurfactant genes in an ecosystem, it becomes possible to develop new biotechnological approaches for bioremediation use. This paper analyzes 46 different metagenome samples, spanning 20 biomes from different geographies obtained from different research projects. A metagenomics bioinformatics pipeline, focused on the biodegradation and biosurfactant-production pathways, genes and organisms, was applied. Our main results show that: (1) surfactation and degradation are correlated events, and therefore should be studied together; (2) terrestrial biomes present more degradation genes, especially cyclic compounds, and less surfactation genes, when compared to water biomes; and (3) latitude has a significant influence on the diversity of genes involved in biodegradation and biosurfactant production. This suggests that microbiomes found near the equator are richer in genes that have a role in these processes and thus have a higher biotechnological potential. In this work we have focused on the biogeographical distribution of hydrocarbon degrading and biosurfactant producing genes. Our principle results can be seen as an important step forward in the application of bioremediation techniques, by considering the biostimulation, optimization or manipulation of a starting microbial consortia from the areas with higher degradation and biosurfactant producing genetic diversity.

Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.

PubMed

Park, Young-Jin; Baek, Jeong Hun; Lee, Seonwook; Kim, Changhoon; Rhee, Hwanseok; Kim, Hyungtae; Seo, Jeong-Sun; Park, Hae-Ran; Yoon, Dae-Eun; Nam, Jae-Young; Kim, Hong-Il; Kim, Jong-Guk; Yoon, Hyeokjun; Kang, Hee-Wan; Cho, Jae-Yong; Song, Eun-Sung; Sung, Gi-Ho; Yoo, Young-Bok; Lee, Chang-Soo; Lee, Byoung-Moo; Kong, Won-Sik

2014-01-01

Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes) and carbohydrate degradation (392 CAZymes), along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi-) cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-)cellulose biomass related industries further increase the significance of F. velutipes as a new model.

Whole Genome and Global Gene Expression Analyses of the Model Mushroom Flammulina velutipes Reveal a High Capacity for Lignocellulose Degradation

PubMed Central

Park, Young-Jin; Baek, Jeong Hun; Lee, Seonwook; Kim, Changhoon; Rhee, Hwanseok; Kim, Hyungtae; Seo, Jeong-Sun; Park, Hae-Ran; Yoon, Dae-Eun; Nam, Jae-Young; Kim, Hong-Il; Kim, Jong-Guk; Yoon, Hyeokjun; Kang, Hee-Wan; Cho, Jae-Yong; Song, Eun-Sung; Sung, Gi-Ho; Yoo, Young-Bok; Lee, Chang-Soo; Lee, Byoung-Moo; Kong, Won-Sik

2014-01-01

Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes) and carbohydrate degradation (392 CAZymes), along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi-) cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-)cellulose biomass related industries further increase the significance of F. velutipes as a new model. PMID:24714189

Improved penicillin amidase production using a genetically engineered mutant of escherichia coli ATCC 11105

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robas, N.; Zouheiry, H.; Branlant, G.

Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, the authors constructed various recombinant E. coli HB 101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic acid (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selectedmore » based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the HindIII fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.« less

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

PubMed Central

Turnbull, Gillian A.; Ousley, Margaret; Walker, Allan; Shaw, Eve; Morgan, J. Alun W.

2001-01-01

Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. coli coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene). PMID:11319111

Biodegradation Ability and Catabolic Genes of Petroleum-Degrading Sphingomonas koreensis Strain ASU-06 Isolated from Egyptian Oily Soil

PubMed Central

Mostafa, Yasser M.; Shoreit, Ahmed

2014-01-01

Polycyclic aromatic hydrocarbons (PAHs) are serious pollutants and health hazards. In this study, 15 PAHs-degrading bacteria were isolated from Egyptian oily soil. Among them, one Gram-negative strain (ASU-06) was selected and biodegradation ability and initial catabolic genes of petroleum compounds were investigated. Comparison of 16S rRNA gene sequence of strain ASU-06 to published sequences in GenBank database as well as phylogenetic analysis identified ASU-06 as Sphingomonas koreensis. Strain ASU-06 degraded 100, 99, 98, and 92.7% of 100 mg/L naphthalene, phenanthrene, anthracene, and pyrene within 15 days, respectively. When these PAHs present in a mixed form, the enhancement phenomenon appeared, particularly in the degradation of pyrene, whereas the degradation rate was 98.6% within the period. This is the first report showing the degradation of different PAHs by this species. PCR experiments with specific primers for catabolic genes alkB, alkB1, nahAc, C12O, and C23O suggested that ASU-06 might possess genes for aliphatic and PAHs degradation, while PAH-RHDαGP gene was not detected. Production of biosurfactants and increasing cell-surface hydrophobicity were investigated. GC/MS analysis of intermediate metabolites of studied PAHs concluded that this strain utilized these compounds via two main pathways, and phthalate was the major constant product that appeared in each day of the degradation period. PMID:25177681

Metadata Analysis of Phanerochaete chrysosporium Gene Expression Data Identified Common CAZymes Encoding Gene Expression Profiles Involved in Cellulose and Hemicellulose Degradation.

PubMed

Kameshwar, Ayyappa Kumar Sista; Qin, Wensheng

2017-01-01

In literature, extensive studies have been conducted on popular wood degrading white rot fungus, Phanerochaete chrysosporium about its lignin degrading mechanisms compared to the cellulose and hemicellulose degrading abilities. This study delineates cellulose and hemicellulose degrading mechanisms through large scale metadata analysis of P. chrysosporium gene expression data (retrieved from NCBI GEO) to understand the common expression patterns of differentially expressed genes when cultured on different growth substrates. Genes encoding glycoside hydrolase classes commonly expressed during breakdown of cellulose such as GH-5,6,7,9,44,45,48 and hemicellulose are GH-2,8,10,11,26,30,43,47 were found to be highly expressed among varied growth conditions including simple customized and complex natural plant biomass growth mediums. Genes encoding carbohydrate esterase class enzymes CE (1,4,8,9,15,16) polysaccharide lyase class enzymes PL-8 and PL-14, and glycosyl transferases classes GT (1,2,4,8,15,20,35,39,48) were differentially expressed in natural plant biomass growth mediums. Based on these results, P. chrysosporium, on natural plant biomass substrates was found to express lignin and hemicellulose degrading enzymes more than cellulolytic enzymes except GH-61 (LPMO) class enzymes, in early stages. It was observed that the fate of P. chrysosporium transcriptome is significantly affected by the wood substrate provided. We believe, the gene expression findings in this study plays crucial role in developing genetically efficient microbe with effective cellulose and hemicellulose degradation abilities.

Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp. Strain HW13 Isolated from a Pristine Environment

PubMed Central

Kitagawa, Wataru; Takami, Sachiko; Miyauchi, Keisuke; Masai, Eiji; Kamagata, Yoichi; Tiedje, James M.; Fukuda, Masao

2002-01-01

The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R. eutropha JMP134. The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134. Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not. Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC. PMID:11751829

Paraoxonase 1 (PON1) gene-108C>T and p.Q192R polymorphisms and arylesterase activity of the enzyme in patients with dementia.

PubMed

Bednarska-Makaruk, Małgorzata Ewa; Krzywkowski, Tomasz; Graban, Alla; Lipczyńska-Łojkowska, Wanda; Bochyńska, Anna; Rodo, Maria; Wehr, Hanna; Ryglewicz, Danuta Krystyna

2013-01-01

Paraoxonase 1 (PON1) activity was determined using phenylacetate as substrate (arylesterase activity) in 304 individuals with dementia--136 recognised as probable Alzheimer's disease (AD), 64 as dementia of vascular origin (VaD) and 104 as mixed dementia (MD) and in 129 persons without symptoms of dementia and in a good general health. -108C>T polymorphism in the PON1 gene promoter and p.Q192R polymorphism in the coding region were identified. PON1 activity was significantly lower in demented patients as compared with controls particularly in dementia of a neurodegenerative character (AD and MD). The prevalence of PON1-108T allele carriers was significantly higher in the AD group than in controls. The frequencies of the p.Q192R genotypes did not differ significantly between the investigated groups. An association of the rare T-R haplotype with dementia, particularly with dementia of the neurodegenerative type, was found. Multivariate regression analysis showed a significant association of PON1 activity with PON1 -108C>T and p.Q192R polymorphisms. The influence not only of promoter -108C>T, but also of p.Q192R polymorphism on PON1 arylesterase activity was observed. One has to admit that this kind of polymorphism does not preclude interference with the enzyme activity. It could be concluded that the PON1 gene promoter polymorphism plays an additional role in Alzheimer's disease development. It seems however that PON1 activity has a dominating influence on the dementia risk.

Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons.

PubMed Central

Lloyd-Jones, G; Lau, P C

1997-01-01

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria. PMID:9251217

Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders

PubMed Central

McGowan, Catherine; Fulthorpe, Roberta; Wright, Alice; Tiedje, J. M.

1998-01-01

Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway, tfdA, was sequenced. The sequences fell into three unique classes found in various members of the beta and gamma subgroups of Proteobacteria. None of the α-Proteobacteria yielded tfdA PCR products. A comparison of the dendrogram of the tfdA genes with that of the SSU rDNA genes demonstrated incongruency in phylogenies, and hence 2,4-D degradation must have originated from gene transfer between species. Only those strains with tfdA sequences highly similar to the tfdA sequence of strain JMP134 (tfdA class I) transferred all the 2,4-D genes and conferred the 2,4-D degradation phenotype to a Burkholderia cepacia recipient. PMID:9758850

De novo prediction of the genomic components and capabilities for microbial plant biomass degradation from (meta-)genomes

PubMed Central

2013-01-01

Background Understanding the biological mechanisms used by microorganisms for plant biomass degradation is of considerable biotechnological interest. Despite of the growing number of sequenced (meta)genomes of plant biomass-degrading microbes, there is currently no technique for the systematic determination of the genomic components of this process from these data. Results We describe a computational method for the discovery of the protein domains and CAZy families involved in microbial plant biomass degradation. Our method furthermore accurately predicts the capability to degrade plant biomass for microbial species from their genome sequences. Application to a large, manually curated data set of microbial degraders and non-degraders identified gene families of enzymes known by physiological and biochemical tests to be implicated in cellulose degradation, such as GH5 and GH6. Additionally, genes of enzymes that degrade other plant polysaccharides, such as hemicellulose, pectins and oligosaccharides, were found, as well as gene families which have not previously been related to the process. For draft genomes reconstructed from a cow rumen metagenome our method predicted Bacteroidetes-affiliated species and a relative to a known plant biomass degrader to be plant biomass degraders. This was supported by the presence of genes encoding enzymatically active glycoside hydrolases in these genomes. Conclusions Our results show the potential of the method for generating novel insights into microbial plant biomass degradation from (meta-)genome data, where there is an increasing production of genome assemblages for uncultured microbes. PMID:23414703

Comparative genome analysis of Bacillus velezensis reveals a potential for degrading lignocellulosic biomass.

PubMed

Chen, Long; Gu, Wei; Xu, Hai-Yan; Yang, Gui-Lian; Shan, Xiao-Feng; Chen, Guang; Kang, Yuan-Huan; Wang, Chun-Feng; Qian, Ai-Dong

2018-05-01

Genomes of 24 sequenced Bacillus velezensis strains were characterized to identity shared and unique genes of lignocellulolytic enzymes and predict potential to degrade lignocellulose. All 24 strains had genes that encoded lignocellulolytic enzymes, with potential to degrade cellulose and hemicelluloses. Several lignocellulosic genes related to cellulose degradation were universally present, including one GH5 (endo-1,4-β-glucanase), one GH30 (glucan endo-1,6-β-glucosidase), two GH4 (6-phospho-β-glucosidase, 6-phospho-α-glucosidase), one GH1 (6-phospho-β-galactosidase), one GH16 (β-glucanase) and three GH32 (two sucrose-6-phosphate hydrolase and levanase). However, in the absence of gene(s) for cellobiohydrolase, it was predicted that none of the 24 strains would be able to directly hydrolyse cellulose. Regarding genes for hemicellulose degradation, four GH43 (1,4-β-xylosidase; except strain 9912D), one GH11 (endo-1,4-β-xylanase), three GH43 (two arabinan endo-1,5-α-L-arabinosidase and one arabinoxylan arabinofuranohydrolase), two GH51 (α-N-arabinofuranosidase), one GH30 (glucuronoxylanase), one GH26 (β-mannosidase) and one GH53 (arabinogalactan endo-1,4-β-galactosidase) were present. In addition, two PL1 (pectate lyase) and one PL9 (pectate lyase) with potential for pectin degradation were conserved among all 24 strains. In addition, all 24 Bacillus velezensis had limited representation of the auxiliary activities super-family, consistent with a limited ability to degrade lignin. Therefore, it was predicted that for these bacteria to degrade lignin, pretreatment of lignocellulosic substrates may be required. Finally, based on in silico studies, we inferred that Bacillus velezensis strains may degrade a range of polysaccharides in lignocellulosic biomasses.

Enhanced Gene Detection Assays for Fumarate-Adding Enzymes Allow Uncovering of Anaerobic Hydrocarbon Degraders in Terrestrial and Marine Systems

PubMed Central

von Netzer, Frederick; Pilloni, Giovanni; Kleindienst, Sara; Krüger, Martin; Knittel, Katrin; Gründger, Friederike

2013-01-01

The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. Fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) have already been established as specific functional marker genes for anaerobic hydrocarbon degraders. Several recent studies based on pure cultures and laboratory enrichments have shown the existence of new and deeply branching FAE gene lineages, such as clostridial benzylsuccinate synthases and homologues, as well as naphthylmethylsuccinate synthases. However, established FAE gene detection assays were not designed to target these novel lineages, and consequently, their detectability in different environments remains obscure. Here, we present a new suite of parallel primer sets for detecting the comprehensive range of FAE markers known to date, including clostridial benzylsuccinate, naphthylmethylsuccinate, and alkylsuccinate synthases. It was not possible to develop one single assay spanning the complete diversity of FAE genes alone. The enhanced assays were tested with a range of hydrocarbon-degrading pure cultures, enrichments, and environmental samples of marine and terrestrial origin. They revealed the presence of several, partially unexpected FAE gene lineages not detected in these environments before: distinct deltaproteobacterial and also clostridial bssA homologues as well as environmental nmsA homologues. These findings were backed up by dual-digest terminal restriction fragment length polymorphism diagnostics to identify FAE gene populations independently of sequencing. This allows rapid insights into intrinsic degrader populations and degradation potentials established in aromatic and aliphatic hydrocarbon-impacted environmental systems. PMID:23124238

Identification of a p-cresol degradation pathway by a GFP-based transposon in Pseudomonas and its dominant expression in colonies.

PubMed

Cho, Ah Ra; Lim, Eun Jin; Veeranagouda, Yaligara; Lee, Kyoung

2011-11-01

In this study, the chromosome-encoded pcuRCAXB genes that are required for p-cresol degradation have been identified by using a newly constructed green fluorescent protein (GFP)-based promoter probe transposon in the long-chain alkylphenol degrader Pseudomonas alkylphenolia. The deduced amino acid sequences of the genes showed the highest identities at the levels of 65-93% compared with those in the databases. The transposon was identified to be inserted in the pcuA gene, with the promoterless gfp gene being under the control of the pcu catabolic gene promoter. The expression of GFP was positively induced by p-cresol and was about 10 times higher by cells grown on agar than those in liquid culture. In addition, phydroxybenzoic acid was detected during p-cresol degradation. These results indicate that P. alkylphenolia additionally possesses a protocatechuate ortho-cleavage route for pcresol degradation that is dominantly expressed in colonies.

Occurrence of Hydrocarbon Degrading Genes in the Soils of the Republic of Tatarstan (Russia)

NASA Astrophysics Data System (ADS)

Biktasheva, L. R.; Shalyamova, R. P.; Guseva, U. A.; Galitskaya, P. Yu

2018-01-01

Oil pollution is one of the most serious environmental problems nowadays. The ability of soils for self-restoration is important, when choosing the strategy of pollution control. This ability depends on the pull of microbes able to decompose hydrocarbons that were present in the nonpolluted soil prior to pollution. In this study, the occurrence of alkane degrading genes in the soils of the Republic of Tatarstan being one of the oil processing regions in Russia, was investigated. It was found that alkane degrading genes belonging to group I were present in 20 of the 25 soil samples, and their abundances ranged between 0.01 and 0.07%. Alkane degrading genes belonging to group II were not detected in the samples investigated, and those belonging to group III were present in all the samples, and their abundances ranged between 0.06 and 7.25%. No correlation between the alkane degrading gene copy numbers and pH and organic carbon content in soils was revealed.

Gene expression metadata analysis reveals molecular mechanisms employed by Phanerochaete chrysosporium during lignin degradation and detoxification of plant extractives.

PubMed

Kameshwar, Ayyappa Kumar Sista; Qin, Wensheng

2017-10-01

Lignin, most complex and abundant biopolymer on the earth's surface, attains its stability from intricate polyphenolic units and non-phenolic bonds, making it difficult to depolymerize or separate from other units of biomass. Eccentric lignin degrading ability and availability of annotated genome make Phanerochaete chrysosporium ideal for studying lignin degrading mechanisms. Decoding and understanding the molecular mechanisms underlying the process of lignin degradation will significantly aid the progressing biofuel industries and lead to the production of commercially vital platform chemicals. In this study, we have performed a large-scale metadata analysis to understand the common gene expression patterns of P. chrysosporium during lignin degradation. Gene expression datasets were retrieved from NCBI GEO database and analyzed using GEO2R and Bioconductor packages. Commonly expressed statistically significant genes among different datasets were further considered to understand their involvement in lignin degradation and detoxification mechanisms. We have observed three sets of enzymes commonly expressed during ligninolytic conditions which were later classified into primary ligninolytic, aromatic compound-degrading and other necessary enzymes. Similarly, we have observed three sets of genes coding for detoxification and stress-responsive, phase I and phase II metabolic enzymes. Results obtained in this study indicate the coordinated action of enzymes involved in lignin depolymerization and detoxification-stress responses under ligninolytic conditions. We have developed tentative network of genes and enzymes involved in lignin degradation and detoxification mechanisms by P. chrysosporium based on the literature and results obtained in this study. However, ambiguity raised due to higher expression of several uncharacterized proteins necessitates for further proteomic studies in P. chrysosporium.

Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice.

PubMed

Tayi, Lavanya; Maku, Roshan V; Patel, Hitendra Kumar; Sonti, Ramesh V

2016-01-01

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.

Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice

PubMed Central

Tayi, Lavanya; Maku, Roshan V.; Patel, Hitendra Kumar; Sonti, Ramesh V.

2016-01-01

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo. PMID:27907079

Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes.

PubMed

Wang, Xiaofu; Chen, Xiaoyun; Xu, Junfeng; Dai, Chen; Shen, Wenbiao

2015-10-01

This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biodegradation of trichloroethylene (TCE) by methanotrophic community.

PubMed

Shukla, Awadhesh K; Vishwakarma, Pranjali; Upadhyay, S N; Tripathi, Anil K; Prasana, H C; Dubey, Suresh K

2009-05-01

Laboratory incubation experiments were carried out to assess the potential of methanotrophic culture for degrading TCE. Measurements of the growth rate and TCE degradation showed that the methanotrophs not only grew in presence of TCE but also degraded TCE. The rate of TCE degradation was found to be 0.19 ppm h(-1). The reverse transcriptase-PCR test was conducted to quantify expression of pmoA and mmoX genes. RT-PCR revealed expression of pmoA gene only. This observation provides evidence that the pmoA gene was functionally active for pMMO enzyme during the study. The diversity of the methanotrophs involved in TCE degradation was assessed by PCR amplification, cloning, restriction fragment length polymorphism and phylogenetic analysis of pmoA genes. Results suggested the occurrence of nine different phylotypes belonging to Type II methanotrophs in the enriched cultures. Out of the nine, five clustered with, genera Methylocystis and rest got clustered in to a separate group.

Hydrocarbon Degradation Pathways used by Coastal Sediment Microbial Communities exposed to Crude Oil

NASA Astrophysics Data System (ADS)

Spaulding-Astudillo, F.; Sharrar, A.; Orcutt, B.

2016-02-01

The site-specific microbial community response to crude oil exposure in marine environments is not well described. Moreover, the abundance of genes implicated in long-chain alkane (LCA) and polycyclic aromatic hydrocarbon (PAH) degradation are not well understood. Coastal sediments from the Beaufort Sea, Gulf of Alaska, and Portland Harbor were treated with crude oil and incubated aerobically. Deep-sea sediments from the Gulf of Mexico were treated with the same crude oil and anaerobically incubated in situ for five months before recovery. Cycloclasticus, a known hydrocarbon-degrader, was abundant in all oiled, aerobic samples regardless of temperature, demonstrating a generalist oil-response strategy. Other hydrocarbon degrading bacteria showed differential response to either site or temperature. Primers for alkB, assA, bssA, and ncr, catabolic gene markers for aerobic LCA degradation, anaerobic LCA degradation, anaerobic LCA & PAH degradation, and anaerobic PAH degradation, respectively, were found in literature and tested on DNA extracts in a QPCR-based assay. Gene abundance was site and condition variable.

Establishment of Bacterial Herbicide Degraders in a Rapid Sand Filter for Bioremediation of Phenoxypropionate-Polluted Groundwater

PubMed Central

Feld, Louise; Nielsen, Tue Kjærgaard; Hansen, Lars Hestbjerg; Aamand, Jens

2015-01-01

In this study, we investigated the establishment of natural bacterial degraders in a sand filter treating groundwater contaminated with the phenoxypropionate herbicides (RS)-2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) and (RS)-2-(2,4-dichlorophenoxy)propanoic acid (DCPP) and the associated impurity/catabolite 4-chlorophenoxypropanoic acid (4-CPP). A pilot facility was set up in a contaminated landfill site. Anaerobic groundwater was pumped up and passed through an aeration basin and subsequently through a rapid sand filter, which is characterized by a short residence time of the water in the filter. For 3 months, the degradation of DCPP, MCPP, and 4-CPP in the sand filter increased to 15 to 30% of the inlet concentration. A significant selection for natural bacterial herbicide degraders also occurred in the sand filter. Using a most-probable-number (MPN) method, we found a steady increase in the number of culturable phenoxypropionate degraders, reaching approximately 5 × 105 degraders per g sand by the end of the study. Using a quantitative PCR targeting the two phenoxypropionate degradation genes, rdpA and sdpA, encoding stereospecific dioxygenases, a parallel increase was observed, but with the gene copy numbers being about 2 to 3 log units higher than the MPN. In general, the sdpA gene was more abundant than the rdpA gene, and the establishment of a significant population of bacteria harboring sdpA occurred faster than the establishment of an rdpA gene-carrying population. The identities of the specific herbicide degraders in the sand filter were assessed by Illumina MiSeq sequencing of 16S rRNA genes from sand filter samples and from selected MPN plate wells. We propose a list of potential degrader bacteria involved in herbicide degradation, including representatives belonging to the Comamonadaceae and Sphingomonadales. PMID:26590282

Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes

PubMed Central

Zhang, Jun; Zhang, Lei; Geng, Alei; Liu, Fanghua; Zhao, Guoping; Wang, Shengyue; Zhou, Zhihua; Yan, Xing

2015-01-01

Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future. PMID:26070087

Chlorophyll metabolism in pollinated vs. parthenocarpic fig fruits throughout development and ripening.

PubMed

Rosianskey, Yogev; Dahan, Yardena; Yadav, Sharawan; Freiman, Zohar E; Milo-Cochavi, Shira; Kerem, Zohar; Eyal, Yoram; Flaishman, Moshe A

2016-08-01

Expression of 13 genes encoding chlorophyll biosynthesis and degradation was evaluated. Chlorophyll degradation was differentially regulated in pollinated and parthenocarpic fig fruits, leading to earlier chlorophyll degradation in parthenocarpic fruits. Varieties of the common fig typically yield a commercial summer crop that requires no pollination, although it can be pollinated. Fig fruit pollination results in larger fruit size, greener skin and darker interior inflorescence color, and slows the ripening process compared to non-pollinated fruits. We evaluated the effect of pollination on chlorophyll content and levels of transcripts encoding enzymes of the chlorophyll metabolism in fruits of the common fig 'Brown Turkey'. We cloned and evaluated the expression of 13 different genes. All 13 genes showed high expression in the fruit skin, inflorescences and leaves, but extremely low expression in roots. Pollination delayed chlorophyll breakdown in the ripening fruit skin and inflorescences. This was correlated with the expression of genes encoding enzymes in the chlorophyll biosynthesis and degradation pathways. Expression of pheophorbide a oxygenase (PAO) was strongly negatively correlated with chlorophyll levels during ripening in pollinated fruits; along with its high expression levels in yellow leaves, this supports a pivotal role for PAO in chlorophyll degradation in figs. Normalizing expression levels of all chlorophyll metabolism genes in the pollinated and parthenocarpic fruit skin and inflorescences showed three synthesis (FcGluTR1, FcGluTR2 and FcCLS1) and three degradation (FcCLH1, FcCLH2 and FcRCCR1) genes with different temporal expression in the pollinated vs. parthenocarpic fruit skin and inflorescences. FcCAO also showed different expressions in the parthenocarpic fruit skin. Thus, chlorophyll degradation is differentially regulated in the pollinated and parthenocarpic fruit skin and inflorescences, leading to earlier and more sustained chlorophyll degradation in the parthenocarpic fruit.

Response of microbial community and catabolic genes to simulated petroleum hydrocarbon spills in soils/sediments from different geographic locations.

PubMed

Liu, Q; Tang, J; Liu, X; Song, B; Zhen, M; Ashbolt, N J

2017-10-01

Study the response of microbial communities and selected petroleum hydrocarbon (PH)-degrading genes on simulated PH spills in soils/sediments from different geographic locations. A microcosm experiment was conducted by spiking mixtures of petroleum hydrocarbons (PHs) to soils/sediments collected from four different regions of China, including the Dagang Oilfield (DG), Sand of Bohai Sea (SS), Northeast China (NE) and Xiamen (XM). Changes in bacterial community and the abundance of PH-degrading genes (alkB, nah and phe) were analysed by denaturing gradient electrophoresis (DGGE) and qPCR, respectively. Degradation of alkanes and PAHs in SS and NE materials were greater (P < 0·05) than those in DG and XM. Clay content was negatively correlated with the degradation of total alkanes by 112 days and PAHs by 56 days, while total organic carbon content was negatively correlated with initial degradation of total alkanes as well as PAHs. Abundances of alkB, nah and phe genes increased 10- to 100-fold and varied by soil type over the incubation period. DGGE fingerprints identified the dominance of α-, β- and γ-Proteobacteria (Gram -ve) and Actinobacteria (Gram +ve) bacteria associated with degradation of PHs in the materials studied. The geographic divergence resulting from the heterogeneity of physicochemical properties of soils/sediments appeared to influence the abundance of metabolic genes and community structure of microbes capable of degrading PHs. When developing practical in-situ bioremediation approaches for PHs contamination of soils/sediment, appropriate microbial community structures and the abundance of PH-degrading genes appear to be influenced by geographic location. © 2017 The Society for Applied Microbiology.

Cytochrome P450 Initiates Degradation of cis-Dichloroethene by Polaromonas sp. Strain JS666

PubMed Central

Nishino, Shirley F.; Shin, Kwanghee A.; Gossett, James M.

2013-01-01

Polaromonas sp. strain JS666 grows on cis-1,2-dichoroethene (cDCE) as the sole carbon and energy source under aerobic conditions, but the degradation mechanism and the enzymes involved are unknown. In this study, we established the complete pathway for cDCE degradation through heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. Several lines of evidence indicate that a cytochrome P450 monooxygenase catalyzes the initial step of cDCE degradation. Both the transient accumulation of dichloroacetaldehyde in cDCE-degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 support a pathway for degradation of cDCE through dichloroacetaldehyde. The mechanism minimizes the formation of cDCE epoxide. The molecular phylogeny of the cytochrome P450 gene and the organization of neighboring genes suggest that the cDCE degradation pathway recently evolved in a progenitor capable of degrading 1,2-dichloroethane either by the recruitment of the cytochrome P450 monooxygenase gene from an alkane catabolic pathway or by selection for variants of the P450 in a preexisting 1,2-dichloroethane catabolic pathway. The results presented here add yet another role to the broad array of productive reactions catalyzed by cytochrome P450 enzymes. PMID:23354711

Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1

PubMed Central

Song, Jinlong; Shi, Yanhua; Li, Kang; Zhao, Bin; Yan, Yanchun

2013-01-01

A novel pyrethroid-degrading esterase gene pytY was isolated from the genomic library of Ochrobactrum anthropi YZ-1. It possesses an open reading frame (ORF) of 897 bp. Blast search showed that its deduced amino acid sequence shares moderate identities (30% to 46%) with most homologous esterases. Phylogenetic analysis revealed that PytY is a member of the esterase VI family. pytY showed very low sequence similarity compared with reported pyrethroid-degrading genes. PytY was expressed, purified, and characterized. Enzyme assay revealed that PytY is a broad-spectrum degrading enzyme that can degrade various pyrethroids. It is a new pyrethroid-degrading gene and enriches genetic resource. Kinetic constants of Km and Vmax were 2.34 mmol·L−1 and 56.33 nmol min−1, respectively, with lambda-cyhalothrin as substrate. PytY displayed good degrading ability and stability over a broad range of temperature and pH. The optimal temperature and pH were of 35°C and 7.5. No cofactors were required for enzyme activity. The results highlighted the potential use of PytY in the elimination of pyrethroid residuals from contaminated environments. PMID:24155944

Interconnection of Key Microbial Functional Genes for Enhanced Benzo[a]pyrene Biodegradation in Sediments by Microbial Electrochemistry.

PubMed

Yan, Zaisheng; He, Yuhong; Cai, Haiyuan; Van Nostrand, Joy D; He, Zhili; Zhou, Jizhong; Krumholz, Lee R; Jiang, He-Long

2017-08-01

Sediment microbial fuel cells (SMFCs) can stimulate the degradation of polycyclic aromatic hydrocarbons in sediments, but the mechanism of this process is poorly understood at the microbial functional gene level. Here, the use of SMFC resulted in 92% benzo[a]pyrene (BaP) removal over 970 days relative to 54% in the controls. Sediment functions, microbial community structure, and network interactions were dramatically altered by the SMFC employment. Functional gene analysis showed that c-type cytochrome genes for electron transfer, aromatic degradation genes, and extracellular ligninolytic enzymes involved in lignin degradation were significantly enriched in bulk sediments during SMFC operation. Correspondingly, chemical analysis of the system showed that these genetic changes resulted in increases in the levels of easily oxidizable organic carbon and humic acids which may have resulted in increased BaP bioavailability and increased degradation rates. Tracking microbial functional genes and corresponding organic matter responses should aid mechanistic understanding of BaP enhanced biodegradation by microbial electrochemistry and development of sustainable bioremediation strategies.

Identification of Carbohydrate Metabolism Genes in the Metagenome of a Marine Biofilm Community Shown to Be Dominated by Gammaproteobacteria, Bacteroidetes

PubMed Central

Edwards, Jennifer L.; Smith, Darren L.; Connolly, John; McDonald, James E.; Cox, Michael J.; Joint, Ian; Edwards, Clive; McCarthy, Alan J.

2010-01-01

Polysaccharides are an important source of organic carbon in the marine environment, degradation of the insoluble, globally abundant cellulose is a major component of the marine carbon cycle. Although a number of species of cultured bacteria are known to degrade crystalline cellulose, little is known of the polysaccharide hydrolases expressed by cellulose-degrading microbial communities, particularly in the marine environment. Next generation 454 Pyrosequencing was applied to analyze the microbial community that colonizes, degrades insoluble polysaccharides in situ in the Irish Sea. The bioinformatics tool MG-RAST was used to examine the randomly sampled data for taxonomic markers, functional genes,, showed that the community was dominated by members of the Gammaproteobacteria, Bacteroidetes. Furthermore, the identification of 211 gene sequences matched to a custom-made database comprising the members of nine glycoside hydrolase families revealed an extensive repertoire of functional genes predicted to be involved in cellulose utilization. This demonstrates that the use of an in situ cellulose baiting method yielded a marine microbial metagenome considerably enriched in functional genes involved in polysaccharide degradation. The research reported here is the first designed to specifically address the bacterial communities that colonize, degrade cellulose in the marine environment, to evaluate the glycoside hydrolase (cellulase, chitinase) gene repertoire of that community, in the absence of the biases associated with PCR-based molecular techniques. PMID:24710093

Delineation of Steroid-Degrading Microorganisms through Comparative Genomic Analysis

PubMed Central

Bergstrand, Lee H.; Cardenas, Erick; Holert, Johannes; Van Hamme, Jonathan D.

2016-01-01

ABSTRACT Steroids are ubiquitous in natural environments and are a significant growth substrate for microorganisms. Microbial steroid metabolism is also important for some pathogens and for biotechnical applications. This study delineated the distribution of aerobic steroid catabolism pathways among over 8,000 microorganisms whose genomes are available in the NCBI RefSeq database. Combined analysis of bacterial, archaeal, and fungal genomes with both hidden Markov models and reciprocal BLAST identified 265 putative steroid degraders within only Actinobacteria and Proteobacteria, which mainly originated from soil, eukaryotic host, and aquatic environments. These bacteria include members of 17 genera not previously known to contain steroid degraders. A pathway for cholesterol degradation was conserved in many actinobacterial genera, particularly in members of the Corynebacterineae, and a pathway for cholate degradation was conserved in members of the genus Rhodococcus. A pathway for testosterone and, sometimes, cholate degradation had a patchy distribution among Proteobacteria. The steroid degradation genes tended to occur within large gene clusters. Growth experiments confirmed bioinformatic predictions of steroid metabolism capacity in nine bacterial strains. The results indicate there was a single ancestral 9,10-seco-steroid degradation pathway. Gene duplication, likely in a progenitor of Rhodococcus, later gave rise to a cholate degradation pathway. Proteobacteria and additional Actinobacteria subsequently obtained a cholate degradation pathway via horizontal gene transfer, in some cases facilitated by plasmids. Catabolism of steroids appears to be an important component of the ecological niches of broad groups of Actinobacteria and individual species of Proteobacteria. PMID:26956583

Effect of milk on the urinary excretion of microbial phenolic acids after cocoa powder consumption in humans.

PubMed

Urpi-Sarda, Mireia; Llorach, Rafael; Khan, Nasiruddin; Monagas, Maria; Rotches-Ribalta, Maria; Lamuela-Raventos, Rosa; Estruch, Ramon; Tinahones, Francisco J; Andres-Lacueva, Cristina

2010-04-28

Health effects of cocoa flavonols depend on their bioavailability, which is strongly influenced by the food matrix and the degree of flavanol polymerization. The effect of milk on the bioavailability of cocoa flavanoids considering phase II metabolites of epicatechin has been the subject of considerable debate. This work studies the effect of milk at the colonic microbial metabolism level of the nonabsorbed flavanol fraction that reaches the colon and is metabolized by the colonic microbiota into various phenolic acids. Twenty-one human volunteers followed a diet low in polyphenols for at least 48 h before taking, in a random order, 40 g of cocoa powder dissolved either in 250 mL of whole milk or in 250 mL of water. Urine samples were collected before the intake and during three different periods (0-6, 6-12, and 12-24 h). Phenolic acids were analyzed by LC-MS/MS after solid-phase extraction. Of the 15 metabolites assessed, the excretion of 9 phenolic acids was affected by the intake of milk. The urinary concentration of 3,4-dihydroxyphenylacetic, protocatechuic, 4-hydroxybenzoic, 4-hydroxyhippuric, hippuric, caffeic, and ferulic acids diminished after the intake of cocoa with milk, whereas urinary concentrations of vanillic and phenylacetic acids increased. In conclusion, milk partially affects the formation of microbial phenolic acids derived from the colonic degradation of procyanidins and other compounds present in cocoa powder.

The Fdb3 transcription factor of the Fusarium Detoxification of Benzoxazolinone gene cluster is required for MBOA but not BOA degradation in Fusarium pseudograminearum.

PubMed

Kettle, Andrew J; Carere, Jason; Batley, Jacqueline; Manners, John M; Kazan, Kemal; Gardiner, Donald M

2016-03-01

A number of cereals produce the benzoxazolinone class of phytoalexins. Fusarium species pathogenic towards these hosts can typically degrade these compounds via an aminophenol intermediate, and the ability to do so is encoded by a group of genes found in the Fusarium Detoxification of Benzoxazolinone (FDB) cluster. A zinc finger transcription factor encoded by one of the FDB cluster genes (FDB3) has been proposed to regulate the expression of other genes in the cluster and hence is potentially involved in benzoxazolinone degradation. Herein we show that Fdb3 is essential for the ability of Fusarium pseudograminearum to efficiently detoxify the predominant wheat benzoxazolinone, 6-methoxy-benzoxazolin-2-one (MBOA), but not benzoxazoline-2-one (BOA). Furthermore, additional genes thought to be part of the FDB gene cluster, based upon transcriptional response to benzoxazolinones, are regulated by Fdb3. However, deletion mutants for these latter genes remain capable of benzoxazolinone degradation, suggesting that they are not essential for this process. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

SEQUENCE SIMILARITIES IN THE GENES ENCODING POLY- CHLORINATED BIPHENYL DEGRADATION BY PSEUDOMONAS STRAIN LB400 AND ALCALIGENES EUTROPHUS H850

EPA Science Inventory

DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains. Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to L...

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants.

PubMed

Fernández-Marín, Hermógenes; Nash, David R; Higginbotham, Sarah; Estrada, Catalina; van Zweden, Jelle S; d'Ettorre, Patrizia; Wcislo, William T; Boomsma, Jacobus J

2015-05-22

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

An update on the use of benzoate, phenylacetate and phenylbutyrate ammonia scavengers for interrogating and modifying liver nitrogen metabolism and its implications in urea cycle disorders and liver disease

PubMed Central

de las Heras, Javier; Aldámiz-Echevarría, Luis; Martínez-Chantar, María-Luz; Delgado, Teresa C.

2017-01-01

Introduction Ammonia-scavenging drugs, benzoate and phenylacetate (PA)/phenylbutyrate (PB), modulate hepatic nitrogen metabolism mainly by providing alternative pathways for nitrogen disposal. Areas Covered We review the major findings and potential novel applications of ammonia-scavenging drugs, focusing on urea cycle disorders and liver disease. Expert Opinion For over 40 years, ammonia-scavenging drugs have been used in the treatment of urea cycle disorders. Recently, the use of these compounds has been advocated in acute liver failure and cirrhosis for reducing hyperammonemic-induced hepatic encephalopathy. The efficacy and mechanisms underlying the antitumor effects of these ammonia-scavenging drugs in liver cancer are more controversial and are discussed in the review. Overall, as ammonia-scavenging drugs are usually safe and well tolerated among cancer patients, further studies should be instigated to explore the role of these drugs in liver cancer. Considering the relevance of glutamine metabolism to the progression and resolution of liver disease, we propose that ammonia-scavenging drugs might also be used to non-invasively probe liver glutamine metabolism in vivo. Finally, novel derivatives of classical ammonia-scavenging drugs with fewer and less severe adverse effects are currently being developed and used in clinical trials for the treatment of acute liver failure and cirrhosis. PMID:27860485

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants

PubMed Central

Fernández-Marín, Hermógenes; Nash, David R.; Higginbotham, Sarah; Estrada, Catalina; van Zweden, Jelle S.; d'Ettorre, Patrizia; Wcislo, William T.; Boomsma, Jacobus J.

2015-01-01

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens. PMID:25925100

An update on the use of benzoate, phenylacetate and phenylbutyrate ammonia scavengers for interrogating and modifying liver nitrogen metabolism and its implications in urea cycle disorders and liver disease.

PubMed

De Las Heras, Javier; Aldámiz-Echevarría, Luis; Martínez-Chantar, María-Luz; Delgado, Teresa C

2017-04-01

Ammonia-scavenging drugs, benzoate and phenylacetate (PA)/phenylbutyrate (PB), modulate hepatic nitrogen metabolism mainly by providing alternative pathways for nitrogen disposal. Areas covered: We review the major findings and potential novel applications of ammonia-scavenging drugs, focusing on urea cycle disorders and liver disease. Expert opinion: For over 40 years, ammonia-scavenging drugs have been used in the treatment of urea cycle disorders. Recently, the use of these compounds has been advocated in acute liver failure and cirrhosis for reducing hyperammonemic-induced hepatic encephalopathy. The efficacy and mechanisms underlying the antitumor effects of these ammonia-scavenging drugs in liver cancer are more controversial and are discussed in the review. Overall, as ammonia-scavenging drugs are usually safe and well tolerated among cancer patients, further studies should be instigated to explore the role of these drugs in liver cancer. Considering the relevance of glutamine metabolism to the progression and resolution of liver disease, we propose that ammonia-scavenging drugs might also be used to non-invasively probe liver glutamine metabolism in vivo. Finally, novel derivatives of classical ammonia-scavenging drugs with fewer and less severe adverse effects are currently being developed and used in clinical trials for the treatment of acute liver failure and cirrhosis.

Molecular Dynamics and Free Energy Simulations of Phenylacetate and CO2 Release from AMDase and Its G74C/C188S Mutant: A Possible Rationale for the Reduced Activity of the Latter.

PubMed

Karmakar, Tarak; Balasubramanian, Sundaram

2016-11-17

Arylmalonate decarboxylase (AMDase) catalyzes the decarboxylation of α-aryl-α-methyl malonates to produce optically pure α-arylpropionates of industrial and medicinal importance. Herein, atomistic molecular dynamics simulations have been carried out to delineate the mechanism of the release of product molecules phenylacetate (PAC) and carbon dioxide (CO 2 ), from the wild-type (WT) and its G74C/C188S mutant enzymes. Both of the product molecules follow a crystallographically characterized solvent-accessible channel to come out of the protein interior. A higher free energy barrier for the release of PAC from G74C/C188S compared to that in the WT is consistent with the experimentally observed compromised efficiency of the mutant. The release of CO 2 precedes that of PAC; free energy barriers for CO 2 and PAC release in the WT enzyme are calculated to be ∼1-2 and ∼23 kcal/mol, respectively. Postdecarboxylation, CO 2 moves toward a hydrophobic pocket formed by Pro 14, Leu 38, Leu 40, Leu 77, and the side chain of Tyr 48 which serves as its temporary "reservoir". CO 2 releases following a channel mainly decorated by apolar residues, unlike in the case of oxalate decarboxylase where polar residues mediate its transport.

The polycyclic aromatic hydrocarbon degradation potential of Gulf of Mexico native coastal microbial communities after the Deepwater Horizon oil spill

PubMed Central

Kappell, Anthony D.; Wei, Yin; Newton, Ryan J.; Van Nostrand, Joy D.; Zhou, Jizhong; McLellan, Sandra L.; Hristova, Krassimira R.

2014-01-01

The Deepwater Horizon (DWH) blowout resulted in oil transport, including polycyclic aromatic hydrocarbons (PAHs) to the Gulf of Mexico shoreline. The microbial communities of these shorelines are thought to be responsible for the intrinsic degradation of PAHs. To investigate the Gulf Coast beach microbial community response to hydrocarbon exposure, we examined the functional gene diversity, bacterial community composition, and PAH degradation capacity of a heavily oiled and non-oiled beach following the oil exposure. With a non-expression functional gene microarray targeting 539 gene families, we detected 28,748 coding sequences. Of these sequences, 10% were uniquely associated with the severely oil-contaminated beach and 6.0% with the non-oiled beach. There was little variation in the functional genes detected between the two beaches; however the relative abundance of functional genes involved in oil degradation pathways, including polycyclic aromatic hydrocarbons (PAHs), were greater in the oiled beach. The microbial PAH degradation potentials of both beaches, were tested in mesocosms. Mesocosms were constructed in glass columns using sands with native microbial communities, circulated with artificial sea water and challenged with a mixture of PAHs. The low-molecular weight PAHs, fluorene and naphthalene, showed rapid depletion in all mesocosms while the high-molecular weight benzo[α]pyrene was not degraded by either microbial community. Both the heavily oiled and the non-impacted coastal communities showed little variation in their biodegradation ability for low molecular weight PAHs. Massively-parallel sequencing of 16S rRNA genes from mesocosm DNA showed that known PAH degraders and genera frequently associated with oil hydrocarbon degradation represented a major portion of the bacterial community. The observed similar response by microbial communities from beaches with a different recent history of oil exposure suggests that Gulf Coast beach communities are primed for PAH degradation. PMID:24847320

[Promotion effects of vitamin B12 on the degradation of 2, 4, 4'-trichlorobiphenyl by Nostoc PD-2].

PubMed

Liu, Jia-Yu; Xiao, Wen-Feng; Lu, Li-Ping; Zhang, Hang-Jun

2014-08-01

Polychlorinated biphenyls are typical persistent chlorinated organic compounds in the environment. Bioremediation of PCB-contaminated environment has become one of the hot issues. In this study, vitamin B12 (VB12) and chlorine-free culture medium were applied to study the effects of VB12 on the degradation of 2,4,4'-trichlorobiphenyl (PCB28) by Nostoc PD-2 and the gene expression during the PCB-degradation process. Results showed that addition of different concentrations of vitamin B12 could improve the PCB-biodegradation rates by Nostoc PD-2. Compared with the control group, the 7-day degradation rate in 10 microg x L(-1), 100 microg x L(-1), and 1 000 microg x L(-1) VB12-treated groups increased by 11.0%, 19.7%, and 21.9% , respectively. The degradation half-time decreased from 5.53 days (treated with 10 microg x L(-1) VB12) to 3.08 days (treated with 100 microg x L(-1) VB12). The expression of cytochrome b6f complex iron-sulfur protein gene and dioxygenase gene showed significant correlation with PCB28-degradation by Nostoc PD-2. While the expression of iron-sulfur protein gene showed more significant correlation with PCB28-degradation. Results in this study indicated that adding VB12 could promote PCB28-degradation by Nostoc PD-2. Moreover, VB12 addition improved the PCB-degradation activity of Nostoc PD-2 at the gene level. The above conclusions could provide a new choice for developing efficient bioremediation technology for PCB-contaminated environment and a new insight into the PCB-biodegradation mechanism by Nostoc PD-2.

Dominant petroleum hydrocarbon-degrading bacteria in the Archipelago Sea in South-West Finland (Baltic Sea) belong to different taxonomic groups than hydrocarbon degraders in the oceans.

PubMed

Reunamo, Anna; Riemann, Lasse; Leskinen, Piia; Jørgensen, Kirsten S

2013-07-15

The natural petroleum hydrocarbon degrading capacity of the Archipelago Sea water in S-W Finland was studied in a microcosm experiment. Pristine and previously oil exposed sites were examined. Bacterial community fingerprinting was performed using terminal restriction fragment length polymorphism (T-RFLP) and samples from selected microcosms were sequenced. The abundance of PAH degradation genes was measured by quantitative PCR. Bacterial communities in diesel exposed microcosms diverged from control microcosms during the experiment. Gram positive PAH degradation genes dominated at both sites in situ, whereas gram negative PAH degrading genes became enriched in diesel microcosms. The dominant bacterial groups after a 14 days of diesel exposure were different depending on the sampling site, belonging to the class Actinobacteria (32%) at a pristine site and Betaproteobacteria (52%) at a previously oil exposed site. The hydrocarbon degrading bacteria in the Baltic Sea differ from those in the oceans, where most hydrocarbon degraders belong to Gammaproteobacteria. Copyright © 2013 Elsevier Ltd. All rights reserved.

NOVEL ORGANIZATION OF THE GENES FOR PHTHALATE DEGRADATION FROM BURKHOLDERIA CEPACIA DBO1

EPA Science Inventory

Burkholderia cepacia DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthala...

Role of TonB1 in pyoverdine-mediated signaling in Pseudomonas aeruginosa.

PubMed

Shirley, Matt; Lamont, Iain L

2009-09-01

Pyoverdines are siderophores secreted by Pseudomonas aeruginosa. Uptake of ferripyoverdine in P. aeruginosa PAO1 occurs via the FpvA receptor protein and requires the energy-transducing protein TonB1. Interaction of (ferri)pyoverdine with FpvA activates pyoverdine gene expression in a signaling process involving the cytoplasmic-membrane-spanning anti-sigma factor FpvR and the sigma factor PvdS. Here, we show that mutation of a region of FpvA that interacts with TonB1 (the TonB box) prevents this signaling process, as well as inhibiting bacterial growth in the presence of the iron-chelating compound ethylenediamine-di(o-hydroxy-phenylacetic acid). Signaling via wild-type FpvA was also eliminated in strains lacking TonB1 but was unaffected in strains lacking either (or both) of two other TonB proteins in P. aeruginosa, TonB2 and TonB3. An absence of pyoverdine-mediated signaling corresponded with proteolysis of PvdS. These data show that interactions between FpvA and TonB1 are required for (ferri)pyoverdine signal transduction, as well as for ferripyoverdine transport, consistent with a mechanistic link between the signaling and transport functions of FpvA.

Role of TonB1 in Pyoverdine-Mediated Signaling in Pseudomonas aeruginosa▿

PubMed Central

Shirley, Matt; Lamont, Iain L.

2009-01-01

Pyoverdines are siderophores secreted by Pseudomonas aeruginosa. Uptake of ferripyoverdine in P. aeruginosa PAO1 occurs via the FpvA receptor protein and requires the energy-transducing protein TonB1. Interaction of (ferri)pyoverdine with FpvA activates pyoverdine gene expression in a signaling process involving the cytoplasmic-membrane-spanning anti-sigma factor FpvR and the sigma factor PvdS. Here, we show that mutation of a region of FpvA that interacts with TonB1 (the TonB box) prevents this signaling process, as well as inhibiting bacterial growth in the presence of the iron-chelating compound ethylenediamine-di(o-hydroxy-phenylacetic acid). Signaling via wild-type FpvA was also eliminated in strains lacking TonB1 but was unaffected in strains lacking either (or both) of two other TonB proteins in P. aeruginosa, TonB2 and TonB3. An absence of pyoverdine-mediated signaling corresponded with proteolysis of PvdS. These data show that interactions between FpvA and TonB1 are required for (ferri)pyoverdine signal transduction, as well as for ferripyoverdine transport, consistent with a mechanistic link between the signaling and transport functions of FpvA. PMID:19592589

Naringenin degradation by the endophytic diazotroph Herbaspirillum seropedicae SmR1.

PubMed

Marin, A M; Souza, E M; Pedrosa, F O; Souza, L M; Sassaki, G L; Baura, V A; Yates, M G; Wassem, R; Monteiro, R A

2013-01-01

Several bacteria are able to degrade flavonoids either to use them as carbon sources or as a detoxification mechanism. Degradation pathways have been proposed for several bacteria, but the genes responsible are not known. We identified in the genome of the endophyte Herbaspirillum seropedicae SmR1 an operon potentially associated with the degradation of aromatic compounds. We show that this operon is involved in naringenin degradation and that its expression is induced by naringenin and chrysin, two closely related flavonoids. Mutation of fdeA, the first gene of the operon, and fdeR, its transcriptional activator, abolished the ability of H. seropedicae to degrade naringenin.

Presence or Absence of mlr Genes and Nutrient Concentrations Co-Determine the Microcystin Biodegradation Efficiency of a Natural Bacterial Community

PubMed Central

Lezcano, María Ángeles; Morón-López, Jesús; Agha, Ramsy; López-Heras, Isabel; Nozal, Leonor; Quesada, Antonio; El-Shehawy, Rehab

2016-01-01

The microcystin biodegradation potential of a natural bacterial community coexisting with a toxic cyanobacterial bloom was investigated in a water reservoir from central Spain. The biodegradation capacity was confirmed in all samples during the bloom and an increase of mlrA gene copies was found with increasing microcystin concentrations. Among the 24 microcystin degrading strains isolated from the bacterial community, only 28% showed presence of mlrA gene, strongly supporting the existence and abundance of alternative microcystin degradation pathways in nature. In vitro degradation assays with both mlr+ and mlr− bacterial genotypes (with presence and absence of the complete mlr gene cluster, respectively) were performed with four isolated strains (Sphingopyxis sp. IM-1, IM-2 and IM-3; Paucibacter toxinivorans IM-4) and two bacterial degraders from the culture collection (Sphingosinicella microcystinivorans Y2; Paucibacter toxinivorans 2C20). Differences in microcystin degradation efficiencies between genotypes were found under different total organic carbon and total nitrogen concentrations. While mlr+ strains significantly improved microcystin degradation rates when exposed to other carbon and nitrogen sources, mlr− strains showed lower degradation efficiencies. This suggests that the presence of alternative carbon and nitrogen sources possibly competes with microcystins and impairs putative non-mlr microcystin degradation pathways. Considering the abundance of the mlr− bacterial population and the increasing frequency of eutrophic conditions in aquatic systems, further research on the diversity of this population and the characterization and conditions affecting non-mlr degradation pathways deserves special attention. PMID:27827872

A cluster of bacterial genes for anaerobic benzene ring biodegradation

PubMed Central

Egland, Paul G.; Pelletier, Dale A.; Dispensa, Marilyn; Gibson, Jane; Harwood, Caroline S.

1997-01-01

A reductive benzoate pathway is the central conduit for the anaerobic biodegradation of aromatic pollutants and lignin monomers. Benzene ring reduction requires a large input of energy and this metabolic capability has, so far, been reported only in bacteria. To determine the molecular basis for this environmentally important process, we cloned and analyzed genes required for the anaerobic degradation of benzoate and related compounds from the phototrophic bacterium, Rhodopseudomonas palustris. A cluster of 24 genes was identified that includes twelve genes likely to be involved in anaerobic benzoate degradation and additional genes that convert the related compounds 4-hydroxybenzoate and cyclohexanecarboxylate to benzoyl-CoA. Genes encoding benzoyl-CoA reductase, a novel enzyme able to overcome the resonance stability of the aromatic ring, were identified by directed mutagenesis. The gene encoding the ring-cleavage enzyme, 2-ketocyclohexanecarboxyl-CoA hydrolase, was identified by assaying the enzymatic activity of the protein expressed in Escherichia coli. Physiological data and DNA sequence analyses indicate that the benzoate pathway consists of unusual enzymes for ring reduction and cleavage interposed among enzymes homologous to those catalyzing fatty acid degradation. The cloned genes should be useful as probes to identify benzoate degradation genes from other metabolically distinct groups of anaerobic bacteria, such as denitrifying bacteria and sulfate-reducing bacteria. PMID:9177244

Role of P450 monooxygenases in the degradation of the endocrine-disrupting chemical nonylphenol by the white rot fungus Phanerochaete chrysosporium.

PubMed

Subramanian, Venkataramanan; Yadav, Jagjit S

2009-09-01

The white rot fungus Phanerochaete chrysosporium extensively degraded the endocrine disruptor chemical nonylphenol (NP; 100% of 100 ppm) in both nutrient-limited cultures and nutrient-sufficient cultures. The P450 enzyme inhibitor piperonyl butoxide caused significant inhibition (approximately 75%) of the degradation activity in nutrient-rich malt extract (ME) cultures but no inhibition in defined low-nitrogen (LN) cultures, indicating an essential role of P450 monooxygenase(s) in NP degradation under nutrient-rich conditions. A genome-wide analysis using our custom-designed P450 microarray revealed significant induction of multiple P450 monooxygenase genes by NP: 18 genes were induced (2- to 195-fold) under nutrient-rich conditions, 17 genes were induced (2- to 6-fold) in LN cultures, and 3 were induced under both nutrient-rich and LN conditions. The P450 genes Pff 311b (corresponding to protein identification number [ID] 5852) and Pff 4a (protein ID 5001) showed extraordinarily high levels of induction (195- and 167-fold, respectively) in ME cultures. The P450 oxidoreductase (POR), glutathione S-transferase (gst), and cellulose metabolism genes were also induced in ME cultures. In contrast, certain metabolic genes, such as five of the peroxidase genes, showed partial downregulation by NP. This study provides the first evidence for the involvement of P450 enzymes in NP degradation by a white rot fungus and the first genome-wide identification of specific P450 genes responsive to an environmentally significant toxicant.

Role of P450 Monooxygenases in the Degradation of the Endocrine-Disrupting Chemical Nonylphenol by the White Rot Fungus Phanerochaete chrysosporium▿

PubMed Central

Subramanian, Venkataramanan; Yadav, Jagjit S.

2009-01-01

The white rot fungus Phanerochaete chrysosporium extensively degraded the endocrine disruptor chemical nonylphenol (NP; 100% of 100 ppm) in both nutrient-limited cultures and nutrient-sufficient cultures. The P450 enzyme inhibitor piperonyl butoxide caused significant inhibition (∼75%) of the degradation activity in nutrient-rich malt extract (ME) cultures but no inhibition in defined low-nitrogen (LN) cultures, indicating an essential role of P450 monooxygenase(s) in NP degradation under nutrient-rich conditions. A genome-wide analysis using our custom-designed P450 microarray revealed significant induction of multiple P450 monooxygenase genes by NP: 18 genes were induced (2- to 195-fold) under nutrient-rich conditions, 17 genes were induced (2- to 6-fold) in LN cultures, and 3 were induced under both nutrient-rich and LN conditions. The P450 genes Pff 311b (corresponding to protein identification number [ID] 5852) and Pff 4a (protein ID 5001) showed extraordinarily high levels of induction (195- and 167-fold, respectively) in ME cultures. The P450 oxidoreductase (POR), glutathione S-transferase (gst), and cellulose metabolism genes were also induced in ME cultures. In contrast, certain metabolic genes, such as five of the peroxidase genes, showed partial downregulation by NP. This study provides the first evidence for the involvement of P450 enzymes in NP degradation by a white rot fungus and the first genome-wide identification of specific P450 genes responsive to an environmentally significant toxicant. PMID:19542331

Mapping the polysaccharide degradation potential of Aspergillus niger

PubMed Central

2012-01-01

Background The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. Results Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. Conclusions The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger. PMID:22799883

Mapping the polysaccharide degradation potential of Aspergillus niger.

PubMed

Andersen, Mikael R; Giese, Malene; de Vries, Ronald P; Nielsen, Jens

2012-07-16

The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger.

Degradation of Bunker C Fuel Oil by White-Rot Fungi in Sawdust Cultures Suggests Potential Applications in Bioremediation

PubMed Central

Young, Darcy; Rice, James; Martin, Rachael; Lindquist, Erika; Lipzen, Anna; Grigoriev, Igor; Hibbett, David

2015-01-01

Fungal lignocellulolytic enzymes are promising agents for oxidizing pollutants. This study investigated degradation of Number 6 “Bunker C” fuel oil compounds by the white-rot fungi Irpex lacteus, Trichaptum biforme, Phlebia radiata, Trametes versicolor, and Pleurotus ostreatus (Basidiomycota, Agaricomycetes). Averaging across all studied species, 98.1%, 48.6%, and 76.4% of the initial Bunker C C10 alkane, C14 alkane, and phenanthrene, respectively were degraded after 180 days of fungal growth on pine media. This study also investigated whether Bunker C oil induces changes in gene expression in the white-rot fungus Punctularia strigosozonata, for which a complete reference genome is available. After 20 days of growth, a monokaryon P. strigosozonata strain degraded 99% of the initial C10 alkane in both pine and aspen media but did not affect the amounts of the C14 alkane or phenanthrene. Differential gene expression analysis identified 119 genes with ≥ log2(2-fold) greater expression in one or more treatment comparisons. Six genes were significantly upregulated in media containing oil; these genes included three enzymes with potential roles in xenobiotic biotransformation. Carbohydrate metabolism genes showing differential expression significantly accumulated transcripts on aspen vs. pine substrates, perhaps reflecting white-rot adaptations to growth on hardwood substrates. The mechanisms by which P. strigosozonata may degrade complex oil compounds remain obscure, but degradation results of the 180-day cultures suggest that diverse white-rot fungi have promise for bioremediation of petroleum fuels. PMID:26111162

Cloning, Characterization and Analysis of cat and ben Genes from the Phenol Degrading Halophilic Bacterium Halomonas organivorans

PubMed Central

Moreno, Maria de Lourdes; Sánchez-Porro, Cristina; Piubeli, Francine; Frias, Luciana; García, María Teresa; Mellado, Encarnación

2011-01-01

Background Extensive use of phenolic compounds in industry has resulted in the generation of saline wastewaters that produce significant environmental contamination; however, little information is available on the degradation of phenolic compounds in saline conditions. Halomonas organivorans G-16.1 (CECT 5995T) is a moderately halophilic bacterium that we isolated in a previous work from saline environments of South Spain by enrichment for growth in different pollutants, including phenolic compounds. PCR amplification with degenerate primers revealed the presence of genes encoding ring-cleaving enzymes of the β-ketoadipate pathway for aromatic catabolism in H. organivorans. Findings The gene cluster catRBCA, involved in catechol degradation, was isolated from H. organivorans. The genes catA, catB, catC and the divergently transcribed catR code for catechol 1,2-dioxygenase (1,2-CTD), cis,cis-muconate cycloisomerase, muconolactone delta-isomerase and a LysR-type transcriptional regulator, respectively. The benzoate catabolic genes (benA and benB) are located flanking the cat genes. The expression of cat and ben genes by phenol and benzoic acid was shown by RT-PCR analysis. The induction of catA gene by phenol and benzoic acid was also probed by the measurement of 1,2-CTD activity in H. organivorans growth in presence of these inducers. 16S rRNA and catA gene-based phylogenies were established among different degrading bacteria showing no phylogenetic correlation between both genes. Conclusions/Significance In this work, we isolated and determined the sequence of a gene cluster from a moderately halophilic bacterium encoding ortho-pathway genes involved in the catabolic metabolism of phenol and analyzed the gene organization, constituting the first report characterizing catabolic genes involved in the degradation of phenol in moderate halophiles, providing an ideal model system to investigate the potential use of this group of extremophiles in the decontamination of saline environments. PMID:21695219

Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle.

PubMed

Patyshakuliyeva, Aleksandrina; Post, Harm; Zhou, Miaomiao; Jurak, Edita; Heck, Albert J R; Hildén, Kristiina S; Kabel, Mirjam A; Mäkelä, Miia R; Altelaar, Maarten A F; de Vries, Ronald P

2015-08-01

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Screening for and isolation and identification of malathion-degrading bacteria: cloning and sequencing a gene that potentially encodes the malathion-degrading enzyme, carboxylestrase in soil bacteria.

PubMed

Goda, Sayed K; Elsayed, Iman E; Khodair, Taha A; El-Sayed, Walaa; Mohamed, Mervat E

2010-11-01

Five malathion-degrading bacterial strains were enriched and isolated from soil samples collected from different agricultural sites in Cairo, Egypt. Malathion was used as a sole source of carbon (50 mg/l) to enumerate malathion degraders, which were designated as IS1, IS2, IS3, IS4, and IS5. They were identified, based on their morphological and biochemical characteristics, as Pseudomonas sp., Pseudomonas putida, Micrococcus lylae, Pseudomonas aureofaciens, and Acetobacter liquefaciens, respectively. IS1 and IS2, which showed the highest degrading activity, were selected for further identification by partial sequence analysis of their 16S rRNA genes. The 16S rRNA gene of IS1 shared 99% similarity with that of Alphaprotoebacterium BAL284, while IS2 scored 100% similarity with that of Pseudomonas putida 32zhy. Malathion residues almost completely disappeared within 6 days of incubation in IS2 liquid cultures. LC/ESI-MS analysis confirmed the degradation of malathion to malathion monocarboxylic and dicarboxylic acids, which formed as a result of carboxylesterase activity. A carboxylesterase gene (CE) was amplified from the IS2 genome by using specifically designed PCR primers. The sequence analysis showed a significant similarity to a known CE gene in different Pseudomonas sp. We report here the isolation of a new malathion-degrading bacteria from soils in Egypt that may be very well adapted to the climatic and environmental conditions of the country. We also report the partial cloning of a new CE gene. Due to their high biodegradation activity, the bacteria isolated from this work merit further study as potential biological agents for the remediation of soil, water, or crops contaminated with the pesticide malathion.

Characterization of a Polycyclic Aromatic Hydrocarbon Degradation Gene Cluster in a Phenanthrene-Degrading Acidovorax Strain▿

PubMed Central

Singleton, David R.; Guzmán Ramirez, Liza; Aitken, Michael D.

2009-01-01

Acidovorax sp. strain NA3 was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil that had been treated in a bioreactor and enriched with phenanthrene. The 16S rRNA gene of the isolate possessed 99.8 to 99.9% similarity to the dominant sequences recovered during a previous stable-isotope probing experiment with [U-13C]phenanthrene on the same soil (D. R. Singleton, S. N. Powell, R. Sangaiah, A. Gold, L. M. Ball, and M. D. Aitken, Appl. Environ. Microbiol. 71:1202-1209, 2005). The strain grew on phenanthrene as a sole carbon and energy source and could mineralize 14C from a number of partially labeled PAHs, including naphthalene, phenanthrene, chrysene, benz[a]anthracene, and benzo[a]pyrene, but not pyrene or fluoranthene. Southern hybridizations of a genomic fosmid library with a fragment of the large subunit of the ring-hydroxylating dioxygenase gene from a naphthalene-degrading Pseudomonas strain detected the presence of PAH degradation genes subsequently determined to be highly similar in both nucleotide sequence and gene organization to an uncharacterized Alcaligenes faecalis gene cluster. The genes were localized to the chromosome of strain NA3. To test for gene induction by selected compounds, RNA was extracted from amended cultures and reverse transcribed, and cDNA associated with the enzymes involved in the first three steps of phenanthrene degradation was quantified by quantitative real-time PCR. Expression of each of the genes was induced most strongly by phenanthene and to a lesser extent by naphthalene, but other tested PAHs and PAH metabolites had negligible effects on gene transcript levels. PMID:19270134

Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription.

PubMed

Marza, Esther; Taouji, Saïd; Barroso, Kim; Raymond, Anne-Aurélie; Guignard, Léo; Bonneu, Marc; Pallares-Lupon, Néstor; Dupuy, Jean-William; Fernandez-Zapico, Martin E; Rosenbaum, Jean; Palladino, Francesca; Dupuy, Denis; Chevet, Eric

2015-03-01

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. The AAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes. © 2015 The Authors.

Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription

PubMed Central

Marza, Esther; Taouji, Saïd; Barroso, Kim; Raymond, Anne-Aurélie; Guignard, Léo; Bonneu, Marc; Pallares-Lupon, Néstor; Dupuy, Jean-William; Fernandez-Zapico, Martin E; Rosenbaum, Jean; Palladino, Francesca; Dupuy, Denis; Chevet, Eric

2015-01-01

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPRER) to restore ER homeostasis. The AAA+ ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPRER genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA+ ATPase, as a novel repressor of a subset of UPRER genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPRER genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes. PMID:25652260

The hmuQ and hmuD Genes from Bradyrhizobium japonicum Encode Heme-Degrading Enzymes

PubMed Central

Puri, Sumant; O'Brian, Mark R.

2006-01-01

Utilization of heme by bacteria as a nutritional iron source involves the transport of exogenous heme, followed by cleavage of the heme macrocycle to release iron. Bradyrhizobium japonicum can use heme as an iron source, but no heme-degrading oxygenase has been described. Here, bioinformatics analyses of the B. japonicum genome identified two paralogous genes renamed hmuQ (bll7075) and hmuD (bll7423) that encode proteins with weak similarity to the heme-degrading monooxygenase IsdG from Staphylococcus aureus. The hmuQ gene is clustered with known heme transport genes in the genome. Recombinant HmuQ bound heme with a Kd value of 0.8 μM and showed spectral properties consistent with a heme oxygenase. In the presence of a reductant, HmuQ catalyzed the degradation of heme and the formation of biliverdin. The hmuQ and hmuD genes complemented a Corynebacterium ulcerans heme oxygenase mutant in trans for utilization of heme as the sole iron source for growth. Furthermore, homologs of hmuQ and hmuD were identified in many bacterial genera, and the recombinant homolog from Brucella melitensis bound heme and catalyzed its degradation. The findings show that hmuQ and hmuD encode heme oxygenases and indicate that the IsdG family of heme-degrading monooxygenases is not restricted to gram-positive pathogenic bacteria. PMID:16952937

No evidential correlation between veterinary antibiotic degradation ability and resistance genes in microorganisms during the biodegradation of doxycycline.

PubMed

Wen, Xin; Wang, Yan; Zou, Yongde; Ma, Baohua; Wu, Yinbao

2018-01-01

Biodegradation of antibiotic residues in the environment by microorganisms may lead to the generation of antibiotic resistance genes (ARGs), which are of great concern to human health. The aim of this study was to determine whether there is a relationship between the ability to degrade antibiotic doxycycline (DOX) and the development of resistance genes in microorganisms. We isolated and identified ten bacterial strains from a vegetable field that had received long-term manure application as fertilizer and were capable of surviving in a series of DOX concentrations (25, 50, 80, and 100mg/L). Our results showed no evidential correlation between DOX degradation ability and the development of resistance genes among the isolated microorganisms that had high DOX degradation capability (P > 0.05). This was based on the fact that Escherichia sp. and Candida sp. were the most efficient bacterial strains to degrade DOX (92.52% and 91.63%, respectively), but their tetracycline resistance genes showed a relatively low risk of antibiotic resistance in a 7-day experiment. Moreover, the tetM of the ribosomal protection protein genes carried by these two preponderant bacteria was five-fold higher than that carried by other isolates (P < 0.05). Pearson correlations between the C t /C 0 of DOX and tet resistance genes of three isolates, except for Escherichia sp. and Candida sp., showed remarkable negative correlations (P < 0.05), mainly because tetG markedly increased during the DOX degradation process. Our results concluded that the biodegradation of antibiotic residues may not necessarily lead to the development of ARGs in the environment. In addition, the two bacteria that we isolated, namely, Escherichia sp. and Candida sp., are potential candidates for the engineering of environmentally friendly bacteria. Copyright © 2017 Elsevier Inc. All rights reserved.

A quantitative PCR approach for quantification of functional genes involved in the degradation of polycyclic aromatic hydrocarbons in contaminated soils.

PubMed

Shahsavari, Esmaeil; Aburto-Medina, Arturo; Taha, Mohamed; Ball, Andrew S

2016-01-01

Polycyclic aromatic hydrocarbons (PAHs) are major pollutants globally and due to their carcinogenic and mutagenic properties their clean-up is paramount. Bioremediation or using PAH degrading microorganisms (mainly bacteria) to degrade the pollutants represents cheap, effective methods. These PAH degraders harbor functional genes which help microorganisms use PAHs as source of food and energy. Most probable number (MPN) and plate counting methods are widely used for counting PAHs degraders; however, as culture based methods only count a small fraction (<1%) of microorganisms capable of carrying out PAH degradation, the use of culture-independent methodologies is desirable.•This protocol presents a robust, rapid and sensitive qPCR method for the quantification of the functional genes involved in the degradation of PAHs in soil samples.•This protocol enables us to screen a vast number of PAH contaminated soil samples in few hours.•This protocol provides valuable information about the natural attenuation potential of contaminated soil and can be used to monitor the bioremediation process.

Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

PubMed

Viggor, Signe; Jõesaar, Merike; Vedler, Eve; Kiiker, Riinu; Pärnpuu, Liis; Heinaru, Ain

2015-12-30

Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains. Copyright © 2015 Elsevier Ltd. All rights reserved.

Presence or Absence of mlr Genes and Nutrient Concentrations Co-Determine the Microcystin Biodegradation Efficiency of a Natural Bacterial Community.

PubMed

Lezcano, María Ángeles; Morón-López, Jesús; Agha, Ramsy; López-Heras, Isabel; Nozal, Leonor; Quesada, Antonio; El-Shehawy, Rehab

2016-11-03

The microcystin biodegradation potential of a natural bacterial community coexisting with a toxic cyanobacterial bloom was investigated in a water reservoir from central Spain. The biodegradation capacity was confirmed in all samples during the bloom and an increase of mlr A gene copies was found with increasing microcystin concentrations. Among the 24 microcystin degrading strains isolated from the bacterial community, only 28% showed presence of mlr A gene, strongly supporting the existence and abundance of alternative microcystin degradation pathways in nature. In vitro degradation assays with both mlr ⁺ and mlr - bacterial genotypes (with presence and absence of the complete mlr gene cluster, respectively) were performed with four isolated strains ( Sphingopyxis sp. IM-1, IM-2 and IM-3; Paucibacter toxinivorans IM-4) and two bacterial degraders from the culture collection ( Sphingosinicella microcystinivorans Y2; Paucibacter toxinivorans 2C20). Differences in microcystin degradation efficiencies between genotypes were found under different total organic carbon and total nitrogen concentrations. While mlr ⁺ strains significantly improved microcystin degradation rates when exposed to other carbon and nitrogen sources, mlr - strains showed lower degradation efficiencies. This suggests that the presence of alternative carbon and nitrogen sources possibly competes with microcystins and impairs putative non- mlr microcystin degradation pathways. Considering the abundance of the mlr - bacterial population and the increasing frequency of eutrophic conditions in aquatic systems, further research on the diversity of this population and the characterization and conditions affecting non- mlr degradation pathways deserves special attention.

Insulin‐degrading enzyme is genetically associated with Alzheimer's disease in the Finnish population

PubMed Central

Vepsäläinen, Saila; Parkinson, Michele; Helisalmi, Seppo; Mannermaa, Arto; Soininen, Hilkka; Tanzi, Rudolph E; Bertram, Lars; Hiltunen, Mikko

2007-01-01

The gene for insulin‐degrading enzyme (IDE), which is located at chromosome 10q24, has been previously proposed as a candidate gene for late‐onset Alzheimer's disease (AD) based on its ability to degrade amyloid β‐protein. Genotyping of single nucleotide polymorphisms (SNPs) in the IDE gene in Finnish patients with AD and controls revealed SNPs rs4646953 and rs4646955 to be associated with AD, conferring an approximately two‐fold increased risk. Single locus findings were corroborated by the results obtained from haplotype analyses. This suggests that genetic alterations in or near the IDE gene may increase the risk for developing AD. PMID:17496198

The role of a groundwater bacterial community in the degradation of the herbicide terbuthylazine.

PubMed

Caracciolo, Anna Barra; Fajardo, Carmen; Grenni, Paola; Saccà, Maria Ludovica; Amalfitano, Stefano; Ciccoli, Roberto; Martin, Margarita; Gibello, Alicia

2010-01-01

A bacterial community in an aquifer contaminated by s-triazines was studied. Groundwater microcosms were treated with terbuthylazine at a concentration of 100 microg L(-1) and degradation of the herbicide was assessed. The bacterial community structure (abundance and phylogenetic composition) and function (carbon production and cell viability) were analysed. The bacterial community was able to degrade the terbuthylazine; in particular, Betaproteobacteria were involved in the herbicide biotransformation. Identification of some bacterial isolates by PCR amplification of the 16S rRNA gene revealed the presence of two Betaproteobacteria species able to degrade the herbicide: Advenella incenata and Janthinobacterium lividum. PCR detection of the genes encoding s-triazine-degrading enzymes indicated the presence of the atzA and atzB genes in A. incenata and the atzB and atzC genes in J. lividum. The nucleotide sequences of the PCR fragments of the atz genes from these strains were 100% identical to the homologous genes of the Pseudomonas sp. strain ADP. In conclusion, the results show the potential for the use of a natural attenuation strategy in the treatment of aquifers polluted with the terbuthylazine. The two bacteria isolated could facilitate the implementation of effective bioremediation protocols, especially in the case of the significant amounts of herbicide that can be found in groundwater as a result of accidental spills.

Insights into the Microbial Degradation of Rubber and Gutta-Percha by Analysis of the Complete Genome of Nocardia nova SH22a

PubMed Central

Luo, Quan; Hiessl, Sebastian; Poehlein, Anja; Daniel, Rolf

2014-01-01

The complete genome sequence of Nocardia nova SH22a was determined in light of the remarkable ability of rubber and gutta-percha (GP) degradation of this strain. The genome consists of a circular chromosome of 8,348,532 bp with a G+C content of 67.77% and 7,583 predicted protein-encoding genes. Functions were assigned to 72.45% of the coding sequences. Among them, a large number of genes probably involved in the metabolism of xenobiotics and hardly degradable compounds, as well as genes that participate in the synthesis of polyketide- and/or nonribosomal peptide-type secondary metabolites, were detected. Based on in silico analyses and experimental studies, such as transposon mutagenesis and directed gene deletion studies, the pathways of rubber and GP degradation were proposed and the relationship between both pathways was unraveled. The genes involved include, inter alia, genes participating in cell envelope synthesis (long-chain-fatty-acid–AMP ligase and arabinofuranosyltransferase), β-oxidation (α-methylacyl-coenzyme A [α-methylacyl-CoA] racemase), propionate catabolism (acyl-CoA carboxylase), gluconeogenesis (phosphoenolpyruvate carboxykinase), and transmembrane substrate uptake (Mce [mammalian cell entry] transporter). This study not only improves our insights into the mechanism of microbial degradation of rubber and GP but also expands our knowledge of the genus Nocardia regarding metabolic diversity. PMID:24747905

Prevalence of the gene trzN and biogeographic patterns among atrazine-degrading bacteria isolated from 13 Colombian agricultural soils.

PubMed

Arbeli, Ziv; Fuentes, Cilia

2010-09-01

The following study evaluated the diversity and biogeography of 83 new atrazine-degrading bacteria and the composition of their atrazine degradation genes. These strains were isolated from 13 agricultural soils and grouped according to rep-PCR genomic fingerprinting into 11 major clusters, which showed biogeographic patterns. Three clusters (54 strains) belonged to the genus Arthrobacter, seven clusters (28 strains) were similar to the genus Nocardioides and only one strain was a gram-negative from the genus Ancylobacter. PCR assays for the detection of the genes atzA, B, C, D, E, F and trzN conducted with each of the 83 strains revealed that 82 strains (all gram positive) possessed trzN, 74 of them possessed the combination of trzN, atzB and atzC, while only the gram-negative strain had atzA. A similar PCR assay for the two analogous genes, atzA and trzN, responsible for the first step of atrazine degradation, was performed with DNA extracted directly from the enrichment cultures and microcosms spiked with atrazine. In these assays, the gene trzN was detected in each culture, while atzA was detected in only six out of 13 soils. These results raise an interesting hypothesis on the evolutionary ecology of the two atrazine chlorohydrolase genes (i.e. atzA and trzN) and about the biogeography of atrazine-degrading bacteria.

Limnobacter spp. as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes.

PubMed

Vedler, Eve; Heinaru, Eeva; Jutkina, Jekaterina; Viggor, Signe; Koressaar, Triinu; Remm, Maido; Heinaru, Ain

2013-12-01

A set of phenol-degrading strains of a collection of bacteria isolated from Baltic Sea surface water was screened for the presence of two key catabolic genes coding for phenol hydroxylases and catechol 2,3-dioxygenases. The multicomponent phenol hydroxylase (LmPH) gene was detected in 70 out of 92 strains studied, and 41 strains among these LmPH(+) phenol-degraders were found to exhibit catechol 2,3-dioxygenase (C23O) activity. Comparative phylogenetic analyses of LmPH and C23O sequences from 56 representative strains were performed. The studied strains were mostly affiliated to the genera Pseudomonas and Acinetobacter. However, the study also widened the range of phenol-degraders by including the genus Limnobacter. Furthermore, using a next generation sequencing approach, the LmPH genes of Limnobacter strains were found to be the most prevalent ones in the microbial community of the Baltic Sea surface water. Four different Limnobacter strains having almost identical 16S rRNA gene sequences (99%) and similar physiological properties formed separate phylogenetic clusters of LmPH and C23O genes in the respective phylogenetic trees. Copyright © 2013 Elsevier GmbH. All rights reserved.

The genes and enzymes for the catabolism of galactitol, D-tagatose, and related carbohydrates in Klebsiella oxytoca M5a1 and other enteric bacteria display convergent evolution.

PubMed

Shakeri-Garakani, A; Brinkkötter, A; Schmid, K; Turgut, S; Lengeler, J W

2004-07-01

Enteric bacteria (Enteriobacteriaceae) carry on their single chromosome about 4000 genes that all strains have in common (referred to here as "obligatory genes"), and up to 1300 "facultative" genes that vary from strain to strain and from species to species. In closely related species, obligatory and facultative genes are orthologous genes that are found at similar loci. We have analyzed a set of facultative genes involved in the degradation of the carbohydrates galactitol, D-tagatose, D-galactosamine and N-acetyl-galactosamine in various pathogenic and non-pathogenic strains of these bacteria. The four carbohydrates are transported into the cell by phosphotransferase (PTS) uptake systems, and are metabolized by closely related or even identical catabolic enzymes via pathways that share several intermediates. In about 60% of Escherichia coli strains the genes for galactitol degradation map to a gat operon at 46.8 min. In strains of Salmonella enterica, Klebsiella pneumoniae and K. oxytoca, the corresponding gat genes, although orthologous to their E. coli counterparts, are found at 70.7 min, clustered in a regulon together with three tag genes for the degradation of D-tagatose, an isomer of D-fructose. In contrast, in all the E. coli strains tested, this chromosomal site was found to be occupied by an aga/kba gene cluster for the degradation of D-galactosamine and N-acetyl-galactosamine. The aga/kba and the tag genes were paralogous either to the gat cluster or to the fru genes for degradation of D-fructose. Finally, in more then 90% of strains of both Klebsiella species, and in about 5% of the E. coli strains, two operons were found at 46.8 min that comprise paralogous genes for catabolism of the isomers D-arabinitol (genes atl or dal) and ribitol (genes rtl or rbt). In these strains gat genes were invariably absent from this location, and they were totally absent in S. enterica. These results strongly indicate that these various gene clusters and metabolic pathways have been subject to convergent evolution among the Enterobacteriaceae. This apparently involved recent horizontal gene transfer and recombination events, as indicated by major chromosomal rearrangements found in their immediate vicinity.

Characterization of para-Nitrophenol-Degrading Bacterial Communities in River Water by Using Functional Markers and Stable Isotope Probing.

PubMed

Kowalczyk, Agnieszka; Eyice, Özge; Schäfer, Hendrik; Price, Oliver R; Finnegan, Christopher J; van Egmond, Roger A; Shaw, Liz J; Barrett, Glyn; Bending, Gary D

2015-10-01

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA-listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [(13)C6]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Enzymatic oxidation of 2-phenylethylamine to phenylacetic acid and 2-phenylethanol with special reference to the metabolism of its intermediate phenylacetaldehyde.

PubMed

Panoutsopoulos, Georgios I; Kouretas, Demetrios; Gounaris, Elias G; Beedham, Christine

2004-12-01

2-phenylethylamine is an endogenous constituent of the human brain and is implicated in cerebral transmission. This bioactive amine is also present in certain foodstuffs such as chocolate, cheese and wine and may cause undesirable side effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalysed by monoamine oxidase B but the oxidation to its acid is usually ascribed to aldehyde dehydrogenase and the contribution of aldehyde oxidase and xanthine oxidase, if any, is ignored. The objective of this study was to elucidate the role of the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, in the metabolism of phenylacetaldehyde derived from its parent biogenic amine. Treatments of 2-phenylethylamine with monoamine oxidase were carried out for the production of phenylacetaldehyde, as well as treatments of synthetic or enzymatic-generated phenylacetaldehyde with aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase. The results indicated that phenylacetaldehyde is metabolised mainly to phenylacetic acid with lower concentrations of 2-phenylethanol by all three oxidising enzymes. Aldehyde dehydrogenase was the predominant enzyme involved in phenylacetaldehyde oxidation and thus it has a major role in 2-phenylethylamine metabolism with aldehyde oxidase playing a less prominent role. Xanthine oxidase does not contribute to the oxidation of phenylacetaldehyde due to low amounts being present in guinea pig. Thus aldehyde dehydrogenase is not the only enzyme oxidising xenobiotic and endobiotic aldehydes and the role of aldehyde oxidase in such reactions should not be ignored.

Ap4A induces apoptosis in human cultured cells.

PubMed

Vartanian, A; Alexandrov, I; Prudowski, I; McLennan, A; Kisselev, L

1999-07-30

Diadenosine oligophosphates (Ap(n)A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5',5'''-P1,P3-triphosphate to diadenosine 5',5'''-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of Ap(n)A into cells was achieved by cold shock. Ap4A at 10 microM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.

Assessment of Anaerobic Toluene Biodegradation Activity by bssA Transcript/Gene Ratios

PubMed Central

Brow, Christina N.; O'Brien Johnson, Reid; Johnson, Richard L.

2013-01-01

Benzylsuccinate synthase (bssA) genes associated with toluene degradation were profiled across a groundwater contaminant plume under nitrate-reducing conditions and were detected in significant numbers throughout the plume. However, differences between groundwater and core sediment samples suggested that microbial transport, rather than local activity, was the underlying cause of the high copy numbers within the downgradient plume. Both gene transcript and reactant concentrations were consistent with this hypothesis. Expression of bssA genes from denitrifying toluene degraders was induced by toluene but only in the presence of nitrate, and transcript abundance dropped rapidly following the removal of either toluene or nitrate. The drop in bssA transcripts following the removal of toluene could be described by an exponential decay function with a half-life on the order of 1 h. Interestingly, bssA transcripts never disappeared completely but were always detected at some level if either inducer was present. Therefore, the detection of transcripts alone may not be sufficient evidence for contaminant degradation. To avoid mistakenly associating basal-level gene expression with actively degrading microbial populations, an integrated approach using the ratio of functional gene transcripts to gene copies is recommended. This approach minimizes the impact of microbial transport on activity assessment and allows reliable assessments of microbial activity to be obtained from water samples. PMID:23811506

Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601T

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oosterkamp, Margreet J.; Veuskens, Teun; Saia, Flavia Talarico

2013-01-01

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacteriummore » that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.« less

Genome Analysis and Physiological Comparison of Alicycliphilus denitrificans Strains BC and K601T

PubMed Central

Talarico Saia, Flávia; Weelink, Sander A. B.; Goodwin, Lynne A.; Daligault, Hajnalka E.; Bruce, David C.; Detter, John C.; Tapia, Roxanne; Han, Cliff S.; Land, Miriam L.; Hauser, Loren J.; Langenhoff, Alette A. M.; Gerritse, Jan; van Berkel, Willem J. H.; Pieper, Dietmar H.; Junca, Howard; Smidt, Hauke; Schraa, Gosse; Davids, Mark; Schaap, Peter J.; Plugge, Caroline M.; Stams, Alfons J. M.

2013-01-01

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far. PMID:23825601

Comparative Genomics of Syntrophic Branched-Chain Fatty Acid Degrading Bacteria

PubMed Central

Narihiro, Takashi; Nobu, Masaru K.; Tamaki, Hideyuki; Kamagata, Yoichi; Sekiguchi, Yuji; Liu, Wen-Tso

2016-01-01

The syntrophic degradation of branched-chain fatty acids (BCFAs) such as 2-methylbutyrate and isobutyrate is an essential step in the production of methane from proteins/amino acids in anaerobic ecosystems. While a few syntrophic BCFA-degrading bacteria have been isolated, their metabolic pathways in BCFA and short-chain fatty acid (SCFA) degradation as well as energy conservation systems remain unclear. In an attempt to identify these pathways, we herein performed comparative genomics of three syntrophic bacteria: 2-methylbutyrate-degrading “Syntrophomonas wolfei subsp. methylbutyratica” strain JCM 14075T (=4J5T), isobutyrate-degrading Syntrophothermus lipocalidus strain TGB-C1T, and non-BCFA-metabolizing S. wolfei subsp. wolfei strain GöttingenT. We demonstrated that 4J5 and TGB-C1 both encode multiple genes/gene clusters involved in β-oxidation, as observed in the Göttingen genome, which has multiple copies of genes associated with butyrate degradation. The 4J5 genome possesses phylogenetically distinct β-oxidation genes, which may be involved in 2-methylbutyrate degradation. In addition, these Syntrophomonadaceae strains harbor various hydrogen/formate generation systems (i.e., electron-bifurcating hydrogenase, formate dehydrogenase, and membrane-bound hydrogenase) and energy-conserving electron transport systems, including electron transfer flavoprotein (ETF)-linked acyl-CoA dehydrogenase, ETF-linked iron-sulfur binding reductase, ETF dehydrogenase (FixABCX), and flavin oxidoreductase-heterodisulfide reductase (Flox-Hdr). Unexpectedly, the TGB-C1 genome encodes a nitrogenase complex, which may function as an alternative H2 generation mechanism. These results suggest that the BCFA-degrading syntrophic strains 4J5 and TGB-C1 possess specific β-oxidation-related enzymes for BCFA oxidation as well as appropriate energy conservation systems to perform thermodynamically unfavorable syntrophic metabolism. PMID:27431485

The structural and functional contributions of β-glucosidase-producing microbial communities to cellulose degradation in composting.

PubMed

Zang, Xiangyun; Liu, Meiting; Fan, Yihong; Xu, Jie; Xu, Xiuhong; Li, Hongtao

2018-01-01

Compost habitats sustain a vast ensemble of microbes that engender the degradation of cellulose, which is an important part of global carbon cycle. β-Glucosidase is the rate-limiting enzyme of degradation of cellulose. Thus, analysis of regulation of β-glucosidase gene expression in composting is beneficial to a better understanding of cellulose degradation mechanism. Genetic diversity and expression of β-glucosidase-producing microbial communities, and relationships of cellulose degradation, metabolic products and the relative enzyme activity during natural composting and inoculated composting were evaluated. Compared with natural composting, adding inoculation agent effectively improved the degradation of cellulose, and maintained high level of the carboxymethyl cellulose (CMCase) and β-glucosidase activities in thermophilic phase. Gene expression analysis showed that glycoside hydrolase family 1 (GH1) family of β-glucosidase genes contributed more to β-glucosidase activity in the later thermophilic phase in inoculated compost. In the cooling phase of natural compost, glycoside hydrolase family 3 (GH3) family of β-glucosidase genes contributed more to β-glucosidase activity. Intracellular β-glucosidase activity played a crucial role in the regulation of β-glucosidase gene expression, and upregulation or downregulation was also determined by extracellular concentration of glucose. At sufficiently high glucose concentrations, the functional microbial community in compost was altered, which may contribute to maintaining β-glucosidase activity despite the high glucose content. This research provides an ecological functional map of microorganisms involved in carbon metabolism in cattle manure-rice straw composting. The performance of the functional microbial groups in the two composting treatments is different, which is related to the cellulase activity and cellulose degradation, respectively.

2,4-Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes.

PubMed Central

Fulthorpe, R R; McGowan, C; Maltseva, O V; Holben, W E; Tiedje, J M

1995-01-01

DNA from 32 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria from diverse locations was probed with the first three genes of the well-known 2,4-D degradation pathway found in Alcaligenes eutrophus JMP134(pJP4). The majority of strains did not show high levels of homology to the first three genes of the 2,4-D degradation pathway, tfdA, -B, and -C. Most strains showed combinations of tfdA-, B-, and C-like elements that exhibited various degrees of homology to the gene probes. Strains having the same genomic fingerprints (as determined by repetitive extragenic palindromic PCR) exhibited the same hybridization pattern regardless of the geographic origin of the strain, with the exception of a strain isolated from Puerto Rico. This strain had the same genomic fingerprint as that of numerous other strains in the collection but differed in its hybridization against the tfdA gene probe. Members of the beta subdivision of the Proteobacteria class, specifically Alcaligenes, Burkholderia, and Rhodoferax species, carried DNA fragments with 60% or more sequence similarity to tfdA of pJP4, and most carried fragments showing at least 60% homology to tfdB. However, many strains did not hybridize with tfdC, although they exhibited chlorocatechol dioxygenase activity. Members of the alpha subdivision of the Proteobacteria class, mostly of the genus Sphingomonas, did not hybridize to either tfdA or tfdC, but some hybridized at low stringency to tfdB. The data suggest that extensive interspecies transfer of a variety of homologous degradative genes has been involved in the evolution of 2,4-D-degrading bacteria. PMID:7574638

A Novel Phenanthrene Dioxygenase from Nocardioides sp. Strain KP7: Expression in Escherichia coli

PubMed Central

Saito, Atsushi; Iwabuchi, Tokuro; Harayama, Shigeaki

2000-01-01

Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the α and β subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8.3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the α and β subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases. PMID:10735855

Gene expression patterns of wood decay fungi Postia placenta and Phanerochaete chrysosporium are influenced by wood substrate composition during degradation

Treesearch

Oleksandr Skyba; Daniel Cullen; Carl J. Douglas; Shawn D. Mansfield

2016-01-01

Identification of the specific genes and enzymes involved in the fungal degradation of lignocellulosic biomass derived from feedstocks with various compositions is essential to the development of improved bioenergy processes. In order to elucidate the effect of substrate composition on gene expression in wood-rotting fungi, we employed microarrays based on the...

Systematic Analysis of the Pleurotus ostreatus Laccase Gene (PoLac) Family and Functional Characterization of PoLac2 Involved in the Degradation of Cotton-Straw Lignin.

PubMed

Jiao, Xiaoyu; Li, Guoqing; Wang, Yan; Nie, Fan; Cheng, Xi; Abdullah, Muhammad; Lin, Yi; Cai, Yongping

2018-04-11

Fungal laccases play important roles in the degradation of lignocellulose. Although some PoLac s have been reported in several studies, still no comprehensive bioinformatics study of the LAC family in Pleurotus ostreatus has been reported. In this study, we identified 12 laccase genes in the whole genome sequence of P. ostreatus and their physical characteristics, gene distribution, phylogenic relationships, gene structure, conserved motifs, and cis-elements were also analyzed. The expression patterns of 12 PoLac genes at different developmental stages and under different culture substrates were also analyzed. The results revealed that PoLac2 and PoLac12 may be involved in the degradation of lignin and the formation of the fruiting body, respectively. Subsequently, we overexpressed PoLac2 in P. ostreatus by the Agrobacterium tumefaciens -mediated transformation (ATMT) method. The transformants' laccase activity increased in varying degrees, and the gene expression level of PoLac2 in transformants was 2-8 times higher than that of the wild-type strain. Furthermore, the lignin degradation rate by transgenic fungus over 30 days was 2.36-6.3% higher than that of wild-type. Our data show that overexpression of PoLac2 significantly enhanced the lignin degradation of cotton-straw. To our knowledge, this study is the first report to demonstrate the functions of PoLac2 in P. ostreatus .

Identification of enzymes involved in oxidation of phenylbutyrate.

PubMed

Palir, Neža; Ruiter, Jos P N; Wanders, Ronald J A; Houtkooper, Riekelt H

2017-05-01

In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the β-oxidation of PB. We used cells deficient in specific steps of fatty acid β-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Detection of arc genes related with the ethyl carbamate precursors in wine lactic acid bacteria.

PubMed

Araque, Isabel; Gil, Joana; Carreté, Ramon; Bordons, Albert; Reguant, Cristina

2009-03-11

Trace amounts of the carcinogen ethyl carbamate can appear in wine by the reaction of ethanol with compounds such as citrulline and carbamyl phosphate, which are produced from arginine degradation by some wine lactic acid bacteria (LAB). In this work, the presence of arc genes for the arginine-deiminase pathway was studied in several strains of different species of LAB. Their ability to degrade arginine was also studied. To detect the presence of arc genes, degenerate primers were designed from the alignment of protein sequences in already sequenced LAB. The usefulness of these degenerate primers has been proven by sequencing some of the amplified PCR fragments and searching for homologies with published sequences of the same species and related ones. Correlation was found between the presence of genes and the ability to degrade arginine. Degrading strains included all heterofermentative lactobacilli, Oenococcus oeni , Pediococcus pentosaceus , and some strains of Leuconostoc mesenteroides and Lactobacillus plantarum .

Glyphosate degradation in glyphosate-resistant and -susceptible crops and weeds.

PubMed

Duke, Stephen O

2011-06-08

High levels of aminomethylphosphonic acid (AMPA), the main glyphosate metabolite, have been found in glyphosate-treated, glyphosate-resistant (GR) soybean, apparently due to plant glyphosate oxidoreductase (GOX)-like activity. AMPA is mildly phytotoxic, and under some conditions the AMPA accumulating in GR soybean correlates with glyphosate-caused phytotoxicity. A bacterial GOX is used in GR canola, and an altered bacterial glyphosate N-acetyltransferase is planned for a new generation of GR crops. In some weed species, glyphosate degradation could contribute to natural resistance. Neither an isolated plant GOX enzyme nor a gene for it has yet been reported in plants. Gene mutation or amplification of plant genes for GOX-like enzyme activity or horizontal transfer of microbial genes from glyphosate-degrading enzymes could produce GR weeds. Yet, there is no evidence that metabolic degradation plays a significant role in evolved resistance to glyphosate. This is unexpected, considering the extreme selection pressure for evolution of glyphosate resistance in weeds and the difficulty in plants of evolving glyphosate resistance via other mechanisms.

Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida.

PubMed

Pradeep, Vijayalakshmi; Subbaiah, Usha Malavalli

2015-01-01

The present study was undertaken with the objective of studying repeated batch and continuous degradation of chlorpyrifos (O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate) using Ca-alginate immobilized cells of Pseudomonas putida isolated from an agricultural soil, and to study the genes and enzymes involved in degradation. The study was carried out to reduce the toxicity of chlorpyrifos by degrading it to less toxic metabolites. Long-term stability of pesticide degradation was studied during repeated batch degradation of chlorpyrifos, which was carried out over a period of 50 days. Immobilized cells were able to show 65% degradation of chlorpyrifos at the end of the 50th cycle with a cell leakage of 112 × 10(3) cfu mL(-1). During continuous treatment, 100% degradation was observed at 100 mL h(-1) flow rate with 2% chlorpyrifos, and with 10% concentration of chlorpyrifos 98% and 80% degradation was recorded at 20 mL h(-1) and 100 mL h(-1) flow rate respectively. The products of degradation detected by liquid chromatography-mass spectrometry analysis were 3,5,6-trichloro-2-pyridinol and chlorpyrifos oxon. Plasmid curing experiments with ethidium bromide indicated that genes responsible for the degradation of chlorpyrifos are present on the chromosome and not on the plasmid. The results of Polymerase chain reaction indicate that a ~890-bp product expected for mpd gene was present in Ps. putida. Enzymatic degradation studies indicated that the enzymes involved in the degradation of chlorpyrifos are membrane-bound. The study indicates that immobilized cells of Ps. putida have the potential to be used in bioremediation of water contaminated with chlorpyrifos.

Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil.

PubMed

Zuo, Zhenqiang; Gong, Ting; Che, You; Liu, Ruihua; Xu, Ping; Jiang, Hong; Qiao, Chuanling; Song, Cunjiang; Yang, Chao

2015-06-01

Agricultural soils are usually co-contaminated with organophosphate (OP) and pyrethroid pesticides. To develop a stable and marker-free Pseudomonas putida for co-expression of two pesticide-degrading enzymes, we constructed a suicide plasmid with expression cassettes containing a constitutive promoter J23119, an OP-degrading gene (mpd), a pyrethroid-hydrolyzing carboxylesterase gene (pytH) that utilizes the upp gene as a counter-selectable marker for upp-deficient P. putida. By introduction of suicide plasmid and two-step homologous recombination, both mpd and pytH genes were integrated into the chromosome of a robust soil bacterium P. putida KT2440 and no selection marker was left on chromosome. Functional expression of mpd and pytH in P. putida KT2440 was demonstrated by Western blot analysis and enzyme activity assays. Degradation experiments with liquid cultures showed that the mixed pesticides including methyl parathion, fenitrothion, chlorpyrifos, permethrin, fenpropathrin, and cypermethrin (0.2 mM each) were degraded completely within 48 h. The inoculation of engineered strain (10(6) cells/g) to soils treated with the above mixed pesticides resulted in a higher degradation rate than in noninoculated soils. All six pesticides could be degraded completely within 15 days in fumigated and nonfumigated soils with inoculation. Theses results highlight the potential of the engineered strain to be used for in situ bioremediation of soils co-contaminated with OP and pyrethroid pesticides.

Ubiquitous Presence and Novel Diversity of Anaerobic Alkane Degraders in Cold Marine Sediments.

PubMed

Gittel, Antje; Donhauser, Johanna; Røy, Hans; Girguis, Peter R; Jørgensen, Bo B; Kjeldsen, Kasper U

2015-01-01

Alkanes are major constituents of crude oil and are released to the marine environment by natural seepage and from anthropogenic sources. Due to their chemical inertness, their removal from anoxic marine sediments is primarily controlled by the activity of anaerobic alkane-degrading microorganisms. To facilitate comprehensive cultivation-independent surveys of the diversity and distribution of anaerobic alkane degraders, we designed novel PCR primers that cover all known diversity of the 1-methylalkyl succinate synthase gene (masD/assA), which catalyzes the initial activation of alkanes. We studied masD/assA gene diversity in pristine and seepage-impacted Danish coastal sediments, as well as in sediments and alkane-degrading enrichment cultures from the Middle Valley (MV) hydrothermal vent system in the Pacific Northwest. MasD/assA genes were ubiquitously present, and the primers captured the diversity of both known and previously undiscovered masD/assA gene diversity. Seepage sediments were dominated by a single masD/assA gene cluster, which is presumably indicative of a substrate-adapted community, while pristine sediments harbored a diverse range of masD/assA phylotypes including those present in seepage sediments. This rare biosphere of anaerobic alkane degraders will likely increase in abundance in the event of seepage or accidental oil spillage. Nanomolar concentrations of short-chain alkanes (SCA) were detected in pristine and seepage sediments. Interestingly, anaerobic alkane degraders closely related to strain BuS5, the only SCA degrader in pure culture, were found in mesophilic MV enrichments, but not in cold sediments from Danish waters. We propose that the new masD/assA gene lineages in these sediments represent novel phylotypes that are either fueled by naturally occurring low levels of SCA or that metabolize medium- to long-chain alkanes. Our study highlights that masD/assA genes are a relevant diagnostic marker to identify seepage and microseepage, e.g., during prospecting for oil and gas, and may act as an indicator of anthropogenic oil spills in marine sediments.

Transgenic plants for enhanced biodegradation and phytoremediation of organic xenobiotics.

PubMed

Abhilash, P C; Jamil, Sarah; Singh, Nandita

2009-01-01

Phytoremediation--the use of plants to clean up polluted soil and water resources--has received much attention in the last few years. Although plants have the inherent ability to detoxify xenobiotics, they generally lack the catabolic pathway for the complete degradation of these compounds compared to microorganisms. There are also concerns over the potential for the introduction of contaminants into the food chain. The question of how to dispose of plants that accumulate xenobiotics is also a serious concern. Hence the feasibility of phytoremediation as an approach to remediate environmental contamination is still somewhat in question. For these reasons, researchers have endeavored to engineer plants with genes that can bestow superior degradation abilities. A direct method for enhancing the efficacy of phytoremediation is to overexpress in plants the genes involved in metabolism, uptake, or transport of specific pollutants. Furthermore, the expression of suitable genes in root system enhances the rhizodegradation of highly recalcitrant compounds like PAHs, PCBs etc. Hence, the idea to amplify plant biodegradation of xenobiotics by genetic manipulation was developed, following a strategy similar to that used to develop transgenic crops. Genes from human, microbes, plants, and animals are being used successfully for this venture. The introduction of these genes can be readily achieved for many plant species using Agrobacterium tumefaciens-mediated plant transformation or direct DNA methods of gene transfer. One of the promising developments in transgenic technology is the insertion of multiple genes (for phase 1 metabolism (cytochrome P450s) and phase 2 metabolism (GSH, GT etc.) for the complete degradation of the xenobiotics within the plant system. In addition to the use of transgenic plants overexpressed with P450 and GST genes, various transgenic plants expressing bacterial genes can be used for the enhanced degradation and remediation of herbicides, explosives, PCBs etc. Another approach to enhancing phytoremediation ability is the construction of plants that secrete chemical degrading enzymes into the rhizosphere. Recent studies revealed that accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as major limitation in improving phytoremediation efficiency. However, this can be overcome by the selective expression of bacterial ACC deaminase (which regulates ethylene levels in plants) in plants together with multiple genes for the different phases of xenobiotic degradation. This review examines the recent developments in use of transgenic-plants for the enhanced metabolism, degradation and phytoremediation of organic xenobiotics and its future directions.

Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin.

PubMed

Bernstein, Jeffrey R; Bulter, Thomas; Liao, James C

2008-01-01

This work demonstrates the transfer of the five-gene cyclohexane carboxylate (CHC) degradation pathway from the high-GC alphaproteobacterium Rhodopseudomonas palustris to Escherichia coli, a gammaproteobacterium. The degradation product of this pathway is pimeloyl-CoA, a key metabolite in E. coli's biotin biosynthetic pathway. This pathway is useful for biotin overproduction in E. coli; however, the expression of GC-rich genes is troublesome in this host. When the native R. palustris CHC degradation pathway is transferred to a DeltabioH pimeloyl-CoA auxotroph of E. coli, it is unable to complement growth in the presence of CHC. To overcome this expression problem we redesigned the operon with decreased GC content and removed stretches of high-GC intergenic DNA which comprise the 5' untranslated region of each gene, replacing these features with shorter low-GC sequences. We show this synthetic construct enables growth of the DeltabioH strain in the presence of CHC. When the synthetic degradation pathway is overexpressed in conjunction with the downstream genes for biotin biosynthesis, we measured significant accumulation of biotin in the growth medium, showing that the pathway transfer is successfully integrated with the host metabolism.

Xylan degradation by the human gut Bacteroides xylanisolvens XB1A(T) involves two distinct gene clusters that are linked at the transcriptional level.

PubMed

Despres, Jordane; Forano, Evelyne; Lepercq, Pascale; Comtet-Marre, Sophie; Jubelin, Gregory; Chambon, Christophe; Yeoman, Carl J; Berg Miller, Margaret E; Fields, Christopher J; Martens, Eric; Terrapon, Nicolas; Henrissat, Bernard; White, Bryan A; Mosoni, Pascale

2016-05-04

Plant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1A(T). Transcriptomic analyses of B. xylanisolvens XB1A(T) grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70. This study shows that xylan degradation by B. xylanisolvens XB1A(T) is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens XB1A(T) has specialized in the degradation of xylans of low complexity. This functional feature may provide a niche to all xylanolytic bacteria harboring similar PULs. Further functional and ecological studies on fibrolytic Bacteroides species are needed to better understand their role in dietary fiber degradation and their impact on intestinal health.

Haloacetic acid-degrading bacterial communities in drinking water systems as determined by cultivation and by terminal restriction fragment length polymorphism of PCR-amplified haloacid dehalogenase gene fragments.

PubMed

Grigorescu, A S; Hozalski, R M; Lapara, T M

2012-04-01

To characterize the HAA-degrading bacteria in drinking water systems. Haloacetic acid (HAA)-degrading bacteria were analysed in drinking water systems by cultivation and by a novel application of terminal restriction fragment length polymorphism (tRFLP). Substantial similarities were observed among the tRFLP patterns of dehI and dehII gene fragments in drinking water samples obtained from three different cities (Minneapolis, MN; St Paul, MN; Bucharest, Romania) and from one biologically active granular activated carbon filter (Hershey, PA). The dominant fragment in the tRFLP profiles of dehI genes from the drinking water samples matched the pattern from an Afipia sp. that was previously isolated from drinking water. In contrast, the dominant fragment in the tRFLP profiles of dehII genes did not match any previously characterized dehII gene fragment. PCR cloning was used to characterize this gene fragment, which had <65% nucleotide sequence identity with any previously characterized dehII gene. Afipia spp. are an appropriate model organism for studying the biodegradation of HAAs in drinking water distribution systems as encoded by dehI genes; the organism that harbours the most prominent dehII gene in drinking water has yet to be cultivated and identified. The development of a novel application of tRFLP targeting dehI and dehII genes could be broadly useful in understanding HAA-degrading bacteria in numerous environments. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients.

PubMed Central

DiGiovanni, G D; Neilson, J W; Pepper, I L; Sinclair, N A

1996-01-01

This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients. The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance. Genes on this plasmid plus chromosomal genes also allow degradation of 2,4-dichloruphenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 micrograms of 2,4-D g-1, significant (10(6) g of soil-1) populations of indigenous recipients or transconjugants arose. These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D. In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR. No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm. These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients. Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred. Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes. PMID:8779592

Characterization and Genomic Analysis of a Highly Efficient Dibutyl Phthalate-Degrading Bacterium Gordonia sp. Strain QH-12.

PubMed

Jin, Decai; Kong, Xiao; Liu, Huijun; Wang, Xinxin; Deng, Ye; Jia, Minghong; Yu, Xiangyang

2016-06-25

A bacterial strain QH-12 isolated from activated sludge was identified as Gordonia sp. based on analysis of 16S rRNA gene sequence and was found to be capable of utilizing dibutyl phthalate (DBP) and other common phthalate esters (PAEs) as the sole carbon and energy source. The degradation kinetics of DBP under different concentrations by the strain QH-12 fit well with the modified Gompertz model (R² > 0.98). However, strain QH-12 could not utilize the major intermediate product phthalate (phthalic acid; PA) as the sole carbon and energy source, and only a little amount of PA was detected. The QH-12 genome analysis revealed the presence of putative hydrolase/esterase genes involved in PAEs-degradation but no phthalic acid catabolic gene cluster was found, suggesting that a novel degradation pathway of PAEs was present in Gordonia sp. QH-12. This information will be valuable for obtaining a more holistic understanding on diverse genetic mechanisms of PAEs-degrading Gordonia sp. strains.

A set of biogenetically interesting polyhalogenated acetogenins from Ptilonia magellanica.

PubMed

Gallardo, Amalia B; Cueto, Mercedes; Díaz-Marrero, Ana R; de la Rosa, José M; Fajardo, Victor; San-Martín, Aurelio; Darias, José

2018-01-01

Ptilonines A-F, pyranosylmagellanicus D-E and magellenediol are previously undescribed acetogenins isolated from the red alga Ptilonia magellanica. Their structures were determined from spectroscopic evidence. The absolute configuration of the known pyranosylmagellanicus A, was established by derivatization with (R)- and (S)-α-methoxy -α-phenylacetic acids (MPA). Ptilonines exhibit an unusual halogenation pattern, that may confer evolutionary advantages to Ptilonia magellanica, for which a biogenetic origin is proposed. The antimicrobial effect of some of these compounds was evaluated. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of Microbial Interaction on Urea Metabolism in Chinese Liquor Fermentation.

PubMed

Wu, Qun; Lin, Jianchun; Cui, Kaixiang; Du, Rubin; Zhu, Yang; Xu, Yan

2017-12-20

Urea is the primary precursor of the carcinogen ethyl carbamate in fermented foods. Understanding urea metabolism is important for controlling ethyl carbamate production. Using Chinese liquor as a model system, we used metatranscriptome analysis to investigate urea metabolism in spontaneous food fermentation processes. Saccharomyces cerevisiae was dominant in gene transcription for urea biosynthesis and degradation. Lysinibacillus sphaericus was dominant for urea degradation. S. cerevisiae degraded 18% and L. sphaericus degraded 13% of urea in their corresponding single cultures, whereas they degraded 56% of urea in coculture after 12 h. Compared to single cultures, transcription of CAR1, DAL2, and argA, which are related to urea biosynthesis, decreased by 51, 36, and 69% in coculture, respectively. Transcription of DUR1 and ureA, which are related to urea degradation, increased by 227 and 70%, respectively. Thus, coexistence of the two strains promoted degradation of urea via transcriptional regulation of genes related to urea metabolism.

Metagenomic discovery of biomass-degrading genes and genomes from cow rumen.

PubMed

Hess, Matthias; Sczyrba, Alexander; Egan, Rob; Kim, Tae-Wan; Chokhawala, Harshal; Schroth, Gary; Luo, Shujun; Clark, Douglas S; Chen, Feng; Zhang, Tao; Mackie, Roderick I; Pennacchio, Len A; Tringe, Susannah G; Visel, Axel; Woyke, Tanja; Wang, Zhong; Rubin, Edward M

2011-01-28

The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.

Screening of SDS-degrading bacteria from car wash wastewater and study of the alkylsulfatase enzyme activity

PubMed Central

Shahbazi, Razieh; Kasra-Kermanshahi, Roha; Gharavi, Sara; Moosavi-Nejad, Zahra; Borzooee, Faezeh

2013-01-01

Background and Objectives Sodium dodecyl sulfate (SDS) is one of the main surfactant components in detergents and cosmetics, used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. Alkylsulfatase is an enzyme that hydrolyses sulfate -ester bonds to give inorganic sulfate and alcohol. The purpose of this study was to isolate SDS–degrading bacteria from Tehran city car wash wastewater, study bacterial alkylsulfatase enzyme activity and identify the alkylsulfatase enzyme coding gene. Materials and Methods Screening of SDS-degrading bacteria was carried out on basal salt medium containing SDS as the sole source of carbon. Amount of SDS degraded was assayed by methylene blue active substance (MBAS). Results and Conclusion Identification of the sdsA gene was carried by PCR and subsequent sequencing of the 16S rDNA gene and biochemical tests identified Pseudomonas aeruginosa. This bacterium is able to degrade 84% of SDS after four days incubation. Bacteria isolated from car wash wastewater were shown to carry the sdsA gene (670bp) and the alkylsulfatase enzyme specific activity expressed from this gene was determined to be 24.3 unit/mg. The results presented in this research indicate that Pseudomonas aeruginosa is a suitable candidate for SDS biodegradation. PMID:23825734

Genome Sequencing Reveals the Potential of Achromobacter sp. HZ01 for Bioremediation

PubMed Central

Hong, Yue-Hui; Ye, Cong-Cong; Zhou, Qian-Zhi; Wu, Xiao-Ying; Yuan, Jian-Ping; Peng, Juan; Deng, Hailin; Wang, Jiang-Hai

2017-01-01

Petroleum pollution is a severe environmental issue. Comprehensively revealing the genetic backgrounds of hydrocarbon-degrading microorganisms contributes to developing effective methods for bioremediation of crude oil-polluted environments. Marine bacterium Achromobacter sp. HZ01 is capable of degrading hydrocarbons and producing biosurfactants. In this study, the draft genome (5.5 Mbp) of strain HZ01 has been obtained by Illumina sequencing, containing 5,162 predicted genes. Genome annotation shows that “amino acid metabolism” is the most abundant metabolic pathway. Strain HZ01 is not capable of using some common carbohydrates as the sole carbon sources, which is due to that it contains few genes associated with carbohydrate transport and lacks some important enzymes related to glycometabolism. It contains abundant proteins directly related to petroleum hydrocarbon degradation. AlkB hydroxylase and its homologs were not identified. It harbors a complete enzyme system of terminal oxidation pathway for n-alkane degradation, which may be initiated by cytochrome P450. The enzymes involved in the catechol pathway are relatively complete for the degradation of aromatic compounds. This bacterium lacks several essential enzymes for methane oxidation, and Baeyer-Villiger monooxygenase involved in the subterminal oxidation pathway and cycloalkane degradation was not identified. These results suggest that strain HZ01 degrades n-alkanes via the terminal oxidation pathway, degrades aromatic compounds primarily via the catechol pathway and cannot perform methane oxidation or cycloalkane degradation. Additionally, strain HZ01 possesses abundant genes related to the metabolism of secondary metabolites, including some genes involved in biosurfactant (such as glycolipids and lipopeptides) synthesis. The genome analysis also reveals its genetic basis for nitrogen metabolism, antibiotic resistance, regulatory responses to environmental changes, cell motility, and material transport. The obtained genome data provide us with a better understanding of hydrocarbon-degrading bacteria, which may contribute to the future design of rational strategies for bioremediation of petroleum-polluted marine environments. PMID:28848520

Contrasting microbial functional genes in two distinct saline-alkali and slightly acidic oil-contaminated sites.

PubMed

Liang, Yuting; Zhao, Huihui; Zhang, Xu; Zhou, Jizhong; Li, Guanghe

2014-07-15

To compare the functional gene structure and diversity of microbial communities in saline-alkali and slightly acidic oil-contaminated sites, 40 soil samples were collected from two typical oil exploration sites in North and South China and analyzed with a comprehensive functional gene array (GeoChip 3.0). The overall microbial pattern was significantly different between the two sites, and a more divergent pattern was observed in slightly acidic soils. Response ratio was calculated to compare the microbial functional genes involved in organic contaminant degradation and carbon, nitrogen, phosphorus, and sulfur cycling. The results indicated a significantly low abundance of most genes involved in organic contaminant degradation and in the cycling of nitrogen and phosphorus in saline-alkali soils. By contrast, most carbon degradation genes and all carbon fixation genes had similar abundance at both sites. Based on the relationship between the environmental variables and microbial functional structure, pH was the major factor influencing the microbial distribution pattern in the two sites. This study demonstrated that microbial functional diversity and heterogeneity in oil-contaminated environments can vary significantly in relation to local environmental conditions. The limitation of nitrogen and phosphorus and the low degradation capacity of organic contaminant should be carefully considered, particularly in most oil-exploration sites with saline-alkali soils. Copyright © 2014 Elsevier B.V. All rights reserved.

Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4).

PubMed Central

Don, R H; Weightman, A J; Knackmuss, H J; Timmis, K N

1985-01-01

Plasmid pJP4 permits its host bacterium, strain JMP134, to degrade and utilize as sole sources of carbon and energy 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981). Mutagenesis of pJP4 by transposons Tn5 and Tn1771 enabled localization of five genes for enzymes involved in these catabolic pathways. Four of the genes, tfdB, tfdC, tfdD, and tfdE, encoded 2,4-dichlorophenol hydroxylase, dichlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and chlorodienelactone hydrolase, respectively. No function has been assigned to the fifth gene, tfdF, although it may encode a trans-chlorodiene-lactone isomerase. Inactivation of genes tfdC, tfdD, and tfdE, which encode the transformation of dichlorocatechol to chloromaleylacetic acid, prevented host strain JMP134 from degrading both 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid, which indicates that the pathways for these two substrates utilize common enzymes for the dissimilation of chlorocatechols. Studies with cloned catabolic genes from pJP4 indicated that whereas all essential steps in the degradation of 2,4-dichlorophenoxyacetic acid are plasmid encoded, the conversion of 3-chlorobenzoate to chlorocatechol is specified by chromosomal genes. PMID:2981813

[Effects of copper on biodegradation mechanism of trichloroethylene by mixed microorganisms].

PubMed

Gao, Yanhui; Zhao, Tiantao; Xing, Zhilin; He, Zhi; Zhang, Lijie; Peng, Xuya

2016-05-25

We isolated and enriched mixed microorganisms SWA1 from landfill cover soils supplemented with trichloroethylene (TCE). The microbial mixture could degrade TCE effectively under aerobic conditions. Then, we investigated the effect of copper ion (0 to 15 μmol/L) on TCE biodegradation. Results show that the maximum TCE degradation speed was 29.60 nmol/min with 95.75% degradation when copper ion was at 0.03 μmol/L. In addition, genes encoding key enzymes during biodegradation were analyzed by Real-time quantitative reverse transcription PCR (RT-qPCR). The relative expression abundance of pmoA gene (4.22E-03) and mmoX gene (9.30E-06) was the highest when copper ion was at 0.03 μmol/L. Finally, we also used MiSeq pyrosequencing to investigate the diversity of microbial community. Methylocystaceae that can co-metabolic degrade TCE were the dominant microorganisms; other microorganisms with the function of direct oxidation of TCE were also included in SWA1 and the microbial diversity decreased significantly along with increasing of copper ion concentration. Based on the above results, variation of copper ion concentration affected the composition of SWA1 and degradation mechanism of TCE. The degradation mechanism of TCE included co-metabolism degradation of methanotrophs and oxidation metabolism directly at copper ion of 0.03 μmol/L. When copper ion at 5 μmol/L (biodegradation was 84.75%), the degradation mechanism of TCE included direct-degradation and co-metabolism degradation of methanotrophs and microorganisms containing phenol hydroxylase. Therefore, biodegradation of TCE by microorganisms was a complicated process, the degradation mechanism included co-metabolism degradation of methanotrophs and bio-oxidation of non-methanotrophs.

Identification of Three Alcohol Dehydrogenase Genes Involved in the Stereospecific Catabolism of Arylglycerol-β-Aryl Ether by Sphingobium sp. Strain SYK-6▿ †

PubMed Central

Sato, Yusuke; Moriuchi, Hideki; Hishiyama, Shojiro; Otsuka, Yuichiro; Oshima, Kenji; Kasai, Daisuke; Nakamura, Masaya; Ohara, Seiji; Katayama, Yoshihiro; Fukuda, Masao; Masai, Eiji

2009-01-01

Degradation of arylglycerol-β-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-β-guaiacyl ether (GGE) to α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (αR,βS)-GGE and (αR,βR)-GGE into (βS)-MPHPV and (βR)-MPHPV, respectively, while LigL and LigN transformed (αS,βR)-GGE and (αS,βS)-GGE to (βR)-MPHPV and (βS)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (αR,βS)-GGE and (αR,βR)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers. PMID:19542348

The Genome of Ganderma lucidum Provide Insights into Triterpense Biosynthesis and Wood Degradation

PubMed Central

Huang, Zhuo; Zhang, Hong-Mei; Liu, Wei; Liu, Le; Ma, Junping; Xia, Zhilan; Chen, Yuxin; Chen, Yuewen; Wang, Depeng; Ni, Peixiang; Guo, An-Yuan; Xiong, Xingyao

2012-01-01

Background Ganoderma lucidum (Reishi or Ling Zhi) is one of the most famous Traditional Chinese Medicines and has been widely used in the treatment of various human diseases in Asia countries. It is also a fungus with strong wood degradation ability with potential in bioenergy production. However, genes, pathways and mechanisms of these functions are still unknown. Methodology/Principal Findings The genome of G. lucidum was sequenced and assembled into a 39.9 megabases (Mb) draft genome, which encoded 12,080 protein-coding genes and ∼83% of them were similar to public sequences. We performed comprehensive annotation for G. lucidum genes and made comparisons with genes in other fungi genomes. Genes in the biosynthesis of the main G. lucidum active ingredients, ganoderic acids (GAs), were characterized. Among the GAs synthases, we identified a fusion gene, the N and C terminal of which are homologous to two different enzymes. Moreover, the fusion gene was only found in basidiomycetes. As a white rot fungus with wood degradation ability, abundant carbohydrate-active enzymes and ligninolytic enzymes were identified in the G. lucidum genome and were compared with other fungi. Conclusions/Significance The genome sequence and well annotation of G. lucidum will provide new insights in function analyses including its medicinal mechanism. The characterization of genes in the triterpene biosynthesis and wood degradation will facilitate bio-engineering research in the production of its active ingredients and bioenergy. PMID:22567134

Evaluation of microbial population and functional genes during the bioremediation of petroleum-contaminated soil as an effective monitoring approach.

PubMed

Shahi, Aiyoub; Aydin, Sevcan; Ince, Bahar; Ince, Orhan

2016-03-01

This study investigated the abundance and diversity of soil n-alkane and polycyclic aromatic hydrocarbon (PAH)-degrading bacterial communities. It also investigated the quantity of the functional genes, the occurrence of horizontal gene transfer (HGT) in the identified bacterial communities and the effect that such HGT can have on biostimulation process. Illumina sequencing was used to detect the microbial diversity of petroleum-polluted soil prior to the biostimulation process, and quantitative real-time PCR was used to determine changes in the bacterial community and functional genes (alkB, phnAc and nah) expressions throughout the biostimulation of petroleum-contaminated soil. The illumine results revealed that γ-proteobacteria, Chloroflexi, Firmicutes, and δ-proteobacteria were the most dominant bacterial phyla in the contaminated site, and that most of the strains were Gram-negative. The results of the gene expression results revealed that gram-negative bacteria and alkB are critical to successful bioremediation. Failure to maintain the stability of hydrocarbon-degrading bacteria and functional gene will reduce the extend to which alkanes and PAHs are degraded. According to the results of the study, the application of a C:N:P ratio of was 100:15:1 in the biodegradation experiment resulted in the highest rate at which petroleum hydrocarbons were biodegraded. The diversity of pollutant-degrading bacteria and the effective transfer of degrading genes among resident microorganisms are essential factors for the successful biostimulation of petroleum hydrocarbons. As such, screening these factors throughout the biostimulation process represents an effective monitoring approach by which the success of the biostimulation can be assessed. Copyright © 2015 Elsevier Inc. All rights reserved.

Lignin-degrading Peroxidases from Genome of Selective Ligninolytic Fungus Ceriporiopsis subverispora

Treesearch

Elena Fernandez-Fueyo; Francisco J. Ruiz-Duenas; Yuta Miki; Marta Jesus Martinez; Kenneth E. Hammel; Angel T. Martinez

2012-01-01

Background: The first genome of a selective lignin degrader is available. Results: Its screening shows 26 peroxidase genes, and 5 genes were heterologously expressed and the catalytic properties investigated. Conclusion: Two new peroxidases oxidize simple and dimeric lignin models and efficiently depolymerize lignin. Significance: Although lignin peroxidase and...

Genomic evaluation of oxalate-degrading transgenic soybean in response to Sclerotinia sclerotiorum infection

USDA-ARS?s Scientific Manuscript database

Oxalate oxidases catalyze the degradation of oxalic acid (OA). Highly resistant transgenic soybean carrying an oxalate oxidase (OxO) gene and its susceptible parent soybean line, AC Colibri, were tested for genome-wide gene expression in response to the necrotrophic, OA producing pathogen Sclerotini...

Isolation of the opdE gene that encodes for a new hydrolase of Enterobacter sp. capable of degrading organophosphorus pesticides.

PubMed

Chino-Flores, Concepción; Dantán-González, Edgar; Vázquez-Ramos, Alejandra; Tinoco-Valencia, Raunel; Díaz-Méndez, Rafael; Sánchez-Salinas, Enrique; Castrejón-Godínez, Maria Luisa; Ramos-Quintana, Fernando; Ortiz-Hernández, Maria Laura

2012-06-01

Microbial enzymes that can hydrolyze organophosphorus compounds have been isolated, identified and characterized from different microbial species in order to use them in biodegradation of organophosphorus compounds. We isolated a bacterial strain Cons002 from an agricultural soil bacterial consortium, which can hydrolyze methyl-parathion (MP) and other organophosphate pesticides. HPLC analysis showed that strain Cons002 is capable of degrading pesticides MP, parathion and phorate. Pulsed-field gel electrophoresis and 16S rRNA amplification were performed for strain characterization and identification, respectively, showing that the strain Cons002 is related to the genus Enterobacter sp. which has a single chromosome of 4.6 Mb and has no plasmids. Genomic library was constructed from DNA of Enterobacter sp. Cons002. A gene called opdE (Organophosphate Degradation from Enterobacter) consists of 753 bp and encodes a protein of 25 kDa, which was isolated using activity methods. This gene opdE had no similarity to any genes reported to degrade organophosphates. When kanamycin-resistance cassette was placed in the gene opdE, hydrolase activity was suppressed and Enterobacter sp. Cons002 had no growth with MP as a nutrients source.

α-Amino acid containing degradable polymers as functional biomaterials: rational design, synthetic pathway, and biomedical applications.

PubMed

Sun, Huanli; Meng, Fenghua; Dias, Aylvin A; Hendriks, Marc; Feijen, Jan; Zhong, Zhiyuan

2011-06-13

Currently, biomedical engineering is rapidly expanding, especially in the areas of drug delivery, gene transfer, tissue engineering, and regenerative medicine. A prerequisite for further development is the design and synthesis of novel multifunctional biomaterials that are biocompatible and biologically active, are biodegradable with a controlled degradation rate, and have tunable mechanical properties. In the past decades, different types of α-amino acid-containing degradable polymers have been actively developed with the aim to obtain biomimicking functional biomaterials. The use of α-amino acids as building units for degradable polymers may offer several advantages: (i) imparting chemical functionality, such as hydroxyl, amine, carboxyl, and thiol groups, which not only results in improved hydrophilicity and possible interactions with proteins and genes, but also facilitates further modification with bioactive molecules (e.g., drugs or biological cues); (ii) possibly improving materials biological properties, including cell-materials interactions (e.g., cell adhesion, migration) and degradability; (iii) enhancing thermal and mechanical properties; and (iv) providing metabolizable building units/blocks. In this paper, recent developments in the field of α-amino acid-containing degradable polymers are reviewed. First, synthetic approaches to prepare α-amino acid-containing degradable polymers will be discussed. Subsequently, the biomedical applications of these polymers in areas such as drug delivery, gene delivery and tissue engineering will be reviewed. Finally, the future perspectives of α-amino acid-containing degradable polymers will be evaluated.

The first genome-level transcriptome of the wood-degrading fungus Phanerochaete chrysosporium grown on red oak.

PubMed

Sato, Shin; Feltus, F Alex; Iyer, Prashanti; Tien, Ming

2009-06-01

As part of an effort to determine all the gene products involved in wood degradation, we have performed massively parallel pyrosequencing on an expression library from the white rot fungus Phanerochaete chrysosporium grown in shallow stationary cultures with red oak as the carbon source. Approximately 48,000 high quality sequence tags (246 bp average length) were generated. 53% of the sequence tags aligned to 4,262 P. chrysosporium gene models, and an additional 18.5% of the tags reliably aligned to the P. chrysosporium genome providing evidence for 961 putative novel fragmented gene models. Due to their role in lignocellulose degradation, the secreted proteins were focused upon. Our results show that the four enzymes required for cellulose degradation: endocellulase, exocellulase CBHI, exocellulase CBHII, and beta-glucosidase are all produced. For hemicellulose degradation, not all known enzymes were produced, but endoxylanases, acetyl xylan esterases and mannosidases were detected. For lignin degradation, the role of peroxidases has been questioned; however, our results show that lignin peroxidase is highly expressed along with the H(2)O(2) generating enzyme, alcohol oxidase. The transcriptome snapshot reveals that H(2)O(2) generation and utilization are central in wood degradation. Our results also reveal new transcripts that encode extracellular proteins with no known function.

A Novel (S)-6-Hydroxynicotine Oxidase Gene from Shinella sp. Strain HZN7

PubMed Central

Qiu, Jiguo; Wei, Yin; Ma, Yun; Wen, Rongti; Wen, Yuezhong

2014-01-01

Nicotine is an important environmental toxicant in tobacco waste. Shinella sp. strain HZN7 can metabolize nicotine into nontoxic compounds via variations of the pyridine and pyrrolidine pathways. However, the catabolic mechanism of this variant pathway at the gene or enzyme level is still unknown. In this study, two 6-hydroxynicotine degradation-deficient mutants, N7-M9 and N7-W3, were generated by transposon mutagenesis. The corresponding mutant genes, designated nctB and tnp2, were cloned and analyzed. The nctB gene encodes a novel flavin adenine dinucleotide-containing (S)-6-hydroxynicotine oxidase that converts (S)-6-hydroxynicotine into 6-hydroxy-N-methylmyosmine and then spontaneously hydrolyzes into 6-hydroxypseudooxynicotine. The deletion and complementation of the nctB gene showed that this enzyme is essential for nicotine or (S)-6-hydroxynicotine degradation. Purified NctB could also convert (S)-nicotine into N-methylmyosmine, which spontaneously hydrolyzed into pseudooxynicotine. The kinetic constants of NctB toward (S)-6-hydroxynicotine (Km = 0.019 mM, kcat = 7.3 s−1) and nicotine (Km = 2.03 mM, kcat = 0.396 s−1) indicated that (S)-6-hydroxynicotine is the preferred substrate in vivo. NctB showed no activities toward the R enantiomer of nicotine or 6-hydroxynicotine. Strain HZN7 could degrade (R)-nicotine into (R)-6-hydroxynicotine without any further degradation. The tnp2 gene from mutant N7-W3 encodes a putative transposase, and its deletion did not abolish the nicotine degradation activity. This study advances the understanding of the microbial diversity of nicotine biodegradation. PMID:25002425

Metagenomic analysis of the bioremediation of diesel-contaminated Canadian high arctic soils.

PubMed

Yergeau, Etienne; Sanschagrin, Sylvie; Beaumier, Danielle; Greer, Charles W

2012-01-01

As human activity in the Arctic increases, so does the risk of hydrocarbon pollution events. On site bioremediation of contaminated soil is the only feasible clean up solution in these remote areas, but degradation rates vary widely between bioremediation treatments. Most previous studies have focused on the feasibility of on site clean-up and very little attention has been given to the microbial and functional communities involved and their ecology. Here, we ask the question: which microorganisms and functional genes are abundant and active during hydrocarbon degradation at cold temperature? To answer this question, we sequenced the soil metagenome of an ongoing bioremediation project in Alert, Canada through a time course. We also used reverse-transcriptase real-time PCR (RT-qPCR) to quantify the expression of several hydrocarbon-degrading genes. Pseudomonas species appeared as the most abundant organisms in Alert soils right after contamination with diesel and excavation (t = 0) and one month after the start of the bioremediation treatment (t = 1m), when degradation rates were at their highest, but decreased after one year (t = 1y), when residual soil hydrocarbons were almost depleted. This trend was also reflected in hydrocarbon degrading genes, which were mainly affiliated with Gammaproteobacteria at t = 0 and t = 1m and with Alphaproteobacteria and Actinobacteria at t = 1y. RT-qPCR assays confirmed that Pseudomonas and Rhodococcus species actively expressed hydrocarbon degradation genes in Arctic biopile soils. Taken together, these results indicated that biopile treatment leads to major shifts in soil microbial communities, favoring aerobic bacteria that can degrade hydrocarbons.

Metagenomic Analysis of the Bioremediation of Diesel-Contaminated Canadian High Arctic Soils

PubMed Central

Yergeau, Etienne; Sanschagrin, Sylvie; Beaumier, Danielle; Greer, Charles W.

2012-01-01

As human activity in the Arctic increases, so does the risk of hydrocarbon pollution events. On site bioremediation of contaminated soil is the only feasible clean up solution in these remote areas, but degradation rates vary widely between bioremediation treatments. Most previous studies have focused on the feasibility of on site clean-up and very little attention has been given to the microbial and functional communities involved and their ecology. Here, we ask the question: which microorganisms and functional genes are abundant and active during hydrocarbon degradation at cold temperature? To answer this question, we sequenced the soil metagenome of an ongoing bioremediation project in Alert, Canada through a time course. We also used reverse-transcriptase real-time PCR (RT-qPCR) to quantify the expression of several hydrocarbon-degrading genes. Pseudomonas species appeared as the most abundant organisms in Alert soils right after contamination with diesel and excavation (t = 0) and one month after the start of the bioremediation treatment (t = 1m), when degradation rates were at their highest, but decreased after one year (t = 1y), when residual soil hydrocarbons were almost depleted. This trend was also reflected in hydrocarbon degrading genes, which were mainly affiliated with Gammaproteobacteria at t = 0 and t = 1m and with Alphaproteobacteria and Actinobacteria at t = 1y. RT-qPCR assays confirmed that Pseudomonas and Rhodococcus species actively expressed hydrocarbon degradation genes in Arctic biopile soils. Taken together, these results indicated that biopile treatment leads to major shifts in soil microbial communities, favoring aerobic bacteria that can degrade hydrocarbons. PMID:22253877

Cloning and Characterization of Benzoate Catabolic Genes in the Gram-Positive Polychlorinated Biphenyl Degrader Rhodococcus sp. Strain RHA1

PubMed Central

Kitagawa, Wataru; Miyauchi, Keisuke; Masai, Eiji; Fukuda, Masao

2001-01-01

Benzoate catabolism is thought to play a key role in aerobic bacterial degradation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabolic genes were cloned from a PCB degrader, Rhodococcus sp. strain RHA1, by using PCR amplification and temporal temperature gradient electrophoresis separation. A nucleotide sequence determination revealed that the deduced amino acid sequences encoded by the RHA1 benzoate catabolic genes, benABCDK, exhibit 33 to 65% identity with those of Acinetobacter sp. strain ADP1. The gene organization of the RHA1 benABCDK genes differs from that of ADP1. The RHA1 benABCDK region was localized on the chromosome, in contrast to the biphenyl catabolic genes, which are located on linear plasmids. Escherichia coli cells containing RHA1 benABCD transformed benzoate to catechol via 2-hydro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoates but did transform 3-chlorobenzoate. The RHA1 benA gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not transform 3-chlorobenzoate. It did, however, exhibit diminished growth on biphenyl and growth repression in the presence of a high concentration of biphenyl (13 mM). These results indicate that the cloned benABCD genes could play an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1. Six rhodococcal benzoate degraders were found to have homologs of RHA1 benABC. In contrast, two rhodococcal strains that cannot transform benzoate were found not to have RHA1 benABC homologs, suggesting that many Rhodococcus strains contain benzoate catabolic genes similar to RHA1 benABC. PMID:11673430

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rojas-Garcia, A.E.; Solis-Heredia, M.J.; Pina-Guzman, B.

Human paraoxonase (PON1) plays a role in detoxification of organophosphorus (OP) compounds by hydrolyzing the bioactive oxons, and in reducing oxidative low-density lipoproteins, which may protect against atherosclerosis. Some PON1 polymorphisms have been found to be responsible for variations in catalytic activity and expression and have been associated with susceptibility to OP poisoning and vascular diseases. Both situations are of public health relevance in Mexico. Therefore, the aim of this study was to evaluate PON1 phenotype and the frequencies of polymorphisms PON1 -162, -108, 55, and 192 in a Mexican population. The studied population consisted of unrelated individuals (n =more » 214) of either gender, 18-52 years old. Serum PON1 activity was assayed using phenylacetate and paraoxon as substrates. PON1 variants, -162, 55, and 192, were determined by real-time PCR using the TaqMan System, and PON1 -108 genotype by PCR-RFLP. We found a wide interindividual variability of PON1 activity with a unimodal distribution; the range of enzymatic activity toward phenylacetate was 84.72 to 422.0 U/mL, and 88.37 to 1645.6 U/L toward paraoxon. All four PON1 polymorphisms showed strong linkage disequilibrium (D% >90). PON1 polymorphisms -108, 55, and 192 were independently associated with arylesterase activity; whereas the activity toward paraoxon was related only with PON1 192 polymorphism, suggesting that this polymorphism is determinant to infer PON1 activity. A better understanding of the phenotype and genotypes of PON1 in Mexican populations will facilitate further epidemiological studies involving PON1 variability in OP poisoning and in the development of atherosclerosis.« less

Effects of aromatic amino acids, phenylacetate and phenylpropionate on fermentation of xylan by the rumen anaerobic fungi, Neocallimastix frontalis and Piromyces communis.

PubMed

Guliye, A Y; Wallace, R J

2007-10-01

Anaerobic fungi are important members of the fibrolytic community of the rumen. The aim of this study was to study their requirement for aromatic amino acids (AA) and related phenyl acids (phenylpropionic and phenylacetic acids) for optimal xylan fermentation. Neocallimastix frontalis RE1 and Piromyces communis P were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia; ammonia plus a complete mixture of 20 AA commonly found in protein; ammonia plus complete AA mixture minus aromatic AA; ammonia plus phenyl acids; ammonia plus complete AA mixture without aromatic AA plus phenyl acids. Both species grew in all the media, indicating no absolute requirement for AA. The complete AA mixture increased (P<0.05) acetate concentration by 18% and 15%, sugar utilization by 33% and 22% and microbial yield by about 22% and 15% in N. frontalis and P. communis, respectively, in comparison with the treatments that had ammonia as the only N source. Neither the supply of aromatic AA or phenol acids, nor their deletion from the complete AA mixture, affected the fermentation rate, products or yield of either species. AA were not essential for N. frontalis and P. communis, but their growth on xylan was stimulated. The effects could not be explained in terms of aromatic AA alone. Ruminant diets should contain sufficient protein to sustain optimal fibre digestion by ruminal fungi. Aromatic AA or phenyl acids alone cannot replace the complete AA mixture.

A diazonium ion cascade from the nitrosation of tolazoline, an imidazoline-containing drug.

PubMed

Loeppky, Richard N; Shi, Jianzheng; Barnes, Charles L; Geddam, Sailaja

2008-02-01

Tolazoline (1-benzylimidazoline), a representative imidazoline-containing drug, reacts readily with nitrite in acetic acid to produce a complex product mixture. Fourteen compounds have been identified as products of this transformation when an 8-fold excess of HNO2 is used. The products, which include N-nitrosoamides, esters, alcohols, and phenylacetic acid, are rationalized as arising from a cascade of reactive diazonium ions. N-Nitrosotolazoline can be isolated from the nitrosation reaction in good yield when the mixture is extracted with CH2Cl2 as the transformation progresses. It nitrosates much more rapidly (50x) than tolazoline to give, among other products, the oxime [1-( N-nitroso-2-imidazolinyl)benzylidene]hydroxylamine, which can also be produced in good yield from the reaction of tolazoline with isopropyl nitrite. At low substrate and nitrite concentrations, the main reaction products are N-nitrosotolazoline, its decomposition product N-2-hydroxyethylphenylacetamide, the above-mentioned oxime, phenyl acetic acid, and 2-hydroxyethyl phenylacetate. The tolazoline nitrosation rate in three buffer systems has been determined at pH 3.4 and 37 degrees C ( kobs = 6.25 x 10 (-5) s (-1) in 0.5 M acetate buffer with a 10 * [NO2(-)] = 250 mM). Because N-nitrosotolazoline exhibits the chemical properties of a direct-acting mutagen and carcinogen, we have used the rate data to estimate its level of formation at nitrite concentrations <3 mM. Cursory examination of the nitrosation chemistry of oxymetazoline, a related drug, is primarily focused at its electron-rich aromatic ring.

Limited mitogenomic degradation in response to a parasitic lifestyle in Orobanchaceae

PubMed Central

Fan, Weishu; Zhu, Andan; Kozaczek, Melisa; Shah, Neethu; Pabón-Mora, Natalia; González, Favio; Mower, Jeffrey P.

2016-01-01

In parasitic plants, the reduction in plastid genome (plastome) size and content is driven predominantly by the loss of photosynthetic genes. The first completed mitochondrial genomes (mitogenomes) from parasitic mistletoes also exhibit significant degradation, but the generality of this observation for other parasitic plants is unclear. We sequenced the complete mitogenome and plastome of the hemiparasite Castilleja paramensis (Orobanchaceae) and compared them with additional holoparasitic, hemiparasitic and nonparasitic species from Orobanchaceae. Comparative mitogenomic analysis revealed minimal gene loss among the seven Orobanchaceae species, indicating the retention of typical mitochondrial function among Orobanchaceae species. Phylogenetic analysis demonstrated that the mobile cox1 intron was acquired vertically from a nonparasitic ancestor, arguing against a role for Orobanchaceae parasites in the horizontal acquisition or distribution of this intron. The C. paramensis plastome has retained nearly all genes except for the recent pseudogenization of four subunits of the NAD(P)H dehydrogenase complex, indicating a very early stage of plastome degradation. These results lend support to the notion that loss of ndh gene function is the first step of plastome degradation in the transition to a parasitic lifestyle. PMID:27808159

High-throughput pyrosequencing used for the discovery of a novel cellulase from a thermophilic cellulose-degrading microbial consortium.

PubMed

Zhao, Chao; Chu, Yanan; Li, Yanhong; Yang, Chengfeng; Chen, Yuqing; Wang, Xumin; Liu, Bin

2017-01-01

To analyze the microbial diversity and gene content of a thermophilic cellulose-degrading consortium from hot springs in Xiamen, China using 454 pyrosequencing for discovering cellulolytic enzyme resources. A thermophilic cellulose-degrading consortium, XM70 that was isolated from a hot spring, used sugarcane bagasse as sole carbon and energy source. DNA sequencing of the XM70 sample resulted in 349,978 reads with an average read length of 380 bases, accounting for 133,896,867 bases of sequence information. The characterization of sequencing reads and assembled contigs revealed that most microbes were derived from four phyla: Geobacillus (Firmicutes), Thermus, Bacillus, and Anoxybacillus. Twenty-eight homologous genes belonging to 15 glycoside hydrolase families were detected, including several cellulase genes. A novel hot spring metagenome-derived thermophilic cellulase was expressed and characterized. The application value of thermostable sugarcane bagasse-degrading enzymes is shown for production of cellulosic biofuel. The practical power of using a short-read-based metagenomic approach for harvesting novel microbial genes is also demonstrated.

Degradable self-assembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake.

PubMed

Barnard, Anna; Posocco, Paola; Pricl, Sabrina; Calderon, Marcelo; Haag, Rainer; Hwang, Mark E; Shum, Victor W T; Pack, Daniel W; Smith, David K

2011-12-21

This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery. © 2011 American Chemical Society

Reliable Gene Expression Measurements from Fine Needle Aspirates of Pancreatic Tumors

PubMed Central

Anderson, Michelle A.; Brenner, Dean E.; Scheiman, James M.; Simeone, Diane M.; Singh, Nalina; Sikora, Matthew J.; Zhao, Lili; Mertens, Amy N.; Rae, James M.

2010-01-01

Background and aims: Biomarker use for pancreatic cancer diagnosis has been impaired by a lack of samples suitable for reliable quantitative RT-PCR (qRT-PCR). Fine needle aspirates (FNAs) from pancreatic masses were studied to define potential causes of RNA degradation and develop methods for accurately measuring gene expression. Methods: Samples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phosphate dehydrogenase, and effects on qRT-PCR were determined by using a 150-bp and a 80-bp amplicon for RPS6. Potential causes of and methods to circumvent RNA degradation were studied by using FNAs from a pancreatic cancer xenograft. Results: RNA extracted from pancreatic mass FNAs was extensively degraded. Fragmentation was related to needle bore diameter and could not be overcome by alterations in aspiration technique. Multiplex PCR for glyceraldehyde-3-phosphate dehydrogenase could distinguish samples that were suitable for qRT-PCR. The use of short PCR amplicons (<100 bp) provided reliable gene expression analysis from FNAs. When appropriate samples were used, the assay was highly reproducible for gene copy number with minimal (0.0003 or about 0.7% of total) variance. Conclusions: The degraded properties of endoscopic FNAs markedly affect the accuracy of gene expression measurements. Our novel approach to designate specimens “informative” for qRT-PCR allowed accurate molecular assessment for the diagnosis of pancreatic diseases. PMID:20709792

Metatranscriptome of an Anaerobic Benzene-Degrading, Nitrate-Reducing Enrichment Culture Reveals Involvement of Carboxylation in Benzene Ring Activation

PubMed Central

Luo, Fei; Gitiafroz, Roya; Devine, Cheryl E.; Gong, Yunchen; Hug, Laura A.; Raskin, Lutgarde

2014-01-01

The enzymes involved in the initial steps of anaerobic benzene catabolism are not known. To try to elucidate this critical step, a metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of benzene and benzoate by an anaerobic benzene-degrading, nitrate-reducing enrichment culture. RNA was extracted from the mixed culture and sequenced without prior mRNA enrichment, allowing simultaneous examination of the active community composition and the differential gene expression between the two treatments. Ribosomal and mRNA sequences attributed to a member of the family Peptococcaceae from the order Clostridiales were essentially only detected in the benzene-amended culture samples, implicating this group in the initial catabolism of benzene. Genes similar to each of two subunits of a proposed benzene-carboxylating enzyme were transcribed when the culture was amended with benzene. Anaerobic benzoate degradation genes from strict anaerobes were transcribed only when the culture was amended with benzene. Genes for other benzoate catabolic enzymes and for nitrate respiration were transcribed in both samples, with those attributed to an Azoarcus species being most abundant. These findings indicate that the mineralization of benzene starts with its activation by a strict anaerobe belonging to the Peptococcaceae, involving a carboxylation step to form benzoate. These data confirm the previously hypothesized syntrophic association between a benzene-degrading Peptococcaceae strain and a benzoate-degrading denitrifying Azoarcus strain for the complete catabolism of benzene with nitrate as the terminal electron acceptor. PMID:24795366

A 2,4-dichlorophenoxyacetic acid degradation plasmid pM7012 discloses distribution of an unclassified megaplasmid group across bacterial species.

PubMed

Sakai, Yoriko; Ogawa, Naoto; Shimomura, Yumi; Fujii, Takeshi

2014-03-01

Analysis of the complete nucleotide sequence of plasmid pM7012 from 2,4-dichlorophenoxyacetic-acid (2,4-D)-degrading bacterium Burkholderia sp. M701 revealed that the plasmid had 582 142 bp, with 541 putative protein-coding sequences and 39 putative tRNA genes for the transport of the standard 20 aa. pM7012 contains sequences homologous to the regions involved in conjugal transfer and plasmid maintenance found in plasmids byi_2p from Burkholderia sp. YI23 and pBVIE01 from Burkholderia sp. G4. No relaxase gene was found in any of these plasmids, although genes for a type IV secretion system and type IV coupling proteins were identified. Plasmids with no relaxase gene have been classified as non-mobile plasmids. However, nucleotide sequences with a high level of similarity to the genes for plasmid transfer, plasmid maintenance, 2,4-D degradation and arsenic resistance contained on pM7012 were also detected in eight other megaplasmids (~600 or 900 kb) found in seven Burkholderia strains and a strain of Cupriavidus, which were isolated as 2,4-D-degrading bacteria in Japan and the United States. These results suggested that the 2,4-D degradation megaplasmids related to pM7012 are mobile and distributed across various bacterial species worldwide, and that the plasmid group could be distinguished from known mobile plasmid groups.

Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples.

PubMed

Reiman, Mario; Laan, Maris; Rull, Kristiina; Sõber, Siim

2017-08-01

RNA degradation is a ubiquitous process that occurs in living and dead cells, as well as during handling and storage of extracted RNA. Reduced RNA quality caused by degradation is an established source of uncertainty for all RNA-based gene expression quantification techniques. RNA sequencing is an increasingly preferred method for transcriptome analyses, and dependence of its results on input RNA integrity is of significant practical importance. This study aimed to characterize the effects of varying input RNA integrity [estimated as RNA integrity number (RIN)] on transcript level estimates and delineate the characteristic differences between transcripts that differ in degradation rate. The study used ribodepleted total RNA sequencing data from a real-life clinically collected set ( n = 32) of human solid tissue (placenta) samples. RIN-dependent alterations in gene expression profiles were quantified by using DESeq2 software. Our results indicate that small differences in RNA integrity affect gene expression quantification by introducing a moderate and pervasive bias in expression level estimates that significantly affected 8.1% of studied genes. The rapidly degrading transcript pool was enriched in pseudogenes, short noncoding RNAs, and transcripts with extended 3' untranslated regions. Typical slowly degrading transcripts (median length, 2389 nt) represented protein coding genes with 4-10 exons and high guanine-cytosine content.-Reiman, M., Laan, M., Rull, K., Sõber, S. Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples. © FASEB.

Coordinated Regulation of Species-Specific Hydroxycinnamic Acid Degradation and Siderophore Biosynthesis Pathways in Agrobacterium fabrum

PubMed Central

Baude, Jessica; Vial, Ludovic; Villard, Camille; Campillo, Tony; Lavire, Céline; Nesme, Xavier

2016-01-01

ABSTRACT The rhizosphere-inhabiting species Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to degrade hydroxycinnamic acids (HCAs), especially ferulic acid and p-coumaric acid, via the novel A. fabrum HCA degradation pathway. Gene expression profiles of A. fabrum strain C58 were investigated in the presence of HCAs, using a C58 whole-genome oligoarray. Both ferulic acid and p-coumaric acid caused variations in the expression of more than 10% of the C58 genes. Genes of the A. fabrum HCA degradation pathway, together with the genes involved in iron acquisition, were among the most highly induced in the presence of HCAs. Two operons coding for the biosynthesis of a particular siderophore, as well as genes of the A. fabrum HCA degradation pathway, have been described as being specific to the species. We demonstrate here their coordinated expression, emphasizing the interdependence between the iron concentration in the growth medium and the rate at which ferulic acid is degraded by cells. The coordinated expression of these functions may be advantageous in HCA-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. The present results confirm that there is cooperation between the A. fabrum-specific genes, defining a particular ecological niche. IMPORTANCE We previously identified seven genomic regions in Agrobacterium fabrum that were specifically present in all of the members of this species only. Here we demonstrated that two of these regions, encoding the hydroxycinnamic acid degradation pathway and the iron acquisition pathway, were regulated in a coordinated manner. The coexpression of these functions may be advantageous in hydroxycinnamic acid-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. These data support the view that bacterial genomic species emerged from a bacterial population by acquiring specific functions that allowed them to outcompete their closest relatives. In conclusion, bacterial species could be defined not only as genomic species but also as ecological species. PMID:27060117

Comparison of the complete genome sequences of four γ-hexachlorocyclohexane-degrading bacterial strains: insights into the evolution of bacteria able to degrade a recalcitrant man-made pesticide.

PubMed

Tabata, Michiro; Ohhata, Satoshi; Nikawadori, Yuki; Kishida, Kouhei; Sato, Takuya; Kawasumi, Toru; Kato, Hiromi; Ohtsubo, Yoshiyuki; Tsuda, Masataka; Nagata, Yuji

2016-12-01

γ-Hexachlorocyclohexane (γ-HCH) is a recalcitrant man-made chlorinated pesticide. Here, the complete genome sequences of four γ-HCH-degrading sphingomonad strains, which are most unlikely to have been derived from one ancestral γ-HCH degrader, were compared. Together with several experimental data, we showed that (i) all the four strains carry almost identical linA to linE genes for the conversion of γ-HCH to maleylacetate (designated "specific" lin genes), (ii) considerably different genes are used for the metabolism of maleylacetate in one of the four strains, and (iii) the linKLMN genes for the putative ABC transporter necessary for γ-HCH utilization exhibit structural divergence, which reflects the phylogenetic relationship of their hosts. Replicon organization and location of the lin genes in the four genomes are significantly different with one another, and that most of the specific lin genes are located on multiple sphingomonad-unique plasmids. Copies of IS6100, the most abundant insertion sequence in the four strains, are often located in close proximity to the specific lin genes. Analysis of the footprints of target duplication upon IS6100 transposition and the experimental detection of IS6100 transposition strongly suggested that the IS6100 transposition has caused dynamic genome rearrangements and the diversification of lin-flanking regions in the four strains. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Plasmid-Mediated Bioaugmentation for the Bioremediation of Contaminated Soils

PubMed Central

Garbisu, Carlos; Garaiyurrebaso, Olatz; Epelde, Lur; Grohmann, Elisabeth; Alkorta, Itziar

2017-01-01

Bioaugmentation, or the inoculation of microorganisms (e.g., bacteria harboring the required catabolic genes) into soil to enhance the rate of contaminant degradation, has great potential for the bioremediation of soils contaminated with organic compounds. Regrettably, cell bioaugmentation frequently turns into an unsuccessful initiative, owing to the rapid decrease of bacterial viability and abundance after inoculation, as well as the limited dispersal of the inoculated bacteria in the soil matrix. Genes that encode the degradation of organic compounds are often located on plasmids and, consequently, they can be spread by horizontal gene transfer into well-established, ecologically competitive, indigenous bacterial populations. Plasmid-mediated bioaugmentation aims to stimulate the spread of contaminant degradation genes among indigenous soil bacteria by the introduction of plasmids, located in donor cells, harboring such genes. But the acquisition of plasmids by recipient cells can affect the host’s fitness, a crucial aspect for the success of plasmid-mediated bioaugmentation. Besides, environmental factors (e.g., soil moisture, temperature, organic matter content) can play important roles for the transfer efficiency of catabolic plasmids, the expression of horizontally acquired genes and, finally, the contaminant degradation activity. For plasmid-mediated bioaugmentation to be reproducible, much more research is needed for a better selection of donor bacterial strains and accompanying plasmids, together with an in-depth understanding of indigenous soil bacterial populations and the environmental conditions that affect plasmid acquisition and the expression and functioning of the catabolic genes of interest. PMID:29062312

Characterization of mechanisms underlying degradation of sclerotia of Sclerotinia sclerotiorum by Aspergillus aculeatus Asp-4 using a combined qRT-PCR and proteomic approach.

PubMed

Hu, Xiaojia; Qin, Lu; Roberts, Daniel P; Lakshman, Dilip K; Gong, Yangmin; Maul, Jude E; Xie, Lihua; Yu, Changbing; Li, Yinshui; Hu, Lei; Liao, Xiangsheng; Liao, Xing

2017-08-31

The biological control agent Aspergillus aculeatus Asp-4 colonizes and degrades sclerotia of Sclerotinia sclerotiorum resulting in reduced germination and disease caused by this important plant pathogen. Molecular mechanisms of mycoparasites underlying colonization, degradation, and reduction of germination of sclerotia of this and other important plant pathogens remain poorly understood. An RNA-Seq screen of Asp-4 growing on autoclaved, ground sclerotia of S. sclerotiorum for 48 h identified 997 up-regulated and 777 down-regulated genes relative to this mycoparasite growing on potato dextrose agar (PDA) for 48 h. qRT-PCR time course experiments characterized expression dynamics of select genes encoding enzymes functioning in degradation of sclerotial components and management of environmental conditions, including environmental stress. This analysis suggested co-temporal up-regulation of genes functioning in these two processes. Proteomic analysis of Asp-4 growing on this sclerotial material for 48 h identified 26 up-regulated and 6 down-regulated proteins relative to the PDA control. Certain proteins with increased abundance had putative functions in degradation of polymeric components of sclerotia and the mitigation of environmental stress. Our results suggest co-temporal up-regulation of genes involved in degradation of sclerotial compounds and mitigation of environmental stress. This study furthers the analysis of mycoparasitism of sclerotial pathogens by providing the basis for molecular characterization of a previously uncharacterized mycoparasite-sclerotial interaction.

The complex physiology of Cellvibrio japonicus xylan degradation relies on a single cytoplasmic β-xylosidase for xylo-oligosaccharide utilization

DOE PAGES

Blake, Andrew D.; Beri, Nina R.; Guttman, Hadassa S.; ...

2017-12-21

Lignocellulose degradation by microbes plays a central role in global carbon cycling, human gut metabolism, and renewable energy technologies. While considerable effort has been put into understanding the biochemical aspects of lignocellulose degradation, much less work has been done to understand how these enzymes work in an in vivo context. Here in this paper, we report a systems level study of xylan degradation in the saprophytic bacterium Cellvibrio japonicus. Transcriptome analysis indicated seven genes that encode carbohydrate active enzymes were up-regulated during growth with xylan containing media. In-frame deletion analysis of these genes found that only gly43F is critical formore » utilization of xylo-oligosaccharides, xylan, and arabinoxylan. Heterologous expression of gly43F was sufficient for the utilization of xylo-oligosaccharides in Escherichia coli. Additional analysis found that the xyn11A, xyn11B, abf43L, abf43K, and abf51A gene products were critical for utilization of arabinoxylan. Furthermore, a predicted transporter (CJA_1315) was required for effective utilization of xylan substrates, and we propose this unannotated gene be called xntA (xylan transporter A). Our major findings are 1) C. japonicus employs both secreted and surface associated enzymes for xylan degradation, which differs from the strategy used for cellulose degradation, and 2) a single cytoplasmic β-xylosidase is essential for the utilization of xylo-oligosaccharides.« less

The complex physiology of Cellvibrio japonicus xylan degradation relies on a single cytoplasmic β-xylosidase for xylo-oligosaccharide utilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blake, Andrew D.; Beri, Nina R.; Guttman, Hadassa S.

Lignocellulose degradation by microbes plays a central role in global carbon cycling, human gut metabolism, and renewable energy technologies. While considerable effort has been put into understanding the biochemical aspects of lignocellulose degradation, much less work has been done to understand how these enzymes work in an in vivo context. Here in this paper, we report a systems level study of xylan degradation in the saprophytic bacterium Cellvibrio japonicus. Transcriptome analysis indicated seven genes that encode carbohydrate active enzymes were up-regulated during growth with xylan containing media. In-frame deletion analysis of these genes found that only gly43F is critical formore » utilization of xylo-oligosaccharides, xylan, and arabinoxylan. Heterologous expression of gly43F was sufficient for the utilization of xylo-oligosaccharides in Escherichia coli. Additional analysis found that the xyn11A, xyn11B, abf43L, abf43K, and abf51A gene products were critical for utilization of arabinoxylan. Furthermore, a predicted transporter (CJA_1315) was required for effective utilization of xylan substrates, and we propose this unannotated gene be called xntA (xylan transporter A). Our major findings are 1) C. japonicus employs both secreted and surface associated enzymes for xylan degradation, which differs from the strategy used for cellulose degradation, and 2) a single cytoplasmic β-xylosidase is essential for the utilization of xylo-oligosaccharides.« less

Effect of benzene and ethylbenzene on the transcription of methyl-tert-butyl ether degradation genes of Methylibium petroleiphilum PM1.

PubMed

Joshi, Geetika; Schmidt, Radomir; Scow, Kate M; Denison, Michael S; Hristova, Krassimira R

2016-09-01

Methyl-tert-butyl ether (MTBE) and its degradation by-product, tert-butyl alcohol (TBA), are widespread contaminants detected frequently in groundwater in California. Since MTBE was used as a fuel oxygenate for almost two decades, leaking underground fuel storage tanks are an important source of contamination. Gasoline components such as BTEX (benzene, toluene, ethylbenzene and xylenes) are often present in mixtures with MTBE and TBA. Investigations of interactions between BTEX and MTBE degradation have not yielded consistent trends, and the molecular mechanisms of BTEX compounds' impact on MTBE degradation are not well understood. We investigated trends in transcription of biodegradation genes in the MTBE-degrading bacterium, Methylibium petroleiphilum PM1 upon exposure to MTBE, TBA, ethylbenzene and benzene as individual compounds or in mixtures. We designed real-time quantitative PCR assays to target functional genes of strain PM1 and provide evidence for induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Delayed induction of mdpA and mdpJ transcription occurred with mixtures of benzene and MTBE or TBA, respectively. bmoA transcription was similar in the presence of MTBE or TBA with benzene as in their absence. Our results also indicate that ethylbenzene, previously proposed as an inhibitor of MTBE degradation in some bacteria, inhibits transcription of mdpA, mdpJ and bmoAgenes in strain PM1.

Effect of benzene and ethylbenzene on the transcription of methyl-tert-butyl ether degradation genes of Methylibium petroleiphilum PM1

PubMed Central

Joshi, Geetika; Schmidt, Radomir; Scow, Kate M.; Denison, Michael S.; Hristova, Krassimira R.

2016-01-01

Methyl-tert-butyl ether (MTBE) and its degradation by-product, tert-butyl alcohol (TBA), are widespread contaminants detected frequently in groundwater in California. Since MTBE was used as a fuel oxygenate for almost two decades, leaking underground fuel storage tanks are an important source of contamination. Gasoline components such as BTEX (benzene, toluene, ethylbenzene and xylenes) are often present in mixtures with MTBE and TBA. Investigations of interactions between BTEX and MTBE degradation have not yielded consistent trends, and the molecular mechanisms of BTEX compounds’ impact on MTBE degradation are not well understood. We investigated trends in transcription of biodegradation genes in the MTBE-degrading bacterium, Methylibium petroleiphilum PM1 upon exposure to MTBE, TBA, ethylbenzene and benzene as individual compounds or in mixtures. We designed real-time quantitative PCR assays to target functional genes of strain PM1 and provide evidence for induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Delayed induction of mdpA and mdpJ transcription occurred with mixtures of benzene and MTBE or TBA, respectively. bmoA transcription was similar in the presence of MTBE or TBA with benzene as in their absence. Our results also indicate that ethylbenzene, previously proposed as an inhibitor of MTBE degradation in some bacteria, inhibits transcription of mdpA, mdpJ and bmoAgenes in strain PM1. PMID:27450417

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community.

PubMed

Posman, Kevin M; DeRito, Christopher M; Madsen, Eugene L

2017-02-15

Investigations of environmental microbial communities are crucial for the discovery of populations capable of degrading hazardous compounds and may lead to improved bioremediation strategies. The goal of this study was to identify microorganisms responsible for aerobic benzene degradation in coal tar-contaminated groundwater. Benzene degradation was monitored in laboratory incubations of well waters using gas chromatography mass spectrometry (GC-MS). Stable isotope probing (SIP) experiments using [ 13 C]benzene enabled us to obtain 13 C-labled community DNA. From this, 16S rRNA clone libraries identified Gammaproteobacteria and Betaproteobacteria as the active benzene-metabolizing microbial populations. Subsequent cultivation experiments yielded nine bacterial isolates that grew in the presence of benzene; five were confirmed in laboratory cultures to grow on benzene. The isolated benzene-degrading organisms were genotypically similar (>97% 16S rRNA gene nucleotide identities) to the organisms identified in SIP experiments. One isolate, Variovorax MAK3, was further investigated for the expression of a putative aromatic ring-hydroxylating dioxygenase (RHD) hypothesized to be involved in benzene degradation. Microcosm experiments using Variovorax MAK3 revealed a 10-fold increase in RHD (Vapar_5383) expression, establishing a link between this gene and benzene degradation. Furthermore, the addition of Variovorax MAK3 to microcosms prepared from site waters accelerated community benzene degradation and correspondingly increased RHD gene expression. In microcosms using uninoculated groundwater, quantitative (q)PCR assays (with 16S rRNA and RDH genes) showed that Variovorax was present and responsive to added benzene. These data demonstrate how the convergence of cultivation-dependent and -independent techniques can boost understandings of active populations and functional genes in complex benzene-degrading microbial communities. Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation. Copyright © 2017 American Society for Microbiology.

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community

PubMed Central

Posman, Kevin M.; DeRito, Christopher M.

2016-01-01

ABSTRACT Investigations of environmental microbial communities are crucial for the discovery of populations capable of degrading hazardous compounds and may lead to improved bioremediation strategies. The goal of this study was to identify microorganisms responsible for aerobic benzene degradation in coal tar-contaminated groundwater. Benzene degradation was monitored in laboratory incubations of well waters using gas chromatography mass spectrometry (GC-MS). Stable isotope probing (SIP) experiments using [13C]benzene enabled us to obtain 13C-labled community DNA. From this, 16S rRNA clone libraries identified Gammaproteobacteria and Betaproteobacteria as the active benzene-metabolizing microbial populations. Subsequent cultivation experiments yielded nine bacterial isolates that grew in the presence of benzene; five were confirmed in laboratory cultures to grow on benzene. The isolated benzene-degrading organisms were genotypically similar (>97% 16S rRNA gene nucleotide identities) to the organisms identified in SIP experiments. One isolate, Variovorax MAK3, was further investigated for the expression of a putative aromatic ring-hydroxylating dioxygenase (RHD) hypothesized to be involved in benzene degradation. Microcosm experiments using Variovorax MAK3 revealed a 10-fold increase in RHD (Vapar_5383) expression, establishing a link between this gene and benzene degradation. Furthermore, the addition of Variovorax MAK3 to microcosms prepared from site waters accelerated community benzene degradation and correspondingly increased RHD gene expression. In microcosms using uninoculated groundwater, quantitative (q)PCR assays (with 16S rRNA and RDH genes) showed that Variovorax was present and responsive to added benzene. These data demonstrate how the convergence of cultivation-dependent and -independent techniques can boost understandings of active populations and functional genes in complex benzene-degrading microbial communities. IMPORTANCE Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation. PMID:27913419

Proliferation of antibiotic resistance genes in microbial consortia of sequencing batch reactors (SBRs) upon exposure to trace erythromycin or erythromycin-H2O.

PubMed

Fan, Caian; He, Jianzhong

2011-05-01

A variety of antibiotics and their metabolites at sub-inhibitory level concentrations are suspected to expand resistance genes in the environment. However, knowledge is limited on the causal correlation of trace antibiotics or their metabolites with resistance proliferation. In this study, erythromycin (ERY) resistance genes were screened on microbial consortia of sequencing batch reactors (SBRs) after one year acclimation to ERY (100 μg/L) or dehydrated erythromycin (ERY-H(2)O, 50 μg/L). The identified esterase gene ereA explains that ERY could be degraded to six products by microbes acclimated to ERY (100 μg/L). However, ERY could not be degraded by microbes acclimated to ERY-H(2)O (50 μg/L), which may be due to the less proliferated ereA gene. Biodegradation of ERY required the presence of exogenous carbon source (e.g., glucose) and nutrients (e.g., nitrogen, phosphorus) for assimilation, but overdosed ammonium-N (>40 mg/L) inhibited degradation of ERY. Zoogloea, a kind of biofilm formation bacteria, became predominant in the ERY degradation consortia, suggesting that the input of ERY could induce biofilm resistance to antibiotics. Our study highlights that lower μg/L level of ERY or ERY-H(2)O in the environment encourages expansion of resistance genes in microbes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Endoplasmic reticulum stress and proteasomal system in amyotrophic lateral sclerosis.

PubMed

Karademir, Betul; Corek, Ceyda; Ozer, Nesrin Kartal

2015-11-01

Protein processing including folding, unfolding and degradation is involved in the mechanisms of many diseases. Unfolded protein response and/or endoplasmic reticulum stress are accepted to be the first steps which should be completed via protein degradation. In this direction, proteasomal system and autophagy play important role as the degradation pathways and controlled via complex mechanisms. Amyotrophic lateral sclerosis is a multifactorial neurodegenerative disease which is also known as the most catastrophic one. Mutation of many different genes are involved in the pathogenesis such as superoxide dismutase 1, chromosome 9 open reading frame 72 and ubiquilin 2. These genes are mainly related to the antioxidant defense systems, endoplasmic reticulum stress related proteins and also protein aggregation, degradation pathways and therefore mutation of these genes cause related disorders.This review focused on the role of protein processing via endoplasmic reticulum and proteasomal system in amyotrophic lateral sclerosis which are the main players in the pathology. In this direction, dysfunction of endoplasmic reticulum associated degradation and related cell death mechanisms that are autophagy/apoptosis have been detailed. Copyright © 2015 Elsevier Inc. All rights reserved.

Biodegradable polymers as non-viral carriers for plasmid DNA delivery.

PubMed

Luten, Jordy; van Nostrum, Cornelus F; De Smedt, Stefaan C; Hennink, Wim E

2008-03-03

Gene therapy holds a great promise for the treatment of acquired and inherited diseases with a genetic origin that are currently incurable. Non-viral gene delivery systems are gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherently associated with the use of viral systems. Many cationic polymers have been studied both in vitro and in vivo for gene delivery purposes. However, in recent years there has been a focus on biodegradable carrier systems. The potential advantage of biodegradable carriers as compared to their non-degradable counterparts is their reduced toxicity and the avoidance of accumulation of the polymer in the cells after repeated administration. Also, the degradation of the polymer can be used as a tool to release the plasmid DNA into the cytosol. In this article the recent results obtained with two classes of degradable gene delivery systems, namely those based on water-soluble cationic polymers and on micro- and nanoparticles will be summarized and discussed.

Formation of mushrooms and lignocellulose degradation encoded in the genome sequence of Schizophyllum commune

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohm, Robin A.; de Jong, Jan F.; Lugones, Luis G.

2010-07-12

The wood degrading fungus Schizophyllum commune is a model system for mushroom development. Here, we describe the 38.5 Mb assembled genome of this basidiomycete and application of whole genome expression analysis to study the 13,210 predicted genes. Comparative analyses of the S. commune genome revealed unique wood degrading machinery and mating type loci with the highest number of reported genes. Gene expression analyses revealed that one third of the 471 identified transcription factor genes were differentially expressed during sexual development. Two of these transcription factor genes were deleted. Inactivation of fst4 resulted in the inability to form mushrooms, whereas inactivationmore » of fst3 resulted in more but smaller mushrooms than wild-type. These data illustrate that mechanisms underlying mushroom formation can be dissected using S. commune as a model. This will impact commercial production of mushrooms and the industrial use of these fruiting bodies to produce enzymes and pharmaceuticals.« less

[Advances in molecular mechanisms of adaptive immunity mediated by type I-E CRISPR/Cas system--A review].

PubMed

Sun, Dongchang; Qiu, Juanping

2016-01-04

To better adapt to the environment, prokaryocyte can take up exogenous genes (from bacteriophages, plasmids or genomes of other species) through horizontal gene transfer. Accompanied by the acquisition of exogenous genes, prokaryocyte is challenged by the invasion of 'selfish genes'. Therefore, to protect against the risk of gene transfer, prokaryocyte needs to establish mechanisms for selectively taking up or degrading exogenous DNA. In recent years, researchers discovered an adaptive immunity, which is mediated by the small RNA guided DNA degradation, prevents the invasion of exogenous genes in prokaryocyte. During the immune process, partial DNA fragments are firstly integrated.to the clustered regularly interspaced short palindromic repeats (CRISPR) located within the genome DNA, and then the mature CRISPR RNA transcript and the CRISPR associated proteins (Cas) form a complex CRISPR/Cas for degrading exogenous DNA. In this review, we will first briefly describe the CRISPR/Cas systems and then mainly focus on the recent advances of the function mechanism and the regulation mechanism of the type I-E CRISPR/Cas system in Escherichia coli.

A comparative genomic analysis of the oxidative enzymes potentially involved in lignin degradation by Agaricus bisporus.

PubMed

Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Fu, Bolei; Cullen, Dan

2013-06-01

The oxidative enzymatic machinery for degradation of organic substrates in Agaricus bisporus (Ab) is at the core of the carbon recycling mechanisms in this fungus. To date, 156 genes have been tentatively identified as part of this oxidative enzymatic machinery, which includes 26 peroxidase encoding genes, nine copper radical oxidase [including three putative glyoxal oxidase-encoding genes (GLXs)], 12 laccases sensu stricto and 109 cytochrome P450 monooxygenases. Comparative analyses of these enzymes in Ab with those of the white-rot fungus, Phanerochaete chrysosporium, the brown-rot fungus, Postia placenta, the coprophilic litter fungus, Coprinopsis cinerea and the ectomychorizal fungus, Laccaria bicolor, revealed enzyme diversity consistent with adaptation to substrates rich in humic substances and partially degraded plant material. For instance, relative to wood decay fungi, Ab cytochrome P450 genes were less numerous (109 gene models), distributed among distinctive families, and lacked extensive duplication and clustering. Viewed together with P450 transcript accumulation patterns in three tested growth conditions, these observations were consistent with the unique Ab lifestyle. Based on tandem gene arrangements, a certain degree of gene duplication seems to have occurred in this fungus in the copper radical oxidase (CRO) and the laccase gene families. In Ab, high transcript levels and regulation of the heme-thiolate peroxidases, two manganese peroxidases and the three GLX-like genes are likely in response to complex natural substrates, including lignocellulose and its derivatives, thereby suggesting an important role in lignin degradation. On the other hand, the expression patterns of the related CROs suggest a developmental role in this fungus. Based on these observations, a brief comparative genomic overview of the Ab oxidative enzyme machinery is presented. Copyright © 2013 Elsevier Inc. All rights reserved.

Response of bacterial pdo1, nah, and C12O genes to aged soil PAH pollution in a coke factory area.

PubMed

Han, Xue-Mei; Liu, Yu-Rong; Zheng, Yuan-Ming; Zhang, Xiao-Xia; He, Ji-Zheng

2014-01-01

Soil pollution caused by polycyclic aromatic hydrocarbons (PAHs) is threatening human health and environmental safety. Investigating the relative prevalence of different PAH-degrading genes in PAH-polluted soils and searching for potential bioindicators reflecting the impact of PAH pollution on microbial communities are useful for microbial monitoring, risk evaluation, and potential bioremediation of soils polluted by PAHs. In this study, three functional genes, pdo1, nah, and C12O, which might be involved in the degradation of PAHs from a coke factory, were investigated by real-time quantitative PCR (qPCR) and clone library approaches. The results showed that the pdo1 and C12O genes were more abundant than the nah gene in the soils. There was a significantly positive relationship between the nah or pdo1 gene abundances and PAH content, while there was no correlation between C12O gene abundance and PAH content. Analyses of clone libraries showed that all the pdo1 sequences were grouped into Mycobacterium, while all the nah sequences were classified into three groups: Pseudomonas, Comamonas, and Polaromonas. These results indicated that the abundances of nah and pdo1 genes were positively influenced by levels of PAHs in soil and could be potential microbial indicators reflecting the impact of soil PAH pollution and that Mycobacteria were one of the most prevalent PAHs degraders in these PAH-polluted soils. Principal component analysis (PCA) and correlation analyses between microbial parameters and environmental factors revealed that total carbon (TC), total nitrogen (TN), and dissolved organic carbon (DOC) had positive effects on the abundances of all PAH-degrading genes. It suggests that increasing TC, TN, and DOC inputs could be a useful way to remediate PAH-polluted soils.

[Microbial degradation of 3-phenoxybenzoic acid--A review].

PubMed

Deng, Weiqin; Liu, Shuliang; Yao, Kai

2015-09-04

3-phenoxybenzoic acid (3-PBA) with estrogen toxicity is one of the intermediate products of most pyrethroid pesticides. 3-PBA is difficult to degrade in the natural environment, and threatens food safety and human health. Microbial degradation of pyrethroids and their intermediate product (3-PBA) has become a hot topic in recent years. Here, we reviewed microbial species, degrading enzymes and degradation genes, degradation pathways of 3-PBA degrading and the application of 3-PBA degradation strains. This article provides references for the study of 3-PBA degradation by microorganisms.

Microbial Metabolic Potential for Carbon Degradation and Nutrient (Nitrogen and Phosphorus) Acquisition in an Ombrotrophic Peatland

PubMed Central

Tfaily, Malak M.; Green, Stefan J.; Steinweg, J. Megan; Chanton, Patrick; Imvittaya, Aopeau; Chanton, Jeffrey P.; Cooper, William; Schadt, Christopher

2014-01-01

This study integrated metagenomic and nuclear magnetic resonance (NMR) spectroscopic approaches to investigate microbial metabolic potential for organic matter decomposition and nitrogen (N) and phosphorus (P) acquisition in soils of an ombrotrophic peatland in the Marcell Experimental Forest (MEF), Minnesota, USA. This analysis revealed vertical stratification in key enzymatic pathways and taxa containing these pathways. Metagenomic analyses revealed that genes encoding laccases and dioxygenases, involved in aromatic compound degradation, declined in relative abundance with depth, while the relative abundance of genes encoding metabolism of amino sugars and all four saccharide groups increased with depth in parallel with a 50% reduction in carbohydrate content. Most Cu-oxidases were closely related to genes from Proteobacteria and Acidobacteria, and type 4 laccase-like Cu-oxidase genes were >8 times more abundant than type 3 genes, suggesting an important and overlooked role for type 4 Cu-oxidase in phenolic compound degradation. Genes associated with sulfate reduction and methanogenesis were the most abundant anaerobic respiration genes in these systems, with low levels of detection observed for genes of denitrification and Fe(III) reduction. Fermentation genes increased in relative abundance with depth and were largely affiliated with Syntrophobacter. Methylocystaceae-like small-subunit (SSU) rRNA genes, pmoA, and mmoX genes were more abundant among methanotrophs. Genes encoding N2 fixation, P uptake, and P regulons were significantly enriched in the surface peat and in comparison to other ecosystems, indicating N and P limitation. Persistence of inorganic orthophosphate throughout the peat profile in this P-limiting environment indicates that P may be bound to recalcitrant organic compounds, thus limiting P bioavailability in the subsurface. Comparative metagenomic analysis revealed a high metabolic potential for P transport and starvation, N2 fixation, and oligosaccharide degradation at MEF relative to other wetland and soil environments, consistent with the nutrient-poor and carbohydrate-rich conditions found in this Sphagnum-dominated boreal peatland. PMID:24682299

Microbial metabolic potential for carbon degradation and nutrient (nitrogen and phosphorus) acquisition in an ombrotrophic peatland.

PubMed

Lin, Xueju; Tfaily, Malak M; Green, Stefan J; Steinweg, J Megan; Chanton, Patrick; Imvittaya, Aopeau; Chanton, Jeffrey P; Cooper, William; Schadt, Christopher; Kostka, Joel E

2014-06-01

This study integrated metagenomic and nuclear magnetic resonance (NMR) spectroscopic approaches to investigate microbial metabolic potential for organic matter decomposition and nitrogen (N) and phosphorus (P) acquisition in soils of an ombrotrophic peatland in the Marcell Experimental Forest (MEF), Minnesota, USA. This analysis revealed vertical stratification in key enzymatic pathways and taxa containing these pathways. Metagenomic analyses revealed that genes encoding laccases and dioxygenases, involved in aromatic compound degradation, declined in relative abundance with depth, while the relative abundance of genes encoding metabolism of amino sugars and all four saccharide groups increased with depth in parallel with a 50% reduction in carbohydrate content. Most Cu-oxidases were closely related to genes from Proteobacteria and Acidobacteria, and type 4 laccase-like Cu-oxidase genes were >8 times more abundant than type 3 genes, suggesting an important and overlooked role for type 4 Cu-oxidase in phenolic compound degradation. Genes associated with sulfate reduction and methanogenesis were the most abundant anaerobic respiration genes in these systems, with low levels of detection observed for genes of denitrification and Fe(III) reduction. Fermentation genes increased in relative abundance with depth and were largely affiliated with Syntrophobacter. Methylocystaceae-like small-subunit (SSU) rRNA genes, pmoA, and mmoX genes were more abundant among methanotrophs. Genes encoding N2 fixation, P uptake, and P regulons were significantly enriched in the surface peat and in comparison to other ecosystems, indicating N and P limitation. Persistence of inorganic orthophosphate throughout the peat profile in this P-limiting environment indicates that P may be bound to recalcitrant organic compounds, thus limiting P bioavailability in the subsurface. Comparative metagenomic analysis revealed a high metabolic potential for P transport and starvation, N2 fixation, and oligosaccharide degradation at MEF relative to other wetland and soil environments, consistent with the nutrient-poor and carbohydrate-rich conditions found in this Sphagnum-dominated boreal peatland.

Enzymatic Degradation of Phenazines Can Generate Energy and Protect Sensitive Organisms from Toxicity

PubMed Central

Costa, Kyle C.; Bergkessel, Megan; Saunders, Scott; Korlach, Jonas

2015-01-01

ABSTRACT Diverse bacteria, including several Pseudomonas species, produce a class of redox-active metabolites called phenazines that impact different cell types in nature and disease. Phenazines can affect microbial communities in both positive and negative ways, where their presence is correlated with decreased species richness and diversity. However, little is known about how the concentration of phenazines is modulated in situ and what this may mean for the fitness of members of the community. Through culturing of phenazine-degrading mycobacteria, genome sequencing, comparative genomics, and molecular analysis, we identified several conserved genes that are important for the degradation of three Pseudomonas-derived phenazines: phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and pyocyanin (PYO). PCA can be used as the sole carbon source for growth by these organisms. Deletion of several genes in Mycobacterium fortuitum abolishes the degradation phenotype, and expression of two genes in a heterologous host confers the ability to degrade PCN and PYO. In cocultures with phenazine producers, phenazine degraders alter the abundance of different phenazine types. Not only does degradation support mycobacterial catabolism, but also it provides protection to bacteria that would otherwise be inhibited by the toxicity of PYO. Collectively, these results serve as a reminder that microbial metabolites can be actively modified and degraded and that these turnover processes must be considered when the fate and impact of such compounds in any environment are being assessed. PMID:26507234

A novel (S)-6-hydroxynicotine oxidase gene from Shinella sp. strain HZN7.

PubMed

Qiu, Jiguo; Wei, Yin; Ma, Yun; Wen, Rongti; Wen, Yuezhong; Liu, Weiping

2014-09-01

Nicotine is an important environmental toxicant in tobacco waste. Shinella sp. strain HZN7 can metabolize nicotine into nontoxic compounds via variations of the pyridine and pyrrolidine pathways. However, the catabolic mechanism of this variant pathway at the gene or enzyme level is still unknown. In this study, two 6-hydroxynicotine degradation-deficient mutants, N7-M9 and N7-W3, were generated by transposon mutagenesis. The corresponding mutant genes, designated nctB and tnp2, were cloned and analyzed. The nctB gene encodes a novel flavin adenine dinucleotide-containing (S)-6-hydroxynicotine oxidase that converts (S)-6-hydroxynicotine into 6-hydroxy-N-methylmyosmine and then spontaneously hydrolyzes into 6-hydroxypseudooxynicotine. The deletion and complementation of the nctB gene showed that this enzyme is essential for nicotine or (S)-6-hydroxynicotine degradation. Purified NctB could also convert (S)-nicotine into N-methylmyosmine, which spontaneously hydrolyzed into pseudooxynicotine. The kinetic constants of NctB toward (S)-6-hydroxynicotine (Km = 0.019 mM, kcat = 7.3 s(-1)) and nicotine (Km = 2.03 mM, kcat = 0.396 s(-1)) indicated that (S)-6-hydroxynicotine is the preferred substrate in vivo. NctB showed no activities toward the R enantiomer of nicotine or 6-hydroxynicotine. Strain HZN7 could degrade (R)-nicotine into (R)-6-hydroxynicotine without any further degradation. The tnp2 gene from mutant N7-W3 encodes a putative transposase, and its deletion did not abolish the nicotine degradation activity. This study advances the understanding of the microbial diversity of nicotine biodegradation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Age-associated microbiome shows the giant panda lives on hemicelluloses, not on cellulose.

PubMed

Zhang, Wenping; Liu, Wenbin; Hou, Rong; Zhang, Liang; Schmitz-Esser, Stephan; Sun, Huaibo; Xie, Junjin; Zhang, Yunfei; Wang, Chengdong; Li, Lifeng; Yue, Bisong; Huang, He; Wang, Hairui; Shen, Fujun; Zhang, Zhihe

2018-05-01

The giant panda feeds almost exclusively on bamboo, a diet highly enriched in lignin and cellulose, but is characterized by a digestive tract similar to carnivores. It is still large unknown if and how the giant panda gut microbiota contributes to lignin and cellulose degradation. Here we show the giant pandas' gut microbiota does not significantly contribute to cellulose and lignin degradation. We found that no operational taxonomic unit had a nearest neighbor identified as a cellulolytic species or strain with a significant higher abundance in juvenile than cubs, a very low abundance of putative lignin and cellulose genes existed in part of analyzing samples but a significant higher abundance of genes involved in starch and hemicellulose degradation in juveniles than cubs. Moreover, a significant lower abundance of putative cellulolytic genes and a significant higher abundance of putative α-amylase and hemicellulase gene families were present in giant pandas than in omnivores or herbivores.

Transcriptome profile of Trichoderma harzianum IOC-3844 induced by sugarcane bagasse.

PubMed

Horta, Maria Augusta Crivelente; Vicentini, Renato; Delabona, Priscila da Silva; Laborda, Prianda; Crucello, Aline; Freitas, Sindélia; Kuroshu, Reginaldo Massanobu; Polikarpov, Igor; Pradella, José Geraldo da Cruz; Souza, Anete Pereira

2014-01-01

Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. In the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. The sequencing generated 14.7 Gbp for downstream analyses. De novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. The present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.

Evolution of Sphingomonad Gene Clusters Related to Pesticide Catabolism Revealed by Genome Sequence and Mobilomics of Sphingobium herbicidovorans MH

PubMed Central

Nielsen, Tue Kjærgaard; Rasmussen, Morten; Demanèche, Sandrine; Cecillon, Sébastien; Vogel, Timothy M.

2017-01-01

Abstract Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology. PMID:28961970

Molecular Biology of Anaerobic Aromatic Biodegradation.

DTIC Science & Technology

1992-08-14

manipulate and clone genes for aromatic acid degradation from the bacterium, Rhodopseudomonas palustris . These tools have enabled us to identify genes...anaerobic degradation of two selected aromatic acids - benzoate and 4-hydroxybenzoate - by one bacterial species - Rhodopseudomonas palustris . Our...PUBLICATIONS. Papers: Gibson, J., J. F. Geissler, and C. S. Harwood. 1990. Benzoate-coenzyme A ligase from Rhodopseudomonas palustris . Methods in Enzymology

Microbial degradation of chloroethenes: a review.

PubMed

Dolinová, Iva; Štrojsová, Martina; Černík, Miroslav; Němeček, Jan; Macháčková, Jiřina; Ševců, Alena

2017-05-01

Contamination by chloroethenes has a severe negative effect on both the environment and human health. This has prompted intensive remediation activity in recent years, along with research into the efficacy of natural microbial communities for degrading toxic chloroethenes into less harmful compounds. Microbial degradation of chloroethenes can take place either through anaerobic organohalide respiration, where chloroethenes serve as electron acceptors; anaerobic and aerobic metabolic degradation, where chloroethenes are used as electron donors; or anaerobic and aerobic co-metabolic degradation, with chloroethene degradation occurring as a by-product during microbial metabolism of other growth substrates, without energy or carbon benefit. Recent research has focused on optimising these natural processes to serve as effective bioremediation technologies, with particular emphasis on (a) the diversity and role of bacterial groups involved in dechlorination microbial processes, and (b) detection of bacterial enzymes and genes connected with dehalogenation activity. In this review, we summarise the different mechanisms of chloroethene bacterial degradation suitable for bioremediation and provide a list of dechlorinating bacteria. We also provide an up-to-date summary of primers available for detecting functional genes in anaerobic and aerobic bacteria degrading chloroethenes metabolically or co-metabolically.

PhdA Catalyzes the First Step of Phenazine-1-Carboxylic Acid Degradation in Mycobacterium fortuitum.

PubMed

Costa, Kyle C; Moskatel, Leon S; Meirelles, Lucas A; Newman, Dianne K

2018-05-15

Phenazines are a class of bacterially produced redox-active metabolites that are found in natural, industrial, and clinical environments. In Pseudomonas spp., phenazine-1-carboxylic acid (PCA)-the precursor of all phenazine metabolites-facilitates nutrient acquisition, biofilm formation, and competition with other organisms. While the removal of phenazines negatively impacts these activities, little is known about the genes or enzymes responsible for phenazine degradation by other organisms. Here, we report that the first step of PCA degradation by Mycobacterium fortuitum is catalyzed by a ph enazine- d egrading decarboxylase (PhdA). PhdA is related to members of the UbiD protein family that rely on a prenylated flavin mononucleotide cofactor for activity. The gene for PhdB, the enzyme responsible for cofactor synthesis, is present in a putative operon with the gene encoding PhdA in a region of the M. fortuitum genome that is essential for PCA degradation. PhdA and PhdB are present in all known PCA-degrading organisms from the Actinobacteria M. fortuitum can also catabolize other Pseudomonas -derived phenazines such as phenazine-1-carboxamide, 1-hydroxyphenazine, and pyocyanin. On the basis of our previous work and the current characterization of PhdA, we propose that degradation converges on a common intermediate: dihydroxyphenazine. An understanding of the genes responsible for degradation will enable targeted studies of phenazine degraders in diverse environments. IMPORTANCE Bacteria from phylogenetically diverse groups secrete redox-active metabolites that provide a fitness advantage for their producers. For example, phenazines from Pseudomonas spp. benefit the producers by facilitating anoxic survival and biofilm formation and additionally inhibit competitors by serving as antimicrobials. Phenazine-producing pseudomonads act as biocontrol agents by leveraging these antibiotic properties to inhibit plant pests. Despite this importance, the fate of phenazines in the environment is poorly understood. Here, we characterize an enzyme from Mycobacterium fortuitum that catalyzes the first step of phenazine-1-carboxylic acid degradation. Knowledge of the genetic basis of phenazine degradation will facilitate the identification of environments where this activity influences the microbial community structure. Copyright © 2018 American Society for Microbiology.

A Novel Aerobic Degradation Pathway for Thiobencarb Is Initiated by the TmoAB Two-Component Flavin Mononucleotide-Dependent Monooxygenase System in Acidovorax sp. Strain T1

PubMed Central

Chu, Cui-Wei; Liu, Bin; Li, Na; Yao, Shi-Gang; Cheng, Dan; Zhao, Jia-Dong; Qiu, Ji-Guo; Yan, Xin; He, Jian

2017-01-01

ABSTRACT Thiobencarb is a thiocarbamate herbicide used in rice paddies worldwide. Microbial degradation plays a crucial role in the dissipation of thiobencarb in the environment. However, the physiological and genetic mechanisms underlying thiobencarb degradation remain unknown. In this study, a novel thiobencarb degradation pathway was proposed in Acidovorax sp. strain T1. Thiobencarb was oxidized and cleaved at the C—S bond, generating diethylcarbamothioic S-acid and 4-chlorobenzaldehyde (4CDA). 4CDA was then oxidized to 4-chlorobenzoic acid (4CBA) and hydrolytically dechlorinated to 4-hydroxybenzoic acid (4HBA). The identification of catabolic genes suggested further hydroxylation to protocatechuic acid (PCA) and finally degradation through the protocatechuate 4,5-dioxygenase pathway. A novel two-component monooxygenase system identified in the strain, TmoAB, was responsible for the initial catabolic reaction. TmoA shared 28 to 32% identity with the oxygenase components of pyrimidine monooxygenase from Agrobacterium fabrum, alkanesulfonate monooxygenase from Pseudomonas savastanoi, and dibenzothiophene monooxygenase from Rhodococcus sp. TmoB shared 25 to 37% identity with reported flavin reductases and oxidized NADH but not NADPH. TmoAB is a flavin mononucleotide (FMN)-dependent monooxygenase and catalyzed the C—S bond cleavage of thiobencarb. Introduction of tmoAB into cells of the thiobencarb degradation-deficient mutant T1m restored its ability to degrade and utilize thiobencarb. A dehydrogenase gene, tmoC, was located 7,129 bp downstream of tmoAB, and its transcription was clearly induced by thiobencarb. The purified TmoC catalyzed the dehydrogenation of 4CDA to 4CBA using NAD+ as a cofactor. A gene cluster responsible for the complete 4CBA metabolic pathway was also cloned, and its involvement in thiobencarb degradation was preliminarily verified by transcriptional analysis. IMPORTANCE Microbial degradation is the main factor in thiobencarb dissipation in soil. In previous studies, thiobencarb was degraded initially via N-deethylation, sulfoxidation, hydroxylation, and dechlorination. However, enzymes and genes involved in the microbial degradation of thiobencarb have not been studied. This study revealed a new thiobencarb degradation pathway in Acidovorax sp. strain T1 and identified a novel two-component FMN-dependent monooxygenase system, TmoAB. Under TmoAB-mediated catalysis, thiobencarb was cleaved at the C—S bond, producing diethylcarbamothioic S-acid and 4CDA. Furthermore, the downstream degradation pathway of thiobencarb was proposed. Our study provides the physiological, biochemical, and genetic foundation of thiobencarb degradation in this microorganism. PMID:28939603

A Novel Aerobic Degradation Pathway for Thiobencarb Is Initiated by the TmoAB Two-Component Flavin Mononucleotide-Dependent Monooxygenase System in Acidovorax sp. Strain T1.

PubMed

Chu, Cui-Wei; Liu, Bin; Li, Na; Yao, Shi-Gang; Cheng, Dan; Zhao, Jia-Dong; Qiu, Ji-Guo; Yan, Xin; He, Qin; He, Jian

2017-12-01

Thiobencarb is a thiocarbamate herbicide used in rice paddies worldwide. Microbial degradation plays a crucial role in the dissipation of thiobencarb in the environment. However, the physiological and genetic mechanisms underlying thiobencarb degradation remain unknown. In this study, a novel thiobencarb degradation pathway was proposed in Acidovorax sp. strain T1. Thiobencarb was oxidized and cleaved at the C-S bond, generating diethylcarbamothioic S -acid and 4-chlorobenzaldehyde (4CDA). 4CDA was then oxidized to 4-chlorobenzoic acid (4CBA) and hydrolytically dechlorinated to 4-hydroxybenzoic acid (4HBA). The identification of catabolic genes suggested further hydroxylation to protocatechuic acid (PCA) and finally degradation through the protocatechuate 4,5-dioxygenase pathway. A novel two-component monooxygenase system identified in the strain, TmoAB, was responsible for the initial catabolic reaction. TmoA shared 28 to 32% identity with the oxygenase components of pyrimidine monooxygenase from Agrobacterium fabrum , alkanesulfonate monooxygenase from Pseudomonas savastanoi , and dibenzothiophene monooxygenase from Rhodococcus sp. TmoB shared 25 to 37% identity with reported flavin reductases and oxidized NADH but not NADPH. TmoAB is a flavin mononucleotide (FMN)-dependent monooxygenase and catalyzed the C-S bond cleavage of thiobencarb. Introduction of tmoAB into cells of the thiobencarb degradation-deficient mutant T1m restored its ability to degrade and utilize thiobencarb. A dehydrogenase gene, tmoC , was located 7,129 bp downstream of tmoAB , and its transcription was clearly induced by thiobencarb. The purified TmoC catalyzed the dehydrogenation of 4CDA to 4CBA using NAD + as a cofactor. A gene cluster responsible for the complete 4CBA metabolic pathway was also cloned, and its involvement in thiobencarb degradation was preliminarily verified by transcriptional analysis. IMPORTANCE Microbial degradation is the main factor in thiobencarb dissipation in soil. In previous studies, thiobencarb was degraded initially via N -deethylation, sulfoxidation, hydroxylation, and dechlorination. However, enzymes and genes involved in the microbial degradation of thiobencarb have not been studied. This study revealed a new thiobencarb degradation pathway in Acidovorax sp. strain T1 and identified a novel two-component FMN-dependent monooxygenase system, TmoAB. Under TmoAB-mediated catalysis, thiobencarb was cleaved at the C-S bond, producing diethylcarbamothioic S -acid and 4CDA. Furthermore, the downstream degradation pathway of thiobencarb was proposed. Our study provides the physiological, biochemical, and genetic foundation of thiobencarb degradation in this microorganism. Copyright © 2017 American Society for Microbiology.

Homology between genes for aromatic hydrocarbon degradation in surface and deep-subsurface sphingomonas strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, E.; Aversano, P.J.; Zylstra, G.J.

The cloned genes for aromatic hydrocarbon degradation from Sphingomonas yanoikuyae B1 were utilized in Southern hybridization experiments with Sphingomonas strains from the surface and deep-subsurface environments. One hybridization pattern was obtained with BamHI-digested genomic DNAs for two surface strains, while a differing pattern was seen for five deep-subsurface strains. The cross-hybridizing genes were located in the chromosomes of the surface strains and on plasmids in the deep-subsurface strains. 31 refs., 3 figs., 1 tab.

Isolation of Inositol Hexaphosphate (IHP)-Degrading Bacteria from Arbuscular Mycorrhizal Fungal Hyphal Compartments Using a Modified Baiting Method Involving Alginate Beads Containing IHP

PubMed Central

Hara, Shintaro; Saito, Masanori

2016-01-01

Phytate (inositol hexaphosphate; IHP)-degrading microbes have been suggested to contribute to arbuscular mycorrhizal fungi (AMF)-mediated P transfer from IHP to plants; however, no IHP degrader involved in AMF-mediated P transfer has been isolated to date. We herein report the isolation of IHP-degrading bacteria using a modified baiting method. We applied alginate beads as carriers of IHP powder, and used them as recoverable IHP in the AM fungal compartment of plant cultivation experiments. P transfer from IHP in alginate beads via AMF was confirmed, and extracted DNA from alginate beads was analyzed by denaturing gradient gel electrophoresis targeting the 16S rRNA gene and a clone library method for the beta-propeller phytase (BPP) gene. The diversities of the 16S rRNA and BPP genes of microbes growing on IHP beads were simple and those of Sphingomonas spp. and Caulobacter spp. dominated. A total of 187 IHP-utilizing bacteria were isolated and identified, and they were consistent with the results of DNA analysis. Furthermore, some isolated Sphingomonas spp. and Caulobacter sp. showed IHP-degrading activity. Therefore, we successfully isolated dominant IHP-degrading bacteria from IHP in an AMF hyphal compartment. These strains may contribute to P transfer from IHP via AMF. PMID:27383681

Transcriptomic analysis of the highly efficient oil-degrading bacterium Acinetobacter venetianus RAG-1 reveals genes important in dodecane uptake and utilization [Transcriptomic analysis of the marine oil-degrading bacterium Acinetobacter venetianus RAG-1 reveals genes important in dodecane uptake and utilization

DOE PAGES

Kothari, Ankita; Charrier, Marimikel; Wu, Yu -Wei; ...

2016-09-22

The hydrocarbonoclastic bacterium Acinetobacter venetianus RAG-1 has attracted substantial attention due to its powerful oil-degrading capabilities and its potential to play an important ecological role in the cleanup of alkanes. In this study, we compare the transcriptome of the strain RAG-1 grown in dodecane, the corresponding alkanol (dodecanol), and sodium acetate for the characterization of genes involved in dodecane uptake and utilization. Comparison of the transcriptional responses of RAG-1 grown on dodecane led to the identification of 1074 genes that were differentially expressed relative to sodium acetate. Of these, 622 genes were upregulated when grown in dodecane. The highly upregulatedmore » genes were involved in alkane catabolism, along with stress response. Our data suggest AlkMb to be primarily involved in dodecane oxidation. Transcriptional response of RAG-1 grown on dodecane relative to dodecanol also led to the identification of permease, outer membrane protein and thin fimbriae coding genes potentially involved in dodecane uptake. As a result, this study provides the first model for key genes involved in alkane uptake and metabolism in A. venetianus RAG-1.« less

In Planta Stage-Specific Fungal Gene Profiling Elucidates the Molecular Strategies of Fusarium graminearum Growing inside Wheat Coleoptiles[W][OA

PubMed Central

Zhang, Xiao-Wei; Jia, Lei-Jie; Zhang, Yan; Jiang, Gang; Li, Xuan; Zhang, Dong; Tang, Wei-Hua

2012-01-01

The ascomycete Fusarium graminearum is a destructive fungal pathogen of wheat (Triticum aestivum). To better understand how this pathogen proliferates within the host plant, we tracked pathogen growth inside wheat coleoptiles and then examined pathogen gene expression inside wheat coleoptiles at 16, 40, and 64 h after inoculation (HAI) using laser capture microdissection and microarray analysis. We identified 344 genes that were preferentially expressed during invasive growth in planta. Gene expression profiles for 134 putative plant cell wall–degrading enzyme genes suggest that there was limited cell wall degradation at 16 HAI and extensive degradation at 64 HAI. Expression profiles for genes encoding reactive oxygen species (ROS)–related enzymes suggest that F. graminearum primarily scavenges extracellular ROS before a later burst of extracellular ROS is produced by F. graminearum enzymes. Expression patterns of genes involved in primary metabolic pathways suggest that F. graminearum relies on the glyoxylate cycle at an early stage of plant infection. A secondary metabolite biosynthesis gene cluster was specifically induced at 64 HAI and was required for virulence. Our results indicate that F. graminearum initiates infection of coleoptiles using covert penetration strategies and switches to overt cellular destruction of tissues at an advanced stage of infection. PMID:23266949

Transcriptomic analysis of the highly efficient oil-degrading bacterium Acinetobacter venetianus RAG-1 reveals genes important in dodecane uptake and utilization [Transcriptomic analysis of the marine oil-degrading bacterium Acinetobacter venetianus RAG-1 reveals genes important in dodecane uptake and utilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kothari, Ankita; Charrier, Marimikel; Wu, Yu -Wei

The hydrocarbonoclastic bacterium Acinetobacter venetianus RAG-1 has attracted substantial attention due to its powerful oil-degrading capabilities and its potential to play an important ecological role in the cleanup of alkanes. In this study, we compare the transcriptome of the strain RAG-1 grown in dodecane, the corresponding alkanol (dodecanol), and sodium acetate for the characterization of genes involved in dodecane uptake and utilization. Comparison of the transcriptional responses of RAG-1 grown on dodecane led to the identification of 1074 genes that were differentially expressed relative to sodium acetate. Of these, 622 genes were upregulated when grown in dodecane. The highly upregulatedmore » genes were involved in alkane catabolism, along with stress response. Our data suggest AlkMb to be primarily involved in dodecane oxidation. Transcriptional response of RAG-1 grown on dodecane relative to dodecanol also led to the identification of permease, outer membrane protein and thin fimbriae coding genes potentially involved in dodecane uptake. As a result, this study provides the first model for key genes involved in alkane uptake and metabolism in A. venetianus RAG-1.« less

Benzene degradation in a denitrifying biofilm reactor: activity and microbial community composition.

PubMed

van der Waals, Marcelle J; Atashgahi, Siavash; da Rocha, Ulisses Nunes; van der Zaan, Bas M; Smidt, Hauke; Gerritse, Jan

2017-06-01

Benzene is an aromatic compound and harmful for the environment. Biodegradation of benzene can reduce the toxicological risk after accidental or controlled release of this chemical in the environment. In this study, we further characterized an anaerobic continuous biofilm culture grown for more than 14 years on benzene with nitrate as electron acceptor. We determined steady state degradation rates, microbial community composition dynamics in the biofilm, and the initial anaerobic benzene degradation reactions. Benzene was degraded at a rate of 0.15 μmol/mg protein/day and a first-order rate constant of 3.04/day which was fourfold higher than rates reported previously. Bacteria belonging to the Peptococcaceae were found to play an important role in this anaerobic benzene-degrading biofilm culture, but also members of the Anaerolineaceae were predicted to be involved in benzene degradation or benzene metabolite degradation based on Illumina MiSeq analysis of 16S ribosomal RNA genes. Biomass retention in the reactor using a filtration finger resulted in reduction of benzene degradation capacity. Detection of the benzene carboxylase encoding gene, abcA, and benzoic acid in the culture vessel indicated that benzene degradation proceeds through an initial carboxylation step.

Regulatory Nexus of Synthesis and Degradation Deciphers Cellular Nrf2 Expression Levels

PubMed Central

Suzuki, Takafumi; Shibata, Tatsuhiro; Takaya, Kai; Shiraishi, Kouya; Kohno, Takashi; Kunitoh, Hideo; Tsuta, Koji; Furuta, Koh; Goto, Koichi; Hosoda, Fumie; Sakamoto, Hiromi; Motohashi, Hozumi

2013-01-01

Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine. PMID:23572560

Growth of Trametes versicolor on phenol.

PubMed

Yemendzhiev, H; Gerginova, M; Krastanov, A; Stoilova, I; Alexieva, Z

2008-11-01

Trametes versicolor 1 was shown to grow on phenol as its sole carbon and energy source. The culture growth and degradation ability dependence on culture medium pH value was observed. The optimal pH value of a liquid Czapek salt medium was 6.5. The investigated strain utilized completely 0.5 g/l phenol in 6 days. The dynamics of the phenol degradation process was investigated. The process was characterized by specific growth rate micromax 0.33 h(-1), metabolic coefficient k=4.4, yield coefficient Yx/s=0.23 and rate of degradation Q=0.506 h(-1). The intracellular activities of phenol hydroxylase (0.333 U/mg protein) and cis,cis-muconate lactonizing enzyme (0.41 U/mg protein) were demonstrated for the first time in this fungus. In an attempt to estimate the occurrence of gene sequences in T. versicolor 1 related to phenol degradation pathway a dot blot analysis with total DNA isolated from this strain was performed. Two synthetic oligonucleotides were used as hybridizing probes. One of the probes was homologous to the 5'end of phyA gene coding for phenol hydroxylase in Trichosporon cutaneum ATCC 46490. The other probe was created on the basis of cis,cis-muconate lactonizing enzyme coding gene in T. cutaneum ATCC 58094. The results of these investigations showed that T. versicolor 1 may carry genes similar to those of Trichosporon cutaneum capable to degrade phenol.

Change of microbial community structure and functional gene abundance in nonylphenol-degrading sediment.

PubMed

Wang, Zhao; Yang, Yuyin; He, Tao; Xie, Shuguang

2015-04-01

Biodegradation by autochthonous microbial community is an important way to clean up nonylphenol (NP) from contaminated river sediment. Knowledge of sediment microbial community can aid in our understanding of biological processes related to NP degradation. However, the change in sediment microbial community associated with NP biodegradation remains unclear. The present study investigated the shift in bacterial community structure and NP-degrading gene abundance in response to NP attenuation in river sediment. Sediment microcosms with different levels of 4-NP (0, 100, or 300 μg/g) were constructed. A nearly complete attenuation of NP occurred in the microcosm with 100 μg/g NP after 9 days' incubation, while a residual NP rate of 8.1 % was observed in the microcosm with 300 μg/g NP after 22 days' incubation. Illumina MiSeq sequencing analysis indicated that Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes predominated in NP-degrading river sediment. Sediment bacterial community structure varied significantly during NP biodegradation and subsequent incubation, which was affected by the level of added NP. The n-alkane biodegradation (alkB) gene abundance showed a significant variation in each NP-amended microcosm (100 or 300 μg/g), while a significant increase in the single component monooxygenase (sMO) gene abundance only occurred in the microcosm spiked with 300 μg/g NP. This study can provide some new insights toward NP-degrading microbial ecology in the environment.

Genome Sequence of Microbulbifer mangrovi DD-13T Reveals Its Versatility to Degrade Multiple Polysaccharides.

PubMed

Imran, Md; Pant, Poonam; Shanbhag, Yogini P; Sawant, Samir V; Ghadi, Sanjeev C

2017-02-01

Microbulbifer mangrovi strain DD-13 T is a novel-type species isolated from the mangroves of Goa, India. The draft genome sequence of strain DD-13 comprised 4,528,106 bp with G+C content of 57.15%. Out of 3479 open reading frames, functions for 3488 protein coding sequences were predicted on the basis of similarity with the cluster of orthologous groups. In addition to protein coding sequences, 34 tRNA genes and 3 rRNA genes were detected. Analysis of nucleotide sequence of predicted gene using a Carbohydrate-Active Enzymes (CAZymes) Analysis Toolkit indicates that strain DD-13 encodes a large set of CAZymes including 255 glycoside hydrolases, 76 carbohydrate esterases, 17 polysaccharide lyases, and 113 carbohydrate-binding modules (CBMs). Many genes from strain DD-13 were annotated as carbohydrases specific for degradation of agar, alginate, carrageenan, chitin, xylan, pullulan, cellulose, starch, β-glucan, pectin, etc. Some of polysaccharide-degrading genes were highly modular and were appended at least with one CBM indicating the versatility of strain DD-13 to degrade complex polysaccharides. The cell growth of strain DD-13 was validated using pure polysaccharides such as agarose or alginate as carbon source as well as by using red and brown seaweed powder as substrate. The homologous carbohydrase produced by strain DD-13 during growth degraded the polysaccharide, ensuring the production of metabolizable reducing sugars. Additionally, several other polysaccharides such as carrageenan, xylan, pullulan, pectin, starch, and carboxymethyl cellulose were also corroborated as growth substrate for strain DD-13 and were associated with concomitant production of homologous carbohydrase.

Rice (Oryza sativa) Laccases Involved in Modification and Detoxification of Herbicides Atrazine and Isoproturon Residues in Plants.

PubMed

Huang, Meng Tian; Lu, Yi Chen; Zhang, Shuang; Luo, Fang; Yang, Hong

2016-08-24

Atrazine (ATR) and isoproturon (IPU) as herbicides have become serious environmental contaminants due to their overuse in crop production. Although ATR and IPU in soils are easily absorbed by many crops, the mechanisms for their degradation or detoxification in plants are poorly understood. This study identified a group of novel genes encoding laccases (EC 1.10.3.2) that are possibly involved in catabolism or detoxification of ATR and IPU residues in rice. Transcriptome profiling shows at least 22 differentially expressed laccase genes in ATR/IPU-exposed rice. Some of the laccase genes were validated by RT-PCR analysis. The biochemical properties of the laccases were analyzed, and their activities in rice were induced under ATR/IPU exposure. To investigate the roles of laccases in degrading or detoxifying ATR/IPU in rice, transgenic yeast cells (Pichia pastoris X-33) expressing two rice laccase genes (LOC_Os01g63180 and LOC_Os12g15680) were generated. Both transformants were found to accumulate less ATR/IPU compared to the control. The ATR/IPU-degraded products in the transformed yeast cells using UPLC-TOF-MS/MS were further characterized. Two metabolites, hydroxy-dehydrogenated atrazine (HDHA) and 2-OH-isopropyl-IPU, catalyzed by laccases were detected in the eukaryotic cells. These results indicate that the laccase-coding genes identified here could confer degradation or detoxification of the herbicides and suggest that the laccases could be one of the important enzymatic pathways responsible for ATR/IPU degradation/detoxification in rice.

Expression of organophosphorus-degradation gene ( opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria

NASA Astrophysics Data System (ADS)

Li, Qiong; Tang, Qing; Xu, Xudong; Gao, Hong

2010-11-01

Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene ( opd) into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, P psbA . From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25°C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.

Characterization of a Gene Cluster Involved in 4-Chlorocatechol Degradation by Pseudomonas reinekei MT1▿

PubMed Central

Cámara, Beatriz; Nikodem, Patricia; Bielecki, Piotr; Bobadilla, Roberto; Junca, Howard; Pieper, Dietmar H.

2009-01-01

Pseudomonas reinekei MT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12OccaA, a novel (chloro)muconate cycloisomerase, MCIccaB, which showed features not yet reported, was induced. This cycloisomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates and transforms 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, suggesting that it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The corresponding genes, ccaA (C12OccaA) and ccaB (MCIccaB), were located in a 5.1-kb genomic region clustered with genes encoding trans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative regulatory gene, ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that C12OccaA and MCIccaB are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCIccaB and the previously identified C12OsalD, rather than C12OccaA, are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding enzymes for channeling salicylates into the ortho-cleavage pathway, form an effective pathway for 4- and 5-chlorosalicylate mineralization. PMID:19465655

Expression pattern of four storage xyloglucan mobilization-related genes during seedling development of the rain forest tree Hymenaea courbaril L.

PubMed

Brandão, A D; Del Bem, L E V; Vincentz, M; Buckeridge, M S

2009-01-01

During seedling establishment, cotyledons of the rain forest tree Hymenaea courbaril mobilize storage cell wall xyloglucan to sustain growth. The polysaccharide is degraded and its products are transported to growing sink tissues. Auxin from the shoot controls the level of xyloglucan hydrolytic enzymes. It is not yet known how important the expression of these genes is for the control of storage xyloglucan degradation. In this work, partial cDNAs of the genes xyloglucan transglycosylase hydrolase (HcXTH1) and beta-galactosidase (HcBGAL1), both related to xyloglucan degradation, and two other genes related to sucrose metabolism [alkaline invertase (HcAlkIN1) and sucrose synthase (HcSUS1)], were isolated. The partial sequences were characterized by comparison with sequences available in the literature, and phylogenetic trees were assembled. Gene expression was evaluated at intervals of 6 h during 24 h in cotyledons, hypocotyl, roots, and leaves, using 45-d-old plantlets. HcXTH1 and HcBGAL1 were correlated to xyloglucan degradation and responded to auxin and light, being down-regulated when transport of auxin was prevented by N-1-naphthylphthalamic acid (NPA) and stimulated by constant light. Genes related to sucrose metabolism, HcAlkIN1 and HcSUS1, responded to inhibition of auxin transport in consonance with storage mobilization in the cotyledons. A model is proposed suggesting that auxin and light are involved in the control of the expression of genes related to storage xyloglucan mobilization in seedlings of H. courbaril. It is concluded that gene expression plays a role in the control of the intercommunication system of the source-sink relationship during seeding growth, favouring its establishment in the shaded environment of the rain forest understorey.

Expression pattern of four storage xyloglucan mobilization-related genes during seedling development of the rain forest tree Hymenaea courbaril L.

PubMed Central

Brandão, A. D.; Del Bem, L. E. V.; Vincentz, M.; Buckeridge, M. S.

2009-01-01

During seedling establishment, cotyledons of the rain forest tree Hymenaea courbaril mobilize storage cell wall xyloglucan to sustain growth. The polysaccharide is degraded and its products are transported to growing sink tissues. Auxin from the shoot controls the level of xyloglucan hydrolytic enzymes. It is not yet known how important the expression of these genes is for the control of storage xyloglucan degradation. In this work, partial cDNAs of the genes xyloglucan transglycosylase hydrolase (HcXTH1) and β-galactosidase (HcBGAL1), both related to xyloglucan degradation, and two other genes related to sucrose metabolism [alkaline invertase (HcAlkIN1) and sucrose synthase (HcSUS1)], were isolated. The partial sequences were characterized by comparison with sequences available in the literature, and phylogenetic trees were assembled. Gene expression was evaluated at intervals of 6 h during 24 h in cotyledons, hypocotyl, roots, and leaves, using 45-d-old plantlets. HcXTH1 and HcBGAL1 were correlated to xyloglucan degradation and responded to auxin and light, being down-regulated when transport of auxin was prevented by N-1-naphthylphthalamic acid (NPA) and stimulated by constant light. Genes related to sucrose metabolism, HcAlkIN1 and HcSUS1, responded to inhibition of auxin transport in consonance with storage mobilization in the cotyledons. A model is proposed suggesting that auxin and light are involved in the control of the expression of genes related to storage xyloglucan mobilization in seedlings of H. courbaril. It is concluded that gene expression plays a role in the control of the intercommunication system of the source–sink relationship during seeding growth, favouring its establishment in the shaded environment of the rain forest understorey. PMID:19221141

A comparative genomic analysis of the oxidative enzymes potentially involved in lignin degradation by Agaricus bisporus

Treesearch

Harshavardhan Doddapaneni; Venkataramanan Subramanian; Bolei Fu; Dan Cullen

2013-01-01

The oxidative enzymatic machinery for degradation of organic substrates in Agaricus bisporus (Ab) is at the core of the carbon recycling mechanisms in this fungus. To date, 156 genes have been tentatively identified as part of this oxidative enzymatic machinery, which includes 26 peroxidase encoding genes, nine copper radical oxidase [including three...

R-carrying genotypes of serum paraoxonase (PON1) 192 polymorphism and higher activity ratio are related to susceptibility against ischemic stroke.

PubMed

Mahrooz, Abdolkarim; Gohari, Ghorban; Hashemi, Mohammad-Bagher; Zargari, Mehryar; Musavi, Hadis; Abedini, Mahmoud; Alizadeh, Ahad

2012-12-01

The polymorphic gene of serum paraoxonase (PON1) and its activity involved in atherosclerosis. The purpose of the study was to analyze PON1 192 Q/R polymorphism and the enzyme activities in ischemic stroke. The polymorphism as the most common polymorphism in PON1 gene coding sequence is associated with variation in the enzyme activity and vascular disease. The study included 85 stroke patients and 71 control subjects. PON1 192 polymorphism was genotyped using PCR protocol. Paraoxonase activity (Para) and arylesterase activity (Aryl) were determined spectrophotometrically using paraoxon and phenylacetate as the substrates. The QR and RR genotypes were more frequent in stroke population compared to controls, resulting in a higher frequency of the R allele in patients (0.24 vs 0.18, OR = 1.41). Patients had significantly higher Para/Aryl ratio than that of controls (P = 0.016). In stroke patients, Para/Aryl and Para/HDL ratios increased with this order: QQ < QR < RR. Hypertension significantly increased the risk of ischemic stroke by 15-fold among R-containing people, while this was significantly increased 4-fold for QQ homozygotes. Smoking increased the risk of having ischemic stroke in both QQ homozygote and QR + RR group (OR = 2.84 and OR = 2.33, respectively). In conclusion, these data highlight the importance of PON1 192 R allele and high Para/Aryl ratio in susceptibility to ischemic stroke in the population. The presence of the 192 R allele potentiates the risk of stroke especially in hypertensive people. Decreased Aryl and increased Para/Aryl, Para/HDL and Aryl/HDL ratios may be markers indicated the increased susceptibility to ischemic stroke in the population.

Gene cloning and in vivo characterization of a dibenzothiophene dioxygenase from Xanthobacter polyaromaticivorans.

PubMed

Hirano, Shin-Ichi; Haruki, Mitsuru; Takano, Kazufumi; Imanaka, Tadayuki; Morikawa, Masaaki; Kanaya, Shigenori

2006-02-01

Xanthobacter polyaromaticivorans sp. nov. 127W is a bacterial strain that is capable of degrading a wide range of cyclic aromatic compounds such as dibenzothiophene, biphenyl, naphthalene, anthracene, and phenanthrene even under extremely low oxygen [dissolved oxygen (DO)< or = 0.2 ppm] conditions (Hirano et al., Biosci Biotechnol Biochem 68:557-564, 2004). A major protein fraction carrying dibenzothiophene degradation activity was purified. Based on its partial amino acid sequences, dbdCa gene encoding alpha subunit terminal oxygenase (DbdCa) and its flanking region were cloned and sequenced. A phylogenetic analysis based on the amino acid sequence demonstrates that DbdCa is a member of a terminal oxygenase component of group IV ring-hydroxylating dioxygenases for biphenyls and monocyclic aromatic hydrocarbons, rather than group III dioxygenases for polycyclic aromatic hydrocarbons. Gene disruption in dbdCa abolished almost of the degradation activity against biphenyl, dibenzothiophene, and anthracene. The gene disruption also impaired degradation activity of the strain under extremely low oxygen conditions (DO< or = 0.2 ppm). These results indicate that Dbd from 127W represents a group IV dioxygenase that is functional even under extremely low oxygen conditions.

Fungal community and cellulose-degrading genes in the composting process of Chinese medicinal herbal residues.

PubMed

Tian, Xueping; Yang, Tao; He, Jingzhong; Chu, Qian; Jia, Xiaojun; Huang, Jun

2017-10-01

The fungal community and the population of 16S rRNA, 18S rRNA and cellulose-degrading genes during the 30-day composting process of Chinese medicinal herbal residues were investigated using Illumina MiSeq and quantitative real-time PCR. An obvious succession of fungal communities occurred during the composting process. Unidentified fungi predominated in the raw materials. As composting progressed, Ascomycota became the most dominant phylum, with Aspergillus being the most dominant genus, and Aspergillus fumigatus making up 99.65% of that genus. Because of the inoculation of cellulolytic fungi in the mature stage, the cellulose degradation rate in inoculation groups was faster and the relative abundances of Aspergillus and the glycoside hydrolase family 7 genes were significantly higher than those in the control groups. These indicated that the fungal inoculants facilitated the degradation of cellulose, increased cellulolytic fungi and optimized the community structure. Copyright © 2017. Published by Elsevier Ltd.

Rhizoremediation of phenanthrene and pyrene contaminated soil using wheat.

PubMed

Shahsavari, Esmaeil; Adetutu, Eric M; Taha, Mohamed; Ball, Andrew S

2015-05-15

Rhizoremediation, the use of the plant rhizosphere and associated microorganisms represents a promising method for the clean up of soils contaminated with polycyclic aromatic hydrocarbons (PAHs) including phenanthrene and pyrene, two model PAHs. Although numerous studies have been published reporting the degradation of phenanthrene and pyrene, very few evaluate the microbial basis of the rhizoremediation process through the application of molecular tools. The aim of this study was to investigate the effect of wheat on the degradation of two model PAHs (alone or in combination) and also on soil bacterial, fungal and nidA gene (i.e. a key gene in the degradation of pyrene) communities. The addition of wheat plants led to a significant enhancement in the degradation of both phenanthrene and pyrene. In pyrene-contaminated soils, the degradation rate increased from 15% (65 mg/kg) and 18% (90 mg/kg) in unplanted soils to 65% (280 mg/kg) and 70% (350 mg/kg) in planted treatments while phenanthrene reduction was enhanced from 97% (394 mg/kg) and 87% (392 mg/kg) for unplanted soils to 100% (406 mg/kg) and 98% (441 mg/kg) in the presence of wheat. PCR-DGGE results showed that the plant root let to some changes in the bacterial and fungal communities; these variations did not reflect any change in hydrocarbon-degrading communities. However, plate counting, traditional MPN and MPN-qPCR of nidA gene revealed that the wheat rhizosphere led to an increase in the total microbial abundance including PAH degrading organisms and these increased activities resulted in enhanced degradation of phenanthrene and pyrene. This clearer insight into the mechanisms underpinning PAH degradation will enable better application of this environmentally friendly technique. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cloning and expression of vgb gene in Bacillus cereus, improve phenol and p-nitrophenol biodegradation

NASA Astrophysics Data System (ADS)

Vélez-Lee, Angel Eduardo; Cordova-Lozano, Felipe; Bandala, Erick R.; Sanchez-Salas, Jose Luis

2016-02-01

In this work, the vgb gene from Vitrocilla stercoraria was used to genetically modify a Bacillus cereus strain isolated from pulp and paper wastewater effluent. The gene was cloned in a multicopy plasmid (pUB110) or uni-copy gene using a chromosome integrative vector (pTrpBG1). B. cereus and its recombinant strains were used for phenol and p-nitrophenol biodegradation using aerobic or micro-aerobic conditions and two different temperatures (i.e. 37 and 25 °C). Complete (100%) phenol degradation was obtained for the strain where the multicopy of vgb gene was present, 98% for the strain where uni-copy gene was present and 45% for wild type strain for the same experimental conditions (i.e. 37 °C and aerobic condition). For p-nitrophenol degradation at the same conditions, the strain with the multi-copy vgb gene was capable to achieve 50% of biodegradation, ˜100% biodegradation was obtained using the uni-copy strain and ˜24% for wild type strain. When the micro-aerobic condition was tested, the biodegradation yield showed a significant decreased. The biodegradation trend observed for aerobic was similar for micro-aerobic assessments: the modified strains showed higher degradation rates when compared with wild type strain. For all experimental conditions, the highest p-nitrophenol degradation was observed using the strain with uni-copy of vgb gene. Besides the increase of biodegradative capability of the strain, insertion of the vgb gene was observed able to modify other morphological characteristics such as avoiding the typical flake formation in the B. cereus culture. In both cases, the modification seems to be related with the enhancement of oxygen supply to the cells generated by the vgb gene insertion. The application of the genetically modified microorganism (GMM) to the biodegradation of pollutants in contaminated water possesses high potential as an environmentally friendly technology to facing this emergent problem.

Long-Term Warming Alters Carbohydrate Degradation Potential in Temperate Forest Soils

DOE PAGES

Pold, Grace; Billings, Andrew F.; Blanchard, Jeff L.; ...

2016-09-02

As Earth's climate warms, soil carbon pools and the microbial communities that process them may change, altering the way in which carbon is recycled in soil. In this study, we used a combination of metagenomics and bacterial cultivation to evaluate the hypothesis that experimentally raising soil temperatures by 5°C for 5, 8, or 20 years increased the potential for temperate forest soil microbial communities to degrade carbohydrates. Warming decreased the proportion of carbohydrate-degrading genes in the organic horizon derived from eukaryotes and increased the fraction of genes in the mineral soil associated with Actinobacteria in all studies. Genes associated withmore » carbohydrate degradation increased in the organic horizon after 5 years of warming but had decreased in the organic horizon after warming the soil continuously for 20 years. However, a greater proportion of the 295 bacteria from 6 phyla (10 classes, 14 orders, and 34 families) isolated from heated plots in the 20-year experiment were able to depolymerize cellulose and xylan than bacterial isolates from control soils. Together, these findings indicate that the enrichment of bacteria capable of degrading carbohydrates could be important for accelerated carbon cycling in a warmer world.« less

Oligogalacturonide-mediated induction of a gene involved in jasmonic acid synthesis in response to the cell-wall-degrading enzymes of the plant pathogen Erwinia carotovora.

PubMed

Norman, C; Vidal, S; Palva, E T

1999-07-01

Identification of Arabidopsis thaliana genes responsive to plant cell-wall-degrading enzymes of Erwinia carotovora subsp. carotovora led to the isolation of a cDNA clone with high sequence homology to the gene for allene oxide synthase, an enzyme involved in the biosynthesis of jasmonates. Expression of the corresponding gene was induced by the extracellular enzymes from this pathogen as well as by treatment with methyl jasmonate and short oligogalacturonides (OGAs). This suggests that OGAs are involved in the induction of the jasmonate pathway during plant defense response to E. carotovora subsp. carotovora attack.

Evolution of Sphingomonad Gene Clusters Related to Pesticide Catabolism Revealed by Genome Sequence and Mobilomics of Sphingobium herbicidovorans MH.

PubMed

Nielsen, Tue Kjærgaard; Rasmussen, Morten; Demanèche, Sandrine; Cecillon, Sébastien; Vogel, Timothy M; Hansen, Lars Hestbjerg

2017-09-01

Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Spatial differentiation of gene expression in Aspergillus niger colony grown for sugar beet pulp utilization

PubMed Central

Benoit, Isabelle; Zhou, Miaomiao; Vivas Duarte, Alexandra; Downes, Damien J.; Todd, Richard B.; Kloezen, Wendy; Post, Harm; Heck, Albert J. R.; Maarten Altelaar, A. F.; de Vries, Ronald P.

2015-01-01

Degradation of plant biomass to fermentable sugars is of critical importance for the use of plant materials for biofuels. Filamentous fungi are ubiquitous organisms and major plant biomass degraders. Single colonies of some fungal species can colonize massive areas as large as five soccer stadia. During growth, the mycelium encounters heterogeneous carbon sources. Here we assessed whether substrate heterogeneity is a major determinant of spatial gene expression in colonies of Aspergillus niger. We analyzed whole-genome gene expression in five concentric zones of 5-day-old colonies utilizing sugar beet pulp as a complex carbon source. Growth, protein production and secretion occurred throughout the colony. Genes involved in carbon catabolism were expressed uniformly from the centre to the periphery whereas genes encoding plant biomass degrading enzymes and nitrate utilization were expressed differentially across the colony. A combined adaptive response of carbon-catabolism and enzyme production to locally available monosaccharides was observed. Finally, our results demonstrate that A. niger employs different enzymatic tools to adapt its metabolism as it colonizes complex environments. PMID:26314379

Spatial differentiation of gene expression in Aspergillus niger colony grown for sugar beet pulp utilization.

PubMed

Benoit, Isabelle; Zhou, Miaomiao; Vivas Duarte, Alexandra; Downes, Damien J; Todd, Richard B; Kloezen, Wendy; Post, Harm; Heck, Albert J R; Maarten Altelaar, A F; de Vries, Ronald P

2015-08-28

Degradation of plant biomass to fermentable sugars is of critical importance for the use of plant materials for biofuels. Filamentous fungi are ubiquitous organisms and major plant biomass degraders. Single colonies of some fungal species can colonize massive areas as large as five soccer stadia. During growth, the mycelium encounters heterogeneous carbon sources. Here we assessed whether substrate heterogeneity is a major determinant of spatial gene expression in colonies of Aspergillus niger. We analyzed whole-genome gene expression in five concentric zones of 5-day-old colonies utilizing sugar beet pulp as a complex carbon source. Growth, protein production and secretion occurred throughout the colony. Genes involved in carbon catabolism were expressed uniformly from the centre to the periphery whereas genes encoding plant biomass degrading enzymes and nitrate utilization were expressed differentially across the colony. A combined adaptive response of carbon-catabolism and enzyme production to locally available monosaccharides was observed. Finally, our results demonstrate that A. niger employs different enzymatic tools to adapt its metabolism as it colonizes complex environments.

CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells.

PubMed

Ito, Saya; Ueno, Akihisa; Ueda, Takashi; Nakagawa, Hideo; Taniguchi, Hidefumi; Kayukawa, Naruhiro; Fujihara-Iwata, Atsuko; Hongo, Fumiya; Okihara, Koji; Ukimura, Osamu

2018-04-03

The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination.

Synthetic Gene Network with Positive Feedback Loop Amplifies Cellulase Gene Expression in Neurospora crassa.

PubMed

Matsu-Ura, Toru; Dovzhenok, Andrey A; Coradetti, Samuel T; Subramanian, Krithika R; Meyer, Daniel R; Kwon, Jaesang J; Kim, Caleb; Salomonis, Nathan; Glass, N Louise; Lim, Sookkyung; Hong, Christian I

2018-05-18

Second-generation or lignocellulosic biofuels are a tangible source of renewable energy, which is critical to combat climate change by reducing the carbon footprint. Filamentous fungi secrete cellulose-degrading enzymes called cellulases, which are used for production of lignocellulosic biofuels. However, inefficient production of cellulases is a major obstacle for industrial-scale production of second-generation biofuels. We used computational simulations to design and implement synthetic positive feedback loops to increase gene expression of a key transcription factor, CLR-2, that activates a large number of cellulases in a filamentous fungus, Neurospora crassa. Overexpression of CLR-2 reveals previously unappreciated roles of CLR-2 in lignocellulosic gene network, which enabled simultaneous induction of approximately 50% of 78 lignocellulosic degradation-related genes in our engineered Neurospora strains. This engineering results in dramatically increased cellulase activity due to cooperative orchestration of multiple enzymes involved in the cellulose degradation pathway. Our work provides a proof of principle in utilizing mathematical modeling and synthetic biology to improve the efficiency of cellulase synthesis for second-generation biofuel production.

CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells

PubMed Central

Ito, Saya; Ueno, Akihisa; Ueda, Takashi; Nakagawa, Hideo; Taniguchi, Hidefumi; Kayukawa, Naruhiro; Fujihara-Iwata, Atsuko; Hongo, Fumiya; Okihara, Koji; Ukimura, Osamu

2018-01-01

The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination. PMID:29707137

Studying the organization of genes encoding plant cell wall degrading enzymes in Chrysomela tremula provides insights into a leaf beetle genome.

PubMed

Pauchet, Y; Saski, C A; Feltus, F A; Luyten, I; Quesneville, H; Heckel, D G

2014-06-01

The ability of herbivorous beetles from the superfamilies Chrysomeloidea and Curculionoidea to degrade plant cell wall polysaccharides has only recently begun to be appreciated. The presence of plant cell wall degrading enzymes (PCWDEs) in the beetle's digestive tract makes this degradation possible. Sequences encoding these beetle-derived PCWDEs were originally identified from transcriptomes and strikingly resemble those of saprophytic and phytopathogenic microorganisms, raising questions about their origin; e.g. are they insect- or microorganism-derived? To demonstrate unambiguously that the genes encoding PCWDEs found in beetle transcriptomes are indeed of insect origin, we generated a bacterial artificial chromosome library from the genome of the leaf beetle Chrysomela tremula, containing 18 432 clones with an average size of 143 kb. After hybridizing this library with probes derived from 12 C. tremula PCWDE-encoding genes and sequencing the positive clones, we demonstrated that the latter genes are encoded by the insect's genome and are surrounded by genes possessing orthologues in the genome of Tribolium castaneum as well as in three other beetle genomes. Our analyses showed that although the level of overall synteny between C. tremula and T. castaneum seems high, the degree of microsynteny between both species is relatively low, in contrast to the more closely related Colorado potato beetle. © 2014 The Royal Entomological Society.

Composting-Like Conditions Are More Efficient for Enrichment and Diversity of Organisms Containing Cellulase-Encoding Genes than Submerged Cultures

PubMed Central

Fayolle-Guichard, Françoise; Lombard, Vincent; Hébert, Agnès; Coutinho, Pedro M.; Groppi, Alexis; Barre, Aurélien; Henrissat, Bernard

2016-01-01

Cost-effective biofuel production from lignocellulosic biomass depends on efficient degradation of the plant cell wall. One of the major obstacles for the development of a cost-efficient process is the lack of resistance of currently used fungal enzymes to harsh conditions such as high temperature. Adapted, thermophilic microbial communities provide a huge reservoir of potentially interesting lignocellulose-degrading enzymes for improvement of the cellulose hydrolysis step. In order to identify such enzymes, a leaf and wood chip compost was enriched on a mixture of thermo-chemically pretreated wheat straw, poplar and Miscanthus under thermophile conditions, but in two different set-ups. Unexpectedly, metagenome sequencing revealed that incubation of the lignocellulosic substrate with compost as inoculum in a suspension culture resulted in an impoverishment of putative cellulase- and hemicellulase-encoding genes. However, mimicking composting conditions without liquid phase yielded a high number and diversity of glycoside hydrolase genes and an enrichment of genes encoding cellulose binding domains. These identified genes were most closely related to species from Actinobacteria, which seem to constitute important players of lignocellulose degradation under the applied conditions. The study highlights that subtle changes in an enrichment set-up can have an important impact on composition and functions of the microcosm. Composting-like conditions were found to be the most successful method for enrichment in species with high biomass degrading capacity. PMID:27936240

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica

PubMed Central

Blackman, Leila M.; Cullerne, Darren P.; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R.

2015-01-01

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response. PMID:26332397

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica.

PubMed

Blackman, Leila M; Cullerne, Darren P; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R

2015-01-01

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response.

GeoChip-Based Analysis of the Functional Gene Diversity and Metabolic Potential of Microbial Communities in Acid Mine Drainage▿ †

PubMed Central

Xie, Jianping; He, Zhili; Liu, Xinxing; Liu, Xueduan; Van Nostrand, Joy D.; Deng, Ye; Wu, Liyou; Zhou, Jizhong; Qiu, Guanzhou

2011-01-01

Acid mine drainage (AMD) is an extreme environment, usually with low pH and high concentrations of metals. Although the phylogenetic diversity of AMD microbial communities has been examined extensively, little is known about their functional gene diversity and metabolic potential. In this study, a comprehensive functional gene array (GeoChip 2.0) was used to analyze the functional diversity, composition, structure, and metabolic potential of AMD microbial communities from three copper mines in China. GeoChip data indicated that these microbial communities were functionally diverse as measured by the number of genes detected, gene overlapping, unique genes, and various diversity indices. Almost all key functional gene categories targeted by GeoChip 2.0 were detected in the AMD microbial communities, including carbon fixation, carbon degradation, methane generation, nitrogen fixation, nitrification, denitrification, ammonification, nitrogen reduction, sulfur metabolism, metal resistance, and organic contaminant degradation, which suggested that the functional gene diversity was higher than was previously thought. Mantel test results indicated that AMD microbial communities are shaped largely by surrounding environmental factors (e.g., S, Mg, and Cu). Functional genes (e.g., narG and norB) and several key functional processes (e.g., methane generation, ammonification, denitrification, sulfite reduction, and organic contaminant degradation) were significantly (P < 0.10) correlated with environmental variables. This study presents an overview of functional gene diversity and the structure of AMD microbial communities and also provides insights into our understanding of metabolic potential in AMD ecosystems. PMID:21097602

Effect of alternative pathway therapy on branched chain amino acid metabolism in urea cycle disorder patients.

PubMed

Scaglia, Fernando; Carter, Susan; O'Brien, William E; Lee, Brendan

2004-04-01

Urea cycle disorders (UCDs) are a group of inborn errors of hepatic metabolism caused by the loss of enzymatic activities that mediate the transfer of nitrogen from ammonia to urea. These disorders often result in life-threatening hyperammonemia and hyperglutaminemia. A combination of sodium phenylbutyrate and sodium phenylacetate/benzoate is used in the clinical management of children with urea cycle defects as a glutamine trap, diverting nitrogen from urea synthesis to alternatives routes of excretion. We have observed that patients treated with these compounds have selective branched chain amino acid (BCAA) deficiency despite adequate dietary protein intake. However, the direct effect of alternative therapy on the steady state levels of plasma branched chain amino acids has not been well characterized. We have measured steady state plasma branched chain and other essential non-branched chain amino acids in control subjects, untreated ornithine transcarbamylase deficiency females and treated null activity urea cycle disorder patients in the fed steady state during the course of stable isotope studies. Steady-state leucine levels were noted to be significantly lower in treated urea cycle disorder patients when compared to either untreated ornithine transcarbamylase deficiency females or control subjects (P<0.0001). This effect was reproduced in control subjects who had depressed leucine levels when treated with sodium phenylacetate/benzoate (P<0.0001). Our studies suggest that this therapeutic modality has a substantial impact on the metabolism of branched chain amino acids in urea cycle disorder patients. These findings suggest that better titration of protein restriction could be achieved with branched chain amino acid supplementation in patients with UCDs who are on alternative route therapy.

Isolation, Identification and Phenotypic Characterization of Microcystin-Degrading Bacteria from Lake Erie

NASA Astrophysics Data System (ADS)

Krishnan, A.; Mou, X. J.

2015-12-01

Lake Erie, the smallest and warmest lake among the Laurentian Great Lakes, is known for its problem of eutrophication and frequent occurrence of harmful cyanobacterial blooms (CyanoHABs). One major harmful effect of CyanoHABs is the production of cyanotoxins, especially microcystins. Microcystins (MC) are a group of hepatotoxins and the predominant variant of them is MC-LR. Field measurements and lab experiments indicate that MC degradation in Lake Erie is mainly carried out by indigenous bacteria. However, our knowledge on taxa involved in this process is very limited. This study aimed to fill this knowledge gap using a culture-dependent approach. Water and surface sediment samples were collected from Lake Erie in 2014 and 2015 and enriched with MC-LR. Cells were plated on a number of culturing media. The obtained pure bacterial cultures were screened for MC degrading abilities by MT2 BIO-LOG assays and by growing cells in liquid media containing MC-LR as the sole carbon source. In the latter experiment, MC concentrations were measured using HPLC. Isolates showing positive MC degradation activities in the screening steps were designated MC+ bacteria and characterized based on their phenotypic properties, including colony pigmentation, elevation, opacity, margin, gram nature and motility. The taxonomic identity of MC+ bacteria was determined by 16S rRNA gene full-length DNA sequencing. The presence of mlrA, a gene encoding MC cleavage pathway, was detected by PCR. Our culturing efforts obtained 520 pure cultures; 44 of them were identified as MC+. These MC+ isolates showed diversity in taxonomic identities and differed in their morphology, gram nature, colony characteristics and motility. PCR amplification of mlrA gene yield negative results for all MC+ isolates, indicating that the primers that were used may not be ubiquitous enough to cover the heterogeneity of mlrA genes or, more likely, alternative degradative genes/pathways were employed by Lake Erie bacteria. The MC+ isolates can serve as models for future identification of MC degradation pathway and used to develop or augment biofilters for effective treatment of MC contaminated water. Key Words: CyanoHAB, microcystins, degradation

[Characterization and microbial community shifts of rice strawdegrading microbial consortia].

PubMed

Wang, Chunfang; Ma, Shichun; Huang, Yan; Liu, Laiyan; Fan, Hui; Deng, Yu

2016-12-04

To study the relationship between microbial community and degradation rate of rice straw, we compared and analyzed cellulose-decomposing ability, microbial community structures and shifts of microbial consortia F1 and F2. We determined exoglucanase activity by 3, 5-dinitrosalicylic acid colorimetry. We determined content of cellulose, hemicellulose and lignin in rice straw by Van Soest method, and calculated degradation rates of rice straw by the weight changes before and after a 10-day incubation. We analyzed and compared the microbial communities and functional microbiology shifts by clone libraries, Miseq analysis and real time-PCR based on the 16S rRNA gene and cel48 genes. Total degradation rate, cellulose, and hemicellulose degradation rate of microbial consortia F1 were significantly higher than that of F2. The variation trend of exoglucanase activity in both microbial consortia F1 and F2 was consistent with that of cel48 gene copies. Microbial diversity of F1 was complex with aerobic bacteria as dominant species, whereas that of F2 was simple with a high proportion of anaerobic cellulose decomposing bacteria in the later stage of incubation. In the first 4 days, unclassified Bacillales and Bacillus were dominant in both F1 and F2. The dominant species and abundance became different after 4-day incubation, Bacteroidetes and Firmicutes were dominant phyla of F1 and F2, respectively. Although Petrimonas and Pusillimonas were common dominant species in F1 and F2, abundance of Petrimonas in F2 (38.30%) was significantly higher than that in F1 (9.47%), and the abundance of Clostridiales OPB54 in F2 increased to 14.85% after 8-day incubation. The abundance of cel48 gene related with cellulose degradation rate and exoglucanase activity, and cel48 gene has the potential as a molecular marker to monitor the process of cellulose degradation. Microbial community structure has a remarkable impact on the degradation efficiency of straw cellulose, and Petrimonas, Paenibacillus, Bacillales, Clostridiales were vital species for microbial consortia F1 and F2 decomposing rice straw.

Cultivation-dependent and cultivation-independent characterization of hydrocarbon-degrading bacteria in Guaymas Basin sediments.

PubMed

Gutierrez, Tony; Biddle, Jennifer F; Teske, Andreas; Aitken, Michael D

2015-01-01

Marine hydrocarbon-degrading bacteria perform a fundamental role in the biodegradation of crude oil and its petrochemical derivatives in coastal and open ocean environments. However, there is a paucity of knowledge on the diversity and function of these organisms in deep-sea sediment. Here we used stable-isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria under aerobic conditions in sediments from Guaymas Basin with uniformly labeled [(13)C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from (13)C-enriched bacterial DNA (from PHE enrichments) were identified to belong to the genus Cycloclasticus. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments. We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization. In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of PAH-degrading bacteria in the deep-sea.

Cultivation-dependent and cultivation-independent characterization of hydrocarbon-degrading bacteria in Guaymas Basin sediments

PubMed Central

Gutierrez, Tony; Biddle, Jennifer F.; Teske, Andreas; Aitken, Michael D.

2015-01-01

Marine hydrocarbon-degrading bacteria perform a fundamental role in the biodegradation of crude oil and its petrochemical derivatives in coastal and open ocean environments. However, there is a paucity of knowledge on the diversity and function of these organisms in deep-sea sediment. Here we used stable-isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria under aerobic conditions in sediments from Guaymas Basin with uniformly labeled [13C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from PHE enrichments) were identified to belong to the genus Cycloclasticus. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments. We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization. In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of PAH-degrading bacteria in the deep-sea. PMID:26217326

Biodegradability of HCH in agricultural soils from Guadeloupe (French West Indies): identification of the lin genes involved in the HCH degradation pathway.

PubMed

Laquitaine, L; Durimel, A; de Alencastro, L F; Jean-Marius, C; Gros, O; Gaspard, S

2016-01-01

Banana has been a main agricultural product in the French West Indies (Guadeloupe and Martinique) since the 1960s. This crop requires the intensive use of pesticides to prevent attacks by insect pests. Chlorinated pesticides, such as hexachlorocyclohexane (HCH), chlordecone and dieldrin, were used until the beginning of the 1990s, resulting in a generalized diffuse contamination of the soil and water in the areas of banana production, hence the need to develop solutions for cleanup of the polluted sites. The aims of this work were (i) to assess lindane degradation in soil slurry microcosms treated with lindane at 10 mg/L and (ii) to detect the catabolic genes involved in the HCH degradation pathway. The soil slurry microcosm system showed a 40% lindane degradation efficiency at the end of a 30-day experiment. Lower lindane removal was also detected in the abiotic controls, probably caused by pesticide adsorption to soil particles. Indeed, the lindane concentration decreased from 6000 to 1330 ng/mL and from 800 to 340 ng/mL for the biotic and abiotic soils, respectively. Nevertheless, some of the genes involved in the HCH degradation pathway were amplified by polymerase chain reaction (PCR) from crude deoxyribonucleic acid (DNA) extracted from the Guadeloupe agricultural soil, suggesting that HCH degradation is probably mediated by bacteria closely related to the family Sphingomonadaceae.

Degradation of phenol via phenylphosphate and carboxylation to 4-hydroxybenzoate by a newly isolated strain of the sulfate-reducing bacterium Desulfobacterium anilini.

PubMed

Ahn, Young-Beom; Chae, Jong-Chan; Zylstra, Gerben J; Häggblom, Max M

2009-07-01

A sulfate-reducing phenol-degrading bacterium, strain AK1, was isolated from a 2-bromophenol-utilizing sulfidogenic estuarine sediment enrichment culture. On the basis of phylogenetic analysis of the 16S rRNA gene and DNA homology, strain AK1 is most closely related to Desulfobacterium anilini strain Ani1 (= DSM 4660(T)). In addition to phenol, this organism degrades a variety of other aromatic compounds, including benzoate, 2-hydroxybenzoate, 4-hydroxybenzoate, 4-hydroxyphenylacetate, 2-aminobenzoate, 2-fluorophenol, and 2-fluorobenzoate, but it does not degrade aniline, 3-hydroxybenzoate, 4-cyanophenol, 2,4-dihydroxybenzoate, monohalogenated phenols, or monohalogenated benzoates. Growth with sulfate as an electron acceptor occurred with acetate and pyruvate but not with citrate, propionate, butyrate, lactate, glucose, or succinate. Strain AK1 is able to use sulfate, sulfite, and thiosulfate as electron acceptors. A putative phenylphosphate synthase gene responsible for anaerobic phenol degradation was identified in strain AK1. In phenol-grown cultures inducible expression of the ppsA gene was verified by reverse transcriptase PCR, and 4-hydroxybenzoate was detected as an intermediate. These results suggest that the pathway for anaerobic degradation of phenol in D. anilini strain AK1 proceeds via phosphorylation of phenol to phenylphosphate, followed by carboxylation to 4-hydroxybenzoate. The details concerning such reaction pathways in sulfidogenic bacteria have not been characterized previously.

Insights into the degradation capacities of Amycolatopsis tucumanensis DSM 45259 guided by microarray data.

PubMed

Bourguignon, Natalia; Bargiela, Rafael; Rojo, David; Chernikova, Tatyana N; de Rodas, Sara A López; García-Cantalejo, Jesús; Näther, Daniela J; Golyshin, Peter N; Barbas, Coral; Ferrero, Marcela; Ferrer, Manuel

2016-12-01

The analysis of catabolic capacities of microorganisms is currently often achieved by cultivation approaches and by the analysis of genomic or metagenomic datasets. Recently, a microarray system designed from curated key aromatic catabolic gene families and key alkane degradation genes was designed. The collection of genes in the microarray can be exploited to indicate whether a given microbe or microbial community is likely to be functionally connected with certain degradative phenotypes, without previous knowledge of genome data. Herein, this microarray was applied to capture new insights into the catabolic capacities of copper-resistant actinomycete Amycolatopsis tucumanensis DSM 45259. The array data support the presumptive ability of the DSM 45259 strain to utilize single alkanes (n-decane and n-tetradecane) and aromatics such as benzoate, phthalate and phenol as sole carbon sources, which was experimentally validated by cultivation and mass spectrometry. Interestingly, while in strain DSM 45259 alkB gene encoding an alkane hydroxylase is most likely highly similar to that found in other actinomycetes, the genes encoding benzoate 1,2-dioxygenase, phthalate 4,5-dioxygenase and phenol hydroxylase were homologous to proteobacterial genes. This suggests that strain DSM 45259 contains catabolic genes distantly related to those found in other actinomycetes. Together, this study not only provided new insight into the catabolic abilities of strain DSM 45259, but also suggests that this strain contains genes uncommon within actinomycetes.

The Draft Genome of the Invasive Walking Stick, Medauroidea extradendata, Reveals Extensive Lineage-Specific Gene Family Expansions of Cell Wall Degrading Enzymes in Phasmatodea

PubMed Central

Brand, Philipp; Lin, Wei; Johnson, Brian R.

2018-01-01

Plant cell wall components are the most abundant macromolecules on Earth. The study of the breakdown of these molecules is thus a central question in biology. Surprisingly, plant cell wall breakdown by herbivores is relatively poorly understood, as nearly all early work focused on the mechanisms used by symbiotic microbes to breakdown plant cell walls in insects such as termites. Recently, however, it has been shown that many organisms make endogenous cellulases. Insects, and other arthropods, in particular have been shown to express a variety of plant cell wall degrading enzymes in many gene families with the ability to break down all the major components of the plant cell wall. Here we report the genome of a walking stick, Medauroidea extradentata, an obligate herbivore that makes uses of endogenously produced plant cell wall degrading enzymes. We present a draft of the 3.3Gbp genome along with an official gene set that contains a diversity of plant cell wall degrading enzymes. We show that at least one of the major families of plant cell wall degrading enzymes, the pectinases, have undergone a striking lineage-specific gene family expansion in the Phasmatodea. This genome will be a useful resource for comparative evolutionary studies with herbivores in many other clades and will help elucidate the mechanisms by which metazoans breakdown plant cell wall components. PMID:29588379

Regulation of Benzoate Degradation in Acinetobacter sp. Strain ADP1 by BenM, a LysR-Type Transcriptional Activator

PubMed Central

Collier, Lauren S.; Gaines, George L.; Neidle, Ellen L.

1998-01-01

In Acinetobacter sp. strain ADP1, benzoate degradation requires the ben genes for converting benzoate to catechol and the cat genes for degrading catechol. Here we describe a novel transcriptional activator, BenM, that regulates the chromosomal ben and cat genes. BenM is homologous to CatM, a LysR-type transcriptional activator of the cat genes. Unusual regulatory features of this system include the abilities of both BenM and CatM to recognize the same inducer, cis,cis-muconate, and to regulate some of the same genes, such as catA and catB. Unlike CatM, BenM responded to benzoate. Benzoate together with cis,cis-muconate increased the BenM-dependent expression of the benABCDE operon synergistically. CatM was not required for this synergism, nor did CatM regulate the expression of a chromosomal benA::lacZ transcriptional fusion. BenM-mediated regulation differs significantly from that of the TOL plasmid-encoded conversion of benzoate to catechol in pseudomonads. The benM gene is immediately upstream of, and divergently transcribed from, benA, and a possible DNA binding site for BenM was identified between the two coding regions. Two mutations in the predicted operator/promoter region rendered ben gene expression either constitutive or inducible by cis,cis-muconate but not benzoate. Mutants lacking BenM, CatM, or both of these regulators degraded aromatic compounds at different rates, and the levels of intermediary metabolites that accumulated depended on the genetic background. These studies indicated that BenM is necessary for ben gene expression but not for expression of the cat genes, which can be regulated by CatM. In a catM-disrupted strain, BenM was able to induce higher levels of catA expression than catB expression. PMID:9573203

Analysis of the xplAB-Containing Gene Cluster Involved in the Bacterial Degradation of the Explosive Hexahydro-1,3,5-Trinitro-1,3,5-Triazine

PubMed Central

Chong, Chun Shiong; Sabir, Dana Khdr; Lorenz, Astrid; Bontemps, Cyril; Andeer, Peter; Stahl, David A.; Strand, Stuart E.; Rylott, Elizabeth L.

2014-01-01

Repeated use of the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military land has resulted in significant soil and groundwater pollution. Rates of degradation of RDX in the environment are low, and accumulated RDX, which the U.S. Environmental Protection Agency has determined is a possible human carcinogen, is now threatening drinking water supplies. RDX-degrading microorganisms have been isolated from RDX-contaminated land; however, despite the presence of these species in contaminated soils, RDX pollution persists. To further understand this problem, we studied RDX-degrading species belonging to four different genera (Rhodococcus, Microbacterium, Gordonia, and Williamsia) isolated from geographically distinct locations and established that the xplA and xplB (xplAB) genes, which encode a cytochrome P450 and a flavodoxin redox partner, respectively, are nearly identical in all these species. Together, the xplAB system catalyzes the reductive denitration of RDX and subsequent ring cleavage under aerobic and anaerobic conditions. In addition to xplAB, the Rhodococcus species studied here share a 14-kb region flanking xplAB; thus, it appears likely that the RDX-metabolizing ability was transferred as a genomic island within a transposable element. The conservation and transfer of xplAB-flanking genes suggest a role in RDX metabolism. We therefore independently knocked out genes within this cluster in the RDX-degrading species Rhodococcus rhodochrous 11Y. Analysis of the resulting mutants revealed that XplA is essential for RDX degradation and that XplB is not the sole contributor of reducing equivalents to XplA. While XplA expression is induced under nitrogen-limiting conditions and further enhanced by the presence of RDX, MarR is not regulated by RDX. PMID:25128343

Genome Sequence Analysis of the Naphthenic Acid Degrading and Metal Resistant Bacterium Cupriavidus gilardii CR3

PubMed Central

Xiao, Jingfa; Hao, Lirui; Crowley, David E.; Zhang, Zhewen; Yu, Jun; Huang, Ning; Huo, Mingxin; Wu, Jiayan

2015-01-01

Cupriavidus sp. are generally heavy metal tolerant bacteria with the ability to degrade a variety of aromatic hydrocarbon compounds, although the degradation pathways and substrate versatilities remain largely unknown. Here we studied the bacterium Cupriavidus gilardii strain CR3, which was isolated from a natural asphalt deposit, and which was shown to utilize naphthenic acids as a sole carbon source. Genome sequencing of C. gilardii CR3 was carried out to elucidate possible mechanisms for the naphthenic acid biodegradation. The genome of C. gilardii CR3 was composed of two circular chromosomes chr1 and chr2 of respectively 3,539,530 bp and 2,039,213 bp in size. The genome for strain CR3 encoded 4,502 putative protein-coding genes, 59 tRNA genes, and many other non-coding genes. Many genes were associated with xenobiotic biodegradation and metal resistance functions. Pathway prediction for degradation of cyclohexanecarboxylic acid, a representative naphthenic acid, suggested that naphthenic acid undergoes initial ring-cleavage, after which the ring fission products can be degraded via several plausible degradation pathways including a mechanism similar to that used for fatty acid oxidation. The final metabolic products of these pathways are unstable or volatile compounds that were not toxic to CR3. Strain CR3 was also shown to have tolerance to at least 10 heavy metals, which was mainly achieved by self-detoxification through ion efflux, metal-complexation and metal-reduction, and a powerful DNA self-repair mechanism. Our genomic analysis suggests that CR3 is well adapted to survive the harsh environment in natural asphalts containing naphthenic acids and high concentrations of heavy metals. PMID:26301592

Pyramidobacter piscolens gen. nov., sp. nov., a member of the phylum 'Synergistetes' isolated from the human oral cavity.

PubMed

Downes, Julia; Vartoukian, Sonia R; Dewhirst, Floyd E; Izard, Jacques; Chen, Tsute; Yu, Wen-Han; Sutcliffe, Iain C; Wade, William G

2009-05-01

Four strains of anaerobic, Gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group distinct from any species with validly published names. 16S rRNA and 23S rRNA gene sequence analyses and DNA-DNA reassociation data revealed that the strains constituted a novel group within the phylum 'Synergistetes' and were most closely related to Jonquetella anthropi. Two libraries of randomly cloned DNA were prepared from strain W5455(T) and were sequenced to provide a genome survey as a resource for metagenomic studies. A new genus and novel species, Pyramidobacter piscolens gen. nov., sp. nov., is proposed to accommodate these strains. The genus Pyramidobacter comprises strains that are anaerobic, non-motile, asaccharolytic bacilli that produce acetic and isovaleric acids and minor to trace amounts of propionic, isobutyric, succinic and phenylacetic acids as end products of metabolism. P. piscolens gen. nov., sp. nov. produced hydrogen sulphide but was otherwise largely biochemically unreactive. Growth was stimulated by the addition of glycine to broth media. The G+C content of the DNA of the type strain was 59 mol%. The type strain of Pyramidobacter piscolens sp. nov. is W5455(T) (=DSM 21147(T)=CCUG 55836(T)).

Pyramidobacter piscolens gen. nov., sp. nov., a member of the phylum ‘Synergistetes’ isolated from the human oral cavity

PubMed Central

Downes, Julia; Vartoukian, Sonia R.; Dewhirst, Floyd E.; Izard, Jacques; Chen, Tsute; Yu, Wen-Han; Sutcliffe, Iain C.; Wade, William G.

2009-01-01

Four strains of anaerobic, Gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group distinct from any species with validly published names. 16S rRNA and 23S rRNA gene sequence analyses and DNA–DNA reassociation data revealed that the strains constituted a novel group within the phylum ‘Synergistetes’ and were most closely related to Jonquetella anthropi. Two libraries of randomly cloned DNA were prepared from strain W5455T and were sequenced to provide a genome survey as a resource for metagenomic studies. A new genus and novel species, Pyramidobacter piscolens gen. nov., sp. nov., is proposed to accommodate these strains. The genus Pyramidobacter comprises strains that are anaerobic, non-motile, asaccharolytic bacilli that produce acetic and isovaleric acids and minor to trace amounts of propionic, isobutyric, succinic and phenylacetic acids as end products of metabolism. P. piscolens gen. nov., sp. nov. produced hydrogen sulphide but was otherwise largely biochemically unreactive. Growth was stimulated by the addition of glycine to broth media. The G+C content of the DNA of the type strain was 59 mol%. The type strain of Pyramidobacter piscolens sp. nov. is W5455T (=DSM 21147T=CCUG 55836T). PMID:19406777

Carbon Catabolite Repression and Impranil Polyurethane Degradation in Pseudomonas protegens Strain Pf-5.

PubMed

Hung, Chia-Suei; Zingarelli, Sandra; Nadeau, Lloyd J; Biffinger, Justin C; Drake, Carrie A; Crouch, Audra L; Barlow, Daniel E; Russell, John N; Crookes-Goodson, Wendy J

2016-10-15

Polyester polyurethane (PU) coatings are widely used to help protect underlying structural surfaces but are susceptible to biological degradation. PUs are susceptible to degradation by Pseudomonas species, due in part to the degradative activity of secreted hydrolytic enzymes. Microorganisms often respond to environmental cues by secreting enzymes or secondary metabolites to benefit their survival. This study investigated the impact of exposing several Pseudomonas strains to select carbon sources on the degradation of the colloidal polyester polyurethane Impranil DLN (Impranil). The prototypic Pseudomonas protegens strain Pf-5 exhibited Impranil-degrading activities when grown in sodium citrate but not in glucose-containing medium. Glucose also inhibited the induction of Impranil-degrading activity by citrate-fed Pf-5 in a dose-dependent manner. Biochemical and mutational analyses identified two extracellular lipases present in the Pf-5 culture supernatant (PueA and PueB) that were involved in degradation of Impranil. Deletion of the pueA gene reduced Impranil-clearing activities, while pueB deletion exhibited little effect. Removal of both genes was necessary to stop degradation of the polyurethane. Bioinformatic analysis showed that putative Cbr/Hfq/Crc-mediated regulatory elements were present in the intergenic sequences upstream of both pueA and pueB genes. Our results confirmed that both PueA and PueB extracellular enzymes act in concert to degrade Impranil. Furthermore, our data showed that carbon sources in the growth medium directly affected the levels of Impranil-degrading activity but that carbon source effects varied among Pseudomonas strains. This study uncovered an intricate and complicated regulation of P. protegens PU degradation activity controlled by carbon catabolite repression. Polyurethane (PU) coatings are commonly used to protect metals from corrosion. Microbiologically induced PU degradation might pose a substantial problem for the integrity of these coatings. Microorganisms from diverse genera, including pseudomonads, possess the ability to degrade PUs via various means. This work identified two extracellular lipases, PueA and PueB, secreted by P. protegens strain Pf-5, to be responsible for the degradation of a colloidal polyester PU, Impranil. This study also revealed that the expression of the degradative activity by strain Pf-5 is controlled by glucose carbon catabolite repression. Furthermore, this study showed that the Impranil-degrading activity of many other Pseudomonas strains could be influenced by different carbon sources. This work shed light on the carbon source regulation of PU degradation activity among pseudomonads and identified the polyurethane lipases in P. protegens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Carbon Catabolite Repression and Impranil Polyurethane Degradation in Pseudomonas protegens Strain Pf-5

PubMed Central

Hung, Chia-Suei; Zingarelli, Sandra; Nadeau, Lloyd J.; Biffinger, Justin C.; Drake, Carrie A.; Crouch, Audra L.; Barlow, Daniel E.; Russell, John N.

2016-01-01

ABSTRACT Polyester polyurethane (PU) coatings are widely used to help protect underlying structural surfaces but are susceptible to biological degradation. PUs are susceptible to degradation by Pseudomonas species, due in part to the degradative activity of secreted hydrolytic enzymes. Microorganisms often respond to environmental cues by secreting enzymes or secondary metabolites to benefit their survival. This study investigated the impact of exposing several Pseudomonas strains to select carbon sources on the degradation of the colloidal polyester polyurethane Impranil DLN (Impranil). The prototypic Pseudomonas protegens strain Pf-5 exhibited Impranil-degrading activities when grown in sodium citrate but not in glucose-containing medium. Glucose also inhibited the induction of Impranil-degrading activity by citrate-fed Pf-5 in a dose-dependent manner. Biochemical and mutational analyses identified two extracellular lipases present in the Pf-5 culture supernatant (PueA and PueB) that were involved in degradation of Impranil. Deletion of the pueA gene reduced Impranil-clearing activities, while pueB deletion exhibited little effect. Removal of both genes was necessary to stop degradation of the polyurethane. Bioinformatic analysis showed that putative Cbr/Hfq/Crc-mediated regulatory elements were present in the intergenic sequences upstream of both pueA and pueB genes. Our results confirmed that both PueA and PueB extracellular enzymes act in concert to degrade Impranil. Furthermore, our data showed that carbon sources in the growth medium directly affected the levels of Impranil-degrading activity but that carbon source effects varied among Pseudomonas strains. This study uncovered an intricate and complicated regulation of P. protegens PU degradation activity controlled by carbon catabolite repression. IMPORTANCE Polyurethane (PU) coatings are commonly used to protect metals from corrosion. Microbiologically induced PU degradation might pose a substantial problem for the integrity of these coatings. Microorganisms from diverse genera, including pseudomonads, possess the ability to degrade PUs via various means. This work identified two extracellular lipases, PueA and PueB, secreted by P. protegens strain Pf-5, to be responsible for the degradation of a colloidal polyester PU, Impranil. This study also revealed that the expression of the degradative activity by strain Pf-5 is controlled by glucose carbon catabolite repression. Furthermore, this study showed that the Impranil-degrading activity of many other Pseudomonas strains could be influenced by different carbon sources. This work shed light on the carbon source regulation of PU degradation activity among pseudomonads and identified the polyurethane lipases in P. protegens. PMID:27496773

Microscopy and bioinformatic analyses of lipid metabolism implicate a sporophytic signaling network supporting pollen development in Arabidopsis.

PubMed

Wang, Yixing; Wu, Hong; Yang, Ming

2008-07-01

The Arabidopsis sporophytic tapetum undergoes a programmed degeneration process to secrete lipid and other materials to support pollen development. However, the molecular mechanism regulating the degeneration process is unknown. To gain insight into this molecular mechanism, we first determined that the most critical period for tapetal secretion to support pollen development is from the vacuolate microspore stage to the early binucleate pollen stage. We then analyzed the expression of enzymes responsible for lipid biosynthesis and degradation with available in-silico data. The genes for these enzymes that are expressed in the stamen but not in the concurrent uninucleate microspore and binucleate pollen are of particular interest, as they presumably hold the clues to unique molecular processes in the sporophytic tissues compared to the gametophytic tissue. No gene for lipid biosynthesis but a single gene encoding a patatin-like protein likely for lipid mobilization was identified based on the selection criterion. A search for genes co-expressed with this gene identified additional genes encoding typical signal transduction components such as a leucine-rich repeat receptor kinase, an extra-large G-protein, other protein kinases, and transcription factors. In addition, proteases, cell wall degradation enzymes, and other proteins were also identified. These proteins thus may be components of a signaling network leading to degradation of a broad range of cellular components. Since a broad range of degradation activities is expected to occur only in the tapetal degeneration process at this stage in the stamen, it is further hypothesized that the signaling network acts in the tapetal degeneration process.

An ethanolic extract of Artemisia dracunculus L. regulates gene expression of ubiquitin-proteasome system enzymes in skeletal muscle: potential role in the treatment of sarcopenic obesity.

PubMed

Kirk-Ballard, Heather; Kilroy, Gail; Day, Britton C; Wang, Zhong Q; Ribnicky, David M; Cefalu, William T; Floyd, Z Elizabeth

2014-01-01

Obesity is linked to insulin resistance, a primary component of metabolic syndrome and type 2 diabetes. The problem of obesity-related insulin resistance is compounded when age-related skeletal muscle loss, called sarcopenia, occurs with obesity. Skeletal muscle loss results from elevated levels of protein degradation and prevention of obesity-related sarcopenic muscle loss will depend on strategies that target pathways involved in protein degradation. An extract from Artemisia dracunculus, termed PMI 5011, improves insulin signaling and increases skeletal muscle myofiber size in a rodent model of obesity-related insulin resistance. The aim of this study was to examine the effect of PMI 5011 on the ubiquitin-proteasome system, a central regulator of muscle protein degradation. Gastrocnemius and vastus lateralis skeletal muscle was obtained from KK-A(y) obese diabetic mice fed a control or 1% (w/w) PMI 5011-supplemented diet. Regulation of genes encoding enzymes of the ubiquitin-proteasome system was determined using real-time quantitative reverse transcriptase polymerase chain reaction. Although MuRF-1 ubiquitin ligase gene expression is consistently down-regulated in skeletal muscle, atrogin-1, Fbxo40, and Traf6 expression is differentially regulated by PMI 5011. Genes encoding other enzymes of the ubiquitin-proteasome system ranging from ubiquitin to ubiquitin-specific proteases are also regulated by PMI 5011. Additionally, expression of the gene encoding the microtubule-associated protein-1 light chain 3 (LC3), a ubiquitin-like protein pivotal to autophagy-mediated protein degradation, is down-regulated by PMI 5011 in the vastus lateralis. PMI 5011 alters the gene expression of ubiquitin-proteasome system enzymes that are essential regulators of skeletal muscle mass. This suggests that PMI 5011 has therapeutic potential in the treatment of obesity-linked sarcopenia by regulating ubiquitin-proteasome-mediated protein degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpinets, Tatiana V; Pelletier, Dale A; Pan, Chongle

Understanding of cellular processes involved in the anaerobic degradation of complex organic compounds by microorganisms is crucial for development of innovative biotechnologies for bioethanol production and for efficient degradation of toxic organic compounds. In natural environment the degradation is usually accomplished by syntrophic consortia comprised of different bacterial species. Here we show that the metabolically versatile phototrophic bacterium Rhodopseudomonas palustris may form its own syntrophic consortia, when it grows anaerobically on p-coumarate or benzoate as a sole carbon source. In the study we reveal the consortia from a comparison of large-scale measurements of mRNA and protein expressions under p-coumarate andmore » benzoate degrading conditions using a novel computational approach referred as phenotype fingerprinting. In this approach marker genes for known R. palustris phenotypes are employed to calculate their expression from the gene and protein expressions in each studied condition. Subpopulations of the consortia are inferred from the expression of phenotypes and known metabolic modes of the R. palustris growth. We find that p-coumarate degrading condition leads to at least three R. palustris subpopulations utilizing p-coumarate, benzoate, and CO2 and H2. Benzoate degrading condition also produces at least three subpopulations utilizing benzoate, CO2 and H2, and N2 and formate. Communication among syntrophs and inter-syntrophic dynamics in each consortium are indicated by up-regulation of transporters and genes involved in the curli formation and chemotaxis. The photoautotrphic subpopulation found in both consortia is characterized by activation of two cbb operons and the uptake hydrogenase system. A specificity of N2-fixing subpopulation in the benzoate degrading consortium is the preferential activation of the vanadium nitrogenase over the molybdenum nitrogenase. The N2-fixing subpopulation in the consortium is confirmed by consumption of dissolved nitrogen gas under the benzoate degrading conditions.« less

Draft Genome Sequence of Paenibacillus sp. Strain DMB20, Isolated from Alang Ship-Breaking Yard, Which Harbors Genes for Xenobiotic Degradation

PubMed Central

Shah, Binal; Jain, Kunal; Patel, Namrata; Pandit, Ramesh; Patel, Anand; Joshi, Chaitanya G.

2015-01-01

Paenibacillus sp. strain DMB20, in cometabolism with other Proteobacteria and Firmicutes, exhibits azoreduction of textile dyes. Here, we report the draft genome sequence of this bacterium, consisting of 6,647,181 bp with 7,668 coding sequences (CDSs). The data presented highlight multiple sets of functional genes associated with xenobiotic compound degradation. PMID:26067950

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus.

PubMed

Wefers, Daniel; Dong, Jia; Abdel-Hamid, Ahmed M; Paul, Hans Müller; Pereira, Gabriel V; Han, Yejun; Dodd, Dylan; Baskaran, Ramiya; Mayer, Beth; Mackie, Roderick I; Cann, Isaac

2017-09-15

The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β-l-arabinopyranosidase (CpAbp27A), and two GH127 β-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para -nitrophenyl ( p NP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries. Copyright © 2017 American Society for Microbiology.

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

PubMed Central

Dong, Jia; Abdel-Hamid, Ahmed M.; Paul, Hans Müller; Pereira, Gabriel V.; Han, Yejun; Dodd, Dylan; Baskaran, Ramiya; Mayer, Beth; Mackie, Roderick I.

2017-01-01

ABSTRACT The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β-l-arabinopyranosidase (CpAbp27A), and two GH127 β-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus. IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus. The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries. PMID:28710263

Advances of naphthalene degradation in Pseudomonas putida ND6

NASA Astrophysics Data System (ADS)

Song, Fu; Shi, Yifei; Jia, Shiru; Tan, Zhilei; Zhao, Huabing

2018-03-01

Naphthalene is one of the most common and simple polycyclic aromatic hydrocarbons. Degradation of naphthalene has been greatly concerned due to its economic, free-pollution and its fine effect in Pseudomonas putida ND6. This review summarizes the development history of naphthalene degradation, the research progress of naphthalene degrading gene and naphthalene degradation pathway of Pseudomonas putida ND6, and the researching path of this strain. Although the study of naphthalene degradation is not consummate in Pseudomonas putida ND6, there is a potential capability for Pseudomonas putida ND6 to degrade the naphthalene in the further research.

New Hydrocarbon Degradation Pathways in the Microbial Metagenome from Brazilian Petroleum Reservoirs

PubMed Central

Sierra-García, Isabel Natalia; Correa Alvarez, Javier; Pantaroto de Vasconcellos, Suzan; Pereira de Souza, Anete; dos Santos Neto, Eugenio Vaz; de Oliveira, Valéria Maia

2014-01-01

Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs. PMID:24587220

An Inducible Propane Monooxygenase Is Responsible for N-Nitrosodimethylamine Degradation by Rhodococcus sp. Strain RHA1▿

PubMed Central

Sharp, Jonathan O.; Sales, Christopher M.; LeBlanc, Justin C.; Liu, Jie; Wood, Thomas K.; Eltis, Lindsay D.; Mohn, William W.; Alvarez-Cohen, Lisa

2007-01-01

Rhodococci are common soil heterotrophs that possess diverse functional enzymatic activities with economic and ecological significance. In this study, the correlation between gene expression and biological removal of the water contaminant N-nitrosodimethylamine (NDMA) is explored. NDMA is a hydrophilic, potent carcinogen that has gained recent notoriety due to its environmental persistence and emergence as a widespread micropollutant in the subsurface environment. In this study, we demonstrate that Rhodococcus sp. strain RHA1 can constitutively degrade NDMA and that activity toward this compound is enhanced by approximately 500-fold after growth on propane. Transcriptomic analysis of RHA1 and reverse transcriptase quantitative PCR assays demonstrate that growth on propane elicits the upregulation of gene clusters associated with (i) the oxidation of propane and (ii) the oxidation of substituted benzenes. Deletion mutagenesis of prmA, the gene encoding the large hydroxylase component of propane monooxygenase, abolished both growth on propane and removal of NDMA. These results demonstrate that propane monooxygenase is responsible for NDMA degradation by RHA1 and explain the enhanced cometabolic degradation of NDMA in the presence of propane. PMID:17873074

Assessment of the horizontal transfer of functional genes as a suitable approach for evaluation of the bioremediation potential of petroleum-contaminated sites: a mini-review.

PubMed

Shahi, Aiyoub; Ince, Bahar; Aydin, Sevcan; Ince, Orhan

2017-06-01

Petroleum sludge contains recalcitrant residuals. These compounds because of being toxic to humans and other organism are of the major concerns. Therefore, petroleum sludge should be safely disposed. Physicochemical methods which are used by this sector are mostly expensive and need complex devices. Bioremediation methods because of being eco-friendly and cost-effective overcome most of the limitations of physicochemical treatments. Microbial strains capable to degrade petroleum hydrocarbons are practically present in all soils and sediments and their population density increases in contact with contaminants. Bacterial strains cannot degrade alone all kinds of petroleum hydrocarbons, rather microbial consortium should collaborate with each other for degradation of petroleum hydrocarbon mixtures. Horizontal transfer of functional genes between bacteria plays an important role in increasing the metabolic potential of the microbial community. Therefore, selecting a suitable degrading gene and tracking its horizontal transfer would be a useful approach to evaluate the bioremediation process and to assess the bioremediation potential of contaminated sites.

Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway.

PubMed Central

Hughes, E J; Bayly, R C; Skurray, R A

1984-01-01

Alcaligenes eutrophus wild-type strain 345 metabolizes m- and p-toluate via a catechol meta-cleavage pathway. DNA analysis, curing studies, and transfer of this phenotype by conjugation and transformation showed that the degradative genes are encoded on a self-transmissible 85-kilobase plasmid, pRA1000. HindIII and XhoI restriction endonuclease analysis of pRA1000 showed it to be similar to the archetypal TOL plasmid, pWWO, differing in the case of HindIII only by the absence of fragments B and D present in pWWO. In strain 345, the presence of pRA1000 prevented the expression of chromosomally encoded enzymes required for the degradation of p-cresol, whereas these enzymes were expressed in strains cured of pRA1000. On the basis of studies with an R68.45-pRA1000 cointegrate plasmid, pRA1001, we conclude that the gene(s) responsible for the effect of p-cresol degradation resides within or near the m- and p-toluate degradative region on pRA1000. Images PMID:6325399

Transcriptomic analysis of genes involved in the biosynthesis, recycling and degradation of L-ascorbic acid in pepper fruits (Capsicum annuum L.).

PubMed

Alós, Enriqueta; Rodrigo, María J; Zacarías, Lorenzo

2013-06-01

Sweet pepper (Capsicum annuum L.) is widely recognized among the vegetables with high content of ascorbic acid (AsA). However, the metabolic pathways involved in the biosynthesis, recycling and degradation of AsA and their relative contribution to the concentration of AsA have not been established yet. In the present work, the expression levels of selected genes involved in the AsA biosynthesis, degradation and recycling pathways were analyzed during development and ripening of pepper fruit cv. Palermo and in mature fruit of four cultivars (Lipari, C-116, Surrentino and Italverde) with different AsA concentrations. An inverse correlation was found between the expression of the biosynthetic genes and AsA concentrations, which could indicate that a feedback mechanism regulates AsA homeostasis in pepper fruits. Interestingly, analysis of mRNA levels of ascorbate oxidase, involved in the degradation of AsA, suggests that this enzyme plays a critical role in the regulation of the AsA pool during fruit development and ripening. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp1[OPEN

PubMed Central

Fang, Fang; Zhang, Xue-lian; Gong, Yi-hui; Li, Wen-jun; Shi, Zhao-wan; He, Quan; Wu, Qing; Li, Lu; Jiang, Lin-lin; Cai, Zhi-gao; Oren-Shamir, Michal; Zhang, Zhao-qi

2015-01-01

In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning. PMID:26514808

Further Concerns About Glutamine: A Case Report on Hyperammonemic Encephalopathy.

PubMed

Cioccari, Luca; Gautschi, Matthias; Etter, Reto; Weck, Anja; Takala, Jukka

2015-10-01

We report a case of a woman with hyperammonemic encephalopathy following glutamine supplementation. Case report. Plasma amino acid analysis suggestive of a urea cycle defect and initiation of a treatment with lactulose and the two ammonia scavenger drugs sodium benzoate and phenylacetate. Together with a restricted protein intake ammonia and glutamine plasma levels decreased with subsequent improvement of the neurological status. Massive catabolism and exogenous glutamine administration may have contributed to hyperammonemia and hyperglutaminemia in this patient. This case adds further concerns regarding glutamine administration to critically ill patients and implies the importance of monitoring ammonia and glutamine serum levels in such patients.

Characterisation of an efficient atrazine-degrading bacterium, Arthrobacter sp. ZXY-2: an attempt to lay the foundation for potential bioaugmentation applications.

PubMed

Zhao, Xinyue; Wang, Li; Ma, Fang; Yang, Jixian

2018-01-01

The isolation of atrazine-degrading microorganisms with specific characteristics is fundamental for bioaugmenting the treatment of wastewater containing atrazine. However, studies describing the specific features of such microorganisms are limited, and further investigation is needed to improve our understanding of bioaugmentation. In this study, strain Arthrobacter sp. ZXY-2, which displayed a strong capacity to degrade atrazine, was isolated and shown to be a potential candidate for bioaugmentation. The factors associated with the biodegrading capacity of strain ZXY-2 were investigated, and how these factors likely govern the metabolic characteristics that control bioaugmentation functionality was determined. The growth pattern of Arthrobacter sp. ZXY-2 followed the Haldane-Andrews model with an inhibition constant ( K i ) of 52.76 mg L -1 , indicating the possible augmentation of wastewater treatment with relatively high atrazine concentrations (> 50 ppm). Real-time quantitative PCR (RT-qPCR) results showed a positive correlation between the atrazine degradation rate and the expression levels of three functional genes ( trzN , atzB , and atzC ), which helped elucidate the role of strain ZXY-2 in bioaugmentation. In addition, multiple copies of the atzB gene were putatively identified, explaining the higher expression levels of this gene than those of the other functional genes. Multiple copies of the atzB gene may represent a compensatory mechanism that ensures the biodegradation of atrazine, a feature that should be exploited in future bioaugmentation applications.

Genetic Linkage of Soil Carbon Pools and Microbial Functions in Subtropical Freshwater Wetlands in Response to Experimental Warming

PubMed Central

Wang, Hang; He, Zhili; Lu, Zhenmei; Zhou, Jizhong; Van Nostrand, Joy D.; Xu, Xinhua

2012-01-01

Rising climate temperatures in the future are predicted to accelerate the microbial decomposition of soil organic matter. A field microcosm experiment was carried out to examine the impact of soil warming in freshwater wetlands on different organic carbon (C) pools and associated microbial functional responses. GeoChip 4.0, a functional gene microarray, was used to determine microbial gene diversity and functional potential for C degradation. Experimental warming significantly increased soil pore water dissolved organic C and phosphorus (P) concentrations, leading to a higher potential for C emission and P export. Such losses of total organic C stored in soil could be traced back to the decomposition of recalcitrant organic C. Warming preferentially stimulated genes for degrading recalcitrant C over labile C. This was especially true for genes encoding cellobiase and mnp for cellulose and lignin degradation, respectively. We confirmed this with warming-enhanced polyphenol oxidase and peroxidase activities for recalcitrant C acquisition and greater increases in recalcitrant C use efficiency than in labile C use efficiency (average percentage increases of 48% versus 28%, respectively). The relative abundance of lignin-degrading genes increased by 15% under warming; meanwhile, soil fungi, as the primary decomposers of lignin, were greater in abundance by 27%. This work suggests that future warming may enhance the potential for accelerated fungal decomposition of lignin-like compounds, leading to greater microbially mediated C losses than previously estimated in freshwater wetlands. PMID:22923398

MPN- and Real-Time-Based PCR Methods for the Quantification of Alkane Monooxygenase Homologous Genes (alkB) in Environmental Samples

NASA Astrophysics Data System (ADS)

Pérez-de-Mora, Alfredo; Schulz, Stephan; Schloter, Michael

Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic-biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays.

Analysis of the core genome and pangenome of Pseudomonas putida.

PubMed

Udaondo, Zulema; Molina, Lázaro; Segura, Ana; Duque, Estrella; Ramos, Juan L

2016-10-01

Pseudomonas putida are strict aerobes that proliferate in a range of temperate niches and are of interest for environmental applications due to their capacity to degrade pollutants and ability to promote plant growth. Furthermore solvent-tolerant strains are useful for biosynthesis of added-value chemicals. We present a comprehensive comparative analysis of nine strains and the first characterization of the Pseudomonas putida pangenome. The core genome of P. putida comprises approximately 3386 genes. The most abundant genes within the core genome are those that encode nutrient transporters. Other conserved genes include those for central carbon metabolism through the Entner-Doudoroff pathway, the pentose phosphate cycle, arginine and proline metabolism, and pathways for degradation of aromatic chemicals. Genes that encode transporters, enzymes and regulators for amino acid metabolism (synthesis and degradation) are all part of the core genome, as well as various electron transporters, which enable aerobic metabolism under different oxygen regimes. Within the core genome are 30 genes for flagella biosynthesis and 12 key genes for biofilm formation. Pseudomonas putida strains share 85% of the coding regions with Pseudomonas aeruginosa; however, in P. putida, virulence factors such as exotoxins and type III secretion systems are absent. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

The Gastrointestinal Tract of the White-Throated Woodrat (Neotoma albigula) Harbors Distinct Consortia of Oxalate-Degrading Bacteria

PubMed Central

Kohl, Kevin D.; Dearing, M. Denise

2014-01-01

The microbiota inhabiting the mammalian gut is a functional organ that provides a number of services for the host. One factor that may regulate the composition and function of gut microbial communities is dietary toxins. Oxalate is a toxic plant secondary compound (PSC) produced in all major taxa of vascular plants and is consumed by a variety of animals. The mammalian herbivore Neotoma albigula is capable of consuming and degrading large quantities of dietary oxalate. We isolated and characterized oxalate-degrading bacteria from the gut contents of wild-caught animals and used high-throughput sequencing to determine the distribution of potential oxalate-degrading taxa along the gastrointestinal tract. Isolates spanned three genera: Lactobacillus, Clostridium, and Enterococcus. Over half of the isolates exhibited significant oxalate degradation in vitro, and all Lactobacillus isolates contained the oxc gene, one of the genes responsible for oxalate degradation. Although diverse potential oxalate-degrading genera were distributed throughout the gastrointestinal tract, they were most concentrated in the foregut, where dietary oxalate first enters the gastrointestinal tract. We hypothesize that unique environmental conditions present in each gut region provide diverse niches that select for particular functional taxa and communities. PMID:24362432

Biodegradation of di-n-Butyl Phthalate by Achromobacter sp. Isolated from Rural Domestic Wastewater.

PubMed

Jin, Decai; Kong, Xiao; Li, Yujie; Bai, Zhihui; Zhuang, Guoqiang; Zhuang, Xuliang; Deng, Ye

2015-10-26

A bacterial strain W-1, isolated from rural domestic wastewater, can utilize the environmental hormone di-n-butyl phthalate (DBP) as the sole carbon and energy source. The isolated bacterium species was confirmed to belong to the genus Achromobacter based on its 16S rRNA gene sequence. The results of substrate utilization tests showed that the strain W-1 could utilize other common phthalates and phenol. High-performance liquid chromatography analysis revealed that the optimal conditions for DBP degradation were pH 7.0, 35 °C, and an agitation rate of 175 rpm. Under these conditions, 500 mg/L of DBP was completely degraded within 30 h. The effects of heavy metals (50 mg/L Cu(2+) and 500 mg/L Pb(2+)) and surfactants (100 mg/L SDS and 500 mg/L Tween 20) on DBP degradation were investigated. The results demonstrated that Cu(2+) and SDS severely inhibited DBP degradation and Pb(2+) weakly inhibited DBP degradation, while Tween 20 greatly enhanced DBP degradation. Furthermore, phthalate degradation genes were found to be located on a plasmid present in Achromobacter sp. W-1.

Characterization of hydrocarbon-degrading and biosurfactant-producing Pseudomonas sp. P-1 strain as a potential tool for bioremediation of petroleum-contaminated soil.

PubMed

Pacwa-Płociniczak, Magdalena; Płaza, Grażyna Anna; Poliwoda, Anna; Piotrowska-Seget, Zofia

2014-01-01

The Pseudomonas sp. P-1 strain, isolated from heavily petroleum hydrocarbon-contaminated soil, was investigated for its capability to degrade hydrocarbons and produce a biosurfactant. The strain degraded crude oil, fractions A5 and P3 of crude oil, and hexadecane (27, 39, 27 and 13% of hydrocarbons added to culture medium were degraded, respectively) but had no ability to degrade phenanthrene. Additionally, the presence of gene-encoding enzymes responsible for the degradation of alkanes and naphthalene in the genome of the P-1 strain was reported. Positive results of blood agar and methylene blue agar tests, as well as the presence of gene rhl, involved in the biosynthesis of rhamnolipid, confirmed the ability of P-1 for synthesis of glycolipid biosurfactant. 1H and 13C nuclear magnetic resonance, Fourier transform infrared spectrum and mass spectrum analyses indicated that the extracted biosurfactant was affiliated with rhamnolipid. The results of this study indicate that the P-1 and/or biosurfactant produced by this strain have the potential to be used in bioremediation of hydrocarbon-contaminated soils.

Impact of sources of environmental degradation on microbial community dynamics in non-polluted and metal-polluted soils.

PubMed

Epelde, Lur; Martín-Sánchez, Iker; González-Oreja, José A; Anza, Mikel; Gómez-Sagasti, María T; Garbisu, Carlos

2012-09-01

Soils are currently being degraded at an alarming rate due to increasing pressure from different sources of environmental degradation. Consequently, we carried out a 4-month microcosm experiment to measure the impact of different sources of environmental degradation (biodiversity loss, nitrogen deposition and climate change) on soil health in a non-polluted (non-degraded) and a heavily metal-polluted (degraded) soil, and to compare their responses. To this aim, we determined a variety of soil microbial properties with potential as bioindicators of soil health: basal respiration; β-glucosaminidase and protease activities; abundance (Q-PCR) of bacterial, fungal and chitinase genes; richness (PCR-DGGE) of fungal and chitinase genes. Non-polluted and metal-polluted soils showed different response microbial dynamics when subjected to sources of environmental degradation. The non-polluted soil appeared resilient to "biodiversity loss" and "climate change" treatments. The metal-polluted soil was probably already too severely affected by the presence of high levels of toxic metals to respond to other sources of stress. Our data together suggests that soil microbial activity and biomass parameters are more sensitive to the applied sources of environmental degradation, showing immediate responses of greater magnitude, while soil microbial diversity parameters do not show such variations. Copyright © 2012 Elsevier B.V. All rights reserved.

Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

PubMed

Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

2013-10-17

Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

RNA interference (RNAI) as a tool to engineer high nutritional value in chicory (Chicorium intybus).

PubMed

Asad, M

2006-01-01

The major component of chicory (Chicorium intybus) root is inulin, which is a polymer of fructose. Inulin production from chicory is hampered by the enzyme fructan 1-exohydrolase (1-FEH) that degrades inulin and limits its yield. Increased FEH activity results in massive breakdown of fructan and production of Fructose and inulo-n-oses. The latter phenomena are to be avoided for industrial fructan production. RNA silencing, which is termed post-transcriptional gene silencing (PTGS) in plants, is an RNA degradation process through sequence specific nucleotide interactions induced by double-stranded RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. We are generating a transgenic chicory plants with suppressed FEH (exohydrolas) genes using RNAi resulting in supressed inulin degradation. A small but important part of the construct is a sequence unique for the target gene (exons) or genes,which were cloned. The hairpin constructs were made and chicory was transformed by Agrobacterium tumifaciense, strain (C58C1). The transgenics should be select and check by means of molecular techniques.

Association of cerebrospinal fluid Aβ42 with A2M gene in cognitively normal subjects

PubMed Central

Millard, Steven P.; Lutz, Franziska; Li, Ge; Galasko, Douglas R.; Farlow, Martin R.; Quinn, Joseph F.; Kaye, Jeffrey A.; Leverenz, James B.; Tsuang, Debby; Yu, Chang-En; Peskind, Elaine R.; Bekris, Lynn M.

2013-01-01

Low cerebrospinal fluid (CSF) Aβ42 levels correlate with increased brain Aβ deposition in Alzheimer’s disease (AD), which suggests a disruption in the degradation and clearance of Aβ from the brain. In addition, APOE ε4 carriers have lower CSF Aβ42 levels than non-carriers. The hypothesis of this investigation was that CSF Aβ42 levels correlate with regulatory region variation in genes that are biologically associated with degradation or clearance of Aβ from the brain. CSF Aβ42 levels were tested for associations with Aβ degradation and clearance genes and APOE ε4. Twenty-four SNPs located within the 5′ and 3′ regions of 12 genes were analyzed. The study sample consisted of 99 AD patients and 168 cognitively normal control subjects. CSF Aβ42 levels were associated with APOE ε4 status in controls but not in AD patients; A2M regulatory region SNPs were also associated with CSF Aβ42 levels in controls, but not in AD patients, even after adjusting for APOE ε4. These results suggest that genetic variation within the A2M gene influences CSF Aβ42 levels. PMID:24011543

Effects of land-use management on soil microbes to degrade organic matter through captured metagenomics and metatranscriptomics

NASA Astrophysics Data System (ADS)

Manoharan, Lokeshwaran; Ahren, Dag; Urich, Tim; Hedlund, Katarina

2017-04-01

The role of microbial communities in different soil ecosystem processes has been hard to determine in the past due to their vast diversity both in terms of taxonomy and functions. Molecular methods such as high-throughput sequencing of environmental communities have made it easier to delve into these diverse ecosystems and understand their functions. Trait-based approaches through quantification of functional genes and their expression have shown to be much more meaningful in explaining ecosystem functioning than the taxonomy based approaches. One such approach is the "captured metagenomics" technique where only the genetic regions of functional enzymes involved in a particular ecosystem process such as carbon metabolism is targeted from the genetic pool and sequenced. This allows focused investigations of ecosystem processes through functional genes in complex environments such as soils. In our study, we have implemented this method to look into the effects of land-use management on the functional genetic diversity of microbial communities to degrade soil organic matter (SOM). Soils from different agricultural and grassland fields in southern Sweden were chosen in this study. Oligonucleotide probes were generated based on the genetic sequences of enzymes involved in organic matter degradation from public databases. On the DNA level, there was a significant shift in the functional genetic diversity of microbes to degrade SOM due to land-use management. Grasslands had a higher abundance and diversity of genes coding for enzymes involved in SOM degradation than agricultural soils. The amount of nitrogen was the main factor that affected the functional diversity of the microbes that degrade SOM in these soils. Interestingly, there was no correlation between the functional diversity of microbes to their taxonomic diversity measured through traditional ribosomal sequencing. In addition, for the first time the capture method was used in large scale, targeting many genes coding for SOM degrading enzymes coupled with RNA/cDNA from the soils to quantify their expressions. For this, the soils from different land-use managements were treated with straw, while the microbial growth rates in these soils were also monitored for a month. RNA was extracted from at three different time points from both treated and untreated soils from different land-use managements. Agricultural soils with straw addition had higher relative microbial growth rates and higher abundance of gene sequences captured compared to the control and grassland soils. Land-use management seems to be the most significant factor in affecting the expression of SOM degrading genes in these soils. Additional analyses of the generated data are expected to provide valuable insights on how land-use management affects the microbial responses during addition of organic matter in soils.

Draft Genome Sequence of Paenibacillus sp. Strain DMB20, Isolated from Alang Ship-Breaking Yard, Which Harbors Genes for Xenobiotic Degradation.

PubMed

Shah, Binal; Jain, Kunal; Patel, Namrata; Pandit, Ramesh; Patel, Anand; Joshi, Chaitanya G; Madamwar, Datta

2015-06-11

Paenibacillus sp. strain DMB20, in cometabolism with other Proteobacteria and Firmicutes, exhibits azoreduction of textile dyes. Here, we report the draft genome sequence of this bacterium, consisting of 6,647,181 bp with 7,668 coding sequences (CDSs). The data presented highlight multiple sets of functional genes associated with xenobiotic compound degradation. Copyright © 2015 Shah et al.

A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect

PubMed Central

Lopez-Serra, Paula; Marcilla, Miguel; Villanueva, Alberto; Ramos-Fernandez, Antonio; Palau, Anna; Leal, Lucía; Wahi, Jessica E.; Setien-Baranda, Fernando; Szczesna, Karolina; Moutinho, Catia; Martinez-Cardus, Anna; Heyn, Holger; Sandoval, Juan; Puertas, Sara; Vidal, August; Sanjuan, Xavier; Martinez-Balibrea, Eva; Viñals, Francesc; Perales, Jose C.; Bramsem, Jesper B.; Ørntoft, Torben F.; Andersen, Claus L.; Tabernero, Josep; McDermott, Ultan; Boxer, Matthew B.; Heiden, Matthew G. Vander; Albar, Juan Pablo; Esteller, Manel

2014-01-01

Cancer cells possess aberrant proteomes that can arise by the disruption of genes involved in physiological protein degradation. Here we demonstrate the presence of promoter CpG island hypermethylation-linked inactivation of DERL3 (Derlin-3), a key gene in the endoplasmic reticulum-associated protein degradation pathway, in human tumours. The restoration of in vitro and in vivo DERL3 activity highlights the tumour suppressor features of the gene. Using the stable isotopic labelling of amino acids in cell culture workflow for differential proteome analysis, we identify SLC2A1 (glucose transporter 1, GLUT1) as a downstream target of DERL3. Most importantly, SLC2A1 overexpression mediated by DERL3 epigenetic loss contributes to the Warburg effect in the studied cells and pinpoints a subset of human tumours with greater vulnerability to drugs targeting glycolysis. PMID:24699711

Regulation of mRNA translation during mitosis.

PubMed

Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

2015-08-25

Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

Distribution and function of carbamate hydrolase genes cehA and mcd in soils: the distinct role of soil pH.

PubMed

Rousidou, Constantina; Karaiskos, Dionysis; Myti, Despoina; Karanasios, Evangelos; Karas, Panagiotis A; Tourna, Maria; Tzortzakakis, Emmanuel A; Karpouzas, Dimitrios G

2017-01-01

Synthetic carbamates constitute a significant pesticide group with oxamyl being a leading compound in the nematicide market. Oxamyl degradation in soil is mainly microbially mediated. However, the distribution and function of carbamate hydrolase genes (cehA, mcd, cahA) associated with the soil biodegradation of carbamates is not yet clear. We studied oxamyl degradation in 16 soils from a potato monoculture area in Greece where oxamyl is regularly used. Oxamyl showed low persistence (DT50 2.4-26.7 days). q-PCR detected the cehA and mcd genes in 10 and three soils, respectively. The abundance of the cehA gene was positively correlated with pH, while both cehA abundance and pH were negatively correlated with oxamyl DT50. Amongst the carbamates used in the study region, oxamyl stimulated the abundance and expression only of the cehA gene, while carbofuran stimulated the abundance and expression of both genes. The cehA gene was also detected in pristine soils upon repeated treatments with oxamyl and carbofuran and only in soils with pH ≥7.2, where the most rapid degradation of oxamyl was observed. These results have major implications regarding the maintenance of carbamate hydrolase genes in soils, have practical implications regarding the agricultural use of carbamates, and provide insights into the evolution of cehA. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Involvement of Two Latex-Clearing Proteins during Rubber Degradation and Insights into the Subsequent Degradation Pathway Revealed by the Genome Sequence of Gordonia polyisoprenivorans Strain VH2

PubMed Central

Hiessl, Sebastian; Schuldes, Jörg; Thürmer, Andrea; Halbsguth, Tobias; Bröker, Daniel; Angelov, Angel; Liebl, Wolfgang; Daniel, Rolf

2012-01-01

The increasing production of synthetic and natural poly(cis-1,4-isoprene) rubber leads to huge challenges in waste management. Only a few bacteria are known to degrade rubber, and little is known about the mechanism of microbial rubber degradation. The genome of Gordonia polyisoprenivorans strain VH2, which is one of the most effective rubber-degrading bacteria, was sequenced and annotated to elucidate the degradation pathway and other features of this actinomycete. The genome consists of a circular chromosome of 5,669,805 bp and a circular plasmid of 174,494 bp with average GC contents of 67.0% and 65.7%, respectively. It contains 5,110 putative protein-coding sequences, including many candidate genes responsible for rubber degradation and other biotechnically relevant pathways. Furthermore, we detected two homologues of a latex-clearing protein, which is supposed to be a key enzyme in rubber degradation. The deletion of these two genes for the first time revealed clear evidence that latex-clearing protein is essential for the microbial utilization of rubber. Based on the genome sequence, we predict a pathway for the microbial degradation of rubber which is supported by previous and current data on transposon mutagenesis, deletion mutants, applied comparative genomics, and literature search. PMID:22327575

Metagenomic Functional Potential Predicts Degradation Rates of a Model Organophosphorus Xenobiotic in Pesticide Contaminated Soils

PubMed Central

Jeffries, Thomas C.; Rayu, Smriti; Nielsen, Uffe N.; Lai, Kaitao; Ijaz, Ali; Nazaries, Loic; Singh, Brajesh K.

2018-01-01

Chemical contamination of natural and agricultural habitats is an increasing global problem and a major threat to sustainability and human health. Organophosphorus (OP) compounds are one major class of contaminant and can undergo microbial degradation, however, no studies have applied system-wide ecogenomic tools to investigate OP degradation or use metagenomics to understand the underlying mechanisms of biodegradation in situ and predict degradation potential. Thus, there is a lack of knowledge regarding the functional genes and genomic potential underpinning degradation and community responses to contamination. Here we address this knowledge gap by performing shotgun sequencing of community DNA from agricultural soils with a history of pesticide usage and profiling shifts in functional genes and microbial taxa abundance. Our results showed two distinct groups of soils defined by differing functional and taxonomic profiles. Degradation assays suggested that these groups corresponded to the organophosphorus degradation potential of soils, with the fastest degrading community being defined by increases in transport and nutrient cycling pathways and enzymes potentially involved in phosphorus metabolism. This was against a backdrop of taxonomic community shifts potentially related to contamination adaptation and reflecting the legacy of exposure. Overall our results highlight the value of using holistic system-wide metagenomic approaches as a tool to predict microbial degradation in the context of the ecology of contaminated habitats. PMID:29515526

[Degradation characteristics of naphthalene with a Pseudomonas aeruginosa strain isolated from soil contaminated by diesel].

PubMed

Liu, Wen-Chao; Wu, Bin-Bin; Li, Xiao-Sen; Lu, Dian-Nan; Liu, Yong-Min

2015-02-01

Abstract: A naphthalene-degrading bacterium (referred as HD-5) was isolated from the diesel-contaminated soil and was assigned to Pseudomonas aeruginosa according to 16S rDNA sequences analysis. Gene nah, which encodes naphthalene dioxygenase, was identified from strain HD-5 by PCR amplification. Different bioremediation approaches, including nature attenuation, bioaugmentation with strain Pseudomonas aeruginosa, biostimulation, and an integrated degradation by bioaugmentation and biostimulation, were evaluated for their effectiveness in the remediating soil containing 5% naphthalene. The degradation rates of naphthalene in the soil were compared among the different bioremediation approaches, the FDA and dehydrogenase activity in bioremediation process were measured, and the gene copy number of 16S rRNA and nah in soil were dynamically monitored using real-time PCR. It was shown that the naphthalene removal rate reached 71.94%, 62.22% and 83.14% in approaches of bioaugmentation (B), biostimulation(S) and integrated degradation composed of bioaugmentation and biostimulation (BS), respectively. The highest removal rate of naphthalene was achieved by using BS protocol, which also gives the highest FDA and dehydrogenase activity. The gene copy number of 16S rRNA and nah in soil increased by about 2.67 x 10(11) g(-1) and 8.67 x 10(8) g(-1) after 31 days treatment using BS protocol. Above-mentioned results also demonstrated that the screened bacterium, Pseudomonas aeruginosa, could grow well in naphthalene-contaminated soil and effectively degrade naphthalene, which is of fundamental importance for bioremediation of naphthalene-contaminated soil.

Distribution of Anaerobic Hydrocarbon-Degrading Bacteria in Soils from King George Island, Maritime Antarctica.

PubMed

Sampaio, Dayanna Souza; Almeida, Juliana Rodrigues Barboza; de Jesus, Hugo E; Rosado, Alexandre S; Seldin, Lucy; Jurelevicius, Diogo

2017-11-01

Anaerobic diesel fuel Arctic (DFA) degradation has already been demonstrated in Antarctic soils. However, studies comparing the distribution of anaerobic bacterial groups and of anaerobic hydrocarbon-degrading bacteria in Antarctic soils containing different concentrations of DFA are scarce. In this study, functional genes were used to study the diversity and distribution of anaerobic hydrocarbon-degrading bacteria (bamA, assA, and bssA) and of sulfate-reducing bacteria (SRB-apsR) in highly, intermediate, and non-DFA-contaminated soils collected during the summers of 2009, 2010, and 2011 from King George Island, Antarctica. Signatures of bamA genes were detected in all soils analyzed, whereas bssA and assA were found in only 4 of 10 soils. The concentration of DFA was the main factor influencing the distribution of bamA-containing bacteria and of SRB in the analyzed soils, as shown by PCR-DGGE results. bamA sequences related to genes previously described in Desulfuromonas, Lautropia, Magnetospirillum, Sulfuritalea, Rhodovolum, Rhodomicrobium, Azoarcus, Geobacter, Ramlibacter, and Gemmatimonas genera were dominant in King George Island soils. Although DFA modulated the distribution of bamA-hosting bacteria, DFA concentration was not related to bamA abundance in the soils studied here. This result suggests that King George Island soils show functional redundancy for aromatic hydrocarbon degradation. The results obtained in this study support the hypothesis that specialized anaerobic hydrocarbon-degrading bacteria have been selected by hydrocarbon concentrations present in King George Island soils.

Bacterial Degraders of Coexisting Dichloromethane, Benzene, and Toluene, Identified by Stable-Isotope Probing.

PubMed

Yoshikawa, Miho; Zhang, Ming; Kurisu, Futoshi; Toyota, Koki

2017-01-01

Most bioremediation studies on volatile organic compounds (VOCs) have focused on a single contaminant or its derived compounds and degraders have been identified under single contaminant conditions. Bioremediation of multiple contaminants remains a challenging issue. To identify a bacterial consortium that degrades multiple VOCs (dichloromethane (DCM), benzene, and toluene), we applied DNA-stable isotope probing. For individual tests, we combined a 13 C-labeled VOC with other two unlabeled VOCs, and prepared three unlabeled VOCs as a reference. Over 11 days, DNA was periodically extracted from the consortia, and the bacterial community was evaluated by next-generation sequencing of bacterial 16S rRNA gene amplicons. Density gradient fractions of the DNA extracts were amplified by universal bacterial primers for the 16S rRNA gene sequences, and the amplicons were analyzed by terminal restriction fragment length polymorphism (T-RFLP) using restriction enzymes: Hha I and Msp I. The T-RFLP fragments were identified by 16S rRNA gene cloning and sequencing. Under all test conditions, the consortia were dominated by Rhodanobacter , Bradyrhizobium / Afipia , Rhizobium , and Hyphomicrobium . DNA derived from Hyphomicrobium and Propioniferax shifted toward heavier fractions under the condition added with 13 C-DCM and 13 C-benzene, respectively, compared with the reference, but no shifts were induced by 13 C-toluene addition. This implies that Hyphomicrobium and Propioniferax were the main DCM and benzene degraders, respectively, under the coexisting condition. The known benzene degrader Pseudomonas sp. was present but not actively involved in the degradation.

Chemical dispersants enhance the activity of oil- and gas condensate-degrading marine bacteria.

PubMed

Tremblay, Julien; Yergeau, Etienne; Fortin, Nathalie; Cobanli, Susan; Elias, Miria; King, Thomas L; Lee, Kenneth; Greer, Charles W

2017-12-01

Application of chemical dispersants to oil spills in the marine environment is a common practice to disperse oil into the water column and stimulate oil biodegradation by increasing its bioavailability to indigenous bacteria capable of naturally metabolizing hydrocarbons. In the context of a spill event, the biodegradation of crude oil and gas condensate off eastern Canada is an essential component of a response strategy. In laboratory experiments, we simulated conditions similar to an oil spill with and without the addition of chemical dispersant under both winter and summer conditions and evaluated the natural attenuation potential for hydrocarbons in near-surface sea water from the vicinity of crude oil and natural gas production facilities off eastern Canada. Chemical analyses were performed to determine hydrocarbon degradation rates, and metagenome binning combined with metatranscriptomics was used to reconstruct abundant bacterial genomes and estimate their oil degradation gene abundance and activity. Our results show important and rapid structural shifts in microbial populations in all three different oil production sites examined following exposure to oil, oil with dispersant and dispersant alone. We found that the addition of dispersant to crude oil enhanced oil degradation rates and favored the abundance and expression of oil-degrading genes from a Thalassolituus sp. (that is, metagenome bin) that harbors multiple alkane hydroxylase (alkB) gene copies. We propose that this member of the Oceanospirillales group would be an important oil degrader when oil spills are treated with dispersant.

Nutrients Can Enhance the Abundance and Expression of Alkane Hydroxylase CYP153 Gene in the Rhizosphere of Ryegrass Planted in Hydrocarbon-Polluted Soil

PubMed Central

Arslan, Muhammad; Afzal, Muhammad; Amin, Imran; Iqbal, Samina; Khan, Qaiser M.

2014-01-01

Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K) in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum) was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination. PMID:25360680

Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii

PubMed Central

Wang, Sen; Zhao, Dong; Bai, Xinfeng; Zhang, Weican

2016-01-01

ABSTRACT Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220, encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii. A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN3. Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220. Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases. IMPORTANCE Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii. PMID:27742681

FibroChip, a Functional DNA Microarray to Monitor Cellulolytic and Hemicellulolytic Activities of Rumen Microbiota

PubMed Central

Comtet-Marre, Sophie; Chaucheyras-Durand, Frédérique; Bouzid, Ourdia; Mosoni, Pascale; Bayat, Ali R.; Peyret, Pierre; Forano, Evelyne

2018-01-01

Ruminants fulfill their energy needs for growth primarily through microbial breakdown of plant biomass in the rumen. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact animal productivity and health. To provide more insight into mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes encoding key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. Eight carbohydrate-active enzyme (CAZyme) families (GH5, GH9, GH10, GH11, GH43, GH48, CE1, and CE6) were selected which represented 392 genes from bacteria, protozoa, and fungi. The DNA microarray, designated as FibroChip, was validated using targets of increasing complexity and demonstrated sensitivity and specificity. In addition, FibroChip was evaluated for its explorative and semi-quantitative potential. Differential expression of CAZyme genes was evidenced in the rumen bacterium Fibrobacter succinogenes S85 grown on wheat straw or cellobiose. FibroChip was used to identify the expressed CAZyme genes from the targeted families in the rumen of a cow fed a mixed diet based on grass silage. Among expressed genes, those encoding GH43, GH5, and GH10 families were the most represented. Most of the F. succinogenes genes detected by the FibroChip were also detected following RNA-seq analysis of RNA transcripts obtained from the rumen fluid sample. Use of the FibroChip also indicated that transcripts of fiber degrading enzymes derived from eukaryotes (protozoa and anaerobic fungi) represented a significant proportion of the total microbial mRNA pool. FibroChip represents a reliable and high-throughput tool that enables researchers to monitor active members of fiber degradation in the rumen. PMID:29487591

FibroChip, a Functional DNA Microarray to Monitor Cellulolytic and Hemicellulolytic Activities of Rumen Microbiota.

PubMed

Comtet-Marre, Sophie; Chaucheyras-Durand, Frédérique; Bouzid, Ourdia; Mosoni, Pascale; Bayat, Ali R; Peyret, Pierre; Forano, Evelyne

2018-01-01

Ruminants fulfill their energy needs for growth primarily through microbial breakdown of plant biomass in the rumen. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact animal productivity and health. To provide more insight into mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes encoding key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. Eight carbohydrate-active enzyme (CAZyme) families (GH5, GH9, GH10, GH11, GH43, GH48, CE1, and CE6) were selected which represented 392 genes from bacteria, protozoa, and fungi. The DNA microarray, designated as FibroChip, was validated using targets of increasing complexity and demonstrated sensitivity and specificity. In addition, FibroChip was evaluated for its explorative and semi-quantitative potential. Differential expression of CAZyme genes was evidenced in the rumen bacterium Fibrobacter succinogenes S85 grown on wheat straw or cellobiose. FibroChip was used to identify the expressed CAZyme genes from the targeted families in the rumen of a cow fed a mixed diet based on grass silage. Among expressed genes, those encoding GH43, GH5, and GH10 families were the most represented. Most of the F. succinogenes genes detected by the FibroChip were also detected following RNA-seq analysis of RNA transcripts obtained from the rumen fluid sample. Use of the FibroChip also indicated that transcripts of fiber degrading enzymes derived from eukaryotes (protozoa and anaerobic fungi) represented a significant proportion of the total microbial mRNA pool. FibroChip represents a reliable and high-throughput tool that enables researchers to monitor active members of fiber degradation in the rumen.

Significant alteration of soil bacterial communities and organic carbon decomposition by different long-term fertilization management conditions of extremely low-productivity arable soil in South China.

PubMed

Xun, Weibing; Zhao, Jun; Xue, Chao; Zhang, Guishan; Ran, Wei; Wang, Boren; Shen, Qirong; Zhang, Ruifu

2016-06-01

Different fertilization managements of red soil, a kind of Ferralic Cambisol, strongly affected the soil properties and associated microbial communities. The association of the soil microbial community and functionality with long-term fertilization management in the unique low-productivity red soil ecosystem is important for both soil microbial ecology and agricultural production. Here, 454 pyrosequencing analysis of 16S recombinant ribonucleic acid genes and GeoChip4-NimbleGen-based functional gene analysis were used to study the soil bacterial community composition and functional genes involved in soil organic carbon degradation. Long-term nitrogen-containing chemical fertilization-induced soil acidification and fertility decline and significantly altered the soil bacterial community, whereas long-term organic fertilization and fallow management improved the soil quality and maintained the bacterial diversity. Short-term quicklime remediation of the acidified soils did not change the bacterial communities. Organic fertilization and fallow management supported eutrophic ecosystems, in which copiotrophic taxa increased in relative abundance and have a higher intensity of labile-C-degrading genes. However, long-term nitrogen-containing chemical fertilization treatments supported oligotrophic ecosystems, in which oligotrophic taxa increased in relative abundance and have a higher intensity of recalcitrant-C-degrading genes but a lower intensity of labile-C-degrading genes. Quicklime application increased the relative abundance of copiotrophic taxa and crop production, although these effects were utterly inadequate. This study provides insights into the interaction of soil bacterial communities, soil functionality and long-term fertilization management in the red soil ecosystem; these insights are important for improving the fertility of unique low-productivity red soil. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Draft genome sequence of Micrococcus luteus strain O'Kane implicates metabolic versatility and the potential to degrade polyhydroxybutyrates.

PubMed

Hanafy, Radwa A; Couger, M B; Baker, Kristina; Murphy, Chelsea; O'Kane, Shannon D; Budd, Connie; French, Donald P; Hoff, Wouter D; Youssef, Noha

2016-09-01

Micrococcus luteus is a predominant member of skin microbiome. We here report on the genomic analysis of Micrococcus luteus strain O'Kane that was isolated from an elevator. The partial genome assembly of Micrococcus luteus strain O'Kane is 2.5 Mb with 2256 protein-coding genes and 62 RNA genes. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of glucose, galactose, fructose, mannose, alanine, aspartate, asparagine, glutamate, glutamine, glycine, serine, cysteine, methionine, arginine, proline, histidine, phenylalanine, and fatty acids. Genomic comparison to other M. luteus representatives identified the potential to degrade polyhydroxybutyrates, as well as several antibiotic resistance genes absent from other genomes.

Role of the ganSPQAB Operon in Degradation of Galactan by Bacillus subtilis.

PubMed

Watzlawick, Hildegard; Morabbi Heravi, Kambiz; Altenbuchner, Josef

2016-10-15

Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the -35 box of the σ(A)-type promoter of Pgan, which is located upstream of ganS IMPORTANCE: Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis Nevertheless, the B. subtilis utilization system of galactan, which is found as the side chain of the rhamnogalacturonan type I complex in pectin, has remained partially studied. Here, we investigated the galactan utilization system consisting of the ganSPQAB operon and its regulator ganR This study improves our knowledge of the carbohydrate degradation systems of B. subtilis, especially the pectin degradation systems. Moreover, the galactan-degrading enzymes may be exploited for the production of galacto-oligosaccharides, which are used as prebiotic substances in the food industry. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Biodegradation of plastics.

PubMed

Shimao, M

2001-06-01

Widespread studies on the biodegradation of plastics have been carried out in order to overcome the environmental problems associated with synthetic plastic waste. Recent work has included studies of the distribution of synthetic polymer-degrading microorganisms in the environment, the isolation of new microorganisms for biodegradation, the discovery of new degradation enzymes, and the cloning of genes for synthetic polymer-degrading enzymes.

The effects of lactate and acid on articular chondrocytes function: Implications for polymeric cartilage scaffold design.

PubMed

Zhang, Xiaolei; Wu, Yan; Pan, Zongyou; Sun, Heng; Wang, Junjuan; Yu, Dongsheng; Zhu, Shouan; Dai, Jun; Chen, Yishan; Tian, Naifeng; Heng, Boon Chin; Coen, Noelle D; Xu, Huazi; Ouyang, Hongwei

2016-09-15

Poly (lactic-co-glycolic acid) (PLGA) and poly-l-lactate acid (PLLA) are biodegradable polymers widely utilized as scaffold materials for cartilage tissue engineering. Their acid degradation products have been widely recognized as being detrimental to cell function. However, the biological effects of lactate, rather than lactic acid, on chondrocytes have never been investigated. This is the major focus of this study. The amounts of lactate and the pH value (acid) of the PLGA and PLLA degradation medium were measured. The effects of PLGA and PLLA degradation medium, as well as different lactate concentrations and timing of exposure on chondrocytes proliferation and cartilage-specific matrix synthesis were investigated by various techniques including global gene expression profiling and gene knockdown experiments. It was shown that PLGA and PLLA degradation medium differentially regulated chondrocyte proliferation and matrix synthesis. Acidic pH caused by lactate inhibited chondrocyte proliferation and matrix synthesis. The effect of lactate on chondrocyte matrix synthesis was both time and dose dependent. A lactate concentration of 100mM and exposure duration of 8h significantly enhanced matrix synthesis. Lactate could also inhibit expression of cartilage matrix degradation genes in osteoarthritic chondrocytes, such as the major aggrecanase ADAMTS5, whilst promoting matrix synthesis simultaneously. Pulsed addition of lactate was shown to be more efficient in promoting COL2A1 expression. Global gene expression data and gene knock down experiments demonstrated that lactate promote matrix synthesis through up-regulation of HIF1A. These observed differential biological effects of lactate on chondrocytes would have implications for the future design of polymeric cartilage scaffolds. Lactic acid is a widely used substrate for polymers synthesis, PLGA and PLLA in particular. Although physical and biological modifications have been made on these polymers to make them be better cartilage scaffolds, little concern has been given on the biological effect of lactic acid, the main degradation product of these polymers, on chondrocytes. Our finding illustrates the differential biological function of lactate and acid on chondrocytes matrix synthesis. These results can facilitate future design of lactate polymers-based cartilage scaffolds. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86

PubMed Central

Mohan, Karishma

2017-01-01

ABSTRACT Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz., veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the β-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz., VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products. IMPORTANCE Pseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the metabolic pathway and gene pool for lignin degradation in bacteria. The biochemical and genetic characterization of phenylpropanoid metabolism makes it a prospective system for its application in producing valuable products, such as vanillin and vanillic acid, from lignocellulose. This study supports the immense potential of P. putida CSV86 as a suitable candidate for bioremediation and biorefinery. PMID:28188206

Leaf carbohydrates influence transcriptional and post-transcriptional regulation of nocturnal carboxylation and starch degradation in the facultative CAM plant, Mesembryanthemum crystallinum.

PubMed

Taybi, Tahar; Cushman, John C; Borland, Anne M

2017-11-01

Nocturnal degradation of transitory starch is a limiting factor for the optimal function of crassulacean acid metabolism and must be coordinated with phosphoenolypyruvate carboxylase (PEPC)-mediated CO 2 uptake to optimise carbon gain over the diel cycle. The aim of this study was to test the hypothesis that nocturnal carboxylation is coordinated with starch degradation in CAM via a mechanism whereby the products of these pathways regulate diel transcript abundance and enzyme activities for both processes. To test this hypothesis, a starch and CAM-deficient mutant of Mesembryanthemum crystallinum was compared with wild type plants under well-watered and saline (CAM-inducing) conditions. Exposure to salinity increased the transcript abundance of genes required for nocturnal carboxylation, starch and sucrose degradation in both wild type and mutant, but the transcript abundance of several of these genes was not sustained over the dark period in the low-carbohydrate, CAM-deficient mutant. The diel pattern of transcript abundance for PEPC mirrored that of PEPC protein, as did the transcripts, protein, and activity of chloroplastic starch phosphorylase in both wild type and mutant, suggesting robust diel coordination of these metabolic processes. Activities of several amylase isoforms were low or lacking in the mutant, whilst the activity of a cytosolic isoform of starch phosphorylase was significantly elevated, indicating contrasting modes of metabolic regulation for the hydrolytic and phosphorylytic routes of starch degradation. Externally supplied sucrose resulted in an increase in nocturnal transcript abundance of genes required for nocturnal carboxylation and starch degradation. These results demonstrate that carbohydrates impact on transcriptional and post-transcriptional regulation of nocturnal carboxylation and starch degradation in CAM. Copyright © 2017 Elsevier GmbH. All rights reserved.

An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp.

PubMed

Fang, Fang; Zhang, Xue-lian; Luo, Hong-hui; Zhou, Jia-jian; Gong, Yi-hui; Li, Wen-jun; Shi, Zhao-wan; He, Quan; Wu, Qing; Li, Lu; Jiang, Lin-lin; Cai, Zhi-gao; Oren-Shamir, Michal; Zhang, Zhao-qi; Pang, Xue-qun

2015-12-01

In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning. © 2015 American Society of Plant Biologists. All Rights Reserved.

Modulation of microbial consortia enriched from different polluted environments during petroleum biodegradation.

PubMed

Omrani, Rahma; Spini, Giulia; Puglisi, Edoardo; Saidane, Dalila

2018-04-01

Environmental microbial communities are key players in the bioremediation of hydrocarbon pollutants. Here we assessed changes in bacterial abundance and diversity during the degradation of Tunisian Zarzatine oil by four indigenous bacterial consortia enriched from a petroleum station soil, a refinery reservoir soil, a harbor sediment and seawater. The four consortia were found to efficiently degrade up to 92.0% of total petroleum hydrocarbons after 2 months of incubation. Illumina 16S rRNA gene sequencing revealed that the consortia enriched from soil and sediments were dominated by species belonging to Pseudomonas and Acinetobacter genera, while in the seawater-derived consortia Dietzia, Fusobacterium and Mycoplana emerged as dominant genera. We identified a number of species whose relative abundances bloomed from small to high percentages: Dietzia daqingensis in the seawater microcosms, and three OTUs classified as Acinetobacter venetianus in all two soils and sediment derived microcosms. Functional analyses on degrading genes were conducted by comparing PCR results of the degrading genes alkB, ndoB, cat23, xylA and nidA1 with inferences obtained by PICRUSt analysis of 16S amplicon data: the two data sets were partly in agreement and suggest a relationship between the catabolic genes detected and the rate of biodegradation obtained. The work provides detailed insights about the modulation of bacterial communities involved in petroleum biodegradation and can provide useful information for in situ bioremediation of oil-related pollution.

Temporal transcriptome analysis of the white-rot fungus Obba rivulosa shows expression of a constitutive set of plant cell wall degradation targeted genes during growth on solid spruce wood.

PubMed

Marinović, Mila; Aguilar-Pontes, Maria Victoria; Zhou, Miaomiao; Miettinen, Otto; de Vries, Ronald P; Mäkelä, Miia R; Hildén, Kristiina

2018-03-01

The basidiomycete white-rot fungus Obba rivulosa, a close relative of Gelatoporia (Ceriporiopsis) subvermispora, is an efficient degrader of softwood. The dikaryotic O. rivulosa strain T241i (FBCC949) has been shown to selectively remove lignin from spruce wood prior to depolymerization of plant cell wall polysaccharides, thus possessing potential in biotechnological applications such as pretreatment of wood in pulp and paper industry. In this work, we studied the time-course of the conversion of spruce by the genome-sequenced monokaryotic O. rivulosa strain 3A-2, which is derived from the dikaryon T241i, to get insight into transcriptome level changes during prolonged solid state cultivation. During 8-week cultivation, O. rivulosa expressed a constitutive set of genes encoding putative plant cell wall degrading enzymes. High level of expression of the genes targeted towards all plant cell wall polymers was detected at 2-week time point, after which majority of the genes showed reduced expression. This implicated non-selective degradation of lignin by the O. rivulosa monokaryon and suggests high variation between mono- and dikaryotic strains of the white-rot fungi with respect to their abilities to convert plant cell wall polymers. Copyright © 2017 Elsevier Inc. All rights reserved.

Land use type significantly affects microbial gene transcription in soil.

PubMed

Nacke, Heiko; Fischer, Christiane; Thürmer, Andrea; Meinicke, Peter; Daniel, Rolf

2014-05-01

Soil microorganisms play an essential role in sustaining biogeochemical processes and cycling of nutrients across different land use types. To gain insights into microbial gene transcription in forest and grassland soil, we isolated mRNA from 32 sampling sites. After sequencing of generated complementary DNA (cDNA), a total of 5,824,229 sequences could be further analyzed. We were able to assign nonribosomal cDNA sequences to all three domains of life. A dominance of bacterial sequences, which were affiliated to 25 different phyla, was found. Bacterial groups capable of aromatic compound degradation such as Phenylobacterium and Burkholderia were detected in significantly higher relative abundance in forest soil than in grassland soil. Accordingly, KEGG pathway categories related to degradation of aromatic ring-containing molecules (e.g., benzoate degradation) were identified in high abundance within forest soil-derived metatranscriptomic datasets. The impact of land use type forest on community composition and activity is evidently to a high degree caused by the presence of wood breakdown products. Correspondingly, bacterial groups known to be involved in lignin degradation and containing ligninolytic genes such as Burkholderia, Bradyrhizobium, and Azospirillum exhibited increased transcriptional activity in forest soil. Higher solar radiation in grassland presumably induced increased transcription of photosynthesis-related genes within this land use type. This is in accordance with high abundance of photosynthetic organisms and plant-infecting viruses in grassland.

Novel Scheme for Biosynthesis of Aryl Metabolites from l-Phenylalanine in the Fungus Bjerkandera adusta

PubMed Central

Lapadatescu, Carmen; Giniès, Christian; Le Quéré, Jean-Luc; Bonnarme, Pascal

2000-01-01

Aryl metabolite biosynthesis was studied in the white rot fungus Bjerkandera adusta cultivated in a liquid medium supplemented with l-phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors (14C- and 13C-labelled l-phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in B. adusta cultures. Metabolite formation required de novo protein biosynthesis. Our results show that l-phenylalanine was deaminated to trans-cinnamic acid by a phenylalanine ammonia lyase and trans-cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from l-phenylalanine to aryl metabolites. Additionally, trans-cinnamic acid can also be transformed via β-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a β-oxidation degradation intermediate. To our knowledge, this is the first time that a β-oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from l-phenylalanine is proposed. PMID:10742235

Comparable dynamics of linuron catabolic genes and IncP-1 plasmids in biopurification systems (BPSs) as a response to linuron spiking.

PubMed

Nour, Eman H; Elsayed, Tarek R; Springael, Dirk; Smalla, Kornelia

2017-06-01

On-farm biopurification systems (BPSs) represent an efficient technology for treating pesticide-contaminated wastewater. Biodegradation by genetically adapted bacteria has been suggested to perform a major contribution to the removal of pesticides in BPSs. Recently, several studies pointed to the role of IncP-1 plasmids in the degradation of pesticides in BPSs but this was never linked with catabolic markers. Therefore, a microcosm experiment was conducted in order to examine whether changes in mobile genetic element (MGE) abundances in response to the application of phenylurea herbicide linuron are linked with changes in catabolic genes. Denaturing gradient gel electrophoresis (DGGE) fingerprints of 16S ribosomal RNA gene fragments amplified from total community (TC)-DNA suggested significant shifts in the bacterial community composition. PCR-Southern blot-based detection of genes involved in linuron hydrolysis (libA and hylA) or degradation of its metabolite 3,4-dichloroaniline (dcaQ I , dcaQ II , and ccdC) in TC-DNA showed that the abundance of the hylA gene was increased faster and stronger in response to linuron application than that of the libA gene, and that the dcaQ II gene was more abundant than the isofunctional gene dcaQ I 20 and 60 days after linuron addition. Furthermore, a significant increase in the relative abundance of the IncP-1-specific korB gene in response to linuron was recorded. Our data suggest that different bacterial populations bearing isofunctional genes coding for enzymes degrading linuron seemed to be enriched in BPSs in response to linuron and that IncP-1 plasmids might be involved in their dissemination.

Bacterial diversity and real-time PCR based assessment of linA and linB gene distribution at hexachlorocyclohexane contaminated sites.

PubMed

Lal, Devi; Jindal, Swati; Kumari, Hansi; Jit, Simran; Nigam, Aeshna; Sharma, Pooja; Kumari, Kirti; Lal, Rup

2015-03-01

The disposal of hexachlorocyclohexane (HCH) muck has created large number of HCH dumpsites all over the world from where the harmful HCH isomers are leaking into the environment. Bacteria have evolved at such contaminated sites that have the ability to degrade HCH. Degradation of various HCH isomers in bacterial strains is mediated primarily by two genes: linA and linB which encode dehydrochlorinase and haloalkane dehalogenase respectively. In this study we explored one such highly contaminated HCH dumpsite located in Lucknow, Uttar Pradesh, India. To assess the biostimulation potential of the contaminated site, microbial diversity study and real-time PCR based quantification of lin genes was carried out. The soil samples from dumpsite and surrounding areas were found to be highly contaminated with HCH residue levels as high as 1.8 × 10(5)  mg kg(-1). The residues were detected in areas upto 13 km from the dumpsite. Sphingomonads, Chromohalobacter, and Marinobacter were the dominant genera present at the dump-site. Role of Sphingomonads in HCH degradation has been well documented. The highest copy numbers of linA and linB genes as determined using real-time PCR were 6.2 × 10(4) and 5.3 × 10(5), respectively, were found in sample from the dump site. The presence of Sphingomonads, linA, and linB genes from HCH contaminated soil indicates the presence of indigenous bacterial communities capable of HCH degradation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microbial community characterization and functional gene quantification in RDX-degrading microcosms derived from sediment and groundwater at two naval sites.

PubMed

Wilson, Fernanda Paes; Cupples, Alison M

2016-08-01

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has long been recognized as a problematic environmental pollutant, and efforts to remediate contaminated soils, sediments, and groundwater have been going on for decades. In recent years, much interest has focused on using bioremediation to clean up these sites. The current study investigated the microorganisms (16S rRNA genes, Illumina) and functional genes (xenA, xenB, and xplA) linked to RDX biodegradation in microcosms composed of sediment or groundwater from two Navy sites. For this, experiments included sediment samples from three depths (5 to 30 ft) from two wells located in one Navy site. In addition, the groundwater upstream and downstream of an emulsified oil biobarrier was examined from another Navy site. Further, for the groundwater experiments, the effect of glucose addition was explored. For the sediment experiments, the most enriched phylotypes during RDX degradation varied over time, by depth and well locations. However, several trends were noted, including the enrichment of Pseudomonas, Rhodococcus, Arthrobacter, and Sporolactobacillus in the sediment microcosms. For the groundwater-based experiments, Pseudomonas, unclassified Rhodocyclaceae, Sphingomonas, and Rhodococcus were also highly abundant during RDX degradation. The abundance of both xplA and xenA significantly increased during RDX degradation compared to the control microcosms for many treatments (both groundwater and sediment microcosms). In a limited number of microcosms, the copy number of the xenB gene increased. Phylotype data were correlated with functional gene data to highlight potentially important biomarkers for RDX biodegradation at these two Navy sites.

Transplantation of transduced chondrocytes protects articular cartilage from interleukin 1-induced extracellular matrix degradation.

PubMed Central

Baragi, V M; Renkiewicz, R R; Jordan, H; Bonadio, J; Hartman, J W; Roessler, B J

1995-01-01

Gene therapy used in the context of delivering a therapeutic gene(s) to chondrocytes offers a new approach for treating chondrocyte-mediated cartilage degradation associated with various human arthropathies including osteoarthritis. In this study, gene delivery to human osteoarthritis chondrocytes in monolayer culture was demonstrated using two adenoviral vectors (Ad.CMVlacZ and Ad.RSVntlacZ) carrying the Escherichia coli beta-galactosidase marker gene, and a third vector (Ad.RSV hIL-1ra) containing the cDNA for human interleukin-1 receptor antagonist. At an moi of 10(3) plaque-forming units/chondrocyte, > 90% of the infected cells stained positive for E. coli beta-galactosidase activity, indicating a high efficiency of transduction. Genetically modified chondrocytes were then transplanted onto the articular surface of osteoarthritic cartilage organ cultures with and without the underlying subchondral bone. Both in situ staining of the cartilage organ cultures for E. coli beta-galactosidase activity and examination by scanning electron microscopy indicated that the transplanted chondrocytes adhered and integrated into the articular surface and continued to express transgenic protein. Chondrocytes transduced with Ad.RSV hIL-1ra and seeded onto the surface of osteoarthritic cartilage secreted high levels of biologically active IL-1 receptor antagonist. The Ad.RSV hIL-1ra-treated cartilage samples were resistant to IL1-induced proteoglycan degradation over 10 d of sustained organ culture. These data demonstrate that transplantation of transduced chondrocytes onto the articular surface protects cartilage from IL-1-induced extracellular matrix degradation. Images PMID:7593634

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fernandez-Fueyo, Elena; Ruiz-Duenas, Francisco J.; Ferreira, Patrica

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We alsomore » observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.« less

Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression

PubMed Central

McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

2016-01-01

The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. PMID:27276034

RNA degradation and models for post-transcriptional gene-silencing.

PubMed

Meins, F

2000-06-01

Post-transcriptional gene silencing (PTGS) is a form of stable but potentially reversible epigenetic modification, which frequently occurs in transgenic plants. The interaction in trans of genes with similar transcribed sequences results in sequence-specific degradation of RNAs derived from the genes involved. Highly expressed single-copy loci, transcribed inverted repeats, and poorly transcribed complex loci can act as sources of signals that trigger PTGS. In some cases, mobile, sequence-specific silencing signals can move from cell to cell or even over long distances in the plant. Several current models hold that silencing signals are 'aberrant' RNAs (aRNA), which differ in some way from normal mRNAs. The most likely candidates are small antisense RNAs (asRNA) and double-stranded RNAs (dsRNA). Direct evidence that these or other aRNAs found in silent tissues can induce PTGS is still lacking. Most current models assume that silencing signals interact with target RNAs in a sequence-specific fashion. This results in degradation, usually in the cytoplasm, by exonucleolytic as well as endonucleolytic pathways, which are not necessarily PTGS-specific. Biochemical-switch models hold that the silent state is maintained by a positive auto-regulatory loop. One possibility is that concentrations of hypothetical silencing signals above a critical threshold trigger their own production by self-replication, by degradation of target RNAs, or by a combination of both mechanisms. These models can account for the stability, reversibility and multiplicity of silent states; the strong influence of transcription rate of target genes on the incidence and stability of silencing, and the amplification and systemic propagation of motile silencing signals.

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis

PubMed Central

Fernandez-Fueyo, Elena; Ruiz-Dueñas, Francisco J.; Ferreira, Patricia; Floudas, Dimitrios; Hibbett, David S.; Canessa, Paulo; Larrondo, Luis F.; James, Tim Y.; Seelenfreund, Daniela; Lobos, Sergio; Polanco, Rubén; Tello, Mario; Honda, Yoichi; Watanabe, Takahito; Watanabe, Takashi; Ryu, Jae San; Kubicek, Christian P.; Schmoll, Monika; Gaskell, Jill; Hammel, Kenneth E.; St. John, Franz J.; Vanden Wymelenberg, Amber; Sabat, Grzegorz; Splinter BonDurant, Sandra; Syed, Khajamohiddin; Yadav, Jagjit S.; Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Lavín, José L.; Oguiza, José A.; Perez, Gumer; Pisabarro, Antonio G.; Ramirez, Lucia; Santoyo, Francisco; Master, Emma; Coutinho, Pedro M.; Henrissat, Bernard; Lombard, Vincent; Magnuson, Jon Karl; Kües, Ursula; Hori, Chiaki; Igarashi, Kiyohiko; Samejima, Masahiro; Held, Benjamin W.; Barry, Kerrie W.; LaButti, Kurt M.; Lapidus, Alla; Lindquist, Erika A.; Lucas, Susan M.; Riley, Robert; Salamov, Asaf A.; Hoffmeister, Dirk; Schwenk, Daniel; Hadar, Yitzhak; Yarden, Oded; de Vries, Ronald P.; Wiebenga, Ad; Stenlid, Jan; Eastwood, Daniel; Grigoriev, Igor V.; Berka, Randy M.; Blanchette, Robert A.; Kersten, Phil; Martinez, Angel T.; Vicuna, Rafael; Cullen, Dan

2012-01-01

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium. PMID:22434909

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

PubMed Central

Dejonghe, Winnie; Goris, Johan; El Fantroussi, Saïd; Höfte, Monica; De Vos, Paul; Verstraete, Willy; Top, Eva M.

2000-01-01

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (105 CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (105 CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon. PMID:10919784

Transcriptome Wide Annotation of Eukaryotic RNase III Reactivity and Degradation Signals

PubMed Central

Gagnon, Jules; Lavoie, Mathieu; Catala, Mathieu; Malenfant, Francis; Elela, Sherif Abou

2015-01-01

Detection and validation of the RNA degradation signals controlling transcriptome stability are essential steps for understanding how cells regulate gene expression. Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome, and encompass a wide variety of sequences, indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes, and their impact on RNA expression is linked to growth conditions. Together, the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions. PMID:25680180

Biochars change the sorption and degradation of thiacloprid in soil: Insights into chemical and biological mechanisms.

PubMed

Zhang, Peng; Sun, Hongwen; Min, Lujuan; Ren, Chao

2018-05-01

One interest of using biochar as soil amendment is to reduce pesticide adverse effects. In this paper, the sorption and degradation of thiacloprid (THI) in a black soil amended by various biochars were systematically investigated, and the mechanisms therein were explored by analyzing the changes in soil physicochemical properties, degrading enzymes and genes and microorganism community. Biochar amendment increased THI sorption in soil, which was associated with an increase in organic carbon and surface area and a decrease in H/C. Amendments of 300-PT (pyrolyzing temperature) biochar promoted the biodegradation of THI by increasing the microbe abundance and improving nitrile hydratase (NHase) activity. In contrast, 500- and 700-PT biochar amendments inhibited biodegradation by reducing THI availability and changing NHase activity and THI-degradative nth gene abundance, and instead promoted chemical degradation mainly through elevated pH, active groups on mineral surface and generation of •OH and other free radicals. Furthermore, THI shifted the soil microbial community, stimulated the NHase activity and elevated nth gene abundance. Biochar amendments also changed soil bacterial community by modulating soil pH, dissolved organic matter and nitrogen and phosphorus levels, which further influenced THI biodegradation. Therefore, the impact of biochars on the fate of a pesticide in soil depends greatly on their type and properties, which should be comprehensively examined when applying biochar to soil. Copyright © 2018 Elsevier Ltd. All rights reserved.

Potential of semiarid soil from Caatinga biome as a novel source for mining lignocellulose-degrading enzymes.

PubMed

Lacerda Júnior, Gileno V; Noronha, Melline F; de Sousa, Sanderson Tarciso P; Cabral, Lucélia; Domingos, Daniela F; Sáber, Mírian L; de Melo, Itamar S; Oliveira, Valéria M

2017-02-01

The litterfall is the major organic material deposited in soil of Brazilian Caatinga biome, thus providing the ideal conditions for plant biomass-degrading microorganisms to thrive. Herein, the phylogenetic composition and lignocellulose-degrading capacity have been explored for the first time from a fosmid library dataset of Caatinga soil by sequence-based screening. A complex bacterial community dominated by Proteobacteria and Actinobacteria was unraveled. SEED subsystems-based annotations revealed a broad range of genes assigned to carbohydrate and aromatic compounds metabolism, indicating microbial ability to utilize plant-derived material. CAZy-based annotation identified 7275 genes encoding 37 glycoside hydrolases (GHs) families related to hydrolysis of cellulose, hemicellulose, oligosaccharides and other lignin-modifying enzymes. Taxonomic affiliation of genes showed high genetic potential of the phylum Acidobacteria for hemicellulose degradation, whereas Actinobacteria members appear to play an important role in celullose hydrolysis. Additionally, comparative analyses revealed greater GHs profile similarity among soils as compared to the digestive tract of animals capable of digesting plant biomass, particularly in the hemicellulases content. Combined results suggest a complex synergistic interaction of community members required for biomass degradation into fermentable sugars. This large repertoire of lignocellulolytic enzymes opens perspectives for mining potential candidates of biochemical catalysts for biofuels production from renewable resources and other environmental applications. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warnecke, Falk; Warnecke, Falk; Luginbuhl, Peter

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial communitymore » resident in the hindgut paunch of a wood-feeding Nasutitermes species to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H{sub 2} metabolism, CO{sub 2}-reductive acetogenesis and N{sub 2} fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-{micro}l environment can be.« less

Variation of nonylphenol-degrading gene abundance and bacterial community structure in bioaugmented sediment microcosm.

PubMed

Wang, Zhao; Yang, Yuyin; Sun, Weimin; Dai, Yu; Xie, Shuguang

2015-02-01

Nonylphenol (NP) can accumulate in river sediment. Bioaugmentation is an attractive option to dissipate heavy NP pollution in river sediment. In this study, two NP degraders were isolated from crude oil-polluted soil and river sediment. Microcosms were constructed to test their ability to degrade NP in river sediment. The shift in the proportion of NP-degrading genes and bacterial community structure in sediment microcosms were characterized using quantitative PCR assay and terminal restriction fragment length polymorphism analysis, respectively. Phylogenetic analysis indicated that the soil isolate belonged to genus Stenotrophomonas, while the sediment isolate was a Sphingobium species. Both of them could almost completely clean up a high level of NP in river sediment (150 mg/kg NP) in 10 or 14 days after inoculation. An increase in the proportion of alkB and sMO genes was observed in sediment microcosms inoculated with Stenotrophomonas strain Y1 and Sphingobium strain Y2, respectively. Moreover, bioaugmentation using Sphingobium strain Y2 could have a strong impact on sediment bacterial community structure, while inoculation of Stenotrophomonas strain Y1 illustrated a weak impact. This study can provide some new insights towards NP biodegradation and bioremediation.

Analysis of genomic regions of Trichoderma harzianum IOC-3844 related to biomass degradation.

PubMed

Crucello, Aline; Sforça, Danilo Augusto; Horta, Maria Augusta Crivelente; dos Santos, Clelton Aparecido; Viana, Américo José Carvalho; Beloti, Lilian Luzia; de Toledo, Marcelo Augusto Szymanski; Vincentz, Michel; Kuroshu, Reginaldo Massanobu; de Souza, Anete Pereira

2015-01-01

Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes.

Analysis of Genomic Regions of Trichoderma harzianum IOC-3844 Related to Biomass Degradation

PubMed Central

Crucello, Aline; Sforça, Danilo Augusto; Horta, Maria Augusta Crivelente; dos Santos, Clelton Aparecido; Viana, Américo José Carvalho; Beloti, Lilian Luzia; de Toledo, Marcelo Augusto Szymanski; Vincentz, Michel; Kuroshu, Reginaldo Massanobu; de Souza, Anete Pereira

2015-01-01

Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes. PMID:25836973

Role of IncP-1β Plasmids pWDL7::rfp and pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses

PubMed Central

Król, J. E.; Penrod, J. T.; McCaslin, H.; Rogers, L. M.; Yano, H.; Stancik, A. D.; Dejonghe, W.; Brown, C. J.; Parales, R. E.; Wuertz, S.

2012-01-01

Broad-host-range catabolic plasmids play an important role in bacterial degradation of man-made compounds. To gain insight into the role of these plasmids in chloroaniline degradation, we determined the first complete nucleotide sequences of an IncP-1 chloroaniline degradation plasmid, pWDL7::rfp and its close relative pNB8c, as well as the expression pattern, function, and bioaugmentation potential of the putative 3-chloroaniline (3-CA) oxidation genes. Based on phylogenetic analysis of backbone proteins, both plasmids are members of a distinct clade within the IncP-1β subgroup. The plasmids are almost identical, but whereas pWDL7::rfp carries a duplicate inverted catabolic transposon, Tn6063, containing a putative 3-CA oxidation gene cluster, dcaQTA1A2BR, pNB8c contains only a single copy of the transposon. No genes for an aromatic ring cleavage pathway were detected on either plasmid, suggesting that only the upper 3-CA degradation pathway was present. The dcaA1A2B gene products expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol in Escherichia coli. Slight differences in the dca promoter region between the plasmids and lack of induction of transcription of the pNB8c dca genes by 3-CA may explain previous findings that pNB8C does not confer 3-CA transformation. Bioaugmentation of activated sludge with pWDL7::rfp accelerated removal of 3-CA, but only in the presence of an additional carbon source. Successful bioaugmentation requires complementation of the upper pathway genes with chlorocatechol cleavage genes in indigenous bacteria. The genome sequences of these plasmids thus help explain the molecular basis of their catabolic activities. PMID:22101050

Complete genome sequence and comparative genome analysis of Klebsiella oxytoca HKOPL1 isolated from giant panda feces.

PubMed

Jiang, Jingwei; Tun, Hein Min; Mauroo, Nathalie France; Ma, Angel Po Yee; Chan, San Yuen; Leung, Frederick C

2014-11-23

The giant panda (Ailuropoda melanoleuca) is an endangered species well-known for ingesting bamboo as a major part of their diet despite the fact that it belongs to order Carnivora. However, the giant panda's draft genome shows no direct evidence of enzymatic genes responsible for cellulose digestion. To explore this phenomenon, we study the giant panda's gut microbiota using genomic approaches in order to better understand their physiological processes as well as any potential microbial cellulose digestion processes. A complete genome of isolated Klebsiella oxytoca HKOPL1 of 5.9 Mb has been successfully sequenced, closed and comprehensively annotated against various databases. Genome comparisons within the Klebsiella genus and K. oxytoca species have also been performed. A total of 5,772 genes were predicted, and among them, 211 potential virulence genes, 35 pathogenicity island-like regions, 1,615 potential horizontal transferring genes, 23 potential antibiotics resistant genes, a potential prophage integrated region, 8 genes in 2,3-Butanediol production pathway and 3 genes in the cellulose degradation pathway could be identified and discussed based on the comparative genomic studies between the complete genome sequence of K. oxytoca HKOPL1 and other Klebsiella strains. A functional study shows that K. oxytoca HKOPL1 can degrade cellulose within 72 hours. Phylogenomic studies indicate that K. oxytoca HKOPL1 is clustered with K. oxytoca strains 1686 and E718. K. oxytoca HKOPL1 is a gram-negative bacterium able to degrade cellulose. We report here the first complete genome sequence of K. oxytoca isolated from giant panda feces. These studies have provided further insight into the role of gut microbiota in giant panda digestive physiology. In addition, K. oxytoca HKOPL1 has the potential for biofuel application in terms of cellulose degradation and potential for the production of 2,3-Butanediol (an important industrial raw material).

Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

PubMed Central

Liu, Kehai; Wang, Xiaoyu; Fan, Wei; Zhu, Qing; Yang, Jingya; Gao, Jing; Gao, Shen

2012-01-01

Background To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed. Methods First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49–57), to yield a bifunctional peptide RGDC-Tat (49–57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in αvβ3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro. Results The vector showed controlled degradation, strong targeting specificity to αvβ3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 μg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa. Conclusion P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy. PMID:22412301

Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii

DOE PAGES

Floudas, Dimitrios; Held, Benjamin W.; Riley, Robert; ...

2015-02-12

Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. In this paper, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot andmore » brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Finally, our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.« less

Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Floudas, Dimitrios; Held, Benjamin W.; Riley, Robert

Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. In this paper, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot andmore » brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Finally, our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.« less

Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii

PubMed Central

Floudas, Dimitrios; Held, Benjamin W.; Riley, Robert; Nagy, Laszlo G.; Koehler, Gage; Ransdell, Anthony S.; Younus, Hina; Chow, Julianna; Chiniquy, Jennifer; Lipzen, Anna; Tritt, Andrew; Sun, Hui; Haridas, Sajeet; LaButti, Kurt; Ohm, Robin A.; Kües, Ursula; Blanchette, Robert A.; Grigoriev, Igor V.; Minto, Robert E.; Hibbett, David S.

2015-01-01

Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present the genome sequences of the white rot fungus Cylindrobasidium torrendii and the brown rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. Cylindrobasidium torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. Fistulina hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition towards a brown rot lifestyle could be an ongoing process in F. hepatica. Our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited. PMID:25683379

Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii.

PubMed

Floudas, Dimitrios; Held, Benjamin W; Riley, Robert; Nagy, Laszlo G; Koehler, Gage; Ransdell, Anthony S; Younus, Hina; Chow, Julianna; Chiniquy, Jennifer; Lipzen, Anna; Tritt, Andrew; Sun, Hui; Haridas, Sajeet; LaButti, Kurt; Ohm, Robin A; Kües, Ursula; Blanchette, Robert A; Grigoriev, Igor V; Minto, Robert E; Hibbett, David S

2015-03-01

Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited. Copyright © 2015 Elsevier Inc. All rights reserved.

Down-regulation of lipoxygenase gene reduces degradation of carotenoids of golden rice during storage.

PubMed

Gayen, Dipak; Ali, Nusrat; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

2015-07-01

Down-regulation of lipoxygenase enzyme activity reduces degradation of carotenoids of bio-fortified rice seeds which would be an effective tool to reduce huge post-harvest and economic losses of bio-fortified rice seeds during storage. Bio-fortified provitamin A-enriched rice line (golden rice) expressing higher amounts of β-carotene in the rice endosperm provides vitamin A for human health. However, it is already reported that degradation of carotenoids during storage is a major problem. The gene responsible for degradation of carotenoids during storage has remained largely unexplored till now. In our previous study, it has been shown that r9-LOX1 gene is responsible for rice seed quality deterioration. In the present study, we attempted to investigate if r9-LOX1 gene has any role in degradation of carotenoids in rice seeds during storage. To establish our hypothesis, the endogenous lipoxygenase (LOX) activity of high-carotenoid golden indica rice seed was silenced by RNAi technology using aleurone layer and embryo-specific Oleosin-18 promoter. To check the storage stability, LOX enzyme down-regulated high-carotenoid T3 transgenic rice seeds were subjected to artificial aging treatment. The results obtained from biochemical assays (MDA, ROS) also indicated that after artificial aging, the deterioration of LOX-RNAi lines was considerably lower compared to β-carotene-enriched transgenic rice which had higher LOX activity in comparison to LOX-RNAi lines. Furthermore, it was also observed by HPLC analysis that down-regulation of LOX gene activity decreases co-oxidation of β-carotene in LOX-RNAi golden rice seeds as compared to the β-carotene-enriched transgenic rice, after artificial aging treatment. Therefore, our study substantially establishes and verifies that LOX is a key enzyme for catalyzing co-oxidation of β-carotene and has a significant role in deterioration of β-carotene levels in the carotenoid-enriched golden rice.

The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

PubMed Central

Seiboth, Bernhard; Karimi, Razieh Aghcheh; Phatale, Pallavi A; Linke, Rita; Hartl, Lukas; Sauer, Dominik G; Smith, Kristina M; Baker, Scott E; Freitag, Michael; Kubicek, Christian P

2012-01-01

Summary Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified. PMID:22554051

Characteristics of microbial community functional structure of a biological coking wastewater treatment system.

PubMed

Joshi, Dev Raj; Zhang, Yu; Zhang, Hong; Gao, Yingxin; Yang, Min

2018-01-01

Nitrogenous heterocyclic compounds are key pollutants in coking wastewater; however, the functional potential of microbial communities for biodegradation of such contaminants during biological treatment is still elusive. Herein, a high throughput functional gene array (GeoChip 5.0) in combination with Illumina HiSeq2500 sequencing was used to compare and characterize the microbial community functional structure in a long run (500days) bench scale bioreactor treating coking wastewater, with a control system treating synthetic wastewater. Despite the inhibitory toxic pollutants, GeoChip 5.0 detected almost all key functional gene (average 61,940 genes) categories in the coking wastewater sludge. With higher abundance, aromatic ring cleavage dioxygenase genes including multi ring1,2diox; one ring2,3diox; catechol represented significant functional potential for degradation of aromatic pollutants which was further confirmed by Illumina HiSeq2500 analysis results. Response ratio analysis revealed that three nitrogenous compound degrading genes- nbzA (nitro-aromatics), tdnB (aniline), and scnABC (thiocyanate) were unique for coking wastewater treatment, which might be strong cause to increase ammonia level during the aerobic process. Additionally, HiSeq2500 elucidated carbozole and isoquinoline degradation genes in the system. These findings expanded our understanding on functional potential of microbial communities to remove organic nitrogenous pollutants; hence it will be useful in optimization strategies for biological treatment of coking wastewater. Copyright © 2017. Published by Elsevier B.V.

Characterization and Genome Analysis of a Nicotine and Nicotinic Acid-Degrading Strain Pseudomonas putida JQ581 Isolated from Marine.

PubMed

Li, Aiwen; Qiu, Jiguo; Chen, Dongzhi; Ye, Jiexu; Wang, Yuhong; Tong, Lu; Jiang, Jiandong; Chen, Jianmeng

2017-05-31

The presence of nicotine and nicotinic acid (NA) in the marine environment has caused great harm to human health and the natural environment. Therefore, there is an urgent need to use efficient and economical methods to remove such pollutants from the environment. In this study, a nicotine and NA-degrading bacterium-strain JQ581-was isolated from sediment from the East China Sea and identified as a member of Pseudomonas putida based on morphology, physio-biochemical characteristics, and 16S rDNA gene analysis. The relationship between growth and nicotine/NA degradation suggested that strain JQ581 was a good candidate for applications in the bioaugmentation treatment of nicotine/NA contamination. The degradation intermediates of nicotine are pseudooxynicotine (PN) and 3-succinoyl-pyridine (SP) based on UV, high performance liquid chromatography, and liquid chromatography-mass spectrometry analyses. However, 6-hydroxy-3-succinoyl-pyridine (HSP) was not detected. NA degradation intermediates were identified as 6-hydroxynicotinic acid (6HNA). The whole genome of strain JQ581 was sequenced and analyzed. Genome sequence analysis revealed that strain JQ581 contained the gene clusters for nicotine and NA degradation. This is the first report where a marine-derived Pseudomonas strain had the ability to degrade nicotine and NA simultaneously.

‘ Candidatus Dichloromethanomonas elyunquensis’ gen. nov., sp. nov., a dichloromethane-degrading anaerobe of the Peptococcaceae family

DOE PAGES

Kleindienst, Sara; Higgins, Steven A.; Tsementzi, Despina; ...

2016-12-21

Taxonomic assignments of anaerobic dichloromethane (DCM)-degrading bacteria remain poorly constrained but are important for understanding the microbial diversity of organisms contributing to DCM turnover in environmental systems. Here, we describe the taxonomic classification of a novel DCM degrader in consortium RM obtained from pristine Rio Mameyes sediment. Phylogenetic analysis of full-length 16S rRNA gene sequences demonstrated that the DCM degrader was most closely related to members of the genera Dehalobacter and Syntrophobotulus, but sequence similarities did not exceed 94% and 93%, respectively. Genome-aggregate average amino acid identities against Peptococcaceae members did not exceed 66%, suggesting that the DCM degrader doesmore » not affiliate with any described genus. Phylogenetic analysis of conserved single-copy functional genes supported that the DCM degrader represents a novel clade. Growth strictly depended on the presence of DCM, which was consumed at a rate of 160 ± 3 μmol L -1 d -1. The DCM degrader attained 5.25 × 10 7 ± 1.0 × 10 7 cells per μmol DCM consumed. Fluorescence in situ hybridization revealed rod-shaped cells 4 ± 0.8 μm long and 0.4 ± 0.1 μm wide. Furthermore, based on the unique phylogenetic, genomic, and physiological characteristics, we propose that the DCM degrader represents a new genus and species, ‘Candidatus Dichloromethanomonas elyunquensis’.« less

‘ Candidatus Dichloromethanomonas elyunquensis’ gen. nov., sp. nov., a dichloromethane-degrading anaerobe of the Peptococcaceae family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kleindienst, Sara; Higgins, Steven A.; Tsementzi, Despina

Taxonomic assignments of anaerobic dichloromethane (DCM)-degrading bacteria remain poorly constrained but are important for understanding the microbial diversity of organisms contributing to DCM turnover in environmental systems. Here, we describe the taxonomic classification of a novel DCM degrader in consortium RM obtained from pristine Rio Mameyes sediment. Phylogenetic analysis of full-length 16S rRNA gene sequences demonstrated that the DCM degrader was most closely related to members of the genera Dehalobacter and Syntrophobotulus, but sequence similarities did not exceed 94% and 93%, respectively. Genome-aggregate average amino acid identities against Peptococcaceae members did not exceed 66%, suggesting that the DCM degrader doesmore » not affiliate with any described genus. Phylogenetic analysis of conserved single-copy functional genes supported that the DCM degrader represents a novel clade. Growth strictly depended on the presence of DCM, which was consumed at a rate of 160 ± 3 μmol L -1 d -1. The DCM degrader attained 5.25 × 10 7 ± 1.0 × 10 7 cells per μmol DCM consumed. Fluorescence in situ hybridization revealed rod-shaped cells 4 ± 0.8 μm long and 0.4 ± 0.1 μm wide. Furthermore, based on the unique phylogenetic, genomic, and physiological characteristics, we propose that the DCM degrader represents a new genus and species, ‘Candidatus Dichloromethanomonas elyunquensis’.« less

Sugar suppresses cell death caused by disruption of fumarylacetoacetate hydrolase in Arabidopsis.

PubMed

Zhi, Tiantian; Zhou, Zhou; Huang, Yi; Han, Chengyun; Liu, Yan; Zhu, Qi; Ren, Chunmei

2016-09-01

Sugar negatively regulates cell death resulting from the loss of fumarylacetoacetate hydrolase that catalyzes the last step in the Tyr degradation pathway in Arabidopsis . Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Previously, we first found that the Tyr degradation pathway plays an important role in plants. Mutation of the SSCD1 gene encoding FAH in Arabidopsis leads to spontaneous cell death under short-day conditions. In this study, we presented that the lethal phenotype of the short-day sensitive cell death1 (sscd1) seedlings was suppressed by sugars including sucrose, glucose, fructose, and maltose in a dose-dependent manner. Real-time quantitative PCR (RT-qPCR) analysis showed the expression of Tyr degradation pathway genes homogentisate dioxygenase and maleylacetoacetate isomerase, and sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G, was up-regulated in the sscd1 mutant, however, this up-regulation could be repressed by sugar. In addition, a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone, an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway. These results indicated that (1) sugar could suppress cell death in sscd1, which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates, resulting in suppression of cell death; and (2) sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G might be involved in the cell death in sscd1. Our work provides insights into the relationship between sugar and sscd1-mediated cell death, and contributes to elucidation of the regulation of cell death resulting from the loss of FAH in plants.

Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms

USGS Publications Warehouse

Miller, L.G.; Warner, K.L.; Baesman, S.M.; Oremland, R.S.; McDonald, I.R.; Radajewski, S.; Murrell, J.C.

2004-01-01

Bacteria in soil microcosm experiments oxidized elevated levels of methyl chloride (MeCl) and methyl bromide (MeBr), the former compound more rapidly than the latter. MeBr was also removed by chemical reactions while MeCl was not. Chemical degradation dominated the early removal of MeBr and accounted for more than half of its total loss. Fractionation of stable carbon isotopes during chemical degradation of MeBr resulted in a kinetic isotope effect (KIE) of 59 ?? 7???. Soil bacterial oxidation dominated the later removal of MeBr and MeCl and was characterized by different KIEs for each compound. The KIE for MeBr oxidation was 69 ?? 9??? and the KIE for MeCl oxidation was 49 ?? 3???. Stable isotope probing revealed that different populations of soil bacteria assimilated added 13C-labeled MeBr and MeCl. The identity of the active MeBr and MeCl degrading bacteria in soil was determined by analysis of 16S rRNA gene sequences amplified from 13C-DNA fractions, which identified a number of sequences from organisms not previously thought to be involved in methyl halide degradation. These included Burkholderia , the major clone type in the 13C-MeBr fraction, and Rhodobacter, Lysobacter and Nocardioides the major clone types in the 13C-MeCl fraction. None of the 16S rRNA gene sequences for methyl halide oxidizing bacteria currently in culture (including Aminobacter strain IMB-1 isolated from fumigated soil) were identified. Functional gene clone types closely related to Aminobacter spp. were identified in libraries containing the sequences for the cmuA gene, which codes for the enzyme known to catalyze the initial step in the oxidation of MeBr and MeCl. The cmuA gene was limited to members of the alpha-Proteobacteria whereas the greater diversity demonstrated by the 16S rRNA gene may indicate that other enzymes catalyze methyl halide oxidation in different groups of bacteria. Copyright ?? 2004 Elsevier Ltd.

Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms

NASA Astrophysics Data System (ADS)

Miller, Laurence G.; Warner, Karen L.; Baesman, Shaun M.; Oremland, Ronald S.; McDonald, Ian R.; Radajewski, Stefan; Murrell, J. Colin

2004-08-01

Bacteria in soil microcosm experiments oxidized elevated levels of methyl chloride (MeCl) and methyl bromide (MeBr), the former compound more rapidly than the latter. MeBr was also removed by chemical reactions while MeCl was not. Chemical degradation dominated the early removal of MeBr and accounted for more than half of its total loss. Fractionation of stable carbon isotopes during chemical degradation of MeBr resulted in a kinetic isotope effect (KIE) of 59 ± 7‰. Soil bacterial oxidation dominated the later removal of MeBr and MeCl and was characterized by different KIEs for each compound. The KIE for MeBr oxidation was 69 ± 9‰ and the KIE for MeCl oxidation was 49 ± 3‰. Stable isotope probing revealed that different populations of soil bacteria assimilated added 13C-labeled MeBr and MeCl. The identity of the active MeBr and MeCl degrading bacteria in soil was determined by analysis of 16S rRNA gene sequences amplified from 13C-DNA fractions, which identified a number of sequences from organisms not previously thought to be involved in methyl halide degradation. These included Burkholderia, the major clone type in the 13C-MeBr fraction, and Rhodobacter, Lysobacter and Nocardioides the major clone types in the 13C-MeCl fraction. None of the 16S rRNA gene sequences for methyl halide oxidizing bacteria currently in culture (including Aminobacter strain IMB-1 isolated from fumigated soil) were identified. Functional gene clone types closely related to Aminobacter spp. were identified in libraries containing the sequences for the cmuA gene, which codes for the enzyme known to catalyze the initial step in the oxidation of MeBr and MeCl. The cmuA gene was limited to members of the alpha-Proteobacteria whereas the greater diversity demonstrated by the 16S rRNA gene may indicate that other enzymes catalyze methyl halide oxidation in different groups of bacteria.

The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring tfd Genes for 2,4-Dichlorophenoxyacetic Acid Degradation

PubMed Central

Vedler, Eve; Vahter, Merle; Heinaru, Ain

2004-01-01

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002 contains plasmid pEST4011. This plasmid ensures its host a stable 2,4-D+ phenotype. We determined the complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a deletion and duplication derivative of pD2M4, the 95-kb highly unstable laboratory ancestor of pEST4011, and was self-generated during different laboratory manipulations performed to increase the stability of the 2,4-D+ phenotype of the original strain, strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a transposon-like structure with identical copies of the hybrid insertion element IS1071::IS1471 at the two ends. The catabolic regions of pEST4011 and pJP4, the best-studied 2,4-D-degradative plasmid, both contain homologous, tfd-like genes for complete 2,4-D degradation, but they have little sequence similarity other than that. The backbone genes of pEST4011 are most similar to the corresponding genes of broad-host-range self-transmissible IncP1 plasmids. The backbones of the other three IncP1 catabolic plasmids that have been sequenced (the 2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1, and the atrazine-catabolic plasmid pADP-1) are nearly identical to the backbone of R751, the archetype plasmid of the IncP1 β subgroup. We show that despite the overall similarity in plasmid organization, the pEST4011 backbone is sufficiently different (51 to 86% amino acid sequence identity between individual backbone genes) from the backbones of members of the three IncP1 subgroups (the α, β, and γ subgroups) that it belongs to a new IncP1subgroup, the δ subgroup. This conclusion was also supported by a phylogenetic analysis of the trfA2, korA, and traG gene products of different IncP1 plasmids. PMID:15489427

Effects of polycyclic aromatic hydrocarbons on microbial community structure and PAH ring hydroxylating dioxygenase gene abundance in soil.

PubMed

Sawulski, Przemyslaw; Clipson, Nicholas; Doyle, Evelyn

2014-11-01

Development of successful bioremediation strategies for environments contaminated with recalcitrant pollutants requires in-depth knowledge of the microorganisms and microbial processes involved in degradation. The response of soil microbial communities to three polycyclic aromatic hydrocarbons, phenanthrene (3-ring), fluoranthene (4-ring) and benzo(a)pyrene (5-ring), was examined. Profiles of bacterial, archaeal and fungal communities were generated using molecular fingerprinting techniques (TRFLP, ARISA) and multivariate statistical tools were employed to interpret the effect of PAHs on community dynamics and composition. The extent and rate of PAH removal was directly related to the chemical structure, with the 5-ring PAH benzo(a)pyrene degraded more slowly than phenathrene or fluoranthene. Bacterial, archaeal and fungal communities were all significantly affected by PAH amendment, time and their interaction. Based on analysis of clone libraries, Actinobacteria appeared to dominate in fluoranthene amended soil, although they also represented a significant portion of the diversity in phenanthrene amended and unamended soils. In addition there appeared to be more γ-Proteobacteria and less Bacteroidetes in soil amended with either PAH compared to the control. The soil bacterial community clearly possessed the potential to degrade PAHs as evidenced by the abundance of PAH ring hydroxylating (PAH-RHDα) genes from both gram negative (GN) and gram positive (GP) bacteria in PAH-amended and control soils. Although the dioxygenase gene from GP bacteria was less abundant in soil than the gene associated with GN bacteria, significant (p < 0.001) increases in the abundance of the GP PAH-RHDα gene were observed during phenanthrene and fluoranthene degradation, whereas there was no significant difference in the abundance of the GN PAH-RHDα gene during the course of the experiment. Few studies to-date have examined the effect of pollutants on more than one microbial community in soil. The current study provides information on the response of soil bacterial, archaeal and fungal communities during the degradation of three priority pollutants and contributes to a knowledge base that can inform the development of effective bioremediation strategies for contaminated sites.

The Role of Monoubiquitination in Endocytic Degradation of Human Ether-a-go-go-related Gene (hERG) Channels under Low K+ Conditions*

PubMed Central

Sun, Tao; Guo, Jun; Shallow, Heidi; Yang, Tonghua; Xu, Jianmin; Li, Wentao; Hanson, Christian; Wu, James G.; Li, Xian; Massaeli, Hamid; Zhang, Shetuan

2011-01-01

A reduction in extracellular K+ concentration ([K+]o) causes cardiac arrhythmias and triggers internalization of the cardiac rapidly activating delayed rectifier potassium channel (IKr) encoded by the human ether-a-go-go-related gene (hERG). We investigated the role of ubiquitin (Ub) in endocytic degradation of hERG channels stably expressed in HEK cells. Under low K+ conditions, UbKO, a lysine-less mutant Ub that only supports monoubiquitination, preferentially interacted and selectively enhanced degradation of the mature hERG channels. Overexpression of Vps24 protein, also known as charged multivesicular body protein 3, significantly accelerated degradation of mature hERG channels, whereas knockdown of Vps24 impeded this process. Moreover, the lysosomal inhibitor bafilomycin A1 inhibited degradation of the internalized mature hERG channels. Thus, monoubiquitination directs mature hERG channels to degrade through the multivesicular body/lysosome pathway. Interestingly, the protease inhibitor lactacystin inhibited the low K+-induced hERG endocytosis and concomitantly led to an accumulation of monoubiquitinated mature hERG channels, suggesting that deubiquitination is also required for the endocytic degradation. Consistently, overexpression of the endosomal deubiquitinating enzyme signal transducing adaptor molecule-binding protein significantly accelerated whereas knockdown of endogenous signal transducing adaptor molecule-binding protein impeded degradation of the mature hERG channels under low K+ conditions. Thus, monoubiquitin dynamically mediates endocytic degradation of mature hERG channels under low K+ conditions. PMID:21177251

Metagenomic Insights into the Fibrolytic Microbiome in Yak Rumen

PubMed Central

Song, Lei; Liu, Di; Liu, Li; Chen, Furong; Wang, Min; Li, Jiabao; Zeng, Xiaowei; Dong, Zhiyang; Hu, Songnian; Li, Lingyan; Xu, Jian; Huang, Li; Dong, Xiuzhu

2012-01-01

The rumen hosts one of the most efficient microbial systems for degrading plant cell walls, yet the predominant cellulolytic proteins and fibrolytic mechanism(s) remain elusive. Here we investigated the cellulolytic microbiome of the yak rumen by using a combination of metagenome-based and bacterial artificial chromosome (BAC)-based functional screening approaches. Totally 223 fibrolytic BAC clones were pyrosequenced and 10,070 ORFs were identified. Among them 150 were annotated as the glycoside hydrolase (GH) genes for fibrolytic proteins, and the majority (69%) of them were clustered or linked with genes encoding related functions. Among the 35 fibrolytic contigs of >10 Kb in length, 25 were derived from Bacteroidetes and four from Firmicutes. Coverage analysis indicated that the fibrolytic genes on most Bacteroidetes-contigs were abundantly represented in the metagenomic sequences, and they were frequently linked with genes encoding SusC/SusD-type outer-membrane proteins. GH5, GH9, and GH10 cellulase/hemicellulase genes were predominant, but no GH48 exocellulase gene was found. Most (85%) of the cellulase and hemicellulase proteins possessed a signal peptide; only a few carried carbohydrate-binding modules, and no cellulosomal domains were detected. These findings suggest that the SucC/SucD-involving mechanism, instead of one based on cellulosomes or the free-enzyme system, serves a major role in lignocellulose degradation in yak rumen. Genes encoding an endoglucanase of a novel GH5 subfamily occurred frequently in the metagenome, and the recombinant proteins encoded by the genes displayed moderate Avicelase in addition to endoglucanase activities, suggesting their important contribution to lignocellulose degradation in the exocellulase-scarce rumen. PMID:22808161

Cysteine degradation gene yhaM, encoding cysteine desulfidase, serves as a genetic engineering target to improve cysteine production in Escherichia coli.

PubMed

Nonaka, Gen; Takumi, Kazuhiro

2017-12-01

Cysteine is an important amino acid for various industries; however, there is no efficient microbial fermentation-based production method available. Owing to its cytotoxicity, bacterial intracellular levels of cysteine are stringently controlled via several modes of regulation, including cysteine degradation by cysteine desulfhydrases and cysteine desulfidases. In Escherichia coli, several metabolic enzymes are known to exhibit cysteine degradative activities, however, their specificity and physiological significance for cysteine detoxification via degradation are unclear. Relaxing the strict regulation of cysteine is crucial for its overproduction; therefore, identifying and modulating the major degradative activity could facilitate the genetic engineering of a cysteine-producing strain. In the present study, we used genetic screening to identify genes that confer cysteine resistance in E. coli and we identified yhaM, which encodes cysteine desulfidase and decomposes cysteine into hydrogen sulfide, pyruvate, and ammonium. Phenotypic characterization of a yhaM mutant via growth under toxic concentrations of cysteine followed by transcriptional analysis of its response to cysteine showed that yhaM is cysteine-inducible, and its physiological role is associated with resisting the deleterious effects of cysteine in E. coli. In addition, we confirmed the effects of this gene on the fermentative production of cysteine using E. coli-based cysteine-producing strains. We propose that yhaM encodes the major cysteine-degrading enzyme and it has the most significant role in cysteine detoxification among the numerous enzymes reported in E. coli, thereby providing a core target for genetic engineering to improve cysteine production in this bacterium.

Effects of different agricultural wastes on the dissipation of PAHs and the PAH-degrading genes in a PAH-contaminated soil.

PubMed

Han, Xuemei; Hu, Hangwei; Shi, Xiuzhen; Zhang, Limei; He, Jizheng

2017-04-01

Land application of agricultural wastes is considered as a promising bioremediation approach for cleaning up soils contaminated by aged polycyclic aromatic hydrocarbons (PAHs). However, it remains largely unknown about how microbial PAH-degraders, which play a key role in the biodegradation of soil PAHs, respond to the amendments of agricultural wastes. Here, a 90-day soil microcosm study was conducted to compare the effects of three agricultural wastes (i.e. WS, wheat stalk; MCSW, mushroom cultivation substrate waste; and CM, cow manure) on the dissipation of aged PAHs and the abundance and community structure of PAH-degrading microorganisms. The results showed that all the three agricultural wastes accelerated the dissipation of aged PAHs and significantly increased abundances of the bacterial 16S rRNA and PAH-degrading genes (i.e. pdo1 and nah). CM and MCSW with lower ratios of C:N eliminated soil PAHs more efficiently than WS with a high ratio of C:N. Low molecular weight PAHs were dissipated more quickly than those with high molecular weight. Phylogenetic analysis revealed that all of the nah and C12O clones were affiliated within Betaproteobacteria and Gammaproteobacteria, and application of agricultural wastes significantly changed the community structure of the microorganisms harboring nah and C12O genes, particularly in the CM treatment. Taken together, our findings suggest that the three tested agricultural wastes could accelerate the degradation of aged PAHs most likely through changing the abundances and community structure of microbial PAH degraders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Stereoselective Degradation and Molecular Ecological Mechanism of Chiral Pesticides Beta-Cypermethrin in Soils with Different pH Values.

PubMed

Yang, Zhong-Hua; Ji, Guo-Dong

2015-12-15

For decades, pesticides have been widely used for agricultural activities around the world, and the environmental problems caused by these compounds have raised widespread concern. However, the different enantioselective behaviors of chiral pesticide enantiomers are often ignored. Here, the selective degradation patterns and mechanisms of chiral pesticide enantiomers were successfully investigated for the first time in the soils of three cultivation areas with different pH values. Beta-cypermethrin was chosen as the target analyte. We found that the degradation rates of the four isomers of beta-cypermethrin were different. We used stepwise regression equations between degradation rates and functional genes to quantitatively study their relationships. Quantitative response analysis revealed that different isomers have different equations even under identical conditions. The results of path analysis showed that a single functional gene can make different direct and indirect contributions to the degradation of different isomers. Finally, the high-throughput technology was used to analysis the genome of the three tested soils and then compared the main microbial communities in them. We have successfully devised a method to investigate the molecular biological mechanisms of the selective degradation behavior of chiral compounds, thus enabling us to better understand these mechanisms.

Falsirhodobacter sp. alg1 Harbors Single Homologs of Endo and Exo-Type Alginate Lyases Efficient for Alginate Depolymerization

PubMed Central

Takahashi, Mami; Tanaka, Reiji; Miyake, Hideo; Shibata, Toshiyuki; Chow, Seinen; Kuroda, Kouichi; Ueda, Mitsuyoshi; Takeyama, Haruko

2016-01-01

Alginate-degrading bacteria play an important role in alginate degradation by harboring highly efficient and unique alginolytic genes. Although the general mechanism for alginate degradation by these bacteria is fairly understood, much is still required to fully exploit them. Here, we report the isolation of a novel strain, Falsirhodobacter sp. alg1, the first report for an alginate-degrading bacterium from the family Rhodobacteraceae. Genome sequencing reveals that strain alg1 harbors a primary alginate degradation pathway with only single homologs of an endo- and exo-type alginate lyase, AlyFRA and AlyFRB, which is uncommon among such bacteria. Subsequent functional analysis showed that both enzymes were extremely efficient to depolymerize alginate suggesting evolutionary interests in the acquirement of these enzymes. The exo-type alginate lyase, AlyFRB in particular could depolymerize alginate without producing intermediate products making it a highly efficient enzyme for the production of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Based on our findings, we believe that the discovery of Falsirhodobacter sp. alg1 and its alginolytic genes hints at the potentiality of a more diverse and unique population of alginate-degrading bacteria. PMID:27176711

Simazine degradation in bioaugmented soil: urea impact and response of ammonia-oxidizing bacteria and other soil bacterial communities.

PubMed

Guo, Qingwei; Wan, Rui; Xie, Shuguang

2014-01-01

The objective of this study was to investigate the impact of exogenous urea nitrogen on ammonia-oxidizing bacteria (AOB) and other soil bacterial communities in soil bioaugmented for simazine remediation. The previously isolated simazine-degrading Arthrobacter sp. strain SD1 was used to degrade the herbicide. The effect of urea on the simazine degradation capacity of the soil bioaugmented with Arthrobacter strain SD1 was assessed using quantitative PCR targeting the s-triazine-degrading trzN and atzC genes. Structures of bacterial and AOB communities were characterized using terminal restriction fragment length polymorphism. Urea fertilizer could affect simazine biodegradation and decreased the proportion of its trzN and atzC genes in soil augmented with Arthrobacter strain SD1. Bioaugmentation process could significantly alter the structures of both bacterial and AOB communities, which were strongly affected by urea amendment, depending on the dosage. This study could provide some new insights towards s-triazine bioremediation and microbial ecology in a bioaugmented system. However, further studies are necessary in order to elucidate the impact of different types and levels of nitrogen sources on s-triazine-degraders and bacterial and AOB communities in bioaugmented soil.

Horizontal Transfer of phnAc Dioxygenase Genes within One of Two Phenotypically and Genotypically Distinctive Naphthalene-Degrading Guilds from Adjacent Soil Environments

PubMed Central

Wilson, Mark S.; Herrick, James B.; Jeon, Che Ok; Hinman, David E.; Madsen, Eugene L.

2003-01-01

Several distinct naphthalene dioxygenases have been characterized to date, which provides the opportunity to investigate the ecological significance, relative distribution, and transmission modes of the different analogs. In this study, we showed that a group of naphthalene-degrading isolates from a polycyclic aromatic hydrocarbon (PAH)-contaminated hillside soil were phenotypically and genotypically distinct from naphthalene-degrading organisms isolated from adjacent, more highly contaminated seep sediments. Mineralization of 14C-labeled naphthalene by soil slurries suggested that the in situ seep community was more acclimated to PAHs than was the in situ hillside community. phnAc-like genes were present in diverse naphthalene-degrading isolates cultured from the hillside soil, while nahAc-like genes were found only among isolates cultured from the seep sediments. The presence of a highly conserved nahAc allele among gram-negative isolates from the coal tar-contaminated seep area provided evidence for in situ horizontal gene transfer and was reported previously (J. B. Herrick, K. G. Stuart-Keil, W. C. Ghiorse, and E. L. Madsen, Appl. Environ. Microbiol. 63:2330-2337, 1997). Natural horizontal transfer of the phnAc sequence was also suggested by a comparison of the phnAc and 16S ribosomal DNA sequences of the hillside isolates. Analysis of metabolites produced by cell suspensions and patterns of amplicons produced by PCR analysis suggested both genetic and metabolic diversity among the naphthalene-degrading isolates of the contaminated hillside. These results provide new insights into the distribution, diversity, and transfer of phnAc alleles and increase our understanding of the acclimation of microbial communities to pollutants. PMID:12676698

Benzene and Naphthalene Degrading Bacterial Communities in an Oil Sands Tailings Pond

PubMed Central

Rochman, Fauziah F.; Sheremet, Andriy; Tamas, Ivica; Saidi-Mehrabad, Alireza; Kim, Joong-Jae; Dong, Xiaoli; Sensen, Christoph W.; Gieg, Lisa M.; Dunfield, Peter F.

2017-01-01

Oil sands process-affected water (OSPW), produced by surface-mining of oil sands in Canada, is alkaline and contains high concentrations of salts, metals, naphthenic acids, and polycyclic aromatic compounds (PAHs). Residual hydrocarbon biodegradation occurs naturally, but little is known about the hydrocarbon-degrading microbial communities present in OSPW. In this study, aerobic oxidation of benzene and naphthalene in the surface layer of an oil sands tailings pond were measured. The potential oxidation rates were 4.3 μmol L−1 OSPW d−1 for benzene and 21.4 μmol L−1 OSPW d−1 for naphthalene. To identify benzene and naphthalene-degrading microbial communities, metagenomics was combined with stable isotope probing (SIP), high-throughput sequencing of 16S rRNA gene amplicons, and isolation of microbial strains. SIP using 13C-benzene and 13C-naphthalene detected strains of the genera Methyloversatilis and Zavarzinia as the main benzene degraders, while strains belonging to the family Chromatiaceae and the genus Thauera were the main naphthalene degraders. Metagenomic analysis revealed a diversity of genes encoding oxygenases active against aromatic compounds. Although these genes apparently belonged to many phylogenetically diverse taxa, only a few of these taxa were predominant in the SIP experiments. This suggested that many members of the community are adapted to consuming other aromatic compounds, or are active only under specific conditions. 16S rRNA gene sequence datasets have been submitted to the Sequence Read Archive (SRA) under accession number SRP109130. The Gold Study and Project submission ID number in Joint Genome Institute IMG/M for the metagenome is Gs0047444 and Gp0055765. PMID:29033909

Genomic organization of a cellulase gene family in Phanerochaete chrysosporium

Treesearch

Sarah F. Covert; Jennifer Bolduc; Daniel Cullen

1992-01-01

Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene (cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were...

Diversity of bacterial dimethylsulfoniopropionate degradation genes in surface seawater of Arctic Kongsfjorden.

PubMed

Zeng, Yin-Xin; Qiao, Zong-Yun; Yu, Yong; Li, Hui-Rong; Luo, Wei

2016-09-08

Dimethylsulfoniopropionate (DMSP), which is the major source of organic sulfur in the world's oceans, plays a significant role in the global sulfur cycle. This compound is rapidly degraded by marine bacteria either by cleavage to dimethylsulfide (DMS) or demethylation to 3-methylmercaptopropionate (MMPA). The diversity of genes encoding bacterial demethylation (dmdA) and DMS production (dddL and dddP) were measured in Arctic Kongsfjorden. Both dmdA and dddL genes were detected in all stations along a transect from the outer to the inner fjord, while dddP gene was only found in the outer and middle parts of the fjord. The dmdA gene was completely confined to the Roseobacter clade, while the dddL gene was confined to the genus Sulfitobacter. Although the dddP gene pool was also dominated by homologs from the Roseobacter clade, there were a few dddP genes showing close relationships to both Alphaproteobacter and Gammaproteobacter. The results of this study suggest that the Roseobacter clade may play an important role in DMSP catabolism via both demethylation and cleavage pathways in surface waters of Kongsfjorden during summer.

Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora.

PubMed

Norman-Setterblad, C; Vidal, S; Palva, E T

2000-04-01

We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone.

Diversity of bacterial dimethylsulfoniopropionate degradation genes in surface seawater of Arctic Kongsfjorden

NASA Astrophysics Data System (ADS)

Zeng, Yin-Xin; Qiao, Zong-Yun; Yu, Yong; Li, Hui-Rong; Luo, Wei

2016-09-01

Dimethylsulfoniopropionate (DMSP), which is the major source of organic sulfur in the world’s oceans, plays a significant role in the global sulfur cycle. This compound is rapidly degraded by marine bacteria either by cleavage to dimethylsulfide (DMS) or demethylation to 3-methylmercaptopropionate (MMPA). The diversity of genes encoding bacterial demethylation (dmdA) and DMS production (dddL and dddP) were measured in Arctic Kongsfjorden. Both dmdA and dddL genes were detected in all stations along a transect from the outer to the inner fjord, while dddP gene was only found in the outer and middle parts of the fjord. The dmdA gene was completely confined to the Roseobacter clade, while the dddL gene was confined to the genus Sulfitobacter. Although the dddP gene pool was also dominated by homologs from the Roseobacter clade, there were a few dddP genes showing close relationships to both Alphaproteobacter and Gammaproteobacter. The results of this study suggest that the Roseobacter clade may play an important role in DMSP catabolism via both demethylation and cleavage pathways in surface waters of Kongsfjorden during summer.

A comparative intracellular proteomic profiling of Pseudomonas aeruginosa strain ASP-53 grown on pyrene or glucose as sole source of carbon and identification of some key enzymes of pyrene biodegradation pathway.

PubMed

Mukherjee, Ashis K; Bhagowati, Pabitra; Biswa, Bhim Bahadur; Chanda, Abhishek; Kalita, Bhargab

2017-09-07

Pseudomonas aeruginosa strain ASP-53, isolated from a petroleum oil-contaminated soil sample, was found to be an efficient degrader of pyrene. PCR amplification of selected hydrocarbon catabolic genes (alkB gene, which encodes for monooxygenase, and the C12O, C23O, and PAH-RHDα genes encoding for the dioxygenase enzyme) from the genomic DNA of P. aeruginosa strain ASP-53 suggested its hydrocarbon degradation potential. The GC-MS analysis demonstrated 30.1% pyrene degradation by P. aeruginosa strain ASP-53 after 144h of incubation at pH6.5, 37°C. Expressions of 115 and 196 intracellular proteins were unambiguously identified and quantitated by shotgun proteomics analysis when the isolate was grown in medium containing pyrene and glucose, respectively. The pyrene-induced uniquely expressed and up-regulated proteins in P. aeruginosa strain ASP-53 in addition to substrate (pyrene) metabolism are also likely to be associated with different cellular functions for example-related to protein folding (molecular chaperone), stress response, metabolism of carbohydrate, proteins and amino acids, and fatty acids; transport of metabolites, energy generation such as ATP synthesis, electron transport and nitrate assimilation, and other oxidation-reduction reactions. Proteomic analyses identified some important enzymes involved in pyrene degradation by P. aeruginosa ASP-53 which shows that this bacterium follows the salicylate pathway of pyrene degradation. This study is the first report on proteomic analysis of pyrene biodegradation pathway by Pseudomonas aeruginosa, isolated from a petroleum-oil contaminated soil sample. The pathway displays partial similarity with deduced pyrene degradation mechanisms of Mycobacterium vanbaalenii PYR-1. The GC-MS analysis as well as PCR amplification of hydrocarbon catabolic genes substantiated the potency of the bacterium under study to effectively degrade high molecular weight, toxic PAH such as pyrene for its filed scale bioremediation experiments. The proteomics approach (LC-MS/MS analysis) identified the differentially regulated intracellular proteins of the isolate P. aeruginosa ASP-53 when grown in pyrene medium. This study identified some important pyrene biodegradation enzymes in Pseudomonas aeruginosa ASP-53 and highlights that the bacterium follows salicylate pathway for pyrene degradation. Copyright © 2017 Elsevier B.V. All rights reserved.

Metagenomic Insights into the RDX-Degrading Potential of the Ovine Rumen Microbiome

PubMed Central

Li, Robert W.; Giarrizzo, Juan Gabriel; Wu, Sitao; Li, Weizhong; Duringer, Jennifer M.; Craig, A. Morrie

2014-01-01

The manufacturing processes of royal demolition explosive (RDX), or hexahydro-1,3,5-trinitro-1,3,5-triazine, have resulted in serious water contamination. As a potential carcinogen, RDX can cause a broad range of harmful effects to humans and animals. The ovine rumen is capable of rapid degradation of nitroaromatic compounds, including RDX. While ruminal RDX-degrading bacteria have been identified, the genes and pathways responsible for RDX degradation in the rumen have yet to be characterized. In this study, we characterized the metabolic potential of the ovine rumen using metagenomic approaches. Sequences homologous to at least five RDX-degrading genes cloned from environmental samples (diaA, xenA, xenB, xplA, and xplB) were present in the ovine rumen microbiome. Among them, diaA was the most abundant, likely reflective of the predominance of the genus Clostridium in the ovine rumen. At least ten genera known to harbor RDX-degrading microorganisms were detectable. Metagenomic sequences were also annotated using public databases, such as Pfam, COG, and KEGG. Five of the six Pfam protein families known to be responsible for RDX degradation in environmental samples were identified in the ovine rumen. However, increased substrate availability did not appear to enhance the proliferation of RDX-degrading bacteria and alter the microbial composition of the ovine rumen. This implies that the RDX-degrading capacity of the ovine rumen microbiome is likely regulated at the transcription level. Our results provide metagenomic insights into the RDX-degrading potential of the ovine rumen, and they will facilitate the development of novel and economic bioremediation strategies. PMID:25383623

Gene expression profiling of Japanese psoriatic skin reveals an increased activity in molecular stress and immune response signals.

PubMed

Kulski, Jerzy K; Kenworthy, William; Bellgard, Matthew; Taplin, Ross; Okamoto, Koichi; Oka, Akira; Mabuchi, Tomotaka; Ozawa, Akira; Tamiya, Gen; Inoko, Hidetoshi

2005-12-01

Gene expression profiling was performed on biopsies of affected and unaffected psoriatic skin and normal skin from seven Japanese patients to obtain insights into the pathways that control this disease. HUG95A Affymetrix DNA chips that contained oligonucleotide arrays of approximately 12,000 well-characterized human genes were used in the study. The statistical analysis of the Affymetrix data, based on the ranking of the Student t-test statistic, revealed a complex regulation of molecular stress and immune gene responses. The majority of the 266 induced genes in affected and unaffected psoriatic skin were involved with interferon mediation, immunity, cell adhesion, cytoskeleton restructuring, protein trafficking and degradation, RNA regulation and degradation, signalling transduction, apoptosis and atypical epidermal cellular proliferation and differentiation. The disturbances in the normal protein degradation equilibrium of skin were reflected by the significant increase in the gene expression of various protease inhibitors and proteinases, including the induced components of the ATP/ubiquitin-dependent non-lysosomal proteolytic pathway that is involved with peptide processing and presentation to T cells. Some of the up-regulated genes, such as TGM1, IVL, FABP5, CSTA and SPRR, are well-known psoriatic markers involved in atypical epidermal cellular organization and differentiation. In the comparison between the affected and unaffected psoriatic skin, the transcription factor JUNB was found at the top of the statistical rankings for the up-regulated genes in affected skin, suggesting that it has an important but as yet undefined role in psoriasis. Our gene expression data and analysis suggest that psoriasis is a chronic interferon- and T-cell-mediated immune disease of the skin where the imbalance in epidermal cellular structure, growth and differentiation arises from the molecular antiviral stress signals initiating inappropriate immune responses.

The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes.

PubMed

Gupta, Deepak K; Rühl, Martin; Mishra, Bagdevi; Kleofas, Vanessa; Hofrichter, Martin; Herzog, Robert; Pecyna, Marek J; Sharma, Rahul; Kellner, Harald; Hennicke, Florian; Thines, Marco

2018-01-15

Agrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology. Here, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery. The genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.

Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of Rhodococcus sp. Strain RHA1†

PubMed Central

Gonçalves, Edmilson R.; Hara, Hirofumi; Miyazawa, Daisuke; Davies, Julian E.; Eltis, Lindsay D.; Mohn, William W.

2006-01-01

Rhodococcus sp. RHA1 grows on a broad range of aromatic compounds and vigorously degrades polychlorinated biphenyls (PCBs). Previous work identified RHA1 genes encoding multiple isozymes for most of the seven steps of the biphenyl (BPH) pathway, provided evidence for coexpression of some of these isozymes, and indicated the involvement of some of these enzymes in the degradation of BPH, ethylbenzene (ETB), and PCBs. To investigate the expression of these isozymes and better understand how they contribute to the robust degradative capacity of RHA1, we comprehensively analyzed the 9.7-Mb genome of RHA1 for BPH pathway genes and characterized the transcriptome of RHA1 growing on benzoate (BEN), BPH, and ETB. Sequence analyses revealed 54 potential BPH pathway genes, including 28 not previously reported. Transcriptomic analysis with a DNA microarray containing 70-mer probes for 8,213 RHA1 genes revealed a suite of 320 genes of diverse functions that were upregulated during growth both on BPH and on ETB, relative to growth on the control substrate, pyruvate. By contrast, only 65 genes were upregulated during growth on BEN. Quantitative PCR assays confirmed microarray results for selected genes and indicated that some of the catabolic genes were upregulated over 10,000-fold. Our analysis suggests that up to 22 enzymes, including 8 newly identified ones, may function in the BPH pathway of RHA1. The relative expression levels of catabolic genes did not differ for BPH and ETB, suggesting a common regulatory mechanism. This study delineated a suite of catabolic enzymes for biphenyl and alkyl-benzenes in RHA1, which is larger than previously recognized and which may serve as a model for catabolism in other environmentally important bacteria having large genomes. PMID:16957245

Comparative Genomics of the Ubiquitous, Hydrocarbon-degrading Genus Marinobacter

NASA Astrophysics Data System (ADS)

Singer, E.; Webb, E.; Edwards, K. J.

2012-12-01

The genus Marinobacter is amongst the most ubiquitous in the global oceans and strains have been isolated from a wide variety of marine environments, including offshore oil-well heads, coastal thermal springs, Antarctic sea water, saline soils and associations with diatoms and dinoflagellates. Many strains have been recognized to be important hydrocarbon degraders in various marine habitats presenting sometimes extreme pH or salinity conditions. Analysis of the genome of M. aquaeolei revealed enormous adaptation versatility with an assortment of strategies for carbon and energy acquisition, sensation, and defense. In an effort to elucidate the ecological and biogeochemical significance of the Marinobacters, seven Marinobacter strains from diverse environments were included in a comparative genomics study. Genomes were screened for metabolic and adaptation potential to elucidate the strategies responsible for the omnipresence of the Marinobacter genus and their remedial action potential in hydrocarbon-polluted waters. The core genome predominantly encodes for key genes involved in hydrocarbon degradation, biofilm-relevant processes, including utilization of external DNA, halotolerance, as well as defense mechanisms against heavy metals, antibiotics, and toxins. All Marinobacter strains were observed to degrade a wide spectrum of hydrocarbon species, including aliphatic, polycyclic aromatic as well as acyclic isoprenoid compounds. Various genes predicted to facilitate hydrocarbon degradation, e.g. alkane 1-monooxygenase, appear to have originated from lateral gene transfer as they are located on gene clusters of 10-20% lower GC-content compared to genome averages and are flanked by transposases. Top ortholog hits are found in other hydrocarbon degrading organisms, e.g. Alcanivorax borkumensis. Strategies for hydrocarbon uptake encoded by various Marinobacter strains include cell surface hydrophobicity adaptation via capsular polysaccharide biosynthesis and attachment using fimbriae and pili. Formation of biofilm with biosurfactant characteristics has been observed in Marinobacter cultures and environmental strains in relation to hydrocarbon degradation. Genomic potential exists for the synthesis of biofilm-related carbon and energy storage compounds, e.g. alginate and isoprenoid wax esters, and quorum sensing encoded by the regulatory luxR gene and N-acyl-L-homoserine lactone (AHL) signals. Halotolerance is predicted to be achieved through biosynthesis and/or import of compatible solutes, including glycine betaine, choline, ectoine, sucrose, periplasmic glucans as well as membrane channel activity regulating intracellular sodium, potassium and chloride concentration balance. Gene abundances concur with those observed in sequenced halophilic Halomonas genomes. Defense mechanisms are plentiful and include arsenate, organic solvent, copper, and mercuric resistance, compounds, which frequently occur in oil refinery wastewater. The Marinobacter genomes reflect dynamic environments and diverse interactions with viruses and other bacteria with similar metabolic strategies, as reflected by the large number of integrases and transposases. This study has provided comprehensive genomic insights into the metabolic versatility and predicted environmental impact potential of one of the most ubiquitous bacterial genera.

Expression-based clustering of CAZyme-encoding genes of Aspergillus niger.

PubMed

Gruben, Birgit S; Mäkelä, Miia R; Kowalczyk, Joanna E; Zhou, Miaomiao; Benoit-Gelber, Isabelle; De Vries, Ronald P

2017-11-23

The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX that were grown on their specific inducing compounds. The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.

Light Modulates Metabolic Pathways and Other Novel Physiological Traits in the Human Pathogen Acinetobacter baumannii

PubMed Central

Müller, Gabriela L.; Tuttobene, Marisel; Altilio, Matías; Martínez Amezaga, Maitena; Nguyen, Meaghan; Cribb, Pamela; Cybulski, Larisa E.; Ramírez, María Soledad; Altabe, Silvia

2017-01-01

ABSTRACT Light sensing in chemotrophic bacteria has been relatively recently ascertained. In the human pathogen Acinetobacter baumannii, light modulates motility, biofilm formation, and virulence through the blue-light-sensing-using flavin (BLUF) photoreceptor BlsA. In addition, light can induce a reduction in susceptibility to certain antibiotics, such as minocycline and tigecycline, in a photoreceptor-independent manner. In this work, we identified new traits whose expression levels are modulated by light in this pathogen, which comprise not only important determinants related to pathogenicity and antibiotic resistance but also metabolic pathways, which represents a novel concept for chemotrophic bacteria. Indeed, the phenylacetic acid catabolic pathway and trehalose biosynthesis were modulated by light, responses that completely depend on BlsA. We further show that tolerance to some antibiotics and modulation of antioxidant enzyme levels are also influenced by light, likely contributing to bacterial persistence in adverse environments. Also, we present evidence indicating that surfactant production is modulated by light. Finally, the expression of whole pathways and gene clusters, such as genes involved in lipid metabolism and genes encoding components of the type VI secretion system, as well as efflux pumps related to antibiotic resistance, was differentially induced by light. Overall, our results indicate that light modulates global features of the A. baumannii lifestyle. IMPORTANCE The discovery that nonphototrophic bacteria respond to light constituted a novel concept in microbiology. In this context, we demonstrated that light could modulate aspects related to bacterial virulence, persistence, and resistance to antibiotics in the human pathogen Acinetobacter baumannii. In this work, we present the novel finding that light directly regulates metabolism in this chemotrophic bacterium. Insights into the mechanism show the involvement of the photoreceptor BlsA. In addition, tolerance to antibiotics and catalase levels are also influenced by light, likely contributing to bacterial persistence in adverse environments, as is the expression of the type VI secretion system and efflux pumps. Overall, a profound influence of light on the lifestyle of A. baumannii is suggested to occur. PMID:28289081

Light Modulates Metabolic Pathways and Other Novel Physiological Traits in the Human Pathogen Acinetobacter baumannii.

PubMed

Müller, Gabriela L; Tuttobene, Marisel; Altilio, Matías; Martínez Amezaga, Maitena; Nguyen, Meaghan; Cribb, Pamela; Cybulski, Larisa E; Ramírez, María Soledad; Altabe, Silvia; Mussi, María Alejandra

2017-05-15

Light sensing in chemotrophic bacteria has been relatively recently ascertained. In the human pathogen Acinetobacter baumannii , light modulates motility, biofilm formation, and virulence through the blue-light-sensing-using flavin (BLUF) photoreceptor BlsA. In addition, light can induce a reduction in susceptibility to certain antibiotics, such as minocycline and tigecycline, in a photoreceptor-independent manner. In this work, we identified new traits whose expression levels are modulated by light in this pathogen, which comprise not only important determinants related to pathogenicity and antibiotic resistance but also metabolic pathways, which represents a novel concept for chemotrophic bacteria. Indeed, the phenylacetic acid catabolic pathway and trehalose biosynthesis were modulated by light, responses that completely depend on BlsA. We further show that tolerance to some antibiotics and modulation of antioxidant enzyme levels are also influenced by light, likely contributing to bacterial persistence in adverse environments. Also, we present evidence indicating that surfactant production is modulated by light. Finally, the expression of whole pathways and gene clusters, such as genes involved in lipid metabolism and genes encoding components of the type VI secretion system, as well as efflux pumps related to antibiotic resistance, was differentially induced by light. Overall, our results indicate that light modulates global features of the A. baumannii lifestyle. IMPORTANCE The discovery that nonphototrophic bacteria respond to light constituted a novel concept in microbiology. In this context, we demonstrated that light could modulate aspects related to bacterial virulence, persistence, and resistance to antibiotics in the human pathogen Acinetobacter baumannii In this work, we present the novel finding that light directly regulates metabolism in this chemotrophic bacterium. Insights into the mechanism show the involvement of the photoreceptor BlsA. In addition, tolerance to antibiotics and catalase levels are also influenced by light, likely contributing to bacterial persistence in adverse environments, as is the expression of the type VI secretion system and efflux pumps. Overall, a profound influence of light on the lifestyle of A. baumannii is suggested to occur. Copyright © 2017 American Society for Microbiology.

Impact of protists on a hydrocarbon-degrading bacterial community from deep-sea Gulf of Mexico sediments: A microcosm study

NASA Astrophysics Data System (ADS)

Beaudoin, David J.; Carmichael, Catherine A.; Nelson, Robert K.; Reddy, Christopher M.; Teske, Andreas P.; Edgcomb, Virginia P.

2016-07-01

In spite of significant advancements towards understanding the dynamics of petroleum hydrocarbon degrading microbial consortia, the impacts (direct or indirect via grazing activities) of bacterivorous protists remain largely unknown. Microcosm experiments were used to examine whether protistan grazing affects the petroleum hydrocarbon degradation capacity of a deep-sea sediment microbial community from an active Gulf of Mexico cold seep. Differences in n-alkane content between native sediment microcosms and those treated with inhibitors of eukaryotes were assessed by comprehensive two-dimensional gas chromatography following 30-90 day incubations and analysis of shifts in microbial community composition using small subunit ribosomal RNA gene clone libraries. More biodegradation was observed in microcosms supplemented with eukaryotic inhibitors. SSU rRNA gene clone libraries from oil-amended treatments revealed an increase in the number of proteobacterial clones (particularly γ-proteobacteria) after spiking sediments with diesel oil. Bacterial community composition shifted, and degradation rates increased, in treatments where protists were inhibited, suggesting protists affect the hydrocarbon degrading capacity of microbial communities in sediments collected at this Gulf of Mexico site.

GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium.

PubMed

Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

2015-01-01

To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium.

GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

PubMed Central

Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

2015-01-01

To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium. PMID:26413044

Regulation of mRNA translation during mitosis

PubMed Central

Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

2015-01-01

Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ∼200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function. DOI: http://dx.doi.org/10.7554/eLife.07957.001 PMID:26305499

Complete genome sequence of Streptomyces sp. strain CFMR 7, a natural rubber degrading actinomycete isolated from Penang, Malaysia.

PubMed

Nanthini, Jayaram; Chia, Kim-Hou; Thottathil, Gincy P; Taylor, Todd D; Kondo, Shinji; Najimudin, Nazalan; Baybayan, Primo; Singh, Siddharth; Sudesh, Kumar

2015-11-20

Streptomyces sp. strain CFMR 7, which naturally degrades rubber, was isolated from a rubber plantation. Whole genome sequencing and assembly resulted in 2 contigs with total genome size of 8.248 Mb. Two latex clearing protein (lcp) genes which are responsible for rubber degrading activities were identified. Copyright © 2015 Elsevier B.V. All rights reserved.

The potato amylase inhibitor gene SbAI regulates cold-induced sweetening in potato tubers by modulating amylase activity.

PubMed

Zhang, Huiling; Liu, Jun; Hou, Juan; Yao, Ying; Lin, Yuan; Ou, Yongbin; Song, Botao; Xie, Conghua

2014-09-01

Potato cold-induced sweetening (CIS) is critical for the postharvest quality of potato tubers. Starch degradation is considered to be one of the key pathways in the CIS process. However, the functions of the genes that encode enzymes related to starch degradation in CIS and the activity regulation of these enzymes have received less attention. A potato amylase inhibitor gene known as SbAI was cloned from the wild potato species Solanum berthaultii. This genetic transformation confirmed that in contrast to the SbAI suppression in CIS-resistant potatoes, overexpressing SbAI in CIS-sensitive potatoes resulted in less amylase activity and a lower rate of starch degradation accompanied by a lower reducing sugar (RS) content in cold-stored tubers. This finding suggested that the SbAI gene may play crucial roles in potato CIS by modulating the amylase activity. Further investigations indicated that pairwise protein-protein interactions occurred between SbAI and α-amylase StAmy23, β-amylases StBAM1 and StBAM9. SbAI could inhibit the activities of both α-amylase and β-amylase in potato tubers primarily by repressing StAmy23 and StBAM1, respectively. These findings provide the first evidence that SbAI is a key regulator of the amylases that confer starch degradation and RS accumulation in cold-stored potato tubers. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition.

PubMed

Huang, Yuhong; Busk, Peter Kamp; Grell, Morten Nedergaard; Zhao, Hai; Lange, Lene

2014-12-01

Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73U/mg) at pH5 and 50°C. The Km and Vmax with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20mM and 2.41U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

Endophytic Bacteria Associated with Hieracium piloselloides: Their Potential for Hydrocarbon-Utilizing and Plant Growth-Promotion.

PubMed

Pawlik, Małgorzata; Piotrowska-Seget, Zofia

2015-01-01

The aim of this study was to assess the potential of 18 crude-oil-degrading endophytic bacteria for removal of hydrocarbons and promotion of plant growth. Strains were isolated from Hieracium piloselloides (tall hawkweed), which grows in soil heavily polluted with petroleum hydrocarbons. Bacteria from the genus Pseudomonas were abundant among the isolates. The potential for hydrocarbon degradation was evaluated by polymerase chain reaction (PCR) analyses of the genes alkB, alkH, C23O, P450, and pah. It was found that 88.89% of the endophytic bacteria contained gene-encoding polycyclic aromatic hydrocarbon (PAH) initial dioxygenase, 61% possessed the 2,3-catechol dioxygenase gene, and 39% of strains that were tested had the cytochrome P-450 hydroxylase gene. All isolates were capable of producing indole-3-acetic acid (1.8-76.4 μg/ml). Only 17% of them were able to produce siderophores, excrete cellulase, and solubilize phosphate. Hydrogen cyanide synthesis occurred in 33% of endophytic bacteria. The 1-aminocyclopropane-1-carboxylate deaminase activity in isolates that were screened was in the range of 2.6 to 74.1 μmol α-ketobutyrate/mg/h. This feature of the bacteria indicated that isolates may enhance the phytoremediation process. Data suggest that crude-oil-degrading endophytic bacteria possess potential to be promising candidates for enhancement of phytoremediation of hydrocarbon-contaminated soil. Further evaluation of these bacteria is needed in order to assess the role played in the degradation of petroleum hydrocarbons.

The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner

PubMed Central

Vicente, Rebeca L.; Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

2016-01-01

Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application. PMID:27977736

Microbial diversity and anaerobic hydrocarbon degradation potential in an oil-contaminated mangrove sediment

PubMed Central

2012-01-01

Background Mangrove forests are coastal wetlands that provide vital ecosystem services and serve as barriers against natural disasters like tsunamis, hurricanes and tropical storms. Mangroves harbour a large diversity of organisms, including microorganisms with important roles in nutrient cycling and availability. Due to tidal influence, mangroves are sites where crude oil from spills farther away can accumulate. The relationship between mangrove bacterial diversity and oil degradation in mangrove sediments remains poorly understood. Results Mangrove sediment was sampled from 0–5, 15–20 and 35–40 cm depth intervals from the Suruí River mangrove (Rio de Janeiro, Brazil), which has a history of oil contamination. DGGE fingerprinting for bamA, dsr and 16S rRNA encoding fragment genes, and qPCR analysis using dsr and 16S rRNA gene fragment revealed differences with sediment depth. Conclusions Analysis of bacterial 16S rRNA gene diversity revealed changes with depth. DGGE for bamA and dsr genes shows that the anaerobic hydrocarbon-degrading community profile also changed between 5 and 15 cm depth, and is similar in the two deeper sediments, indicating that below 15 cm the anaerobic hydrocarbon-degrading community appears to be well established and homogeneous in this mangrove sediment. qPCR analysis revealed differences with sediment depth, with general bacterial abundance in the top layer (0–5 cm) being greater than in both deeper sediment layers (15–20 and 35–40 cm), which were similar to each other. PMID:22935169

Microbial diversity and anaerobic hydrocarbon degradation potential in an oil-contaminated mangrove sediment.

PubMed

Andrade, Luiza L; Leite, Deborah C A; Ferreira, Edir M; Ferreira, Lívia Q; Paula, Geraldo R; Maguire, Michael J; Hubert, Casey R J; Peixoto, Raquel S; Domingues, Regina M C P; Rosado, Alexandre S

2012-08-30

Mangrove forests are coastal wetlands that provide vital ecosystem services and serve as barriers against natural disasters like tsunamis, hurricanes and tropical storms. Mangroves harbour a large diversity of organisms, including microorganisms with important roles in nutrient cycling and availability. Due to tidal influence, mangroves are sites where crude oil from spills farther away can accumulate. The relationship between mangrove bacterial diversity and oil degradation in mangrove sediments remains poorly understood. Mangrove sediment was sampled from 0-5, 15-20 and 35-40 cm depth intervals from the Suruí River mangrove (Rio de Janeiro, Brazil), which has a history of oil contamination. DGGE fingerprinting for bamA, dsr and 16S rRNA encoding fragment genes, and qPCR analysis using dsr and 16S rRNA gene fragment revealed differences with sediment depth. Analysis of bacterial 16S rRNA gene diversity revealed changes with depth. DGGE for bamA and dsr genes shows that the anaerobic hydrocarbon-degrading community profile also changed between 5 and 15 cm depth, and is similar in the two deeper sediments, indicating that below 15 cm the anaerobic hydrocarbon-degrading community appears to be well established and homogeneous in this mangrove sediment. qPCR analysis revealed differences with sediment depth, with general bacterial abundance in the top layer (0-5 cm) being greater than in both deeper sediment layers (15-20 and 35-40 cm), which were similar to each other.

Comparative genomic analysis of nine Sphingobium strains: Insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways

DOE PAGES

Verma, Helianthous; Kumar, Roshan; Oldach, Phoebe; ...

2014-11-23

Background: Sphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes ( S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6). Results: Efficient HCH degradersmore » phylogenetically clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. In addition, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity. In conclusion, the bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their arrangement with respect to IS 6100 and evidence for potential plasmid content elucidate possible evolutionary acquisition mechanisms for this pathway. This study further opens the horizon for selection of bacterial strains for inclusion in an HCH bioremediation consortium and suggests that HDIPO4, IP26 and B90A would be appropriate candidates for inclusion.« less

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

PubMed Central

Condemine, G; Hugouvieux-Cotte-Pattat, N; Robert-Baudouy, J

1986-01-01

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis. PMID:3949717

Biocatalytic Desulfurization Capabilities of a Mixed Culture during Non-Destructive Utilization of Recalcitrant Organosulfur Compounds

PubMed Central

Ismail, Wael; El-Sayed, Wael S.; Abdul Raheem, Abdul Salam; Mohamed, Magdy E.; El Nayal, Ashraf M.

2016-01-01

We investigated the biodesulfurization potential of a mixed culture AK6 enriched from petroleum hydrocarbons-polluted soil with dibenzothiophene (DBT) as a sulfur source. In addition to DBT, AK6 utilized the following compounds as sulfur sources: 4-methyldibenzothiophene (4-MDBT), benzothiophene (BT), and 4,6- dimethyldibenzothiophene (4,6-DM-DBT). None of these compounds supported the growth of AK6 as the sole carbon and sulfur source. AK6 could not grow on dibenzylsulfide (DBS) as a sulfur source. The AK6 community structure changed according to the provided sulfur source. The major DGGE bands represented members of the genera Sphingobacterium, Klebsiella, Pseudomonas, Stenotrophomonas, Arthrobacter, Mycobacterium, and Rhodococcus. Sphingobacterium sp. and Pseudomonas sp. were abundant across all cultures utilizing any of the tested thiophenic S-compounds. Mycobacterium/Rhodococcus spp. were restricted to the 4-MDBT culture. The 4-MDBT culture had the highest species richness and diversity. Biodesulfurization of DBT by resting cells of AK6 produced 2-hydroxybiphenyl (2-HBP) in addition to trace amounts of phenylacetate. AK6 transformed DBT to 2-hydroxybiphenyl with a specific activity of 9 ± 0.6 μM 2-HBP g dry cell weight−1 h−1. PCR confirmed the presence in the AK6 community of the sulfur-specific (4S) pathway genes dszB and dszC. Mixed cultures hold a better potential than axenic ones for the development of a biodesulfurization technology. PMID:26973637

Hepatic encephalopathy in patients with acute decompensation of cirrhosis and acute-on-chronic liver failure.

PubMed

Romero-Gómez, Manuel; Montagnese, Sara; Jalan, Rajiv

2015-02-01

Hepatic encephalopathy in a hospitalized cirrhotic patient is associated with a high mortality rate and its presence adds further to the mortality of patients with acute-on-chronic liver failure (ACLF). The exact pathophysiological mechanisms of HE in this group of patients are unclear but hyperammonemia, systemic inflammation (including sepsis, bacterial translocation, and insulin resistance) and oxidative stress, modulated by glutaminase gene alteration, remain as key factors. Moreover, alcohol misuse, hyponatremia, renal insufficiency, and microbiota are actively explored. HE diagnosis requires exclusion of other causes of neurological, metabolic and psychiatric dysfunction. Hospitalization in the ICU should be considered in every patient with overt HE, but particularly if this is associated with ACLF. Precipitating factors should be identified and treated as required. Evidence-based specific management options are limited to bowel cleansing and non-absorbable antibiotics. Ammonia lowering drugs, such as glycerol phenylbutyrate and ornithine phenylacetate show promise but are still in clinical trials. Albumin dialysis may be useful in refractory cases. Antibiotics, prebiotics, and treatment of diabetes reduce systemic inflammation. Where possible and not contraindicated, large portal-systemic shunts may be embolized but liver transplantation is the most definitive step in the management of HE in this setting. HE in patients with ACLF appears to be clinically and pathophysiologically distinct from that of acute decompensation and requires further studies and characterization. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Appetite for danger - genetic potential for PCP degradation at historically polluted groundwater sites

NASA Astrophysics Data System (ADS)

Mikkonen, Anu; Yläranta, Kati; Tiirola, Marja; Romantschuk, Martin; Sinkkonen, Aki

2016-04-01

Pentachlorophenol (PCP) is a priority pollutant of exclusively anthropogenic origin. Formerly used commonly in timber preservatives, PCP has persisted at polluted groundwater sites decades after its use was banned, typically as the last detectable contaminant component. Notorious for its toxicity and poor biodegradability, little is known about the genetic potential and pathways for PCP degradation in the environment. The only fully characterized mineralization pathway is initiated by the enzyme coded by chromosomal pcpB gene, previously detected in PCP degrading Sphingomonadaceae bacteria isolated at two continents. However, there is no information about the abundance or diversity of any PCP degradation related gene at contaminated sites in situ. Our aim was to assess whether pcpB and/or sphingomonads seem to play a role in in situ degradation of PCP, by studying whether pcpB i) is detectable at chlorophenol-polluted groundwater sediments, ii) responds to PCP concentration changes, and iii) shows correlation with the abundance of sphingomonads or a specific sphingomonad genus. Novel protocols for quantification and profiling of pcpB, with primers covering full known diversity, were developed and tested at two sites in Finland with well-documented long-term chlorophenol contamination history: Kärkölä and Pursiala. High throughput sequencing complemented characterization of the total bacterial community and pcpB gene pool. The relative abundance of pcpB in bacterial community was associated with spatial variability in groundwater PCP concentration in Pursiala, and with temporal differences in groundwater PCP concentration in Kärkölä. T-RFLP fingerprinting results indicated and Ion Torrent PGM and Sanger sequencing confirmed the presence of a single phylotype of pcpB at both geographically distant, historically contaminated sites, matching the one detected previously in Canadian bioreactor clones and Kärkölä bioreactor isolates. Sphingomonad abundance generally correlated positively with pcpB abundance. Sphingomonad and pcpB ranges in the same sample were comparable regardless of differences in sphingomonad community composition between different groundwater wells. These first cultivation-independent results of pcpB abundance and diversity at contaminated sites indicate that pcpB confers competitive advantage in environments contaminated with the priority pollutant PCP and may be related to its degradation in situ; its relative abundance amongst bacteria reflected PCP concentration, and it seemed to be widely shared among various sphingomonad genera. Interestingly, decades under contamination pressure have led to no diversification of pcpB gene at these sites, suggesting constrained distribution and evolution of genetic potential for PCP degradation. It remains open whether the present form of the gene or pathway is the single most efficient one in these environments, or whether inoculation with diverse degrader strains isolated elsewhere could further enhance PCP bioremediation.

Benzoate Metabolism Intermediate Benzoyl Coenzyme A Affects Gentisate Pathway Regulation in Comamonas testosteroni

PubMed Central

Chen, Dong-Wei; Zhang, Yun; Jiang, Cheng-Ying

2014-01-01

A previous study showed that benzoate was catabolized via a coenzyme A (CoA)-dependent epoxide pathway in Azoarcus evansii (R. Niemetz, U. Altenschmidt, S. Brucker, and G. Fuchs, Eur. J. Biochem. 227:161-168, 1995), but gentisate 1,2-dioxygenase was induced. Similarly, we found that the Comamonas testosteroni strain CNB-1 degraded benzoate via a CoA-dependent epoxide pathway and that gentisate 1,2-dioxygenase (GenA) was also induced when benzoate or 3-hydroxybenzoate served as a carbon source for growth. Genes encoding the CoA-dependent epoxide (box genes) and gentisate (gen genes) pathways were identified. Genetic disruption revealed that the gen genes were not involved in benzoate and 3-hydroxybenzoate degradation. Hence, we investigated gen gene regulation in the CNB-1 strain. The PgenA promoter, a MarR-type regulator (GenR), and the GenR binding site were identified. We found that GenR took gentisate, 3-hydroxybenzoate, and benzoyl-CoA as effectors and that binding of GenR to its target DNA sequence was prohibited when these effectors were present. In vivo studies showed that the CNB-1 mutant that lost benzoyl-CoA synthesis was not able to activate PgenA promoter, while transcription of genA was upregulated in another CNB-1 mutant that lost the ability to degrade benzoyl-CoA. The finding that benzoyl-CoA (a metabolic intermediate of benzoate degradation) and 3-hydroxybenzoate function as GenR effectors explains why GenA was induced when CNB-1 grew on benzoate or 3-hydroxybenzoate. Regulation of gentisate pathways by MarR-, LysR-, and IclR-type regulators in diverse bacterial groups is discussed in detail. PMID:24771026

Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.

In this study, the evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil andmore » symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.« less

Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression

DOE PAGES

Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.; ...

2016-06-08

In this study, the evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil andmore » symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.« less

Oxalate-Metabolising Genes of the White-Rot Fungus Dichomitus squalens Are Differentially Induced on Wood and at High Proton Concentration

PubMed Central

de Vries, Ronald P.; Timonen, Sari; Hildén, Kristiina

2014-01-01

Oxalic acid is a prevalent fungal metabolite with versatile roles in growth and nutrition, including degradation of plant biomass. However, the toxicity of oxalic acid makes regulation of its intra- and extracellular concentration crucial. To increase the knowledge of fungal oxalate metabolism, a transcriptional level study on oxalate-catabolising genes was performed with an effective lignin-degrading white-rot fungus Dichomitus squalens, which has demonstrated particular abilities in production and degradation of oxalic acid. The expression of oxalic-acid decomposing oxalate decarboxylase (ODC) and formic-acid decomposing formate dehydrogenase (FDH) encoding genes was followed during the growth of D. squalens on its natural spruce wood substrate. The effect of high proton concentration on the regulation of the oxalate-catabolising genes was determined after addition of organic acid (oxalic acid) and inorganic acid (hydrochloric acid) to the liquid cultures of D. squalens. In order to evaluate the co-expression of oxalate-catabolising and manganese peroxidase (MnP) encoding genes, the expression of one MnP encoding gene, mnp1, of D. squalens was also surveyed in the solid state and liquid cultures. Sequential action of ODC and FDH encoding genes was detected in the studied cultivations. The odc1, fdh2 and fdh3 genes of D. squalens showed constitutive expression, whereas ODC2 and FHD1 most likely are the main responsible enzymes for detoxification of high concentrations of oxalic and formic acids. The results also confirmed the central role of ODC1 when D. squalens grows on coniferous wood. Phylogenetic analysis revealed that fungal ODCs have evolved from at least two gene copies whereas FDHs have a single ancestral gene. As a conclusion, the multiplicity of oxalate-catabolising genes and their differential regulation on wood and in acid-amended cultures of D. squalens point to divergent physiological roles for the corresponding enzymes. PMID:24505339

Dynamics of Multiple lin Gene Expression in Sphingomonas paucimobilis B90A in Response to Different Hexachlorocyclohexane Isomers

PubMed Central

Suar, Mrutyunjay; van der Meer, Jan Roelof; Lawlor, Kirsten; Holliger, Christof; Lal, Rup

2004-01-01

Sphingomonas paucimobilis B90A is able to degrade the α-, β-, γ-, and δ-isomers of hexachlorocyclohexane (HCH). It contains the genes linA, linB, linC, linD, linE, and linR, which have been implicated in HCH degradation. In this study, dynamic expression of the lin genes was measured in chemostat-grown S. paucimobilis B90A by RNA dot blot hybridization and real-time reverse transcriptase PCR upon exposure to a pulse of different HCH isomers. Irrespective of the addition of HCH, linA, linB, and linC were all expressed constitutively. In contrast, linD and linE were induced with α-HCH (2 mg/liter) and γ-HCH (7 mg/liter). A sharp increase in mRNA levels for linD and linE was observed from 10 to 45 min after the addition of α- or γ-HCH. Induction of linD and linE was not detectable upon the addition of 0.7 mg of γ-HCH per liter, although the compound was degraded by the cells. The addition of β-HCH (5 mg/liter) or δ-HCH (20 mg/liter) did not lead to linE and linD induction, despite the fact that 50% of the compounds were degraded. This suggests that degradation of β- and δ-HCH proceeds by a different pathway than that of α- and γ-HCH. PMID:15528530

Mendel's green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway

PubMed Central

Sato, Yutaka; Morita, Ryouhei; Nishimura, Minoru; Yamaguchi, Hiroyasu; Kusaba, Makoto

2007-01-01

Mutants that retain greenness of leaves during senescence are known as “stay-green” mutants. The most famous stay-green mutant is Mendel's green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR. Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a) and chlorophyll b (Chl b) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl–protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1, which encode the first enzymes in the degrading pathways of Chl a and Chl b, respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes. PMID:17709752

Mendel's green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway.

PubMed

Sato, Yutaka; Morita, Ryouhei; Nishimura, Minoru; Yamaguchi, Hiroyasu; Kusaba, Makoto

2007-08-28

Mutants that retain greenness of leaves during senescence are known as "stay-green" mutants. The most famous stay-green mutant is Mendel's green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR. Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a) and chlorophyll b (Chl b) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl-protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1, which encode the first enzymes in the degrading pathways of Chl a and Chl b, respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes.

Transcription of lignocellulose-decomposition associated genes, enzyme activities and production of ethanol upon bioconversion of waste substrate by Phlebia radiata.

PubMed

Mäkinen, Mari A; Risulainen, Netta; Mattila, Hans; Lundell, Taina K

2018-05-04

Previously identified twelve plant cell wall degradation-associated genes of the white rot fungus Phlebia radiata were studied by RT-qPCR in semi-aerobic solid-state cultures on lignocellulose waste material, and on glucose-containing reference medium. Wood-decay-involved enzyme activities and ethanol production were followed to elucidate both the degradative and fermentative processes. On the waste lignocellulose substrate, P. radiata carbohydrate-active enzyme (CAZy) genes encoding cellulolytic and hemicellulolytic activities were significantly upregulated whereas genes involved in lignin modification displayed a more complex response. Two lignin peroxidase genes were differentially expressed on waste lignocellulose compared to glucose medium, whereas three manganese peroxidase-encoding genes were less affected. On the contrary, highly significant difference was noticed for three cellulolytic genes (cbhI_1, eg1, bgl1) with higher expression levels on the lignocellulose substrate than on glucose. This indicates expression of the wood-attacking degradative enzyme system by the fungus also on the recycled, waste core board material. During the second week of cultivation, ethanol production increased on the core board to 0.24 g/L, and extracellular activities against cellulose, xylan, and lignin were detected. Sugar release from the solid lignocellulose resulted with concomitant accumulation of ethanol as fermentation product. Our findings confirm that the fungus activates its white rot decay system also on industrially processed lignocellulose adopted as growth substrate, and under semi-aerobic cultivation conditions. Thus, P. radiata is a good candidate for lignocellulose-based renewable biotechnology to make biofuels and biocompounds from materials with less value for recycling or manufacturing.

Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis

PubMed Central

Liu, Xian-De; Yao, Jun; Tripathi, Durga Nand; Ding, Zhiyong; Xu, Yi; Sun, Mianen; Zhang, Jiangwei; Bai, Shanshan; German, Peter; Hoang, Anh; Zhou, Lijun; Jonasch, Darius; Zhang, Xuesong; Conti, Claudio J.; Efstathiou, Eleni; Tannir, Nizar M; Eissa, N. Tony; Mills, Gordon B.; Walker, Cheryl Lyn; Jonasch, Eric

2014-01-01

Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin-proteasome system (UPS). Here we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy-lysosome system components. Inhibition of autophagy increases HIF2α, while induction of autophagy decreases HIF2α. The E3 ligase von Hippel Lindau (VHL) and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, while proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction. Importantly, clear cell renal cell carcinoma (ccRCC) is frequently associated with mono-allelic loss and/or mutation of autophagy related gene ATG7, and low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy plays an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism. PMID:24998849

A physical mechanism of cancer heterogeneity

NASA Astrophysics Data System (ADS)

Chen, Cong; Wang, Jin

2016-02-01

We studied a core cancer gene regulatory network motif to uncover possible source of cancer heterogeneity from epigenetic sources. When the time scale of the protein regulation to the gene is faster compared to the protein synthesis and degradation (adiabatic regime), normal state, cancer state and an intermediate premalignant state emerge. Due to the epigenetics such as DNA methylation and histone remodification, the time scale of the protein regulation to the gene can be slower or comparable to the protein synthesis and degradation (non-adiabatic regime). In this case, many more states emerge as possible phenotype alternations. This gives the origin of the heterogeneity. The cancer heterogeneity is reflected from the emergence of more phenotypic states, larger protein concentration fluctuations, wider kinetic distributions and multiplicity of kinetic paths from normal to cancer state, higher energy cost per gene switching, and weaker stability.

Robust transcriptional tumor signatures applicable to both formalin-fixed paraffin-embedded and fresh-frozen samples

PubMed Central

Cheng, Jun; He, Jun; Liu, Huaping; Cai, Hao; Hong, Guini; Zhang, Jiahui; Li, Na; Ao, Lu; Guo, Zheng

2017-01-01

Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable resource for clinical researches. However, FFPE samples are usually considered an unreliable source for gene expression analysis due to the partial RNA degradation. In this study, through comparing gene expression profiles between FFPE samples and paired fresh-frozen (FF) samples for three cancer types, we firstly showed that expression measurements of thousands of genes had at least two-fold change in FFPE samples compared with paired FF samples. Therefore, for a transcriptional signature based on risk scores summarized from the expression levels of the signature genes, the risk score thresholds trained from FFPE (or FF) samples could not be applied to FF (or FFPE) samples. On the other hand, we found that more than 90% of the relative expression orderings (REOs) of gene pairs in the FF samples were maintained in their paired FFPE samples and largely unaffected by the storage time. The result suggested that the REOs of gene pairs were highly robust against partial RNA degradation in FFPE samples. Finally, as a case study, we developed a REOs-based signature to distinguish liver cirrhosis from hepatocellular carcinoma (HCC) using FFPE samples. The signature was validated in four datasets of FFPE samples and eight datasets of FF samples. In conclusion, the valuable FFPE samples can be fully exploited to identify REOs-based diagnostic and prognostic signatures which could be robustly applicable to both FF samples and FFPE samples with degraded RNA. PMID:28036264

Genome analysis of Daldinia eschscholtzii strains UM 1400 and UM 1020, wood-decaying fungi isolated from human hosts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan, Chai Ling; Yew, Su Mei; Ngeow, Yun Fong

Background: Daldinia eschscholtzii is a wood-inhabiting fungus that causes wood decay under certain conditions. It has a broad host range and produces a large repertoire of potentially bioactive compounds. However, there is no extensive genome analysis on this fungal species. Results: Two fungal isolates (UM 1400 and UM 1020) from human specimens were identified as Daldinia eschscholtzii by morphological features and ITS-based phylogenetic analysis. Both genomes were similar in size with 10,822 predicted genes in UM 1400 (35.8 Mb) and 11,120 predicted genes in UM 1020 (35.5 Mb). A total of 751 gene families were shared among both UM isolates,more » including gene families associated with fungus-host interactions. In the CAZyme comparative analysis, both genomes were found to contain arrays of CAZyme related to plant cell wall degradation. Genes encoding secreted peptidases were found in the genomes, which encode for the peptidases involved in the degradation of structural proteins in plant cell wall. In addition, arrays of secondary metabolite backbone genes were identified in both genomes, indicating of their potential to produce bioactive secondary metabolites. Both genomes also contained an abundance of gene encoding signaling components, with three proposed MAPK cascades involved in cell wall integrity, osmoregulation, and mating/filamentation. Besides genomic evidence for degrading capability, both isolates also harbored an array of genes encoding stress response proteins that are potentially significant for adaptation to living in the hostile environments. In conclusion: Our genomic studies provide further information for the biological understanding of the D. eschscholtzii and suggest that these wood-decaying fungi are also equipped for adaptation to adverse environments in the human host.« less

Genome-scale analysis of anaerobic benzoate and phenol metabolism in the hyperthermophilic archaeon Ferroglobus placidus

PubMed Central

Holmes, Dawn E; Risso, Carla; Smith, Jessica A; Lovley, Derek R

2012-01-01

Insight into the mechanisms for the anaerobic metabolism of aromatic compounds by the hyperthermophilic archaeon Ferroglobus placidus is expected to improve understanding of the degradation of aromatics in hot (>80° C) environments and to identify enzymes that might have biotechnological applications. Analysis of the F. placidus genome revealed genes predicted to encode enzymes homologous to those previously identified as having a role in benzoate and phenol metabolism in mesophilic bacteria. Surprisingly, F. placidus lacks genes for an ATP-independent class II benzoyl-CoA (coenzyme A) reductase (BCR) found in all strictly anaerobic bacteria, but has instead genes coding for a bzd-type ATP-consuming class I BCR, similar to those found in facultative bacteria. The lower portion of the benzoate degradation pathway appears to be more similar to that found in the phototroph Rhodopseudomonas palustris, than the pathway reported for all heterotrophic anaerobic benzoate degraders. Many of the genes predicted to be involved in benzoate metabolism were found in one of two gene clusters. Genes for phenol carboxylation proceeding through a phenylphosphate intermediate were identified in a single gene cluster. Analysis of transcript abundance with a whole-genome microarray and quantitative reverse transcriptase polymerase chain reaction demonstrated that most of the genes predicted to be involved in benzoate or phenol metabolism had higher transcript abundance during growth on those substrates vs growth on acetate. These results suggest that the general strategies for benzoate and phenol metabolism are highly conserved between microorganisms living in moderate and hot environments, and that anaerobic metabolism of aromatic compounds might be analyzed in a wide range of environments with similar molecular targets. PMID:21776029

Microbial community functional structure in response to antibiotics in pharmaceutical wastewater treatment systems.

PubMed

Zhang, Yu; Xie, Jianping; Liu, Miaomiao; Tian, Zhe; He, Zhili; van Nostrand, Joy D; Ren, Liren; Zhou, Jizhong; Yang, Min

2013-10-15

It is widely demonstrated that antibiotics in the environment affect microbial community structure. However, direct evidence regarding the impacts of antibiotics on microbial functional structures in wastewater treatment systems is limited. Herein, a high-throughput functional gene array (GeoChip 3.0) in combination with quantitative PCR and clone libraries were used to evaluate the microbial functional structures in two biological wastewater treatment systems, which treat antibiotic production wastewater mainly containing oxytetracycline. Despite the bacteriostatic effects of antibiotics, the GeoChip detected almost all key functional gene categories, including carbon cycling, nitrogen cycling, etc., suggesting that these microbial communities were functionally diverse. Totally 749 carbon-degrading genes belonging to 40 groups (24 from bacteria and 16 from fungi) were detected. The abundance of several fungal carbon-degrading genes (e.g., glyoxal oxidase (glx), lignin peroxidase or ligninase (lip), manganese peroxidase (mnp), endochitinase, exoglucanase_genes) was significantly correlated with antibiotic concentrations (Mantel test; P < 0.05), showing that the fungal functional genes have been enhanced by the presence of antibiotics. However, from the fact that the majority of carbon-degrading genes were derived from bacteria and diverse antibiotic resistance genes were detected in bacteria, it was assumed that many bacteria could survive in the environment by acquiring antibiotic resistance and may have maintained the position as a main player in nutrient removal. Variance partitioning analysis showed that antibiotics could explain 24.4% of variations in microbial functional structure of the treatment systems. This study provides insights into the impacts of antibiotics on microbial functional structure of a unique system receiving antibiotic production wastewater, and reveals the potential importance of the cooperation between fungi and bacteria with antibiotic resistance in maintaining the stability and performance of the systems. Copyright © 2013 Elsevier Ltd. All rights reserved.

Genome analysis of Daldinia eschscholtzii strains UM 1400 and UM 1020, wood-decaying fungi isolated from human hosts

DOE PAGES

Chan, Chai Ling; Yew, Su Mei; Ngeow, Yun Fong; ...

2015-11-18

Background: Daldinia eschscholtzii is a wood-inhabiting fungus that causes wood decay under certain conditions. It has a broad host range and produces a large repertoire of potentially bioactive compounds. However, there is no extensive genome analysis on this fungal species. Results: Two fungal isolates (UM 1400 and UM 1020) from human specimens were identified as Daldinia eschscholtzii by morphological features and ITS-based phylogenetic analysis. Both genomes were similar in size with 10,822 predicted genes in UM 1400 (35.8 Mb) and 11,120 predicted genes in UM 1020 (35.5 Mb). A total of 751 gene families were shared among both UM isolates,more » including gene families associated with fungus-host interactions. In the CAZyme comparative analysis, both genomes were found to contain arrays of CAZyme related to plant cell wall degradation. Genes encoding secreted peptidases were found in the genomes, which encode for the peptidases involved in the degradation of structural proteins in plant cell wall. In addition, arrays of secondary metabolite backbone genes were identified in both genomes, indicating of their potential to produce bioactive secondary metabolites. Both genomes also contained an abundance of gene encoding signaling components, with three proposed MAPK cascades involved in cell wall integrity, osmoregulation, and mating/filamentation. Besides genomic evidence for degrading capability, both isolates also harbored an array of genes encoding stress response proteins that are potentially significant for adaptation to living in the hostile environments. In conclusion: Our genomic studies provide further information for the biological understanding of the D. eschscholtzii and suggest that these wood-decaying fungi are also equipped for adaptation to adverse environments in the human host.« less

Characterization of the two intracellular lipases of Y. lipolytica encoded by TGL3 and TGL4 genes: new insights into the role of intracellular lipases and lipid body organisation.

PubMed

Dulermo, Thierry; Tréton, Brigitte; Beopoulos, Athanasios; Kabran Gnankon, Affoué Philomène; Haddouche, Ramdane; Nicaud, Jean-Marc

2013-09-01

Eukaryotes store lipids in a specialised organelle, the lipid body (LB), mainly as triglycerides (TAGs). Both the rates of synthesis and degradation contribute to the control of the accumulation of TAGs. The synthesis of TAGs in yeasts has been well documented, especially in the model yeast Saccharomyces cerevisiae and in the oleaginous yeast Yarrowia lipolytica. However, descriptions of the processes involved in TAG degradation are more scarce and mostly for S. cerevisiae. Here, we report the characterisation of two Y. lipolytica genes, YlTGL3 and YlTGL4, encoding intracellular lipases involved in TAG degradation. The two proteins are localised in lipid bodies, and YlTgl4 was mainly found at the interface between LBs. Surprisingly, the spatial organisation of YlTgl3 and YlTgl4 depends on the culture medium and on the physiological phase of the cell. Inactivation of one or both genes doubles the lipid accumulation capacity of Y. lipolytica, increasing the cell's capacity to accumulate TAGs. The amino acid sequence of YlTgl4 contains the consensus sequence motif (G/A)XSXG, typical of serine hydrolases, whereas YlTgl3 does not. Single and double mutants are unable to degrade TAGs, and higher expression of YlTgl4 correlates with TAG degradation. Therefore, we propose that YlTgl4 is the main lipase responsible for TAG degradation and that YlTgl3 may act as a positive regulator of YlTgl4 rather than a functional lipase. Thus, contrary to S. cerevisiae, Y. lipolytica possesses two intracellular lipases with distinct roles and with distinct localisations in the LB. © 2013. Published by Elsevier B.V. All rights reserved.

DNA-based stable isotope probing coupled with cultivation methods implicates Methylophaga in hydrocarbon degradation

PubMed Central

Mishamandani, Sara; Gutierrez, Tony; Aitken, Michael D.

2014-01-01

Marine hydrocarbon-degrading bacteria perform a fundamental role in the oxidation and ultimate removal of crude oil and its petrochemical derivatives in coastal and open ocean environments. Those with an almost exclusive ability to utilize hydrocarbons as a sole carbon and energy source have been found confined to just a few genera. Here we used stable isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate hydrocarbon-degrading bacteria in coastal North Carolina sea water (Beaufort Inlet, USA) with uniformly labeled [13C]n-hexadecane. The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from n-hexadecane enrichments) were identified to belong to the genus Alcanivorax, with ≤98% sequence identity to the closest type strain—thus representing a putative novel phylogenetic taxon within this genus. Unexpectedly, we also identified 13C-enriched sequences in heavy DNA fractions that were affiliated to the genus Methylophaga. This is a contentious group since, though some of its members have been proposed to degrade hydrocarbons, substantive evidence has not previously confirmed this. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Alcanivorax and Methylophaga to determine their abundance in incubations amended with unlabeled n-hexadecane. Both showed substantial increases in gene copy number during the experiments. Subsequently, we isolated a strain representing the SIP-identified Methylophaga sequences (99.9% 16S rRNA gene sequence identity) and used it to show, for the first time, direct evidence of hydrocarbon degradation by a cultured Methylophaga sp. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of hydrocarbon-degrading bacteria in the marine environment. PMID:24578702

Combining the auxin-inducible degradation system with CRISPR/Cas9-based genome editing for the conditional depletion of endogenous Drosophila melanogaster proteins.

PubMed

Bence, Melinda; Jankovics, Ferenc; Lukácsovich, Tamás; Erdélyi, Miklós

2017-04-01

Inducible protein degradation techniques have considerable advantages over classical genetic approaches, which generate loss-of-function phenotypes at the gene or mRNA level. The plant-derived auxin-inducible degradation system (AID) is a promising technique which enables the degradation of target proteins tagged with the AID motif in nonplant cells. Here, we present a detailed characterization of this method employed during the adult oogenesis of Drosophila. Furthermore, with the help of CRISPR/Cas9-based genome editing, we improve the utility of the AID system in the conditional elimination of endogenously expressed proteins. We demonstrate that the AID system induces efficient and reversible protein depletion of maternally provided proteins both in the ovary and the early embryo. Moreover, the AID system provides a fine spatiotemporal control of protein degradation and allows for the generation of different levels of protein knockdown in a well-regulated manner. These features of the AID system enable the unraveling of the discrete phenotypes of genes with highly complex functions. We utilized this system to generate a conditional loss-of-function allele which allows for the specific degradation of the Vasa protein without affecting its alternative splice variant (solo) and the vasa intronic gene (vig). With the help of this special allele, we demonstrate that dramatic decrease of Vasa protein in the vitellarium does not influence the completion of oogenesis as well as the establishment of proper anteroposterior and dorsoventral polarity in the developing oocyte. Our study suggests that both the localization and the translation of gurken mRNA in the vitellarium is independent from Vasa. © 2017 Federation of European Biochemical Societies.

Uremic Toxins Inhibit Transport by Breast Cancer Resistance Protein and Multidrug Resistance Protein 4 at Clinically Relevant Concentrations

PubMed Central

Mutsaers, Henricus A. M.; van den Heuvel, Lambertus P.; Ringens, Lauke H. J.; Dankers, Anita C. A.; Russel, Frans G. M.; Wetzels, Jack F. M.; Hoenderop, Joost G.; Masereeuw, Rosalinde

2011-01-01

During chronic kidney disease (CKD), there is a progressive accumulation of toxic solutes due to inadequate renal clearance. Here, the interaction between uremic toxins and two important efflux pumps, viz. multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP) was investigated. Membrane vesicles isolated from MRP4- or BCRP-overexpressing human embryonic kidney cells were used to study the impact of uremic toxins on substrate specific uptake. Furthermore, the concentrations of various uremic toxins were determined in plasma of CKD patients using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Our results show that hippuric acid, indoxyl sulfate and kynurenic acid inhibit MRP4-mediated [3H]-methotrexate ([3H]-MTX) uptake (calculated Ki values: 2.5 mM, 1 mM, 25 µM, respectively) and BCRP-mediated [3H]-estrone sulfate ([3H]-E1S) uptake (Ki values: 4 mM, 500 µM and 50 µM, respectively), whereas indole-3-acetic acid and phenylacetic acid reduce [3H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC50 value: 7 mM, respectively). In contrast, p-cresol, p-toluenesulfonic acid, putrescine, oxalate and quinolinic acid did not alter transport mediated by MRP4 or BCRP. In addition, our results show that hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid and phenylacetic acid accumulate in plasma of end-stage CKD patients with mean concentrations of 160 µM, 4 µM, 129 µM, 1 µM and 18 µM, respectively. Moreover, calculated Ki values are below the maximal plasma concentrations of the tested toxins. In conclusion, this study shows that several uremic toxins inhibit active transport by MRP4 and BCRP at clinically relevant concentrations. PMID:21483698

Elucidating the role of the phenylacetic acid metabolic complex in the pathogenic activity of Rhizoctonia solani anastomosis group 3.

PubMed

Bartz, Faith E; Glassbrook, Norman J; Danehower, David A; Cubeta, Marc A

2012-01-01

The soil fungus Rhizoctonia solani produces phytotoxic phenylacetic acid (PAA) and hydroxy (OH-) and methoxy (MeO-) derivatives of PAA. However, limited information is available on the specific role that these compounds play in the development of Rhizoctonia disease symptoms and concentration(s) required to induce a host response. Reports that PAA inhibits the growth of R. solani conflict with the established ability of the fungus to produce and metabolize PAA. Experiments were conducted to clarify the role of the PAA metabolic complex in Rhizoctonia disease. In this study the concentration of PAA and derivatives required to induce tomato root necrosis and stem canker, in the absence of the fungus, and the concentration that inhibits mycelial growth of R. solani were determined. The effect of exogenous PAA and derivatives of PAA on tomato seedling growth also was investigated. Growth of tomato seedlings in medium containing 0.1-7.5 mM PAA and derivatives induced necrosis of up to 85% of root system. Canker development resulted from injection of tomato seedling stems with 7.5 mM PAA, 3-OH-PAA, or 3-MeO-PAA. PAA in the growth medium reduced R. solani biomass, with 50% reduction observed at 7.5 mM. PAA, and derivatives were quantified from the culture medium of 14 isolates of R. solani belonging to three distinct anastomosis groups by GC-MS. The quantities ranged from below the limit of detection to 678 nM, below the concentrations experimentally determined to be phytotoxic. Correlation analyses revealed that isolates of R. solani that produced high PAA and derivatives in vitro also caused high mortality on tomato seedlings. The results of this investigation add to the body of evidence that the PAA metabolic complex is involved in Rhizoctonia disease development but do not indicate that production of these compounds is the primary or the only determinant of pathogenicity.

Ammonia control in children ages 2 months through 5 years with urea cycle disorders: comparison of sodium phenylbutyrate and glycerol phenylbutyrate.

PubMed

Smith, Wendy; Diaz, George A; Lichter-Konecki, Uta; Berry, Susan A; Harding, Cary O; McCandless, Shawn E; LeMons, Cindy; Mauney, Joe; Dickinson, Klara; Coakley, Dion F; Moors, Tristen; Mokhtarani, Masoud; Scharschmidt, Bruce F; Lee, Brendan

2013-06-01

To examine ammonia levels, pharmacokinetics, and safety of glycerol phenylbutyrate (GPB; also referred to as HPN-100) and sodium phenylbutyrate (NaPBA) in young children with urea cycle disorders (UCDs). This open label switch-over study enrolled patients ages 29 days to under 6 years taking NaPBA. Patients underwent 24-hour blood and urine sampling on NaPBA and again on a phenylbutyric acid-equimolar dose of GPB and completed questionnaires regarding signs and symptoms associated with NaPBA and/or their UCD. Fifteen patients (8 argininosuccinate lyase deficiency, 3 argininosuccinic acid synthetase deficiency, 3 ornithine transcarbamylase deficiency, 1 arginase deficiency) ages 2 months through 5 years enrolled in and completed the study. Daily ammonia exposure (24-hour area under the curve) was lower on GPB and met predefined noninferiority criteria (ratio of means 0.79; 95% CI 0.593-1.055; P=.03 Wilcoxon; 0.07 t test). Six patients experienced mild adverse events on GPB; there were no serious adverse events or significant laboratory changes. Liver tests and argininosuccinic acid levels among patients with argininosuccinate lyase deficiency were unchanged or improved on GPB. Eleven of 15 patients reported 35 symptoms on day 1; 23 of these 35 symptoms improved or resolved on GPB. Mean systemic exposure to phenylbutyric acid, phenylacetic acid, and phenylacetylglutamine (PAGN) were similar and phenylacetic acid exposure tended to be higher in the youngest children on both drugs. Urinary PAGN concentration was greater on morning voids and varied less over 24 hours on GPB versus NaPBA. GPB results in more evenly distributed urinary output of PAGN over 24 hours were associated with fewer symptoms and offers ammonia control comparable with that observed with NaPBA in young children with UCDs. Copyright © 2013 Mosby, Inc. All rights reserved.

Changes in volatile compound composition of Antrodia camphorata during solid state fermentation.

PubMed

Xia, Yongjun; Zhang, Baorong; Li, Weijiang; Xu, Ganrong

2011-10-01

Although the volatiles present in mushrooms and fungi have been investigated by many researchers, including Antrodia camphorata in submerged fermentation, there are few data available regarding changes in volatile compounds during fermentation. Our research has revealed that solid state fermentation of A. camphorata is highly odiferous compared with submerged cultures and the odor changed with increasing culture time. Therefore the aim of this study was to investigate the changes in volatile compound composition of A. camphorata during solid state fermentation. Altogether, 124 major volatile compounds were identified. The volatile compounds produced by A. camphorata during growth in solid state fermentation were quite different. Oct-1-en-3-ol, octan-3-one and methyl 2-phenylacetate were predominant in exponential growth phase production, while the dominant volatiles produced in stationary phase were octan-3-one and methyl 2-phenylacetate. In stationary phase, lactone compounds in A. camphorata, such as 5-butyloxolan-2-one, 5-heptyloxolan-2-one, 6-heptyloxan-2-one, contributed greatly to peach and fruit-like flavor. Terpene and terpene alcohol compounds, such as 1-terpineol, L-linalool, T-cadinol, (E, E)-farnesol, β-elemene, cis-α-bisabolene and α-muurolene, made different contributions to herbal fresh aroma in A. camphorata. Nineteen volatile sesquiterpenes were detected from solid state fermentation of A. camphorata. The compounds 5-n-butyl-5H-furan-2-one, β-ionone, (-)-caryophyllene oxide, aromadendrene oxide, diepi-α-cedrene epoxide, β-elemene, α-selinene, α-muurolene, azulene, germacrene D, γ-cadinene and 2-methylpyrazine have not hitherto been reported in A. camphorata. The preliminary results suggest that the aroma-active compounds produced by A camphorata in solid state fermentation might serve as an important source of natural aroma compounds for the food and cosmetic industries or antibiotic activity compounds. The sesquiterpenes could be identified as possible taxonomic markers for A. camphorata. Copyright © 2011 Society of Chemical Industry.

Transcriptome analysis of the digestive system of a wood-feeding termite (Coptotermes formosanus) revealed a unique mechanism for effective biomass degradation.

PubMed

Geng, Alei; Cheng, Yanbing; Wang, Yongli; Zhu, Daochen; Le, Yilin; Wu, Jian; Xie, Rongrong; Yuan, Joshua S; Sun, Jianzhong

2018-01-01

Wood-feeding termite, Coptotermes formosanus Shiraki, represents a highly efficient system for biomass deconstruction and utilization. However, the detailed mechanisms of lignin modification and carbohydrate degradation in this system are still largely elusive. In order to reveal the inherent mechanisms for efficient biomass degradation, four different organs (salivary glands, foregut, midgut, and hindgut) within a complete digestive system of a lower termite, C. formosanus , were dissected and collected. Comparative transcriptomics was carried out to analyze these organs using high-throughput RNA sequencing. A total of 71,117 unigenes were successfully assembled, and the comparative transcriptome analyses revealed significant differential distributions of GH (glycosyl hydrolase) genes and auxiliary redox enzyme genes in different digestive organs. Among the GH genes in the salivary glands, the most abundant were GH9, GH22, and GH1 genes. The corresponding enzymes may have secreted into the foregut and midgut to initiate the hydrolysis of biomass and to achieve a lignin-carbohydrate co-deconstruction system. As the most diverse GH families, GH7 and GH5 were primarily identified from the symbiotic protists in the hindgut. These enzymes could play a synergistic role with the endogenous enzymes from the host termite for biomass degradation. Moreover, twelve out of fourteen genes coding auxiliary redox enzymes from the host termite origin were induced by the feeding of lignin-rich diets. This indicated that these genes may be involved in lignin component deconstruction with its redox network during biomass pretreatment. These findings demonstrate that the termite digestive system synergized the hydrolysis and redox reactions in a programmatic process, through different parts of its gut system, to achieve a maximized utilization of carbohydrates. The detailed unique mechanisms identified from the termite digestive system may provide new insights for advanced design of future biorefinery.

The Gene Cluster for para-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

PubMed Central

Min, Jun; Zhang, Jun-Jie

2014-01-01

Burkholderia sp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) or para-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of the pnp genes in the pnpABA1CDEF cluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activity in vitro in the conversion of 2C4NP to CBQ. Genetic analyses indicated that pnpA plays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels. PMID:25085488

A HIF-LIMD1 negative feedback mechanism mitigates the pro-tumorigenic effects of hypoxia.

PubMed

Foxler, Daniel E; Bridge, Katherine S; Foster, John G; Grevitt, Paul; Curry, Sean; Shah, Kunal M; Davidson, Kathryn M; Nagano, Ai; Gadaleta, Emanuela; Rhys, Hefin I; Kennedy, Paul T; Hermida, Miguel A; Chang, Ting-Yu; Shaw, Peter E; Reynolds, Louise E; McKay, Tristan R; Wang, Hsei-Wei; Ribeiro, Paulo S; Plevin, Michael J; Lagos, Dimitris; Lemoine, Nicholas R; Rajan, Prabhakar; Graham, Trevor A; Chelala, Claude; Hodivala-Dilke, Kairbaan M; Spendlove, Ian; Sharp, Tyson V

2018-06-21

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-β subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1 -negative lung cancers. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Monitoring the degradation capability of novel haloalkaliphilic tributyltin chloride (TBTCl) resistant bacteria from butyltin-polluted site.

PubMed

Hassan, Hamdy A; Dawah, Somya E; El-Sheekh, Mostafa M

2018-03-28

Tributyltin (TBT) is recognized as a major environmental problem at a global scale. Haloalkaliphilic tributyltin (TBT)-degrading bacteria may be a key factor in the remediation of TBT polluted sites. In this work, three haloalkaliphilic bacteria strains were isolated from a TBT-contaminated site in the Mediterranean Sea. After analysis of the 16S rRNA gene sequences the isolates were identified as Sphingobium sp. HS1, Stenotrophomonas chelatiphaga HS2 and Rhizobium borbori HS5. The optimal growth conditions for biodegradation of TBT by the three strains were pH 9 and 7% (w/v) salt concentration. S. chelatiphaga HS2 was the most effective TBT degrader and has the ability to transform most TBT into dibutyltin and monobutyltin (DBT and MBT). A gene was amplified from strain HS2 and identified as TBTB-permease-like, that encodes an ArsB-permease. A reverse transcription polymerase chain reaction analysis in the HS2 strain confirmed that the TBTB-permease-like gene contributes to TBT resistance. The three novel haloalkaliphilic TBT degraders have never been reported previously. Copyright © 2018 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Method for regulation of plant lignin composition

DOEpatents

Chapple, Clint

1999-01-01

A method is disclosed for the regulation of lignin composition in plant tissue. Plants are transformed with a gene encoding an active F5H gene. The expression of the F5H gene results in increased levels of syringyl monomer providing a lignin composition more easily degraded with chemicals and enzymes.

Polynucleotide phosphorlyase (PNPase) is required for Salmonella enterica serovar Typhimurium colonization in swine

USDA-ARS?s Scientific Manuscript database

The pnp gene encodes polynucleotide phosphorylase, an exoribonuclease involved in RNA degradation. A mutation in the pnp gene was previously identified by our group in a signature-tagged mutagenesis screen designed to search for Salmonella enterica serovar Typhimurium genes required for survival in...

The role of gigaxonin in the degradation of the glial-specific intermediate filament protein GFAP

PubMed Central

Lin, Ni-Hsuan; Huang, Yu-Shan; Opal, Puneet; Goldman, Robert D.; Messing, Albee; Perng, Ming-Der

2016-01-01

Alexander disease (AxD) is a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding the intermediate filament (IF) protein GFAP. This disease is characterized by excessive accumulation of GFAP, known as Rosenthal fibers, within astrocytes. Abnormal GFAP aggregation also occurs in giant axon neuropathy (GAN), which is caused by recessive mutations in the gene encoding gigaxonin. Given that one of the functions of gigaxonin is to facilitate proteasomal degradation of several IF proteins, we sought to determine whether gigaxonin is involved in the degradation of GFAP. Using a lentiviral transduction system, we demonstrated that gigaxonin levels influence the degradation of GFAP in primary astrocytes and in cell lines that express this IF protein. Gigaxonin was similarly involved in the degradation of some but not all AxD-associated GFAP mutants. In addition, gigaxonin directly bound to GFAP, and inhibition of proteasome reversed the clearance of GFAP in cells achieved by overexpressing gigaxonin. These studies identify gigaxonin as an important factor that targets GFAP for degradation through the proteasome pathway. Our findings provide a critical foundation for future studies aimed at reducing or reversing pathological accumulation of GFAP as a potential therapeutic strategy for AxD and related diseases. PMID:27798231

Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment

PubMed Central

Yang, Fu-Chun; Chen, Yi-Lung; Tang, Sen-Lin; Yu, Chang-Ping; Wang, Po-Hsiang; Ismail, Wael; Wang, Chia-Hsiang; Ding, Jiun-Yan; Yang, Cheng-Yu; Yang, Chia-Ying; Chiang, Yin-Ru

2016-01-01

Steroid hormones, such as androgens, are common surface-water contaminants. However, literature on the ecophysiological relevance of steroid-degrading organisms in the environment, particularly in anoxic ecosystems, is extremely limited. We previously reported that Steroidobacter denitrificans anaerobically degrades androgens through the 2,3-seco pathway. In this study, the genome of Sdo. denitrificans was completely sequenced. Transcriptomic data revealed gene clusters that were distinctly expressed during anaerobic growth on testosterone. We isolated and characterized the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, were used as biomarkers to investigate androgen biodegradation in the largest sewage treatment plant in Taipei, Taiwan. Androgen metabolite analysis indicated that denitrifying bacteria in anoxic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assays showed androgen degradation in anoxic sewage by Thauera spp. through the action of 1-testosterone hydratase/dehydrogenase. Our integrative ‘omics' approach can be used for culture-independent investigations of the microbial degradation of structurally complex compounds where isotope-labeled substrates are not easily available. PMID:26872041

The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem.

PubMed

Samach, A; Klenz, J E; Kohalmi, S E; Risseeuw, E; Haughn, G W; Crosby, W L

1999-11-01

Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ-identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F-box protein that interacts with Arabidopsis SKP1-like proteins, both in the yeast two-hybrid system and in vitro. In yeast and other organisms both F-box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F-box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.

Functional gene diversity of soil microbial communities from five oil-contaminated fields in China.

PubMed

Liang, Yuting; Van Nostrand, Joy D; Deng, Ye; He, Zhili; Wu, Liyou; Zhang, Xu; Li, Guanghe; Zhou, Jizhong

2011-03-01

To compare microbial functional diversity in different oil-contaminated fields and to know the effects of oil contaminant and environmental factors, soil samples were taken from typical oil-contaminated fields located in five geographic regions of China. GeoChip, a high-throughput functional gene array, was used to evaluate the microbial functional genes involved in contaminant degradation and in other major biogeochemical/metabolic processes. Our results indicated that the overall microbial community structures were distinct in each oil-contaminated field, and samples were clustered by geographic locations. The organic contaminant degradation genes were most abundant in all samples and presented a similar pattern under oil contaminant stress among the five fields. In addition, alkane and aromatic hydrocarbon degradation genes such as monooxygenase and dioxygenase were detected in high abundance in the oil-contaminated fields. Canonical correspondence analysis indicated that the microbial functional patterns were highly correlated to the local environmental variables, such as oil contaminant concentration, nitrogen and phosphorus contents, salt and pH. Finally, a total of 59% of microbial community variation from GeoChip data can be explained by oil contamination, geographic location and soil geochemical parameters. This study provided insights into the in situ microbial functional structures in oil-contaminated fields and discerned the linkages between microbial communities and environmental variables, which is important to the application of bioremediation in oil-contaminated sites.

Functional gene diversity of soil microbial communities from five oil-contaminated fields in China

PubMed Central

Liang, Yuting; Van Nostrand, Joy D; Deng, Ye; He, Zhili; Wu, Liyou; Zhang, Xu; Li, Guanghe; Zhou, Jizhong

2011-01-01

To compare microbial functional diversity in different oil-contaminated fields and to know the effects of oil contaminant and environmental factors, soil samples were taken from typical oil-contaminated fields located in five geographic regions of China. GeoChip, a high-throughput functional gene array, was used to evaluate the microbial functional genes involved in contaminant degradation and in other major biogeochemical/metabolic processes. Our results indicated that the overall microbial community structures were distinct in each oil-contaminated field, and samples were clustered by geographic locations. The organic contaminant degradation genes were most abundant in all samples and presented a similar pattern under oil contaminant stress among the five fields. In addition, alkane and aromatic hydrocarbon degradation genes such as monooxygenase and dioxygenase were detected in high abundance in the oil-contaminated fields. Canonical correspondence analysis indicated that the microbial functional patterns were highly correlated to the local environmental variables, such as oil contaminant concentration, nitrogen and phosphorus contents, salt and pH. Finally, a total of 59% of microbial community variation from GeoChip data can be explained by oil contamination, geographic location and soil geochemical parameters. This study provided insights into the in situ microbial functional structures in oil-contaminated fields and discerned the linkages between microbial communities and environmental variables, which is important to the application of bioremediation in oil-contaminated sites. PMID:20861922

Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood

PubMed Central

Hori, Chiaki; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Suzuki, Hitoshi; Master, Emma; Ferreira, Patricia; Ruiz-Dueñas, Francisco J.; Held, Benjamin; Canessa, Paulo; Larrondo, Luis F.; Schmoll, Monika; Druzhinina, Irina S.; Kubicek, Christian P.; Gaskell, Jill A.; Kersten, Phil; St. John, Franz; Glasner, Jeremy; Sabat, Grzegorz; Splinter BonDurant, Sandra; Syed, Khajamohiddin; Yadav, Jagjit; Mgbeahuruike, Anthony C.; Kovalchuk, Andriy; Asiegbu, Fred O.; Lackner, Gerald; Hoffmeister, Dirk; Rencoret, Jorge; Gutiérrez, Ana; Sun, Hui; Lindquist, Erika; Barry, Kerrie; Riley, Robert; Grigoriev, Igor V.; Henrissat, Bernard; Kües, Ursula; Berka, Randy M.; Martínez, Angel T.; Covert, Sarah F.; Blanchette, Robert A.; Cullen, Daniel

2014-01-01

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes. PMID:25474575

Effects of triploidy on growth and protein degradation in skeletal muscle during recovery from feed deprivation in juvenile rainbow trout (Oncorhynchus mykiss).

PubMed

Cleveland, Beth M; Weber, Gregory M

2013-09-01

Identifying physiological differences between diploid and triploid rainbow trout will help define how ploidy affects mechanisms that impact growth and nutrient utilization. Juvenile diploid and triploid female rainbow trout (Oncorhynchus mykiss) were either continually fed or fasted for one week, followed by four weeks of refeeding, and indices of growth and proteolysis-related gene expression in skeletal muscle were measured. Fasting reduced growth, and based on gene expression analysis, increased capacity for protein degradation. Regardless of feeding treatment, triploids displayed slightly greater feed intake and specific growth rates than diploids. Continually fed triploids displayed lower expression of several autophagy-related genes than diploids, suggesting that reduced rates of protein degradation contributed to their faster growth. Reduced expression of ubiquitin ligases fbxo32 and fbxo25 and autophagy-related genes during refeeding implicates reduced proteolysis in recovery growth. At one week of refeeding triploids exhibited greater gains in eviscerated body weight and length, whereas diploids exhibited greater gains in gastrointestinal tract weights. During refeeding two autophagy-related genes, atg4b and lc3b, decreased within one week to continually fed levels in the triploids, but in diploids overshot in expression at one and two weeks of refeeding then rebounding above continually fed levels by week four, suggesting a delayed return to basal levels of proteolysis. Published by Elsevier Inc.

Uncovering Hidden Layers of Cell Cycle Regulation through Integrative Multi-omic Analysis

PubMed Central

Aviner, Ranen; Shenoy, Anjana; Elroy-Stein, Orna; Geiger, Tamar

2015-01-01

Studying the complex relationship between transcription, translation and protein degradation is essential to our understanding of biological processes in health and disease. The limited correlations observed between mRNA and protein abundance suggest pervasive regulation of post-transcriptional steps and support the importance of profiling mRNA levels in parallel to protein synthesis and degradation rates. In this work, we applied an integrative multi-omic approach to study gene expression along the mammalian cell cycle through side-by-side analysis of mRNA, translation and protein levels. Our analysis sheds new light on the significant contribution of both protein synthesis and degradation to the variance in protein expression. Furthermore, we find that translation regulation plays an important role at S-phase, while progression through mitosis is predominantly controlled by changes in either mRNA levels or protein stability. Specific molecular functions are found to be co-regulated and share similar patterns of mRNA, translation and protein expression along the cell cycle. Notably, these include genes and entire pathways not previously implicated in cell cycle progression, demonstrating the potential of this approach to identify novel regulatory mechanisms beyond those revealed by traditional expression profiling. Through this three-level analysis, we characterize different mechanisms of gene expression, discover new cycling gene products and highlight the importance and utility of combining datasets generated using different techniques that monitor distinct steps of gene expression. PMID:26439921

Biodegradation analyses of trichloroethylene (TCE) by bacteria and its use for biosensing of TCE.

PubMed

Chee, Gab-Joo

2011-09-30

Trichloroethylene (TCE) is a toxic, recalcitrant groundwater pollutant. TCE-degrading microorganisms were isolated from various environments. The aerobic bacteria isolated from toluene- and tryptophan-containing media were Pseudomonas sp. strain ASA86 and Burkholderia sp. strain TAM17, respectively; these are necessary for inducing TCE biodegradation in a selective medium. The half-degradation time of TCE to a concentration of 1mg/L was 18 h for strain ASA86 and 7 days for strain TAM17. While identifying toluene/TCE degradation genes, we found that in strain ASA86, the gene was the same as the todC1 gene product encoding toluene dioxygenase identified in Pseudomonas putida F1, and that in strain TAM17, the gene was similar to the tecA1 gene product encoding chlorobenzene dioxygenase identified in Burkholderia sp. PS12. A novel TCE biosensor was developed using strain ASA86 as the inducer of toluene under aerobic conditions. The TCE biosensor exhibited a linear relationship below 3 ppm TCE. Detection limit of the biosensor was 0.05 ppm TCE. The response time of the biosensor was less than 10 min. The biosensor response displayed a constant level during a 2 day period. The TCE biosensor displayed sufficient sensitivity for monitoring TCE in environmental systems. Copyright © 2011 Elsevier B.V. All rights reserved.

Phanerochaete chrysosporium genomics

Treesearch

Luis F. Larrondo; Rafael Vicuna; Dan Cullen

2005-01-01

A high quality draft genome sequence has been generated for the lignocellulose-degrading basidiomycete Phanerochaete chrysosporium (Martinez et al. 2004). Analysis of the genome in the context of previously established genetics and physiology is presented. Transposable elements and their potential relationship to genes involved in lignin degradation are systematically...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Isanapong, Jantiya; Goodwin, Lynne A.; Bruce, David

Microbial communities in the termite hindgut are essential for degrading plant material. We present the high-quality draft genome sequence of the Opitutaceae bacterium strain TAV1, the first member of the phylum Verrucomicrobia to be isolated from wood-feeding termites. The genomic analysis reveals genes coding for lignocellulosic degradation and nitrogen fixation.

von Hippel–Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

PubMed Central

Mousnier, Aurélie; Kubat, Nicole; Massias-Simon, Aurélie; Ségéral, Emmanuel; Rain, Jean-Christophe; Benarous, Richard; Emiliani, Stéphane; Dargemont, Catherine

2007-01-01

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin–proteasome pathway. Here, we identify von Hippel–Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel–Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin–VHL–proteasome pathway in the integration–transcription transition of the viral replication cycle. PMID:17698809

Stuxnet Facilitates the Degradation of Polycomb Protein during Development.

PubMed

Du, Juan; Zhang, Junzheng; He, Tao; Li, Yajuan; Su, Ying; Tie, Feng; Liu, Min; Harte, Peter J; Zhu, Alan Jian

2016-06-20

Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. Here, we report a Drosophila gene, stuxnet (stx), that controls Pc protein stability. We find that heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Our biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; Stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. Our results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of methyl parathion degradation by a Burkholderia zhejiangensis strain, CEIB S4-3, isolated from agricultural soils.

PubMed

Popoca-Ursino, Elida C; Martínez-Ocampo, Fernando; Dantán-González, Edgar; Sánchez-Salinas, Enrique; Ortiz-Hernández, Ma Laura

2017-12-01

Through the use of an enrichment technique, we isolated from the agricultural soils of Morelos in central México a strain of Burkholderia zhejiangensis identified as CEIB S4-3, it's could use the pesticide methyl parathion (MP) as the only source of carbon and degrade completely p-nitrophenol (PNP). For more efficient MP and PNP degradation by the CEIB S4-3 strain, the absence of an extra carbon source, a large inoculum and an MP concentration up to 50 mg/l are required. Sequence and annotation analysis of the draft genome, showed presence of mpd functional gene, which was expressed and its activity on the MP was confirmed. Additionally, the genes coding for enzymes in the benzoquinone pathway (conducted by Gram-negative bacteria) and the benzenotriol pathway (conducted by Gram-positive bacteria) were found, which was corroborated by identification of intermediary metabolites by HPLC. Thus, we propose that B. zhejiangensis CEIB S4-3 uses both degradation pathways.

Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation

PubMed Central

Costaglioli, Patricia; Barthe, Christophe; Claverol, Stephane; Brözel, Volker S; Perrot, Michel; Crouzet, Marc; Bonneu, Marc; Garbay, Bertrand; Vilain, Sebastien

2012-01-01

Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole-genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm-specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs. PMID:23170231

Genome analysis of crude oil degrading Franconibacter pulveris strain DJ34 revealed its genetic basis for hydrocarbon degradation and survival in oil contaminated environment.

PubMed

Pal, Siddhartha; Kundu, Anirban; Banerjee, Tirtha Das; Mohapatra, Balaram; Roy, Ajoy; Manna, Riddha; Sar, Pinaki; Kazy, Sufia K

2017-10-01

Franconibacter pulveris strain DJ34, isolated from Duliajan oil fields, Assam, was characterized in terms of its taxonomic, metabolic and genomic properties. The bacterium showed utilization of diverse petroleum hydrocarbons and electron acceptors, metal resistance, and biosurfactant production. The genome (4,856,096bp) of this strain contained different genes related to the degradation of various petroleum hydrocarbons, metal transport and resistance, dissimilatory nitrate, nitrite and sulfite reduction, chemotaxy, biosurfactant synthesis, etc. Genomic comparison with other Franconibacter spp. revealed higher abundance of genes for cell motility, lipid transport and metabolism, transcription and translation in DJ34 genome. Detailed COG analysis provides deeper insights into the genomic potential of this organism for degradation and survival in oil-contaminated complex habitat. This is the first report on ecophysiology and genomic inventory of Franconibacter sp. inhabiting crude oil rich environment, which might be useful for designing the strategy for bioremediation of oil contaminated environment. Copyright © 2017 Elsevier Inc. All rights reserved.

Isolation and characterization of a novel native Bacillus thuringiensis strain BRC-HZM2 capable of degrading chlorpyrifos.

PubMed

Wu, Songqing; Peng, Yan; Huang, Zhangmin; Huang, Zhipeng; Xu, Lei; Ivan, Gelbič; Guan, Xiong; Zhang, Lingling; Zou, Shuangquan

2015-03-01

Studies were carried out to isolate chlorpyrifos degrading Bacillus thuringiensis (Bt) strains from chlorpyrifos-contaminated samples. Six Bt strains (isolation rate 2.7%) were isolated by modified sodium acetate antibiotic heat treatment, and one novel strain (BRC-HZM2) was selected for further analysis. Phenotype and phylogeny analysis of this strain was conducted on the basis of biochemical reactions, antibiotic sensitivity, 16s rRNA genes, plasmid profile, insecticidal crystal protein profiles, and PCR-RFLP for cry and cyt genes. The degradation rate of chlorpyrifos in liquid culture was estimated during 48 h of incubation for the isolate BRC-HZM2. More than 50% of the initial chlorpyrifos concentration degraded within 12 h, 88.9% after 48 h. These results highlight the potential of the Bt strain for biological control and the bioremediation of environments contaminated with chlorpyrifos. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Working your SOCS off: The role of ASB10 and protein degradation pathways in glaucoma.

PubMed

Keller, Kate E; Wirtz, Mary K

2017-05-01

Evidence is accumulating to suggest that mutations in the Ankyrin and SOCS Box-containing protein-10 (ASB10) gene are associated with glaucoma. Since its identification in a large Oregon family with primary open-angle glaucoma (POAG), ASB10 variants have been associated with disease in US, German and Pakistani cohorts. ASB10 is a member of the ASB family of proteins, which have a common structure including a unique N-terminus, a variable number of central ankyrin (ANK) repeat domains and a suppressor of cytokine signaling (SOCS) box at the C-terminus. Mutations in ASB10 are distributed throughout the entire length of the gene including the two alternatively spliced variants of exon 1. A homozygous mutation in a Pakistani individual with POAG, which lies in the center of the SOCS box, is associated with a particularly severe form of the disease. Like other SOCS box-containing proteins, ASB10 functions in ubiquitin-mediated degradation pathways. The ANK repeats bind to proteins destined for degradation. The SOCS box recruits ubiquitin ligase proteins to form a complex to transfer ubiquitin to a substrate bound to the ANK repeats. The ubiquitin-tagged protein then enters either the proteasomal degradation pathway or the autophagic-lysosomal pathway. The choice of pathway appears to be dependent on which lysine residues are used to build polyubiquitin chains. However, these reciprocal pathways work in tandem to degrade proteins because inhibition of one pathway increases degradation via the other pathway. In this publication, we will review the literature that supports identification of ASB10 as a glaucoma-associated gene and the current knowledge of the function of the ASB10 protein. In addition, we present new data that indicates ASB10 expression is up-regulated by the inflammatory cytokines tumor necrosis factor-α and interleukin-1α. Finally, we will describe the emerging role of other SOCS box-containing proteins in protein degradation pathways in ocular cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative transcriptome analysis reveals different strategies for degradation of steam-exploded sugarcane bagasse by Aspergillus niger and Trichoderma reesei.

PubMed

Borin, Gustavo Pagotto; Sanchez, Camila Cristina; de Santana, Eliane Silva; Zanini, Guilherme Keppe; Dos Santos, Renato Augusto Corrêa; de Oliveira Pontes, Angélica; de Souza, Aline Tieppo; Dal'Mas, Roberta Maria Menegaldo Tavares Soares; Riaño-Pachón, Diego Mauricio; Goldman, Gustavo Henrique; Oliveira, Juliana Velasco de Castro

2017-06-30

Second generation (2G) ethanol is produced by breaking down lignocellulosic biomass into fermentable sugars. In Brazil, sugarcane bagasse has been proposed as the lignocellulosic residue for this biofuel production. The enzymatic cocktails for the degradation of biomass-derived polysaccharides are mostly produced by fungi, such as Aspergillus niger and Trichoderma reesei. However, it is not yet fully understood how these microorganisms degrade plant biomass. In order to identify transcriptomic changes during steam-exploded bagasse (SEB) breakdown, we conducted a RNA-seq comparative transcriptome profiling of both fungi growing on SEB as carbon source. Particular attention was focused on CAZymes, sugar transporters, transcription factors (TFs) and other proteins related to lignocellulose degradation. Although genes coding for the main enzymes involved in biomass deconstruction were expressed by both fungal strains since the beginning of the growth in SEB, significant differences were found in their expression profiles. The expression of these enzymes is mainly regulated at the transcription level, and A. niger and T. reesei also showed differences in TFs content and in their expression. Several sugar transporters that were induced in both fungal strains could be new players on biomass degradation besides their role in sugar uptake. Interestingly, our findings revealed that in both strains several genes that code for proteins of unknown function and pro-oxidant, antioxidant, and detoxification enzymes were induced during growth in SEB as carbon source, but their specific roles on lignocellulose degradation remain to be elucidated. This is the first report of a time-course experiment monitoring the degradation of pretreated bagasse by two important fungi using the RNA-seq technology. It was possible to identify a set of genes that might be applied in several biotechnology fields. The data suggest that these two microorganisms employ different strategies for biomass breakdown. This knowledge can be exploited for the rational design of enzymatic cocktails and 2G ethanol production improvement.

Hydrocarbon-Degrading Bacteria and the Bacterial Community Response in Gulf of Mexico Beach Sands Impacted by the Deepwater Horizon Oil Spill▿†‡

PubMed Central

Kostka, Joel E.; Prakash, Om; Overholt, Will A.; Green, Stefan J.; Freyer, Gina; Canion, Andy; Delgardio, Jonathan; Norton, Nikita; Hazen, Terry C.; Huettel, Markus

2011-01-01

A significant portion of oil from the recent Deepwater Horizon (DH) oil spill in the Gulf of Mexico was transported to the shoreline, where it may have severe ecological and economic consequences. The objectives of this study were (i) to identify and characterize predominant oil-degrading taxa that may be used as model hydrocarbon degraders or as microbial indicators of contamination and (ii) to characterize the in situ response of indigenous bacterial communities to oil contamination in beach ecosystems. This study was conducted at municipal Pensacola Beach, FL, where chemical analysis revealed weathered oil petroleum hydrocarbon (C8 to C40) concentrations ranging from 3.1 to 4,500 mg kg−1 in beach sands. A total of 24 bacterial strains from 14 genera were isolated from oiled beach sands and confirmed as oil-degrading microorganisms. Isolated bacterial strains were primarily Gammaproteobacteria, including representatives of genera with known oil degraders (Alcanivorax, Marinobacter, Pseudomonas, and Acinetobacter). Sequence libraries generated from oiled sands revealed phylotypes that showed high sequence identity (up to 99%) to rRNA gene sequences from the oil-degrading bacterial isolates. The abundance of bacterial SSU rRNA gene sequences was ∼10-fold higher in oiled (0.44 × 107 to 10.2 × 107 copies g−1) versus clean (0.024 × 107 to 1.4 × 107 copies g−1) sand. Community analysis revealed a distinct response to oil contamination, and SSU rRNA gene abundance derived from the genus Alcanivorax showed the largest increase in relative abundance in contaminated samples. We conclude that oil contamination from the DH spill had a profound impact on the abundance and community composition of indigenous bacteria in Gulf beach sands, and our evidence points to members of the Gammaproteobacteria (Alcanivorax, Marinobacter) and Alphaproteobacteria (Rhodobacteraceae) as key players in oil degradation there. PMID:21948834

Activity and determinants of cholinesterases and paraoxonase-1 in blood of workers exposed to non-cholinesterase inhibiting pesticides.

PubMed

Lozano-Paniagua, David; Gómez-Martín, Antonio; Gil, Fernando; Parrón, Tesifón; Alarcón, Raquel; Requena, Mar; Lacasaña, Marina; Hernández, Antonio F

2016-11-25

Pesticide exposure has been associated with different adverse health effects which may be modulated to some extent by paraoxonase-1 (PON1) activity and genetic polymorphisms. This study assessed seasonal variations in PON1 activity (using paraoxon -POase-, phenylacetate -AREase-, diazoxon -DZOase- and dihydrocoumarin -DHCase- as substrates), erythrocyte acetylcholinesterase (AChE) and plasma cholinesterase (using butyrylthiocholine -BuChE- and benzoylcholine -BeChE- as substrates. The study population consisted of intensive agriculture workers regularly exposed to pesticides other than organophosphates and non-exposed controls from Almería (Southeastern Spain). The effect of common genetic polymorphisms of PON1 and BCHE on paraoxonase-1 and cholinesterase activities toward different substrates was also assessed. Linear mixed models were used to compare esterase activities in agricultural workers and control subjects over the two study periods (high and low exposure to pesticides). The significant decrease in AChE and increase in BuChE and BeChE activities observed in workers with respect to control subjects was attributed to pesticide exposure. Workers also had higher levels of AREase, DZOase and, to a lesser extent, of POase, but showed decreased DHCase activity. While PON1 Q192R and PON1 -108C/T gene polymorphisms were significantly associated with all PON1 activities, PON1 L55M showed a significant association with AREase, DZOase and DHCase. BCHE-K (Karlow variant) was significantly associated with lower BeChE activity (but not with BuChE) and BCHE-A (atypical variant) showed no significant association with any cholinesterase activity. These findings suggest that increased PON1, BuChE and BeChE activities in exposed workers might result from an adaptive response against pesticide exposure to compensate for adverse effects at the biochemical level. This response appears to be modulated by PON1 and BCHE gene polymorphisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Functional Genomics of Lignocellulose Degradation in the Basidiomycete White Rot Schizophyllum commune

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohm, Robin A.; Tegelaar, Martin; Henrissat, Bernard

2013-03-01

White and brown rot fungi are among the most important wood decayers in nature. Although more than 50 genomes of Basidiomycete white and brown rots have been sequenced by the Joint Genome Institute, there is still a lot to learn about how these fungi degrade the tough polymers present in wood. In particular, very little is known about how these fungi regulate the expression of genes involved in lignocellulose degradation. Here, we used transcriptomics, proteomics, and promoter analysis in an effort to gain insight into the process of lignocellulose degradation.

Effects of heat and other inducers of the stress response on protein degradation in Chinese hamster and Drosophila cells.

PubMed

Westwood, J T; Steinhardt, R A

1989-04-01

Many recent studies have suggested that heat and other inducers of the heat shock (stress) response in eukaryotic cells might result in the generation of abnormal proteins which would result in the overloading of protein degradation systems and the stabilization of proteins involved in positively regulating heat shock (hs) gene expression. In this study we have examined the effects different heat treatments and other hs inducers have on protein degradation in Chinese hamster ovary (CHO) and Drosophila Kc and Schneider cells. We have found that intermediate temperatures which induced the hs response (42 degrees C in CHO and 34 degrees C in Kc cells) did increase protein degradation rates whereas, higher temperatures which also induced the hs response (45 degrees C in CHO and 37 degrees C in Kc cells) initially increased but then decreased protein degradation rates. While these results are consistent with a model in which the protein degradation system is being overloaded and/or components of it are being depleted, we have found several conditions which induce hs proteins which rule out this mechanism. Exposure of either cell type to amino acid analogs (5 mM canavanine or 5 mM S-aminoethyl cysteine) resulted in the rapid degradation of those proteins which had incorporated the analogs in both CHO and Drosophila cells. However, the addition of analogs had little or no effect on the degradation of preexisting proteins, indicating that the introduction of abnormal proteins probably didn't overload the protein degradation system(s). The addition of 100 microM cadmium sulfate or 100 microM sodium arsenite had little or no effect on protein degradation rates in CHO cells even though both were good inducers of the hs proteins. Thus, exposure to inducers of the hs response does not universally increase protein degradation rates nor does it stabilize preexisting proteins. Therefore, the degradation of abnormal proteins is probably not involved in inducing the hs genes.

Enzymes involved in a novel anaerobic cyclohexane carboxylic acid degradation pathway.

PubMed

Kung, Johannes W; Meier, Anne-Katrin; Mergelsberg, Mario; Boll, Matthias

2014-10-01

The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Enzymes Involved in a Novel Anaerobic Cyclohexane Carboxylic Acid Degradation Pathway

PubMed Central

Kung, Johannes W.; Meier, Anne-Katrin; Mergelsberg, Mario

2014-01-01

The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways. PMID:25112478

Occurrence, diversity and community structure of culturable atrazine degraders in industrial and agricultural soils exposed to the herbicide in Shandong Province, P.R. China.

PubMed

Bazhanov, Dmitry P; Li, Chengyun; Li, Hongmei; Li, Jishun; Zhang, Xinjian; Chen, Xiangfeng; Yang, Hetong

2016-11-08

Soil populations of bacteria rapidly degrading atrazine are critical to the environmental fate of the herbicide. An enrichment bias from the routine isolation procedure prevents studying the diversity of atrazine degraders. In the present work, we analyzed the occurrence, diversity and community structure of soil atrazine-degrading bacteria based on their direct isolation. Atrazine-degrading bacteria were isolated by direct plating on a specially developed SM agar. The atrazine degradation genes trzN and atzABC were detected by multiplex PCR. The diversity of atrazine degraders was characterized by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) genotyping followed by 16S rRNA gene phylogenetic analysis. The occurrence of atrazine-degrading bacteria was also assessed by conventional PCR targeting trzN and atzABC in soil DNA. A total of 116 atrazine-degrading isolates were recovered from bulk and rhizosphere soils sampled near an atrazine factory and from geographically distant maize fields. Fifteen genotypes were distinguished among 56 industrial isolates, with 13 of them representing eight phylogenetic groups of the genus Arthrobacter. The remaining two were closely related to Pseudomonas alcaliphila and Gulosibacter molinativorax and constituted major components of the atrazine-degrading community in the most heavily contaminated industrial plantless soil. All isolates from the adjacent sites inhabited by cogon grass or common reed were various Arthrobacter spp. with a strong prevalence of A. aurescens group. Only three genotypes were distinguished among 60 agricultural strains. Genetically similar Arthrobacter ureafaciens bacteria which occurred as minor inhabitants of cogon grass roots in the industrial soil were ubiquitous and predominant atrazine degraders in the maize rhizosphere. The other two genotypes represented two distant Nocardioides spp. that were specific to their geographic origins. Direct plating on SM agar enabled rapid isolation of atrazine-degrading bacteria and analysis of their natural diversity in soil. The results obtained provided evidence that contaminated soils harbored communities of genetically distinct bacteria capable of individually degrading and utilizing atrazine. The community structures of culturable atrazine degraders were habitat-specific. Bacteria belonging to the genus Arthrobacter were the predominant degraders of atrazine in the plant rhizosphere.

Global transcriptomic analysis of the response of Corynebacterium glutamicum to ferulic acid.

PubMed

Chen, Can; Pan, Junfeng; Yang, Xiaobing; Xiao, He; Zhang, Yaoling; Si, Meiru; Shen, Xihui; Wang, Yao

2017-03-01

Corynebacterium glutamicum can survive by using ferulic acid as the sole carbon source. In this study, we assessed the response of C. glutamicum to ferulic acid stress by means of a global transcriptional response analysis. The transcriptional data showed that several genes involved in degradation of ferulic acid were affected. Moreover, several genes related to the stress response; protein protection or degradation and DNA repair; replication, transcription and translation; and the cell envelope were differentially expressed. Deletion of the katA or sigE gene in C. glutamicum resulted in a decrease in cell viability under ferulic acid stress. These insights will facilitate further engineering of model industrial strains, with enhanced tolerance to ferulic acid to enable easy production of biofuels from lignocellulose.

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

DOE PAGES

Peterson, Joseph R.; Thor, ShengShee; Kohler, Lars; ...

2016-11-16

Here, while a few studies on the variations in mRNA expression and half-lives measured under different growth conditions have been used to predict patterns of regulation in bacterial organisms, the extent to which this information can also play a role in defining metabolic phenotypes has yet to be examined systematically. Here we present the first comprehensive study for a model methanogen. As a result, we use expression and half-life data for the methanogen Methanosarcina acetivorans growing on fast- and slow-growth substrates to examine the regulation of its genes. Unlike Escherichia coli where only small shifts in half-lives were observed, wemore » found that most mRNA have significantly longer half-lives for slow growth on acetate compared to fast growth on methanol or trimethylamine. Interestingly, half-life shifts are not uniform across functional classes of enzymes, suggesting the existence of a selective stabilization mechanism for mRNAs. Using the transcriptomics data we determined whether transcription or degradation rate controls the change in transcript abundance. Degradation was found to control abundance for about half of the metabolic genes underscoring its role in regulating metabolism. Genes involved in half of the metabolic reactions were found to be differentially expressed among the substrates suggesting the existence of drastically different metabolic phenotypes that extend beyond just the methanogenesis pathways. By integrating expression data with an updated metabolic model of the organism (iST807) significant differences in pathway flux and production of metabolites were predicted for the three growth substrates. In conclusion, this study provides the first global picture of differential expression and half-lives for a class II methanogen, as well as provides the first evidence in a single organism that drastic genome-wide shifts in RNA half-lives can be modulated by growth substrate. We determined which genes in each metabolic pathway control the flux and classified them as regulated by transcription (e.g. transcription factor) or degradation (e.g. post-transcriptional modification). We found that more than half of genes in metabolism were controlled by degradation. Our results suggest that M. acetivorans employs extensive post-transcriptional regulation to optimize key metabolic steps, and more generally that degradation could play a much greater role in optimizing an organism’s metabolism than previously thought.« less

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peterson, Joseph R.; Thor, ShengShee; Kohler, Lars

Here, while a few studies on the variations in mRNA expression and half-lives measured under different growth conditions have been used to predict patterns of regulation in bacterial organisms, the extent to which this information can also play a role in defining metabolic phenotypes has yet to be examined systematically. Here we present the first comprehensive study for a model methanogen. As a result, we use expression and half-life data for the methanogen Methanosarcina acetivorans growing on fast- and slow-growth substrates to examine the regulation of its genes. Unlike Escherichia coli where only small shifts in half-lives were observed, wemore » found that most mRNA have significantly longer half-lives for slow growth on acetate compared to fast growth on methanol or trimethylamine. Interestingly, half-life shifts are not uniform across functional classes of enzymes, suggesting the existence of a selective stabilization mechanism for mRNAs. Using the transcriptomics data we determined whether transcription or degradation rate controls the change in transcript abundance. Degradation was found to control abundance for about half of the metabolic genes underscoring its role in regulating metabolism. Genes involved in half of the metabolic reactions were found to be differentially expressed among the substrates suggesting the existence of drastically different metabolic phenotypes that extend beyond just the methanogenesis pathways. By integrating expression data with an updated metabolic model of the organism (iST807) significant differences in pathway flux and production of metabolites were predicted for the three growth substrates. In conclusion, this study provides the first global picture of differential expression and half-lives for a class II methanogen, as well as provides the first evidence in a single organism that drastic genome-wide shifts in RNA half-lives can be modulated by growth substrate. We determined which genes in each metabolic pathway control the flux and classified them as regulated by transcription (e.g. transcription factor) or degradation (e.g. post-transcriptional modification). We found that more than half of genes in metabolism were controlled by degradation. Our results suggest that M. acetivorans employs extensive post-transcriptional regulation to optimize key metabolic steps, and more generally that degradation could play a much greater role in optimizing an organism’s metabolism than previously thought.« less

Response of Functional Structure of Soil Microbial Community to Multi-level Nitrogen Additions on the Central Tibetan Plateau

NASA Astrophysics Data System (ADS)

Zhang, G.; Yuan, Y.

2015-12-01

The use of fossil fuels and fertilizers has increased the amount of biologically reactive nitrogen in the atmosphere over the past century. Tibet is the one of the most threatened regions by nitrogen deposition, thus understanding how its microbial communities function maybe of high importance to predicting microbial responses to nitrogen deposition. Here we describe a short-time nitrogen addition conducted in an alpine steppe ecosystem to investigate the response of functional structure of soil microbial community to multi-level nitrogen addition. Using a GeoChip 4.0, we showed that functional diversities and richness of functional genes were unchanged at low level of nitrogen fertilizer inputs (<20 kg N ha-1 yr-1), but significantly decreased at higher nitrogen fertilizer inputs (>=40 kg N ha-1 yr-1). Detrended correspondence analysis indicated that the functional structure of microbial communities was markedly different across the nitrogen gradients. Most C degradation genes whose abundances significantly increased under elevated N fertilizer were those involved in the degradation of relatively labile C (starch, hemicellulose, cellulose), whereas the abundance of certain genes involved in the degradation of recalcitrant C (i.e. lignin) was largely decreased (such as manganese peroxidase, mnp). The results suggest that the elevated N fertilization rates might significantly accelerate the labile C degradation, but might not spur recalcitrant C degradation. The combined effect of gdh and ureC genes involved in N cycling appeared to shift the balance between ammonia and organic N toward organic N ammonification and hence increased the N mineralization potential. Moreover, Urease directly involved in urea mineralization significantly increased. Lastly, Canonical correspondence analysis showed that soil (TOC+NH4++NO3-+NO2-+pH) and plant (Aboveground plant productivity + Shannon Diversity) variables could explain 38.9% of the variation of soil microbial community composition. On the basis of above observations, we predict that increasing of nitrogen deposition on the Tibetan steppe ecosystem is very likely to change soil microbial community functional structure, with particular effects on microbial C and N-cycling genes and consequently microbe-mediated soil C and N dynamics.

EIN3 and ORE1 Accelerate Degreening during Ethylene-Mediated Leaf Senescence by Directly Activating Chlorophyll Catabolic Genes in Arabidopsis

PubMed Central

Qiu, Kai; Li, Zhongpeng; Yang, Zhen; Chen, Junyi; Wu, Shouxin; Zhu, Xiaoyu; Gao, Shan; Gao, Jiong; Ren, Guodong; Kuai, Benke; Zhou, Xin

2015-01-01

Degreening, caused by chlorophyll degradation, is the most obvious symptom of senescing leaves. Chlorophyll degradation can be triggered by endogenous and environmental cues, and ethylene is one of the major inducers. ETHYLENE INSENSITIVE3 (EIN3) is a key transcription factor in the ethylene signaling pathway. It was previously reported that EIN3, miR164, and a NAC (NAM, ATAF, and CUC) transcription factor ORE1/NAC2 constitute a regulatory network mediating leaf senescence. However, how this network regulates chlorophyll degradation at molecular level is not yet elucidated. Here we report a feed-forward regulation of chlorophyll degradation that involves EIN3, ORE1, and chlorophyll catabolic genes (CCGs). Gene expression analysis showed that the induction of three major CCGs, NYE1, NYC1 and PAO, by ethylene was largely repressed in ein3 eil1 double mutant. Dual-luciferase assay revealed that EIN3 significantly enhanced the promoter activity of NYE1, NYC1 and PAO in Arabidopsis protoplasts. Furthermore, Electrophoretic mobility shift assay (EMSA) indicated that EIN3 could directly bind to NYE1, NYC1 and PAO promoters. These results reveal that EIN3 functions as a positive regulator of CCG expression during ethylene-mediated chlorophyll degradation. Interestingly, ORE1, a senescence regulator which is a downstream target of EIN3, could also activate the expression of NYE1, NYC1 and PAO by directly binding to their promoters in EMSA and chromatin immunoprecipitation (ChIP) assays. In addition, EIN3 and ORE1 promoted NYE1 and NYC1 transcriptions in an additive manner. These results suggest that ORE1 is also involved in the direct regulation of CCG transcription. Moreover, ORE1 activated the expression of ACS2, a major ethylene biosynthesis gene, and subsequently promoted ethylene production. Collectively, our work reveals that EIN3, ORE1 and CCGs constitute a coherent feed-forward loop involving in the robust regulation of ethylene-mediated chlorophyll degradation during leaf senescence in Arabidopsis. PMID:26218222

Stable carbon isotope fractionation of chlorinated ethenes by a microbial consortium containing multiple dechlorinating genes.

PubMed

Liu, Na; Ding, Longzhen; Li, Haijun; Zhang, Pengpeng; Zheng, Jixing; Weng, Chih-Huang

2018-08-01

The study aimed to determine the possible contribution of specific growth conditions and community structures to variable carbon enrichment factors (Ɛ- carbon ) values for the degradation of chlorinated ethenes (CEs) by a bacterial consortium with multiple dechlorinating genes. Ɛ- carbon values for trichloroethylene, cis-1,2-dichloroethylene, and vinyl chloride were -7.24% ± 0.59%, -14.6% ± 1.71%, and -21.1% ± 1.14%, respectively, during their degradation by a microbial consortium containing multiple dechlorinating genes including tceA and vcrA. The Ɛ- carbon values of all CEs were not greatly affected by changes in growth conditions and community structures, which directly or indirectly affected reductive dechlorination of CEs by this consortium. Stability analysis provided evidence that the presence of multiple dechlorinating genes within a microbial consortium had little effect on carbon isotope fractionation, as long as the genes have definite, non-overlapping functions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Gene delivery through the use of a hyaluronate-associated intracellularly degradable cross-linked polyethyleneimine

PubMed Central

Xu, Peisheng; Quick, Griffin; Yeo, Yoon

2009-01-01

For a non-viral gene delivery system to be clinically effective, it should be non-toxic, compatible with biological components, and highly efficient in gene transfection. With this goal in mind, we investigated the gene delivery efficiency of a ternary complex consisting of DNA, an intracellularly degradable polycation, and sodium hyaluronate (DPH complex). Here, we report that the DPH ternary complex achieved significantly higher transfection efficiency than other polymer systems, especially in the presence of serum. The high transfection efficiency and serum tolerance of DPH are attributed to a unique interplay between CLPEI and HA, which leads to (i) the improved stability of DNA in the extracellular environment and at the early stage of intracellular trafficking and (ii) timely dissociation of the DNA-polymer complex. This study reinforces findings of earlier studies that emphasized each step as a bottleneck for efficient gene delivery; yet, it is the first to show that it is possible to overcome these obstacles simultaneously by taking advantage of two distinctive approaches. PMID:19631979

Exposure to Solute Stress Affects Genome-Wide Expression but Not the Polycyclic Aromatic Hydrocarbon-Degrading Activity of Sphingomonas sp. Strain LH128 in Biofilms

PubMed Central

Fida, Tekle Tafese; Breugelmans, Philip; Lavigne, Rob; Coronado, Edith; Johnson, David R.; van der Meer, Jan Roelof; Mayer, Antonia P.; Heipieper, Hermann J.; Hofkens, Johan

2012-01-01

Members of the genus Sphingomonas are important catalysts for removal of polycyclic aromatic hydrocarbons (PAHs) in soil, but their activity can be affected by various stress factors. This study examines the physiological and genome-wide transcription response of the phenanthrene-degrading Sphingomonas sp. strain LH128 in biofilms to solute stress (invoked by 450 mM NaCl solution), either as an acute (4-h) or a chronic (3-day) exposure. The degree of membrane fatty acid saturation was increased as a response to chronic stress. Oxygen consumption in the biofilms and phenanthrene mineralization activities of biofilm cells were, however, not significantly affected after imposing either acute or chronic stress. This finding was in agreement with the transcriptomic data, since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to nonstressed conditions. The transcriptomic data suggest that LH128 adapts to NaCl stress by (i) increasing the expression of genes coping with osmolytic and ionic stress such as biosynthesis of compatible solutes and regulation of ion homeostasis, (ii) increasing the expression of genes involved in general stress response, (iii) changing the expression of general and specific regulatory functions, and (iv) decreasing the expression of protein synthesis such as proteins involved in motility. Differences in gene expression between cells under acute and chronic stress suggest that LH128 goes through changes in genome-wide expression to fully adapt to NaCl stress, without significantly changing phenanthrene degrading activity. PMID:23001650

Hydrocarbon activation under sulfate-reducing and methanogenic conditions proceeds by different mechanisms.

NASA Astrophysics Data System (ADS)

Head, Ian; Gray, Neil; Aitken, Caroline; Sherry, Angela; Jones, Martin; Larter, Stephen

2010-05-01

Microbial degradation of alkanes typically involves their conversion to fatty acids which are then catabolised by beta-oxidation. The critical step in this process is activation of the hydrocarbon. Under oxic conditions this is catalyzed by monooxygenase enzymes with the formation of long chain alcohols. In the absence of oxygen alternative alkane activation mechanisms have been observed or proposed. Fumarate addition to alkanes to form alkyl succinates is considered a central process in anaerobic hydrocarbon degradation. Comparative studies of crude oil degradation under sulphate-reducing and methanogenic conditions revealed distinctive patterns of compound class removal and metabolite formation. Alkyl succinates derived from C7 to C26 n-alkanes and branched chain alkanes were found in abundance in sulfate-reducing systems but these were not detected during methanogenic crude oil degradation. Only one other mechanism of alkane activation has been elucidated to date. This involves addition of carbon derived from bicarbonate/CO2 to C-3 of an alkane chain to form a 2-ethylalkane with subsequent removal of the ethyl group leading to the formation of a fatty acid 1 carbon shorter than the original alkane. 2-ethylalkanes have never been detected as metabolites of anaerobic alkane degradation and were not detected in crude oil-degrading methanogenic systems. Due to the range of alkanes present in crude oil it was not possible to infer the generation of C-odd acids from C-even alkanes which is characteristic of the C-3 carboxylation mechanism. Furthermore genes homologous to alkysuccinate synthetases were not detected in the methanogenic hydrocarbon degrading community by pyrosequencing of total DNA extracted from methanogenic enrichments cultures. beta-oxidation genes were detected and intriguingly, alcohol and aldehyde dehydrogenase genes were present. This offers the possibility that alkane activation in the methanogenic system does not proceed via acid metabolites, but may be initiated by an anaerobic hydroxylation reaction. This is not unprecedented and hydroxylation of ethylbenzene has been demonstrated. However the C-H bond dissociation energy of alkanes is typically considered too high to readily permit alkane hydroxylation. It is however clear that alkane activation in these methanogenic crude oil-degrading systems involves mechanisms other than the well-known fumarate-addition reactions.

The AhR Ligand, TCDD, Regulates Androgen Receptor Activity Differently in Androgen-Sensitive versus Castration-Resistant Human Prostate Cancer Cells.

PubMed

Ghotbaddini, Maryam; Powell, Joann B

2015-07-06

The reported biological effects of TCDD include induction of drug metabolizing enzymes, wasting syndrome and tumor promotion. TCDD elicits most of its effects through binding the aryl hydrocarbon receptor (AhR). TCDD induced degradation of AhR has been widely reported and requires ubiquitination of the protein. The rapid depletion of AhR following TCDD activation serves as a mechanism to modulate AhR mediated gene induction. In addition to inducing AhR degradation, TCDD has been reported to induce degradation of hormone receptors. The studies reported here, evaluate the effect of TCDD exposure on androgen receptor (AR) expression and activity in androgen-sensitive LNCaP and castration-resistant C4-2 prostate cancer cells. Our results show that TCDD exposure does not induce AhR or AR degradation in C4-2 cells. However, both AhR and AR are degraded in LNCaP cells following TCDD exposure. In addition, TCDD enhances AR phosphorylation and induces expression of AR responsive genes in LNCaP cells. Our data reveals that TCDD effect on AR expression and activity differs in androgen-sensitive and castration-resistant prostate cancer cell models.

Genetics Home Reference: spinal muscular atrophy

MedlinePlus

... atrophy types I, II, III, and IV. SMN1 gene mutations lead to a shortage of the SMN protein. ... to be broken down (degraded) within cells. UBA1 gene mutations lead to reduced or absent levels of functional ...

Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus.

PubMed Central

Giardina, P; Cannio, R; Martirani, L; Marzullo, L; Palmieri, G; Sannia, G

1995-01-01

The gene (pox1) encoding a phenol oxidase from Pleurotus ostreatus, a lignin-degrading basidiomycete, was cloned and sequenced, and the corresponding pox1 cDNA was also synthesized and sequenced. The isolated gene consists of 2,592 bp, with the coding sequence being interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA consensus sequences could be identified at positions -174 and -84, respectively. The isolation of a second cDNA (pox2 cDNA), showing 84% similarity, and of the corresponding truncated genomic clones demonstrated the existence of a multigene family coding for isoforms of laccase in P. ostreatus. PCR amplifications of specific regions on the DNA of isolated monokaryons proved that the two genes are not allelic forms. The POX1 amino acid sequence deduced was compared with those of other known laccases from different fungi. PMID:7793961

Complete Genome Sequence of Akkermansia glycaniphila Strain PytT, a Mucin-Degrading Specialist of the Reticulated Python Gut

PubMed Central

Ouwerkerk, Janneke P.; Schaap, Peter J.; Ritari, Jarmo; Paulin, Lars; Belzer, Clara

2017-01-01

ABSTRACT Akkermansia glycaniphila is a novel Akkermansia species that was isolated from the intestine of the reticulated python and shares the capacity to degrade mucin with the human strain Akkermansia muciniphila MucT. Here, we report the complete genome sequence of strain PytT of 3,074,121 bp. The genomic analysis reveals genes for mucin degradation and aerobic respiration. PMID:28057747

Assessment of solvent capsule-based healing for woven E-glass fibre-reinforced polymers

NASA Astrophysics Data System (ADS)

Manfredi, Erica; Cohades, Amaël; Richard, Inès; Michaud, Véronique

2015-01-01

Vacuum Assisted Resin Infusion Molding (VARIM) with low vacuum pressure difference was used to manufacture woven glass fibre-reinforced epoxy resin plates, with a fibre volume fraction of approx. 50 vol% and containing ethyl phenylacetate (EPA)-filled capsules for self-healing purposes. Capsules were introduced by functionalising the fabrics through manual dispersion. We investigated the capability of autonomously healing delaminations induced by static loading in Mode I and II. Healing did not take place for composite samples; this was attributed to the presence of bare fibres on the crack plane and to the reduction of EPA diffusion into the matrix in the presence of fibres both of which hinder the swelling mechanism responsible for healing the cracks.

Microbial Evolution Is in the Cards: Horizontal Gene Transfer in the Classroom

ERIC Educational Resources Information Center

Kagle, Jeanne; Hay, Anthony G.

2007-01-01

Horizontal gene transfer, the exchange of genetic material between bacteria, is a potentially important factor in the degradation of synthetic compounds introduced to the environment and in the acquisition of other characteristics including antibiotic resistance. This game-based activity illustrates the role of horizontal gene transfer in the…

Gene expression analysis of copper tolerance and wood decay in the brown rot fungus Fibroporia radiculosa

USDA-ARS?s Scientific Manuscript database

Many brown rot fungi are capable of rapidly degrading wood and are copper-tolerant. To better understand the genes that control these processes, we examined gene expression of Fibroporia radiculosa growing on wood treated with a copper-based preservative that combined copper carbonate with dimethyld...

Current Strategies in the Modification of PLGA-based Gene Delivery System.

PubMed

Ramezani, Mohammad; Ebrahimian, Mahboubeh; Hashemi, Maryam

2017-01-01

Successful gene therapy has been limited by safe and efficient delivery of nucleic acid to the target cells. Poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) are able to deliver drugs and genes efficiently. This formulation has several advantages in comparison with other formulations including improvement in solubility, stability, controlling of degradation and release of the entrapped agents. For application of PLGA as a gene carrier, there exist many challenges. PLGA NPs could protect the encapsulated DNA from in vivo degradation but the DNA release is slow and the negative charge acts as a barrier to DNA incorporation and delivery. Also, during the preparation process, DNA could be exposed to high shear stress and organic solvents which could result in its inactivation. Moreover, PLGA NPs could be modified with different agents to reduce cytotoxicity, to enhance delivery efficiency and to target specific tissues/cells. This review summarizes different methods used for the preparation of PLGA NPs as gene carriers and recent strategies for the modification of PLGA particles applied in gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Succession of microbial functional communities in response to a pilot-scale ethanol-blended fuel release throughout the plume life cycle.

PubMed

Ma, Jie; Deng, Ye; Yuan, Tong; Zhou, Jizhong; Alvarez, Pedro J J

2015-03-01

GeoChip, a comprehensive gene microarray, was used to examine changes in microbial functional gene structure throughout the 4-year life cycle of a pilot-scale ethanol blend plume, including 2-year continuous released followed by plume disappearance after source removal. Canonical correlation analysis (CCA) and Mantel tests showed that dissolved O2 (which was depleted within 5 days of initiating the release and rebounded 194 days after source removal) was the most influential environmental factor on community structure. Initially, the abundance of anaerobic BTEX degradation genes increased significantly while that of aerobic BTEX degradation genes decreased. Gene abundance for N fixation, nitrification, P utilization, sulfate reduction and S oxidation also increased, potentially changing associated biogeochemical cycle dynamics. After plume disappearance, most genes returned to pre-release abundance levels, but the final functional structure significantly differed from pre-release conditions. Overall, observed successions of functional structure reflected adaptive responses that were conducive to biodegradation of ethanol-blend releases. Copyright © 2015. Published by Elsevier Ltd.

Catabolism of 4-alkylphenols by Acinetobacter sp. OP5: genetic organization of the oph gene cluster and characterization of alkylcatechol 2, 3-dioxygenase.

PubMed

Tuan, Nguyen Ngoc; Lin, Yi-Wen; Huang, Shir-Ly

2013-03-01

In this study, a specific PCR primer set was successfully designed for alkylcatechol 2, 3-dioxygenase genes and applied to detect the presence of this biomarker in 4-t-octylphenol-degrading Acinetobacter sp. strain OP5. A gene cluster (ophRBA1A2A3A4A5A6CEH) encoding multicomponent phenol hydroxylase and alkylcatechol 2, 3-dioxygenase was then cloned from this strain and showed the highest homology to those involved in the published medium-chain alkylphenol gene clusters. The pure enzyme of recombinant cell harboring ophB showed meta-cleavage activities for 4-methylcatechol (1,435%), 4-ethylcatechol (982%), catechol (100%), 4-t-butylcatechol (16.6%), and 4-t-octylcatechol (3.2%). The results suggest that the developed molecular technique is useful and easy in detection of medium/long-chain alkylphenol degradation gene cluster. In addition, it also provides a better understanding of the distribution of biodegradative genes and pathway for estrogenic-active long-chain alkylphenols in bacteria. Copyright © 2013 Elsevier Ltd. All rights reserved.

Depth-Resolved Quantification of Anaerobic Toluene Degraders and Aquifer Microbial Community Patterns in Distinct Redox Zones of a Tar Oil Contaminant Plume▿

PubMed Central

Winderl, Christian; Anneser, Bettina; Griebler, Christian; Meckenstock, Rainer U.; Lueders, Tillmann

2008-01-01

Microbial degradation is the only sustainable component of natural attenuation in contaminated groundwater environments, yet its controls, especially in anaerobic aquifers, are still poorly understood. Hence, putative spatial correlations between specific populations of key microbial players and the occurrence of respective degradation processes remain to be unraveled. We therefore characterized microbial community distribution across a high-resolution depth profile of a tar oil-impacted aquifer where benzene, toluene, ethylbenzene, and xylene (BTEX) degradation depends mainly on sulfate reduction. We conducted depth-resolved terminal restriction fragment length polymorphism fingerprinting and quantitative PCR of bacterial 16S rRNA and benzylsuccinate synthase genes (bssA) to quantify the distribution of total microbiota and specific anaerobic toluene degraders. We show that a highly specialized degrader community of microbes related to known deltaproteobacterial iron and sulfate reducers (Geobacter and Desulfocapsa spp.), as well as clostridial fermenters (Sedimentibacter spp.), resides within the biogeochemical gradient zone underneath the highly contaminated plume core. This zone, where BTEX compounds and sulfate—an important electron acceptor—meet, also harbors a surprisingly high abundance of the yet-unidentified anaerobic toluene degraders carrying the previously detected F1-cluster bssA genes (C. Winderl, S. Schaefer, and T. Lueders, Environ. Microbiol. 9:1035-1046, 2007). Our data suggest that this biogeochemical gradient zone is a hot spot of anaerobic toluene degradation. These findings show that the distribution of specific aquifer microbiota and degradation processes in contaminated aquifers are tightly coupled, which may be of value for the assessment and prediction of natural attenuation based on intrinsic aquifer microbiota. PMID:18083871