Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth.

PubMed

Vu, Huong N; Subuyuj, Gabriel A; Vijayakumar, Srividhya; Good, Nathan M; Martinez-Gomez, N Cecilia; Skovran, Elizabeth

2016-04-01

Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH requires pyrroloquinoline quinone (PQQ) and Ca in its active site, while XoxF-MeDH requires PQQ and lanthanides, such as Ce and La. Using MeDH mutant strains to conduct growth analysis and MeDH activity assays, we demonstrate that M. extorquens AM1 has at least one additional lanthanide-dependent methanol oxidation system contributing to methanol growth. Additionally, the abilities of different lanthanides to support growth were tested and strongly suggest that both XoxF and the unknown methanol oxidation system are able to use La, Ce, Pr, Nd, and, to some extent, Sm. Further, growth analysis using increasing La concentrations showed that maximum growth rate and yield were achieved at and above 1 μM La, while concentrations as low as 2.5 nM allowed growth at a reduced rate. Contrary to published data, we show that addition of exogenous lanthanides results in differential expression from the xox1 and mxa promoters, upregulating genes in the xox1 operon and repressing genes in the mxa operon. Using transcriptional reporter fusions, intermediate expression from both the mxa and xox1 promoters was detected when 50 to 100 nM La was added to the growth medium, suggesting that a condition may exist under which M. extorquens AM1 is able to utilize both enzymes simultaneously. Together, these results suggest that M. extorquens AM1 actively senses and responds to lanthanide availability, preferentially utilizing the lanthanide-dependent MeDHs when possible. The biological role of lanthanides is a nascent field of study with tremendous potential to impact many areas in biology. Our studies demonstrate that there is at least one additional lanthanide-dependent methanol oxidation system, distinct from the MxaFI and XoxF MeDHs, that may aid in classifying additional environmental organisms as methylotrophs. Further

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

PubMed Central

Vu, Huong N.; Subuyuj, Gabriel A.; Vijayakumar, Srividhya; Good, Nathan M.; Martinez-Gomez, N. Cecilia

2016-01-01

ABSTRACT Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH requires pyrroloquinoline quinone (PQQ) and Ca in its active site, while XoxF-MeDH requires PQQ and lanthanides, such as Ce and La. Using MeDH mutant strains to conduct growth analysis and MeDH activity assays, we demonstrate that M. extorquens AM1 has at least one additional lanthanide-dependent methanol oxidation system contributing to methanol growth. Additionally, the abilities of different lanthanides to support growth were tested and strongly suggest that both XoxF and the unknown methanol oxidation system are able to use La, Ce, Pr, Nd, and, to some extent, Sm. Further, growth analysis using increasing La concentrations showed that maximum growth rate and yield were achieved at and above 1 μM La, while concentrations as low as 2.5 nM allowed growth at a reduced rate. Contrary to published data, we show that addition of exogenous lanthanides results in differential expression from the xox1 and mxa promoters, upregulating genes in the xox1 operon and repressing genes in the mxa operon. Using transcriptional reporter fusions, intermediate expression from both the mxa and xox1 promoters was detected when 50 to 100 nM La was added to the growth medium, suggesting that a condition may exist under which M. extorquens AM1 is able to utilize both enzymes simultaneously. Together, these results suggest that M. extorquens AM1 actively senses and responds to lanthanide availability, preferentially utilizing the lanthanide-dependent MeDHs when possible. IMPORTANCE The biological role of lanthanides is a nascent field of study with tremendous potential to impact many areas in biology. Our studies demonstrate that there is at least one additional lanthanide-dependent methanol oxidation system, distinct from the MxaFI and XoxF MeDHs, that may aid in classifying additional environmental organisms as

Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

PubMed

Zhu, Wen-Liang; Cui, Jin-Yu; Cui, Lan-Yu; Liang, Wei-Fan; Yang, Song; Zhang, Chong; Xing, Xin-Hui

2016-03-01

Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

Distribution of Tetrahydromethanopterin-Dependent Enzymes in Methylotrophic Bacteria and Phylogeny of Methenyl Tetrahydromethanopterin Cyclohydrolases

PubMed Central

Vorholt, Julia A.; Chistoserdova, Ludmila; Stolyar, Sergei M.; Thauer, Rudolf K.; Lidstrom, Mary E.

1999-01-01

The methylotrophic proteobacterium Methylobacterium extorquens AM1 possesses tetrahydromethanopterin (H4MPT)-dependent enzymes, which are otherwise specific to methanogenic and sulfate-reducing archaea and which have been suggested to be involved in formaldehyde oxidation to CO2 in M. extorquens AM1. The distribution of H4MPT-dependent enzyme activities in cell extracts of methylotrophic bacteria from 13 different genera are reported. H4MPT-dependent activities were detected in all of the methylotrophic and methanotrophic proteobacteria tested that assimilate formaldehyde by the serine or ribulose monophosphate pathway. H4MPT-dependent activities were also found in autotrophic Xanthobacter strains. However, no H4MPT-dependent enzyme activities could be detected in other autotrophic α-proteobacteria or in gram-positive methylotrophic bacteria. Genes encoding methenyl H4MPT cyclohydrolase (mch genes) were cloned and sequenced from several proteobacteria. Bacterial and archaeal Mch sequences have roughly 35% amino acid identity and form distinct groups in phylogenetic analysis. PMID:10482517

Genetic and Phenotypic Comparison of Facultative Methylotrophy between Methylobacterium extorquens Strains PA1 and AM1

PubMed Central

Nayak, Dipti D.; Marx, Christopher J.

2014-01-01

Methylobacterium extorquens AM1, a strain serendipitously isolated half a century ago, has become the best-characterized model system for the study of aerobic methylotrophy (the ability to grow on reduced single-carbon compounds). However, with 5 replicons and 174 insertion sequence (IS) elements in the genome as well as a long history of domestication in the laboratory, genetic and genomic analysis of M. extorquens AM1 face several challenges. On the contrary, a recently isolated strain - M. extorquens PA1- is closely related to M. extorquens AM1 (100% 16S rRNA identity) and contains a streamlined genome with a single replicon and only 20 IS elements. With the exception of the methylamine dehydrogenase encoding gene cluster (mau), genes known to be involved in methylotrophy are well conserved between M. extorquens AM1 and M. extorquens PA1. In this paper we report four primary findings regarding methylotrophy in PA1. First, with a few notable exceptions, the repertoire of methylotrophy genes between PA1 and AM1 is extremely similar. Second, PA1 grows faster with higher yields compared to AM1 on C1 and multi-C substrates in minimal media, but AM1 grows faster in rich medium. Third, deletion mutants in PA1 throughout methylotrophy modules have the same C1 growth phenotypes observed in AM1. Finally, the precision of our growth assays revealed several unexpected growth phenotypes for various knockout mutants that serve as leads for future work in understanding their basis and generality across Methylobacterium strains. PMID:25232997

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1.

PubMed

Martinez-Gomez, N Cecilia; Good, Nathan M; Lidstrom, Mary E

2015-06-15

During an environmental perturbation, the survival of a cell and its response to the perturbation depend on both the robustness and functionality of the metabolic network. The regulatory mechanisms that allow the facultative methylotrophic bacterium Methylobacterium extorquens AM1 to effect the metabolic transition from succinate to methanol growth are not well understood. Methenyl-dephosphotetrahydromethanopterin (methenyl-dH4MPT), an early intermediate during methanol metabolism, transiently accumulated 7- to 11-fold after addition of methanol to a succinate-limited culture. This accumulation partially inhibited the activity of the methylene-H4MPT dehydrogenase, MtdA, restricting carbon flux to the assimilation cycles. A strain overexpressing the gene (mch) encoding the enzyme that consumes methenyl-dH4MPT did not accumulate methenyl-dH4MPT and had a growth rate that was 2.7-fold lower than that of the wild type. This growth defect demonstrates the physiological relevance of this enzymatic regulatory mechanism during the acclimation period. Changes in metabolites and enzymatic activities were analyzed in the strain overexpressing mch. Under these conditions, the activity of the enzyme coupling formaldehyde with dH4MPT (Fae) remained constant, with concomitant formaldehyde accumulation. Release of methenyl-dH4MPT regulation did not affect the induction of the serine cycle enzyme activities immediately after methanol addition, but after 1 h, the activity of these enzymes decreased, likely due to the toxicity of formaldehyde accumulation. Our results support the hypothesis that in a changing environment, the transient accumulation of methenyl-dH4MPT and inhibition of MtdA activity are strategies that permit flexibility and acclimation of the metabolic network while preventing the accumulation of the toxic compound formaldehyde. The identification and characterization of regulatory mechanisms for methylotrophy are in the early stages. We report a nontranscriptional

Methylobacterium hispanicum sp. nov. and Methylobacterium aquaticum sp. nov., isolated from drinking water.

PubMed

Gallego, Virginia; García, María Teresa; Ventosa, Antonio

2005-01-01

Members of the genus Methylobacterium are ubiquitous in nature and can be isolated from almost any freshwater environment where dissolved oxygen exists. This genus is composed of a variety of pink-pigmented, facultatively methylotrophic (PPFM) bacteria. During a screening programme to monitor the bacterial population present in the drinking water of a municipal water supply in Seville (Spain) during the year 2003, five strains of PPFM bacteria were isolated and characterized. Analysis of their complete 16S rRNA gene sequences revealed that they constituted two separate phylogenetic groups (strains GP34T and GR18, and strains GR16T, GP22 and GP32, respectively) showing highest similarity to members of the genus Methylobacterium. The highest 16S rRNA sequence similarities of strain GP34T were found with respect to the type strains of Methylobacterium radiotolerans (96.6 %) and Methylobacterium fujisawaense (96.4 %) and the highest 16S rRNA sequence similarities of strain GR16T were to the type strains of Methylobacterium extorquens (96.0 %) and Methylobacterium rhodesianum (95.8 %). The G+C content of their DNA ranged from 66.5 to 67.8 mol%. DNA-DNA hybridization studies confirmed that they constituted two separate genospecies. On the basis of this phenotypic, phylogenetic and genotypic study, two novel species of the genus Methylobacterium are proposed: Methylobacterium hispanicum sp. nov., with type strain GP34T (CECT 5997T=CCM 7219T=DSM 16372T=CIP 108332T), and Methylobacterium aquaticum sp. nov., with type strain GR16T (CECT 5998T=CCM 7218T=DSM 16371T=CIP 108333T).

Identification and growth characteristics of pink pigmented oxidative bacteria, Methylobacterium mesophilicum and biovars isolated from chlorinated and raw water supplies.

PubMed

O'Brien, J R; Murphy, J M

1993-01-01

Pink pigmented bacteria were isolated from a blood bank water purification unit, a municipal town water supply (tap water), and an island (untreated) ground water source. A total of thirteen strains including two reference strains of pink pigmented bacteria were compared in a numerical phenotypic study using 119 binary characters. Three clusters were derived, one major cluster of eleven strains was subdivided into two sub-clusters on the basis of methanol utilization. Five strains were facultative methylotrophs and were classified as Methylobacterium mesophilicum biovar 1. The other six strains did not utilize methanol, but on the basis of high phenotypic similarity of 83.6% were classified as M. mesophilicum biovar 2. The single reference strain comprising cluster 2 Pseudomonas extorquens NCIB 9399 was assigned to the genus Methylobacterium and classified as M. extorquens. Cluster 3 was the single reference strain Rhizobium CB 376.

Methylobacterium gossipiicola sp. nov., a pink-pigmented, facultatively methylotrophic bacterium isolated from the cotton phyllosphere.

PubMed

Madhaiyan, Munusamy; Poonguzhali, Selvaraj; Senthilkumar, Murugaiyan; Lee, Jung-Sook; Lee, Keun-Chul

2012-01-01

A pink, aerobic, facultatively methylotrophic, motile, Gram-negative rod, designated Gh-105(T), was isolated from the phyllosphere of cotton from Coimbatore (Tamilnadu, India). 16S rRNA gene sequence analysis showed clearly that the isolate belonged to the Methylobacterium cluster. Strain Gh-105(T) was most closely related to Methylobacterium adhaesivum AR27(T) (99% 16S rRNA gene sequence similarity) and Methylobacterium iners 5317S-33(T) (97.5%). The isolate grew with C(1) compounds such as methanol and dichloromethane, but not with formaldehyde, formate, methylamine, trimethylamine or methane, as sole carbon sources and carried mxaF, which encodes methanol dehydrogenase and supports methylotrophic metabolism. The major fatty acid was C(18:1)ω7c and the G+C content of the genomic DNA was 64.2 mol%. Physiological and biochemical data and DNA-DNA relatedness with M. adhaesivum KACC 12195(T) and M. iners KACC 11765(T) revealed clear phenotypic and genotypic differences. For this reason, we propose that strain Gh-105(T) (=CCM 7572(T) =NRRL B-51692(T)) represents the type strain of a novel species, with the name Methylobacterium gossipiicola sp. nov.

Metabolomics revealed an association of metabolite changes and defective growth in Methylobacterium extorquens AM1 overexpressing ecm during growth on methanol

DOE PAGES

Cui, Jinyu; Good, Nathan M.; Hu, Bo; ...

2016-04-26

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multi-carbon compounds. The ethylmalonyl-CoA (EMC) pathway is one of the central assimilatory pathways in M. extorquens during growth on C1 and C2 substrates. Previous studies had shown that ethylmalonyl-CoA mutase functioned as a control point during the transition from growth on succinate to growth on ethylamine. In this study we overexpressed ecm, phaA, mcmAB and found that upregulating ecm by expressing it from the strong constitutive mxaF promoter caused a 27% decrease in growth rate on methanol compared to the strain with an empty vector. Targetedmore » metabolomics demonstrated that most of the central intermediates in the ecm over-expressing strain did not change significantly compared to the control strain; However, poly-β-hydroxybutyrate (PHB) was 4.5-fold lower and 3-hydroxybutyryl-CoA was 1.6-fold higher. Moreover, glyoxylate, a toxic and highly regulated essential intermediate, was determined to be 2.6-fold higher when ecm was overexpressed. These results demonstrated that overexpressing ecm can manipulate carbon flux through the EMC pathway and divert it from the carbon and energy storage product PHB, leading to an accumulation of glyoxylate. Furthermore, untargeted metabolomics discovered two unusual metabolites, alanine (Ala)-meso-diaminopimelic acid (mDAP) and Ala-mDAP-Ala, each over 45-fold higher in the ecm overexpressing strain. These two peptides were also found to be highly produced in a dose-dependent manner when glyoxylate was added to the control strain. Overall, this work has explained a direct association of ecm overexpression with glyoxylate accumulation up to a toxic level, which inhibits cell growth on methanol. Lastly, this research provides useful insight for manipulating the EMC pathway for efficiently producing high-value chemicals in M. extorquens.« less

Metabolomics revealed an association of metabolite changes and defective growth in Methylobacterium extorquens AM1 overexpressing ecm during growth on methanol

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

Cui, Jinyu; Good, Nathan M.; Hu, Bo

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multi-carbon compounds. The ethylmalonyl-CoA (EMC) pathway is one of the central assimilatory pathways in M. extorquens during growth on C1 and C2 substrates. Previous studies had shown that ethylmalonyl-CoA mutase functioned as a control point during the transition from growth on succinate to growth on ethylamine. In this study we overexpressed ecm, phaA, mcmAB and found that upregulating ecm by expressing it from the strong constitutive mxaF promoter caused a 27% decrease in growth rate on methanol compared to the strain with an empty vector. Targetedmore » metabolomics demonstrated that most of the central intermediates in the ecm over-expressing strain did not change significantly compared to the control strain; However, poly-β-hydroxybutyrate (PHB) was 4.5-fold lower and 3-hydroxybutyryl-CoA was 1.6-fold higher. Moreover, glyoxylate, a toxic and highly regulated essential intermediate, was determined to be 2.6-fold higher when ecm was overexpressed. These results demonstrated that overexpressing ecm can manipulate carbon flux through the EMC pathway and divert it from the carbon and energy storage product PHB, leading to an accumulation of glyoxylate. Furthermore, untargeted metabolomics discovered two unusual metabolites, alanine (Ala)-meso-diaminopimelic acid (mDAP) and Ala-mDAP-Ala, each over 45-fold higher in the ecm overexpressing strain. These two peptides were also found to be highly produced in a dose-dependent manner when glyoxylate was added to the control strain. Overall, this work has explained a direct association of ecm overexpression with glyoxylate accumulation up to a toxic level, which inhibits cell growth on methanol. Lastly, this research provides useful insight for manipulating the EMC pathway for efficiently producing high-value chemicals in M. extorquens.« less

Methylobacterium pseudosasae sp. nov., a pink-pigmented, facultatively methylotrophic bacterium isolated from the bamboo phyllosphere.

PubMed

Madhaiyan, Munusamy; Poonguzhali, Selvaraj

2014-02-01

A pink-pigmented, Gram negative, aerobic, facultatively methylotrophic bacterium, strain BL44(T), was isolated from bamboo leaves and identified as a member of the genus Methylobacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed similarity values of 98.7-97.0 % with closely related type strains and showed highest similarity to Methylobacterium zatmanii DSM 5688(T) (98.7 %) and Methylobacterium thiocyanatum DSM 11490(T) (98.7 %). Methylotrophic metabolism in this strain was confirmed by PCR amplification and sequencing of the mxaF gene coding for the α-subunit of methanol dehydrogenase. Strain BL44(T) produced three known quorum sensing signal molecules with similar retention time to C8, C10 and C12-HSLs when characterized by GC-MS. The fatty acid profiles contained major amounts of C18:1 ω7c, iso-3OH C17:0 and summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH), which supported the grouping of the isolate in the genus Methylobacterium. The DNA G+C content was 66.9 mol%. DNA relatedness of the strain BL44(T) to its most closely related strains ranged from 12-43.3 %. On the basis of the phenotypic, phylogenetic and DNA-DNA hybridization data, strain BL44(T) is assigned to a novel species of the genus Methylobacterium for which the name Methylobacterium pseudosasae sp. nov. is proposed (type strain BL44(T) = NBRC 105205(T) = ICMP 17622(T)).

Genes of the N-Methylglutamate Pathway Are Essential for Growth of Methylobacterium extorquens DM4 with Monomethylamine

PubMed Central

Gruffaz, Christelle; Muller, Emilie E. L.; Louhichi-Jelail, Yousra; Nelli, Yella R.; Guichard, Gilles

2014-01-01

Monomethylamine (MMA, CH3NH2) can be used as a carbon and nitrogen source by many methylotrophic bacteria. Methylobacterium extorquens DM4 lacks the MMA dehydrogenase encoded by mau genes, which in M. extorquens AM1 is essential for growth on MMA. Identification and characterization of minitransposon mutants with an MMA-dependent phenotype showed that strain DM4 grows with MMA as the sole source of carbon, energy, and nitrogen by the N-methylglutamate (NMG) pathway. Independent mutations were found in a chromosomal region containing the genes gmaS, mgsABC, and mgdABCD for the three enzymes of the pathway, γ-glutamylmethylamide (GMA) synthetase, NMG synthase, and NMG dehydrogenase, respectively. Reverse transcription-PCR confirmed the operonic structure of the two divergent gene clusters mgsABC-gmaS and mgdABCD and their induction during growth with MMA. The genes mgdABCD and mgsABC were found to be essential for utilization of MMA as a carbon and nitrogen source. The gene gmaS was essential for MMA utilization as a carbon source, but residual growth of mutant DM4gmaS growing with succinate and MMA as a nitrogen source was observed. Plasmid copies of gmaS and the gmaS homolog METDI4690, which encodes a protein 39% identical to GMA synthetase, fully restored the ability of mutants DM4gmaS and DM4gmaSΔmetdi4690 to use MMA as a carbon and nitrogen source. Similarly, chemically synthesized GMA, the product of GMA synthetase, could be used as a nitrogen source for growth in the wild-type strain, as well as in DM4gmaS and DM4gmaSΔmetdi4690 mutants. The NADH:ubiquinone oxidoreductase respiratory complex component NuoG was also found to be essential for growth with MMA as a carbon source. PMID:24682302

Bioelectrochemical conversion of CO2 to value added product formate using engineered Methylobacterium extorquens.

PubMed

Jang, Jungho; Jeon, Byoung Wook; Kim, Yong Hwan

2018-05-08

The conversion of carbon dioxide to formate is a fundamental step for building C1 chemical platforms. Methylobacterium extorquens AM1 was reported to show remarkable activity converting carbon dioxide into formate. Formate dehydrogenase 1 from M. extorquens AM1 (MeFDH1) was verified as the key responsible enzyme for the conversion of carbon dioxide to formate in this study. Using a 2% methanol concentration for induction, microbial harboring the recombinant MeFDH1 expressing plasmid produced the highest concentration of formate (26.6 mM within 21 hours) in electrochemical reactor. 60 μM of sodium tungstate in the culture medium was optimal for the expression of recombinant MeFDH1 and production of formate (25.7 mM within 21 hours). The recombinant MeFDH1 expressing cells showed maximum formate productivity of 2.53 mM/g-wet cell/hr, which was 2.5 times greater than that of wild type. Thus, M. extorquens AM1 was successfully engineered by expressing MeFDH1 as recombinant enzyme to elevate the production of formate from CO 2 after elucidating key responsible enzyme for the conversion of CO 2 to formate.

Design and production of functionalized biopolyesters by Methylobacterium extorquens ATCC 55366: Toward new tissue engineering materials

NASA Astrophysics Data System (ADS)

Hoefer, Heinrich Friedrich Philipp Till Nikolaus

Vascular networks are required to support the formation and function of three-dimensional tissues. Biodegradable scaffolds are being considered in order to promote vascularization where natural regeneration of lost or destroyed vascular networks fails. Particularly; composite materials are expected to fulfill the complex demands of a patient's body to support wound healing. Microbial biopolyesters are being regarded as such second and third generation biomaterials. Methylobacterium extorquens is one of several microorganisms that should be considered for the production of advanced polyhydroxyalkanoates (PHAs). M. extorquens displays a distinct advantage in that it is able to utilize methanol as an inexpensive substrate for growth and biopolyester production. The design of functionalized PHAs, which would be made of both saturated short-chain-length (scl, C ≤ 5) and unsaturated medium-chain-length (mcl, 6 ≤ C ≤ 14) monomeric units, aimed at combining desirable material properties of inert scl/mcl-PHAs with those of functionalized mcl-PHAs. By independently inserting the phaC1 or the phaC2 gene from Pseudomonas fluorescens GK13, recombinant M. extorquens strains were obtained which were capable of producing PHAs containing C-C double bonds. A fermentation process was developed to obtain gram quantities of biopolyesters employing the recombinant M. extorquens ATCC 55366 strain which harbored the phaC2 gene of P. fluorescens GK13, the better one of the two strains at incorporating unsaturated monomeric units. The PHAs produced were found in a blend of scl-PHAs and functionalized scl/mcl-PHAs (4 ≤ C ≤ 6), which were the products of the native and of the recombinant PHA synthase, respectively. Thermo-mechanical analysis confirmed that the functionalized scl/mcl-PHAs exhibited the desirable material properties expected. This project contributed to current research on polyhydroxyalkanoates at different levels. The terminal double bonds of the functionalized scl

An engineered Calvin-Benson-Bassham cycle for carbon dioxide fixation in Methylobacterium extorquens AM1.

PubMed

Schada von Borzyskowski, Lennart; Carrillo, Martina; Leupold, Simeon; Glatter, Timo; Kiefer, Patrick; Weishaupt, Ramon; Heinemann, Matthias; Erb, Tobias J

2018-04-04

Organisms are either heterotrophic or autotrophic, meaning that they cover their carbon requirements by assimilating organic compounds or by fixing inorganic carbon dioxide (CO 2 ). The conversion of a heterotrophic organism into an autotrophic one by metabolic engineering is a long-standing goal in synthetic biology and biotechnology, because it ultimately allows for the production of value-added compounds from CO 2 . The heterotrophic Alphaproteobacterium Methylobacterium extorquens AM1 is a platform organism for a future C1-based bioeconomy. Here we show that M. extorquens AM1 provides unique advantages for establishing synthetic autotrophy, because energy metabolism and biomass formation can be effectively separated from each other in the organism. We designed and realized an engineered strain of M. extorquens AM1 that can use the C1 compound methanol for energy acquisition and forms biomass from CO 2 by implementation of a heterologous Calvin-Benson-Bassham (CBB) cycle. We demonstrate that the heterologous CBB cycle is active, confers a distinct phenotype, and strongly increases viability of the engineered strain. Metabolic 13 C-tracer analysis demonstrates the functional operation of the heterologous CBB cycle in M. extorquens AM1 and comparative proteomics of the engineered strain show that the host cell reacts to the implementation of the CBB cycle in a plastic way. While the heterologous CBB cycle is not able to support full autotrophic growth of M. extorquens AM1, our study represents a further advancement in the design and realization of synthetic autotrophic organisms. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Methylobacterium phyllosphaerae sp. nov., a pink-pigmented, facultative methylotroph from the phyllosphere of rice.

PubMed

Madhaiyan, Munusamy; Poonguzhali, Selvaraj; Kwon, Soon-Wo; Sa, Tong-Min

2009-01-01

A pink-pigmented, aerobic, facultatively methylotrophic bacterial strain, CBMB27T, isolated from leaf tissues of rice (Oryza sativa L. 'Dong-Jin'), was analysed using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Methylobacterium oryzae, Methylobacterium fujisawaense and Methylobacterium mesophilicum; strain CBMB27T showed sequence similarities of 98.3, 98.5 and 97.3 %, respectively, to the type strains of these three species. DNA-DNA hybridization experiments revealed low levels (<38 %) of DNA-DNA relatedness between strain CBMB27T and its closest relatives. The sequence of the 1-aminocyclopropane-1-carboxylate deaminase gene (acdS) in strain CBMB27T differed from those of close relatives. The major fatty acid of the isolate was C(18 : 1)omega7c and the G+C content of the genomic DNA was 66.8 mol%. Based on the results of 16S rRNA gene sequence analysis, DNA-DNA hybridization, and physiological and biochemical characterization, which enabled the isolate to be differentiated from all recognized species of the genus Methylobacterium, it was concluded that strain CBMB27T represents a novel species in the genus Methylobacterium for which the name Methylobacterium phyllosphaerae sp. nov. is proposed (type strain CBMB27T =LMG 24361T =KACC 11716T =DSM 19779T).

[Distribution of bacteria of Methylobacterium genus in the terrestrial biotopes of the Antarctic region].

PubMed

Romanovskaia, V A; Rokitko, P V; Shilin, S O; Chernaia, N A; Tashirev, A B

2009-01-01

Methylotrophs distribution has been studied in the terrestrial biotopes (moss, lichen, grass, soil, sludge of lakes) on the islands of Galindez, Barkhans, Irizar, Uruguay, Jalour, Petermann, Berthelot, Cruls, King George, Corner, Skua located in the Pacific sector of the Antarctic Region, as well as in analogous biotopes on the western shore of the Antarctic peninsula Basing on a complex of diagnosis features the isolated pink-pigmented strains, which facultatively use methanol and realize the serine cycle of assimilation of one-carbon compounds, are attributed to Methylobacterium genus. Methylobacterium strains occur more often in mosses, grass Deschampsia antarctica and lichens, than in the soil and lake sludge. Some regions ofAntarctica are comparable by the number of Methylobacterium cells with the same in the regions with moderate climate. An analysis of gene sequences 16S rRNA of the Antarctic methylobacteria with those of GenBank has shown a high extent of similarity with Methylobacterium extorquens (99.4-99.7%). Notwithstanding that the strains of Methylobacterium are resistant to the broad range of extreme factors (gamma-irradiation, UV-irradiation, dehydration), the Antarctic and collection strains of the genus were sensitive to the ions of such heavy metals as Cu, Hg, Cd, Cr (10 mg/l).

Growth and enzymological characteristics of a pink-pigmented facultative methylotroph Methylobacterium sp. MB1.

PubMed

Baev, M V; Kuznetsov, E V; Skladnev, D A; Govorukhina, N I; Sterkin, V E; Tsygankov, Y D

1992-01-01

Growth characteristics of batch and continuous cultures of the pink facultative methylotroph Methylobacterium sp. MB1 were determined. The response of a chemostat culture to a pulse increase of methanol concentration was studied. Malate, succinate and oxaloacetate additions to the methanol-supplemented medium decreased batch culture growth inhibition by methanol. The carotenoid content in cells grown in a chemostat decreased with increasing growth rate. The key enzyme activities of C1-metabolism were measured in a chemostat culture at different dilution rates.

Methylobacterium oryzae sp. nov., an aerobic, pink-pigmented, facultatively methylotrophic, 1-aminocyclopropane-1-carboxylate deaminase-producing bacterium isolated from rice.

PubMed

Madhaiyan, Munusamy; Kim, Byung-Yong; Poonguzhali, Selvaraj; Kwon, Soon-Wo; Song, Myung-Hee; Ryu, Jeoung-Hyun; Go, Seung-Joo; Koo, Bon-Sung; Sa, Tong-Min

2007-02-01

A pink-pigmented, facultatively methylotrophic bacterium, strain CBMB20T, isolated from stem tissues of rice, was analysed by a polyphasic approach. Strain CBMB20T utilized 1-aminocyclopropane 1-carboxylate (ACC) as a nitrogen source and produced ACC deaminase. It was related phylogenetically to members of the genus Methylobacterium. 16S rRNA gene sequence analysis indicated that strain CBMB20T was most closely related to Methylobacterium fujisawaense, Methylobacterium radiotolerans and Methylobacterium mesophilicum; however, DNA-DNA hybridization values were less than 70 % with the type strains of these species. The DNA G+C content of strain CBMB20T was 70.6 mol%. The study presents a detailed phenotypic characterization of strain CBMB20T that allows its differentiation from other Methylobacterium species. In addition, strain CBMB20T is the only known member of the genus Methylobacterium to be described from the phyllosphere of rice. Based on the data presented, strain CBMB20T represents a novel species in the genus Methylobacterium, for which the name Methylobacterium oryzae sp. nov. is proposed, with strain CBMB20T (=DSM 18207T=LMG 23582T=KACC 11585T) as the type strain.

Biosynthetic Approaches to Isotope Enrichment for Applications in Neutron Scattering and High Field NMR Spectroscopy: Methylotrophic

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

Mary E. lidstrom

Limitations in current isotopic labeling methods present a substantial bottleneck for the application of advanced structural techniques to many important biochemical problems. New tools are required to efficiently produce the necessary labeling patterns in biochemical precursors and incorporate them into protein molecules for structural studies. This project proposed involved one aspect of this problem, the development of expression vectors for a methylotrophic bacterium, Methylobacterium extorquens AM1. If high-level, efficient expression could be obtained in such a bacterium, it would be possible to use low-cost {sup 2}H- and/or {sup 13}C-labeled substrates such as methanol to label proteins. The Lidstrom laboratory atmore » the University of Washington worked closely with the collaborators at Los Alamos National Laboratories in the development and use of these vectors. (1) Overexpression of a target gene, bacterial dehalogenase--This enzyme was expressed in Methylobacterium extorquens AM1 using a high level methanol-inducible promoter, the mxaF promoter. High expression was achieved, but most was in an insoluble form. They expressed this protein in a mutant lacking polybetahydroxybutyrate granules, and high expression was achieved, up to 10% of the total soluble protein. The recombinant protein was purified and shown to be active, with characteristics similar to the enzyme produced in E. coli. (2) Development of regulated expression systems--A number of regulated promoters were tested in M. extorquens AM1, the most promising of which appeared to be the E. coli lac promoter coupled to the Laciq regulator. The repressor was shown to be active and a chromosomal insertion construct was generated that repressed the low-level lac promoter activity in M. extorquens AM1. However, IPTG induced this system only poorly. A number of studies were carried out leading to the conclusion that IPTG entered the cell but was exported by one or more export pumps. Target genes for

Methylobacterium marchantiae sp. nov., a pink-pigmented, facultatively methylotrophic bacterium isolated from the thallus of a liverwort.

PubMed

Schauer, S; Kämpfer, P; Wellner, S; Spröer, C; Kutschera, U

2011-04-01

A pink-pigmented, facultatively methylotrophic bacterium, designated strain JT1(T), was isolated from a thallus of the liverwort Marchantia polymorpha L. and was analysed by using a polyphasic approach. Comparative 16S rRNA gene sequence analysis placed the strain in a clade with Methylobacterium adhaesivum AR27(T), Methylobacterium fujisawaense DSM 5686(T), Methylobacterium radiotolerans JCM 2831(T) and Methylobacterium jeotgali S2R03-9(T), with which it showed sequence similarities of 97.8, 97.7, 97.2 and 97.4 %, respectively. However, levels of DNA-DNA relatedness between strain JT1(T) and these and the type strains of other closely related species were lower than 70 %. Cells of JT1(T) stained Gram-negative and were motile, rod-shaped and characterized by numerous fimbriae-like appendages on the outer surface of their wall (density up to 200 µm(-2)). Major fatty acids were C(18 : 1)ω7c and C(16 : 0). Based on the morphological, physiological and biochemical data presented, strain JT1(T) is considered to represent a novel species of the genus Methylobacterium, for which the name Methylobacterium marchantiae sp. nov. is proposed. The type strain is JT1(T) ( = DSM 21328(T)  = CCUG 56108(T)).

Cloning of a methanol-inducible moxF promoter and its analysis in moxB mutants of Methylobacterium extorquens AM1rif.

PubMed Central

Morris, C J; Lidstrom, M E

1992-01-01

In Methylobacterium extorquens AM1, gene encoding methanol dehydrogenase polypeptides are transcriptionally regulated in response to C1 compounds, including methanol (M. E. Lidstrom and D. I. Stirling, Annu. Rev. Microbiol. 44:27-57, 1990). In order to study this regulation, a transcriptional fusion has been constructed between a beta-galactosidase reporter gene and a 1.55-kb XhoI-SalI fragment of M. extorquens AM1rif DNA encoding the N terminus of the methanol dehydrogenase large subunit (moxF) and 1,289 bp of upstream DNA. The fusion exhibited orientation-specific promoter activity in M. extorquens AM1rif but was expressed constitutively when the transcriptional fusion was located on the plasmid. However, correct regulation was restored when the construction was inserted in the M. extorquens AM1rif chromosome. This DNA fragment was shown to contain both the moxFJGI promoter and the sequences necessary in cis for its transcriptional regulation by methanol. Transcription from this promoter was studied in the M. extorquens AM1rif moxB mutant strains UV4rif and UV25rif, which have a pleiotropic phenotype with regard to the components of methanol oxidation. In these mutants, beta-galactosidase activity from the fusion was reduced to a level equal to that of the vector background when the fusion was present in both plasmid and chromosomal locations. Since both constitutive and methanol-inducible promoter activities were lost in the mutants, moxB appears to be required for transcription of the genes encoding the methanol dehydrogenase polypeptides. Images PMID:1624436

Oxalyl-Coenzyme A Reduction to Glyoxylate Is the Preferred Route of Oxalate Assimilation in Methylobacterium extorquens AM1

PubMed Central

Schneider, Kathrin; Skovran, Elizabeth

2012-01-01

Oxalate catabolism is conducted by phylogenetically diverse organisms, including Methylobacterium extorquens AM1. Here, we investigate the central metabolism of this alphaproteobacterium during growth on oxalate by using proteomics, mutant characterization, and 13C-labeling experiments. Our results confirm that energy conservation proceeds as previously described for M. extorquens AM1 and other characterized oxalotrophic bacteria via oxalyl-coenzyme A (oxalyl-CoA) decarboxylase and formyl-CoA transferase and subsequent oxidation to carbon dioxide via formate dehydrogenase. However, in contrast to other oxalate-degrading organisms, the assimilation of this carbon compound in M. extorquens AM1 occurs via the operation of a variant of the serine cycle as follows: oxalyl-CoA reduction to glyoxylate and conversion to glycine and its condensation with methylene-tetrahydrofolate derived from formate, resulting in the formation of C3 units. The recently discovered ethylmalonyl-CoA pathway operates during growth on oxalate but is nevertheless dispensable, indicating that oxalyl-CoA reductase is sufficient to provide the glyoxylate required for biosynthesis. Analysis of an oxalyl-CoA synthetase- and oxalyl-CoA-reductase-deficient double mutant revealed an alternative, although less efficient, strategy for oxalate assimilation via one-carbon intermediates. The alternative process consists of formate assimilation via the tetrahydrofolate pathway to fuel the serine cycle, and the ethylmalonyl-CoA pathway is used for glyoxylate regeneration. Our results support the notion that M. extorquens AM1 has a plastic central metabolism featuring multiple assimilation routes for C1 and C2 substrates, which may contribute to the rapid adaptation of this organism to new substrates and the eventual coconsumption of substrates under environmental conditions. PMID:22493020

High-Throughput Identification and Screening of Novel Methylobacterium Species Using Whole-Cell MALDI-TOF/MS Analysis

PubMed Central

Tani, Akio; Sahin, Nurettin; Matsuyama, Yumiko; Enomoto, Takashi; Nishimura, Naoki; Yokota, Akira; Kimbara, Kazuhide

2012-01-01

Methylobacterium species are ubiquitous α-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing. PMID:22808262

Genetics in methylotrophic bacteria: Appendix. Final report

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

Lidstrom, M.E.

This research has focused primarily on promoters in Methylobacterium extorquens AM1 and in methanotrophic bacteria. In Methylobacterium extorquens work continued on the moxF promoter. The author constructed chromosomal lacZ fusions of this promoter to avoid the regulation problems of plasmid-borne fragments and has shown that this is regulated normally in the chromosome. She has constructed lacZ fusions to some of the mox genes involved in the synthesis of the cofactor, PQQ, in order to carry out similar analysis of transcription of PQQ genes. The author has continued to isolate mox genes in methanotrophs for the purpose of studying their promotersmore » and transcriptional regulation.« less

Taxonomy of oxalotrophic Methylobacterium strains.

PubMed

Sahin, Nurettin; Kato, Yuko; Yilmaz, Ferah

2008-10-01

Most of the oxalotrophic bacteria are facultative methylotrophs and play important ecological roles in soil fertility and cycling of elements. This study gives a detailed picture of the taxonomy and diversity of these bacteria and provides new information about the taxonomical variability within the genus Methylobacterium. Twelve mesophilic, pink-pigmented, and facultatively methylotrophic oxalate-oxidizing strains were included in this work that had been previously isolated from the soil and some plant tissues by the potassium oxalate enrichment method. The isolates were characterized using biochemical tests, cellular lipid profiles, spectral characteristics of carotenoid pigments, G+C content of the DNA, and 16S rDNA sequencing. The taxonomic similarities among the strains were analyzed using the simple matching (S (SM)) and Jaccard (S (J)) coefficients, and the UPGMA clustering algorithm. The phylogenetic position of the strains was inferred by the neighbor-joining method on the basis of the 16S rDNA sequences. All isolates were Gram-negative, facultatively methylotrophic, oxidase and catalase positive, and required no growth factors. Based on the results of numerical taxonomy, the strains formed four closely related clusters sharing > or =85% similarity. Analysis of the 16S rDNA sequences demonstrated that oxalotrophic, pink-pigmented, and facultatively methylotrophic strains could be identified as members of the genus Methylobacterium. Except for M. variabile and M. aquaticum, all of the Methylobacterium type strains tested had the ability of oxalate utilization. Our results indicate that the capability of oxalate utilization seems to be an uncommon trait and could be used as a valuable taxonomic criterion for differentiation of Methylobacterium species.

Taxonomy of oxalotrophic Methylobacterium strains

NASA Astrophysics Data System (ADS)

Sahin, Nurettin; Kato, Yuko; Yilmaz, Ferah

2008-10-01

Most of the oxalotrophic bacteria are facultative methylotrophs and play important ecological roles in soil fertility and cycling of elements. This study gives a detailed picture of the taxonomy and diversity of these bacteria and provides new information about the taxonomical variability within the genus Methylobacterium. Twelve mesophilic, pink-pigmented, and facultatively methylotrophic oxalate-oxidizing strains were included in this work that had been previously isolated from the soil and some plant tissues by the potassium oxalate enrichment method. The isolates were characterized using biochemical tests, cellular lipid profiles, spectral characteristics of carotenoid pigments, G+C content of the DNA, and 16S rDNA sequencing. The taxonomic similarities among the strains were analyzed using the simple matching ( S SM) and Jaccard ( S J) coefficients, and the UPGMA clustering algorithm. The phylogenetic position of the strains was inferred by the neighbor-joining method on the basis of the 16S rDNA sequences. All isolates were Gram-negative, facultatively methylotrophic, oxidase and catalase positive, and required no growth factors. Based on the results of numerical taxonomy, the strains formed four closely related clusters sharing ≥85% similarity. Analysis of the 16S rDNA sequences demonstrated that oxalotrophic, pink-pigmented, and facultatively methylotrophic strains could be identified as members of the genus Methylobacterium. Except for M. variabile and M. aquaticum, all of the Methylobacterium type strains tested had the ability of oxalate utilization. Our results indicate that the capability of oxalate utilization seems to be an uncommon trait and could be used as a valuable taxonomic criterion for differentiation of Methylobacterium species.

Methylobacterium pseudosasicola sp. nov. and Methylobacterium phyllostachyos sp. nov., isolated from bamboo leaf surfaces.

PubMed

Madhaiyan, Munusamy; Poonguzhali, Selvaraj

2014-07-01

Two strains of Gram-negative, methylotrophic bacteria, isolated because of their abilities to promote plant growth, were subjected to a polyphasic taxonomic study. The isolates were strictly aerobic, motile, pink-pigmented, facultatively methylotrophic, non-spore-forming rods. The chemotaxonomic characteristics of the isolates included the presence of C18 : 1ω7c as the major cellular fatty acid. The DNA G+C contents of strains BL36(T) and BL47(T) were 69.4 and 69.8 mol%, respectively. 16S rRNA gene sequence analysis of strains BL36(T) and BL47(T) placed them under the genus Methylobacterium, with the pairwise sequence similarity between them and the type strains of closely related species ranging from 97.2 to 99.0%. On the basis of their phenotypic and phylogenetic distinctiveness and the results of DNA-DNA hybridization analysis, the isolates represent two novel species within the genus Methylobacterium, for which the names Methylobacterium pseudosasicola sp. nov. (type strain BL36(T) = NBRC 105203(T) = ICMP 17621(T)) and Methylobacterium phyllostachyos sp. nov. (type strain BL47(T) = NBRC 105206(T) = ICMP 17619(T)) are proposed. © 2014 IUMS.

Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2.

PubMed

Yezza, A; Fournier, D; Halasz, A; Hawari, J

2006-11-01

A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at alpha concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.

Chloride-associated adaptive response in aerobic methylotrophic dichloromethane-utilising bacteria.

PubMed

Torgonskaya, Maria L; Doronina, Nina V; Hourcade, Edith; Trotsenko, Yuri A; Vuilleumier, Stéphane

2011-06-01

Aerobic methylotrophic bacteria able to grow with dichloromethane (DCM) as the sole carbon and energy source possess a specific glutathione S-transferase, DCM dehalogenase, which transforms DCM to formaldehyde, used for biomass and energy production, and hydrochloric acid, which is excreted. Evidence is presented for chloride-specific responses for three DCM-degrading bacteria, Methylobacterium extorquens DM4, Methylopila helvetica DM6 and Albibacter methylovorans DM10. Chloride release into the medium was inhibited by sodium azide and m -chlorophenylhydrazone, suggesting an energy-dependent process. In contrast, only nigericin affected chloride excretion in Mb. extorquens DM4 and Mp. helvetica DM6, while valinomycin had the same effect in A. methylovorans DM10 only. Chloride ions stimulated DCM-dependent induction of DCM dehalogenase expression for Mp. helvetica DM6 and A. methylovorans DM10, and shortened the time for onset of chloride release into the medium. Striking chloride-containing structures were observed by electron microscopy and X-ray microanalysis on the cell surface of Mp. helvetica DM6 and A. methylovorans DM10 during growth with DCM, and with methanol in medium supplemented with sodium chloride. Taken together, these data suggest the existence of both general and specific chloride-associated adaptations in aerobic DCM-degrading bacteria. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Parallel and Divergent Evolutionary Solutions for the Optimization of an Engineered Central Metabolism in Methylobacterium extorquens AM1

PubMed Central

Carroll, Sean Michael; Chubiz, Lon M.; Agashe, Deepa; Marx, Christopher J.

2015-01-01

Bioengineering holds great promise to provide fast and efficient biocatalysts for methanol-based biotechnology, but necessitates proven methods to optimize physiology in engineered strains. Here, we highlight experimental evolution as an effective means for optimizing an engineered Methylobacterium extorquens AM1. Replacement of the native formaldehyde oxidation pathway with a functional analog substantially decreased growth in an engineered Methylobacterium, but growth rapidly recovered after six hundred generations of evolution on methanol. We used whole-genome sequencing to identify the basis of adaptation in eight replicate evolved strains, and examined genomic changes in light of other growth and physiological data. We observed great variety in the numbers and types of mutations that occurred, including instances of parallel mutations at targets that may have been “rationalized” by the bioengineer, plus other “illogical” mutations that demonstrate the ability of evolution to expose unforeseen optimization solutions. Notably, we investigated mutations to RNA polymerase, which provided a massive growth benefit but are linked to highly aberrant transcriptional profiles. Overall, we highlight the power of experimental evolution to present genetic and physiological solutions for strain optimization, particularly in systems where the challenges of engineering are too many or too difficult to overcome via traditional engineering methods. PMID:27682084

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing (R)-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

PubMed Central

Rohde, Maria-Teresa; Tischer, Sylvi; Harms, Hauke

2016-01-01

ABSTRACT The biotechnological production of the methyl methacrylate precursor 2-hydroxyisobutyric acid (2-HIBA) via bacterial poly-3-hydroxybutyrate (PHB) overflow metabolism requires suitable (R)-3-hydroxybutyryl coenzyme A (CoA)-specific coenzyme B12-dependent mutases (RCM). Here, we characterized a predicted mutase from Bacillus massiliosenegalensis JC6 as a mesophilic RCM closely related to the thermophilic enzyme previously identified in Kyrpidia tusciae DSM 2912 (M.-T. Weichler et al., Appl Environ Microbiol 81:4564–4572, 2015, https://doi.org/10.1128/AEM.00716-15). Using both RCM variants, 2-HIBA production from methanol was studied in fed-batch bioreactor experiments with recombinant Methylobacterium extorquens AM1. After complete nitrogen consumption, the concomitant formation of PHB and 2-HIBA was achieved, indicating that both sets of RCM genes were successfully expressed. However, although identical vector systems and incubation conditions were chosen, the metabolic activity of the variant bearing the RCM genes from strain DSM 2912 was severely inhibited, likely due to the negative effects caused by heterologous expression. In contrast, the biomass yield of the variant expressing the JC6 genes was close to the wild-type performance, and 2-HIBA titers of 2.1 g liter−1 could be demonstrated. In this case, up to 24% of the substrate channeled into overflow metabolism was converted to the mutase product, and maximal combined 2-HIBA plus PHB yields from methanol of 0.11 g g−1 were achieved. Reverse transcription-quantitative PCR analysis revealed that metabolic genes, such as methanol dehydrogenase and acetoacetyl-CoA reductase genes, are strongly downregulated after exponential growth, which currently prevents a prolonged overflow phase, thus preventing higher product yields with strain AM1. IMPORTANCE In this study, we genetically modified a methylotrophic bacterium in order to channel intermediates of its overflow metabolism to the C4 carboxylic

Evaluation of pink-pigmented facultative methylotrophic bacteria for phosphate solubilization.

PubMed

Jayashree, Shanmugam; Vadivukkarasi, Ponnusamy; Anand, Kirupanithi; Kato, Yuko; Seshadri, Sundaram

2011-08-01

Thirteen pink-pigmented facultative methylotrophic (PPFM) strains isolated from Adyar and Cooum rivers in Chennai and forest soil samples in Tamil Nadu, India, along with Methylobacterium extorquens, M. organophilum, M. gregans, and M. komagatae were screened for phosphate solubilization in plates. P-solubilization index of the PPFMs grown on NBRIP-BPB plates for 7 days ranged from 1.1 to 2.7. The growth of PPFMs in tricalcium phosphate amended media was found directly proportional to the glucose concentration. Higher phosphate solubilization was observed in four strains MSF 32 (415 mg l(-l)), MDW 80 (301 mg l(-l)), M. komagatae (279 mg l(-l)), and MSF 34 (202 mg l(-l)), after 7 days of incubation. A drop in the media pH from 6.6 to 3.4 was associated with an increase in titratable acidity. Acid phosphatase activity was more pronounced in the culture filtrate than alkaline phosphatase activity. Adherence of phosphate to densely grown bacterial surface was observed under scanning electron microscope after 7-day-old cultures. Biochemical characterization and screening for methanol dehydrogenase gene (mxaF) confirmed the strains as methylotrophs. The mxaF gene sequence from MSF 32 clustered towards M. lusitanum sp. with 99% similarity. This study forms the first detailed report on phosphate solubilization by the PPFMs.

A novel pink-pigmented facultative methylotroph, Methylobacterium thiocyanatum sp. nov., capable of growth on thiocyanate or cyanate as sole nitrogen sources.

PubMed

Wood, A P; Kelly, D P; McDonald, I R; Jordan, S L; Morgan, T D; Khan, S; Murrell, J C; Borodina, E

1998-02-01

The isolation and properties of a novel species of pink-pigmented methylotroph, Methylobacterium thiocyanatum, are described. This organism satisfied all the morphological, biochemical, and growth-substrate criteria to be placed in the genus Methylobacterium. Sequencing of the gene encoding its 16S rRNA confirmed its position in this genus, with its closest phylogenetic relatives being M. rhodesianum, M. zatmanii and M. extorquens, from which it differed in its ability to grow on several diagnostic substrates. Methanol-grown organisms contained high activities of hydroxypyruvate reductase -3 micromol NADH oxidized min-1 (mg crude extract protein)-1], showing that the serine pathway was used for methylotrophic growth. M. thiocyanatum was able to use thiocyanate or cyanate as the sole source of nitrogen for growth, and thiocyanate as the sole source of sulfur in the absence of other sulfur compounds. It tolerated high concentrations (at least 50 mM) of thiocyanate or cyanate when these were supplied as nitrogen sources. Growing cultures degraded thiocyanate to produce thiosulfate as a major sulfur end product, apparently with the intermediate formation of volatile sulfur compounds (probably hydrogen sulfide and carbonyl sulfide). Enzymatic hydrolysis of thiocyanate by cell-free extracts was not demonstrated. Cyanate was metabolized by means of a cyanase enzyme that was expressed at approximately sevenfold greater activity during growth on thiocyanate [Vmax 634 +/- 24 nmol NH3 formed min-1 (mg protein)-1] than on cyanate [89 +/- 9 nmol NH3 min-1 (mg protein)-1]. Kinetic study of the cyanase in cell-free extracts showed the enzyme (1) to exhibit high affinity for cyanate (Km 0.07 mM), (2) to require bicarbonate for activity, (3) to be subject to substrate inhibition by cyanate and competitive inhibition by thiocyanate (Ki 0.65 mM), (4) to be unaffected by 1 mM ammonium chloride, (5) to be strongly inhibited by selenocyanate, and (6) to be slightly inhibited by 5 m

Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources.

PubMed

Vuilleumier, Stéphane; Chistoserdova, Ludmila; Lee, Ming-Chun; Bringel, Françoise; Lajus, Aurélie; Zhou, Yang; Gourion, Benjamin; Barbe, Valérie; Chang, Jean; Cruveiller, Stéphane; Dossat, Carole; Gillett, Will; Gruffaz, Christelle; Haugen, Eric; Hourcade, Edith; Levy, Ruth; Mangenot, Sophie; Muller, Emilie; Nadalig, Thierry; Pagni, Marco; Penny, Christian; Peyraud, Rémi; Robinson, David G; Roche, David; Rouy, Zoé; Saenampechek, Channakhone; Salvignol, Grégory; Vallenet, David; Wu, Zaining; Marx, Christopher J; Vorholt, Julia A; Olson, Maynard V; Kaul, Rajinder; Weissenbach, Jean; Médigue, Claudine; Lidstrom, Mary E

2009-01-01

Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name "island integration determinant" (iid). These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

PubMed Central

Lee, Ming-Chun; Bringel, Françoise; Lajus, Aurélie; Zhou, Yang; Gourion, Benjamin; Barbe, Valérie; Chang, Jean; Cruveiller, Stéphane; Dossat, Carole; Gillett, Will; Gruffaz, Christelle; Haugen, Eric; Hourcade, Edith; Levy, Ruth; Mangenot, Sophie; Muller, Emilie; Nadalig, Thierry; Pagni, Marco; Penny, Christian; Peyraud, Rémi; Robinson, David G.; Roche, David; Rouy, Zoé; Saenampechek, Channakhone; Salvignol, Grégory; Vallenet, David; Wu, Zaining; Marx, Christopher J.; Vorholt, Julia A.; Olson, Maynard V.; Kaul, Rajinder; Weissenbach, Jean; Médigue, Claudine; Lidstrom, Mary E.

2009-01-01

Background Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. Methodology/Principal Findings The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid). Conclusion/Significance These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive

Epiphytic pink-pigmented methylotrophic bacteria enhance germination and seedling growth of wheat (Triticum aestivum) by producing phytohormone.

PubMed

Meena, Kamlesh K; Kumar, Manish; Kalyuzhnaya, Marina G; Yandigeri, Mahesh S; Singh, Dhananjaya P; Saxena, Anil K; Arora, Dilip K

2012-05-01

Methylotrophic bacteria were isolated from the phyllosphere of different crop plants such as sugarcane, pigeonpea, mustard, potato and radish. The methylotrophic isolates were differentiated based on growth characteristics and colony morphology on methanol supplemented ammonium mineral salts medium. Amplification of the mxaF gene helped in the identification of the methylotrophic isolates as belonging to the genus Methylobacterium. Cell-free culture filtrates of these strains enhanced seed germination of wheat (Triticum aestivum) with highest values of 98.3% observed using Methylobacterium sp. (NC4). Highest values of seedling length and vigour were recorded with Methylobacterium sp. (NC28). HPLC analysis of production by bacterial strains ranged from 1.09 to 9.89 μg ml(-1) of cytokinins in the culture filtrate. Such cytokinin producing beneficial methylotrophs can be useful in developing bio-inoculants through co-inoculation of pink-pigmented facultative methylotrophs with other compatible bacterial strains, for improving plant growth and productivity, in an environment-friendly manner.

Difference in C3-C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1.

PubMed

Fu, Yanfen; Beck, David A C; Lidstrom, Mary E

2016-07-19

Two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. (13)C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. The major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways. Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, (13)C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.

Genome information of Methylobacterium oryzae, a plant-probiotic methylotroph in the phyllosphere.

PubMed

Kwak, Min-Jung; Jeong, Haeyoung; Madhaiyan, Munusamy; Lee, Yi; Sa, Tong-Min; Oh, Tae Kwang; Kim, Jihyun F

2014-01-01

Pink-pigmented facultative methylotrophs in the Rhizobiales are widespread in the environment, and many Methylobacterium species associated with plants produce plant growth-promoting substances. To gain insights into the life style at the phyllosphere and the genetic bases of plant growth promotion, we determined and analyzed the complete genome sequence of Methylobacterium oryzae CBMB20T, a strain isolated from rice stem. The genome consists of a 6.29-Mb chromosome and four plasmids, designated as pMOC1 to pMOC4. Among the 6,274 coding sequences in the chromosome, the bacterium has, besides most of the genes for the central metabolism, all of the essential genes for the assimilation and dissimilation of methanol that are either located in methylotrophy islands or dispersed. M. oryzae is equipped with several kinds of genes for adaptation to plant surfaces such as defense against UV radiation, oxidative stress, desiccation, or nutrient deficiency, as well as high proportion of genes related to motility and signaling. Moreover, it has an array of genes involved in metabolic pathways that may contribute to promotion of plant growth; they include auxin biosynthesis, cytokine biosynthesis, vitamin B12 biosynthesis, urea metabolism, biosorption of heavy metals or decrease of metal toxicity, pyrroloquinoline quinone biosynthesis, 1-aminocyclopropane-1-carboxylate deamination, phosphate solubilization, and thiosulfate oxidation. Through the genome analysis of M. oryzae, we provide information on the full gene complement of M. oryzae that resides in the aerial parts of plants and enhances plant growth. The plant-associated lifestyle of M. oryzae pertaining to methylotrophy and plant growth promotion, and its potential as a candidate for a bioinoculant targeted to the phyllosphere and focused on phytostimulation are illuminated.

Biotechnological and agronomic potential of endophytic pink-pigmented methylotrophic Methylobacterium spp.

PubMed

Dourado, Manuella Nóbrega; Camargo Neves, Aline Aparecida; Santos, Daiene Souza; Araújo, Welington Luiz

2015-01-01

The genus Methylobacterium is composed of pink-pigmented facultative methylotrophic (PPFM) bacteria, which are able to synthesize carotenoids and grow on reduced organic compounds containing one carbon (C1), such as methanol and methylamine. Due to their high phenotypic plasticity, these bacteria are able to colonize different habitats, such as soil, water, and sediment, and different host plants as both endophytes and epiphytes. In plant colonization, the frequency and distribution may be influenced by plant genotype or by interactions with other associated microorganisms, which may result in increasing plant fitness. In this review, different aspects of interactions with the host plant are discussed, including their capacity to fix nitrogen, nodule the host plant, produce cytokinins, auxin and enzymes involved in the induction of systemic resistance, such as pectinase and cellulase, and therefore plant growth promotion. In addition, bacteria belonging to this group can be used to reduce environmental contamination because they are able to degrade toxic compounds, tolerate high heavy metal concentrations, and increase plant tolerance to these compounds. Moreover, genome sequencing and omics approaches have revealed genes related to plant-bacteria interactions that may be important for developing strains able to promote plant growth and protection against phytopathogens.

tRNA Is the Source of Low-Level trans-Zeatin Production in Methylobacterium spp.†‡

PubMed Central

Koenig, Robbin L.; Morris, Roy O.; Polacco, Joe C.

2002-01-01

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs. PMID:11889088

Methylopila helvetica sp. nov. and Methylobacterium dichloromethanicum sp. nov.--novel aerobic facultatively methylotrophic bacteria utilizing dichloromethane.

PubMed

Doronina, N V; Trotsenko, Y A; Tourova, T P; Kuznetsov, B B; Leisinger, T

2000-06-01

Eight strains of Gram-negative, aerobic, asporogenous, neutrophilic, mesophilic, facultatively methylotrophic bacteria are taxonomically described. These icl- serine pathway methylobacteria utilize dichloromethane, methanol and methylamine as well as a variety of polycarbon compounds as the carbon and energy source. The major cellular fatty acids of the non-pigmented strains DM1, DM3, and DM5 to DM9 are C18:1, C16:0, C18:0, Ccy19:0 and that of the pink-pigmented strain DM4 is C18:1. The main quinone of all the strains is Q-10. The non-pigmented strains have similar phenotypic properties and a high level of DNA-DNA relatedness (81-98%) as determined by hybridization. All strains belong to the alpha-subgroup of the alpha-Proteobacteria. 16S rDNA sequence analysis led to the classification of these dichloromethane-utilizers in the genus Methylopila as a new species - Methylopila helvetica sp.nov. with the type strain DM9 (=VKM B-2189). The pink-pigmented strain DM4 belongs to the genus Methylobacterium but differs from the known members of this genus by some phenotypic properties, DNA-DNA relatedness (14-57%) and 16S rDNA sequence. Strain DM4 is named Methylobacterium dichloromethanicum sp. nov. (VKM B-2191 = DSMZ 6343).

Genomic and Transcriptomic Analysis of Growth-Supporting Dehalogenation of Chlorinated Methanes in Methylobacterium

PubMed Central

Chaignaud, Pauline; Maucourt, Bruno; Weiman, Marion; Alberti, Adriana; Kolb, Steffen; Cruveiller, Stéphane; Vuilleumier, Stéphane; Bringel, Françoise

2017-01-01

Bacterial adaptation to growth with toxic halogenated chemicals was explored in the context of methylotrophic metabolism of Methylobacterium extorquens, by comparing strains CM4 and DM4, which show robust growth with chloromethane and dichloromethane, respectively. Dehalogenation of chlorinated methanes initiates growth-supporting degradation, with intracellular release of protons and chloride ions in both cases. The core, variable and strain-specific genomes of strains CM4 and DM4 were defined by comparison with genomes of non-dechlorinating strains. In terms of gene content, adaptation toward dehalogenation appears limited, strains CM4 and DM4 sharing between 75 and 85% of their genome with other strains of M. extorquens. Transcript abundance in cultures of strain CM4 grown with chloromethane and of strain DM4 grown with dichloromethane was compared to growth with methanol as a reference C1 growth substrate. Previously identified strain-specific dehalogenase-encoding genes were the most transcribed with chlorinated methanes, alongside other genes encoded by genomic islands (GEIs) and plasmids involved in growth with chlorinated compounds as carbon and energy source. None of the 163 genes shared by strains CM4 and DM4 but not by other strains of M. extorquens showed higher transcript abundance in cells grown with chlorinated methanes. Among the several thousand genes of the M. extorquens core genome, 12 genes were only differentially abundant in either strain CM4 or strain DM4. Of these, 2 genes of known function were detected, for the membrane-bound proton translocating pyrophosphatase HppA and the housekeeping molecular chaperone protein DegP. This indicates that the adaptive response common to chloromethane and dichloromethane is limited at the transcriptional level, and involves aspects of the general stress response as well as of a dehalogenation-specific response to intracellular hydrochloric acid production. Core genes only differentially abundant in either

Genomic and Transcriptomic Analysis of Growth-Supporting Dehalogenation of Chlorinated Methanes in Methylobacterium.

PubMed

Chaignaud, Pauline; Maucourt, Bruno; Weiman, Marion; Alberti, Adriana; Kolb, Steffen; Cruveiller, Stéphane; Vuilleumier, Stéphane; Bringel, Françoise

2017-01-01

Bacterial adaptation to growth with toxic halogenated chemicals was explored in the context of methylotrophic metabolism of Methylobacterium extorquens , by comparing strains CM4 and DM4, which show robust growth with chloromethane and dichloromethane, respectively. Dehalogenation of chlorinated methanes initiates growth-supporting degradation, with intracellular release of protons and chloride ions in both cases. The core, variable and strain-specific genomes of strains CM4 and DM4 were defined by comparison with genomes of non-dechlorinating strains. In terms of gene content, adaptation toward dehalogenation appears limited, strains CM4 and DM4 sharing between 75 and 85% of their genome with other strains of M. extorquens . Transcript abundance in cultures of strain CM4 grown with chloromethane and of strain DM4 grown with dichloromethane was compared to growth with methanol as a reference C 1 growth substrate. Previously identified strain-specific dehalogenase-encoding genes were the most transcribed with chlorinated methanes, alongside other genes encoded by genomic islands (GEIs) and plasmids involved in growth with chlorinated compounds as carbon and energy source. None of the 163 genes shared by strains CM4 and DM4 but not by other strains of M. extorquens showed higher transcript abundance in cells grown with chlorinated methanes. Among the several thousand genes of the M. extorquens core genome, 12 genes were only differentially abundant in either strain CM4 or strain DM4. Of these, 2 genes of known function were detected, for the membrane-bound proton translocating pyrophosphatase HppA and the housekeeping molecular chaperone protein DegP. This indicates that the adaptive response common to chloromethane and dichloromethane is limited at the transcriptional level, and involves aspects of the general stress response as well as of a dehalogenation-specific response to intracellular hydrochloric acid production. Core genes only differentially abundant in

Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

DOE PAGES

Gonzalez, Javier M.; Marti-Arbona, Ricardo; Chen, Julian C. -H.; ...

2017-01-27

Malyl-CoA lyase (MCL) is an Mg 2+-dependent enzyme that catalyzes the reversible cleavage of (2 S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg 2+is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg 2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding.more » Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. Furthermore, this domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.« less

Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

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

Gonzalez, Javier M.; Marti-Arbona, Ricardo; Chen, Julian C. -H.

Malyl-CoA lyase (MCL) is an Mg 2+-dependent enzyme that catalyzes the reversible cleavage of (2 S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg 2+is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg 2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding.more » Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. Furthermore, this domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.« less

Genome Information of Methylobacterium oryzae, a Plant-Probiotic Methylotroph in the Phyllosphere

PubMed Central

Madhaiyan, Munusamy; Lee, Yi; Sa, Tong-Min; Oh, Tae Kwang; Kim, Jihyun F.

2014-01-01

Pink-pigmented facultative methylotrophs in the Rhizobiales are widespread in the environment, and many Methylobacterium species associated with plants produce plant growth-promoting substances. To gain insights into the life style at the phyllosphere and the genetic bases of plant growth promotion, we determined and analyzed the complete genome sequence of Methylobacterium oryzae CBMB20T, a strain isolated from rice stem. The genome consists of a 6.29-Mb chromosome and four plasmids, designated as pMOC1 to pMOC4. Among the 6,274 coding sequences in the chromosome, the bacterium has, besides most of the genes for the central metabolism, all of the essential genes for the assimilation and dissimilation of methanol that are either located in methylotrophy islands or dispersed. M. oryzae is equipped with several kinds of genes for adaptation to plant surfaces such as defense against UV radiation, oxidative stress, desiccation, or nutrient deficiency, as well as high proportion of genes related to motility and signaling. Moreover, it has an array of genes involved in metabolic pathways that may contribute to promotion of plant growth; they include auxin biosynthesis, cytokine biosynthesis, vitamin B12 biosynthesis, urea metabolism, biosorption of heavy metals or decrease of metal toxicity, pyrroloquinoline quinone biosynthesis, 1-aminocyclopropane-1-carboxylate deamination, phosphate solubilization, and thiosulfate oxidation. Through the genome analysis of M. oryzae, we provide information on the full gene complement of M. oryzae that resides in the aerial parts of plants and enhances plant growth. The plant-associated lifestyle of M. oryzae pertaining to methylotrophy and plant growth promotion, and its potential as a candidate for a bioinoculant targeted to the phyllosphere and focused on phytostimulation are illuminated. PMID:25211235

Cellulase production by pink pigmented facultative methylotrophic strains (PPFMs).

PubMed

Jayashree, Shanmugam; Lalitha, Rajendran; Vadivukkarasi, Ponnusamy; Kato, Yuko; Seshadri, Sundaram

2011-07-01

Pink pigmented facultative methylotrophs (PPFM) isolated from water samples of Cooum and Adyar rivers in Chennai and soil samples of forests located in various districts of Tamil Nadu, India were screened for cellulase production using carboxymethylcellulose agar (CMC agar) medium. The strains showed wide variations in the production of clearing zones around the colonies on CMC agar medium flooded with Congo red. CMCase and filter paper assays were used to quantitatively measure the cellulase activity of 13 PPFM strains. Among the strains, Methylobacterium gregans, MNW 60, MHW 109, MSF 34, and MSF 40 showed cellulolytic activity ranging from 0.73 to 1.16 U mL(-1) with wide temperature (35-65°C) and pH (5 to 8) tolerance. SDS-PAGE analysis of the crude enzyme of PPFM strain MNW 60 exhibited several protein bands, and zymogram analysis revealed two dimeric cellulase bands with molecular mass of ~92 and 42 kDa. Scanning electron microscopic studies revealed significant morphological differences between the cells grown in normal and CMC amended medium. The strain MNW 60 was identified as Methylobacterium sp. based on biochemical, physiological, and morphological analyses, and the methylotrophic nature was authenticated by the presence of mxaF gene, encoding methanol dehydrogenase as a key indicator enzyme of methylotrophs, with 99% similarity to Methylobacterium lusitanum. With the 16S ribosomal RNA sequence showing 97% similarity to M. lusitanum strain MP2, this can be proposed as a novel taxon of the genus Methylobacterium. The study forms the first detailed report on the extracellular cellulase production by pink pigmented Methylobacterium sp., and it is expected that this might be the basis for further studies on cellulase production by PPFMs to explore the molecular mechanism, strain improvement, and large-scale cellulase production for its application.

Biotechnological and Agronomic Potential of Endophytic Pink-Pigmented Methylotrophic Methylobacterium spp.

PubMed Central

Dourado, Manuella Nóbrega; Aparecida Camargo Neves, Aline; Santos, Daiene Souza; Araújo, Welington Luiz

2015-01-01

The genus Methylobacterium is composed of pink-pigmented facultative methylotrophic (PPFM) bacteria, which are able to synthesize carotenoids and grow on reduced organic compounds containing one carbon (C1), such as methanol and methylamine. Due to their high phenotypic plasticity, these bacteria are able to colonize different habitats, such as soil, water, and sediment, and different host plants as both endophytes and epiphytes. In plant colonization, the frequency and distribution may be influenced by plant genotype or by interactions with other associated microorganisms, which may result in increasing plant fitness. In this review, different aspects of interactions with the host plant are discussed, including their capacity to fix nitrogen, nodule the host plant, produce cytokinins, auxin and enzymes involved in the induction of systemic resistance, such as pectinase and cellulase, and therefore plant growth promotion. In addition, bacteria belonging to this group can be used to reduce environmental contamination because they are able to degrade toxic compounds, tolerate high heavy metal concentrations, and increase plant tolerance to these compounds. Moreover, genome sequencing and omics approaches have revealed genes related to plant-bacteria interactions that may be important for developing strains able to promote plant growth and protection against phytopathogens. PMID:25861650

Difference in C3–C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1

DOE PAGES

Fu, Yanfen; Beck, David A. C.; Lidstrom, Mary E.

2016-07-19

In this study, two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. As a result, the major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways.more » Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.« less

Difference in C3–C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1

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

Fu, Yanfen; Beck, David A. C.; Lidstrom, Mary E.

In this study, two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. As a result, the major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways.more » Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.« less

Interactions of Methylotrophs with Plants and Other Heterotrophic Bacteria

PubMed Central

Iguchi, Hiroyuki; Yurimoto, Hiroya; Sakai, Yasuyoshi

2015-01-01

Methylotrophs, which can utilize methane and/or methanol as sole carbon and energy sources, are key players in the carbon cycle between methane and CO2, the two most important greenhouse gases. This review describes the relationships between methylotrophs and plants, and between methanotrophs (methane-utilizers, a subset of methylotrophs) and heterotrophic bacteria. Some plants emit methane and methanol from their leaves, and provide methylotrophs with habitats. Methanol-utilizing methylotrophs in the genus Methylobacterium are abundant in the phyllosphere and have the ability to promote the growth of some plants. Methanotrophs also inhabit the phyllosphere, and methanotrophs with high methane oxidation activities have been found on aquatic plants. Both plant and environmental factors are involved in shaping the methylotroph community on plants. Methanotrophic activity can be enhanced by heterotrophic bacteria that provide growth factors (e.g., cobalamin). Information regarding the biological interaction of methylotrophs with other organisms will facilitate a better understanding of the carbon cycle that is driven by methylotrophs. PMID:27682083

Plant growth-promoting Methylobacterium induces defense responses in groundnut (Arachis hypogaea L.) compared with rot pathogens.

PubMed

Madhaiyan, M; Suresh Reddy, B V; Anandham, R; Senthilkumar, M; Poonguzhali, S; Sundaram, S P; Sa, Tongmin

2006-10-01

This study, framed in two different phases, studied the plant-growth promotion and the induction of systemic resistance in groundnut by Methylobacterium. Seed imbibition with Methylobacterium sp. increased germination by 19.5% compared with controls. Combined inoculation of Methylobacterium sp. with Rhizobium sp. also significantly increased plant growth, nodulation, and yield attributes in groundnut compared with individual inoculation of Rhizobium sp. Methylobacterium sp. challenge-inoculated with Aspergillus niger/Sclerotium rolfsii in groundnut significantly enhanced germination percentage and seedling vigour and showed increased phenylalanine ammonia lyase (PAL), beta-1,3-glucanase, and peroxidase (PO) activities. Under pot-culture conditions, in Methylobacterium sp. seed-treated groundnut plants challenge-inoculated with A. niger/S. rolfsii through foliar sprays on day 30, the activities of enzymes PO, PAL, and beta-1,3-glucanase increased constantly from 24 to 72 hours, after which decreased activity was noted. Five isozymes of polyphenol oxidase and PO could be detected in Methylobacterium-treated plants challenged with A. niger/S. rolfsii. Induced systemic resistance activity in groundnut against rot pathogens in response to methylotrophic bacteria suggests the possibility that pink-pigmented facultative methylotrophic bacteria might be used as a means of biologic disease control.

Methylotrophic Methylobacterium Bacteria Nodulate and Fix Nitrogen in Symbiosis with Legumes

PubMed Central

Sy, Abdoulaye; Giraud, Eric; Jourand, Philippe; Garcia, Nelly; Willems, Anne; de Lajudie, Philippe; Prin, Yves; Neyra, Marc; Gillis, Monique; Boivin-Masson, Catherine; Dreyfus, Bernard

2001-01-01

Rhizobia described so far belong to three distinct phylogenetic branches within the α-2 subclass of Proteobacteria. Here we report the discovery of a fourth rhizobial branch involving bacteria of the Methylobacterium genus. Rhizobia isolated from Crotalaria legumes were assigned to a new species, “Methylobacterium nodulans,” within the Methylobacterium genus on the basis of 16S ribosomal DNA analyses. We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of Methylobacterium spp. but a unique feature among rhizobia. Genes encoding two key enzymes of methylotrophy and nodulation, the mxaF gene, encoding the α subunit of the methanol dehydrogenase, and the nodA gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060. Plant tests and nodA amplification assays showed that “M. nodulans” is the only nodulating Methylobacterium sp. identified so far. Phylogenetic sequence analysis showed that “M. nodulans” NodA is closely related to Bradyrhizobium NodA, suggesting that this gene was acquired by horizontal gene transfer. PMID:11114919

Facile Arsenazo III-Based Assay for Monitoring Rare Earth Element Depletion from Cultivation Media for Methanotrophic and Methylotrophic Bacteria

PubMed Central

Hogendoorn, Carmen; Roszczenko-Jasińska, Paula; Martinez-Gomez, N. Cecilia; de Graaff, Johann; Grassl, Patrick; Pol, Arjan; Op den Camp, Huub J. M.

2018-01-01

ABSTRACT Recently, methanotrophic and methylotrophic bacteria were found to utilize rare earth elements (REEs). To monitor the REE content in culture media of these bacteria, we have developed a rapid screening method using the Arsenazo III (AS III) dye for spectrophotometric REE detection in the low μM (0.1 to 10 μM) range. We designed this assay to follow LaIII and EuIII depletion from the culture medium by the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV. The assay can also be modified to screen the uptake of other REEs, such as PrIII, or to monitor the depletion of LaIII from growth media in neutrophilic methylotrophs such as Methylobacterium extorquens strain AM1. The AS III assay presents a convenient and fast detection method for REE levels in culture media and is a sensitive alternative to inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS). IMPORTANCE REE-dependent bacterial metabolism is a quickly emerging field, and while the importance of REEs for both methanotrophic and methylotrophic bacteria is now firmly established, many important questions, such as how these insoluble elements are taken up into cells, are still unanswered. Here, an Arsenazo III dye-based assay has been developed for fast, specific, and sensitive determination of REE content in different culture media. This assay presents a useful tool for optimizing cultivation protocols, as well as for routine REE monitoring during bacterial growth without the need for specialized analytical instrumentation. Furthermore, this assay has the potential to promote the discovery of other REE-dependent microorganisms and can help to elucidate the mechanisms for acquisition of REEs by methanotrophic and methylotrophic bacteria. PMID:29453257

Facile Arsenazo III-Based Assay for Monitoring Rare Earth Element Depletion from Cultivation Media for Methanotrophic and Methylotrophic Bacteria.

PubMed

Hogendoorn, Carmen; Roszczenko-Jasińska, Paula; Martinez-Gomez, N Cecilia; de Graaff, Johann; Grassl, Patrick; Pol, Arjan; Op den Camp, Huub J M; Daumann, Lena J

2018-04-15

Recently, methanotrophic and methylotrophic bacteria were found to utilize rare earth elements (REEs). To monitor the REE content in culture media of these bacteria, we have developed a rapid screening method using the Arsenazo III (AS III) dye for spectrophotometric REE detection in the low μM (0.1 to 10 μM) range. We designed this assay to follow La III and Eu III depletion from the culture medium by the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV. The assay can also be modified to screen the uptake of other REEs, such as Pr III , or to monitor the depletion of La III from growth media in neutrophilic methylotrophs such as Methylobacterium extorquens strain AM1. The AS III assay presents a convenient and fast detection method for REE levels in culture media and is a sensitive alternative to inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS). IMPORTANCE REE-dependent bacterial metabolism is a quickly emerging field, and while the importance of REEs for both methanotrophic and methylotrophic bacteria is now firmly established, many important questions, such as how these insoluble elements are taken up into cells, are still unanswered. Here, an Arsenazo III dye-based assay has been developed for fast, specific, and sensitive determination of REE content in different culture media. This assay presents a useful tool for optimizing cultivation protocols, as well as for routine REE monitoring during bacterial growth without the need for specialized analytical instrumentation. Furthermore, this assay has the potential to promote the discovery of other REE-dependent microorganisms and can help to elucidate the mechanisms for acquisition of REEs by methanotrophic and methylotrophic bacteria. Copyright © 2018 Hogendoorn et al.

Characterization of new plasmids from methylotrophic bacteria.

PubMed

Brenner, V; Holubová, I; Benada, O; Hubácek, J

1991-07-01

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.

Methylobacterium trifolii sp. nov. and Methylobacterium thuringiense sp. nov., methanol-utilizing, pink-pigmented bacteria isolated from leaf surfaces.

PubMed

Wellner, S; Lodders, N; Glaeser, S P; Kämpfer, P

2013-07-01

Three pink-pigmented, aerobic, Gram-stain-negative, rod-shaped and facultatively methylotrophic strains were isolated from the phyllosphere of Trifolium repens and Cerastium holosteoides. 16S rRNA gene sequence analysis support the affiliation of all strains to the genus Methylobacterium. The closest relatives of strains C34(T) and T5 were Methylobacterium gnaphalii 23e(T) (98.0 and 98.5 % sequence similarity, respectively) and Methylobacterium organophilum JCM 2833(T) (97.0 and 97.2 %, respectively). Strain TA73(T) showed the highest sequence similarities to Methylobacterium marchantiae JT1(T) and Methylobacterium bullatum F3.2(T) (both 97.9 %), followed by Methylobacterium phyllosphaerae CBMB27(T) and Methylobacterium brachiatum DSM 19569(T) (both 97.8 %), Methylobacterium cerastii C15(T) and Methylobacterium radiotolerans JCM 2831(T) (both 97.7 %). The major components in the fatty acid profiles were C18 : 1ω7c, C16 : 0 and one unknown fatty acid for strain TA73(T) and C18 : 1ω7c, C16 : 1ω7c/iso-C15 : 0 2-OH, C18 : 0 and C16 : 0 for strains C34(T) and T5. Physiological and biochemical analysis, including DNA-DNA hybridization, revealed clear differences between the investigated strains and their closest phylogenetic neighbours. DNA-DNA hybridization studies also showed high similarities between strains C34(T) and T5 (59.6-100 %). Therefore, the isolates represent two novel species within the genus Methylobacterium, for which the names Methylobacterium trifolii sp. nov. (type strain TA73(T) = LMG 25778(T) = CCM 7786(T)) and Methylobacterium thuringiense sp. nov. (type strain C34(T) = LMG 25777(T) = CCM 7787(T)) are proposed.

Methylobacterium tarhaniae sp. nov., isolated from arid soil.

PubMed

Veyisoglu, Aysel; Camas, Mustafa; Tatar, Demet; Guven, Kiymet; Sazak, Anil; Sahin, Nevzat

2013-08-01

A reddish-orange-pigmented, Gram-stain-negative, aerobic, facultatively methylotrophic strain, N4211(T), isolated from arid soil, collected from Abuja, Nigeria, was analysed by using a polyphasic approach. Phylogenetic analysis, based on 16S rRNA gene sequences, showed that strain N4211(T) belonged to the genus Methylobacterium. Strain N4211(T) was most closely related to Methylobacterium aquaticum GR16(T) (98.56 %), Methylobacterium platani PMB02(T) (97.95 %) and Methylobacterium variabile GR3(T) (97.2 %), and the phylogenetic similarities to all other species of the genus Methylobacterium with validly published names were less than 97.0 %. The major ubiquinones detected were Q-10. The major fatty acids were summed feature 7 (C18 : 1 cis11/t9/t6). The DNA G+C content was 67.3 mol%. DNA-DNA relatedness of strain N4211(T) and the most closely related strains M. aquaticum DSM 16371(T) and M. platani KCTC 12901(T) were 60.0 and 48.2 %, respectively. On the basis of phenotypic, phylogenetic and DNA-DNA hybridization data, strain N4211(T) is assigned to a novel species of the genus Methylobacterium for which the name Methylobacterium tarhaniae sp. nov. is proposed. The type strain is N4211(T)( = KCTC 23615(T) = DSM 25844(T)).

Hydrogen and carbon isotope fractionation during degradation of chloromethane by methylotrophic bacteria

PubMed Central

Nadalig, Thierry; Greule, Markus; Bringel, Françoise; Vuilleumier, Stéphane; Keppler, Frank

2013-01-01

Chloromethane (CH3Cl) is a widely studied volatile halocarbon involved in the destruction of ozone in the stratosphere. Nevertheless, its global budget still remains debated. Stable isotope analysis is a powerful tool to constrain fluxes of chloromethane between various environmental compartments which involve a multiplicity of sources and sinks, and both biotic and abiotic processes. In this study, we measured hydrogen and carbon isotope fractionation of the remaining untransformed chloromethane following its degradation by methylotrophic bacterial strains Methylobacterium extorquens CM4 and Hyphomicrobium sp. MC1, which belong to different genera but both use the cmu pathway, the only pathway for bacterial degradation of chloromethane characterized so far. Hydrogen isotope fractionation for degradation of chloromethane was determined for the first time, and yielded enrichment factors (ε) of −29‰ and −27‰ for strains CM4 and MC1, respectively. In agreement with previous studies, enrichment in 13C of untransformed CH3Cl was also observed, and similar isotope enrichment factors (ε) of −41‰ and −38‰ were obtained for degradation of chloromethane by strains CM4 and MC1, respectively. These combined hydrogen and carbon isotopic data for bacterial degradation of chloromethane will contribute to refine models of the global atmospheric budget of chloromethane. PMID:24019296

Genes related to antioxidant metabolism are involved in Methylobacterium mesophilicum-soybean interaction.

PubMed

Araújo, Welington Luiz; Santos, Daiene Souza; Dini-Andreote, Francisco; Salgueiro-Londoño, Jennifer Katherine; Camargo-Neves, Aline Aparecida; Andreote, Fernando Dini; Dourado, Manuella Nóbrega

2015-10-01

The genus Methylobacterium is composed of pink-pigmented methylotrophic bacterial species that are widespread in natural environments, such as soils, stream water and plants. When in association with plants, this genus colonizes the host plant epiphytically and/or endophytically. This association is known to promote plant growth, induce plant systemic resistance and inhibit plant infection by phytopathogens. In the present study, we focused on evaluating the colonization of soybean seedling-roots by Methylobacterium mesophilicum strain SR1.6/6. We focused on the identification of the key genes involved in the initial step of soybean colonization by methylotrophic bacteria, which includes the plant exudate recognition and adaptation by planktonic bacteria. Visualization by scanning electron microscopy revealed that M. mesophilicum SR1.6/6 colonizes soybean roots surface effectively at 48 h after inoculation, suggesting a mechanism for root recognition and adaptation before this period. The colonization proceeds by the development of a mature biofilm on roots at 96 h after inoculation. Transcriptomic analysis of the planktonic bacteria (with plant) revealed the expression of several genes involved in membrane transport, thus confirming an initial metabolic activation of bacterial responses when in the presence of plant root exudates. Moreover, antioxidant genes were mostly expressed during the interaction with the plant exudates. Further evaluation of stress- and methylotrophic-related genes expression by qPCR showed that glutathione peroxidase and glutathione synthetase genes were up-regulated during the Methylobacterium-soybean interaction. These findings support that glutathione (GSH) is potentially a key molecule involved in cellular detoxification during plant root colonization. In addition to methylotrophic metabolism, antioxidant genes, mainly glutathione-related genes, play a key role during soybean exudate recognition and adaptation, the first step in

Methylobacterium indicum sp. nov., a facultative methylotrophic bacterium isolated from rice seed.

PubMed

Chaudhry, Vasvi; Baindara, Piyush; Pal, Vijay Kumar; Chawla, Niharika; Patil, Prabhu B; Korpole, Suresh

2016-02-01

Two pink pigmented, Gram-negative, motile, aerobic, rod shaped endophytic bacteria designated as SE2.11(T) and SE3.6 were isolated in different experiments from surface sterilized rice seeds. Both strains grew optimally at 28°C temperature. They were positive for catalase and nitrate reduction. The 16S rRNA gene sequence of the strains SE2.11(T) and SE3.6 displayed between 98.1% and 97.2% similarities with the validly published species of the genus Methylobacterium. The major cellular fatty acid was C18:1 ω7c in both the strains, a characteristic feature observed for members of the genus Methylobacterium. The predominant polar lipids were phospholipids including phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG). Phylogenetic analysis of 16S rRNA gene sequences resulted in the formation of a coherent cluster of strains SE2.11(T) and SE3.6 with closest relative Methylobacterium platani JCM 14648(T). However, digital DNA-DNA hybridization (dDDH) of strains SE2.11(T) and SE3.6 with the closest type strain M. platani JCM 14648(T) revealed similarity of 35.5% and 35.4%, respectively. Further, the ANI analysis of strains SE2.11(T) and SE3.6 genomes revealed only 87.9% identity with M. platani JCM 14648(T). Based on differences in biochemical, chemotaxonomic characteristics along with low identity at whole genome level we conclude that both strains represent a novel species of the genus Methylobacterium, for which the name Methylobacterium indicum sp. nov., is proposed. The type strain Methylobacterium indicum is SE2.11(T) (=MTCC 12298(T)=JCM 30761(T)) and SE3.6 (=MTCC 12299=JCM 30762) is another strain. Copyright © 2015 Elsevier GmbH. All rights reserved.

Methylobacterium variabile sp. nov., a methylotrophic bacterium isolated from an aquatic environment.

PubMed

Gallego, Virginia; García, Maria Teresa; Ventosa, Antonio

2005-07-01

Strain GR3(T) was isolated from drinking water during a screening programme to monitor the bacterial population present in the distribution system of Seville (Spain), and it was studied phenotypically, genotypically and phylogenetically. This pink-pigmented bacterium was identified as a Methylobacterium sp. Members of this genus are distributed in a wide variety of natural habitats, including soil, dust, air, freshwater and aquatic sediments. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain GR3(T) was closely related to Methylobacterium aquaticum (97.4% sequence similarity), whereas sequence similarity values with respect to the rest of the species belonging to this genus were lower than 96%. Furthermore, the DNA-DNA hybridization data and its phenotypic characteristics clearly indicate that the isolate represents a novel Methylobacterium species, for which the name Methylobacterium variabile sp. nov. is proposed. GR3(T) (=DSM 16961(T)=CCM 7281(T)=CECT 7045(T)) is the type strain; the DNA G+C content of this strain is 69.2 mol%.

The intracellular Scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins.

PubMed

Koskimäki, Janne J; Pirttilä, Anna Maria; Ihantola, Emmi-Leena; Halonen, Outi; Frank, A Carolin

2015-03-24

Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside

Characterization of Ni-tolerant methylobacteria associated with the hyperaccumulating plant Thlaspi goesingense and description of Methylobacterium goesingense sp. nov.

PubMed

Idris, Rughia; Kuffner, Melanie; Bodrossy, Levente; Puschenreiter, Markus; Monchy, Sebastien; Wenzel, Walter W; Sessitsch, Angela

2006-12-01

Various pink-pigmented facultative methylotrophic (PPFM) bacteria (strains iEII3, iEIV1, iEI6, iEII1, iEIII3 iEIII4, iEIII5, iRII1, iRII2, iRIII1, iRIV1 and iRIV2) were obtained from the rhizosphere and endosphere of hyperaccumulating plant Thlaspi goesingense grown in Redschlag, Austria [R. Idris, R. Trifonova, M. Puschenreiter, W.W. Wenzel, A. Sessitsch, Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense, Appl. Environ. Microbiol. 70 (2004) 2667-2677]. Due to their unexpected diversity, abundance and nickel tolerance they were further characterized by detailed 16S rRNA gene analysis, DNA-DNA hybridization, fatty acid analysis, heavy metal tolerance, screening for known Ni resistance genes and phenotypic analysis. These strains were found to exhibit different multiple heavy metal resistance characteristics to Ni, Cd, Co, Zn and Cr. On the basis of their physiological and genotypic properties, strains could be grouped with Methylobacterium extorquens and M. mesophilicum. One endophyte, strain iEII3, was found to belong to a novel species for which the name M. goesingense is proposed.

DNA:DNA hybridization studies on the pink-pigmented facultative methylotrophs.

PubMed

Hood, D W; Dow, C S; Green, P N

1987-03-01

The genomic relatedness among 36 strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) was estimated by determination of DNA base composition and by DNA:DNA hybridization studies. A reproducible hybridization system was developed for the rapid analysis of multiple DNA samples. Results indicated that the PPFMs comprise four major and several minor homology groups, and that they should remain grouped in a single genus, Methylobacterium.

77 FR 49793 - Ortho-Phthalaldehyde; Receipt of Application for Emergency Exemption, Solicitation of Public Comment

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-17

... parapaucimobilis, Stenotrophomonas maltophilia, Methylobacterium extorquens, and unidentified gram negative rods..., Stenotrophomonas maltophilia, Methylobacterium extorquens, and unidentified gram negative rods. Information in...

Phenotypic and genetic diversity of chlorine-resistant Methylobacterium strains isolated from various environments.

PubMed Central

Hiraishi, A; Furuhata, K; Matsumoto, A; Koike, K A; Fukuyama, M; Tabuchi, K

1995-01-01

Strains of pink-pigmented facultative methylotrophs which were isolated previously from various environments and assigned tentatively to the genus Methylobacterium were characterized in comparison with authentic strains of previously known species of this genus. Most of the isolates derived from chlorinated water supplies exhibited resistance to chlorine, whereas 29 to 40% of the isolates from air, natural aquatic environments, and clinical materials were chlorine resistant. None of the tested authentic strains of Methylobacterium species obtained from culture collections exhibited chlorine resistance. Numerical analysis of phenotypic profiles showed that the test organisms tested were separated from each other except M. organophilum and M. rhodesianum. The chlorine-resistant isolates were randomly distributed among all clusters. The 16S ribosomal DNA (rDNA) sequence-based phylogenetic analyses showed that representatives of the isolates together with known Methylobacterium species formed a line of descent distinct from that of members of related genera in the alpha-2 subclass of the Proteobacteria and were divided into three subclusters within the Methylobacterium group. These results demonstrate that there is phenotypic and genetic diversity among chlorine-resistant Methylobacterium strains within the genus. PMID:7793931

Phenotypic and genetic diversity of chlorine-resistant Methylobacterium strains isolated from various environments.

PubMed

Hiraishi, A; Furuhata, K; Matsumoto, A; Koike, K A; Fukuyama, M; Tabuchi, K

1995-06-01

Strains of pink-pigmented facultative methylotrophs which were isolated previously from various environments and assigned tentatively to the genus Methylobacterium were characterized in comparison with authentic strains of previously known species of this genus. Most of the isolates derived from chlorinated water supplies exhibited resistance to chlorine, whereas 29 to 40% of the isolates from air, natural aquatic environments, and clinical materials were chlorine resistant. None of the tested authentic strains of Methylobacterium species obtained from culture collections exhibited chlorine resistance. Numerical analysis of phenotypic profiles showed that the test organisms tested were separated from each other except M. organophilum and M. rhodesianum. The chlorine-resistant isolates were randomly distributed among all clusters. The 16S ribosomal DNA (rDNA) sequence-based phylogenetic analyses showed that representatives of the isolates together with known Methylobacterium species formed a line of descent distinct from that of members of related genera in the alpha-2 subclass of the Proteobacteria and were divided into three subclusters within the Methylobacterium group. These results demonstrate that there is phenotypic and genetic diversity among chlorine-resistant Methylobacterium strains within the genus.

Unusual Methylobacterium fujisawaense Infection in a Patient with Acute Leukaemia Undergoing Hematopoietic Stem Cell Transplantation: First Case Report

PubMed Central

Fanci, Rosa; Corti, Giampaolo; Bartoloni, Alessandro; Tortoli, Enrico; Mariottini, Alessandro; Pecile, Patrizia

2010-01-01

Microorganisms of the genus Methylobacterium are facultative methylotrophic, gram-negative rods that are ubiquitous in nature and rarely cause human disease, mostly in subjects with preexisting causes of immune depression. Methylobacterium fujisawaense, first proposed as a new species in 1988, has never been reported as a bacterial agent of human infections so far. Here we describe a case of M. fujisawaense infection in a relapsed acute leukaemia undergoing unrelated allogeneic hematopoietic stem cell transplantation. Molecular identification of an M. fujisawaense strain was obtained from multiple mycobacterial blood cultures. PMID:20396386

Metagenomic Analysis Revealed Methylamine and Ureide Utilization of Soybean-Associated Methylobacterium

PubMed Central

Minami, Tomoyuki; Anda, Misue; Mitsui, Hisayuki; Sugawara, Masayuki; Kaneko, Takakazu; Sato, Shusei; Ikeda, Seishi; Okubo, Takashi; Tsurumaru, Hirohito; Minamisawa, Kiwamu

2016-01-01

Methylobacterium inhabits the phyllosphere of a large number of plants. We herein report the results of comparative metagenome analyses on methylobacterial communities of soybean plants grown in an experimental field in Tohoku University (Kashimadai, Miyagi, Japan). Methylobacterium was identified as the most dominant genus (33%) among bacteria inhabiting soybean stems. We classified plant-derived Methylobacterium species into Groups I, II, and III based on 16S rRNA gene sequences, and found that Group I members (phylogenetically close to M. extorquens) were dominant in soybean-associated Methylobacterium. By comparing 29 genomes, we found that all Group I members possessed a complete set of genes for the N-methylglutamate pathway for methylamine utilization, and genes for urea degradation (urea carboxylase, urea amidolyase, and conventional urease). Only Group I members and soybean methylobacterial isolates grew in a culture supplemented with methylamine as the sole carbon source. They utilized urea or allantoin (a urea-related compound in legumes) as the sole nitrogen source; however, group III also utilized these compounds. The utilization of allantoin may be crucial in soybean-bacterial interactions because allantoin is a transported form of fixed nitrogen in legume plants. Soybean-derived Group I strain AMS5 colonized the model legume Lotus japonicus well. A comparison among the 29 genomes of plant-derived and other strains suggested that several candidate genes are involved in plant colonization such as csgG (curli fimbriae). Genes for the N-methylglutamate pathway and curli fimbriae were more abundant in soybean microbiomes than in rice microbiomes in the field. Based on these results, we discuss the lifestyle of Methylobacterium in the legume phyllosphere. PMID:27431374

Dominant colonization and inheritance of Methylobacterium sp. strain OR01 on perilla plants.

PubMed

Mizuno, Masayuki; Yurimoto, Hiroya; Iguchi, Hiroyuki; Tani, Akio; Sakai, Yasuyoshi

2013-01-01

Pink-pigmented facultative methylotrophs (PPFMs) are major inhabitants of the phyllosphere. In a preceding study, we found that perilla plants harbor a dominant population of PPFMs on their leaves and seeds, and that the closest relative of PPFMs (Methylobacterium sp. strain OR01 as representative strain) isolated from red perilla seeds was M. fujisawaense DSM5686(T). In the present study, the specific interaction between red perilla and Methylobacterium species was investigated. All the PPFMs isolated from red perilla seeds harvested in the Ohara area of Kyoto, Japan in 2009, 2010, and 2011 and the PPFMs isolated from red perilla leaves planted at four geographically different places in Japan had 16S rRNA sequences identical to that of strain OR01. Direct transmission of PPFMs from seeds to leaves and the competitiveness of strain OR01 were confirmed. This report is the first step toward understanding the species-level specificity of the interaction between perilla plants and Methylobacterium species.

The One-carbon Carrier Methylofuran from Methylobacterium extorquens AM1 Contains a Large Number of α- and γ-Linked Glutamic Acid Residues*

PubMed Central

Hemmann, Jethro L.; Saurel, Olivier; Ochsner, Andrea M.; Stodden, Barbara K.; Kiefer, Patrick; Milon, Alain; Vorholt, Julia A.

2016-01-01

Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a methanofuran analog was proposed to be involved in formaldehyde oxidation in Alphaproteobacteria. Here, we report the structure of the cofactor, which we termed methylofuran. Using an in vitro enzyme assay and LC-MS, methylofuran was identified in cell extracts and further purified. From the exact mass and MS-MS fragmentation pattern, the structure of the cofactor was determined to consist of a polyglutamic acid side chain linked to a core structure similar to the one present in archaeal methanofuran variants. NMR analyses showed that the core structure contains a furan ring. However, instead of the tyramine moiety that is present in methanofuran cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a 13C-labeled precursor. Methylofuran was present as a mixture of different species with varying numbers of glutamic acid residues in the side chain ranging from 12 to 24. Notably, the glutamic acid residues were not solely γ-linked, as is the case for all known methanofurans, but were identified by NMR as a mixture of α- and γ-linked amino acids. Considering the unusual peptide chain, the elucidation of the structure presented here sets the basis for further research on this cofactor, which is probably the largest cofactor known so far. PMID:26895963

Cultivable Methylobacterium species diversity in rice seeds identified with whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis.

PubMed

Okumura, Marie; Fujitani, Yoshiko; Maekawa, Masahiko; Charoenpanich, Jittima; Murage, Hunja; Kimbara, Kazuhide; Sahin, Nurettin; Tani, Akio

2017-02-01

Methylobacterium species are methylotrophic bacteria that widely inhabit plant surfaces. In addition to studies on methylotrophs as model organisms, research has also been conducted on their mechanism of plant growth promotion as well as the species-species specificity of plant-microbe interaction. We employed whole-cell matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (WC-MS) analysis, which enables the rapid and accurate identification of bacteria at the species level, to identify Methylobacterium isolates collected from the rice seeds of different cultivars harvested in Japan, Thailand, and Kenya. Rice seeds obtained from diverse geographical locations showed different communities of Methylobacterium species. We found that M. fujisawaense, M. aquaticum, M. platani, and M. radiotolerans are the most frequently isolated species, but none were isolated as common species from 18 seed samples due to the highly biased communities in some samples. These findings will contribute to the development of formulations containing selected species that promote rice growth, though it may be necessary to customize the formulations depending on the cultivars and farm conditions. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Computational Platform for Flux Analysis Using 13C-Label Tracing- Phase I SBIR Final Report

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

Van Dien, Stephen J.

Isotopic label tracing is a powerful experimental technique that can be combined with metabolic models to quantify metabolic fluxes in an organism under a particular set of growth conditions. In this work we constructed a genome-scale metabolic model of Methylobacterium extorquens, a facultative methylotroph with potential application in the production of useful chemicals from methanol. A series of labeling experiments were performed using 13C-methanol, and the resulting distribution of labeled carbon in the proteinogenic amino acids was determined by mass spectrometry. Algorithms were developed to analyze this data in context of the metabolic model, yielding flux distributions for wild-type andmore » several engineered strains of M. extorquens. These fluxes were compared to those predicted by model simulation alone, and also integrated with microarray data to give an improved understanding of the metabolic physiology of this organism.« less

Laboratory divergence of Methylobacterium extorquens AM1 through unintended domestication and past selection for antibiotic resistance

PubMed Central

2014-01-01

Background A common assumption of microorganisms is that laboratory stocks will remain genetically and phenotypically constant over time, and across laboratories. It is becoming increasingly clear, however, that mutations can ruin strain integrity and drive the divergence or “domestication” of stocks. Since its discovery in 1960, a stock of Methylobacterium extorquens AM1 (“AM1”) has remained in the lab, propagated across numerous growth and storage conditions, researchers, and facilities. To explore the extent to which this lineage has diverged, we compared our own “Modern” stock of AM1 to a sample archived at a culture stock center shortly after the strain’s discovery. Stored as a lyophilized sample, we hypothesized that this Archival strain would better reflect the first-ever isolate of AM1 and reveal ways in which our Modern stock has changed through laboratory domestication or other means. Results Using whole-genome re-sequencing, we identified some 29 mutations – including single nucleotide polymorphisms, small indels, the insertion of mobile elements, and the loss of roughly 36 kb of DNA - that arose in the laboratory-maintained Modern lineage. Contrary to our expectations, Modern was both slower and less fit than Archival across a variety of growth substrates, and showed no improvement during long-term growth and storage. Modern did, however, outperform Archival during growth on nutrient broth, and in resistance to rifamycin, which was selected for by researchers in the 1980s. Recapitulating selection for rifamycin resistance in replicate Archival populations showed that mutations to RNA polymerase B (rpoB) substantially decrease growth in the absence of antibiotic, offering an explanation for slower growth in Modern stocks. Given the large number of genomic changes arising from domestication (28), it is somewhat surprising that the single other mutation attributed to purposeful laboratory selection accounts for much of the phenotypic

A Systems Biology Approach Uncovers Cellular Strategies Used by Methylobacterium extorquens AM1 During the Switch from Multi- to Single-Carbon Growth

PubMed Central

Skovran, Elizabeth; Crowther, Gregory J.; Guo, Xiaofeng; Yang, Song; Lidstrom, Mary E.

2010-01-01

Background When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? Methylobacterium extorquens is a bacterium capable of growth on both multi- and single-carbon compounds. These different modes of growth utilize dramatically different central metabolic pathways with limited pathway overlap. Methodology/Principal Findings This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate growth (predicted to be energy-limited) to methanol growth (predicted to be reducing-power-limited), analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylene tetrahydrofolate dehydrogenase as one control point. This “downstream priming” mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints. Conclusions/Significance This multi-level approach has resulted in new insights into the metabolic strategies carried out to effect this shift between two dramatically different modes of growth and identified a number of potential flux control and regulatory check points as a further step toward understanding metabolic adaptation and the cellular strategies employed to maintain metabolic setpoints. PMID:21124828

A systems biology approach uncovers cellular strategies used by Methylobacterium extorquens AM1 during the switch from multi- to single-carbon growth.

PubMed

Skovran, Elizabeth; Crowther, Gregory J; Guo, Xiaofeng; Yang, Song; Lidstrom, Mary E

2010-11-24

When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? Methylobacterium extorquens is a bacterium capable of growth on both multi- and single-carbon compounds. These different modes of growth utilize dramatically different central metabolic pathways with limited pathway overlap. This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate growth (predicted to be energy-limited) to methanol growth (predicted to be reducing-power-limited), analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylene tetrahydrofolate dehydrogenase as one control point. This "downstream priming" mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints. This multi-level approach has resulted in new insights into the metabolic strategies carried out to effect this shift between two dramatically different modes of growth and identified a number of potential flux control and regulatory check points as a further step toward understanding metabolic adaptation and the cellular strategies employed to maintain metabolic setpoints.

The One-carbon Carrier Methylofuran from Methylobacterium extorquens AM1 Contains a Large Number of α- and γ-Linked Glutamic Acid Residues.

PubMed

Hemmann, Jethro L; Saurel, Olivier; Ochsner, Andrea M; Stodden, Barbara K; Kiefer, Patrick; Milon, Alain; Vorholt, Julia A

2016-04-22

Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a methanofuran analog was proposed to be involved in formaldehyde oxidation in Alphaproteobacteria. Here, we report the structure of the cofactor, which we termed methylofuran. Using an in vitro enzyme assay and LC-MS, methylofuran was identified in cell extracts and further purified. From the exact mass and MS-MS fragmentation pattern, the structure of the cofactor was determined to consist of a polyglutamic acid side chain linked to a core structure similar to the one present in archaeal methanofuran variants. NMR analyses showed that the core structure contains a furan ring. However, instead of the tyramine moiety that is present in methanofuran cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a (13)C-labeled precursor. Methylofuran was present as a mixture of different species with varying numbers of glutamic acid residues in the side chain ranging from 12 to 24. Notably, the glutamic acid residues were not solely γ-linked, as is the case for all known methanofurans, but were identified by NMR as a mixture of α- and γ-linked amino acids. Considering the unusual peptide chain, the elucidation of the structure presented here sets the basis for further research on this cofactor, which is probably the largest cofactor known so far. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

[Isolation and preliminary characterization of carotenoids from pink-pigmented methylotrophs].

PubMed

Konovalova, A M; Shylin, S O; Rokytko, P V

2006-01-01

An effective method was developed for complete removal of pigments from the cells and solvent mixture for further separation of pigments using thin layer chromatography on silica gel. Carotenoid samples that have been obtained in this way are of good purity for further investigations. Carotenoid pigments of pink-pigmented facultative methylotrophic bacteria Methylobacterium have been characterized. These carotenoids are represented mainly by xanthophylls, particularly hydroxycarotenoids. Strains M. fujisawaense B-3365 and M. mesophilicum B-3352 also have nonpolar carotenes in a small amount. Physico-chemical properties of carotenoids have been studied.

Production of acyl-homoserine lactone quorum-sensing signals is widespread in gram-negative Methylobacterium.

PubMed

Poonguzhali, Selvaraj; Madhaiyan, Munusamy; Sa, Tongmin

2007-02-01

Members of Methylobacterium, referred as pink-pigmented facultative methylotrophic bacteria, are frequently associated with terrestrial and aquatic plants, tending to form aggregates on the phyllosphere. We report here that the production of autoinducer molecules involved in the cell-to-cell signaling process, which is known as quorum sensing, is common among Methylobacterium species. Several strains of Methylobacterium were tested for their ability to produce N-acyl-homoserine lactone (AHL) signal molecules using different indicators. Most strains of Methylobacterium tested could elicit a positive response in Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. The synthesis of these compounds was cell-density dependent, and the maximal activity was reached during the late exponential to stationary phases. The bacterial extracts were separated by thin-layer chromatography and bioassayed with A. tumefaciens NT1 (traR, tra::lacZ749). They revealed the production of various patterns of the signal molecules, which are strain dependent. At least two signal molecules could be detected in most of the strains tested, and comparison of their relative mobilities suggested that they are homologs of N-octanoyl-DL-homoserine lactone (C8-HSL) and N-decanoyl-DL-homoserine lactone (C10-HSL).

Methylobacterium frigidaeris sp. nov., isolated from an air conditioning system.

PubMed

Lee, Yunho; Jeon, Che Ok

2018-01-01

A reddish pink-pigmented, Gram-stain-negative, aerobic and methylotrophic bacterial strain, designated strain IER25-16 T , was isolated from a laboratory air conditioning system in the Republic of Korea. Cells were motile rods showing catalase- and oxidase-positive reactions. Strain IER25-16 T grew at 10-40 °C (optimum, 30 °C), at pH 4.0-7.0 (optimum, pH 5.0-7.0) and in the presence of 0-1.0 % (w/v) NaCl (optimum, 0 %). The major respiratory quinone was ubiquinone-10 and ubiquinone-9 was also detected as the minor respiratory quinone. Summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c) was detected as the predominant fatty acids. The genomic DNA G+C content of strain IER25-16 T was 70.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain IER25-16 T belonged to the genus Methylobacterium of the class Alphaproteobacteria. Strain IER25-16 T was most closely related to Methylobacterium platani PMB02 T (97.9 %), Methylobacterium aquaticum GR16 T (97.9 %) and Methylobacterium tarhaniae N4211 T (97.5 %). The average nucleotide identity and in silico DNA-DNA hybridization values between strain IER25-16 T and M. platani, M. aquaticum and M. tarhaniae were 88.3, 88.8 and 89.6 % and 36.2, 37.3 and 39.3 %, respectively. The phenotypic and chemotaxonomic features and the phylogenetic inference clearly suggested that strain IER25-16 T represents a novel species of the genus Methylobacterium, for which the name Methylobacteriumfrigidaeris sp. nov. is proposed. The type strain is strain IER25-16 T (=KACC 19280 T =JCM 32048 T ).

Ergothioneine production using Methylobacterium species, yeast, and fungi.

PubMed

Fujitani, Yoshiko; Alamgir, Kabir Md; Tani, Akio

2018-06-14

Ergothioneine (EGT) is a sulfur-containing, anti-oxidative amino acid derived from histidine. EGT is synthesized in bacteria and fungi but not in animals and plants, and is now recognized as important for human health. Its cost-effective fermentative production has not been elucidated due to the lack of information for productive microorganisms. In this study, we doubled the gene copy for EGT synthesis and deleted the histidine ammonia-lyase gene in a potent EGT-producing methylotrophic bacterium Methylobacterium aquaticum strain 22A, and optimized its culture conditions, resulting in increased EGT production of 7.0 mg EGT/g dry cell weight and 100 μg EGT/5 mL/7 days. In addition, through screening we found EGT-producing eukaryotic strains of Aureobasidium pullulans and Rhodotorula mucilaginosa, which can produce 1.0 and 3.2 mg EGT/g dry cell weight, 70 and 120 μg EGT/5 mL/7 days, respectively. This study proposes practical uses of potent EGT-producing recombinant Methylobacterium species and non-recombinant yeast and fungal strains. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The genome of Methylobacillus flagellatus, the molecular basis forobligate methylotrophy, and the polyphyletic origin ofmethylotrophy

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

Chistoserdova, Ludmila; Lapidus, Alla; Han, Cliff

Along with methane, methanol and methylated amines representimportant biogenic atmospheric constituents, thus not only methanotrophs,but also non-methanotrophic methylotrophs play a significant role inglobal carbon cycling. The complete genome of a model obligate methanoland methylamine utilizer, Methylobacillus flagellatus (strain KT) wassequenced. The genome is represented by a single circular chromosome ofapproximately 3 Mb pairs, potentially encoding a total of 2,766 proteins.Based on genome analysis as well as the results from previous genetic andmutational analyses, methylotrophy is enabled by methanol- andmethylamine dehydrogenases, the tetrahydromethanopterin-linkedformaldehyde oxidation pathway, the assimilatory and dissimilatorybranches of the ribulose monophosphate cycle, and by formatedehydrogenases. Some of the methylotrophymore » genes are present in more thanone (identical or non-identical) copy. The obligate dependence on singlecarbon compounds appears to be due to the incomplete tricarboxylic acidcycle, as no genes potentially encoding alpha ketoglutarate, malate orsuccinate dehydrogenases are identifiable. The genome of M. flagellatuswas compared, in terms of methylotrophy functions, to the previouslysequenced genomes of three methylotrophs: Methylobacterium extorquens(Alphaproteobacterium, 7 Mbp), Methylibium petroleophilum(Betaproteobacterium, 4 Mbp), and Methylococcus capsulatus(Gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/orphylogenetically, methylotrophy functions in M. flagellatus were moresimilar to the ones in M. capsulatus and M. extorquens than to the onesin the more closely related M. petroleophilum, providing the firstgenomic evidence for the polyphyletic origin of methylotrophy inBetaproteobacteria.« less

Ethylene emission and PR protein synthesis in ACC deaminase producing Methylobacterium spp. inoculated tomato plants (Lycopersicon esculentum Mill.) challenged with Ralstonia solanacearum under greenhouse conditions.

PubMed

Yim, Woojong; Seshadri, Sundaram; Kim, Kiyoon; Lee, Gillseung; Sa, Tongmin

2013-06-01

Bacteria of genus Methylobacterium have been found to promote plant growth and regulate the level of ethylene in crop plants. This work is aimed to test the induction of defense responses in tomato against bacterial wilt by stress ethylene level reduction mediated by the ACC deaminase activity of Methylobacterium strains. Under greenhouse conditions, the disease index value in Methylobacterium sp. inoculated tomato plants was lower than control plants. Plants treated with Methylobacterium sp. challenge inoculated with Ralstonia solanacearum (RS) showed significantly reduced disease symptoms and lowered ethylene emission under greenhouse condition. The ACC and ACO (1-aminocyclopropane-1-carboxylate oxidase) accumulation in tomato leaves were significantly reduced with Methylobacterium strains inoculation. While ACC oxidase gene expression was found higher in plants treated with R. solanacearum than Methylobacterium sp. treatment, PR proteins related to induced systemic resistance like β-1,3-glucanase, PAL, PO and PPO were increased in Methylobacterium sp. inoculated plants. A significant increase in β-1,3-glucanase and PAL gene expression was found in all the Methylobacterium spp. treatments compared to the R. solanacearum treatment. This study confirms the activity of Methylobacterium sp. in increasing the defense enzymes by modulating the ethylene biosynthesis pathway and suggests the use of methylotrophic bacteria as potential biocontrol agents in tomato cultivation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Lanthanide-Dependent Regulation of Methylotrophy in Methylobacterium aquaticum Strain 22A

PubMed Central

Masuda, Sachiko; Suzuki, Yutaka; Fujitani, Yoshiko; Mitsui, Ryoji; Nakagawa, Tomoyuki

2018-01-01

ABSTRACT Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca2+-dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF. This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La3+. We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La3+; this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides. IMPORTANCE Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place

Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association

PubMed Central

Dourado, Manuella Nóbrega; Andreote, Fernando Dini; Dini-Andreote, Francisco; Conti, Raphael; Araújo, Janete Magali; Araújo, Welington Luiz

2012-01-01

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant. PMID:22481887

Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association.

PubMed

Dourado, Manuella Nóbrega; Andreote, Fernando Dini; Dini-Andreote, Francisco; Conti, Raphael; Araújo, Janete Magali; Araújo, Welington Luiz

2012-01-01

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.

Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1

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

Nunn, D.N.; Lidstrom, M.E.

A method has been developed for the direct selection of methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 (formerly Pseudomonas sp. strain AM1). Using this direct selection technique, we have isolated mutants of Methylobacterium sp. strain AM1 that are no longer capable of growth on methanol but retain the ability to grow on methylamine. These methanol oxidation (Mox) mutants were complemented with a genomic clone bank of this organism constructed in the broad-host-range cosmid pVK100, and subcloning and Tn5 mutagenesis experiments have assigned the Mox mutants to 10 distinct complementation groups. Using an open reading frame beta-galactosidasemore » fusion vector and antibodies specific for Methylobacterium sp. strain AM1 methanol dehydrogenase, we have identified the methanol dehydrogenase structural gene and determined the direction of transcription. The results suggest that the synthesis and utilization of an active methanol dehydrogenase in this organism requires at least 10 different gene functions.« less

Methylobacterium sp. resides in unculturable state in potato tissues in vitro and becomes culturable after induction by Pseudomonas fluorescens IMGB163.

PubMed

Podolich, O; Laschevskyy, V; Ovcharenko, L; Kozyrovska, N; Pirttilä, A M

2009-03-01

To induce growth of endophytic bacteria residing in an unculturable state in tissues of in vitro-grown potato plantlets. To isolate and identify the induced bacteria and to localize the strains in tissues of in vitro-grown potato plantlets. The inoculation of in vitro-grown potato plants with Pseudomonas fluorescens IMBG163 led to induction of another bacterium, a pink-pigmented facultative methylotroph that was identified as Methylobacterium sp. using phylogenetic 16S rDNA approach. Two molecular methods were used for localizing methylobacteria in potato plantlets: PCR and in situ hybridization (ISH/FISH). A PCR product specific for the Methylobacterium genus was found in DNA isolated from the surface-sterilized plantlet leaves. Presence of Methylobacterium rRNA was detected by ISH/FISH in leaves and stems of inoculated as well as axenic potato plantlets although the bacterium cannot be isolated from the axenic plants. Methylobacterium sp. resides in unculturable state within tissues of in vitro-grown potato plants and becomes culturable after inoculation with P. fluorescens IMBG163. In order to develop endophytic biofertilizers and biocontrol agents, a detailed knowledge of the life-style of endophytes is essential. To our knowledge, this is the first report on increase of the culturability of endophytes in response to inoculation by nonpathogenic bacteria.

Identification of putative methanol dehydrogenase (moxF) structural genes in methylotrophs and cloning of moxF genes from methylococcus capsulatus bath and Methylomonas albus BG8

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

Stephens, R.L.; Haygood, M.G.; Lidstrom, M.E.

An open-reading-frame fragment of a Methylobacterium sp. strain AM1 gene (moxF) encoding a portion of the methanol dehydrogenase structural protein has been used as a hybridization probe to detect similar sequences in a variety of methylotrophic bacteria. This hybridization was used to isolate clones containing putative moxF genes from two obligate methanotrophic bacteria, Methylococcus capsulatus Bath and Methylomonas albus BG8. The identity of these genes was confirmed in two ways. A T7 expression vector was used to produce methanol dehydrogenase protein in Escherichia coli from the cloned genes,a and in each case the protein was identified by immunoblotting with antiserummore » against the Methylomonas albus methanol dehydrogenase. In addition, a moxF mutant of Methylobacterium strain AM1 was complemented to a methanol-positive phenotype that partially restored methanol dehydrogenase activity, using broad-host-range plasmids containing the moxF genes from each methanotroph. The partial complementation of a moxF mutant in a facultative serine pathway methanol utilizer by moxF genes from type I and type X obligate methane utilizers suggests broad functional conservation of the methanol oxidation system among gram-negative methylotrophs.« less

The Diversity of Endophytic Methylotrophic Bacteria in an Oil-Contaminated and an Oil-Free Mangrove Ecosystem and Their Tolerance to Heavy Metals

PubMed Central

Dourado, Manuella Nobrega; Ferreira, Anderson; Araújo, Welington Luiz; Azevedo, João Lúcio; Lacava, Paulo Teixeira

2012-01-01

Methylobacterium strains were isolated from mangrove samples collected in Bertioga, SP, Brazil, from locations either contaminated or uncontaminated by oil spills. The tolerances of the strains to different heavy metals were assessed by exposing them to different concentrations of cadmium, lead, and arsenic (0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM). Additionally, the genetic diversity of Methylobacterium spp. was determined by sequence analysis of the 16S rRNA genes. The isolates from the contaminated locations were grouped, suggesting that oil can select for microorganisms that tolerate oil components and can change the methylotrophic bacterial community. Cadmium is the most toxic heavy metal assessed in this work, followed by arsenic and lead, and two isolates of Methylobacterium were found to be tolerant to all three metals. These isolates have the potential to bioremediate mangrove environments contaminated by oil spills by immobilizing the heavy metals present in the oil. PMID:22482056

Genome of Methylobacillus flagellatus, Molecular Basis for Obligate Methylotrophy, and Polyphyletic Origin of Methylotrophy

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

Chistoserdova, L; Lapidus, A; Han, C

Along with methane, methanol and methylated amines represent important biogenic atmospheric constituents; thus, not only methanotrophs but also nonmethanotrophic methylotrophs play a significant role in global carbon cycling. The complete genome of a model obligate methanol and methylamine utilizer, Methylobacillus flagellatus (strain KT) was sequenced. The genome is represented by a single circular chromosome of approximately 3 Mbp, potentially encoding a total of 2,766 proteins. Based on genome analysis as well as the results from previous genetic and mutational analyses, methylotrophy is enabled by methanol and methylamine dehydrogenases and their specific electron transport chain components, the tetrahydromethanopterin-linked formaldehyde oxidation pathwaymore » and the assimilatory and dissimilatory ribulose monophosphate cycles, and by a formate dehydrogenase. Some of the methylotrophy genes are present in more than one (identical or nonidentical) copy. The obligate dependence on single-carbon compounds appears to be due to the incomplete tricarboxylic acid cycle, as no genes potentially encoding alpha-ketoglutarate, malate, or succinate dehydrogenases are identifiable. The genome of M. flagellatus was compared in terms of methylotrophy functions to the previously sequenced genomes of three methylotrophs, Methylobacterium extorquens (an alphaproteobacterium, 7 Mbp), Methylibium petroleiphilum (a betaproteobacterium, 4 Mbp), and Methylococcus capsulatus (a gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/or phylogenetically, the methylotrophy functions in M. flagellatus were more similar to those in M. capsulatus and M. extorquens than to the ones in the more closely related M. petroleiphilum species, providing the first genomic evidence for the polyphyletic origin of methylotrophy in Betaproteobacteria.« less

Organization of Genes Required for the Oxidation of Methanol to Formaldehyde in Three Type II Methylotrophs

PubMed Central

Bastien, C.; Machlin, S.; Zhang, Y.; Donaldson, K.; Hanson, R. S.

1989-01-01

Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome cL, an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closely linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome cL structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed. PMID:16348074

Phenotypic characterization of ten methanol oxidation (Mox) mutant classes in methylobacterium AM1

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

Nunn, D.N.; Lidstrom, M.E.

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium strain AM1 have been characterized by complementation analysis and assigned to ten complementation groups, Mox A1,A2,A3 and B-H. We have characterized each of the mutants belonging to the ten Mox complementation groups by PMS-DCPIP dye linked methanol dehydrogenase activity, by methanol-dependent whole cell oxygen consumption, by the presence or absence of methanol dehydrogenase protein by SDS-polyacrylamide gels and Western blotting, by the absorption spectra of purified mutant methanol dehydrogenase proteins and by the presence or absence of the soluble cytochrome c proteins of Methylobacterium AM1. We propose functions for each ofmore » the genes deficient in the mutants of the ten Mox complementation groups. These functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome c/sub L/, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome c/sub L/, three gene functions responsible for the proper association of the PQQ prosthetic group with the methanol dehydrogenase apoprotein and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol. 24 refs., 5 figs., 2 tabs.« less

DE-FG02-96ER20226 FinalTechnicalReport

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

Lidstrom, M E

In the future, environmental concerns will mandate that manufacturing processes shift towards the use of renewable resources and the minimization of wastes, especially hazardous wastes. One-carbon compounds are of interest as feedstocks for synthesis of chemicals and materials, because they represent a relatively inexpensive, abundant and renewable resource. In addition, the environmentally-benign characteristics of microbial processes make them of interest as part of a long-term waste-minimization strategy for industry. The concept that methylotrophic bacteria could serve as non-polluting multistage catalysts to generate chemicals and materials using C1 compounds as feedstocks is a highly attractive one. In order to develop productionmore » strains of methylotrophs, it is necessary to understand and manipulate central methylotrophic pathways. One of the most important of these is the methanol oxidation, or Mox system. In this project, we are studying the promoters and transcriptional regulation of this 25-gene system in Methylobacterium extorquens AM1, a facultative methanol-utilizer. We have addressed the significance of a hexanucleotide sequence upstream of all mox promoters and have shown that it is required for activity of these promoters using both deletion and mutational analyses. In addition, we have identified a putative hairpin structure in the RNA leader region of the mxa promoter that is also essential for transcriptional, and have assessed the mechanism of action of this regulatory region. This work is providing the foundation for development of methylotrophic strains to convert methanol into higher value added products.« less

Organization of genes required for the oxidation of methanol to formaldehyde in three type II methylotrophs

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

Bastien, C.; Machlin, S.; Zhang, Y.

Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome c{sub L}, an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closelymore » linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome c{sub L} structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed.« less

Distribution of pink-pigmented facultative methylotrophs on leaves of vegetables.

PubMed

Mizuno, Masayuki; Yurimoto, Hiroya; Yoshida, Naoko; Iguchi, Hiroyuki; Sakai, Yasuyoshi

2012-01-01

The distribution of pink-pigmented facultative methylotrophs (PPFMs) on the leaves of various vegetables was studied. All kinds of vegetable leaves tested gave pink-pigmented colonies on agar plates containing methanol as sole carbon source. The numbers of PPFMs on the leaves, colony-forming units (CFU)/g of fresh leaves, differed among the plants, although they were planted and grown at the same farm. Commercial green perilla, Perilla frutescens viridis (Makino) Makino, gave the highest counts of PPFMs (2.0-4.1×10(7) CFU/g) of all the commercial vegetable leaves tested, amounting to 15% of total microbes on the leaves. The PPFMs isolated from seeds of two varieties of perilla, the red and green varieties, exhibited high sequence similarity as to the 16S rRNA gene to two different Methylobacterium species, M. fujisawaense DSM5686(T) and M. radiotolerans JCM2831(T) respectively, suggesting that there is specific interaction between perilla and the PPFMs.

Methylotrophic bacteria in sustainable agriculture.

PubMed

Kumar, Manish; Tomar, Rajesh Singh; Lade, Harshad; Paul, Diby

2016-07-01

Excessive use of chemical fertilizers to increase production from available land has resulted in deterioration of soil quality. To prevent further soil deterioration, the use of methylotrophic bacteria that have the ability to colonize different habitats, including soil, sediment, water, and both epiphytes and endophytes as host plants, has been suggested for sustainable agriculture. Methylotrophic bacteria are known to play a significant role in the biogeochemical cycle in soil ecosystems, ultimately fortifying plants and sustaining agriculture. Methylotrophs also improve air quality by using volatile organic compounds such as dichloromethane, formaldehyde, methanol, and formic acid. Additionally, methylotrophs are involved in phosphorous, nitrogen, and carbon cycling and can help reduce global warming. In this review, different aspects of the interaction between methylotrophs and host plants are discussed, including the role of methylotrophs in phosphorus acquisition, nitrogen fixation, phytohormone production, iron chelation, and plant growth promotion, and co-inoculation of these bacteria as biofertilizers for viable agriculture practices.

Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1

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

Nunn, D.N.; Lidstrom, M.E.

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 have been characterized by complementation analysis and assigned to 10 complementation groups, Mox A1, A2, A3, and B through H. In this study we have characterized each of the mutants belonging to the 10 Mox complementation groups for the following criteria: (i) phenazine methosulfate-dichlorophenolindophenol dye-linked methanol dehydrogenase activity; (ii) methanol-dependent whole-cell oxygen consumption; (iii) the presence or absence of methanol dehydrogenase protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; (iv) the absorption spectra of purified mutant methanol dehydrogenase proteins; and (v) the presence or absence ofmore » the soluble cytochrome c proteins of Methylobacterium sp. strain AM1, as determined by reduced-oxidized difference spectra and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this information, we have proposed functions for each of the genes deficient in the mutants of the 10 Mox complementation groups. These proposed gene functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome c/sub L/, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome c/sub L/, three gene functions responsible for the proper association of the pyrrolo-quinoline quinone prosthetic group with the methanol dehydrogenase apoprotein, and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol.« less

Plant colonization by pink-pigmented facultative methylotrophic bacteria (PPFMs).

PubMed

Omer, Zahra S; Tombolini, Riccardo; Gerhardson, Berndt

2004-03-01

Bacteria belonging to the genus Methylobacterium are characterized by being able to rely on methanol as a sole carbon and energy source and by presenting a more or less intense pink reddish pigmentation. These bacteria, also referred to as pink-pigmented methylotrophic bacteria (PPFMs), are common inhabitants of the phyllosphere and are found in many other environmental samples. Since they grow slowly they are often overlooked and their impact on phyllosphere microbial communities and on the plants harboring them is not well studied nor has their ecology been elucidated. In a survey of PPFM colonization in three different agricultural sites, PPFM populations were identified on both red clover and winter wheat, but red clover was more consistently colonized. Isolations from collected leaves showed PPFM populations to decrease from spring towards summer, but they increased again towards the end of the cropping season. Isolates from red clover readily colonized winter wheat leaves and vice versa in greenhouse experiments, but population sizes were dependent on the application procedure. Tested isolates had also good potential to colonize the rhizosphere, especially after seed inoculations. Confocal scanning laser microscopy showed gfp-tagged isolates to colonize the surface of clover leaves by forming large aggregates.

Methylotrophic bacteria on the surfaces of field-grown sunflower plants: a biogeographic perspective.

PubMed

Schauer, S; Kutschera, U

2008-03-01

Plant-associated methylobacteria of the genus Methylobacterium colonize the foliage and roots of embryophytes, living on the volatile compound methanol emitted from the cells of their host organism. In this study we analyzed these surface-dwelling pink-pigmented epiphytes in three contrasting habitats of field-grown sunflower plants (Helianthus annuus). Using the methanol-ammonium salts agar surface impression method and a polymerase chain reaction (PCR)-based assay, we document the occurrence and characterize the composition of the methylobacteria in these epiphytic habitats. In both the sun-exposed phylloplane (yellow ligulate florets; green leaves) and the moist, dark rhizoplane pink-pigmented methylobacteria were detected that are assigned to the taxa M. mesophilicum, M. extorquens, M. radiotolerans and M. sp. (un-identifiable by our methods). Considerable differences in relative species compositions were found. These data are discussed with respect to a biogeographic model of the plant surface and microbial population dynamics on leaves. In addition, methylobacteria were analyzed by microscopic techniques. We document that in sedentary colonies extracellular polymers are secreted. However, flagella, which were observed in single cells maintained in liquid cultures, are absent in these bacterial aggregates.

Toblerols: Cyclopropanol-Containing Polyketide Modulators of Antibiosis in Methylobacteria.

PubMed

Ueoka, Reiko; Bortfeld-Miller, Miriam; Morinaka, Brandon I; Vorholt, Julia A; Piel, Jörn

2018-01-22

Trans-AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular type I PKSs that generate bioactive polyketides of impressive structural diversity. In this study, we detected, in the genome of several bacteria a cryptic, architecturally unusual trans-AT PKS gene cluster which eluded automated PKS prediction. Genomic mining of one of these strains, the model methylotroph Methylobacterium extorquens AM1, revealed unique epoxide- and cyclopropanol-containing polyketides named toblerols. Relative and absolute stereochemistry were determined by NMR experiments, chemical derivatization, and the comparison of CD data between the derivatized natural product and a synthesized model compound. Biosynthetic data suggest that the cyclopropanol moiety is generated by carbon-carbon shortening of a more extended precursor. Surprisingly, a knock-out strain impaired in polyketide production showed strong inhibitory activity against other methylobacteria in contrast to the wild-type producer. The activity was inhibited by complementation with toblerols, thus suggesting that these compounds modulate an as-yet unknown methylobacterial antibiotic. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methylobacterium and Its Role in Health Care-Associated Infection

PubMed Central

Degener, John E.; van der Mei, Henny C.

2014-01-01

Methylobacterium species are a cause of health care-associated infection, including infections in immunocompromised hosts. The ability of Methylobacterium species to form biofilms and to develop resistance to high temperatures, drying, and disinfecting agents may explain the colonization of Methylobacterium in the hospital environment in, e.g., endoscopes. Due to its slow growth, it can be easily missed during microbiological surveillance of endoscope reprocessing. The purpose of this minireview is to present an overview of documented infections and cross-contaminations with Methylobacterium related to endoscopic procedures and to illustrate the health care-associated relevance of this slow-growing bacterium. PMID:24430456

Control of Wilt and Rot Pathogens of Tomato by Antagonistic Pink Pigmented Facultative Methylotrophic Delftia lacustris and Bacillus spp.

PubMed

Janahiraman, Veeranan; Anandham, Rangasamy; Kwon, Soon W; Sundaram, Subbiah; Karthik Pandi, Veeranan; Krishnamoorthy, Ramasamy; Kim, Kiyoon; Samaddar, Sandipan; Sa, Tongmin

2016-01-01

The studies on the biocontrol potential of pink pigmented facultative methylotrophic (PPFM) bacteria other than the genus Methylobacterium are scarce. In the present study, we report three facultative methylotrophic isolates; PPO-1, PPT-1, and PPB-1, respectively, identified as Delftia lacustris, Bacillus subtilis , and Bacillus cereus by 16S rRNA gene sequence analysis. Hemolytic activity was tested to investigate the potential pathogenicity of isolates to plants and humans, the results indicates that the isolates PPO-1, PPT-1, and PPB-1 are not pathogenic strains. Under in vitro conditions, D. lacustris PPO-1, B . subtilis PPT-1, and B . cereus PPB-1 showed direct antagonistic effect by inhibiting the mycelial growth of fungal pathogens; Fusarium oxysporum f. sp. lycopersici (2.15, 2.05, and 1.95 cm), Sclerotium rolfsii (2.14, 2.04, and 1.94 cm), Pythium ultimum (2.12, 2.02, and 1.92 cm), and Rhizoctonia solani (2.18, 2.08, and 1.98 cm) and also produced volatile inhibitory compounds. Under plant growth chamber condition methylotrophic bacterial isolates; D . lacustris PPO-1, B . subtilis PPT-1, and B. cereus PPB-1 significantly reduced the disease incidence of tomato. Under greenhouse condition, D . lacustris PPO-1, B . subtilis PPT-1, and B . cereus PPB-1 inoculated tomato plants, when challenged with F . oxysporum f. sp. lycopersici, S . rolfsii, P . ultimum , and R . solani , increased the pathogenesis related proteins (β-1,3-glucanase and chitinase) and defense enzymes (phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and catalase) on day 5 after inoculation. In the current study, we first report the facultative methylotrophy in pink pigmented D. lacustris, B . subtilis , and B . cereus and their antagonistic potential against fungal pathogens. Direct antagonistic and ISR effects of these isolates against fungal pathogens of tomato evidenced their possible use as a biocontrol agent.

Control of Wilt and Rot Pathogens of Tomato by Antagonistic Pink Pigmented Facultative Methylotrophic Delftia lacustris and Bacillus spp.

PubMed Central

Janahiraman, Veeranan; Anandham, Rangasamy; Kwon, Soon W.; Sundaram, Subbiah; Karthik Pandi, Veeranan; Krishnamoorthy, Ramasamy; Kim, Kiyoon; Samaddar, Sandipan; Sa, Tongmin

2016-01-01

The studies on the biocontrol potential of pink pigmented facultative methylotrophic (PPFM) bacteria other than the genus Methylobacterium are scarce. In the present study, we report three facultative methylotrophic isolates; PPO-1, PPT-1, and PPB-1, respectively, identified as Delftia lacustris, Bacillus subtilis, and Bacillus cereus by 16S rRNA gene sequence analysis. Hemolytic activity was tested to investigate the potential pathogenicity of isolates to plants and humans, the results indicates that the isolates PPO-1, PPT-1, and PPB-1 are not pathogenic strains. Under in vitro conditions, D. lacustris PPO-1, B. subtilis PPT-1, and B. cereus PPB-1 showed direct antagonistic effect by inhibiting the mycelial growth of fungal pathogens; Fusarium oxysporum f. sp. lycopersici (2.15, 2.05, and 1.95 cm), Sclerotium rolfsii (2.14, 2.04, and 1.94 cm), Pythium ultimum (2.12, 2.02, and 1.92 cm), and Rhizoctonia solani (2.18, 2.08, and 1.98 cm) and also produced volatile inhibitory compounds. Under plant growth chamber condition methylotrophic bacterial isolates; D. lacustris PPO-1, B. subtilis PPT-1, and B. cereus PPB-1 significantly reduced the disease incidence of tomato. Under greenhouse condition, D. lacustris PPO-1, B. subtilis PPT-1, and B. cereus PPB-1 inoculated tomato plants, when challenged with F. oxysporum f. sp. lycopersici, S. rolfsii, P. ultimum, and R. solani, increased the pathogenesis related proteins (β-1,3-glucanase and chitinase) and defense enzymes (phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and catalase) on day 5 after inoculation. In the current study, we first report the facultative methylotrophy in pink pigmented D. lacustris, B. subtilis, and B. cereus and their antagonistic potential against fungal pathogens. Direct antagonistic and ISR effects of these isolates against fungal pathogens of tomato evidenced their possible use as a biocontrol agent. PMID:27872630

PQQ: Biosynthetic studies in Methylobacterium AM1 and Hyphomicrobium X using specific TC labeling and NMR. [Pyrroloquinoline quinones

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

Houck, D.R.; Hanners, J.L.; Unkefer, C.J.

Using TC labeling and NMR spectroscopy we have determined biosynthetic precursors of pyrroloquinoline quinone (PQQ) in two closely related serine-type methylotrophs, Methylobacterium AM1 and Hyphomicrobium X. Analysis of the TC-labeling data revealed that PQQ is constructed from two amino acids: the portion containing N-6, C-7,8,9 and the two carboxylic acid groups, C-7' and 9', is derived-intact-from glutamate. The remaining portion is derived from tyrosine; the phenol side chain provides the six carbons of the ring containing the orthoquinone, whereas internal cyclization of the amino acid backbone forms the pyrrole-2-carboxylic acid moiety. This is analogous to the cyclization of dopaquinone tomore » form dopachrome. Dopaquinone is a product of the oxidation of tyrosine (via dopa) in reactions catalyzed by monophenol monooxygenase (EC 1.14.18.1). Starting with tyrosine and glutamate, we will discuss possible biosynthetic routes to PQQ. 29 refs., 4 figs., 2 tabs.« less

Stress Tolerance of Methylobacterium Biofilms in Bathrooms

PubMed Central

Yano, Takehisa; Kubota, Hiromi; Hanai, Junya; Hitomi, Jun; Tokuda, Hajime

2013-01-01

A comprehensive survey of microbial flora within pink biofilms in bathrooms was performed. Pink biofilms develop relatively rapidly in bathrooms, can be difficult to remove, and are quick to recur. Bacterium-sized cells were found to be predominant in 42 pink biofilms in Japan using a scanning electron microscope. Methylobacterium strains were detected from all samples in bathrooms by an isolation method. To explain this predominance, 14 biofilm samples were analyzed by fluorescence in situ hybridization. Methylobacterium was indicated to be the major genus in all biofilms. The isolated Methylobacterium survived after contact with 1.0% cleaning agents, including benzalkonium chloride for 24 h. Their tolerance did not differ under biofilm-like conditions on fiber reinforced plastics (FRP), a general material of bath tubs, floors, and walls. Also, the strains exhibited higher tolerance to desiccation than other isolated species on FRP. Some Methylobacterium survived and exhibited potential to grow after four weeks of desiccation without any nutrients. These specific characteristics could be a cause of their predominance in bathrooms, an environment with rapid flowing water, drying, low nutrients, and occasional exposure to cleaning agents. PMID:23207727

A novel growth-promoting microbe, Methylobacterium funariae sp. nov., isolated from the leaf surface of a common moss.

PubMed

Schauer, S; Kutschera, U

2011-04-01

Land plants (embryophytes) evolved in the presence of prokaryotic microbes. As a result, numerous mutually beneficial associations (symbioses) developed that can be analyzed using a variety of methods. Here we describe the isolation and characterization of a new pink-pigmented facultatively methylotrophic symbiotic bacterium of the genus Methylobacterium (laboratory strain F3.2) that was isolated from the gametophytic phylloids of the common cord moss Funaria hygrometrica Hedw. Plantlets were collected in the field and analyzed in the laboratory. Colonies of methylobacteria were obtained by the agar-impression-method. Based on its unique phenotype (the bacterial cells are characterized by fimbriae-like appendages), a comparative 16S rRNA gene (DNA) sequence analysis, and an average DNA-DNA hybridization value of 8,4 %, compared with its most closely related sister taxon, this isolate is described as a new species, Methylobacterium funariae sp. nov. (type strain F3.2). This new epiphytic bacterium inhabits the leaf surface of "primitive" land plants such as mosses and interacts with its host organism via the secretion of phytohormones (cytokinines, auxins). These external signals are perceived by the plant cells that divide and grow more rapidly than in the absence of their prokaryotic phytosymbionts. We suggest that M. funariae sp. nov. uses methanol emitted from the stomatal pores as principal carbon source for cell metabolism. However, our novel data indicate that, in this unique symbiotic plant-microbe interaction, the uptake of amino acids leached from the surface of the epidermal cells of the green host organism may be of importance as microbial carbon- and nitrogen-source.

Specific plant induced biofilm formation in Methylobacterium species.

PubMed

Rossetto, Priscilla B; Dourado, Manuella N; Quecine, Maria C; Andreote, Fernando D; Araújo, Welington L; Azevedo, João L; Pizzirani-Kleiner, Aline A

2011-07-01

Two endophytic strains of Methylobacterium spp. were used to evaluate biofilm formation on sugarcane roots and on inert wooden sticks. Results show that biofilm formation is variable and that plant surface and possibly root exudates have a role in Methylobacterium spp. host recognition, biofilm formation and successful colonization as endophytes.

Physiological, evolutionary, and genetic experiments with hopanoids in Methylobacterium: probing the function of geologically stable molecules

NASA Astrophysics Data System (ADS)

Bradley, A. S.; Muller, E.; Bringel, F.; Vuilleumier, S.; Pearson, A.; Marx, C. J.

2010-12-01

Hopanoids are geologically stable triterpenoids with a rock record extending to the Archean (1), but little information exists regarding their physiological role in modern organisms. Determining the physiological role of hopanoids is a key step in deciphering their geological and evolutionary history. To this end, we are investigating the function of hopanoids in the facultative methylotrophic bacterium Methylobacterium through a series of experiments in which we compare the behavior of wild type strains to mutants deficient in key genes associated with hopanoid biosynthesis. Mutant strains of bacteria deficient in the gene shc for squalene-hopene cyclase (SHC) lack hopanoids, but show only a subtle growth defect under pH and temperature stress in Rhodopseudomonas (2), and no growth defect in Streptomyces (3). In contrast, mutant strains of Methylobacterium deficient in SHC show a severe growth defect under usual growth conditions, with slower growth rates, alterations in cell morphology, increased sensitivity to toxic compounds, and severe flocculation during growth in liquid media. This severe phenotype offered an opportunity to investigate the function of hopanoids through an experimental evolution protocol. By serial passage through batch culture, sixteen replicate populations of the mutant strain were evolved in liquid media for approximately 120 generations. Populations evolved on each substrate show improved growth rates, approaching that of wild type strains. Current work is aimed at characterizing the physiology, and resequencing genomes of evolved isolates to determine the adaptations corresponding improved fitness. We predict that these adaptations will lead to hypotheses regarding hopanoid function. Mutations of other hopanoid-associated genes in Methylobacterium produce an altered suite of hopanoid compounds. Through mutation of hopanoid-associated genes, we have identified the first steps of hopanoid side chain biosynthesis (4). These mutant strains

Specific plant induced biofilm formation in Methylobacterium species

PubMed Central

Rossetto, Priscilla B.; Dourado, Manuella N.; Quecine, Maria C.; Andreote, Fernando D.; Araújo, Welington L.; Azevedo, João L.; Pizzirani-Kleiner, Aline A.

2011-01-01

Two endophytic strains of Methylobacterium spp. were used to evaluate biofilm formation on sugarcane roots and on inert wooden sticks. Results show that biofilm formation is variable and that plant surface and possibly root exudates have a role in Methylobacterium spp. host recognition, biofilm formation and successful colonization as endophytes. PMID:24031703

Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

PubMed Central

Stacheter, Astrid; Noll, Matthias; Lee, Charles K; Selzer, Mirjam; Glowik, Beate; Ebertsch, Linda; Mertel, Ralf; Schulz, Daria; Lampert, Niclas; Drake, Harold L; Kolb, Steffen

2013-01-01

The role of soil methylotrophs in methanol exchange with the atmosphere has been widely overlooked. Methanol can be derived from plant polymers and be consumed by soil microbial communities. In the current study, methanol-utilizing methylotrophs of 14 aerated soils were examined to resolve their comparative diversities and capacities to utilize ambient concentrations of methanol. Abundances of cultivable methylotrophs ranged from 106–108 gsoilDW−1. Methanol dissimilation was measured based on conversion of supplemented 14C-methanol, and occurred at concentrations down to 0.002 μmol methanol gsoilDW−1. Tested soils exhibited specific affinities to methanol (a0s=0.01 d−1) that were similar to those of other environments suggesting that methylotrophs with similar affinities were present. Two deep-branching alphaproteobacterial genotypes of mch responded to the addition of ambient concentrations of methanol (⩽0.6 μmol methanol gsoilDW−1) in one of these soils. Methylotroph community structures were assessed by amplicon pyrosequencing of genes of mono carbon metabolism (mxaF, mch and fae). Alphaproteobacteria-affiliated genotypes were predominant in all investigated soils, and the occurrence of novel genotypes indicated a hitherto unveiled diversity of methylotrophs. Correlations between vegetation type, soil pH and methylotroph community structure suggested that plant–methylotroph interactions were determinative for soil methylotrophs. PMID:23254514

Methylopila capsulata gen. nov., sp. nov., a novel non-pigmented aerobic facultatively methylotrophic bacterium.

PubMed

Doronina, N V; Trotsenko, Y A; Krausova, V I; Boulygina, E S; Tourova, T P

1998-10-01

A new genus, Methylopila, and one new species are described for a group of seven strains of facultatively methylotrophic bacteria with the serine pathway of C1 assimilation. These bacteria are aerobic, Gram-negative, non-spore--forming, motile, colourless rods that multiply by binary fission. Their DNA base content ranges from 66 to 70 mol % G + C. Their cellular fatty acid profile consists primarily of C18:1 omega 7 cis-vaccenic and C19:0 cyclopropane acids. The major hydroxy acid is 3-OH C14:0. The main ubiquinone is Q-10. The dominant cellular phospholipids are phosphatidylethanolamine and phosphatidylcholine. The new isolates have a low level of DNA-DNA homology (5-10%) with the type strains of the serine pathway methylobacteria belonging to the genera Methylobacterium, Aminobacter, Hyphomicrobium and Methylorhabdus. Another approach, involving 16S rRNA gene sequence analysis of strain IM1T, has shown that the new isolates represent a separate branch within the alpha-2 subclass of the Proteobacteria. The type species of the new genus is Methylopila capsulata sp. nov., with the type strain IM1T (= VKM B-1606T).

Metabolism of the aliphatic nitramine 4-nitro-2,4-diazabutanal by Methylobacterium sp. strain JS178.

PubMed

Fournier, Diane; Trott, Sandra; Hawari, Jalal; Spain, Jim

2005-08-01

The aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB; C2H5N3O3) is a ring cleavage metabolite that accumulates during the aerobic degradation of the energetic compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by various Rhodococcus spp. NDAB is also produced during the alkaline hydrolysis of either RDX or octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and during the photolysis of RDX. Traces of NDAB were observed in a soil sampled from an ammunition-manufacturing facility contaminated with both HMX and RDX, suggesting natural attenuation. In this study, we report the isolation of a soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink-pigmented facultative methylotroph affiliated with the genus Methylobacterium. The strain, named Methylobacterium sp. strain JS178, degrades NDAB as a sole nitrogen source, with concomitant growth and formation of 1 molar equivalent of nitrous oxide (N2O). Comparison of the growth yield of strain JS178 grown on NDAB, nitrite (NO2-), or ammonium (NH4+) as a nitrogen source revealed that 1 N equivalent is assimilated from each mole of NDAB, which completes the nitrogen mass balance. In radiotracer experiments, strain JS178 mineralized 1 C of the [14C]NDAB produced in situ from [14C]RDX by Rhodococcus sp. strain DN22. Studies on the regulation of NDAB degradation indicated that allantoin, an intermediate in the purine catabolic pathway and a central molecule in the storage and transport of nitrogen in plants, up-regulated the enzyme(s) involved in the degradation of the nitramine. The results reveal the potential for the sequential participation of rhodococci and methylobacteria to effect the complete degradation of RDX.

Metabolism of the Aliphatic Nitramine 4-Nitro-2,4-Diazabutanal by Methylobacterium sp. Strain JS178

PubMed Central

Fournier, Diane; Trott, Sandra; Hawari, Jalal; Spain, Jim

2005-01-01

The aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB; C2H5N3O3) is a ring cleavage metabolite that accumulates during the aerobic degradation of the energetic compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by various Rhodococcus spp. NDAB is also produced during the alkaline hydrolysis of either RDX or octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and during the photolysis of RDX. Traces of NDAB were observed in a soil sampled from an ammunition-manufacturing facility contaminated with both HMX and RDX, suggesting natural attenuation. In this study, we report the isolation of a soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink-pigmented facultative methylotroph affiliated with the genus Methylobacterium. The strain, named Methylobacterium sp. strain JS178, degrades NDAB as a sole nitrogen source, with concomitant growth and formation of 1 molar equivalent of nitrous oxide (N2O). Comparison of the growth yield of strain JS178 grown on NDAB, nitrite (NO2−), or ammonium (NH4+) as a nitrogen source revealed that 1 N equivalent is assimilated from each mole of NDAB, which completes the nitrogen mass balance. In radiotracer experiments, strain JS178 mineralized 1 C of the [14C]NDAB produced in situ from [14C]RDX by Rhodococcus sp. strain DN22. Studies on the regulation of NDAB degradation indicated that allantoin, an intermediate in the purine catabolic pathway and a central molecule in the storage and transport of nitrogen in plants, up-regulated the enzyme(s) involved in the degradation of the nitramine. The results reveal the potential for the sequential participation of rhodococci and methylobacteria to effect the complete degradation of RDX. PMID:16085803

A novel growth-promoting microbe, Methylobacterium funariae sp. nov., isolated from the leaf surface of a common moss

PubMed Central

Schauer, S

2011-01-01

Land plants (embryophytes) evolved in the presence of prokaryotic microbes. As a result, numerous mutually beneficial associations (symbioses) developed that can be analyzed using a variety of methods. Here we describe the isolation and characterization of a new pink-pigmented facultatively methylotrophic symbiotic bacterium of the genus Methylobacterium (laboratory strain F3.2) that was isolated from the gametophytic phylloids of the common cord moss Funaria hygrometrica Hedw. Plantlets were collected in the field and analyzed in the laboratory. Colonies of methylobacteria were obtained by the agar-impression-method. Based on its unique phenotype (the bacterial cells are characterized by fimbriae-like appendages), a comparative 16S rRNA gene (DNA) sequence analysis and an average DNA-DNA hybridization value of 8.4%, compared with its most closely related sister taxon, this isolate is described as a new species, Methylobacterium funariae sp. nov. (type strain F3.2). This new epiphytic bacterium inhabits the leaf surface of “primitive” land plants such as mosses and interacts with its host organism via the secretion of phytohormones (cytokinines, auxins). These external signals are perceived by the plant cells that divide and grow more rapidly than in the absence of their prokaryotic phytosymbionts. We suggest that M. funariae sp. nov. uses methanol emitted from the stomatal pores as principal carbon source for cell metabolism. However, our novel data indicate that, in this unique symbiotic plant-microbe interaction, the uptake of amino acids leached from the surface of the epidermal cells of the green host organism may be of importance as microbial carbon- and nitrogen-source. PMID:21673511

Production of ergothioneine by Methylobacterium species

PubMed Central

Alamgir, Kabir M.; Masuda, Sachiko; Fujitani, Yoshiko; Fukuda, Fumio; Tani, Akio

2015-01-01

Metabolomic analysis revealed that Methylobacterium cells accumulate a large amount of ergothioneine (EGT), which is a sulfur-containing, non-proteinogenic, antioxidative amino acid derived from histidine. EGT biosynthesis and its role in methylotrophy and physiology for plant surface-symbiotic Methylobacterium species were investigated in this study. Almost all Methylobacterium type strains can synthesize EGT. We selected one of the most productive strains (M. aquaticum strain 22A isolated from a moss), and investigated the feasibility of fermentative EGT production through optimization of the culture condition. Methanol as a carbon source served as the best substrate for production. The productivity reached up to 1000 μg/100 ml culture (1200 μg/g wet weight cells, 6.3 mg/g dry weight) in 38 days. Next, we identified the genes (egtBD) responsible for EGT synthesis, and generated a deletion mutant defective in EGT production. Compared to the wild type, the mutant showed better growth on methanol and on the plant surface as well as severe susceptibility to heat treatment and irradiation of ultraviolet (UV) and sunlight. These results suggested that EGT is not involved in methylotrophy, but is involved in their phyllospheric lifestyle fitness of the genus in natural conditions. PMID:26579093

Production of ergothioneine by Methylobacterium species.

PubMed

Alamgir, Kabir M; Masuda, Sachiko; Fujitani, Yoshiko; Fukuda, Fumio; Tani, Akio

2015-01-01

Metabolomic analysis revealed that Methylobacterium cells accumulate a large amount of ergothioneine (EGT), which is a sulfur-containing, non-proteinogenic, antioxidative amino acid derived from histidine. EGT biosynthesis and its role in methylotrophy and physiology for plant surface-symbiotic Methylobacterium species were investigated in this study. Almost all Methylobacterium type strains can synthesize EGT. We selected one of the most productive strains (M. aquaticum strain 22A isolated from a moss), and investigated the feasibility of fermentative EGT production through optimization of the culture condition. Methanol as a carbon source served as the best substrate for production. The productivity reached up to 1000 μg/100 ml culture (1200 μg/g wet weight cells, 6.3 mg/g dry weight) in 38 days. Next, we identified the genes (egtBD) responsible for EGT synthesis, and generated a deletion mutant defective in EGT production. Compared to the wild type, the mutant showed better growth on methanol and on the plant surface as well as severe susceptibility to heat treatment and irradiation of ultraviolet (UV) and sunlight. These results suggested that EGT is not involved in methylotrophy, but is involved in their phyllospheric lifestyle fitness of the genus in natural conditions.

Inhibition of Adherence of Mycobacterium avium to Plumbing Surface Biofilms of Methylobacterium spp.

PubMed Central

Ji, Pan; Pruden, Amy; Falkinham, Joseph O.

2017-01-01

Both Mycobacterium spp. and Methylobacterium spp. are opportunistic premise plumbing pathogens that are found on pipe surfaces in households. However, examination of data published in prior microbiological surveys indicates that Methylobacterium spp. and Mycobacterium spp. tend not to coexist in the same household plumbing biofilms. That evidence led us to test the hypothesis that Methylobacterium spp. in biofilms could inhibit the adherence of Mycobacterium avium. Measurements of adherence of M. avium cells to stainless steel coupons using both culture and PCR-based methods showed that the presence of Methylobacterium spp. biofilms substantially reduced M. avium adherence and vice versa. That inhibition of M. avium adherence was not reduced by UV-irradiation, cyanide/azide exposure, or autoclaving of the Methylobacterium spp. biofilms. Further, there was no evidence of the production of anti-mycobacterial compounds by biofilm-grown Methylobacterium spp. cells. The results add to understanding of the role of microbial interactions in biofilms as a driving force in the proliferation or inhibition of opportunistic pathogens in premise plumbing, and provide a potential new avenue by which M. avium exposures may be reduced for at-risk individuals. PMID:28906463

Practical Application of Methanol-Mediated Mutualistic Symbiosis between Methylobacterium Species and a Roof Greening Moss, Racomitrium japonicum

PubMed Central

Tani, Akio; Takai, Yuichiro; Suzukawa, Ikko; Akita, Motomu; Murase, Haruhiko; Kimbara, Kazuhide

2012-01-01

Bryophytes, or mosses, are considered the most maintenance-free materials for roof greening. Racomitrium species are most often used due to their high tolerance to desiccation. Because they grow slowly, a technology for forcing their growth is desired. We succeeded in the efficient production of R. japonicum in liquid culture. The structure of the microbial community is crucial to stabilize the culture. A culture-independent technique revealed that the cultures contain methylotrophic bacteria. Using yeast cells that fluoresce in the presence of methanol, methanol emission from the moss was confirmed, suggesting that it is an important carbon and energy source for the bacteria. We isolated Methylobacterium species from the liquid culture and studied their characteristics. The isolates were able to strongly promote the growth of some mosses including R. japonicum and seed plants, but the plant-microbe combination was important, since growth promotion was not uniform across species. One of the isolates, strain 22A, was cultivated with R. japonicum in liquid culture and in a field experiment, resulting in strong growth promotion. Mutualistic symbiosis can thus be utilized for industrial moss production. PMID:22479445

Detection of Intracellular Bacteria in the Buds of Scotch Pine (Pinus sylvestris L.) by In Situ Hybridization

PubMed Central

Pirttilä, Anna Maria; Laukkanen, Hanna; Pospiech, Helmut; Myllylä, Raili; Hohtola, Anja

2000-01-01

Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified as Methylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts. PMID:10877808

Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060.

PubMed

Rozova, O N; Khmelenina, V N; Trotsenko, Y A

2012-03-01

The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His(6)-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.

IDENTITY OF THE PINK-PIGMENTED METHANOL-OXIDIZING BACTERIA AS VIBRIO EXTORQUENS

PubMed Central

Stocks, Peter K.; McCleskey, C. S.

1964-01-01

Stocks, Peter K. (Louisiana State University, Baton Rouge), and C. S. McCleskey. Identity of the pink-pigmented methanol-oxidizing bacteria as Vibrio extorquens. J. Bacteriol. 88:1065–1070. 1964.—Pink-pigmented bacteria isolated from enrichment cultures of methane oxidizers were found to possess similar morphological, cultural, and physiological characteristics. All the strains utilized methanol, formate, oxalate, succinate, glycerol, and benzene as sole carbon sources; methanol, formate, and glycerol afforded best growth. Most strains utilized fructose and ribose; other carbohydrates tested were not available as carbon and energy sources. There was strain variation in the use of hexane, heptane, n-propanol, n-butanol, acetate, and propionate. Methane, ethane, n-propane, and n-butane were not utilized. Our isolates, and Pseudomonas methanica of Harrington and Kallio (not the methane-dependent P. methanica of Dworkin and Foster), Pseudomonas AM1 of Peele and Quayle, Pseudomonas PRL-W4 of Kaneda and Roxburgh, and Protaminobacter ruber den Dooren de Jong are nearly identical with Vibrio extorquens (Bassalik) Bhat and Barker, and should be considered the same species. Images PMID:14219020

IDENTITY OF THE PINK-PIGMENTED METHANOL-OXIDIZING BACTERIA AS VIBRIO EXTORQUENS.

PubMed

STOCKS, P K; MCCLESKEY, C S

1964-10-01

Stocks, Peter K. (Louisiana State University, Baton Rouge), and C. S. McCleskey. Identity of the pink-pigmented methanol-oxidizing bacteria as Vibrio extorquens. J. Bacteriol. 88:1065-1070. 1964.-Pink-pigmented bacteria isolated from enrichment cultures of methane oxidizers were found to possess similar morphological, cultural, and physiological characteristics. All the strains utilized methanol, formate, oxalate, succinate, glycerol, and benzene as sole carbon sources; methanol, formate, and glycerol afforded best growth. Most strains utilized fructose and ribose; other carbohydrates tested were not available as carbon and energy sources. There was strain variation in the use of hexane, heptane, n-propanol, n-butanol, acetate, and propionate. Methane, ethane, n-propane, and n-butane were not utilized. Our isolates, and Pseudomonas methanica of Harrington and Kallio (not the methane-dependent P. methanica of Dworkin and Foster), Pseudomonas AM1 of Peele and Quayle, Pseudomonas PRL-W4 of Kaneda and Roxburgh, and Protaminobacter ruber den Dooren de Jong are nearly identical with Vibrio extorquens (Bassalik) Bhat and Barker, and should be considered the same species.

Biodegradation of nitro-substituted explosives 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine by a phytosymbiotic Methylobacterium sp. associated with poplar tissues (Populus deltoides x nigra DN34).

PubMed

Van Aken, Benoit; Yoon, Jong Moon; Schnoor, Jerald L

2004-01-01

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides x nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-(14)C]TNT (25 mg liter(-1)) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of (14)CO(2) was recorded from [(14)C]TNT. In addition, the isolated methylotroph was shown to transform [U-(14)C]RDX (20 mg liter(-1)) and [U-(14)C]HMX (2.5 mg liter(-1)) in less than 40 days. After 55 days of incubation, 58.0% of initial [(14)C]RDX and 61.4% of initial [(14)C]HMX were mineralized into (14)CO(2). The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [(14)C]RDX and [(14)C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.

Biodegradation of Nitro-Substituted Explosives 2,4,6-Trinitrotoluene, Hexahydro-1,3,5-Trinitro-1,3,5-Triazine, and Octahydro-1,3,5,7-Tetranitro-1,3,5-Tetrazocine by a Phytosymbiotic Methylobacterium sp. Associated with Poplar Tissues (Populus deltoides × nigra DN34)

PubMed Central

Van Aken, Benoit; Yoon, Jong Moon; Schnoor, Jerald L.

2004-01-01

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides × nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-14C]TNT (25 mg liter−1) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of 14CO2 was recorded from [14C]TNT. In addition, the isolated methylotroph was shown to transform [U-14C]RDX (20 mg liter−1) and [U-14C]HMX (2.5 mg liter−1) in less than 40 days. After 55 days of incubation, 58.0% of initial [14C]RDX and 61.4% of initial [14C]HMX were mineralized into 14CO2. The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [14C]RDX and [14C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites. PMID:14711682

Phyllosphere Methylobacterium bacteria contain UVA-absorbing compounds.

PubMed

Yoshida, Shigenobu; Hiradate, Syuntaro; Koitabashi, Motoo; Kamo, Tsunashi; Tsushima, Seiya

2017-02-01

Microbes inhabiting the phyllosphere encounter harmful ultraviolet rays, and must develop adaptive strategies against this irradiation. In this study, we screened bacterial isolates originating from the phyllosphere of various plants which harbored absorbers of ultraviolet A (UVA), a wavelength range which is recognized as harmful to human skin. Of the 200 phyllosphere bacterial isolates we screened, methanol extracts from bacterial cells of seventeen isolates absorbed wavelengths in the range of 315-400nm. All of the UVA-absorbing strains belonged to Methylobacterium species based on 16S ribosomal RNA gene sequences, suggesting that cells of this bacterial genus contain specific UVA-absorbing compounds. When cells of a representative Methylobacterium strain were extracted using various solvents, UVA absorption was observed in the extracts obtained using several aqueous solvents, indicating that the UVA-absorbing compounds were highly polar. A compound was purified using solid columns and HPLC separation, and comparative analysis revealed that the absorption strength and spectrum of the compound were similar to those of the known UVA filter, avobenzone. The compound was also verified to be stable under UVA exposure for at least 480min. Based on these results, the UVA-absorbing compound harbored by Methylobacterium has potential to be used as a novel sunscreen ingredient. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of intraspecies interactions in biofilm formation by Methylobacterium species isolated from pink-pigmented household biofilms.

PubMed

Xu, Fang-Fang; Morohoshi, Tomohiro; Wang, Wen-Zhao; Yamaguchi, Yuka; Liang, Yan; Ikeda, Tsukasa

2014-01-01

Concern regarding household biofilms has grown due to their widespread existence and potential to threaten human health by serving as pathogen reservoirs. Previous studies identified Methylobacterium as one of the dominant genera found in household biofilms. In the present study, we examined the mechanisms underlying biofilm formation by using the bacterial consortium found in household pink slime. A clone library analysis revealed that Methylobacterium was the predominant genus in household pink slime. In addition, 16 out of 21 pink-pigmented bacterial isolates were assigned to the genus Methylobacterium. Although all of the Methylobacterium isolates formed low-level biofilms, the amount of the biofilms formed by Methylobacterium sp. P-1M and P-18S was significantly increased by co-culturing with other Methylobacterium strains that belonged to a specific phylogenetic group. The single-species biofilm was easily washed from the glass surface, whereas the dual-species biofilm strongly adhered after washing. A confocal laser scanning microscopy analysis showed that the dual-species biofilms were significantly thicker and tighter than the single-species biofilms.

Evaluation of Intraspecies Interactions in Biofilm Formation by Methylobacterium Species Isolated from Pink-Pigmented Household Biofilms

PubMed Central

Xu, Fang-Fang; Morohoshi, Tomohiro; Wang, Wen-Zhao; Yamaguchi, Yuka; Liang, Yan; Ikeda, Tsukasa

2014-01-01

Concern regarding household biofilms has grown due to their widespread existence and potential to threaten human health by serving as pathogen reservoirs. Previous studies identified Methylobacterium as one of the dominant genera found in household biofilms. In the present study, we examined the mechanisms underlying biofilm formation by using the bacterial consortium found in household pink slime. A clone library analysis revealed that Methylobacterium was the predominant genus in household pink slime. In addition, 16 out of 21 pink-pigmented bacterial isolates were assigned to the genus Methylobacterium. Although all of the Methylobacterium isolates formed low-level biofilms, the amount of the biofilms formed by Methylobacterium sp. P-1M and P-18S was significantly increased by co-culturing with other Methylobacterium strains that belonged to a specific phylogenetic group. The single-species biofilm was easily washed from the glass surface, whereas the dual-species biofilm strongly adhered after washing. A confocal laser scanning microscopy analysis showed that the dual-species biofilms were significantly thicker and tighter than the single-species biofilms. PMID:25381715

Cultivation-Independent Characterization of Methylobacterium Populations in the Plant Phyllosphere by Automated Ribosomal Intergenic Spacer Analysis▿ †

PubMed Central

Knief, Claudia; Frances, Lisa; Cantet, Franck; Vorholt, Julia A.

2008-01-01

Bacteria of the genus Methylobacterium are widespread in the environment, but their ecological role in ecosystems, such as the plant phyllosphere, is not very well understood. To gain better insight into the distribution of different Methylobacterium species in diverse ecosystems, a rapid and specific cultivation-independent method for detection of these organisms and analysis of their community structure is needed. Therefore, 16S rRNA gene-targeted primers specific for this genus were designed and evaluated. These primers were used in PCR in combination with a reverse primer that binds to the tRNAAla gene, which is located upstream of the 23S rRNA gene in the 16S-23S intergenic spacer (IGS). PCR products that were of different lengths were obtained due to the length heterogeneity of the IGS of different Methylobacterium species. This length variation allowed generation of fingerprints of Methylobacterium communities in environmental samples by automated ribosomal intergenic spacer analysis. The Methylobacterium communities on leaves of different plant species in a natural field were compared using this method. The new method allows rapid comparisons of Methylobacterium communities and is thus a useful tool to study Methylobacterium communities in different ecosystems. PMID:18263752

Assessing metabolic heterogeneity in genetically homogeneous populations of bacteria using SIMS

NASA Astrophysics Data System (ADS)

McClelland, H. L. O.; Fike, D. A.; Jones, C.; Bradley, A. S.

2016-12-01

Biogeochemical cycles of elements are catalyzed by microbes, and can be assessed using a wide array of geochemical techniques. As the spatial resolution of these analytical techniques improves over time, it has become apparent that spatial heterogeneity of geochemical processes may impose noise on a range of geochemical signals. This spatial heterogeneity may reflect population structure, as well as metabolic heterogeneity among cells. New analytical approaches are required to understand, at the cellular level, differences in biogeochemical cycling of elements. We are developing such approaches by applying secondary-ion mass spectrometry (SIMS) techniques to populations of model organisms. In this work we report initial results from the analysis of genetically homogeneous cultures of Methylobacterium extorquens PA1, a facultative methylotrophic Alphaproteobacterium that has been extensively studied growing on both single carbon (e.g., methanol) and multi-carbon (e.g., succinate) substrates. PA1 cultures acclimated to succinate exhibited a more pronounced lag when grown on methanol compared with populations acclimated to methanol. However neither acclimation condition results in a pronounced lag during growth on succinate. When grown on a mixture of methanol and succinate, Methylobacterium co-utilize these substrates on a population level. We investigated the degree to which this apparent coutilisation is representative of individual cells, or whether it is a superposition of distinct metabolically specialized subpopulations. To explore this metabolic heterogeneity, we have grown populations of PA1 in liquid media containing a mixture of both methanol and succinate with one or the other substrate labelled with 13C. SIMS analysis of the isotopic composition of each cell allows us to infer the substrate, or mix of substrates, used for anabolic processes in each cell, along with cell-specfic growth rates via the exponential dilution of a 15N label.

In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

DOEpatents

Young, Travis; Schultz, Peter G.

2017-08-15

The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris are also provided.

Methylobacterium platani sp. nov., isolated from a leaf of the tree Platanus orientalis.

PubMed

Kang, Yoon-Suk; Kim, Juhyun; Shin, Hyeon-Dong; Nam, Young-Do; Bae, Jin-Woo; Jeon, Che Ok; Park, Woojun

2007-12-01

A novel bacterial strain, designated PMB02(T), was isolated from a leaf of the tree Platanus orientalis. Colonies grown on TYG agar plates were circular, pink-pigmented and slow-growing, being 0.2-1.5 mm in diameter after 3 days growth. The cells of strain PMB02(T) were Gram-negative, aerobic, motile rods that possessed oxidase and catalase activities and grew at 20-30 degrees C, pH 6-8 and in media containing less than 1 % NaCl. The major respiratory quinone was identified as Q-10. A phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain PMB02(T) was related to members of the genus Methylobacterium. A comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Methylobacterium aquaticum and Methylobacterium variabile, with which it showed sequence similarities of 97.7 and 97.4 %, respectively. The values for DNA-DNA hybridization between strain PMB02(T) and M. aquaticum CCM 7218(T) and M. variabile GR3(T) were less than 32 %. On the basis of the phenotypic characterization, the phylogenetic analysis and the DNA-DNA relatedness data, strain PMB02(T) is considered to represent a novel species of the genus Methylobacterium, for which the name Methylobacterium platani sp. nov. is proposed. The type strain is PMB02(T) (=KCTC 12901(T)=JCM 14648(T)).

Investigation of XoxF methanol dehydrogenases reveals new methylotrophic bacteria in pelagic marine and freshwater ecosystems.

PubMed

Ramachandran, Arthi; Walsh, David A

2015-10-01

The diversity and distribution of methylotrophic bacteria have been investigated in the oceans and lakes using the methanol dehydrogenase mxaF gene as a functional marker. However, pelagic marine (OM43) and freshwater (LD28 and PRD01a001B) methylotrophs within the Betaproteobacteria lack mxaF, instead possessing a related xoxF4-encoded methanol dehydrogenase. Here, we developed and employed xoxF4 as a complementary functional gene marker to mxaF for studying methylotrophs in aquatic environment. Using xoxF4, we detected OM43-related and LD28-related methylotrophs in the ocean and freshwaters of North America, respectively, and showed the coexistence of these two lineages in a large estuarine system (St Lawrence Estuary). Gene expression patterns of xoxF4 supported a positive relationship between xoxF4-containing methylotroph activity and spring time productivity, suggesting phytoplankton blooms are a source of methylotrophic substrates. Further investigation of methanol dehydrogenase diversity in pelagic ecosystems using comparative metagenomics provided strong support for a widespread distribution of xoxF4 (as well as several distinct xoxF5) containing methylotrophs in marine and freshwater surface waters. In total, these results demonstrate a geographical distribution of OM43/LD28-related methylotrophs that includes marine and freshwaters and suggest that methylotrophy occurring in the water column is an important component of lake and estuary carbon cycling and biogeochemistry. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Kinetic and molecular analyses reveal isoprene degradation potential of Methylobacterium sp.

PubMed

Srivastva, Navnita; Vishwakarma, P; Bhardwaj, Y; Singh, A; Manjunath, K; Dubey, Suresh K

2017-10-01

Efforts were made to isolate and characterize bacteria capable of growing on methane and organic compounds, and to achieve the simultaneous degradation of more than one pollutant. Among the methanotrophs, species of Methylobacterium was able to catabolize a variety of hydrocarbons, including the branched-chain alkenes. Therefore, laboratory incubations experiments were carried out in batch mode to assess the potential of Methylobacterium sp. PV1 for degrading isoprene, the low-molecular-weight alkene, the most abundant non-methane volatile hydrocarbon present in the environment. Methylobacterium sp. PV1, isolated from paddy field soil, was characterized by pmoA and 16S rRNA gene sequencing and FAME analysis, and used for isoprene degradation. The kinetics of biodegradation is studied using the Michaelis-Menten model. The optimum degradation (80%) with maximum average relative degradation rate was observed at 150ppm isoprene. The degradation products were also analyzed using FTIR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies.

PubMed

Taubert, Martin; Grob, Carolina; Howat, Alexandra M; Burns, Oliver J; Chen, Yin; Neufeld, Josh D; Murrell, J Colin

2016-01-01

Methylotrophs are microorganisms ubiquitous in the environment that can metabolize one-carbon (C1) compounds as carbon and/or energy sources. The activity of these prokaryotes impacts biogeochemical cycles within their respective habitats and can determine whether these habitats act as sources or sinks of C1 compounds. Due to the high importance of C1 compounds, not only in biogeochemical cycles, but also for climatic processes, it is vital to understand the contributions of these microorganisms to carbon cycling in different environments. One of the most challenging questions when investigating methylotrophs, but also in environmental microbiology in general, is which species contribute to the environmental processes of interest, or "who does what, where and when?" Metabolic labeling with C1 compounds substituted with (13)C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of (13)C into the biomass of active methylotrophs leads to an increase in the molecular mass of their biomolecules. For DNA-based stable isotope probing (DNA-SIP), labeled and unlabeled DNA is separated by isopycnic ultracentrifugation. The ability to specifically analyze DNA of active methylotrophs from a complex background community by high-throughput sequencing techniques, i.e. targeted metagenomics, is the hallmark strength of DNA-SIP for elucidating ecosystem functioning, and a protocol is detailed in this chapter.

[Adaptation of aerobic methylobacteria to dichloromethane degradation].

PubMed

Torgonskaia, M L; Firsova, Iu E; Doronina, N V; Trotsenko, Iu A

2007-01-01

A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCI. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.

Metabolism of the Aliphatic Nitramine 4-Nitro-2,4-Diazabutanal by Methylobacterium sp. Strain JS178

DTIC Science & Technology

2005-08-01

soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink - pigmented facultative methylotroph affiliated with the...colonization by pink - pigmented facultative methylotrophic bacteria (PPFMs). FEMS Micro- bioi. Ecol. 47:319-326. 17. Schubert, K. R., and M. J. Boland... pink - pigmented , facultatively methylotrophic, bacterium isolated from pop- lar trees (Populus deltoides X nigra DN34). J. Syst. Evol. MicrobiaL 54

Complete Genome Sequence of Methylobacterium aquaticum Strain 22A, Isolated from Racomitrium japonicum Moss

PubMed Central

Ogura, Yoshitoshi; Hayashi, Tetsuya; Kimbara, Kazuhide

2015-01-01

Methylobacterium species colonize plant surfaces and utilize methanol emitted from plants. Methylobacterium aquaticum strain 22A was isolated from a hydroponic culture of a moss, Racomitrium japonicum, and is a potent plant growth promoter. The complete genome sequencing of the strain confirmed the presence of genes related to plant growth promotion and methylotrophy. PMID:25858842

Complete Genome Sequence of Methylobacterium aquaticum Strain 22A, Isolated from Racomitrium japonicum Moss.

PubMed

Tani, Akio; Ogura, Yoshitoshi; Hayashi, Tetsuya; Kimbara, Kazuhide

2015-04-09

Methylobacterium species colonize plant surfaces and utilize methanol emitted from plants. Methylobacterium aquaticum strain 22A was isolated from a hydroponic culture of a moss, Racomitrium japonicum, and is a potent plant growth promoter. The complete genome sequencing of the strain confirmed the presence of genes related to plant growth promotion and methylotrophy. Copyright © 2015 Tani et al.

Site and plant species are important determinants of the Methylobacterium community composition in the plant phyllosphere.

PubMed

Knief, Claudia; Ramette, Alban; Frances, Lisa; Alonso-Blanco, Carlos; Vorholt, Julia A

2010-06-01

The plant phyllosphere constitutes a habitat for numerous microorganisms; among them are members of the genus Methylobacterium. Owing to the ubiquitous occurrence of methylobacteria on plant leaves, they represent a suitable target for studying plant colonization patterns. The influence of the factor site, host plant species, time and the presence of other phyllosphere bacteria on Methylobacterium community composition and population size were evaluated in this study. Leaf samples were collected from Arabidopsis thaliana or Medicago truncatula plants and from the surrounding plant species at several sites. The abundance of cultivable Methylobacterium clearly correlated with the abundance of other phyllosphere bacteria, suggesting that methylobacteria constitute a considerable and rather stable fraction of the phyllosphere microbiota under varying environmental conditions. Automated ribosomal intergenic spacer analysis (ARISA) was applied to characterize the Methylobacterium community composition and showed the presence of similar communities on A. thaliana plants at most sites in 2 consecutive years of sampling. A substantial part of the observed variation in the community composition was explained by site and plant species, especially in the case of the plants collected at the Arabidopsis sites (50%). The dominating ARISA peaks that were detected on A. thaliana plants were found on other plant species grown at the same site, whereas some different peaks were detected on A. thaliana plants from other sites. This indicates that site-specific factors had a stronger impact on the Methylobacterium community composition than did plant-specific factors and that the Methylobacterium-plant association is not highly host plant species specific.

Isolation and identification of methylobacterium species from the tap water in hospitals in Japan and their antibiotic susceptibility.

PubMed

Furuhata, Katsunori; Kato, Yuko; Goto, Keiichi; Hara, Motonobu; Yoshida, Shin-ichi; Fukuyama, Masafumi

2006-01-01

Contamination of tap water by Methylobacterium species has become a serious concern in hospitals. This study was planned to examine the distribution of Methylobacterium species inhabiting tap water used in Japanese hospitals and antibiotic sensitivity of the isolates in 2004. Species identification of 58 isolates was performed based on the homology of a partial sequence of 16S rDNA. The dominant Methylobacterium species in hospital water were M. aquaticum and M. fujisawaense. To examine the biochemical properties of these isolates, a carbon source utilization was tested using an API50CH kit. The phenotypic character varied widely, and was not necessarily consistent with the results of phylogenic analysis based on the partial 16S rDNA sequence, suggesting that the biochemical properties are not suitable for identification of Methylobacterium species. The isolates were also subjected to antibiotic sensitivity tests. They were resistant to 8 antibiotics, but highly sensitive to imipenem (MIC90 = 1 microg/ml) and tetracycline (MIC90 = 8 microg/ml). These findings concerning the isolates revealed the presence of Methylobacterium species with resistance to multiple antibiotics in hospital tap water.

Complete Genome Sequences of Six Strains of the Genus Methylobacterium

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

Marx, Christopher J; Bringel, Francoise O.; Christoserdova, Ludmila

The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.

Complete genome sequences of six strains of the genus methylobacterium

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

Marx, Christopher J; Bringel, Francoise O.; Christoserdova, Ludmila

The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.

Isolation and genetic characterization of Aurantimonas and Methylobacterium strains from stems of hypernodulated soybeans.

PubMed

Anda, Mizue; Ikeda, Seishi; Eda, Shima; Okubo, Takashi; Sato, Shusei; Tabata, Satoshi; Mitsui, Hisayuki; Minamisawa, Kiwamu

2011-01-01

The aims of this study were to isolate Aurantimonas and Methylobacterium strains that responded to soybean nodulation phenotypes and nitrogen fertilization rates in a previous culture-independent analysis (Ikeda et al. ISME J. 4:315-326, 2010). Two strategies were adopted for isolation from enriched bacterial cells prepared from stems of field-grown, hypernodulated soybeans: PCR-assisted isolation for Aurantimonas and selective cultivation for Methylobacterium. Thirteen of 768 isolates cultivated on Nutrient Agar medium were identified as Aurantimonas by colony PCR specific for Aurantimonas and 16S rRNA gene sequencing. Meanwhile, among 187 isolates on methanol-containing agar media, 126 were identified by 16S rRNA gene sequences as Methylobacterium. A clustering analysis (>99% identity) of the 16S rRNA gene sequences for the combined datasets of the present and previous studies revealed 4 and 8 operational taxonomic units (OTUs) for Aurantimonas and Methylobacterium, respectively, and showed the successful isolation of target bacteria for these two groups. ERIC- and BOX-PCR showed the genomic uniformity of the target isolates. In addition, phylogenetic analyses of Aurantimonas revealed a phyllosphere-specific cluster in the genus. The isolates obtained in the present study will be useful for revealing unknown legume-microbe interactions in relation to the autoregulation of nodulation.

Methylobacterium spp. as an indicator for the presence or absence of Mycobacterium spp.

PubMed

Falkinham, Joseph O; Williams, Myra D; Kwait, Rebecca; Lande, Leah

2016-06-01

A published survey of bacteria in showerhead biofilm samples revealed that Methylobacterium spp. and Mycobacterium spp. seldom coexisted in biofilms. To confirm that information, biofilm samples were collected from household plumbing of Mycobacterium avium patients and Methylobacterium spp. and M. avium numbers were measured by direct colony counts. The results demonstrated that if Methylobacterium spp. were present, Mycobacterium spp. were absent, and the opposite. The data demonstrate that microbial populations in biofilms can influence the presence or absence of opportunistic premise plumbing pathogens and, thereby, increase the range of strategies to reduce exposure to waterborne pathogens. Finally, by assessing for the visual presence of methylobacteria as pink pigmentation on showers and shower curtains, homeowners and managers of hospitals and other buildings can quickly determine whether a premise plumbing biofilm sample has mycobacteria with a high degree of assurance. Copyright © 2016 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

Complete Genome Sequences of Six Strains of the Genus Methylobacterium

PubMed Central

Bringel, Françoise; Chistoserdova, Ludmila; Moulin, Lionel; Farhan Ul Haque, Muhammad; Fleischman, Darrell E.; Gruffaz, Christelle; Jourand, Philippe; Knief, Claudia; Lee, Ming-Chun; Muller, Emilie E. L.; Nadalig, Thierry; Peyraud, Rémi; Roselli, Sandro; Russ, Lina; Goodwin, Lynne A.; Ivanova, Natalia; Kyrpides, Nikos; Lajus, Aurélie; Land, Miriam L.; Médigue, Claudine; Mikhailova, Natalia; Nolan, Matt; Woyke, Tanja; Stolyar, Sergey; Vorholt, Julia A.

2012-01-01

The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints. PMID:22887658

Regulation of root development in Arabidopsis thaliana by phytohormone-secreting epiphytic methylobacteria.

PubMed

Klikno, Jana; Kutschera, Ulrich

2017-09-01

In numerous experimental studies, seedlings of the model dicot Arabidopsis thaliana have been raised on sterile mineral salt agar. However, under natural conditions, no plant has ever grown in an environment without bacteria. Here, we document that germ-free (gnotobiotic) seedlings, raised on mineral salt agar without sucrose, develop very short root hairs. In the presence of a soil extract that contains naturally occurring microbes, root hair elongation is promoted; this effect can be mimicked by the addition of methylobacteria to germ-free seedlings. Using five different bacterial species (Methylobacterium mesophilicum, Methylobacterium extorquens, Methylobacterium oryzae, Methylobacterium podarium, and Methylobacterium radiotolerans), we show that, over 9 days of seedling development in a light-dark cycle, root development (hair elongation, length of the primary root, branching patterns) is regulated by these epiphytic microbes that occur in the rhizosphere of field-grown plants. In a sterile liquid culture test system, auxin (IAA) inhibited root growth with little effect on hair elongation and significantly stimulated hypocotyl enlargement. Cytokinins (trans-zeatin, kinetin) and ethylene (application of the precursor ACC) likewise exerted an inhibitory effect on root growth but, in contrast to IAA, drastically stimulated root hair elongation. Methylobacteria are phytosymbionts that produce/secrete cytokinins. We conclude that, under real-world conditions (soil), the provision of these phytohormones by methylobacteria (and other epiphytic microbes) regulates root development during seedling establishment.

Metabolic footprint of epiphytic bacteria on Arabidopsis thaliana leaves

PubMed Central

Ryffel, Florian; Helfrich, Eric JN; Kiefer, Patrick; Peyriga, Lindsay; Portais, Jean-Charles; Piel, Jörn; Vorholt, Julia A

2016-01-01

The phyllosphere, which is defined as the parts of terrestrial plants above the ground, is a large habitat for different microorganisms that show a high extent of adaption to their environment. A number of hypotheses were generated by culture-independent functional genomics studies to explain the competitiveness of specialized bacteria in the phyllosphere. In contrast, in situ data at the metabolome level as a function of bacterial colonization are lacking. Here, we aimed to obtain new insights into the metabolic interplay between host and epiphytes upon colonization of Arabidopsis thaliana leaves in a controlled laboratory setting using environmental metabolomics approaches. Quantitative nuclear magnetic resonance (NMR) and imaging high-resolution mass spectrometry (IMS) methods were used to identify Arabidopsis leaf surface compounds and their possible involvement in the epiphytic lifestyle by relative changes in compound pools. The dominant carbohydrates on the leaf surfaces were sucrose, fructose and glucose. These sugars were significantly and specifically altered after epiphytic leaf colonization by the organoheterotroph Sphingomonas melonis or the phytopathogen Pseudomonas syringae pv. tomato, but only to a minor extent by the methylotroph Methylobacterium extorquens. In addition to carbohydrates, IMS revealed surprising alterations in arginine metabolism and phytoalexin biosynthesis that were dependent on the presence of bacteria, which might reflect the consequences of bacterial activity and the recognition of not only pathogens but also commensals by the plant. These results highlight the power of environmental metabolomics to aid in elucidating the molecular basis underlying plant–epiphyte interactions in situ. PMID:26305156

A new cofactor in prokaryotic enzyme: Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

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

McIntire, W.S.; Wemmer, D.E.; Chistoserdov, A.

Methylamine dehydrogenase (MADH), an {alpha}{sub 2}{beta}{sub 2} enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small {beta} subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting {sup 1}H and {sup 13}C nuclear magnetic resonance, and mass spectralmore » data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, of pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.« less

[Comparative characteristics of biosynthesis of polyhydroxybutyrate from methanol by Methylobacteria extorquens G10 and Methyloligella halotolerans C2].

PubMed

Poroshina, M N; Doronina, N V; Ezhov, V A; Trotsenko, Iu A

2014-01-01

The biosynthesis of polyhydroxybutyrate by Methylobacteria extorquens G10 and Methyloligella halotolerans C2 via the serine pathway of C1 metabolism was comparatively studied. Nitrogen limitation stimulated synthesis of the biopolymer in both cultures. It was shown that, despite the similarity of the pathways of methanol metabolism and those of polyhydroxybutyrate biosynthesis, the methylobacteria synthesized polymers of different molecular weights. In the case of M. extorquens G10, an increase in the content of the residual nitrogen in the culture medium was found to result in a reduction of the molecular weight of the polymer from 250 to 85 kDa, whereas M. halotolerans C2 synthesized a polymer of high molecular weight (approximately 3000 kDa) regardless of the residual content of the nitrogen source. It was established that the examined methylobacteria can utilize not only pure methanol but also a crude one, a feature that made it possible to significantly reduce the cost of the resulting polyhydroxybutyrate.

Methylotrophic methanogenesis discovered in the archaeal phylum Verstraetearchaeota

DOE PAGES

Vanwonterghem, Inka; Evans, Paul N.; Parks, Donovan H.; ...

2016-10-03

Methanogenesis is the primary biogenic source of methane in the atmosphere and a key contributor to climate change. The long-standing dogma that methanogenesis originated within the Euryarchaeota was recently challenged by the discovery of putative methane-metabolizing genes in members of the Bathyarchaeota, suggesting that methanogenesis may be more phylogenetically widespread than currently appreciated. Here, we present the discovery of divergent methyl-coenzyme M reductase genes in population genomes recovered from anoxic environments with high methane flux that belong to a new archaeal phylum, the Verstraetearchaeota. These archaea encode the genes required for methylotrophic methanogenesis, and may conserve energy using a mechanismmore » similar to that proposed for the obligate H 2-dependent methylotrophic Methanomassiliicoccales and recently described Candidatus ‘Methanofastidiosa’. Our findings indicate that we are only beginning to understand methanogen diversity and support an ancient origin for methane metabolism in the Archaea, which is changing our understanding of the global carbon cycle.« less

Methylotrophic methanogenesis discovered in the archaeal phylum Verstraetearchaeota

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

Vanwonterghem, Inka; Evans, Paul N.; Parks, Donovan H.

Methanogenesis is the primary biogenic source of methane in the atmosphere and a key contributor to climate change. The long-standing dogma that methanogenesis originated within the Euryarchaeota was recently challenged by the discovery of putative methane-metabolizing genes in members of the Bathyarchaeota, suggesting that methanogenesis may be more phylogenetically widespread than currently appreciated. Here, we present the discovery of divergent methyl-coenzyme M reductase genes in population genomes recovered from anoxic environments with high methane flux that belong to a new archaeal phylum, the Verstraetearchaeota. These archaea encode the genes required for methylotrophic methanogenesis, and may conserve energy using a mechanismmore » similar to that proposed for the obligate H 2-dependent methylotrophic Methanomassiliicoccales and recently described Candidatus ‘Methanofastidiosa’. Our findings indicate that we are only beginning to understand methanogen diversity and support an ancient origin for methane metabolism in the Archaea, which is changing our understanding of the global carbon cycle.« less

[Aerobic methylobacteria as the basis for a biosensor for dichloromethane detection].

PubMed

Plekhanova, Iu V; Firsova, Iu E; Doronina, N V; Trotsenko, Iu A; Reshetilov, A N

2013-01-01

Cells of dichloromethane (DChM) bacteria-destructors were immobilized by sorption on different types of membranes, which were fixed on the measuring surface of a pH-sensitive field transistor. The presence of DChM in the medium (0.6-8.8 mM) led to a change in the transistor's output signal, which was determined by the appearance of H+ ions in the medium due to DChM utilization by methylobateria. Among four strains of methylobacteria--Methylobacterium dichloromethanicum DM4, Methylobacterium extorquens DM 17, Methylopila helvetica DM6, and Ancylobacter dichloromethanicus DM 16--the highest and most stable activity toward DChM degradation was observed in the strain M. dichloromethanicum DM4. Among 11 types of membranes for cell immobilization, Millipore nitrocellulose membranes and chromatographic fiber paper GF/A, which allow one to obtain stable biosensor signals for 2 weeks without a bioreceptor change, were chosen as optimal carriers.

[Effect of DNA-damaging agents on the aerobic methylobacteria capable and incapable of utilizing dichloromethane].

PubMed

Firsova, Iu E; Torgonskaia, M L; Doronina, N V; Trotsenko, Iu A

2005-01-01

Methylobacterium dichloromethanicum DM4, a degrader of dichloromethane (DCM), was more tolerant to the effect of H2O2 and UV irradiation than Methylobacterium extorquens AM1, which does not consume DCM. Addition of CH2Cl2 to methylobacteria with active serine, ribulose monophosphate, and ribulose bisphosphate pathways of C1 metabolism, grown on methanol, resulted in a 1.1- to 2.5-fold increase in the incorporation of [alpha-32P]dATP into DNA Klenow fragment (exo-). As DCM dehalogenase was not induced in this process, the increase in total lengths of DNA gaps resulted from the action of DCM rather than S-chloromethylglutathione (intermediate of primary dehalogenation). The degree of DNA damage in the presence of CH2Cl2 was lower in DCM degraders than methylobacteria incapable of degrading this pollutant. This suggests that DCM degraders possess a more efficient mechanism of DNA repair.

Large carbon isotope fractionation associated with oxidation of methyl halides by methylotrophic bacteria

USGS Publications Warehouse

Miller, L.G.; Kalin, Robert M.; McCauley, S.E.; Hamilton, John T.G.; Harper, D.B.; Millet, D.B.; Oremland, R.S.; Goldstein, Allen H.

2001-01-01

The largest biological fractionations of stable carbon isotopes observed in nature occur during production of methane by methanogenic archaea. These fractionations result in substantial (as much as ???70???) shifts in ??13C relative to the initial substrate. We now report that a stable carbon isotopic fractionation of comparable magnitude (up to 70???) occurs during oxidation of methyl halides by methylotrophic bacteria. We have demonstrated biological fractionation with whole Cells of three methylotrophs (strain IMB-1, strain CC495, and strain MB2) and, to a lesser extent, with the purified cobalamin-dependent methyltransferase enzyme obtained from strain CC495. Thus, the genetic similarities recently reported between methylotrophs, and methanogens with respect to their pathways for C1-unit metabolism are also reflected in the carbon isotopic fractionations achieved by these organisms. We found that only part of the observed fractionation of carbon isotopes could be accounted for by the activity of the corrinoid methyltransferase enzyme, suggesting fractionation by enzymes further along the degradation pathway. These observations are of potential biogeochemical significance in the application of stable carbon isotope ratios to constrain the tropospheric budgets for the ozone-depleting halocarbons, methyl bromide and methyl chloride.

Draft Genome Sequence of Methylobacterium sp. ME121, Isolated from Soil as a Mixed Single Colony with Kaistia sp. 32K

PubMed Central

Fujinami, Shun; Takeda-Yano, Kiyoko; Onodera, Takefumi; Satoh, Katsuya; Shimizu, Tetsu; Wakabayashi, Yuu; Narumi, Issay; Nakamura, Akira

2015-01-01

Methylobacterium sp. ME121 was isolated from soil as a mixed single colony with Kaistia sp. 32K, and its growth was enhanced by coculture. Here, we report the draft genome sequence of Methylobacterium sp. ME121, which may contribute to the study of the molecular mechanisms underlying this phenomenon. PMID:26337893

Methylobacterium Species Promoting Rice and Barley Growth and Interaction Specificity Revealed with Whole-Cell Matrix-Assisted Laser Desorption / Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) Analysis

PubMed Central

Tani, Akio; Sahin, Nurettin; Fujitani, Yoshiko; Kato, Akiko; Sato, Kazuhiro; Kimbara, Kazuhide

2015-01-01

Methylobacterium species frequently inhabit plant surfaces and are able to utilize the methanol emitted from plants as carbon and energy sources. As some of the Methylobacterium species are known to promote plant growth, significant attention has been paid to the mechanism of growth promotion and the specificity of plant–microbe interactions. By screening our Methylobacterium isolate collection for the high growth promotion effect in vitro, we selected some candidates for field and pot growth tests for rice and barley, respectively. We found that inoculation resulted in better ripening of rice seeds, and increased the size of barley grains but not the total yield. In addition, using whole-cell matrix-assister laser desorption/ionization- time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, we identified and classified Methylobacterium isolates from Methylobacterium-inoculated rice plants. The inoculated species could not be recovered from the rice plants, and in some cases, the Methylobacterium community structure was affected by the inoculation, but not with predomination of the inoculated species. The isolates from non-inoculated barley of various cultivars grown in the same field fell into just two species. These results suggest that there is a strong selection pressure at the species level of Methylobacterium residing on a given plant species, and that selection of appropriate species that can persist on the plant is important to achieve growth promotion. PMID:26053875

Relative importance of methylotrophic methanogenesis in sediments of the Western Mediterranean Sea

NASA Astrophysics Data System (ADS)

Zhuang, Guang-Chao; Heuer, Verena B.; Lazar, Cassandre S.; Goldhammer, Tobias; Wendt, Jenny; Samarkin, Vladimir A.; Elvert, Marcus; Teske, Andreas P.; Joye, Samantha B.; Hinrichs, Kai-Uwe

2018-03-01

Microbial production of methane is an important terminal metabolic process during organic matter degradation in marine sediments. It is generally acknowledged that hydrogenotrophic and acetoclastic methanogenesis constitute the dominant pathways of methane production; the importance of methanogenesis from methylated compounds remains poorly understood. We conducted various biogeochemical and molecular genetic analyses to characterize substrate availability, rates of methanogenesis, and methanogen community composition, and further evaluated the contribution of different substrates and pathways for methane production in deltaic surface and subsurface sediments of the Western Mediterranean Sea. Major substrates representing three methanogenic pathways, including H2, acetate, and methanol, trimethylamine (TMA), and dimethylsulfide (DMS), were detected in the pore waters and sediments, and exhibited variability over depth and between sites. In accompanying incubation experiments, methanogenesis rates from various 14C labeled substrates varied as well, suggesting that environmental factors, such as sulfate concentration and organic matter quality, could significantly influence the relative importance of individual pathway. In particular, methylotrophic and hydrogenotrophic methanogenesis contributed to the presence of micromolar methane concentrations in the sulfate reduction zone, with methanogenesis from methanol accounting for up to 98% of the total methane production in the topmost surface sediment. In the sulfate-depleted zone, hydrogenotrophic methanogenesis was the dominant methanogenic pathway (67-98%), and enhanced methane production from acetate was observed in organic-rich sediment (up to 31%). Methyl coenzyme M reductase gene (mcrA) analysis revealed that the composition of methanogenic communities was generally consistent with the distribution of methanogenic activity from different substrates. This study provides the first quantitative assessment of

A Methylobacterium-like organism from algal crusts covering silicone rubber electric insulators in Africa.

PubMed

Zarnowski, R; Felske, A; Ellis, R J; Geuns, J M C; Pietr, S J

2002-01-01

The primary goals of this study were to isolate, identify and characterize culturable bacteria living in a close association with microalgae within green crusts covering silicone rubber electric insulators in Tanzania. Twenty-four bacterial colonies were isolated from an Apatococcus crust. Characterization by statistical analyses of total cellular protein profiles demonstrated that they were highly similar to one another. Final identification was achieved using 16S rDNA sequencing and fatty acid methyl ester profiling. These analyses revealed the presence of microbes with high similarity to Methylobacterium radiotolerans. The selected isolate, A1, displayed strong inhibitory activity against Rhizoctonia solani and was found to be resistant to relatively high concentrations of zinc in the growth medium. This study revealed the presence of M. radiotolerans bacteria in a novel environment--within algal crusts formed on electrical insulators in Africa. Moreover, this bacterium was found to be a predominant culturable species within those complex algal-microbial associations. The isolate also shared some traits of biotechnological importance with other members of the Methylobacterium genus. The data presented provide a valuable contribution concerning the formation and function of associations between green microalgae and bacteria. This study also provides some information about the utility of bacteria from the genus Methylobacterium in biotechnological applications, such as biocontrol of rhizoctoniosis and bioremediation of heavy metal-contaminated soils.

Genetic and physical analyses of Methylobacterium organophilum XX genes encoding methanol oxidation

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

Machlin, S.M.; Tam, P.E.; Bastien, C.A.

When allyl alcohol was used as a suicide substrate, spontaneous mutants and UV light- and nitrous acid-generated mutants of Methylobacterium organophilum XX were selected which grew on methylamine but not on methanol. There was no detectable methanol dehydrogenase (MDH) activity in crude extracts of these mutants, yet Western blots revealed that some mutants still produced MDH protein. Complementation of 50 mutants by a cosmid gene bank of M. organophilum XX demonstrated that three major regions of the genome, each of which was separated by a minimum of 40 kilobases, were required for expression of active MDH. By subcloning and Tn5more » insertion mutagenesis of subcloned fragments, at least 11 genes clustered within these three regions were subsequently identified. The identity of the MDH structural gene, which was initially determined by hybridization to the structural gene of Methylobacterium sp. strain AM1, was confirmed by Western blot analysis of an MDH-..beta..-galactosidase fusion protein.« less

Production of amino acids using auxotrophic mutants of methylotrophic bacillus

DOEpatents

Hanson, Richard S.; Flickinger, Michael C.; Schendel, Frederick J.; Guettler, Michael V.

2001-07-17

A method of producing amino acids by culturing an amino acid auxotroph of a biologically pure strain of a type I methylotrophic bacterium of the genus Bacillus which exhibits sustained growth at 50.degree. C. using methanol as a carbon and energy source and requiring vitamin B.sub.12 and biotin is provided.

Electro-biocatalytic production of formate from carbon dioxide using an oxygen-stable whole cell biocatalyst.

PubMed

Hwang, Hyojin; Yeon, Young Joo; Lee, Sumi; Choe, Hyunjun; Jang, Min Gee; Cho, Dae Haeng; Park, Sehkyu; Kim, Yong Hwan

2015-06-01

The use of biocatalysts to convert CO2 into useful chemicals is a promising alternative to chemical conversion. In this study, the electro-biocatalytic conversion of CO2 to formate was attempted with a whole cell biocatalyst. Eight species of Methylobacteria were tested for CO2 reduction, and one of them, Methylobacterium extorquens AM1, exhibited an exceptionally higher capability to synthesize formate from CO2 by supplying electrons with electrodes, which produced formate concentrations of up to 60mM. The oxygen stability of the biocatalyst was investigated, and the results indicated that the whole cell catalyst still exhibited CO2 reduction activity even after being exposed to oxygen gas. From the results, we could demonstrate the electro-biocatalytic conversion of CO2 to formate using an obligate aerobe, M. extorquens AM1, as a whole cell biocatalyst without providing extra cofactors or hydrogen gas. This electro-biocatalytic process suggests a promising approach toward feasible way of CO2 conversion to formate. Copyright © 2015 Elsevier Ltd. All rights reserved.

Multiple evidence for methylotrophic methanogenesis as the dominant methanogenic pathway in hypersaline sediments from the Orca Basin, Gulf of Mexico

NASA Astrophysics Data System (ADS)

Zhuang, Guang-Chao; Elling, Felix J.; Nigro, Lisa M.; Samarkin, Vladimir; Joye, Samantha B.; Teske, Andreas; Hinrichs, Kai-Uwe

2016-08-01

Among the most extreme habitats on Earth, dark, deep, anoxic brines host unique microbial ecosystems that remain largely unexplored. As the terminal step of anaerobic degradation of organic matter, methanogenesis is a potentially significant but poorly constrained process in deep-sea hypersaline environments. We combined biogeochemical and phylogenetic analyses with incubation experiments to unravel the origin of methane in the hypersaline sediments of Orca Basin in the northern Gulf of Mexico. Substantial concentrations of methane, up to 3.4 mM, coexisted with high concentrations of sulfate from 16 to 43 mM in two sediment cores retrieved from the northern and southern parts of Orca Basin. The strong depletion of 13C in methane (-77‰ to -89‰) points towards a biological source. While low concentrations of competitive substrates limited the significance of hydrogenotrophic and acetoclastic methanogenesis, the presence of non-competitive methylated substrates (methanol, trimethylamine, dimethyl sulfide, dimethylsulfoniopropionate) supported the potential for methane generation through methylotrophic methanogenesis. Thermodynamic calculations demonstrated that hydrogenotrophic and acetoclastic methanogenesis were unlikely to occur under in situ conditions, while methylotrophic methanogenesis from a variety of substrates was highly favorable. Likewise, carbon isotope relationships between methylated substrates and methane suggested methylotrophic methanogenesis was the major source of methane. Stable and radio-isotope tracer experiments with 13C-labeled bicarbonate, acetate and methanol and 14C-labeled methylamine indicated that methylotrophic methanogenesis was the predominant methanogenic pathway. Based on 16S rRNA gene sequences, halophilic methylotrophic methanogens related to the genus Methanohalophilus dominated the benthic archaeal community in the northern basin and also occurred in the southern basin. High abundances of methanogen lipid biomarkers such as

Methylotrophic bacteria symbiosis with the higher plants as means of minimization of the lower hydrocarbons concentration during artificial ecosystem gas exchange

NASA Astrophysics Data System (ADS)

Berkovich, Yuliy; Smolyanina, Svetlana; Moukhamedieva, Lana; Mardanov, Robert; Doronina, Nina; Ivanova, Ekaterina

Plant growth unit should be included in the LSS for the space vehicles for vitamin greens supply and psychological support of cosmonauts during long-term missions. The lower hydrocarbons such as methane, methanol, methylated sulfuric compounds and methylated amines, ethylene and so on, are the natural products of human and plant metabolism and usually considered as the air pollutions. It is shown, that one way to decrease the lower hydrocarbons concentration in the artificial ecosystems could be colonization of the plants by methylotrophic bacteria. The aerobic methylotrophic bacteria possess unique ability to use methane and its oxidized or replaced derivatives without food damage and human, animals or plants infection. We have found that methylotrophic bacteria are the phyto-symbiotic bacteria: they stimulate growth and development of the colonized plants because of synthesizing cytokinins and auxins, and vitamin B12.Two collection strains of the obligate methylotrophic bacteria - Methylovorus mays C and Methylomonas metanica S - were chosen because of their high activity to assimilate the lower hydrocarbons due to functioning of methanoldehydrogenase, methanmonooxigenase and ribulose monophosphate cycle enzymes system.Colonization of the leaf cabbage Brassica chinensis L. by these strains led to approximately 30 % reduce of methanol and methane concentration in the air inside phytotron. Experimental estimations of the influence of methylotrophic bacteria on leafy greens growth and development are obtained.

Quantitative metabolomics of the thermophilic methylotroph Bacillus methanolicus.

PubMed

Carnicer, Marc; Vieira, Gilles; Brautaset, Trygve; Portais, Jean-Charles; Heux, Stephanie

2016-06-01

The gram-positive bacterium Bacillus methanolicus MGA3 is a promising candidate for methanol-based biotechnologies. Accurate determination of intracellular metabolites is crucial for engineering this bacteria into an efficient microbial cell factory. Due to the diversity of chemical and cell properties, an experimental protocol validated on B. methanolicus is needed. Here a systematic evaluation of different techniques for establishing a reliable basis for metabolome investigations is presented. Metabolome analysis was focused on metabolites closely linked with B. methanolicus central methanol metabolism. As an alternative to cold solvent based procedures, a solvent-free quenching strategy using stainless steel beads cooled to -20 °C was assessed. The precision, the consistency of the measurements, and the extent of metabolite leakage from quenched cells were evaluated in procedures with and without cell separation. The most accurate and reliable performance was provided by the method without cell separation, as significant metabolite leakage occurred in the procedures based on fast filtration. As a biological test case, the best protocol was used to assess the metabolome of B. methanolicus grown in chemostat on methanol at two different growth rates and its validity was demonstrated. The presented protocol is a first and helpful step towards developing reliable metabolomics data for thermophilic methylotroph B. methanolicus. This will definitely help for designing an efficient methylotrophic cell factory.

Genome sequence of the dark pink pigmented Listia bainesii microsymbiont Methylobacterium sp. WSM2598

PubMed Central

2014-01-01

Strains of a pink-pigmented Methylobacterium sp. are effective nitrogen- (N2) fixing microsymbionts of species of the African crotalarioid genus Listia. Strain WSM2598 is an aerobic, motile, Gram-negative, non-spore-forming rod isolated in 2002 from a Listia bainesii root nodule collected at Estcourt Research Station in South Africa. Here we describe the features of Methylobacterium sp. WSM2598, together with information and annotation of a high-quality draft genome sequence. The 7,669,765 bp draft genome is arranged in 5 scaffolds of 83 contigs, contains 7,236 protein-coding genes and 18 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 G enomic E ncyclopedia for B acteria and A rchaea- R oot N odule B acteria (GEBA-RNB) project. PMID:25780498

Genome sequence of the dark pink pigmented Listia bainesii microsymbiont Methylobacterium sp. WSM2598.

PubMed

Ardley, Julie; Tian, Rui; Howieson, John; Yates, Ron; Bräu, Lambert; Han, James; Lobos, Elizabeth; Huntemann, Marcel; Chen, Amy; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Pati, Amrita; Goodwin, Lynne; Woyke, Tanja; Kyrpides, Nikos; Reeve, Wayne

2014-01-01

Strains of a pink-pigmented Methylobacterium sp. are effective nitrogen- (N2) fixing microsymbionts of species of the African crotalarioid genus Listia. Strain WSM2598 is an aerobic, motile, Gram-negative, non-spore-forming rod isolated in 2002 from a Listia bainesii root nodule collected at Estcourt Research Station in South Africa. Here we describe the features of Methylobacterium sp. WSM2598, together with information and annotation of a high-quality draft genome sequence. The 7,669,765 bp draft genome is arranged in 5 scaffolds of 83 contigs, contains 7,236 protein-coding genes and 18 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 G enomic E ncyclopedia for B acteria and A rchaea- R oot N odule B acteria (GEBA-RNB) project.

Isolation and characterization of two new methanesulfonic acid-degrading bacterial isolates from a Portuguese soil sample.

PubMed

De Marco, P; Murrell, J C; Bordalo, A A; Moradas-Ferreira, P

2000-02-01

Two novel bacterial strains that can utilize methanesulfonic acid as a source of carbon and energy were isolated from a soil sample collected in northern Portugal. Morphological, physiological, biochemical and molecular biological characterization of the two isolates indicate that strain P1 is a pink-pigmented facultative methylotroph belonging to the genus Methylobacterium, while strain P2 is a restricted methylotroph belonging to the genus Hyphomicrobium. Both strains are strictly aerobic, degrade methanesulfonate, and release small quantities of sulfite into the medium. Growth on methanesulfonate induces a specific polypeptide profile in each strain. This, together with the positive hybridization to a DNA probe that carries the msm genes of Methylosulfonomonas methylovora strain M2, strongly endorses the contention that a methanesulfonic acid monooxygenase related to that found in the previously known methanesulfonate-utilizing bacteria is present in strains P1 and P2. The isolation of bacteria containing conserved msm genes from diverse environments and geographical locations supports the hypothesis that a common enzyme may be globally responsible for the oxidation of methanesulfonate by natural methylotrophic communities.

Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria.

PubMed

Yim, W J; Kim, K Y; Lee, Y W; Sundaram, S P; Lee, Y; Sa, T M

2014-07-15

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato. Copyright © 2014 Elsevier GmbH. All rights reserved.

[Methanotrophs and methylobacteria are found in woody plant tissues within a winter period].

PubMed

Doronina, N V; Ivanova, E G; Suzina, N F; Trotsenko, Iu A

2004-01-01

Samples of tree seeds, buds, and needles collected within a winter period at ambient temperatures from -11 to -17 degrees C were analyzed for the presence of methylotrophic microflora. Thin sections of blue spruce needles were found to contain bacteria morphologically close to pink-pigmented methylobacteria. The methylobacteria that were isolated in pure cultures from samples of linden seeds and buds, pine and blue spruce needles, as well as of lilac, maple, and apple buds, were classified into the genera Methylobacterium and Paracoccus based on the data of morphological studies, enzyme assay, and DNA-DNA hybridization analysis. The methanotrophs that were isolated in pure cultures from samples of linden buds and blue spruce needles were identified into the genus Methylocystis based on the data of morphological studies, enzyme assay, DNA-DNA hybridization, and the phylogenetic analysis of the particulate methane monooxygenase gene pmoA sequences. The inference is made that aerobic methylotrophic bacteria are permanently associated with plants. At the beginning of the vegetative period in spring, the phyllosphere of coniferous and deciduous trees is colonized by the methylotrophic bacteria that have wintered inside plant tissues.

[Facultative and obligate aerobic methylobacteria synthesize cytokinins].

PubMed

Ivanova, E G; Doronina, N V; Shepeliakovskaia, A O; Laman, A G; Brovko, F A; Trotsenko, Iu A

2000-01-01

The presence and expression of genes controlling the synthesis and secretion of cytokinins by the pink-pigmented facultative methylotroph Methylobacterium mesophilicum VKM B-2143 with the serine pathway and nonpigmented obligate methylotroph Methylovorus mays VKM B-2221 with the ribulose monophosphate pathway of C1 metabolism were shown using the polymerase chain reaction (PCR) and reverse transcription-PCR methods. The presence of the corresponding mRNA in M. mesophilicum cells grown on methanol or succinate suggests that the expression of these genes is constitutive. The cytokinin activity of culture liquid and its fractions was determined by a biotest with Amarantus caudatus L. seedlings. Using enzyme-linked immunosorbent analysis, we detected zeatin (riboside) in the culture liquid of both bacteria studied. The data obtained show that the aerobic methylobacteria are phytosymbionts that are able to utilize the single- and polycarbon compounds secreted by symbiotic plants and to synthesize cytokinins.

Interaction between endophytic bacteria from citrus plants and the phytopathogenic bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis.

PubMed

Lacava, P T; Araújo, W L; Marcon, J; Maccheroni, W; Azevedo, J L

2004-01-01

To isolate endophytic bacteria and Xylella fastidiosa and also to evaluate whether the bacterial endophyte community contributes to citrus-variegated chlorosis (CVC) status in sweet orange (Citrus sinensis [L.] Osbeck cv. Pera). The presence of Xylella fastidiosa and the population diversity of culturable endophytic bacteria in the leaves and branches of healthy, CVC-asymptomatic and CVC-symptomatic sweet orange plants and in tangerine (Citrus reticulata cv. Blanco) plants were assessed, and the in vitro interaction between endophytic bacteria and X. fastidiosa was investigated. There were significant differences in endophyte incidence between leaves and branches, and among healthy, CVC-asymptomatic and CVC-symptomatic plants. Bacteria identified as belonging to the genus Methylobacterium were isolated only from branches, mainly from those sampled from healthy and diseased plants, from which were also isolated X. fastidiosa. The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens. This work provides the first evidence of an interaction between citrus endophytic bacteria and X. fastidiosa and suggests a promising approach that can be used to better understand CVC disease.

Efficient methanol-degrading aerobic bacteria isolated from a wetland ecosystem.

PubMed

Thulasi, Kavitha; Jayakumar, Arjun; Balakrishna Pillai, Aneesh; Gopalakrishnapillai Sankaramangalam, Vinod Kumar; Kumarapillai, Harikrishnan

2018-04-10

Methylotrophs present in the soil play an important role in the regulation of one carbon compounds in the environment, and thereby aid in mitigating global warming. The study envisages the isolation and characterization of methanol-degrading bacteria from Kuttanad wetland ecosystem, India. Three methylotrophs, viz. Achromobacter spanius KUT14, Acinetobacter sp. KUT26 and Methylobacterium radiotolerans KUT39 were isolated and their phylogenetic positions were determined by constructing a phylogenetic tree based on 16S rDNA sequences. In vitro activity of methanol dehydrogenase enzyme, responsible for methanol oxidation was evaluated and the genes involved in methanol metabolism, mxaF and xoxF were partially amplified and sequenced. The specific activity of methanol dehydrogenase (451.9 nmol min -1 mg -1 ) observed in KUT39 is the highest, reported ever to our knowledge from a soil bacterium. KUT14 recorded the least activity of 50.15 nmol min -1 mg -1 and is the first report on methylotrophy in A. spanius.

Deep sequencing of Myxilla (Ectyomyxilla) methanophila, an epibiotic sponge on cold-seep tubeworms, reveals methylotrophic, thiotrophic, and putative hydrocarbon-degrading microbial associations.

PubMed

Arellano, Shawn M; Lee, On On; Lafi, Feras F; Yang, Jiangke; Wang, Yong; Young, Craig M; Qian, Pei-Yuan

2013-02-01

The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ(13)C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

Biochemical and chemical characterization of pink-pigmented oxidative bacteria.

PubMed Central

Wallace, P L; Hollis, D G; Weaver, R E; Moss, C W

1990-01-01

The biochemical and chemical characteristics were determined for 156 clinical isolates of pink-pigmented bacteria that are similar to but distinct from Methylobacterium extorquens (synonymous with Pseudomonas mesophilica). These isolates were gram-negative, nonfermentative, usually nonvacuolated, coccoid rods; all grew at 35 degrees C and were catalase and urease positive; the majority grew on MacConkey agar and were variable for oxidase production and motility. On the basis of oxidation of xylose and mannitol and hydrolysis of esculin, these 156 strains were subdivided into four groups that were designated "pink coccoid" groups I, II, III, and IV. Groups I, II, and III are similar to an unnamed taxon described by Gilardi and Faur in 1984; only strains of group IV hydrolyze esculin. The cellular fatty acid compositions of strains of groups I, II, and III were essentially identical and differed from strains of group IV by the absence of 3-OH-C14:0 and the presence of C19:0 delta and 2-OH-C19:0 delta. The fatty acid composition of group IV strains was most similar to that of M. extorquens but differed by the presence of small amounts of two C17:1 acids, 3-OH-C16:0, and 2-OH-C18:1. PMID:2332467

Biochemical and chemical characterization of pink-pigmented oxidative bacteria.

PubMed

Wallace, P L; Hollis, D G; Weaver, R E; Moss, C W

1990-04-01

The biochemical and chemical characteristics were determined for 156 clinical isolates of pink-pigmented bacteria that are similar to but distinct from Methylobacterium extorquens (synonymous with Pseudomonas mesophilica). These isolates were gram-negative, nonfermentative, usually nonvacuolated, coccoid rods; all grew at 35 degrees C and were catalase and urease positive; the majority grew on MacConkey agar and were variable for oxidase production and motility. On the basis of oxidation of xylose and mannitol and hydrolysis of esculin, these 156 strains were subdivided into four groups that were designated "pink coccoid" groups I, II, III, and IV. Groups I, II, and III are similar to an unnamed taxon described by Gilardi and Faur in 1984; only strains of group IV hydrolyze esculin. The cellular fatty acid compositions of strains of groups I, II, and III were essentially identical and differed from strains of group IV by the absence of 3-OH-C14:0 and the presence of C19:0 delta and 2-OH-C19:0 delta. The fatty acid composition of group IV strains was most similar to that of M. extorquens but differed by the presence of small amounts of two C17:1 acids, 3-OH-C16:0, and 2-OH-C18:1.

Complete Genome Sequence of Methylobacterium populi P-1M, Isolated from Pink-Pigmented Household Biofilm

PubMed Central

Morohoshi, Tomohiro

2016-01-01

Methylobacterium populi P-1M is isolated from the pink-pigmented household biofilm. Here, we present the complete genome sequence of P-1M, consisting of one chromosome of 5,705,640 bp and five plasmids of 64,864 bp, 59,879 bp, 42,569 bp, 41,417 bp, and 29,506 bp. PMID:27313289

Genetic and metabolic diversity of pink-pigmented facultative methylotrophs in phyllosphere of tropical plants.

PubMed

Balachandar, D; Raja, P; Sundaram, Sp

2008-01-01

Diversity of Pink-Pigmented Facultative Methylotrophs (PPFMs) in phyllosphere of cotton, maize and sunflower was determined based on differential carbon-substrate utilization profile and Random Amplified Polymorphic DNA data. Results indicate that six diversified groups of PPFMs are found in these crops. Sunflower and maize phyllosphere harbor four different groups of methylobacteria while cotton has only two groups.

STOICHIOMETRIC MODEL FOR EVALUATING THE METABOLIC CAPABILITIES OF THE FACULTATIVE METHYLOTROPH METHYLOBACTERIUM EXTORQUENS AM1, WITH APPLICATION TO RECONSTRUCTION OF C(3) AND C(4) METABOLISM. (R826729)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Lanthanide-Dependent Regulation of Methylotrophy in Methylobacteriumaquaticum Strain 22A.

PubMed

Masuda, Sachiko; Suzuki, Yutaka; Fujitani, Yoshiko; Mitsui, Ryoji; Nakagawa, Tomoyuki; Shintani, Masaki; Tani, Akio

2018-01-01

Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca 2+ -dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF . This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La 3+ . We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La 3+ ; this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides. IMPORTANCE Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place

Complete Genome Sequence of Methylobacterium populi P-1M, Isolated from Pink-Pigmented Household Biofilm.

PubMed

Morohoshi, Tomohiro; Ikeda, Tsukasa

2016-06-16

Methylobacterium populi P-1M is isolated from the pink-pigmented household biofilm. Here, we present the complete genome sequence of P-1M, consisting of one chromosome of 5,705,640 bp and five plasmids of 64,864 bp, 59,879 bp, 42,569 bp, 41,417 bp, and 29,506 bp. Copyright © 2016 Morohoshi and Ikeda.

Structure prediction and analysis of MxaF from obligate, facultative and restricted facultative methylobacterium.

PubMed

Singh, Raghvendra Pratap; Singh, Ram Nageena; Srivastava, Manish K; Srivastava, Alok Kumar; Kumar, Sudheer; Dubey, Ramesh Chandra; Sharma, Arun Kumar

2012-01-01

Methylobacteria are ubiquitous in the biosphere which are capable of growing on C1 compounds such as formate, formaldehyde, methanol and methylamine as well as on a wide range of multi-carbon growth substrates such as C2, C3 and C4 compounds due to the methylotrophic enzymes methanol dehydrogenase (MDH). MDH is performing these functions with the help of a key protein mxaF. Unfortunately, detailed structural analysis and homology modeling of mxaF is remains undefined. Hence, the objective of this research is the characterization and three dimensional modeling of mxaF protein from three different methylotrophs by using I-TASSER server. The predicted model were further optimize and validate by Profile 3D, Errat, Verifiy3-D and PROCHECK server. Predicted and best evaluated models have been successfully deposited to PMDB database with PMDB ID PM0077505, PM0077506 and PM0077507. Active site identification revealed 11, 13 and 14 putative functional site residues in respected models. It may play a major role during protein-protein, and protein-cofactor interactions. This study can provide us an ab-initio and detail information to understand the structure, mechanism of action and regulation of mxaF protein.

Genetic and metabolic diversity of pink-pigmented facultative methylotrophs in phyllosphere of tropical plants

PubMed Central

Balachandar, D.; Raja, P.; Sundaram, SP.

2008-01-01

Diversity of Pink-Pigmented Facultative Methylotrophs (PPFMs) in phyllosphere of cotton, maize and sunflower was determined based on differential carbon-substrate utilization profile and Random Amplified Polymorphic DNA data. Results indicate that six diversified groups of PPFMs are found in these crops. Sunflower and maize phyllosphere harbor four different groups of methylobacteria while cotton has only two groups. PMID:24031182

Effects of Eu and Sm on Methylobacterium sp.

NASA Astrophysics Data System (ADS)

Hibi, Yoshihisa; Okuda, Masayo; Sakuma, Ryusuke; Iwama, Tomonori; Kawai, Keiichi

Eu and Sm have been widely used in high technology products. In this study the authors isolated a soil bacterium, identified as Methylobacterium sp. MAFF211642, which exhibited colonies on 1/100 nutrient agar, supplemented by 30µM Eu and Sm; the soil bacterium was found to exhibit larger colonies than those in the absence of these elements. However, when 0.5% methanol was added to the nutrient agar, only Sm was found to stimulate the growth. Other rare earth and metal elements did not affect or inhibit, regardless of the presence of methanol. Addition of both Sm and methanol to the nutrient broth increased the growth of this strain 10-fold in colony forming unit larger than when both were absent. When both methanol and Sm were added to the nutrient broth, specific activity of methanol dehydrogenase in a crude extract of the bacterium increased approximately 5.4-fold.

Expression, purification and preliminary X-ray characterization of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1

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

Omi, Rie; Department of Chemistry, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585; Jitsumori, Keiji

A recombinant form of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1 has been expressed in E. coli, purified and crystallized. The crystal belongs to space group P6{sub 3}. Diffraction data have been collected to 1.75 Å resolution. dl-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (dl-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of dl-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P6{sub 3}, with unit-cell parameters a = b = 186.2, c =more » 114.4 Å. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V{sub M} value of 4.20–2.10 Å{sup 3} Da{sup −1}. A self-rotation function revealed peaks on the χ = 180° section. X-ray data have been collected to 1.75 Å resolution.« less

Systemic Infection of an Immunocompromised Patient with Methylobacterium zatmanii

PubMed Central

Hornei, B.; Lüneberg, E.; Schmidt-Rotte, H.; Maaß, M.; Weber, K.; Heits, F.; Frosch, M.; Solbach, W.

1999-01-01

We describe the identification of Methylobacterium zatmanii as the causative agent of bacteremia and fever in an immunocompromised patient. The patient, a 60-year-old man, had a 5-month history of acute myeloid leukemia and had been on chemotherapy throughout this period. Seven days after the onset of neutropenia, the patient developed fever. The combination of ciprofloxacin, co-trimoxazole, imipenem, amikacin, and vancomycin led to a complete defervescence. On subculture from six positive blood cultures, the organism grew only on buffered charcoal yeast extract agar and not on standard agars. Identification by universal PCR and subsequent sequence analysis of the amplified 16S rRNA gene segment was achieved. This identification by molecular biology techniques was confirmed by conventional biochemical tests. To our knowledge, this is the first description of M. zatmanii isolated from patient material. PMID:9854105

Methylobacteria isolated from bryophytes and the 2-fold description of the same microbial species.

PubMed

Schauer, S; Kutschera, U

2013-02-01

On the surface of healthy land plants (embryophytes), numerous non-pathogenic bacteria have been discovered and described. Among these epiphytic microbes, pink-pigmented facultative methylotrophic microbes of the genus Methylobacterium are of special significance, because these microorganisms consume methanol emitted via the stomatal pores and secrete growth-promoting phytohormones. Methylobacterium funariae, Schauer and Kutschera 2011, a species isolated in our lab from the common cord moss, described as a nova species in this journal, was recently characterized for a second time as a "new taxon" under a different name, "M. bullatum." Based on a phylogenetic analysis, we show that these taxa are identical. In addition, we provide novel information on the exact cell size, and describe the correct type locality of this bacterial species, which was classified as a phytosymbiont. Finally, we discuss the hypothesis that certain methylobacteria may preferentially colonize bryophytes. With reference to our recent discovery that thalli of ferns form, like liverworts and moss protonemata, associations with methylobacteria, we argue that the haploid phase of cryptogames are preferred host organisms of these pink-pigmented microbial phytosymbionts.

Methylarcula marina gen. nov., sp. nov. and Methylarcula terricola sp. nov.: novel aerobic, moderately halophilic, facultatively methylotrophic bacteria from coastal saline environments.

PubMed

Doronina, N V; Trotsenko, Y A; Tourova, T P

2000-09-01

A new genus, Methylarcula, with two new species, Methylarcula marina and Methylarcula terricola, are proposed for strains h1T and h37T of moderately halophilic facultatively methylotrophic bacteria isolated from the coastal saline habitats. These methylobacteria are aerobic, Gram-negative, asporogenous, non-motile, colourless rods that multiply by binary fission. Their cellular fatty acids profiles consist primarily of straight-chain unsaturated (C18:1; 70-80%), saturated (C18:0; 14-16%) and cyclopropane (C19:0; 5-6%) acids. The major ubiquinone is Q-10. The dominant phospholipids are phosphatidylethanolamine and phosphatidylcholine. Both strains could use methylamine, some sugars and organic acids as carbon and energy sources. They grew well under optimal conditions (29-35 degrees C, pH 7.5-8.5, 0.5-1.0 M NaCl) and accumulated intracellularly poly-beta-hydroxybutyrate and the compatible solute ectoine. The ectoine pool was found to increase upon increasing the external NaCl concentration and accounted for 18% of the dry cellular weight. Both strains oxidized methylamine by the N-methylglutamate (N-MG) pathway enzymes (gamma-glutamylmethylamide synthetase/lyase and N-MG synthetase/lyase) to formaldehyde and assimilated it via the icl- serine pathway. The DNA G+C content was 60-4 mol% for Methylarcula marina h1T and 57.1 mol% for Methylarcula terricola h37T. The DNA-DNA hybridization value between strains hl and h37 was 25-30%, although they had a low level of DNA relatedness (5-7%) with the type strains of the serine pathway methylobacteria belonging to the genera Methylobacterium, Aminobacter, Methylorhabdus and Methylopila. A comparative 16S rDNA sequence-based phylogenetic analysis placed the two species of Methylarcula into a separate branch of the alpha-3 subclass of the Proteobacteria. The type strains of the new species are Methylarcula marina h1T (= VKM B-2159T) and Methylarcula terricola h37T (= VKM B-2160T).

Structure prediction and analysis of MxaF from obligate, facultative and restricted facultative methylobacterium

PubMed Central

Singh, Raghvendra Pratap; Singh, Ram Nageena; Srivastava, Manish K; Srivastava, Alok Kumar; Kumar, Sudheer; Dubey, Ramesh Chandra; Sharma, Arun Kumar

2012-01-01

Methylobacteria are ubiquitous in the biosphere which are capable of growing on C1 compounds such as formate, formaldehyde, methanol and methylamine as well as on a wide range of multi-carbon growth substrates such as C2, C3 and C4 compounds due to the methylotrophic enzymes methanol dehydrogenase (MDH). MDH is performing these functions with the help of a key protein mxaF. Unfortunately, detailed structural analysis and homology modeling of mxaF is remains undefined. Hence, the objective of this research is the characterization and three dimensional modeling of mxaF protein from three different methylotrophs by using I-TASSER server. The predicted model were further optimize and validate by Profile 3D, Errat, Verifiy3-D and PROCHECK server. Predicted and best evaluated models have been successfully deposited to PMDB database with PMDB ID PM0077505, PM0077506 and PM0077507. Active site identification revealed 11, 13 and 14 putative functional site residues in respected models. It may play a major role during protein-protein, and protein-cofactor interactions. This study can provide us an ab-initio and detail information to understand the structure, mechanism of action and regulation of mxaF protein. PMID:23275704

[Biotechnological potential of methylotrophic bacteria: a review of current status and future prospects].

PubMed

Trotsenko, Iu A; Doronina, N V; Khmelenina, V N

2005-01-01

Major results of the authors' findings on the implementation of biotechnological potential of aerobic methylobacteria and methanotrophs for obtaining forage proteins, biopolymers (polybutyrate and polysaccharides), enzymes (oxidoreductases), and bioprotectors (ectoin), as well as for degrading toxic C1 and Cn compounds have been reviewed. Unique features of the structural and functional organization of the metabolism of extremophilic (tolerant) methylotrophs are discussed, with a view for their prospective use in various fields of modern biotechnology, including biocatalysis and nanotechnology.

Culturable Facultative Methylotrophic Bacteria from the Cactus Neobuxbaumia macrocephala Possess the Locus xoxF and Consume Methanol in the Presence of Ce3+ and Ca2+

PubMed Central

del Rocío Bustillos-Cristales, María; Corona-Gutierrez, Ivan; Castañeda-Lucio, Miguel; Águila-Zempoaltécatl, Carolina; Seynos-García, Eduardo; Hernández-Lucas, Ismael; Muñoz-Rojas, Jesús; Medina-Aparicio, Liliana; Fuentes-Ramírez, Luis Ernesto

2017-01-01

Methanol-consuming culturable bacteria were isolated from the plant surface, rhizosphere, and inside the stem of Neobuxbaumia macrocephala. All 38 isolates were facultative methylotrophic microorganisms. Their classification included the Classes Actinobacteria, Sphingobacteriia, Alpha-, Beta-, and Gammaproteobacteria. The deduced amino acid sequences of methanol dehydrogenase obtained by PCR belonging to Actinobacteria, Alpha-, Beta-, and Gammaproteobacteria showed high similarity to rare-earth element (REE)-dependent XoxF methanol dehydrogenases, particularly the group XoxF5. The sequences included Asp301, the REE-coordinating amino acid, present in all known XoxF dehydrogenases and absent in MxaF methanol dehydrogenases. The quantity of the isolates showed positive hybridization with a xoxF probe, but not with a mxaF probe. Isolates of all taxonomic groups showed methylotrophic growth in the presence of Ce3+ or Ca2+. The presence of xoxF-like sequences in methylotrophic bacteria from N. macrocephala and its potential relationship with their adaptability to xerophytic plants are discussed. PMID:28855445

Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph

DOE PAGES

Bennett, R. Kyle; Gonzalez, Jacqueline E.; Whitaker, W. Brian; ...

2017-12-05

Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene ( pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determinedmore » by 13C-labeling in intracellular metabolites. As a result, introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.« less

Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph

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

Bennett, R. Kyle; Gonzalez, Jacqueline E.; Whitaker, W. Brian

Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene ( pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determinedmore » by 13C-labeling in intracellular metabolites. As a result, introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.« less

Draft Genome Sequence of Methylobacterium radiotolerans Strain MAMP 4754, a Bacterial Endophyte Isolated from Combretum erythrophyllum in South Africa

PubMed Central

Photolo, Mampolelo M.; Mavumengwana, Vuyo; Serepa-Dlamini, Mahloro H.

2017-01-01

ABSTRACT We announce here the draft genome sequence of Methylobacterium radiotolerans strain MAMP 4754, isolated from the roots of the medicinal plant Combretum erythrophyllum. M. radiotolerans has a genome size of 7,389,282 bp with 7,166 genes and a G+C content of 70.5%. PMID:28982992

Influence of plant species and environmental conditions on epiphytic and endophytic pink-pigmented facultative methylotrophic bacterial populations associated with field-grown rice cultivars.

PubMed

Madhaiyan, Munusamy; Poonguzhali, Selvaraj; Sa, Tongmin

2007-10-01

The total methylotrophic population associated with rice plants from different cultivars was enumerated at three different stages: vegetative, flowering, and harvesting. The bacterial population in the leaf, rhizosphere soil, endophytic in the stem and roots, and epiphytic in the florets and grains were determined from four rice cultivars, Il-mi, Nam-pyeoung, O-dae, and Dong-jin, sampled from three different field sites. The methylotrophic bacteria isolated on AMS media containing 0.5% methanol as the sole carbon source uniformly showed three distinct morphologies, which were recorded as separate groups and their distribution among the various samples was determined using the ecophysiological index. The growth stage at the time of sampling had a more significant effect on the methylotrophic population and their distribution than the field site or cultivar. A similar effect was also observed for the PPFMs, where their population in different plant parts increased from V10 to R4 and then decreased towards stage R9. A canonical discriminant analysis of the PPFM population from different parts of rice showed clear variations among the cultivars, sampled sites, and growth stages, although the variations were more prominent among the growth stages.

Identification of Methyl Halide-Utilizing Genes in the Methyl Bromide-Utilizing Bacterial Strain IMB-1 Suggests a High Degree of Conservation of Methyl Halide-Specific Genes in Gram-Negative Bacteria

USGS Publications Warehouse

Woodall, C.A.; Warner, K.L.; Oremland, R.S.; Murrell, J.C.; McDonald, I.R.

2001-01-01

Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the Proteobacteria, can grow with methyl bromide as a sole carbon and energy source. A single cmu gene cluster was identified in IMB-1 that contained six open reading frames: cmuC, cmuA, orf146, paaE, hutI, and partial metF. CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyl-transferase motif. However, cmuB, identified in M. chloromethanicum and H. chloromethanicum, was not detected in IMB-1.

Requirement of carbon dioxide for initial growth of facultative methylotroph, Acidomonas methanolica MB58.

PubMed

Mitsui, Ryoji; Katayama, Hiroko; Tanaka, Mitsuo

2015-07-01

The facultative methylotrophic bacterium Acidomonas methanolica MB58 can utilize C1 compounds via the ribulose monophosphate pathway. A large gene cluster comprising three components related to C1 metabolism was found in the genome. From upstream, the first was an mxa cluster encoding proteins for oxidation of methanol to formaldehyde; the second was the rmp cluster encoding enzymes for formaldehyde fixation; and the third was the cbb gene cluster encoding proteins for carbon dioxide (CO2) fixation. Examination of CO2 requirements for growth of A. methanolica MB58 cells demonstrated that it did not grow on any carbon source under CO2-free conditions. Measurement of ribulose-1,5-bisphosphate carboxylase activity and RT-PCR analysis demonstrated enzymatic activity was detected in A. methanolica MB58 at growth phase, regardless of carbon sources. However, methanol dehydrogenase and 3-hexlose-6-phosphate synthase expression was regulated by methanol or formaldehyde; it were detected during growth and apparently differed from ribulose-1,5-bisphosphate carboxylase expression. These results suggested that A. methanolica MB58 may be initially dependent on autotrophic growth and that carbon assimilation was subsequently coupled with the ribulose monophosphate pathway at early- to mid-log phases during methylotrophic growth. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Expression of a codon-optimized Aspergillus niger pectin methylesterase gene in the methylotrophic yeast Candida boidinii.

PubMed

Kawaguchi, Kosuke; Yurimoto, Hiroya; Sakai, Yasuyoshi

2014-01-01

A codon-optimized Aspergillus niger pectin methylesterase (PME) gene was expressed in the methylotrophic yeast Canidia boidinii. The PME-producing strains showed better growth on pectin than the wild-type strains, suggesting that the PME-producing strains could efficiently utilize methyl ester moieties of pectin. On the other hand, overproduction of PME negatively affected the proliferation of C. boidinii on leaves of Arabidopsis thaliana.

Bacillus subtilis yckG and yckF Encode Two Key Enzymes of the Ribulose Monophosphate Pathway Used by Methylotrophs, and yckH Is Required for Their Expression

PubMed Central

Yasueda, Hisashi; Kawahara, Yoshio; Sugimoto, Shin-ichi

1999-01-01

The ribulose monophosphate (RuMP) pathway is one of the metabolic pathways for the synthesis of compounds containing carbon-carbon bonds from one-carbon units and is found in many methane- and methanol-utilizing bacteria, which are known as methylotrophs. The characteristic enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), neither of which was thought to exist outside methylotrophs. However, the presumed yckG gene product (YckG) of Bacillus subtilis shows a primary structure similar to that of methylotroph HPS (F. Kunst et al., Nature 390:249–256, 1997). We have also investigated the sequence similarity between the yckF gene product (YckF) and methylotroph PHI (Y. Sakai, R. Mitsui, Y. Katayama, H. Yanase, and N. Kato, FEMS Microbiol. Lett. 176:125–130, 1999) and found that the yckG and yckF genes of B. subtilis express enzymatic activities of HPS and PHI, respectively. Both of these activities were concomitantly induced in B. subtilis by formaldehyde, with induction showing dependence on the yckH gene, but were not induced by methanol, formate, or methylamine. Disruption of either gene caused moderate sensitivity to formaldehyde, suggesting that these enzymes may act as a detoxification system for formaldehyde in B. subtilis. In conclusion, we found an active yckG (for HPS)-yckF (for PHI) gene structure (now named hxlA-hxlB) in a nonmethylotroph, B. subtilis, which inherently preserves the RuMP pathway. PMID:10572115

[Aerobic methylobacteria are capable of synthesizing auxins].

PubMed

Ivanova, E G; Doronina, N V; Trotsenko, Iu A

2001-01-01

Obligately and facultatively methylotrophic bacteria with different pathways of C1 metabolism were found to be able to produce auxins, particularly indole-3-acetic acid (IAA), in amounts of 3-100 micrograms/ml. Indole-3-pyruvic acid and indole-3-acetamide were detected only in methylobacteria with the serine pathway of C1 metabolism, Methylobacterium mesophilicum and Aminobacter aminovorans. The production of auxins by methylobacteria was stimulated by the addition of tryptophan to the growth medium and was inhibited by ammonium ions. The methylobacteria under study lacked tryptophan decarboxylase and tryptophan side-chain oxidase. At the same time, they were found to contain several aminotransferases. IAA is presumably synthesized by methylobacteria through indole-3-pyruvic acid.

Methanohalophilus zhilinae sp. nov., an alkaliphilic, halophilic, methylotrophic methanogen

NASA Technical Reports Server (NTRS)

Mathrani, I. M.; Boone, D. R.; Mah, R. A.; Fox, G. E.; Lau, P. P.

1988-01-01

Methanohalophilus zhilinae, a new alkaliphilic, halophilic, methylotrophic species of methanogenic bacteria, is described. Strain WeN5T (T = type strain) from Bosa Lake of the Wadi el Natrun in Egypt was designated the type strain and was further characterized. This strain was nonmotile, able to catabolize dimethylsulfide, and able to grow in medium with a methyl group-containing substrate (such as methanol or trimethylamine) as the sole organic compound added. Sulfide (21 mM) inhibited cultures growing on trimethylamine. The antibiotic susceptibility pattern of strain WeN5T was typical of the pattern for archaeobacteria, and the guanine-plus-cytosine content of the deoxyribonucleic acid was 38 mol%. Characterization of the 16S ribosomal ribonucleic acid sequence indicated that strain WeN5T is phylogenetically distinct from members of previously described genera other than Methanohalophilus and supported the partition of halophilic methanogens into their own genus.

Genetics of bacteria that oxidize one-carbon compounds. Progress report, March 1, 1991--June 30, 1993

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

Hanson, R.S.

In the past several years researchers have identified at least 20 genes whose products were required for the oxidation of methanol to formaldehyde in three different facultative methylotrophic bacteria. These genes include structural genes for a cytochrome c{sub L} (mox G) and is a specific electron acceptor for methanol dehydrogenase (MDH), and the two structural genes that encode the large subunit (mox F) and smaller subunit (mox I) of MDH. Other genes are required for the synthesis of the prosthetic group of MDH, Pyrroloquinoline quinone (PQQ), and proteins required for assembly of the active MDH in the periplasm. Three genesmore » are believed to be required for incorporation of calcium into the MDH tetramer. The principal investigator`s group has studied the regulation of methanol oxidation in the pink-pigmented-facultative methylotroph Methylobacterium organophilum XX. The authors have mapped several genes and have sequenced the mox F gene and sequences upstream of mox F. The authors had tentatively identified several genes required for the transcription of the MDH structural genes in three methylotrophs. In the previous proposal, the P.I. proposed to establish an in-vitro transcription/translation system to study the function of the regulatory gene products. Further studies demonstrated that the regulation of transcription of these genes was far more complex than imagined at that time and the research plan was modified to determine the number and function of the regulatory genes using genetic approaches.« less

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Hwang, Jung-Shan; Yamada, Kazunori; Honda, Ayae; Nakade, Kohji; Ishihama, Akira

2000-01-01

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni2+-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris. PMID:10756019

Moss-associated methylobacteria as phytosymbionts: an experimental study

NASA Astrophysics Data System (ADS)

Hornschuh, M.; Grotha, R.; Kutschera, U.

2006-10-01

Methylotrophic bacteria inhabit the surface of plant organs, but the interaction between these microbes and their host cells is largely unknown. Protonemata (gametophytes) of the moss Funaria hygrometrica were cultivated in vitro under axenic conditions and the growth of the protonemal filaments recorded. In the presence of methylobacteria (different strains of Methylobacterium), average cell length and the number of cells per filament were both enhanced. We tested the hypothesis that auxin (indole-3-acetic acid, IAA), secreted by the epiphytic bacteria and taken up by the plant cells, may in part be responsible for this promotion of protonema development. The antiauxin parachlorophenoxyisobutyric acid (PCIB) was used as a tool to analyze the role of IAA and methylobacteria in the regulation of cell growth. In the presence of PCIB, cell elongation and protonema differentiation were both inhibited. This effect was compensated for by the addition of different Methylobacterium strains to the culture medium. Biosynthesis and secretion of IAA by methylobacteria maintained in liquid culture was documented via a colorimetric assay and thin layer chromatography. Our results support the hypothesis that the development of Funaria protonemata is promoted by beneficial phytohormone-producing methylobacteria, which can be classified as phytosymbionts.

Moss-associated methylobacteria as phytosymbionts: an experimental study.

PubMed

Hornschuh, M; Grotha, R; Kutschera, U

2006-10-01

Methylotrophic bacteria inhabit the surface of plant organs, but the interaction between these microbes and their host cells is largely unknown. Protonemata (gametophytes) of the moss Funaria hygrometrica were cultivated in vitro under axenic conditions and the growth of the protonemal filaments recorded. In the presence of methylobacteria (different strains of Methylobacterium), average cell length and the number of cells per filament were both enhanced. We tested the hypothesis that auxin (indole-3-acetic acid, IAA), secreted by the epiphytic bacteria and taken up by the plant cells, may in part be responsible for this promotion of protonema development. The antiauxin parachlorophenoxyisobutyric acid (PCIB) was used as a tool to analyze the role of IAA and methylobacteria in the regulation of cell growth. In the presence of PCIB, cell elongation and protonema differentiation were both inhibited. This effect was compensated for by the addition of different Methylobacterium strains to the culture medium. Biosynthesis and secretion of IAA by methylobacteria maintained in liquid culture was documented via a colorimetric assay and thin layer chromatography. Our results support the hypothesis that the development of Funaria protonemata is promoted by beneficial phytohormone-producing methylobacteria, which can be classified as phytosymbionts.

Methylobacteria isolated from bryophytes and the 2-fold description of the same microbial species

PubMed Central

Schauer, S.; Kutschera, U.

2013-01-01

On the surface of healthy land plants (embryophytes), numerous non-pathogenic bacteria have been discovered and described. Among these epiphytic microbes, pink-pigmented facultative methylotrophic microbes of the genus Methylobacterium are of special significance, because these microorganisms consume methanol emitted via the stomatal pores and secrete growth-promoting phytohormones. Methylobacterium funariae, Schauer and Kutschera 2011, a species isolated in our lab from the common cord moss, described as a nova species in this journal, was recently characterized for a second time as a “new taxon” under a different name, “M. bullatum.” Based on a phylogenetic analysis, we show that these taxa are identical. In addition, we provide novel information on the exact cell size, and describe the correct type locality of this bacterial species, which was classified as a phytosymbiont. Finally, we discuss the hypothesis that certain methylobacteria may preferentially colonize bryophytes. With reference to our recent discovery that thalli of ferns form, like liverworts and moss protonemata, associations with methylobacteria, we argue that the haploid phase of cryptogames are preferred host organisms of these pink-pigmented microbial phytosymbionts. PMID:23299423

Methane-oxidizing seawater microbial communities from an Arctic shelf

NASA Astrophysics Data System (ADS)

Uhlig, Christiane; Kirkpatrick, John B.; D'Hondt, Steven; Loose, Brice

2018-06-01

Marine microbial communities can consume dissolved methane before it can escape to the atmosphere and contribute to global warming. Seawater over the shallow Arctic shelf is characterized by excess methane compared to atmospheric equilibrium. This methane originates in sediment, permafrost, and hydrate. Particularly high concentrations are found beneath sea ice. We studied the structure and methane oxidation potential of the microbial communities from seawater collected close to Utqiagvik, Alaska, in April 2016. The in situ methane concentrations were 16.3 ± 7.2 nmol L-1, approximately 4.8 times oversaturated relative to atmospheric equilibrium. The group of methane-oxidizing bacteria (MOB) in the natural seawater and incubated seawater was > 97 % dominated by Methylococcales (γ-Proteobacteria). Incubations of seawater under a range of methane concentrations led to loss of diversity in the bacterial community. The abundance of MOB was low with maximal fractions of 2.5 % at 200 times elevated methane concentration, while sequence reads of non-MOB methylotrophs were 4 times more abundant than MOB in most incubations. The abundances of MOB as well as non-MOB methylotroph sequences correlated tightly with the rate constant (kox) for methane oxidation, indicating that non-MOB methylotrophs might be coupled to MOB and involved in community methane oxidation. In sea ice, where methane concentrations of 82 ± 35.8 nmol kg-1 were found, Methylobacterium (α-Proteobacteria) was the dominant MOB with a relative abundance of 80 %. Total MOB abundances were very low in sea ice, with maximal fractions found at the ice-snow interface (0.1 %), while non-MOB methylotrophs were present in abundances similar to natural seawater communities. The dissimilarities in MOB taxa, methane concentrations, and stable isotope ratios between the sea ice and water column point toward different methane dynamics in the two environments.

Methylobacterium populi VP2: Plant Growth-Promoting Bacterium Isolated from a Highly Polluted Environment for Polycyclic Aromatic Hydrocarbon (PAH) Biodegradation

PubMed Central

Piccolo, Alessandro; Carotenuto, Rita; Pepe, Olimpia

2014-01-01

The use of microorganisms to accelerate the natural detoxification processes of toxic substances in the soil represents an alternative ecofriendly and low-cost method of environmental remediation compared to harmful incineration and chemical treatments. Fourteen strains able to grow on minimal selective medium with a complex mixture of different classes of xenobiotic compounds as the sole carbon source were isolated from the soil of the ex-industrial site ACNA (Aziende Chimiche Nazionali Associate) in Cengio (Savona, Italy). The best putative degrading isolate, Methylobacterium populi VP2, was identified using a polyphasic approach on the basis of its phenotypic, biochemical, and molecular characterisation. Moreover, this strain also showed multiple plant growth promotion activities: it was able to produce indole-3-acetic acid (IAA) and siderophores, solubilise phosphate, and produce a biofilm in the presence of phenanthrene and alleviate phenanthrene stress in tomato seeds. This is the first report on the simultaneous occurrence of the PAH-degrading ability by Methylobacterium populi and its multiple plant growth-promoting activities. Therefore, the selected indigenous strain, which is naturally present in highly contaminated soils, is good candidate for plant growth promotion and is capable of biodegrading xenobiotic organic compounds to remediate contaminated soil alone and/or soil associated with plants. PMID:25152928

Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

PubMed Central

Oda, Saori; Yurimoto, Hiroya; Nitta, Nobuhisa; Sasano, Yu

2015-01-01

We identified genes encoding components of the Hap complex, CbHAP2, CbHAP3, and CbHAP5, as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. We found that the Cbhap2Δ, Cbhap3Δ, and Cbhap5Δ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae, the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii. Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts. PMID:25595445

High molecular weight dissolved organic matter enrichment selects for methylotrophs in dilution to extinction cultures

PubMed Central

Sosa, Oscar A; Gifford, Scott M; Repeta, Daniel J; DeLong, Edward F

2015-01-01

The role of bacterioplankton in the cycling of marine dissolved organic matter (DOM) is central to the carbon and energy balance in the ocean, yet there are few model organisms available to investigate the genes, metabolic pathways, and biochemical mechanisms involved in the degradation of this globally important carbon pool. To obtain microbial isolates capable of degrading semi-labile DOM for growth, we conducted dilution to extinction cultivation experiments using seawater enriched with high molecular weight (HMW) DOM. In total, 93 isolates were obtained. Amendments using HMW DOM to increase the dissolved organic carbon concentration 4x (280 μM) or 10x (700 μM) the ocean surface water concentrations yielded positive growth in 4–6% of replicate dilutions, whereas <1% scored positive for growth in non-DOM-amended controls. The majority (71%) of isolates displayed a distinct increase in cell yields when grown in increasing concentrations of HMW DOM. Whole-genome sequencing was used to screen the culture collection for purity and to determine the phylogenetic identity of the isolates. Eleven percent of the isolates belonged to the gammaproteobacteria including Alteromonadales (the SAR92 clade) and Vibrio. Surprisingly, 85% of isolates belonged to the methylotrophic OM43 clade of betaproteobacteria, bacteria thought to metabolically specialize in degrading C1 compounds. Growth of these isolates on methanol confirmed their methylotrophic phenotype. Our results indicate that dilution to extinction cultivation enriched with natural sources of organic substrates has a potential to reveal the previously unsuspected relationships between naturally occurring organic nutrients and the microorganisms that consume them. PMID:25978545

[Enhanced Resistance of Pea Plants to Oxidative: Stress Caused by Paraquat during Colonization by Aerobic Methylobacteria].

PubMed

Agafonova, N V; Doronina, N Y; Trotsenko, Yu A

2016-01-01

The influence of colonization of the pea (Pisum sativum L.) by aerobic methylobacteria of five different species (Methylophilus flavus Ship, Methylobacterium extorquens G10, Methylobacillus arboreus Iva, Methylopila musalis MUSA, Methylopila turkiensis Sidel) on plant resistance to paraquat-induced stresses has been studied. The normal conditions of pea colonization by methylobacteria were characterized by a decrease in the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) and in the concentrations of endogenous H2O2, proline, and malonic dialdehyde, which is a product of lipid peroxidation and indicator of damage to plant cell membranes, and an increase in the activity of the photosynthetic apparatus (the content of chlorophylls a, b and carotenoids). In the presence of paraquat, the colonized plants had higher activities of antioxidant enzymes, stable photosynthetic indices, and a less intensive accumulation of the products of lipid peroxidation as compared to noncolonized plants. Thus, colonization by methylobacteria considerably increased the adaptive protection of pea plants to the paraquat-induced oxidative stress.

[Microbial synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium Brevibacterium methylicum with RMP assimilation of carbon].

PubMed

Mosin, O V; Shvets, V I; Skladnev, D A; Ignatov, I

2014-01-01

The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent crystallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions.

Nucleotide sequence and transcriptional start site of the Methylobacterium organophilum XX methanol dehydrogenase structural gene

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

Machlin, S.M.; Hanson, R.S.

The nucleotide sequence of a cloned 2.5-kilobase-pair SmaI fragment containing the methanol dehydrogenase (MDH) structural gene from Methylobacterium organophilum XX was determined. A single open reading frame with a coding capacity of 626 amino acids (molecular weight, 66,000) was identified on one stand, and N-terminal sequencing of purified MDH revealed that 27 of these residues constituted a putative signal peptide. Primer extension mapping of in vivo transcripts indicated that the start of mRNA synthesis was 160 to 170 base pairs upstream of the ATG codon. Northern (RNA) blot analysis further demonstrated that the transcript was 2.1 kilobase pairs in lengthmore » and therefore appeared to encode only MDH.« less

Development of alginate-based aggregate inoculants of Methylobacterium sp. and Azospirillum brasilense tested under in vitro conditions to promote plant growth.

PubMed

Joe, M M; Saravanan, V S; Islam, M R; Sa, T

2014-02-01

To develop co-aggregated bacterial inoculant comprising of Methylobacterium oryzae CBMB20/Methylobacterium suomiense CBMB120 strains with Azospirillum brasilense (CW903) strain and testing their efficiency as inoculants for plant growth promotion (PGP). Biofilm formation and co-aggregation efficiency was studied between A. brasilense CW903 and methylobacterial strains M. oryzae CBMB20 and M. suomiense CBMB120. Survival and release of these co-aggregated bacterial strains entrapped in alginate beads were assessed. PGP attributes of the co-aggregated bacterial inoculant were tested in tomato plants under water-stressed conditions. Results suggest that the biofilm formation efficiency of the CBMB20 and CBMB120 strains increased by 15 and 34%, respectively, when co-cultivated with CW903. Co-aggregation with CW903 enhanced the survivability of CBMB20 strain in alginate beads. Water stress index score showed least stress index in plants inoculated with CW903 and CBMB20 strains maintained as a co-aggregated inoculant. This study reports the development of co-aggregated cell inoculants containing M. oryzae CBMB20 and A. brasilense CW903 strains conferred better shelf life and stress abatement in inoculated tomato plants. These findings could be extended to other PGP bacterial species to develop multigeneric bioinoculants with multiple benefits for various crops. © 2013 The Society for Applied Microbiology.

[Physiological and biochemical analysis of the transformants of aerobic methylobacteria expressing the dcm A gene of dichloromethane dehydrogenase].

PubMed

Firsova, Iu E; Doronina, N V; Trotsenko, Iu A

2004-01-01

The transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr-/pME8220 and DM4-2cr-/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl- ions, CH2O, and HCOOH, nor to the impairment of glutathione synthesis or to the maintenance of intracellular pH. The (exo-) Klenow fragment-mediated incorporation of [alpha-32P]dATP into the DNA of the transformants DM4-2cr-/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH2Cl2 than when they were incubated with CH3OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH2Cl2 showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH2Cl2 degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.

moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1

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

Anderson, D.J.; Lidstrom, M.E.

The polypeptides encoded by a putative methanol oxidation (mox) operon of Methylobacterium sp. strain AM1 were expressed in Escherichia coli, using a coupled in vivo T7 RNA polymerase/promoter gene expression system. Two mox genes had been previously mapped to this region: moxF, the gene encoding the methanol dehydrogenase (MeDH) polypeptide; and moxG, a gene believed to encode a soluble type c cytochrome, cytochrome c/sub L/. In this study, four polypeptides of M/sub r/, 60,000, 30,000, 20,000, and 12,000 were found to be encoded by the moxFG region and were tentatively designated moxF, -J, -G, and -I, respectively. The arrangement ofmore » the genes (5' to 3') was found to be moxFJGI. The identities of three of the four polypeptides were determined by protein immunoblot analysis. The product of moxF, the M/sub r/-60,000 polypeptide, was confirmed to be the MeDH polypeptide. The product of moxG, the M/sub r/-20,000 polypeptide, was identified as mature cytochrome c/sub L/, and the product of moxI, the M/sub r/-12,000 polypeptide, was identified as a MeDH-associated polypeptide that copurifies with the holoenzyme. The identity of the M/sub r/-30,000 polypeptide (the moxJ gene product) could not be determined. The function of the M/sub r/-12,000 MeDH-associated polypeptide is not yet clear. However, it is not present in mutants that lack the M/sub r/-60,000 MeDH subunit, and it appears that the stability of the MeDH-associated polypeptide is dependent on the presence of the M/sub r/-60,000 MeDH polypeptide. Our data suggest that both the M/sub r/-30,000 and -12,000 polypeptides are involved in methanol oxidation, which would bring to 12 the number of mox genes in Methylobacterium sp. strain AM1.« less

Poly(3-hydroxybutyrate) production in an integrated electromicrobial setup: Investigation under stress-inducing conditions.

PubMed

Al Rowaihi, Israa Salem; Paillier, Alexis; Rasul, Shahid; Karan, Ram; Grötzinger, Stefan Wolfgang; Takanabe, Kazuhiro; Eppinger, Jörg

2018-01-01

Poly(3-hydroxybutyrate) (PHB), a biodegradable polymer, can be produced by different microorganisms. The PHB belongs to the family of polyhydroxyalkanoate (PHA) that mostly accumulates as a granule in the cytoplasm of microorganisms to store carbon and energy. In this study, we established an integrated one-pot electromicrobial setup in which carbon dioxide is reduced to formate electrochemically, followed by sequential microbial conversion into PHB, using the two model strains, Methylobacterium extorquens AM1 and Cupriavidus necator H16. This setup allows to investigate the influence of different stress conditions, such as coexisting electrolysis, relatively high salinity, nutrient limitation, and starvation, on the production of PHB. The overall PHB production efficiency was analyzed in reasonably short reaction cycles typically as short as 8 h. As a result, the PHB formation was detected with C. necator H16 as a biocatalyst only when the electrolysis was operated in the same solution. The specificity of the source of PHB production is discussed, such as salinity, electricity, concurrent hydrogen production, and the possible involvement of reactive oxygen species (ROS).

Poly(3-hydroxybutyrate) production in an integrated electromicrobial setup: Investigation under stress-inducing conditions

PubMed Central

Al Rowaihi, Israa Salem; Paillier, Alexis; Rasul, Shahid; Karan, Ram; Grötzinger, Stefan Wolfgang; Eppinger, Jörg

2018-01-01

Poly(3-hydroxybutyrate) (PHB), a biodegradable polymer, can be produced by different microorganisms. The PHB belongs to the family of polyhydroxyalkanoate (PHA) that mostly accumulates as a granule in the cytoplasm of microorganisms to store carbon and energy. In this study, we established an integrated one-pot electromicrobial setup in which carbon dioxide is reduced to formate electrochemically, followed by sequential microbial conversion into PHB, using the two model strains, Methylobacterium extorquens AM1 and Cupriavidus necator H16. This setup allows to investigate the influence of different stress conditions, such as coexisting electrolysis, relatively high salinity, nutrient limitation, and starvation, on the production of PHB. The overall PHB production efficiency was analyzed in reasonably short reaction cycles typically as short as 8 h. As a result, the PHB formation was detected with C. necator H16 as a biocatalyst only when the electrolysis was operated in the same solution. The specificity of the source of PHB production is discussed, such as salinity, electricity, concurrent hydrogen production, and the possible involvement of reactive oxygen species (ROS). PMID:29698424

Methylobacillus pratensis sp. nov., a novel non-pigmented, aerobic, obligately methylotrophic bacterium isolated from meadow grass.

PubMed

Doronina, Nina V; Trotsenko, Yuri A; Kolganova, Tatjana V; Tourova, Tatjana P; Salkinoja-Salonen, Mirja S

2004-09-01

Strain F31T was isolated from meadow grass (Poa trivialis L.) sampled from the city park in Helsinki. Analysis of phenotypic and genotypic properties showed the strain to be related to the group of obligately methylotrophic non-methane utilizing bacteria (methylobacteria) with the ribulose monophosphate pathway of formaldehyde assimilation. Phylogenetic analysis showed the strain to be closely related to the genus Methylobacillus, and analysis of fatty acid composition confirmed this association. Thus, on the basis of its genotypic and phenotypic properties, the isolate is proposed as a novel species of the genus Methylobacillus, Methylobacillus pratensis sp. nov., with F31T as the type strain (= VKM B-2247T = NCIMB 13994T).

Genotyping and chlorine-resistance of Methylobacterium aquaticum isolated from water samples in Japan.

PubMed

Furuhata, Katsunori; Banzai, Azusa U; Kawakami, Yasushi; Ishizaki, Naoto; Yoshida, Yoshihiro; Goto, Keiichi; Fukuyama, Masafumi

2011-09-01

For microbial ecological analysis, 14 strains of Methylobacterium aquaticum isolated from water samples were subjected to clustering analysis on the basis of ribotyping and RAPD-PCR tests. The ribopatterns after digestion with EcoRI obtained from 14 strains of M. aquaticum were used to divide the strains into two groups (Groups I and II) with a similarity of 55%. From the analysis of RAPD patterns using primer 208, the 14 strains were divided into 3 groups (A-C) based on a homology of 45% or greater, and from that using primer 272, there were 4 groups (A-D) based on a homology of 50% or greater. The chlorine resistance (99.9% CT values) of these isolates was also experimentally confirmed, and we attempted to define the connection between chlorine resistance and the geno-cluster. The average CT value of group I was 0.89 mg•min/l and the average of group II was 0.69 mg•min/l. No remarkable differences in the CT values for the groups were found.

GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Liachko, Ivan; Youngblood, Rachel A.; Tsui, Kyle; Bubb, Kerry L.; Queitsch, Christine; Raghuraman, M. K.; Nislow, Corey; Brewer, Bonita J.; Dunham, Maitreya J.

2014-01-01

The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins—a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation. PMID:24603708

GC-rich DNA elements enable replication origin activity in the methylotrophic yeast Pichia pastoris.

PubMed

Liachko, Ivan; Youngblood, Rachel A; Tsui, Kyle; Bubb, Kerry L; Queitsch, Christine; Raghuraman, M K; Nislow, Corey; Brewer, Bonita J; Dunham, Maitreya J

2014-03-01

The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins--a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation.

Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol.

PubMed

Liu, Yiqi; Tu, Xiaohu; Xu, Qin; Bai, Chenxiao; Kong, Chuixing; Liu, Qi; Yu, Jiahui; Peng, Qiangqiang; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2018-01-01

As a promising one-carbon renewable substrate for industrial biotechnology, methanol has attracted much attention. However, engineering of microorganisms for industrial production of pharmaceuticals using a methanol substrate is still in infancy. In this study, the methylotrophic yeast Pichia pastoris was used to produce anti-hypercholesterolemia pharmaceuticals, lovastatin and its precursor monacolin J, from methanol. The biosynthetic pathways for monacolin J and lovastatin were first assembled and optimized in single strains using single copies of the relevant biosynthetic genes, and yields of 60.0mg/L monacolin J and 14.4mg/L lovastatin were obtained using methanol following pH controlled monoculture. To overcome limitations imposed by accumulation of intermediates and metabolic stress in monoculture, approaches using pathway splitting and co-culture were developed. Two pathway splitting strategies for monacolin J, and four for lovastatin were tested at different metabolic nodes. Biosynthesis of monacolin J and lovastatin was improved by 55% and 71%, respectively, when the upstream and downstream modules were separately accommodated in two different fluorescent strains, split at the metabolic node of dihydromonacolin L. However, pathway distribution at monacolin J blocked lovastatin biosynthesis in all designs, mainly due to its limited ability of crossing cellular membranes. Bioreactor fermentations were tested for the optimal co-culture strategies, and yields of 593.9mg/L monacolin J and 250.8mg/L lovastatin were achieved. This study provides an alternative method for production of monacolin J and lovastatin and reveals the potential of a methylotrophic yeast to produce complicated pharmaceuticals from methanol. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Degradation of formaldehyde at high concentrations by phenol-adapted Ralstonia eutropha closely related to pink-pigmented facultative methylotrophs.

PubMed

Habibi, Alireza; Vahabzadeh, Farzaneh

2013-01-01

The ability of the phenol-adapted Ralstonia eutropha to utilize formaldehyde (FD) as the sole source of carbon and energy was studied. Adaptation to FD was accomplished by substituting FD for glucose in a stepwise manner. The bacterium in the liquid test culture could tolerate concentrations of FD up to 900 mg L(-1). Degradation of FD was complete in 528 h at 30°C with shaking at 150 rpm (r = 1.67 mg L(-1) h(-1)), q = 0.035 g(FD) g(cell) (-1) h(-1). Substrate inhibition kinetics (Haldane and Luong equations) are used to describe the experimental data. At non-inhibitory concentrations of FD, the Monod equation was used. According to the Luong model, the values of the maximum specific growth rate (μ(max)), half-saturation coefficient (k(S)), the maximum allowable formaldehyde concentration (S(m)), and the shape factor (n) were 0.117 h(-1), 47.6 mg L(-1), 900 mg L(-1), and 2.2, respectively. The growth response of the test bacterium to consecutive FD feedings was examined, and the FD-adapted R. eutropha cells were able to degrade 1000 mg L(-1) FD in 150 h through 4 cycles of FD feeds. During FD degradation, formic acid metabolite was formed. Assimilation of FD, methanol, formic acid, and oxalate by the test bacterium was accompanied by the formation of a pink pigment. The carotenoid nature of the cellular pigment has been confirmed and the test bacterium appeared to be closely related to pink-pigmented facultative methylotrophs (PPFM). The extent of harm to soil exposed to biotreated wastewaters containing FD may be moderated due to the association between methylotrophic/oxalotrophic bacteria and plants.

[Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

PubMed

Morozkina, E V; Vavilova, E A; Zatsepin, S S; Klyachko, E V; Yagudin, T A; Chulkin, A M; Dudich, I V; Semenkova, L N; Churilova, I V; Benevolenskii, S V

2016-01-01

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

Salt tolerant Methylobacterium mesophilicum showed viable colonization abilities in the plant rhizosphere

PubMed Central

Egamberdieva, Dilfuza; Wirth, Stephan; Alqarawi, Abdulaziz A.; Abd_Allah, E.F.

2015-01-01

The source of infection has always been considered as an important factor in epidemiology and mostly linked to environmental source such as surface water, soil, plants and also animals. The activity of the opportunistic pathogens associated with plant root, their adaptation and survival under hostile environmental condition is poorly understood. In this study the salt tolerance ability of Methylobacterium mesophilicum and its colonization in the root and shoot of plants under severe drought and salt stress conditions were investigated. The colonization of plant by M. mesophilicum was investigated in a gnotobiotic sand system, and their survival in pots with saline soil. Bacterial strain was found to colonize rhizosphere of cucumber, tomato and paprika grown under normal and salt stress condition and reached up to 6.4 × 104 and 2.6 × 104 CFU/g root. The strain was resistant to Gentamicin, Ampicillin, Amoxicillin plus Clavulanic acid, Cefotaxime, neomycin, penicillin and was also tolerant to salinity stress (up to 6% NaCl). These abilities play important roles in enabling persistent colonization of the plant surface by M. mesophilicum strains. In conclusion, this study provides background information on the behaviour of opportunistic pathogen M. mesophilicum on plants and their survival in harsh environmental conditions. PMID:26288563

SPRUCE Methylotrophic Methanogenesis in Sphagnum-dominated Peatland Soils – CH4 and CO2 Production in Laboratory Incubations

DOE Data Explorer

Zalman, Cassandra A.; Meade, N.; Chanton, J.; Kostka, J. E.; Bridgham, S. D.; Keller, J. K.

2017-12-01

This study investigated the potential for methylotrophic methanogenesis in three Sphagnum-dominated peatland soils in northern Minnesota. Collected soils were amended with 13C-labeled traditional substrates (acetate and sodium bicarbonate/ H2) and methylated substrates (methanol, monomethylamine (“MMA”), dimethylsulfide (“DMS”)) and monitored for δ13C-CH4, δ 13C-CO2, and net CH4 and CO2 production in laboratory incubations. The peatlands included in the study were (1) the S1 Bog, home to the SPRUCE Experiment and located at the Marcell Experimental Forest (MEF, U.S. Forest Service), (2) Bog Lake Fen, also located at the MEF, and (3) Zim Bog. These sites have been described in detail previously (Medvedeff et al., 2015)

Combining amplicon sequencing and metabolomics in cirrhotic patients highlights distinctive microbiota features involved in bacterial translocation, systemic inflammation and hepatic encephalopathy.

PubMed

Iebba, Valerio; Guerrieri, Francesca; Di Gregorio, Vincenza; Levrero, Massimo; Gagliardi, Antonella; Santangelo, Floriana; Sobolev, Anatoly P; Circi, Simone; Giannelli, Valerio; Mannina, Luisa; Schippa, Serena; Merli, Manuela

2018-05-29

In liver cirrhosis (LC), impaired intestinal functions lead to dysbiosis and possible bacterial translocation (BT). Bacteria or their byproducts within the bloodstream can thus play a role in systemic inflammation and hepatic encephalopathy (HE). We combined 16S sequencing, NMR metabolomics and network analysis to describe the interrelationships of members of the microbiota in LC biopsies, faeces, peripheral/portal blood and faecal metabolites with clinical parameters. LC faeces and biopsies showed marked dysbiosis with a heightened proportion of Enterobacteriaceae. Our approach showed impaired faecal bacterial metabolism of short-chain fatty acids (SCFAs) and carbon/methane sources in LC, along with an enhanced stress-related response. Sixteen species, mainly belonging to the Proteobacteria phylum, were shared between LC peripheral and portal blood and were functionally linked to iron metabolism. Faecal Enterobacteriaceae and trimethylamine were positively correlated with blood proinflammatory cytokines, while Ruminococcaceae and SCFAs played a protective role. Within the peripheral blood and faeces, certain species (Stenotrophomonas pavanii, Methylobacterium extorquens) and metabolites (methanol, threonine) were positively related to HE. Cirrhotic patients thus harbour a 'functional dysbiosis' in the faeces and peripheral/portal blood, with specific keystone species and metabolites related to clinical markers of systemic inflammation and HE.

Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol

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

Allen, L.N.; Hanson, R.S.

Four new cloning vectors have been constructed from the broad-host-range cloning vector pRK290. These vectors, pLA2901, pLA2905, pLA2910, and pLA2917, confer resistance to kanamycin and tetracycline. The latter two are cosmid derivatives of pLA2901. The new vectors can be mobilized into, and are stably maintained in, a variety of gram-negative bacteria. A Sau3A genomic bank of Methylobacterium organophilum strain xx DNA has been constructed in pLA2917, and complementation analysis, with a variety of mutants unable to grow on methanol, revealed at least five separate regions necessary for growth on methanol. Complementation analysis and Tn5 mutagenesis data suggest that at leastmore » three genes are responsible for expression of active methanol dehydrogenase.« less

Leisingera methylohalidivorans gen. nov., sp. nov., a marine methylotroph that grows on methyl bromide

USGS Publications Warehouse

Schaefer, J.K.; Goodwin, K.D.; McDonald, I.R.; Murrell, J.C.; Oremland, R.S.

2002-01-01

A marine methylotroph, designated strain MB2T, was isolated for its ability to grow on methyl bromide as a sole carbon and energy source. Methyl chloride and methyl iodide also supported growth, as did methionine and glycine betaine. A limited amount of growth was observed with dimethyl sulfide. Growth was also noted with unidentified components of the complex media marine broth 2216, yeast extract and Casamino acids. No growth was observed on methylated amines, methanol, formate, acetate, glucose or a variety of other substrates. Growth on methyl bromide and methyl iodide resulted in their oxidation to CO2 with stoichiometric release of bromide and iodide, respectively. Strain MB2T exhibited growth optima at NaCl and Mg2+ concentrations similar to that of seawater. Phylogenetic analysis of the 16S rDNA sequence placed this strain in the ??-Proteobacteria in proximity to the genera Ruegeria and Roseobacter. It is proposed that strain MB2T (= ATCC BAA-92T = DSM 14336T) be designated Leisingera methylohalidivorans gen. nov., sp. nov.

Biodecoloration of Reactive Black 5 by the methylotrophic yeast Candida boidinii MM 4035.

PubMed

Martorell, María M; Pajot, Hipólito F; Ahmed, Pablo M; de Figueroa, Lucía I C

2017-03-01

Azo dyes are extensively used in textile dyeing and other industries. Effluents of dying industries are specially colored and could cause severe damage to the environment. The anaerobic treatment of textile dying effluents is nowadays the preferred option, but it could generate carcinogenic aromatic amines. Recently, yeasts have become a promising alternative, combining unicellular growth with oxidative mechanisms. This work reports the characterization of the first methylotrophic yeast with dye decolorizing ability, Candida boidinii MM 4035 and some insights into its decoloration mechanism. The analysis of two selected media revealed a possible two stages mechanism of Reactive Black 5 decoloration. In glucose poor media, decoloration is incomplete and only the first stage proceeds, leading to the accumulation of a purple compound. In media with higher glucose concentrations, the yeast is able to decolorize totally an initial concentration of 200mg/L. The entire process is co-metabolic, being largely dependent on glucose concentration but being able to proceed with several nitrogen sources. Manganese dependent peroxidase but not laccase activity could be detected during decoloration. Aromatic amines do not accumulate in culture media, supporting an oxidative decoloration mechanism of unknown ecophysiological relevance. Copyright © 2016. Published by Elsevier B.V.

Proteomic analysis of the thermophilic methylotroph Bacillus methanolicus MGA3.

PubMed

Müller, Jonas E N; Litsanov, Boris; Bortfeld-Miller, Miriam; Trachsel, Christian; Grossmann, Jonas; Brautaset, Trygve; Vorholt, Julia A

2014-03-01

Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram-positive bacterium possesses a soluble NAD(+) -dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label-free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50°C and 37°C). From a total of approximately 1200 different detected proteins, approximately 1000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested, the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the synthesis of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional tricarboxylic acid cycle, the proteins of which are differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. All MS data have been deposited in the ProteomeXchange with the identifiers PXD000637 and PXD000638 (http://proteomecentral.proteomexchange.org/dataset/PXD000637, http://proteomecentral.proteomexchange.org/dataset/PXD000638). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Advenella kashmirensis subsp. methylica PK1, a facultative methylotroph from carex rhizosphere].

PubMed

Poroshina, M N; Doronina, N V; Kaparullina, E N; Trotsenko, Iu A

2015-01-01

A strain (PK1) of facultative methylobacteria growing on methanol as a carbon and energy source was isolated from carex rhizosphere (Pamukkale National Park, Turkey). The cells were nonmotile gram-negative rods propagating by binary fission. The organism was a strict anaerobe, oxidase- and catalase-positive. Optimal growth occurred at 29°C, pH 8.0-8.5, and 0.5% NaCl; no growth occurred at 2% NaCl. The organism used the ribulose bisphosphate pathway of C1 assimilation. Predominant fatty acids were 11-octodecenoic (18:1ω7) and cis-hexadecenoic (16:1ω7c). Phosphatidylethanolamine and diphosphatidylglycerol were the dominant phospholipids. Q8 was the main ubiquinone. DNA G+C content was 55.4 mol % (mp). Sequencing of the 16S rRNA gene revealed that strain PK1 belonged to the genus Advenella with 98.8 and 99.2% similarity to the type strains A. incenata CCUG 45225T and A. kashmirensis WT001T, respectively. DNA-DNA homology of strain PK1 and A. kashmirensis WT001T was 70%. While MALDI analysis confirmed their close clusterization, RAPD analysis revealed the differences between strain PKI and other Advenella strains. Based on its geno- and phenotypic properties, the isolate PK1 was classified as A. kashmirensis subsp. methylica PK1 (VKM-B 2850 = DSM 27514), the first known methylotroph of the genus Advenella.

Prevention of pink-pigmented methylotrophic bacteria (Methylohacterium mesophilicum) contamination of plant tissue cultures.

PubMed

Chanprame, S; Todd, J J; Widholm, J M

1996-12-01

Pink-pigmented facultative methylotrophic bacteria (PPFMs) have been found on the surfaces of leaves of most plants tested. We found PPFMs on the leaf surfaces of all 40 plants (38 species) tested and on soybean pods by pressing onto AMS medium with methanol as the sole carbon source. The abundance ranged from 0.5 colony forming unit (cfu) /cm(2) to 69.4 cfu/cm(2) on the leaf surfaces. PPFMs were found in homogenized leaf tissues of only 4 of the species after surface disinfestation with 1.05% sodium hypochlorite and were rarely found in cultures initiated from surface disinfested Datura innoxia leaves or inside surface disinfested soybean pods. Of 20 antibiotics tested for PPFM growth inhibition, rifampicin was the most effective and of seven others which also inhibited PPFM growth, cefotaxime should be the most useful due to the expected low plant cell toxicity. These antibiotics could be used in concert with common surface sterilization procedures to prevent the introduction or to eliminate PPFM bacteria in tissue cultures. Thus, while PPFMs are present on the surfaces of most plant tissues, surface disinfestation alone can effectively remove them so that uncontaminated tissue cultures can be initiated in most cases.

Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1.

PubMed

Mauffrey, Florian; Cucaita, Alexandra; Constant, Philippe; Villemur, Richard

2017-01-01

Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate ([Formula: see text]) to nitrite ([Formula: see text]). These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (Nar). Strain JAM1 also contains gene clusters encoding two nitric oxide (NO) reductases and one nitrous oxide (N 2 O) reductase, suggesting that NO and N 2 O can be reduced by strain JAM1. Here we characterized further the denitrifying activities of M. nitratireducenticrescens JAM1. Series of oxic and anoxic cultures of strain JAM1 were performed with N 2 O, [Formula: see text] or sodium nitroprusside, and growth and N 2 O, [Formula: see text], [Formula: see text] and N 2 concentrations were measured. Ammonium ([Formula: see text])-free cultures were also tested to assess the dynamics of N 2 O, [Formula: see text] and [Formula: see text]. Isotopic labeling of N 2 O was performed in 15 NH 4 + -amended cultures. Cultures with the JAM1Δ narG1narG2 double mutant were performed to assess the involvement of the Nar systems on N 2 O production. Finally, RT-qPCR was used to measure the gene expression levels of the denitrification genes cytochrome bc -type nitric oxide reductase ( cnorB1 and cnorB2 ) and nitrous oxide reductase ( nosZ ), and also nnrS and norR that encode NO-sensitive regulators. Strain JAM1 can reduce NO to N 2 O and N 2 O to N 2 and can sustain growth under anoxic conditions by reducing N 2 O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory [Formula: see text] reductase, [Formula: see text]-amended cultures produce N 2 O, representing up to 6% of the N-input. [Formula: see text] was shown to be the key intermediate of this production process. Upregulation in the expression of c norB1 , cnorB2, nnrS and nor

The development of an unconventional food regeneration process: Quantifying the nutritional components of a model methylotrophic yeast

NASA Technical Reports Server (NTRS)

Petersen, G. R.; Stokes, B. O.

1986-01-01

A hybrid chemical/biological approach to unconventional food regeneration is discussed. Carbon dioxide and water, the major wastes of human metabolism would be converted to methanol by one of several physiochemical processes available (thermal, photocatalytic, etc.). Methanol is then used to supply carbon and energy for the culture of microorganisms which in turn produce biological useful basic food stuffs for human nutrition. Our work has focused on increasing the carbohydrate levels of a candidate methylotrophic yeast to more nearly coincide with human nutritional requirements. Yeasts were chosen due to their high carbohydrate levels compared to bacteria and their present familiarity in the human diet. The initial candidate yeast studied was a thermotolerant strain of Hansenula polymor pha, DL-1. The quantitative results that permit an evaluation of the overall efficiency in hybrid chemical/biological food production schemes are discussed. A preliminary evaluation of the overall efficiency of such schemes is also discussed.

Oxidation of methyl halides by the facultative methylotroph strain IMB-1

USGS Publications Warehouse

Schaefer, J.K.; Oremland, R.S.

1999-01-01

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 ??M MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [14C]formaldehyde to 14CO2 but had only a small capacity for oxidation of [14C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [14C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K(s) values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO2. The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

PubMed Central

Cos, Oriol; Ramón, Ramón; Montesinos, José Luis; Valero, Francisco

2006-01-01

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail. PMID:16600031

Transhydrogenase Promotes the Robustness and Evolvability of E. coli Deficient in NADPH Production

PubMed Central

Chou, Hsin-Hung; Marx, Christopher J.; Sauer, Uwe

2015-01-01

Metabolic networks revolve around few metabolites recognized by diverse enzymes and involved in myriad reactions. Though hub metabolites are considered as stepping stones to facilitate the evolutionary expansion of biochemical pathways, changes in their production or consumption often impair cellular physiology through their system-wide connections. How does metabolism endure perturbations brought immediately by pathway modification and restore hub homeostasis in the long run? To address this question we studied laboratory evolution of pathway-engineered Escherichia coli that underproduces the redox cofactor NADPH on glucose. Literature suggests multiple possibilities to restore NADPH homeostasis. Surprisingly, genetic dissection of isolates from our twelve evolved populations revealed merely two solutions: (1) modulating the expression of membrane-bound transhydrogenase (mTH) in every population; (2) simultaneously consuming glucose with acetate, an unfavored byproduct normally excreted during glucose catabolism, in two subpopulations. Notably, mTH displays broad phylogenetic distribution and has also played a predominant role in laboratory evolution of Methylobacterium extorquens deficient in NADPH production. Convergent evolution of two phylogenetically and metabolically distinct species suggests mTH as a conserved buffering mechanism that promotes the robustness and evolvability of metabolism. Moreover, adaptive diversification via evolving dual substrate consumption highlights the flexibility of physiological systems to exploit ecological opportunities. PMID:25715029

Synthesis and biosynthesis of {sup 13}C-, {sup 15}N-labeled deoxynucleosides useful for biomolecular structural determinations

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

Ashburn, D.A.; Garcia, K.; Hanners, J.L.

Currently, there is a great emphasis on elucidating the structure, function, and dynamics of DNA. Much of the research involved in this study uses nuclear magnetic resonance (NMR) spectroscopy. Effective use of NMR spectroscopy for DNA molecules with mw > 10,000 requires stable isotope enrichment. We present strategies for site-specific isotopic labeling of the purine bases adenosine and guanosine and the biosynthesis of (U-{sup 13}C, {sup 15}N) DNA from methylotropic bacteria. With commercially available 6-chloropurine, an effective two-step route leads to 2{prime}-deoxy-(amino-{sup 15}N)adenosine (dA). The resulting d(amino-{sup 15}N)A is used in a series of reactions to synthesize 2{prime}-deoxy-(2-{sup 13}C,1,amino-{sup 15}N{submore » 2})guanosine or any combination thereof. An improved biosynthesis of labeled DNA has been accomplished using Methylobacterium extorquens AS1. Each liter of growth medium contains 4 g of methanol to yield 1 g of lyophilized cells. As much as 200 mg of RNA per liter of culture has been obtained. We are currently developing large-scale isolation protocols. General synthetic pathways to oligomeric DNA will be presented.« less

Study of the genetics and regulation of methane oxidation. Progress report, second year and a half, August 1, 1981-January 31, 1983

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

Not Available

The purpose is to develop mutagenesis, gene transfer and cloning systems in methanotrophic bacteria, and use these techniques to study the methane oxidation genes. Although we have been successful in the first part of these objectives, the study of methane oxidation genes has proven difficult. Problems arose due to the discovery that the culture, Methylobacterium ethanolicum, is in reality a stable coculture between two methylotrophs. These partners are Methylocystis POC, an obligate methanotroph and Xanthobacter H4.14, and autotrophic methanolutilizer. The Methylocystis strain contains the three plasmids we had observed previously in methane-grown cultures, while the Xanthobacter strain contains no detectiblemore » plasmids. Therefore, our original approach to studying the methane oxidation genes, that of isolating plasmid mutants, is no longer valid. However, our discovery of the nature of this culture has led to some interesting results which show promise in elucidating the genetic structure of the methane oxidation genes in obligate methanotrophs. In addition, we have been successful in developing mutagenesis, gene transfer and cloning systems that are applicable to a wide variety of methanotrophs.« less

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

PubMed Central

Cue, D; Lam, H; Dillingham, R L; Hanson, R S; Flickinger, M C

1997-01-01

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus. PMID:9097439

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

PubMed

Cue, D; Lam, H; Dillingham, R L; Hanson, R S; Flickinger, M C

1997-04-01

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.

Regulation of intracellular formaldehyde toxicity during methanol metabolism of the methylotrophic yeast Pichia methanolica.

PubMed

Wakayama, Keishi; Yamaguchi, Sakiko; Takeuchi, Akihito; Mizumura, Tasuku; Ozawa, Shotaro; Tomizuka, Noboru; Hayakawa, Takashi; Nakagawa, Tomoyuki

2016-11-01

In this study we found that the methylotrophic yeast Pichia methanolica showed impaired growth on high methanol medium (>5%, or 1.56 M, methanol). In contrast, P. methanolica grew well on glucose medium containing 5% methanol, but the growth defects reappeared on glucose medium supplemented with 5 mM formaldehyde. During methanol growth of P. methanolica, formaldehyde accumulated in the medium up to 0.3 mM before it was consumed rapidly based on cell growth. These findings indicate that the growth defect of P. methanolica on high methanol media is not caused directly by methanol toxicity, but rather by formaldehyde, which is a key toxic intermediate of methanol metabolism. Moreover, during methanol growth of P. methanolica, expression of enzymes in the methanol-oxidation pathway were induced before the alcohol oxidase isozymes Mod1p and Mod2p, and Mod1p expression was induced before Mod2p. These results suggest that to avoid excess accumulation of formaldehyde-the toxic intermediate of methanol metabolism-P. methanolica grown on methanol strictly regulates the order in which methanol-metabolizing enzymes are expressed. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Novel Cysteine-Centered Sulfur Metabolic Pathway in the Thermotolerant Methylotrophic Yeast Hansenula polymorpha

PubMed Central

Oh, Doo-Byoung; Kwon, Ohsuk; Lee, Sang Yup; Sibirny, Andriy A.; Kang, Hyun Ah

2014-01-01

In yeast and filamentous fungi, sulfide can be condensed either with O-acetylhomoserine to generate homocysteine, the precursor of methionine, or with O-acetylserine to directly generate cysteine. The resulting homocysteine and cysteine can be interconverted through transsulfuration pathway. Here, we systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast Hansenula polymorpha, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of H. polymorpha, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of de novo synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in H. polymorpha, despite the presence of both directions of transsulfuration pathway Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of H. polymorpha sulfur metabolism. 35S-Cys was more efficiently incorporated into intracellular sulfur compounds such as glutathione than 35S-Met in H. polymorpha, further supporting the cysteine-centered sulfur pathway. This is the first report on the novel features of H. polymorpha sulfur metabolic pathway, which are noticeably distinct from those of other yeast and filamentous fungal species. PMID:24959887

Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris.

PubMed

Kim, Tae-Kang; Zhang, Rundong; Feng, Wenke; Cai, Jian; Pierce, William; Song, Zhao-Hui

2005-03-01

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.

Isolation and characterization of an N-methylcarbamate insecticide-degrading methylotrophic bacterium.

PubMed Central

Topp, E; Hanson, R S; Ringelberg, D B; White, D C; Wheatcroft, R

1993-01-01

A gram-negative bacterium which hydrolyzed aryl N-methylcarbamate insecticides was isolated from an agricultural soil which quickly degraded these pesticides. This organism, designated strain ER2, grew on carbofuran as a sole source of carbon and nitrogen with a doubling time of 3 h in a mineral salts medium. The aromatic nucleus of the molecule was not metabolized, and carbofuran 7-phenol accumulated as the end product of metabolism. The insecticides carbaryl, bendiocarb, and propoxur were similarly hydrolyzed, with each yielding the corresponding phenol. Strain ER2 contained two plasmids (120 and 130 kb). A probe cloned from the pDL11 plasmid of Achromobacter sp. strain WM111, which encodes the carbofuran hydrolase (mcd) gene (P. H. Tomasek and J. S. Karns, J. Bacteriol. 171:4038-4044, 1989), hybridized to the 120-kb plasmid. Restriction fragment profiles of pDL11 and strain ER2 plasmid DNAs suggested that the 120-kb plasmid of strain ER2 is very similar to pDL11. On the basis of the results of biochemical tests, 16S rRNA sequence analysis, and membrane lipid analyses, strain ER2 was found to be a phylogenetically unique type II methylotroph. The constitutive carbofuran hydrolase activity in glucose-grown cells increased sevenfold when strain ER2 was grown in the presence of 100 mg of carbofuran per liter as the sole source of carbon and nitrogen or as the sole nitrogen source in the presence of glucose. Growth on carbofuran resulted in the induction of enzymes required for methylamine-dependent respiration and the serine pathway of formaldehyde assimilation. These results indicate that the carbofuran hydrolase mcd gene is conserved on a plasmid found in organisms from different geographic areas and that the specific activity of carbofuran degradation may increase in response to carbofuran treatment. Images PMID:7504430

[Methylophaga murata sp. nov.: a haloalkaliphilic aerobic methylotroph from deteriorating marble].

PubMed

Doronina, N V; Li, Ts D; Ivanova, E G; Trotsenko, Iu A

2005-01-01

The haloalkaliphilic methylotrophic bacterium (strain Kr3) isolated from material scraped off the deteriorating marble of the Moscow Kremlin masonry has been found to be able to utilize methanol, methylamine, trimethylamine, and fructose as carbon and energy sources. Its cells are gram-negative motile rods multiplying by binary fission. Spores are not produced. The isolate is strictly aerobic and requires vitamin B12 and Na+ ions for growth. It is oxidase- and catalase-positive and reduces nitrates to nitrites. Growth occurs at temperatures between 0 and 42 degrees C (with the optimum temperatures being 20-32 degrees C), pH values between 6 and 11 (with the optimum at 8-9), and NaCl concentrations between 0.05 and 3 M (with the optimum at 0.5-1.5 M). The dominant cellular phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. The major cellular fatty acids are palmitic (C16:0), palmitoleic (C16:1), and octadecenoic (C18:1) acids. The major ubiquinone is Q8. The isolate accumulates ectoine and glutamate, as well as a certain amount of sucrose, to function as osmoprotectants and synthesizes an exopolysaccharide composed of carbohydrate and protein components. It is resistant to heating at 70 degrees C, freezing, and drying; utilizes methanol, with the resulting production of formic acid, which is responsible for the marble-degrading activity of the isolate; and implements the 2-keto-3-deoxy-6-phosphogluconate variant of the ribulose monophosphate pathway. The G+C content of its DNA is 44.6 mol%. Based on 16S rRNA gene sequencing and DNA-DNA homology levels (23-41%) with neutrophilic and alkaliphilic methylobacteria from the genus Methylophaga, the isolate has been identified as a new species, Methylophaga murata (VKM B-2303T = NCIMB 13993T).

High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

PubMed

Sakai, Y; Goh, T K; Tani, Y

1993-06-01

We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.

Characterization of micro-organisms isolated from dairy industry after cleaning and fogging disinfection with alkyl amine and peracetic acid.

PubMed

Bore, E; Langsrud, S

2005-01-01

To characterize micro-organisms isolated from Norwegian dairy production plants after cleaning and fogging disinfection with alkyl amine/peracetic acid and to indicate reasons for survival. Microbial samples were collected from five dairy plants after cleaning and fogging disinfection. Isolates from two of these production plants, which used fogging with alkylamino acetate (plant A), and peracetic acid (plant B), were chosen for further characterization. The sequence of the 16S ribosomal DNA, fatty acid analysis and biochemical characteristics were used to identify isolates. Three isolates identified as Rhodococcus erythropolis, Methylobacterium rhodesianum and Rhodotorula mucilaginosa were isolated from plant A and one Sphingomonas sp. and two M. extorquens from plant B. Different patterns of resistance to seven disinfectants in a bactericidal suspension test and variable degree of attachment to stainless steel were found. The strains with higher disinfectant resistance showed lower degree of attachment than susceptible strains. The study identifies and characterizes micro-organisms present after cleaning and fogging disinfection. Both surface attachment and resistance were shown as possible reasons for the presence of the isolates after cleaning and disinfection. These results contribute to the awareness of disinfectant resistance as well as attachment as mechanisms of survival in dairy industry. It also strengthens the argument of frequent alternation of disinfectants in the food processing industry to avoid the establishment of resistant house strains.

Diversity, Bacterial Symbionts and Antibacterial Potential of Gut-Associated Fungi Isolated from the Pantala flavescens Larvae in China

PubMed Central

Shao, Ming-Wei; Lu, Yi-Hui; Miao, Shuang; Zhang, Yun; Chen, Ting-Ting; Zhang, Ying-Lao

2015-01-01

The diversity of fungi associated with the gut of Pantala flavescens larvae was investigated using a culture-dependent method and molecular identification based on an analysis of the internally transcribed spacer sequence. In total, 48 fungal isolates were obtained from P. flavescens larvae. Based on phylogenetic analyses, the fungal isolates were grouped in 5 classes and 12 different genera. Fourteen bacterial 16S rDNA sequences derived from total genomic DNA extractions of fungal mycelia were obtained. The majority of the sequences were associated with Proteobacteria (13/14), and one Bacillaceae (1/14) was included. Leclercia sp., Oceanobacillus oncorhynchi and Methylobacterium extorquens, were reported for the first time as bacterial endosymbionts in fungi. High-performance liquid chromatography (HPLC) analysis indicated that bacterial symbionts produced specific metabolites and also exerted an inhibitory effect on fungal metabolites. The biological activity of the fungal culture extracts against the pathogenic bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) was investigated, and 20 extracts (42%) exhibited antibacterial activity against at least one of the tested bacterial strains. This study is the first report on the diversity and antibacterial activity of symbiotic fungi residing in the gut of P. flavescens larvae, and the results show that these fungi are highly diverse and could be exploited as a potential source of bioactive compounds. PMID:26221957

Diversity, Bacterial Symbionts and Antibacterial Potential of Gut-Associated Fungi Isolated from the Pantala flavescens Larvae in China.

PubMed

Shao, Ming-Wei; Lu, Yi-Hui; Miao, Shuang; Zhang, Yun; Chen, Ting-Ting; Zhang, Ying-Lao

2015-01-01

The diversity of fungi associated with the gut of Pantala flavescens larvae was investigated using a culture-dependent method and molecular identification based on an analysis of the internally transcribed spacer sequence. In total, 48 fungal isolates were obtained from P. flavescens larvae. Based on phylogenetic analyses, the fungal isolates were grouped in 5 classes and 12 different genera. Fourteen bacterial 16S rDNA sequences derived from total genomic DNA extractions of fungal mycelia were obtained. The majority of the sequences were associated with Proteobacteria (13/14), and one Bacillaceae (1/14) was included. Leclercia sp., Oceanobacillus oncorhynchi and Methylobacterium extorquens, were reported for the first time as bacterial endosymbionts in fungi. High-performance liquid chromatography (HPLC) analysis indicated that bacterial symbionts produced specific metabolites and also exerted an inhibitory effect on fungal metabolites. The biological activity of the fungal culture extracts against the pathogenic bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) was investigated, and 20 extracts (42%) exhibited antibacterial activity against at least one of the tested bacterial strains. This study is the first report on the diversity and antibacterial activity of symbiotic fungi residing in the gut of P. flavescens larvae, and the results show that these fungi are highly diverse and could be exploited as a potential source of bioactive compounds.

Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization*

PubMed Central

Müller, Daniel B.; Schubert, Olga T.; Röst, Hannes; Aebersold, Ruedi; Vorholt, Julia A.

2016-01-01

Plants are colonized by a diverse community of microorganisms, the plant microbiota, exhibiting a defined and conserved taxonomic structure. Niche separation based on spatial segregation and complementary adaptation strategies likely forms the basis for coexistence of the various microorganisms in the plant environment. To gain insights into organism-specific adaptations on a molecular level, we selected two exemplary community members of the core leaf microbiota and profiled their proteomes upon Arabidopsis phyllosphere colonization. The highly quantitative mass spectrometric technique SWATH MS was used and allowed for the analysis of over two thousand proteins spanning more than three orders of magnitude in abundance for each of the model strains. The data suggest that Sphingomonas melonis utilizes amino acids and hydrocarbon compounds during colonization of leaves whereas Methylobacterium extorquens relies on methanol metabolism in addition to oxalate metabolism, aerobic anoxygenic photosynthesis and alkanesulfonate utilization. Comparative genomic analyses indicates that utilization of oxalate and alkanesulfonates is widespread among leaf microbiota members whereas, aerobic anoxygenic photosynthesis is almost exclusively found in Methylobacteria. Despite the apparent niche separation between these two strains we also found a relatively small subset of proteins to be coregulated, indicating common mechanisms, underlying successful leaf colonization. Overall, our results reveal for two ubiquitous phyllosphere commensals species-specific adaptations to the host environment and provide evidence for niche separation within the plant microbiota. PMID:27457762

Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

PubMed

Lueders, Tillmann; Manefield, Mike; Friedrich, Michael W

2004-01-01

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

The Arabidopsis leaf transcriptome reveals distinct but also overlapping responses to colonization by phyllosphere commensals and pathogen infection with impact on plant health.

PubMed

Vogel, Christine; Bodenhausen, Natacha; Gruissem, Wilhelm; Vorholt, Julia A

2016-10-01

Plants are colonized by a variety of bacteria, most of which are not pathogenic. Currently, the plant responses to phyllosphere commensals or to pathogen infection in the presence of commensals are not well understood. Here, we examined the transcriptional response of Arabidopsis thaliana leaves to colonization by common commensal bacteria in a gnotobiotic system using RNA sequencing and conducted plant mutant assays. Arabidopsis responded differently to the model bacteria Sphingomonas melonis Fr1 (S.Fr1) and Methylobacterium extorquens PA1 (M.PA1). Whereas M.PA1 only marginally affected the expression of plant genes (< 10), S.Fr1 colonization changed the expression of almost 400 genes. For the latter, genes related to defense responses were activated and partly overlapped with those elicited by the pathogen Pseudomonas syringae DC3000 (Pst). As S.Fr1 is able to mediate plant protective activity against Pst, we tested plant immunity mutants and found that the pattern-recognition co-receptor mutant bak1/bkk1 showed attenuated S.Fr1-dependent plant protection. The experiments demonstrate that the plant responds differently to members of its natural phyllosphere microbiota. A subset of commensals trigger expression of defense-related genes and thereby may contribute to plant health upon pathogen encounter. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

PubMed Central

Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

2016-01-01

Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

Genetics of bacteria that utilize one carbon compounds: Final report, March 1, 1982-February 29, 1988

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

Hanson, R.S.

Broad host range plasmid vectors useful for cloning genes from bacteria that grow on methane and methanol were constructed. We have cloned and mapped nineteen genes required for the growth of Methylobacterium organophilum strain XX on methanol. Nineteen genes were found in seven linkage groups on the M. organophilum genome and were separated by 40 kb or more. Eleven genes were required for the synthesis of methanol dehydrogenase (MDH) and were located in three unlinked gene clusters. The MDH structural gene was localized on a 2.5 kb DNA fragment. The gene was sequenced and contains a 175 bp untranslated leadermore » sequence, a signal sequence and the structural gene. MDH messenger RNA (mRNA) has a half life of approximately 20 min. and is present at approximately 2% of the cellular mRNA. The structural gene for the ..gamma.. subunit of methane monoxygenases has been cloned from Methylosporovibrio. Methane monooxygenase subunits have been purified by Prof. J. Lipscomb's laboratory and are being sequenced to construct DNA probes to identify cloned subunit genes. New facultative methylotrophic bacteria were isolated and characterized. Several amino acid auxotrophs have been isolated. 11 refs.« less

The kinetic study of hydrogen bacteria and methanotrophs in pure and defined mixed cultures

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

Arora, D.K.

The kinetics of pure and mixed cultures of Alcaligenes eutrophus H 16 and Methylobacterium organophilum CRL 26 under double substrate limited conditions were studied. In pure culture growth kinetics, a non-interactive model was found to fit the experimental data best. The yield of biomass on limiting substrate was found to vary with the dilution rate. The variation in the biomass yield may be attributed to the change in metabolic pathways resulting from a shift in the limiting substrates. Both species exhibited wall growth in the chemostat under dark conditions. However, under illuminated conditions, there was significant reduction in wall growth.more » Poly-{beta}-hydroxybutyric acid was synthesized by both species under ammonia and oxygen limiting conditions. The feed gas mixture was optimized to achieve the steady-state coexistence of these two species in a chemostate for the first time. In mixed cultures, the biomass species assays were differentiated on the basis of their selective growth on particular compounds: Sarcosine and D-arabinose were selected for hydrogen bacteria and methylotrophs, respectively. The kinetics parameters estimated from pure cultures were used to predict the growth kinetics of these species in defined mixed cultures.« less

Degradation of 1,2-dichloroethane by Ancylobacter aquaticus and other facultative methylotrophs.

PubMed Central

van den Wijngaard, A J; van der Kamp, K W; van der Ploeg, J; Pries, F; Kazemier, B; Janssen, D B

1992-01-01

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains of Ancylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhlA) genes of strains AD20 and AD25 were the same as the sequence of dhlA from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission. Images PMID:1575500

Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

PubMed Central

Zahn, J A; Duncan, C; DiSpirito, A A

1994-01-01

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline. Images PMID:7928947

Molecular Analysis of a Novel Methanesulfonic Acid Monooxygenase from the Methylotroph Methylosulfonomonas methylovora

PubMed Central

de Marco, Paolo; Moradas-Ferreira, Pedro; Higgins, Timothy P.; McDonald, Ian; Kenna, Elizabeth M.; Murrell, J. Colin

1999-01-01

Methylosulfonomonas methylovora M2 is an unusual gram-negative methylotrophic bacterium that can grow on methanesulfonic acid (MSA) as the sole source of carbon and energy. Oxidation of MSA by this bacterium is carried out by a multicomponent MSA monooxygenase (MSAMO). Cloning and sequencing of a 7.5-kbp SphI fragment of chromosomal DNA revealed four tightly linked genes encoding this novel monooxygenase. Analysis of the deduced MSAMO polypeptide sequences indicated that the enzyme contains a two-component hydroxylase of the mononuclear-iron-center type. The large subunit of the hydroxylase, MsmA (48 kDa), contains a typical Rieske-type [2Fe–2S] center with an unusual iron-binding motif and, together with the small subunit of the hydroxylase, MsmB (20 kDa), showed a high degree of identity with a number of dioxygenase enzymes. However, the other components of the MSAMO, MsmC, the ferredoxin component, and MsmD, the reductase, more closely resemble those found in other classes of oxygenases. MsmC has a high degree of identity to ferredoxins from toluene and methane monooxygenases, which are enzymes characterized by possessing hydroxylases containing μ-oxo bridge binuclear iron centers. MsmD is a reductase of 38 kDa with a typical chloroplast-like [2Fe–2S] center and conserved flavin adenine dinucleotide- and NAD-binding motifs and is similar to a number of mono- and dioxygenase reductase components. Preliminary analysis of the genes encoding MSAMO from a marine MSA-degrading bacterium, Marinosulfonomonas methylotropha, revealed the presence of msm genes highly related to those found in Methylosulfonomonas, suggesting that MSAMO is a novel type of oxygenase that may be conserved in all MSA-utilizing bacteria. PMID:10094704

Structural and functional characterization of recombinant napin-like protein of Momordica charantia expressed in methylotrophic yeast Pichia pastoris.

PubMed

Yadav, Shailesh Kumar R; Sahu, Tejram; Dixit, Aparna

2016-08-01

Napin and napin-like proteins belong to the 2S albumin seed storage family of proteins and have been shown to display a variety of biological activities. However, due to a high degree of polymorphism, purification of a single napin or napin-like protein exhibiting biological activity is extremely difficult. In the present study, we have produced the napin-like protein of Momordica charantia using the methylotrophic Pichia pastoris expression system. The recombinant napin-like protein (rMcnapin) secreted in the extracellular culture supernatant was enriched by ammonium sulfate precipitation, and purified using size exclusion chromatography at a yield of ∼290 mg/L of culture. Secondary structure analysis of the purified rMcnapin revealed it to be predominantly α-helical with minimal β strand content. CD spectroscopic and fluorescence spectroscopic analyses revealed the rMcnapin to be stable at a wide range of temperatures and pH. The rMcnapin exhibited antifungal activity against Trichoderma viride with an IC50 of ∼3.7 μg/ml and trypsin inhibitor activity with an IC50 of 4.2 μM. Thus, large amounts of homogenous preparations of the biologically active rMcnapin could be obtained at shake flask level, which is otherwise difficult from its natural source.

The methylotrophic Bacillus methanolicus MGA3 possesses two distinct fructose 1,6-bisphosphate aldolases.

PubMed

Stolzenberger, Jessica; Lindner, Steffen N; Wendisch, Volker F

2013-08-01

The thermotolerant Gram-positive methylotroph Bacillus methanolicus is able to grow with methanol, glucose or mannitol as a sole carbon and energy source. Fructose 1,6-bisphosphate aldolase (FBA), a key enzyme of glycolysis and gluconeogenesis, is encoded in the genome of B. methanolicus by two putative fba genes, the chromosomally located fba(C) and fba(P) on the naturally occurring plasmid pBM19. Their amino acid sequences share 75 % identity and suggest a classification as class II aldolases. Both enzymes were purified from recombinant Escherichia coli and were found to be active as homotetramers. Both enzymes were activated by either manganese or cobalt ions, and inhibited by ADP, ATP and EDTA. The kinetic parameters allowed us to distinguish the chromosomally encoded FBA(C) from the plasmid encoded FBA(P), since FBA(C) showed higher affinity towards fructose 1,6-bisphosphate (Km of 0.16±0.01 mM as compared to 2±0.08 mM) as well as higher glycolytic catalytic efficiency (31.3 as compared to 0.8 s(-1) mM(-1)) than FBA(P). However, FBA(P) exhibited a higher catalytic efficiency in gluconeogenesis (50.4 as compared to 1.4 s(-1) mM(-1) with dihydroxyacetone phosphate and 4 as compared to 0.4 s(-1) mM(-1) with glyceraldehyde 3-phosphate as limiting substrate). The aldolase-negative Corynebacterium glutamicum mutant Δfda could be complemented with both FBA genes from B. methanolicus. Based on the kinetic data, we propose that FBA(C) acts as major aldolase in glycolysis, whereas FBA(P) acts as major aldolase in gluconeogenesis in B. methanolicus.

Albibacter methylovorans gen. nov., sp. nov., a novel aerobic, facultatively autotrophic and methylotrophic bacterium that utilizes dichloromethane.

PubMed

Doronina, N V; Trotsenko, Y A; Tourova, T P; Kuznetsov, B B; Leisinger, T

2001-05-01

A novel genus, Albibacter, with one species, Albibacter methylovorans sp. nov., is proposed for a facultatively chemolithotrophic and methylotrophic bacterium (strain DM10T) with the ribulose bisphosphate (RuBP) pathway of C1 assimilation. The bacterium is a Gram-negative, aerobic, asporogenous, nonmotile, colourless rod that multiplies by binary fission. The organism utilizes dichloromethane, methanol, methylamine, formate and CO2/H2, as well as a variety of polycarbon compounds, as carbon and energy sources. It is neutrophilic and mesophilic. The major cellular fatty acids are straight-chain unsaturated C18:1, saturated C16:0 and cyclopropane C19:0 acids. The main ubiquinone is Q-10. The dominant phospholipids are phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl choline and cardiolipin. The DNA G+C content is 66.7 mol%. Strain DM10T has a very low degree of DNA-DNA hybridization (4-7%) with the type species of the genera Paracoccus, Xanthobacter, Blastobacter, Angulomicrobium, Ancylobacter and Ralstonia of RuBP pathway methylobacteria. Another approach, involving comparative 16S rDNA analysis, has shown that the novel isolate represents a separate branch within the alpha-2 subgroup of the Proteobacteria. The type species of the new genus is Albibacter methylovorans sp. nov.; the type strain is DM10T (= VKM B-2236T = DSM 13819T).

Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization.

PubMed

Müller, Daniel B; Schubert, Olga T; Röst, Hannes; Aebersold, Ruedi; Vorholt, Julia A

2016-10-01

Plants are colonized by a diverse community of microorganisms, the plant microbiota, exhibiting a defined and conserved taxonomic structure. Niche separation based on spatial segregation and complementary adaptation strategies likely forms the basis for coexistence of the various microorganisms in the plant environment. To gain insights into organism-specific adaptations on a molecular level, we selected two exemplary community members of the core leaf microbiota and profiled their proteomes upon Arabidopsis phyllosphere colonization. The highly quantitative mass spectrometric technique SWATH MS was used and allowed for the analysis of over two thousand proteins spanning more than three orders of magnitude in abundance for each of the model strains. The data suggest that Sphingomonas melonis utilizes amino acids and hydrocarbon compounds during colonization of leaves whereas Methylobacterium extorquens relies on methanol metabolism in addition to oxalate metabolism, aerobic anoxygenic photosynthesis and alkanesulfonate utilization. Comparative genomic analyses indicates that utilization of oxalate and alkanesulfonates is widespread among leaf microbiota members whereas, aerobic anoxygenic photosynthesis is almost exclusively found in Methylobacteria. Despite the apparent niche separation between these two strains we also found a relatively small subset of proteins to be coregulated, indicating common mechanisms, underlying successful leaf colonization. Overall, our results reveal for two ubiquitous phyllosphere commensals species-specific adaptations to the host environment and provide evidence for niche separation within the plant microbiota. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

PubMed Central

2014-01-01

Background Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt C ) and one located on the natural occurring plasmid pBM19 (tkt P ). Results Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn2+ and Mg2+. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni2+, ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKTC: kcat/KM: 264 s-1 mM-1; TKTP: kcat/KM: 231 s-1 mM) and ribulose 5-phosphate (TKTC: kcat/KM: 109 s-1 mM; TKTP: kcat/KM: 84 s-1 mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKTC: kcat/KM: 108 s-1 mM; TKTP: kcat/KM: 71 s-1 mM) and fructose 6-phosphate (TKTC kcat/KM: 115 s-1 mM; TKTP: kcat/KM: 448 s-1 mM). Conclusions Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt P , but not of tkt C during growth with methanol [J Bacteriol 188:3063–3072, 2006] argues for TKTP being the major TKT relevant in the RuMP pathway

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus.

PubMed

Markert, Benno; Stolzenberger, Jessica; Brautaset, Trygve; Wendisch, Volker F

2014-01-09

Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt(C)) and one located on the natural occurring plasmid pBM19 (tkt(P)). Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn(2+) and Mg(2+). Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni(2+), ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKT(C): kcat/KM: 264 s(-1) mM(-1); TKT(P): kcat/KM: 231 s(-1) mM) and ribulose 5-phosphate (TKT(C): kcat/KM: 109 s(-1) mM; TKT(P): kcat/KM: 84 s(-1) mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKT(C): kcat/KM: 108 s(-1) mM; TKT(P): kcat/KM: 71 s(-1) mM) and fructose 6-phosphate (TKT(C) kcat/KM: 115 s(-1) mM; TKT(P): kcat/KM: 448 s(-1) mM). Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt(P), but not of tkt(C) during growth with methanol [J Bacteriol 188:3063-3072, 2006] argues for TKT(P) being the major TKT relevant in the RuMP pathway. Neither

Ogataea phyllophila sp. nov., Candida chumphonensis sp. nov. and Candida mattranensis sp. nov., three methylotrophic yeast species from phylloplane in Thailand.

PubMed

Koowadjanakul, Nampueng; Jindamorakot, Sasitorn; Yongmanitchai, Wichien; Limtong, Savitree

2011-08-01

Five strains (LN12, LN14(T), LN15(T), LN16 and LN17(T)) representing three novel methylotrophic yeast species were isolated from the external surface of plant leaves by three-consecutive enrichments. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene and the phylogenetic analysis, the five strains were assigned to be one novel Ogataea species and two novel Candida species. Three strains (LN12, LN14(T) and LN16) represent a single novel species of the genus Ogataea, for which the name Ogataea phyllophila sp. nov. is proposed. The type strain is LN14(T) (= BCC 42666(T) = NBRC 107780(T) = CBS 12095(T)). Strain LN15(T) was assigned to be Candida chumphonensis sp. nov. (type strain LN15(T) = BCC 42667(T) = NBRC 107781(T) = CBS 12096(T)). Strain LN17(T) represented another novel species of Candida that was named Candida mattranensis sp. nov. (type strain LN17(T) = BCC 42668(T) = NBRC 107782(T) = CBS 12097(T)).

Differential gene expression in Xylella fastidiosa 9a5c during co-cultivation with the endophytic bacterium Methylobacterium mesophilicum SR1.6/6.

PubMed

Dourado, Manuella Nóbrega; Santos, Daiene Souza; Nunes, Luiz Roberto; Costa de Oliveira, Regina Lúcia Batista da; de Oliveira, Marcus Vinicius; Araújo, Welington Luiz

2015-12-01

Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC), colonizes plant xylem, reducing sap flow, and inducing internerval chlorosis, leaf size reduction, necrosis, and harder and smaller fruits. This bacterium may be transmitted from plant to plant by sharpshooter insects, including Bucephalogonia xanthopis. The citrus endophytic bacterium Methylobacterium mesophilicum SR1.6/6 colonizes citrus xylem and previous studies showed that this strain is also transferred from plant to plant by B. xanthopis (Insecta), suggesting that this endophytic bacterium may interact with X. fastidiosa in planta and inside the insect vector during co-transmission by the same insect vector. To better understand the X. fastidiosa behavior in the presence of M. mesophilicum, we evaluated the X. fastidiosa transcriptional profile during in vitro interaction with M. mesophilicum SR1.6/6. The results showed that during co-cultivation, X. fastidiosa down-regulated genes related to growth and up-regulated genes related to energy production, stress, transport, and motility, suggesting the existence of a specific adaptive response to the presence of M. mesophilicum in the culture medium. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methylophaga natronica sp. nov., a new alkaliphilic and moderately halophilic, restricted-facultatively methylotrophic bacterium from soda lake of the Southern Transbaikal region.

PubMed

Doronina, Nina; Darmaeva, Tsyregma; Trotsenko, Yuri

2003-09-01

A new, moderately haloalkaliphilic and restricted-facultatively methylotrophic bacterium (strain Bur2T) with the ribulose monophosphate pathway of carbon assimilation is described. The isolate, which utilizes methanol, methylamine and fructose, is an aerobic, Gram-negative, asporogenous, motile short rod multiplying by binary fission. It is auxotrophic for vitamin B12, and requires NaHCO3 or NaCl for growth in alkaline medium. Cellular fatty acids profile consists primarily of straight-chain saturated C16:0, unsaturated C16:1 and C18:1 acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. Diphosphatidylglycerol is also present. Optimal growth conditions are 25-29 degrees C, pH 8.5-9.0 and 2-3% (w/v) NaCl. Cells accumulate ectoine and glutamate as the main osmoprotectants. The G + C content of the DNA is 45.0 mol%. Based on 16S rDNA sequence analysis and DNA-DNA relatedness (25-35%) with type strains of marine and soda lake methylobacteria belonging to the genus Methylophaga, the novel isolate was classified as a new species of this genus and named Methylophaga natronica (VKM B-2288T).

Effects of Bacterial Host and Dichloromethane Dehalogenase on the Competitiveness of Methylotrophic Bacteria Growing with Dichloromethane

PubMed Central

Gisi, Daniel; Willi, Laurent; Traber, Hubert; Leisinger, Thomas; Vuilleumier, Stéphane

1998-01-01

Methylobacterium sp. strain DM4 and Methylophilus sp. strain DM11 can grow with dichloromethane (DCM) as the sole source of carbon and energy by virtue of homologous glutathione-dependent DCM dehalogenases with markedly different kinetic properties (the kcat values of the enzymes of these strains are 0.6 and 3.3 s−1, respectively, and the Km values are 9 and 59 μM, respectively). These strains, as well as transconjugant bacteria expressing the DCM dehalogenase gene (dcmA) from DM11 or DM4 on a broad-host-range plasmid in the background of dcmA mutant DM4-2cr, were investigated by growing them under growth-limiting conditions and in the presence of an excess of DCM. The maximal growth rates and maximal levels of dehalogenase for chemostat-adapted bacteria were higher than the maximal growth rates and maximal levels of dehalogenase for batch-grown bacteria. The substrate saturation constant of strain DM4 was much lower than the Km of its associated dehalogenase, suggesting that this strain is adapted to scavenge low concentrations of DCM. Strains and transconjugants expressing the DCM dehalogenase from strain DM11, on the other hand, had higher growth rates than bacteria expressing the homologous dehalogenase from strain DM4. Competition experiments performed with pairs of DCM-degrading strains revealed that a strain expressing the dehalogenase from DM4 had a selective advantage in continuous culture under substrate-limiting conditions, while strains expressing the DM11 dehalogenase were superior in batch culture when there was an excess of substrate. Only DCM-degrading bacteria with a dcmA gene similar to that from strain DM4, however, were obtained in batch enrichment cultures prepared with activated sludge from sewage treatment plants. PMID:9546153

Expression and characterization of the antimicrobial peptide ABP-dHC-cecropin A in the methylotrophic yeast Pichia pastoris.

PubMed

Sang, Ming; Wei, Hui; Zhang, Jiaxin; Wei, Zhiheng; Wu, Xiaolong; Chen, Yan; Zhuge, Qiang

2017-12-01

ABP-dHC-cecropin A is a linear cationic peptide that exhibits antimicrobial properties. To explore a new approach for expression of ABP-dHC-cecropin A using the methylotrophic yeast Pichia pastoris, we cloned the ABP-dHC-cecropin A gene into the vector pPICZαA. The SacI-linearized plasmid pPICZαA-ABP-dHC-cecropin A was then transformed into P. pastoris GS115 by electroporation. Expression was induced after a 96-h incubation with 0.5% methanol at 20 °C in a culture supplied with 2% casamino acids to avoid proteolysis. Under these conditions, approximately 48 mg of ABP-dHC-cecropin A was secreted into 1L (4 × 250-mL)of medium. Recombinant ABP-dHC-cecropin A was purified using size-exclusion chromatography, and 21 mg of pure active ABP-dHC-cecropin A was obtained from 1L (4 × 250-mL)of culture. Electrophoresis on 4-20% gradient gels indicated that recombinant ABP-dHC-cecropin A was secreted as a protein approximately 4 kDa in size. Recombinant ABP-dHC-cecropin A was successfully expressed, as the product displayed antibacterial and antifungal activities (based on an antibacterial assay, scanning electron microscopy, and antifungal assay) indistinguishable from those of the synthesized protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Cluster formation in liverwort-associated methylobacteria and its implications.

PubMed

Kutschera, U; Thomas, J; Hornschuh, M

2007-08-01

Pink-pigmented methylotropic bacteria of the genus Methylobacterium inhabit the surfaces of plant organs. In bryophytes, these methylobacteria enhance cell growth, but the nature of this plant-microbe interaction is largely unknown. In this study, methylobacteria were isolated from the upper surface of the free-living thalli of the liverwort Marchantia polymorpha L. Identification of one strain by 16S ribosomal RNA (rRNA) gene-targeted polymerase chain reaction (PCR) and other data show that these microbes represent an undescribed species of the genus Methylobacterium (Methylobacterium sp.). The growth-promoting activity of these wild-type methylobacteria was tested and compared with that of the type strain Methylobacterium mesophilicum. Both types of methylobacteria stimulated surface expansion of isolated gemmae from Marchantia polymorpha by about 350%. When suspended in water, the liverwort-associated bacteria (Methylobacterium sp.) formed dense clusters of up to 600 cells. In liquid cultures of Methylobacterium mesophilicum, single cells were observed, but no clustering occurred. We suggest that the liverwort-associated methylobacteria are co-evolved symbionts of the plants: Cluster formation may be a behavior that enhances the survival of the epiphytic microbes during periods of drought of these desiccation-tolerant lower plants.

Cluster formation in liverwort-associated methylobacteria and its implications

NASA Astrophysics Data System (ADS)

Kutschera, U.; Thomas, J.; Hornschuh, M.

2007-08-01

Pink-pigmented methylotropic bacteria of the genus Methylobacterium inhabit the surfaces of plant organs. In bryophytes, these methylobacteria enhance cell growth, but the nature of this plant-microbe interaction is largely unknown. In this study, methylobacteria were isolated from the upper surface of the free-living thalli of the liverwort Marchantia polymorpha L. Identification of one strain by 16S ribosomal RNA (rRNA) gene-targeted polymerase chain reaction (PCR) and other data show that these microbes represent an undescribed species of the genus Methylobacterium ( Methylobacterium sp.). The growth-promoting activity of these wild-type methylobacteria was tested and compared with that of the type strain Methylobacterium mesophilicum. Both types of methylobacteria stimulated surface expansion of isolated gemmae from Marchantia polymorpha by about 350%. When suspended in water, the liverwort-associated bacteria ( Methylobacterium sp.) formed dense clusters of up to 600 cells. In liquid cultures of Methylobacterium mesophilicum, single cells were observed, but no clustering occurred. We suggest that the liverwort-associated methylobacteria are co-evolved symbionts of the plants: Cluster formation may be a behavior that enhances the survival of the epiphytic microbes during periods of drought of these desiccation-tolerant lower plants.

The Abundance of Pink-Pigmented Facultative Methylotrophs in the Root Zone of Plant Species in Invaded Coastal Sage Scrub Habitat

PubMed Central

Irvine, Irina C.; Brigham, Christy A.; Suding, Katharine N.; Martiny, Jennifer B. H.

2012-01-01

Pink-pigmented facultative methylotrophic bacteria (PPFMs) are associated with the roots, leaves and seeds of most terrestrial plants and utilize volatile C1 compounds such as methanol generated by growing plants during cell division. PPFMs have been well studied in agricultural systems due to their importance in crop seed germination, yield, pathogen resistance and drought stress tolerance. In contrast, little is known about the PPFM abundance and diversity in natural ecosystems, let alone their interactions with non-crop species. Here we surveyed PPFM abundance in the root zone soil of 5 native and 5 invasive plant species along ten invasion gradients in Southern California coastal sage scrub habitat. PPFMs were present in every soil sample and ranged in abundance from 102 to 105 CFU/g dry soil. This abundance varied significantly among plant species. PPFM abundance was 50% higher in the root zones of annual or biennial species (many invasives) than perennial species (all natives). Further, PPFM abundance appears to be influenced by the plant community beyond the root zone; pure stands of either native or invasive species had 50% more PPFMs than mixed species stands. In sum, PPFM abundance in the root zone of coastal sage scrub plants is influenced by both the immediate and surrounding plant communities. The results also suggest that PPFMs are a good target for future work on plant-microorganism feedbacks in natural ecosystems. PMID:22383990

The abundance of pink-pigmented facultative methylotrophs in the root zone of plant species in invaded coastal sage scrub habitat.

PubMed

Irvine, Irina C; Brigham, Christy A; Suding, Katharine N; Martiny, Jennifer B H

2012-01-01

Pink-pigmented facultative methylotrophic bacteria (PPFMs) are associated with the roots, leaves and seeds of most terrestrial plants and utilize volatile C(1) compounds such as methanol generated by growing plants during cell division. PPFMs have been well studied in agricultural systems due to their importance in crop seed germination, yield, pathogen resistance and drought stress tolerance. In contrast, little is known about the PPFM abundance and diversity in natural ecosystems, let alone their interactions with non-crop species. Here we surveyed PPFM abundance in the root zone soil of 5 native and 5 invasive plant species along ten invasion gradients in Southern California coastal sage scrub habitat. PPFMs were present in every soil sample and ranged in abundance from 10(2) to 10(5) CFU/g dry soil. This abundance varied significantly among plant species. PPFM abundance was 50% higher in the root zones of annual or biennial species (many invasives) than perennial species (all natives). Further, PPFM abundance appears to be influenced by the plant community beyond the root zone; pure stands of either native or invasive species had 50% more PPFMs than mixed species stands. In sum, PPFM abundance in the root zone of coastal sage scrub plants is influenced by both the immediate and surrounding plant communities. The results also suggest that PPFMs are a good target for future work on plant-microorganism feedbacks in natural ecosystems.

OK, thanks! A new mutualism between Chlamydomonas and methylobacteria facilitates growth on amino acids and peptides.

PubMed

Calatrava, Victoria; Hom, Erik F Y; Llamas, Ángel; Fernández, Emilio; Galván, Aurora

2018-04-01

Nitrogen is a key nutrient for land plants and phytoplankton in terrestrial and aquatic ecosystems. The model alga Chlamydomonas reinhardtii can grow efficiently on several inorganic nitrogen sources (e.g. ammonium, nitrate, nitrite) as well as many amino acids. In this study, we show that Chlamydomonas is unable to use proline, hydroxyproline and peptides that contain these amino acids. However, we discovered that algal growth on these substrates is supported in association with Methylobacterium spp., and that a mutualistic carbon-nitrogen metabolic exchange between Chlamydomonas and Methylobacterium spp. is established. Specifically, the mineralization of these amino acids and peptides by Methylobacterium spp. produces ammonium that can be assimilated by Chlamydomonas, and CO2 photosynthetically fixed by Chlamydomonas yields glycerol that can be assimilated by Methylobacterium. As Chlamydomonas is an algal ancestor to land plants and Methylobacterium is a plant growth-promoting bacterium, this new model of mutualism may facilitate insights into the ecology and evolution of plant-bacterial interactions and design principles of synthetic ecology.

Nature, nomenclature and taxonomy of obligate methanol utilizing strains.

PubMed

Cercel, M

1999-01-01

In a screening program, a number of different bacterial strains with the ability to utilize methanol as a sole carbon and energy source were isolated and described. They are well known methanol utilizing genera Pseudomonas, Klebsiella, Micrococcus, Methylomonas or, on the contrary, the new, unknown genera and species of methylotrophic bacteria. In the last category, Acinetobacter and Alcaligenes are the new reported genera of organisms able to use methanol as a sole carbon and energy source. The present paper reports the very complex physiological and biochemical modifications when very versatile bacteria such as Pseudomonas aeruginosa and Acinetobacter calcoaceticus are cultured on methanol and when the obligate methylotrophic state is compared with the facultative methylotrophic state of the same bacterial strain. Based on experiments and comparisons with literature data, it seems that Methylomonas methanica is the obligate methylotrophic state of Pseudomonas aeruginosa and that Acinetobacter calcoaceticus is the facultative methylotrophic state of Methylococcus capsulatus, an obligate methylotroph. The relationship of the obligate to the facultative and of the facultative to the obligate methylotrophy were established. These new methylotrophic genera and species, the profound physiological and biochemical modifications as well as the new data concerning nature, nomenclature and taxonomy of methanol utilizing bateria were reported for the first time in 1983.

Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha.

PubMed

Kim, Moo Woong; Rhee, Sang Ki; Kim, Jeong-Yoon; Shimma, Yoh-ichi; Chiba, Yasunori; Jigami, Yoshifumi; Kang, Hyun Ah

2004-03-01

Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins. In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H. polymorpha. Oligosaccharide profiling showed that the major oligosaccharide species derived from the H. polymorpha-secreted recombinant GOD (rGOD) had core-type structures (Man(8-12)GlcNAc(2)). Analyses using anti-alpha 1,3-mannose antibody and exoglycosidases specific for alpha 1,2- or alpha 1,6-mannose linkages revealed that the mannose outer chains of N-glycans on the rGOD have very short alpha 1,6 extensions and are mainly elongated in alpha 1,2-linkages without a terminal alpha 1,3-linked mannose addition. The N-glycans released from the H. polymorpha mannoproteins were shown to contain mostly mannose in their outer chains, which displayed almost identical size distribution and structure to those of H. polymorpha-derived rGOD. These results strongly indicate that the outer chain processing of N-glycans by H. polymorpha significantly differs from that by Saccharomyces cerevisiae, thus generating much shorter mannose outer chains devoid of terminal alpha 1,3-linked mannoses.

Mating-type switching by chromosomal inversion in methylotrophic yeasts suggests an origin for the three-locus Saccharomyces cerevisiae system

PubMed Central

Hanson, Sara J.; Byrne, Kevin P.; Wolfe, Kenneth H.

2014-01-01

Saccharomyces cerevisiae has a complex system for switching the mating type of haploid cells, requiring the genome to have three mating-type (MAT)–like loci and a mechanism for silencing two of them. How this system originated is unknown, because the three-locus system is present throughout the family Saccharomycetaceae, whereas species in the sister Candida clade have only one locus and do not switch. Here we show that yeasts in a third clade, the methylotrophs, have a simpler two-locus switching system based on reversible inversion of a section of chromosome with MATa genes at one end and MATalpha genes at the other end. In Hansenula polymorpha the 19-kb invertible region lies beside a centromere so that, depending on the orientation, either MATa or MATalpha is silenced by centromeric chromatin. In Pichia pastoris, the orientation of a 138-kb invertible region puts either MATa or MATalpha beside a telomere and represses transcription of MATa2 or MATalpha2. Both species are homothallic, and inversion of their MAT regions can be induced by crossing two strains of the same mating type. The three-locus system of S. cerevisiae, which uses a nonconservative mechanism to replace DNA at MAT, likely evolved from a conservative two-locus system that swapped genes between expression and nonexpression sites by inversion. The increasing complexity of the switching apparatus, with three loci, donor bias, and cell lineage tracking, can be explained by continuous selection to increase sporulation ability in young colonies. Our results provide an evolutionary context for the diversity of switching and silencing mechanisms. PMID:25349420

Mating-type switching by chromosomal inversion in methylotrophic yeasts suggests an origin for the three-locus Saccharomyces cerevisiae system.

PubMed

Hanson, Sara J; Byrne, Kevin P; Wolfe, Kenneth H

2014-11-11

Saccharomyces cerevisiae has a complex system for switching the mating type of haploid cells, requiring the genome to have three mating-type (MAT)-like loci and a mechanism for silencing two of them. How this system originated is unknown, because the three-locus system is present throughout the family Saccharomycetaceae, whereas species in the sister Candida clade have only one locus and do not switch. Here we show that yeasts in a third clade, the methylotrophs, have a simpler two-locus switching system based on reversible inversion of a section of chromosome with MATa genes at one end and MATalpha genes at the other end. In Hansenula polymorpha the 19-kb invertible region lies beside a centromere so that, depending on the orientation, either MATa or MATalpha is silenced by centromeric chromatin. In Pichia pastoris, the orientation of a 138-kb invertible region puts either MATa or MATalpha beside a telomere and represses transcription of MATa2 or MATalpha2. Both species are homothallic, and inversion of their MAT regions can be induced by crossing two strains of the same mating type. The three-locus system of S. cerevisiae, which uses a nonconservative mechanism to replace DNA at MAT, likely evolved from a conservative two-locus system that swapped genes between expression and nonexpression sites by inversion. The increasing complexity of the switching apparatus, with three loci, donor bias, and cell lineage tracking, can be explained by continuous selection to increase sporulation ability in young colonies. Our results provide an evolutionary context for the diversity of switching and silencing mechanisms.

Electrochemical synthesis of formic acid from CO2 catalyzed by Shewanella oneidensis MR-1 whole-cell biocatalyst.

PubMed

Le, Quang Anh Tuan; Kim, Hee Gon; Kim, Yong Hwan

2018-09-01

The electro-biocatalytic conversion of CO 2 into formic acid using whole-cell and isolated biocatalysts is useful as an alternative route for CO 2 sequestration. In this study, Shewanella oneidensis MR-1 (S. oneidensis MR-1), a facultative aerobic bacterium that has been extensively studied for its utility as biofuel cells as well as for the detoxification of heavy metal oxides (i.e., MnO 2 , uranium), has been applied for the first time as a whole-cell biocatalyst for formic acid synthesis from gaseous CO 2 and electrons supplied from an electrode. S. oneidensis MR-1, when aerobically grown in Luria-Bertani (LB) medium, exhibited its ability as a whole-cell biocatalyst for the conversion of CO 2 into formic acid with moderate productivity of 0.59 mM h -1 for 24 h. In addition, an optimization of growth conditions of S. oneidensis MR-1 resulted in a remarkable increase in productivity. The CO 2 reduction reaction catalyzed by S. oneidensis MR-1, when anaerobically grown in newly optimized LB medium supplemented with fumarate and nitrate, exhibited 3.2-fold higher productivity (1.9 mM h -1 for 72 h) compared to that grown aerobically in only LB medium. Furthermore, the average conversion rate of formic acid synthesis catalyzed by S. oneidensis MR-1 when grown in the optimal medium over a period of 72 h was 3.8 mM h -1  g -1 wet-cell, which is 9.6-fold higher than that catalyzed by Methylobacterium extorquens AM1 whole-cells in our previous study. Copyright © 2018 Elsevier Inc. All rights reserved.

Linkage of the Nit1C gene cluster to bacterial cyanide assimilation as a nitrogen source.

PubMed

Jones, Lauren B; Ghosh, Pallab; Lee, Jung-Hyun; Chou, Chia-Ni; Kunz, Daniel A

2018-05-21

A genetic linkage between a conserved gene cluster (Nit1C) and the ability of bacteria to utilize cyanide as the sole nitrogen source was demonstrated for nine different bacterial species. These included three strains whose cyanide nutritional ability has formerly been documented (Pseudomonas fluorescens Pf11764, Pseudomonas putida BCN3 and Klebsiella pneumoniae BCN33), and six not previously known to have this ability [Burkholderia (Paraburkholderia) xenovorans LB400, Paraburkholderia phymatum STM815, Paraburkholderia phytofirmans PsJN, Cupriavidus (Ralstonia) eutropha H16, Gluconoacetobacter diazotrophicus PA1 5 and Methylobacterium extorquens AM1]. For all bacteria, growth on or exposure to cyanide led to the induction of the canonical nitrilase (NitC) linked to the gene cluster, and in the case of Pf11764 in particular, transcript levels of cluster genes (nitBCDEFGH) were raised, and a nitC knock-out mutant failed to grow. Further studies demonstrated that the highly conserved nitB gene product was also significantly elevated. Collectively, these findings provide strong evidence for a genetic linkage between Nit1C and bacterial growth on cyanide, supporting use of the term cyanotrophy in describing what may represent a new nutritional paradigm in microbiology. A broader search of Nit1C genes in presently available genomes revealed its presence in 270 different bacteria, all contained within the domain Bacteria, including Gram-positive Firmicutes and Actinobacteria, and Gram-negative Proteobacteria and Cyanobacteria. Absence of the cluster in the Archaea is congruent with events that may have led to the inception of Nit1C occurring coincidentally with the first appearance of cyanogenic species on Earth, dating back 400-500 million years.

Characterization of two new facultative methoantrophs

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

Lynch, M.J.; Wopat, A.E.; O'Connor, M.L.

Two new facultative methane-oxidizing bacteria have been isolated from lake water enrichments. The organisms have been characterized in terms of colony types, growth characteristics, the guanine plus cytosine content of their deoxyribonucleic acid, thin sections, oxidation rates, and carbon assimilation pathways. Methane-grown cells of both organisms contained intracytoplasmic membranes similar to those described as type II in other methanotrophic bacteria. Both organisms assimilated methane by way of the isocitrate lyase-negative serine pathway for formaldehyde incorporation. It is proposed that both organisms be classified in the genus Methylobacterium as two new species, Methylobacterium ethanolicum and Methylobacterium hypolimneticum.

Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

PubMed Central

Stolzenberger, Jessica; Lindner, Steffen N.; Persicke, Marcus; Brautaset, Trygve

2013-01-01

The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpXC) and one on plasmid pBM19 (GlpXP), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum Δfbp mutant could be phenotypically complemented with glpXC and glpXP from B. methanolicus. GlpXP and GlpXC share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn2+ ions and inhibited by Li+, but differed in terms of the kinetic parameters. GlpXC showed a much higher catalytic efficiency and a lower Km for fructose 1,6-bisphosphate (86.3 s−1 mM−1 and 14 ± 0.5 μM, respectively) than GlpXP (8.8 s−1 mM−1 and 440 ± 7.6 μM, respectively), indicating that GlpXC is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpXP but not for GlpXC. Based on these in vitro data, GlpXP is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus. PMID:24013630

Characterization of fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase from the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus.

PubMed

Stolzenberger, Jessica; Lindner, Steffen N; Persicke, Marcus; Brautaset, Trygve; Wendisch, Volker F

2013-11-01

The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpX(C)) and one on plasmid pBM19 (GlpX(P)), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum Δfbp mutant could be phenotypically complemented with glpX(C) and glpX(P) from B. methanolicus. GlpX(P) and GlpX(C) share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn(2+) ions and inhibited by Li(+), but differed in terms of the kinetic parameters. GlpX(C) showed a much higher catalytic efficiency and a lower Km for fructose 1,6-bisphosphate (86.3 s(-1) mM(-1) and 14 ± 0.5 μM, respectively) than GlpX(P) (8.8 s(-1) mM(-1) and 440 ± 7.6 μM, respectively), indicating that GlpX(C) is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpX(P) but not for GlpX(C). Based on these in vitro data, GlpX(P) is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus.

Comprehensive Genomic Analyses of the OM43 Clade, Including a Novel Species from the Red Sea, Indicate Ecotype Differentiation among Marine Methylotrophs

PubMed Central

Jimenez-Infante, Francy; Ngugi, David Kamanda; Vinu, Manikandan; Alam, Intikhab; Kamau, Allan Anthony; Blom, Jochen; Bajic, Vladimir B.

2015-01-01

The OM43 clade within the family Methylophilaceae of Betaproteobacteria represents a group of methylotrophs that play important roles in the metabolism of C1 compounds in marine environments and other aquatic environments around the globe. Using dilution-to-extinction cultivation techniques, we successfully isolated a novel species of this clade (here designated MBRS-H7) from the ultraoligotrophic open ocean waters of the central Red Sea. Phylogenomic analyses indicate that MBRS-H7 is a novel species that forms a distinct cluster together with isolate KB13 from Hawaii (Hawaii-Red Sea [H-RS] cluster) that is separate from the cluster represented by strain HTCC2181 (from the Oregon coast). Phylogenetic analyses using the robust 16S-23S internal transcribed spacer revealed a potential ecotype separation of the marine OM43 clade members, which was further confirmed by metagenomic fragment recruitment analyses that showed trends of higher abundance in low-chlorophyll and/or high-temperature provinces for the H-RS cluster but a preference for colder, highly productive waters for the HTCC2181 cluster. This potential environmentally driven niche differentiation is also reflected in the metabolic gene inventories, which in the case of the H-RS cluster include those conferring resistance to high levels of UV irradiation, temperature, and salinity. Interestingly, we also found different energy conservation modules between these OM43 subclades, namely, the existence of the NADH:quinone oxidoreductase complex I (NUO) system in the H-RS cluster and the nonhomologous NADH:quinone oxidoreductase (NQR) system in the HTCC2181 cluster, which might have implications for their overall energetic yields. PMID:26655752

Analysis of the MT1/MT2 Systems Involved in the Metabolism of One-Carbon Compounds in "Methanosarcina acetivorans" C2A

ERIC Educational Resources Information Center

Opulencia, Rina Bagsic

2009-01-01

Methanogens are strictly anaerobic Archaea that derive their energy for growth by reducing a limited number of substrates to methane. "Methanosarcina" spp. utilize the methylotrophic pathway to grow on methanol, methylamines and methylsulfides. These compounds enter the methylotrophic pathway as methyl-coenzyme M, the synthesis of which is…

[The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene Mth1 in methylotrophic yeast Pichia methanolica].

PubMed

Leonovich, O A; Kurales, Iu A; Dutova, T A; Isakova, E P; Deriabina, Iu I; Rabinovich, Ia M

2009-01-01

Two independent mutant strains of methylotrophic yeast Pichia methanolica (mth1 arg1 and mth2 arg4) from the initial line 616 (ade1 ade5) were investigated. The mutant strains possessed defects in genes MTH1 and MTH2 which resulted in the inability to assimilate methanol as a sole carbon source and the increased activity of alcohol oxidase (AO). The function of the AUG2 gene encoding one of the subunits of AO and CTA1, a probable homolog of peroxisomal catalase of Saccharomyces cereviseae, was investigated by analyses of the molecular forms of isoenzymes. It was shown that optimal conditions for the expression of the AUG2 gene on a medium supplemented with 3% of methanol leads to an increasing synthesis of peroxisomal catalase. The mutant mth1 possessed a dominant formation of AO isoform with electrophoretic mobility which is typical for isogenic form 9, the product of the AUG2 gene, and a decreased level of peroxisomal catalase. The restoration of growth of four spontaneous revertants of the mutant mth1 (Rmth1) on the methanol containing medium was accompanied by an increase in activity of AO isogenic form 9 and peroxisomal catalase. The obtained results confirmed the functional continuity of the structural gene AUG2 in mutant mth1. The correlation of activity of peroxisomal catalase and AO isogenic form 1 in different conditions evidenced the existence of common regulatory elements for genes AUG2 and CTA1 in methilotrophic yeast Pichia methanolica.

Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1

PubMed Central

2011-01-01

Background Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast Hansenula polymorpha is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of H. polymorpha DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (GSH2) and its precursor cysteine (MET4) was studied. Results Glutathione producing capacity of H. polymorpha DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSGin, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSGex, per liter of the culture medium. Conclusions H. polymorpha is an competitive glutathione producer as compared to other known yeast and bacteria strains (Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione. PMID:21255454

Complete Genome Sequence of Methanohalophilus halophilus DSM 3094T, Isolated from a Cyanobacterial Mat and Bottom Deposits at Hamelin Pool, Shark Bay, Northwestern Australia.

PubMed

L'Haridon, Stéphane; Corre, Erwan; Guan, Yue; Vinu, Manikandan; La Cono, Violetta; Yakimov, Mickail; Stingl, Ulrich; Toffin, Laurent; Jebbar, Mohamed

2017-02-16

The complete genome sequence of Methanohalophilus halophilus DSM 3094 T , a member of the Methanosarcinaceae family and the Methanosarcianales order, consists of 2,022,959 bp in one contig and contains 2,137 predicted genes. The genome is consistent with a halophilic methylotrophic anaerobic lifestyle, including the methylotrophic and CO 2 -H 2 methanogensis pathways. Copyright © 2017 L’Haridon et al.

Genomics of Methylotrophy in Gram-Positive Methylamine-Utilizing Bacteria

PubMed Central

McTaggart, Tami L.; Beck, David A. C.; Setboonsarng, Usanisa; Shapiro, Nicole; Woyke, Tanja; Lidstrom, Mary E.; Kalyuzhnaya, Marina G.; Chistoserdova, Ludmila

2015-01-01

Gram-positive methylotrophic bacteria have been known for a long period of time, some serving as model organisms for characterizing the specific details of methylotrophy pathways/enzymes within this group. However, genome-based knowledge of methylotrophy within this group has been so far limited to a single species, Bacillus methanolicus (Firmicutes). The paucity of whole-genome data for Gram-positive methylotrophs limits our global understanding of methylotrophy within this group, including their roles in specific biogeochemical cycles, as well as their biotechnological potential. Here, we describe the isolation of seven novel strains of Gram-positive methylotrophs that include two strains of Bacillus and five representatives of Actinobacteria classified within two genera, Arthrobacter and Mycobacterium. We report whole-genome sequences for these isolates and present comparative analysis of the methylotrophy functional modules within these genomes. The genomic sequences of these seven novel organisms, all capable of growth on methylated amines, present an important reference dataset for understanding the genomic basis of methylotrophy in Gram-positive methylotrophic bacteria. This study is a major contribution to the field of methylotrophy, aimed at closing the gap in the genomic knowledge of methylotrophy within this diverse group of bacteria. PMID:27682081

Inter individual variations of the fish skin microbiota: host genetics basis of mutualism?

PubMed

Boutin, Sébastien; Sauvage, Christopher; Bernatchez, Louis; Audet, Céline; Derome, Nicolas

2014-01-01

The commensal microbiota of fish skin is suspected to provide a protection against opportunist infections. The skin of fish harbors a complex and diverse microbiota that closely interacts with the surrounding water microbial communities. Up to now there is no clear evidence as to whether the host regulates the recruitment of environmental bacteria to build a specific skin microbiota. To address this question, we detected Quantitative Trait Loci (QTL) associated with the abundance of specific skin microbiota bacterial strains in brook charr (Salvelinus fontinalis), combining 16S RNA tagged-amplicon 454 pyrosequencing with genetic linkage analysis. Skin microbiota analysis revealed high inter-individual variation among 86 F2 fish progeny based upon the relative abundance of bacterial operational taxonomic units (OTUs). Out of those OTUs, the pathogenic strain Flavobacterium psychrophilum and the non-pathogenic strain Methylobacterium rhodesianum explained the majority of inter-individual distances. Furthermore, a strong negative correlation was found between Flavobacterium and Methylobacterium, suggesting a mutually competitive relationship. Finally, after considering a total of 266 markers, genetic linkage analysis highlighted three major QTL associated with the abundance of Lysobacter, Rheinheimera and Methylobacterium. All these three genera are known for their beneficial antibacterial activity. Overall, our results provide evidence that host genotype may regulate the abundance of specific genera among their surface microbiota. They also indicate that Lysobacter, Rheinheimera and Methylobacterium are potentially important genera in providing protection against pathogens.

Phylogenetic position and emended description of the genus Methylovorus.

PubMed

Doronina, Nina V; Ivanova, Ekaterina G; Trotsenko, Yuri A

2005-03-01

The genus Methylovorus, currently represented by the restricted facultative methylotroph Methylovorus glucosotrophus Govorukhina and Trotsenko 1991 and the obligate methylotroph Methylovorus mays Doronina et al. 2001, is here established by direct sequencing of amplified 16S rRNA genes and DNA-DNA hybridization to be clearly separated from the extant ribulose monophosphate (RuMP) pathway methylobacteria and to form a distinct branch within the beta-Proteobacteria.

[Changes of chlorine isotope composition characterize bacterial dehalogenation of dichloromethane].

PubMed

Ziakun, A M; Firsova, Iu E; Torgonskaia, M L; Doronina, N V; Trotsenko, Iu A

2007-01-01

Fractionation of dichloromethane (DCM) molecules with different chlorine isotopes by aerobic methylobacteria Methylobacterium dichloromethanicum DM4 and Albibacter nethylovorans DM10; cell-free extract of strain DM4; and transconjugant Methylobacterium evtorquens Al1/pME 8220, expressing the dcmA gene for DCM dehalogenase but unable to grow on DCM, was studied. Kinetic indices of DCM isotopomers for chlorine during bacterial dehalogenation and diffusion were compared. A two-step model is proposed, which suggests diffusional DCM transport to bacterial cells.

Inter Individual Variations of the Fish Skin Microbiota: Host Genetics Basis of Mutualism?

PubMed Central

Boutin, Sébastien; Sauvage, Christopher; Bernatchez, Louis; Audet, Céline; Derome, Nicolas

2014-01-01

The commensal microbiota of fish skin is suspected to provide a protection against opportunist infections. The skin of fish harbors a complex and diverse microbiota that closely interacts with the surrounding water microbial communities. Up to now there is no clear evidence as to whether the host regulates the recruitment of environmental bacteria to build a specific skin microbiota. To address this question, we detected Quantitative Trait Loci (QTL) associated with the abundance of specific skin microbiota bacterial strains in brook charr (Salvelinus fontinalis), combining 16S RNA tagged-amplicon 454 pyrosequencing with genetic linkage analysis. Skin microbiota analysis revealed high inter-individual variation among 86 F2 fish progeny based upon the relative abundance of bacterial operational taxonomic units (OTUs). Out of those OTUs, the pathogenic strain Flavobacterium psychrophilum and the non-pathogenic strain Methylobacterium rhodesianum explained the majority of inter-individual distances. Furthermore, a strong negative correlation was found between Flavobacterium and Methylobacterium, suggesting a mutually competitive relationship. Finally, after considering a total of 266 markers, genetic linkage analysis highlighted three major QTL associated with the abundance of Lysobacter, Rheinheimera and Methylobacterium. All these three genera are known for their beneficial antibacterial activity. Overall, our results provide evidence that host genotype may regulate the abundance of specific genera among their surface microbiota. They also indicate that Lysobacter, Rheinheimera and Methylobacterium are potentially important genera in providing protection against pathogens. PMID:25068850

Methanococcoides vulcani sp. nov., a marine methylotrophic methanogen that uses betaine, choline and N,N-dimethylethanolamine for methanogenesis, isolated from a mud volcano, and emended description of the genus Methanococcoides.

PubMed

L'Haridon, Stéphane; Chalopin, Morgane; Colombo, Delphine; Toffin, Laurent

2014-06-01

A novel, strictly anaerobic, methylotrophic marine methanogen, strain SLH33(T), was isolated from deep sediment samples covered by an orange microbial mat collected from the Napoli Mud Volcano. Cells of strain SLH33(T) were Gram-stain-negative, motile, irregular cocci that occurred singly. Cells utilized trimethylamine, dimethylamine, monomethylamine, methanol, betaine, N,N-dimethylethanolamine and choline (N,N,N-trimethylethanolamine) as substrates for growth and methanogenesis. The optimal growth temperature was 30 °C; maximum growth rate was obtained at pH 7.0 in the presence of 0.5 M Na(+). The DNA G+C content of strain SLH33(T) was 43.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain SLH33(T) within the genus Methanococcoides. The novel isolate was related most closely to Methanococcoides methylutens TMA-10(T) (98.8% 16S rRNA gene sequence similarity) but distantly related to Methanococcoides burtonii DSM 6242(T) (97.6%) and Methanococcoides alaskense AK-5(T) (97.6%). DNA-DNA hybridization studies indicated that strain SLH33(T) represents a novel species, given that it shared less than 16% DNA-DNA relatedness with Methanococcoides methylutens TMA-10(T). The name Methanococcoides vulcani sp. nov. is proposed for this novel species, with strain SLH33(T) ( = DSM 26966(T) = JCM 19278(T)) as the type strain. An emended description of the genus Methanococcoides is also proposed. © 2014 IUMS.

Gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the Atlantic Ocean

PubMed Central

Dixon, Joanna L; Sargeant, Stephanie; Nightingale, Philip D; Colin Murrell, J

2013-01-01

Methanol biogeochemistry and its importance as a carbon source in seawater is relatively unexplored. We report the first microbial methanol carbon assimilation rates (k) in productive coastal upwelling waters of up to 0.117±0.002 d−1 (∼10 nmol l−1 d−1). On average, coastal upwelling waters were 11 times greater than open ocean northern temperate (NT) waters, eight times greater than gyre waters and four times greater than equatorial upwelling (EU) waters; suggesting that all upwelling waters upon reaching the surface (⩽20 m), contain a microbial population that uses a relatively high amount of carbon (0.3–10 nmol l−1 d−1), derived from methanol, to support their growth. In open ocean Atlantic regions, microbial uptake of methanol into biomass was significantly lower, ranging between 0.04–0.68 nmol l−1 d−1. Microbes in the Mauritanian coastal upwelling used up to 57% of the total methanol for assimilation of the carbon into cells, compared with an average of 12% in the EU, and 1% in NT and gyre waters. Several methylotrophic bacterial species were identified from open ocean Atlantic waters using PCR amplification of mxaF encoding methanol dehydrogenase, the key enzyme in bacterial methanol oxidation. These included Methylophaga sp., Burkholderiales sp., Methylococcaceae sp., Ancylobacter aquaticus, Paracoccus denitrificans, Methylophilus methylotrophus, Methylobacterium oryzae, Hyphomicrobium sp. and Methylosulfonomonas methylovora. Statistically significant correlations for upwelling waters between methanol uptake into cells and both chlorophyll a concentrations and methanol oxidation rates suggest that remotely sensed chlorophyll a images, in these productive areas, could be used to derive total methanol biological loss rates, a useful tool for atmospheric and marine climatically active gas modellers, and air–sea exchange scientists. PMID:23178665

Methylotroph cloning vehicle

DOEpatents

Hanson, R.S.; Allen, L.N.

1989-04-25

A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C[sub 1]-utilizing host and in a C[sub 1]-utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C[sub 1]-utilizing host to the C[sub 1]-utilizing host; DNA providing resistance to two antibiotics to which the wild-type C[sub 1]-utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C[sub 1]-utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C[sub 1]-utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C[sub 1]-utilizing (e.g., E. coli) host, and then conjugated with a selected C[sub 1]-utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C[sub 1] gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields. 3 figs.

Methylotroph cloning vehicle

DOEpatents

Hanson, Richard S.; Allen, Larry N.

1989-04-25

A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C.sub.1 -utilizing host and in a C.sub.1 -utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C.sub.1 -utilizing host to the C.sub.1 -utilizing host; DNA providing resistance to two antibiotics to which the wild-type C.sub.1 -utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C.sub.1 -utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C.sub.1 -utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C.sub.1 -utilizing (e.g., E. coli) host, and then conjugated with a selected C.sub.1 -utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C.sub.1 gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields.

Methylamine as a nitrogen source for microorganisms from a coastal marine environment.

PubMed

Taubert, Martin; Grob, Carolina; Howat, Alexandra M; Burns, Oliver J; Pratscher, Jennifer; Jehmlich, Nico; von Bergen, Martin; Richnow, Hans H; Chen, Yin; Murrell, J Colin

2017-06-01

Nitrogen is a key limiting resource for biomass production in the marine environment. Methylated amines, released from the degradation of osmolytes, could provide a nitrogen source for marine microbes. Thus far, studies in aquatic habitats on the utilization of methylamine, the simplest methylated amine, have mainly focussed on the fate of the carbon from this compound. Various groups of methylotrophs, microorganisms that can grow on one-carbon compounds, use methylamine as a carbon source. Non-methylotrophic microorganisms may also utilize methylamine as a nitrogen source, but little is known about their diversity, especially in the marine environment. In this proof-of-concept study, stable isotope probing (SIP) was used to identify microorganisms from a coastal environment that assimilate nitrogen from methylamine. SIP experiments using 15 N methylamine combined with metagenomics and metaproteomics facilitated identification of active methylamine-utilizing Alpha- and Gammaproteobacteria. The draft genomes of two methylamine utilizers were obtained and their metabolism with respect to methylamine was examined. Both bacteria identified in these SIP experiments used the γ-glutamyl-methylamide pathway, found in both methylotrophs and non-methylotrophs, to metabolize methylamine. The utilization of 15 N methylamine also led to the release of 15 N ammonium that was used as nitrogen source by other microorganisms not directly using methylamine. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris.

PubMed

Sahu, Umakant; Rangarajan, Pundi N

2016-09-23

Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) + glutamate (YNB + Glu), or YNB + aspartate (YNB + Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases, respectively; and (iv) GLN1 encoding glutamine synthetase. Synthesis of all these enzymes is regulated by Mxr1p at the level of transcription except GDH2, whose synthesis is regulated at the level of translation. Mxr1p activates the transcription of AAT1, AAT2, and GLN1 in cells cultured in YP as well as in YNB + Glu media, whereas transcription of MDH1 and MDH2 is activated in cells cultured in YNB + Glu but not in YP. A truncated Mxr1p composed of 400 N-terminal amino acids activates transcription of target genes in cells cultured in YP but not in YNB + Glu. Mxr1p binds to Mxr1p response elements present in the promoters of AAT2, MDH2, and GLN1 We conclude that Mxr1p is essential for utilization of amino acids as the sole source of carbon and nitrogen, and it is a global regulator of multiple metabolic pathways in P. pastoris. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcriptome analysis of thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape.

PubMed

Irla, Marta; Neshat, Armin; Brautaset, Trygve; Rückert, Christian; Kalinowski, Jörn; Wendisch, Volker F

2015-02-14

Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5'-ends. Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5'-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are

Prerequisites for amplicon pyrosequencing of microbial methanol utilizers in the environment

PubMed Central

Kolb, Steffen; Stacheter, Astrid

2013-01-01

The commercial availability of next generation sequencing (NGS) technologies facilitated the assessment of functional groups of microorganisms in the environment with high coverage, resolution, and reproducibility. Soil methylotrophs were among the first microorganisms in the environment that were assessed with molecular tools, and nowadays, as well with NGS technologies. Studies in the past years re-attracted notice to the pivotal role of methylotrophs in global conversions of methanol, which mainly originates from plants, and is involved in oxidative reactions and ozone formation in the atmosphere. Aerobic methanol utilizers belong to Bacteria, yeasts, Ascomycota, and molds. Numerous bacterial methylotrophs are facultatively aerobic, and also contribute to anaerobic methanol oxidation in the environment, whereas strict anaerobic methanol utilizers belong to methanogens and acetogens. The diversity of enzymes catalyzing the initial oxidation of methanol is considerable, and comprises at least five different enzyme types in aerobes, and one in strict anaerobes. Only the gene of the large subunit of pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH; mxaF) has been analyzed by environmental pyrosequencing. To enable a comprehensive assessment of methanol utilizers in the environment, new primers targeting genes of the PQQ MDH in Methylibium (mdh2), of the nicotinamide adenine dinucleotide-dependent MDH (mdh), of the methanol oxidoreductase of Actinobacteria (mdo), of the fungal flavin adenine nucleotide-dependent alcohol oxidase (mod1, mod2, and homologs), and of the gene of the large subunit of the methanol:corrinoid methyltransferases (mtaC) in methanogens and acetogens need to be developed. Combined stable isotope probing of nucleic acids or proteins with amplicon-based NGS are straightforward approaches to reveal insights into functions of certain methylotrophic taxa in the global methanol cycle. PMID:24046766

Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast.

PubMed

Zepeda, Andrea B; Pessoa, Adalberto; Farías, Jorge G

2018-05-19

Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production. Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Spatial Variations of the Methanogenic Communities in the Sediments of Tropical Mangroves

PubMed Central

Jing, Hongmei; Cheung, Shunyan; Zhou, Zhi; Wu, Chen; Nagarajan, Sanjay; Liu, Hongbin

2016-01-01

Methane production by methanogens in mangrove sediments is known to contribute significantly to global warming, but studies on the shift of methanogenic community in response to anthropogenic contaminations were still limited. In this study, the effect of anthropogenic activities in the mangrove sediments along the north and south coastlines of Singapore were investigated by pyrosequencing of the mcrA gene. Our results showed that hydrogenotrophic, acetoclastic and methylotrophic methanogens coexist in the sediments. The predominance of the methylotrophic Methanosarcinales reflects the potential for high methane production as well as the possible availability of low acetate and high methylated C-1 compounds as substrates. A decline in the number of acetoclastic/methylotrophic methanogens in favor of hydrogenotrophic methanogens was observed along a vertical profile in Sungei Changi, which was contaminated by heavy metals. The diversity of methanogens in the various contaminated stations was significantly different from that in a pristine St. John’s Island. The spatial variation in the methanogenic communities among the different stations was more distinct than those along the vertical profiles at each station. We suggest that the overall heterogeneity of the methanogenic communities residing in the tropical mangrove sediments might be due to the accumulated effects of temperature and concentrations of nitrate, cobalt, and nickel. PMID:27684479

Methanogenic diversity and activity in hypersaline sediments of the centre of the Napoli mud volcano, Eastern Mediterranean Sea.

PubMed

Lazar, Cassandre Sara; Parkes, R John; Cragg, Barry A; L'Haridon, Stéphane; Toffin, Laurent

2011-08-01

Submarine mud volcanoes are a significant source of methane to the atmosphere. The Napoli mud volcano, situated in the brine-impacted Olimpi Area of the Eastern Mediterranean Sea, emits mainly biogenic methane particularly at the centre of the mud volcano. Temperature gradients support the suggestion that Napoli is a cold mud volcano with moderate fluid flow rates. Biogeochemical and molecular genetic analyses were carried out to assess the methanogenic activity rates, pathways and diversity in the hypersaline sediments of the centre of the Napoli mud volcano. Methylotrophic methanogenesis was the only significant methanogenic pathway in the shallow sediments (0-40 cm) but was also measured throughout the sediment core, confirming that methylotrophic methanogens could be well adapted to hypersaline environments. Hydrogenotrophic methanogenesis was the dominant pathway below 50 cm; however, low rates of acetoclastic methanogenesis were also present, even in sediment layers with the highest salinity, showing that these methanogens can thrive in this extreme environment. PCR-DGGE and methyl coenzyme M reductase gene libraries detected sequences affiliated with anaerobic methanotrophs (mainly ANME-1) as well as Methanococcoides methanogens. Results show that the hypersaline conditions in the centre of the Napoli mud volcano influence active biogenic methane fluxes and methanogenic/methylotrophic diversity. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization.

PubMed

Whitaker, William B; Sandoval, Nicholas R; Bennett, Robert K; Fast, Alan G; Papoutsakis, Eleftherios T

2015-06-01

Synthetic methylotrophy is the development of non-native methylotrophs that can utilize methane and methanol as sole carbon and energy sources or as co-substrates with carbohydrates to produce metabolites as biofuels and chemicals. The availability of methane (from natural gas) and its oxidation product, methanol, has been increasing, while prices have been decreasing, thus rendering them as attractive fermentation substrates. As they are more reduced than most carbohydrates, methane and methanol, as co-substrates, can enhance the yields of biologically produced metabolites. Here we discuss synthetic biology and metabolic engineering strategies based on the native biology of aerobic methylotrophs for developing synthetic strains grown on methanol, with Escherichia coli as the prototype. Copyright © 2015. Published by Elsevier Ltd.

Using "Leaf Prints" To Investigate a Common Bacterium.

ERIC Educational Resources Information Center

Holland, Mark A.; Davis, Rebecca; Moffitt, Stephanie; O'Laughlin, Kristinae; Peach, Denise; Sussan, Stacy; Wimbrow, Lori; Tayman, Bryan

2000-01-01

Describes a laboratory activity investigating important concepts in microbiology and ecology in which students design and carry out their own experiments and learn about microorganisms. Points out that all microorganisms are not dangerous and studies the relationship between Methylobacterium and plants. (YDS)

The effects of metabolite molecules produced by drinking water-isolated bacteria on their single and multispecies biofilms.

PubMed

Simões, Lúcia Chaves; Simões, Manuel; Vieira, Maria João

2011-08-01

The elucidation of the mechanisms by which diverse species survive and interact in drinking water (DW) biofilm communities may allow the identification of new biofilm control strategies. The purpose of the present study was to investigate the effects of metabolite molecules produced by bacteria isolated from DW on biofilm formation. Six opportunistic bacteria, viz. Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp. isolated from a drinking water distribution systems (DWDS) were used to form single and multispecies biofilms in the presence and absence of crude cell-free supernatants produced by the partner bacteria. Biofilms were characterized in terms of mass and metabolic activity. Additionally, several physiological aspects regulating interspecies interactions (sessile growth rates, antimicrobial activity of cell-free supernatants, and production of iron chelators) were studied to identify bacterial species with biocontrol potential in DWDS. Biofilms of Methylobacterium sp. had the highest growth rate and M. mucogenicum biofilms the lowest. Only B. cepacia was able to produce extracellular iron-chelating molecules. A. calcoaceticus, B. cepacia, Methylobacterium sp. and M. mucogenicum biofilms were strongly inhibited by crude cell-free supernatants from the other bacteria. The crude cell-free supernatants of M. mucogenicum and S. capsulata demonstrated a high potential for inhibiting the growth of counterpart biofilms. Multispecies biofilm formation was strongly inhibited in the absence of A. calcoaceticus. Only crude cell-free supernatants produced by B. cepacia and A. calcoaceticus had no inhibitory effects on multispecies biofilm formation, while metabolite molecules of M. mucogenicum showed the most significant biocontrol potential.

Consumption of Tropospheric Levels of Methyl Bromide by C1 Compound-Utilizing Bacteria and Comparison to Saturation Kinetics

USGS Publications Warehouse

Goodwin, K.D.; Varner, R.K.; Crill, P.M.; Oremland, R.S.

2001-01-01

Pure cultures of methylotrophs and methanotrophs are known to oxidize methyl bromide (MeBr); however, their ability to oxidize tropospheric concentrations (parts per trillion by volume [pptv]) has not been tested. Methylotrophs and methanotrophs were able to consume MeBr provided at levels that mimicked the tropospheric mixing ratio of MeBr (12 pptv) at equilibrium with surface waters (???2 pM). Kinetic investigations using picomolar concentrations of MeBr in a continuously stirred tank reactor (CSTR) were performed using strain IMB-1 and Leisingeria methylohalidivorans strain MB2T - terrestrial and marine methylotrophs capable of halorespiration. First-order uptake of MeBr with no indication of threshold was observed for both strains. Strain MB2T displayed saturation kinetics in batch experiments using micromolar MeBr concentrations, with an apparent Ks of 2.4 ??M MeBr and a Vmax of 1.6 nmol h-1 (106 cells)-1. Apparent first-order degradation rate constants measured with the CSTR were consistent with kinetic parameters determined in batch experiments, which used 35- to 1 ?? 107-fold-higher MeBr concentrations. Ruegeria algicola (a phylogenetic relative of strain MB2T), the common heterotrophs Escherichia coli and Bacillus pumilus, and a toluene oxidizer, Pseudomonas mendocina KR1, were also tested. These bacteria showed no significant consumption of 12 pptv MeBr; thus, the ability to consume ambient mixing ratios of MeBr was limited to C1 compound-oxidizing bacteria in this study. Aerobic C1 bacteria may provide model organisms for the biological oxidation of tropospheric MeBr in soils and waters.

Assessment of the endogenous respiration rate and the observed biomass yield for methanol-fed denitrifying bacteria under anoxic and aerobic conditions.

PubMed

Alikhani, Jamal; Al-Omari, Ahmed; De Clippeleir, Haydee; Murthy, Sudhir; Takacs, Imre; Massoudieh, Arash

2017-01-01

In this study, the endogenous respiration rate and the observed biomass yield of denitrifying methylotrophic biomass were estimated through measuring changes in denitrification rates (DNR) as a result of maintaining the biomass under methanol deprived conditions. For this purpose, activated sludge biomass from a full-scale wastewater treatment plant was kept in 10-L batch reactors for 8 days under fully aerobic and anoxic conditions at 20 °C without methanol addition. To investigate temperature effects, another biomass sample was placed under starvation conditions over a period of 10 days under aerobic conditions at 25 °C. A series of secondary batch tests were conducted to measure DNR and observed biomass yields. The decline in DNR over the starvation period was used as a surrogate to biomass decay rate in order to infer the endogenous respiration rates of the methylotrophs. The regression analysis on the declining DNR data shows 95% confidence intervals of 0.130 ± 0.017 day -1 for endogenous respiration rate under aerobic conditions at 20 °C, 0.102 ± 0.013 day -1 under anoxic conditions at 20 °C, and 0.214 ± 0.044 day -1 under aerobic conditions at 25 °C. Results indicated that the endogenous respiration rate of methylotrophs is 20% slower under anoxic conditions than under aerobic conditions, and there is a significant temperature dependency, with an Arrhenius coefficient of 1.10. The observed biomass yield value showed an increasing trend from approximately 0.2 to 0.6 when the starvation time increased from 0 to 10 days.

Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1.

PubMed

Wu, Tung-Yun; Chen, Chang-Ting; Liu, Jessica Tse-Jin; Bogorad, Igor W; Damoiseaux, Robert; Liao, James C

2016-06-01

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the southern African crotalarioid clade Lotononis s.l.

PubMed Central

Ardley, Julie K.; Reeve, Wayne G.; O'Hara, Graham W.; Yates, Ron J.; Dilworth, Michael J.; Howieson, John G.

2013-01-01

Background and Aims The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. Methods Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. Key Results Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. Conclusions Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of

Biofilm-forming activity of bacteria isolated from toilet bowl biofilms and the bactericidal activity of disinfectants against the isolates.

PubMed

Mori, Miho; Gomi, Mitsuhiro; Matsumune, Norihiko; Niizeki, Kazuma; Sakagami, Yoshikazu

2013-01-01

To evaluate the sanitary conditions of toilets, the bacterial counts of the toilet bowl biofilms in 5 Kansai area and 11 Kansai and Kanto area homes in Japan were measured in winter and summer seasons, respectively. Isolates (128 strains) were identified by analyzing 16S ribosomal RNA sequences. The number of colonies and bacterial species from biofilms sampled in winter tended to be higher and lower, respectively, than those in summer. Moreover, the composition of bacterial communities in summer and winter samples differed considerably. In summer samples, biofilms in Kansai and Kanto areas were dominated by Blastomonas sp. and Mycobacterium sp., respectively. Methylobacterium sp. was detected in all toilet bowl biofilms except for one sample. Methylobacterium sp. constituted the major presence in biofilms along with Brevundimonas sp., Sphingomonas sp., and/or Pseudomonas sp. The composition ratio of the sum of their genera was 88.0 from 42.9% of the total bacterial flora. The biofilm formation abilities of 128 isolates were investigated, and results suggested that Methylobacterium sp. and Sphingomonas sp. were involved in biofilm formation in toilet bowls. The biofilm formation of a mixed bacteria system that included bacteria with the highest biofilm-forming ability in a winter sample was greater than mixture without such bacteria. This result suggests that isolates possessing a high biofilm-forming activity are involved in the biofilm formation in the actual toilet bowl. A bactericidal test against 25 strains indicated that the bactericidal activities of didecyldimethylammonium chloride (DDAC) tended to be higher than those of polyhexamethylene biguanide (PHMB) and N-benzyl-N,N-dimethyldodecylammonium chloride (ADBAC). In particular, DDAC showed high bactericidal activity against approximately 90% of tested strains under the 5 h treatment.

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli

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

Whitaker, W. Brian; Jones, J. Andrew; Bennett, R. Kyle

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolyticmore » intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. In conclusion, by incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.« less

Evidence of active methanogen communities in shallow sediments of the sonora margin cold seeps.

PubMed

Vigneron, Adrien; L'Haridon, Stéphane; Godfroy, Anne; Roussel, Erwan G; Cragg, Barry A; Parkes, R John; Toffin, Laurent

2015-05-15

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Diffusional limits to the consumption of atmospheric methane by soils

USGS Publications Warehouse

Striegl, Robert G.

1993-01-01

Net transport of atmospheric gases into and out of soil systems is primarily controlled by diffusion along gas partial pressure gradients. Gas fluxes between soil and the atmosphere can therefore be estimated by a generalization of the equation for ordinary gaseous diffusion in porous unsaturated media. Consumption of CH4 by methylotrophic bacteria in the top several centimeters of soil causes the uptake of atmospheric CH4 by aerated soils. The capacity of the methylotrophs to consume CH4 commonly exceeds the potential of CH4 to diffuse from the atmosphere to the consumers. The maximum rate of uptake of atmospheric CH4 by soil is, therefore, limited by diffusion and can be calculated from soil physical properties and the CH4 concentration gradient. The CH4 concentration versus depth profile is theoretically described by the equation for gaseous diffusion with homogeneous chemical reaction in porous unsaturated media. This allows for calculation of the in situ rate of CH4 consumption within specified depth intervals.

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli

DOE PAGES

Whitaker, W. Brian; Jones, J. Andrew; Bennett, R. Kyle; ...

2016-11-01

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolyticmore » intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. In conclusion, by incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.« less

Microbial communities of deep-sea methane seeps at Hikurangi continental margin (New Zealand).

PubMed

Ruff, S Emil; Arnds, Julia; Knittel, Katrin; Amann, Rudolf; Wegener, Gunter; Ramette, Alban; Boetius, Antje

2013-01-01

The methane-emitting cold seeps of Hikurangi margin (New Zealand) are among the few deep-sea chemosynthetic ecosystems of the Southern Hemisphere known to date. Here we compared the biogeochemistry and microbial communities of a variety of Hikurangi cold seep ecosystems. These included highly reduced seep habitats dominated by bacterial mats, partially oxidized habitats populated by heterotrophic ampharetid polychaetes and deeply oxidized habitats dominated by chemosynthetic frenulate tubeworms. The ampharetid habitats were characterized by a thick oxic sediment layer that hosted a diverse and biomass-rich community of aerobic methanotrophic Gammaproteobacteria. These bacteria consumed up to 25% of the emanating methane and clustered within three deep-branching groups named Marine Methylotrophic Group (MMG) 1-3. MMG1 and MMG2 methylotrophs belong to the order Methylococcales, whereas MMG3 methylotrophs are related to the Methylophaga. Organisms of the groups MMG1 and MMG3 are close relatives of chemosynthetic endosymbionts of marine invertebrates. The anoxic sediment layers of all investigated seeps were dominated by anaerobic methanotrophic archaea (ANME) of the ANME-2 clade and sulfate-reducing Deltaproteobacteria. Microbial community analysis using Automated Ribosomal Intergenic Spacer Analysis (ARISA) showed that the different seep habitats hosted distinct microbial communities, which were strongly influenced by the seep-associated fauna and the geographic location. Despite outstanding features of Hikurangi seep communities, the organisms responsible for key ecosystem functions were similar to those found at seeps worldwide. This suggests that similar types of biogeochemical settings select for similar community composition regardless of geographic distance. Because ampharetid polychaetes are widespread at cold seeps the role of aerobic methanotrophy may have been underestimated in seafloor methane budgets.

Metagenomic survey of methanesulfonic acid (MSA) catabolic genes in an Atlantic Ocean surface water sample and in a partial enrichment

PubMed Central

Henriques, Ana C.; Azevedo, Rui M.S.

2016-01-01

Methanesulfonic acid (MSA) is a relevant intermediate of the biogeochemical cycle of sulfur and environmental microorganisms assume an important role in the mineralization of this compound. Several methylotrophic bacterial strains able to grow on MSA have been isolated from soil or marine water and two conserved operons, msmABCD coding for MSA monooxygenase and msmEFGH coding for a transport system, have been repeatedly encountered in most of these strains. Homologous sequences have also been amplified directly from the environment or observed in marine metagenomic data, but these showed a base composition (G + C content) very different from their counterparts from cultivated bacteria. The aim of this study was to understand which microorganisms within the coastal surface oceanic microflora responded to MSA as a nutrient and how the community evolved in the early phases of an enrichment by means of metagenome and gene-targeted amplicon sequencing. From the phylogenetic point of view, the community shifted significantly with the disappearance of all signals related to the Archaea, the Pelagibacteraceae and phylum SAR406, and the increase in methylotroph-harboring taxa, accompanied by other groups so far not known to comprise methylotrophs such as the Hyphomonadaceae. At the functional level, the abundance of several genes related to sulfur metabolism and methylotrophy increased during the enrichment and the allelic distribution of gene msmA diagnostic for MSA monooxygenase altered considerably. Even more dramatic was the disappearance of MSA import-related gene msmE, which suggests that alternative transporters must be present in the enriched community and illustrate the inadequacy of msmE as an ecofunctional marker for MSA degradation at sea. PMID:27761315

Microbial Communities of Deep-Sea Methane Seeps at Hikurangi Continental Margin (New Zealand)

PubMed Central

Ruff, S. Emil; Arnds, Julia; Knittel, Katrin; Amann, Rudolf; Wegener, Gunter; Ramette, Alban; Boetius, Antje

2013-01-01

The methane-emitting cold seeps of Hikurangi margin (New Zealand) are among the few deep-sea chemosynthetic ecosystems of the Southern Hemisphere known to date. Here we compared the biogeochemistry and microbial communities of a variety of Hikurangi cold seep ecosystems. These included highly reduced seep habitats dominated by bacterial mats, partially oxidized habitats populated by heterotrophic ampharetid polychaetes and deeply oxidized habitats dominated by chemosynthetic frenulate tubeworms. The ampharetid habitats were characterized by a thick oxic sediment layer that hosted a diverse and biomass-rich community of aerobic methanotrophic Gammaproteobacteria. These bacteria consumed up to 25% of the emanating methane and clustered within three deep-branching groups named Marine Methylotrophic Group (MMG) 1-3. MMG1 and MMG2 methylotrophs belong to the order Methylococcales, whereas MMG3 methylotrophs are related to the Methylophaga . Organisms of the groups MMG1 and MMG3 are close relatives of chemosynthetic endosymbionts of marine invertebrates. The anoxic sediment layers of all investigated seeps were dominated by anaerobic methanotrophic archaea (ANME) of the ANME-2 clade and sulfate-reducing Deltaproteobacteria. Microbial community analysis using Automated Ribosomal Intergenic Spacer Analysis (ARISA) showed that the different seep habitats hosted distinct microbial communities, which were strongly influenced by the seep-associated fauna and the geographic location. Despite outstanding features of Hikurangi seep communities, the organisms responsible for key ecosystem functions were similar to those found at seeps worldwide. This suggests that similar types of biogeochemical settings select for similar community composition regardless of geographic distance. Because ampharetid polychaetes are widespread at cold seeps the role of aerobic methanotrophy may have been underestimated in seafloor methane budgets. PMID:24098632

Rapid quantitative estimation of chlorinated methane utilizing bacteria in drinking water and the effect of nanosilver on biodegradation of the trichloromethane in the environment.

PubMed

Zamani, Isaac; Bouzari, Majid; Emtiazi, Giti; Fanaei, Maryam

2015-03-01

Halomethanes are toxic and carcinogenic chemicals, which are widely used in industry. Also they can be formed during water disinfection by chlorine. Biodegradation by methylotrophs is the most important way to remove these pollutants from the environment. This study aimed to represent a simple and rapid method for quantitative study of halomethanes utilizing bacteria in drinking water and also a method to facilitate the biodegradation of these compounds in the environment compared to cometabolism. Enumeration of chlorinated methane utilizing bacteria in drinking water was carried out by most probable number (MPN) method in two steps. First, the presence and the number of methylotroph bacteria were confirmed on methanol-containing medium. Then, utilization of dichloromethane was determined by measuring the released chloride after the addition of 0.04 mol/L of it to the growth medium. Also, the effect of nanosilver particles on biodegradation of multiple chlorinated methanes was studied by bacterial growth on Bushnell-Haas Broth containing chloroform (trichloromethane) that was treated with 0.2 ppm nanosilver. Most probable number of methylotrophs and chlorinated methane utilizing bacteria in tested drinking water were 10 and 4 MPN Index/L, respectively. Chloroform treatment by nanosilver leads to dechlorination and the production of formaldehyde. The highest growth of bacteria and formic acid production were observed in the tubes containing 1% chloroform treated with nanosilver. By combining the two tests, a rapid approach to estimation of most probable number of chlorinated methane utilizing bacteria is introduced. Treatment by nanosilver particles was resulted in the easier and faster biodegradation of chloroform by bacteria. Thus, degradation of these chlorinated compounds is more efficient compared to cometabolism.

Isolation and characterization of aerobic anoxygenic phototrophs from exposed soils from the Sør Rondane Mountains, East Antarctica.

PubMed

Tahon, Guillaume; Willems, Anne

2017-09-01

This study investigated the culturable aerobic phototrophic bacteria present in soil samples collected in the proximity of the Belgian Princess Elisabeth Station in the Sør Rondane Mountains, East Antarctica. Until recently, only oxygenic phototrophic bacteria (Cyanobacteria) were well known from Antarctic soils. However, more recent non-cultivation-based studies have demonstrated the presence of anoxygenic phototrophs and, particularly, aerobic anoxygenic phototrophic bacteria in these areas. Approximately 1000 isolates obtained after prolonged incubation under different growth conditions were studied and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Representative strains were identified by sequence analysis of 16S rRNA genes. More than half of the isolates grouped among known aerobic anoxygenic phototrophic taxa, particularly with Sphingomonadaceae, Methylobacterium and Brevundimonas. In addition, a total of 330 isolates were tested for the presence of key phototrophy genes. While rhodopsin genes were not detected, multiple isolates possessed key genes of the bacteriochlorophyll synthesis pathway. The majority of these potential aerobic anoxygenic phototrophic strains grouped with Alphaproteobacteria (Sphingomonas, Methylobacterium, Brevundimonas and Polymorphobacter). Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Roseomonas, a new genus associated with bacteremia and other human infections.

PubMed Central

Rihs, J D; Brenner, D J; Weaver, R E; Steigerwalt, A G; Hollis, D G; Yu, V L

1993-01-01

In the 1980s, a pink bacterium different from species of the genus Methylobacterium was implicated in human infection. Using biochemical tests and DNA hybridization, we examined 42 strains of pink-pigmented, gram-negative bacteria that were not members of the genus Methylobacterium. The isolates included 6 strains each of CDC "pink coccoid" groups I, II, III, and IV; 10 isolates from Gilardi's "unnamed taxon"; and 8 blood isolates from ill, debilitated, or immunosuppressed patients. The DNA hybridization studies supported the creation of six genomospecies encompassing the 42 strains. Reactions for esculin hydrolysis, glycerol oxidation, and D-mannose oxidation enabled separation of genomospecies 1 through 4. These tests, as well as motility, nitrate reduction, citrate utilization, and oxidation of L-arabinose, D-galactose, and D-xylose, differentiated genomospecies 5 and 6 from each other and from genomospecies 1 through 4. These organisms were susceptible in vitro to the aminoglycosides, tetracycline, and imipenem and generally susceptible to the quinolones. We propose the new genus, Roseomonas, for these bacteria to include three named species, Roseomonas gilardii sp. nov., Roseomonas cervicalis sp. nov., and Roseomonas fauriae sp. nov., and three unnamed genomospecies. Images PMID:8308122

Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase.

Treesearch

Richard J. Baerends; Grietje J. Sulter; Thomas W. Jeffries; James M. Cregg; Marten Veenhuis

2002-01-01

Glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the catabolism of methanol as a carbon source and certain primary amines, such as methylamine as nitrogen sources in methylotrophic yeasts. Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha. Unlike the recently described Pichia pastoris...

Obligate methylotrophy: evaluation of dimethyl ether as a C1 compound.

PubMed Central

Meyers, A J

1982-01-01

The suitability of dimethyl ether as a C1 compound was examined with the obligate methylobacterium Methylococcus capsulatus (Texas). The ether did not support growth and was not formed during growth on methane; it was an inhibitor of growth and oxidation of methane and a poor oxidation substrate for cell suspensions. NADH stimulation of methane, but not dimethyl ether, oxidation occurred in cell extracts. PMID:6802804

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli.

PubMed

Whitaker, W Brian; Jones, J Andrew; Bennett, R Kyle; Gonzalez, Jacqueline E; Vernacchio, Victoria R; Collins, Shannon M; Palmer, Michael A; Schmidt, Samuel; Antoniewicz, Maciek R; Koffas, Mattheos A; Papoutsakis, Eleftherios T

2017-01-01

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13 C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Formation of methane and carbon dioxide from dimethylselenide in anoxic sediments and by a methanogenic bacterium

USGS Publications Warehouse

Oremland, Ronald S.; Zehr, Jon P.

1986-01-01

Anaerobic San Francisco Bay salt marsh sediments rapidly metabolized [14C]dimethylselenide (DMSe) to 14CH4 and 14CO2. Addition of selective inhibitors (2-bromoethanesulfonic acid or molybdate) to these sediments indicated that both methanogenic and sulfate-respiring bacteria could degrade DMSe to gaseous products. However, sediments taken from the selenium-contaminated Kesterson Wildlife Refuge produced only 14CO2 from [14C]DMSe, implying that methanogens were not important in the Kesterson samples. A pure culture of a dimethylsulfide (DMS)-grown methylotrophic methanogen converted [14C]DMSe to 14CH4 and14CO2. However, the organism could not grow on DMSe. Addition of DMS to either sediments or the pure culture retarded the metabolism of DMSe. This effect appeared to be caused by competitive inhibition, thereby indicating a common enzyme system for DMS and DMSe metabolism. DMSe appears to be degraded as part of the DMS pool present in anoxic environments. These results suggest that methylotrophic methanogens may demethylate methylated forms of other metals and metalloids found in nature.

Diversity and physiology of culturable bacteria associated with a coastal Antarctic ice core.

PubMed

Antony, Runa; Krishnan, K P; Laluraj, C M; Thamban, Meloth; Dhakephalkar, P K; Engineer, Anupama S; Shivaji, S

2012-06-20

Microbiological studies of polar ice at different depths may provide important comparisons, as they preserve records of microbial cells and past climate. In this study, we examined bacterial abundance, diversity and glaciochemical composition from three depths of an ice core from coastal Dronning Maud Land, East Antarctica. Higher bacterial abundance corresponded with high in situ sea-salt Na(+) and dust concentration, suggesting that bacteria might have been transported and deposited into ice along with dust particles and marine aerosols. Fourteen bacterial isolates belonging to the genera Methylobacterium, Brevundimonas, Paenibacillus, Bacillus and Micrococcus were retrieved. Frequent isolation of similar bacterial genera from different cold environments suggests that they possess features that enable survival and metabolism for extended periods of time at sub-zero temperatures. The highest number and diversity of recoverable bacteria was obtained from 49 m depth corresponding to 1926 AD and consisted of bacteria from 4 different genera whereas at 11 m (1989 AD) and 33 m (1953 AD) samples only species belonging to the genera Bacillus was recovered. Among the Bacillus species, Bacillus aryabhattai which has been reported only from the upper stratosphere, was isolated and is the first record from the Earth's surface. Methylobacterium was the most dominant genera at 49 m depth and its prevalence is attributable to a combination of high in situ methanesulfonate concentration, specialized metabolism and environmental hardiness of Methylobacterium. Some of the isolated bacteria were found to respire and grow using methanesulfonate, suggesting that they may utilize this substrate to sustain growth in ice. In addition, NO(3)(-) (2.93-3.69 μM), NH(4)(+) (1.45-3.90 μM) and PO(4)(3-) (0.01-0.75 μM) present in the ice could be potential sources fueling bacterial metabolism in this environment. It could be deduced from the study that variation in bacterial abundance and

Development and use of fluorescent 16S rRNA-targeted probes for the specific detection of Methylophaga species by in situ hybridization in marine sediments.

PubMed

Janvier, Monique; Regnault, Béatrice; Grimont, Patrick

2003-09-01

Methylotrophic bacteria are widespread in nature. They may play an important role in the cycling of carbon and in the metabolism of dimethylsulfide in a marine environment. Bacteria belonging to the genus Methylophaga are a unique group of aerobic, halophilic, non-methane-utilizing methylotrophs. Two 16S rRNA-targeted oligonucleotide probes were developed for the specific detection of Methylophaga species, marine methylobacteria, by fluorescence in situ hybridization. Probe MPH-730 was highly specific for all members of the genus Methylophaga while probe MPHm-994 targeted exclusively M. marina. The application of these probes were demonstrated by the detection of Methylophaga species in enrichment cultures from various marine sediments. All isolates recovered were visualized by using the genus specific probe MPH-730. The results were confirmed by 16S rDNA sequencing which demonstrated that all selected isolates belong to Methylophaga. Five isolates could be detected by the M. marina-specific probe MPHm-994 and were confirmed by rRNA gene restriction pattern (ribotyping). With the development of these specific probes, fluorescence in situ hybridization shows that the genus Methylophaga is widespread in marine samples.

Microbial megacities fueled by methane oxidation in a mineral spring cave

PubMed Central

Karwautz, Clemens; Kus, Günter; Stöckl, Michael; Neu, Thomas R; Lueders, Tillmann

2018-01-01

Massive biofilms have been discovered in the cave of an iodine-rich former medicinal spring in southern Germany. The biofilms completely cover the walls and ceilings of the cave, giving rise to speculations about their metabolism. Here we report on first insights into the structure and function of the biofilm microbiota, combining geochemical, imaging and molecular analytics. Stable isotope analysis indicated that thermogenic methane emerging into the cave served as an important driver of biofilm formation. The undisturbed cavern atmosphere contained up to 3000 p.p.m. methane and was microoxic. A high abundance and diversity of aerobic methanotrophs primarily within the Methylococcales (Gammaproteobacteria) and methylotrophic Methylophilaceae (Betaproteobacteria) were found in the biofilms, along with a surprising diversity of associated heterotrophic bacteria. The highest methane oxidation potentials were measured for submerged biofilms on the cavern wall. Highly organized globular structures of the biofilm matrix were revealed by fluorescent lectin staining. We propose that the extracellular matrix served not only as an electron sink for nutrient-limited biofilm methylotrophs but potentially also as a diffusive barrier against volatilized iodine species. Possible links between carbon and iodine cycling in this peculiar habitat are discussed. PMID:28949325

Diversity and Function of Methanotrophic Bacteria in Caves

NASA Astrophysics Data System (ADS)

Webster, K.; Schimmelmann, A.; Lennon, J. T.

2016-12-01

Despite representing the second largest sink for the atmospheric greenhouse gas methane (CH4), the methanotrophic organisms responsible for atmospheric CH4 consumption have eluded cultivation. High-throughput studies of methanotrophic communities present an opportunity to learn novel details about the organisms responsible, yet such studies have rarely been conducted. Recent observations of subatmospheric CH4 concentrations in cave-air have led to the hypothesis that methanotrophs are active over large spatial scales in the subsurface. Karst terrains cover between 10 - 20 % of the terrestrial surface area and offer abundant cave-related methanotrophic habitat due to the exchange of air with the atmosphere. We collected 42 cave soil samples from 20 caves to test the hypothesis that subterranean methanotrophy removes CH4 from cave-air. Methanotrophs were found in 90 % of samples, notably in locations with subatmospheric CH4­ concentrations. Methylocystaceae were present in caves and accounted for 92 % of the methanotrophic community on average (median), however almost all of the observations were of unidentified Methylocystaceae. Abundances of uncultured and unidentified members of the Methylococcales were correlated with cave-air CH4 concentrations suggesting that some Methylococcales may contribute to atmospheric CH4 oxidation. Individual caves had a strong influence on the observed methanotrophic community composition accounting for 77 % of the variance in the assemblage. Nevertheless, cave-air CH4 concentrations were predictive of the methanotrophic community composition accounting for 5 % of the variation. Our findings also are suggestive of CH4-fueled microbial food webs. For example, abundances of known methylotrophic organisms were correlated with cave-air CH4 concentrations. This may suggest that some methylotrophs contribute to atmospheric CH4 oxidation or that molecules produced in the CH4 oxidation pathway, like methanol, are leaked from methanotrophic

Influence of one or two Bt genes transgenic cotton free living nitrogen fixers and p-solubilising microorganisms in vertisols and alfisols

NASA Astrophysics Data System (ADS)

Sudha, T.; Babu, R.; Biradar, D. P.; Patil, V. C.; Shirnalli, G.

2012-04-01

India, the largest cotton grower in the world benefited from the cultivation of genetically modified Bt transgenic cotton. Bt cotton with the single gene (cry 1Ac) contributed to increased productivity over the last eight years. But in the recent years, there has been an increasing trend to adopt two genes (cry 1Ac and cry 2Ab) transgenic cotton in India. The two gene Bt cotton hybrids were planted over a large area (57%) during 2009 than the single gene Bt cotton hybrids. In this context, the field experiments were conducted in farmers field in both Vertisols and Alfisols during monsoon season of 2009 to study the effect of a single gene Bt hybrid (RCH-2Bt, JK-99Bt, Mallika Bt, MRC-6918 Bt, Brahma Bt, RCH-708 Bt, Bunny Bt) as well as two gene Bt hybrids (RCH-2 BGII Bt, Bunny BGII Bt) compared with the non genetically modified (non-Bt) hybrid (DHH-11) on the population of free living nitrogen fixing microorganisms (Azospirillum and methylotrophs) and P-solubilizers in two different soil types under rainfed situation. Observations on microbial population were recorded at flowering and at harvest in both the soil types. Results indicated a higher population of Azospirillum, methylotrophs and P-solubilisers in the rhizosphere grown with single or two gene Bt hybrid and non-Bt hybrid at flowering stage in both the soil types. In Vertisol, significantly higher population of methylotrophs in MRC-6918 Bt (30 x 102/g of soil), P-solubilizers in RCH-2 Bt (31x103/g of soil) and Azospirillum in RCH-708 Bt (0.79 x 106 /g of soil) was recorded as compared to non-Bt hybrid DHH-11 (2 x 102/g of soil, 12 x 103/g of soil, 0.54 x 106/g of soil), respectively. Whereas, in Alfisol, significantly higher population of methylotrophs in RCH-2 Bt (13x 102/g of soil), P-solubilisers in JK-99 Bt (38 x 103/g of soil) and Azospirillum in RCH-2Bt (0.57 x 106/g of soil) was recorded over non Bt hybrid DHH-11 (2x 102/g of soil, 13x 103/g of soil and 0.17 x106/g of soil) respectively. Our results

Assembly and loss of the polar flagellum in plant-associated methylobacteria

NASA Astrophysics Data System (ADS)

Doerges, L.; Kutschera, U.

2014-04-01

On the leaf surfaces of numerous plant species, inclusive of sunflower ( Helianthus annuus L.), pink-pigmented, methanol-consuming, phytohormone-secreting prokaryotes of the genus Methylobacterium have been detected. However, neither the roles, nor the exact mode of colonization of these epiphytic microbes have been explored in detail. Using germ-free sunflower seeds, we document that, during the first days of seedling development, methylobacteria exert no promotive effect on organ growth. Since the microbes are evenly distributed over the outer surface of the above-ground phytosphere, we analyzed the behavior of populations taken from two bacterial strains that were cultivated as solid, biofilm-like clones on agar plates in different aqueous environments ( Methylobacterium mesophilicum and M. marchantiae, respectively). After transfer into liquid medium, the rod-shaped, immobile methylobacteria assembled a flagellum and developed into planktonic microbes that were motile. During the linear phase of microbial growth in liquid cultures, the percentage of swimming, flagellated bacteria reached a maximum, and thereafter declined. In stationary populations, living, immotile bacteria, and isolated flagella were observed. Hence, methylobacteria that live in a biofilm, transferred into aqueous environments, assemble a flagellum that is lost when cell density has reached a maximum. This swimming motility, which appeared during ontogenetic development within growing microbial populations, may be a means to colonize the moist outer surfaces of leaves.

Hospital microbial surface colonization revealed during monitoring of Klebsiella spp., Pseudomonas aeruginosa, and non-tuberculous mycobacteria.

PubMed

Geadas Farias, Pedro; Gama, Fernando; Reis, Diogo; Alarico, Susana; Empadinhas, Nuno; Martins, José Carlos; de Almeida, Ana Figueiredo; Morais, Paula Vasconcelos

2017-07-01

Hospital environmental conditions, human occupancy, and the characteristics of the equipment influence the survival of microbial communities and raise a concern with regard to nosocomial infections. The objective of the present work was to use the monitoring of Pseudomonas aeruginosa, Klebsiella spp. and non-tuberculous mycobacteria as a strategy to improve knowledge on microbial colonization of non-critical equipment and surfaces, in a tertiary hospital from Central Portugal. A 3-month microbiological survey was performed in a district teaching hospital. A total of 173 samples were obtained from the wards Hematology, Urology, Medicine, and Renal Transplants, and 102 presumptive strains recovered. Per sampling, Pseudomonas Isolation agar showed 42.8 to 73.3% of presumptive P. aeruginosa colonies and MacConkey agar recovered mostly Staphylococcus. Most of the colonies recovered in Middlebrook 7H10-PANTA belonged to the genus Methylobacterium. Taps and WC shower curtains carry high bacterial species diversity. The Redundancy Analysis grouped the samples in those mostly handled by patients, and those mostly handled by healthcare staff or of mixed use. This study shows that the preferential users of the space and equipment seem to be important contributors to the microbial community. The most recovered genus was Methylobacterium, known as colonizer of the water distribution system therefore, it is possible that the water points and biofilms in taps also contribute as dispersion hotspots.

Assembly and loss of the polar flagellum in plant-associated methylobacteria.

PubMed

Doerges, L; Kutschera, U

2014-04-01

On the leaf surfaces of numerous plant species, inclusive of sunflower (Helianthus annuus L.), pink-pigmented, methanol-consuming, phytohormone-secreting prokaryotes of the genus Methylobacterium have been detected. However, neither the roles, nor the exact mode of colonization of these epiphytic microbes have been explored in detail. Using germ-free sunflower seeds, we document that, during the first days of seedling development, methylobacteria exert no promotive effect on organ growth. Since the microbes are evenly distributed over the outer surface of the above-ground phytosphere, we analyzed the behavior of populations taken from two bacterial strains that were cultivated as solid, biofilm-like clones on agar plates in different aqueous environments (Methylobacterium mesophilicum and M. marchantiae, respectively). After transfer into liquid medium, the rod-shaped, immobile methylobacteria assembled a flagellum and developed into planktonic microbes that were motile. During the linear phase of microbial growth in liquid cultures, the percentage of swimming, flagellated bacteria reached a maximum, and thereafter declined. In stationary populations, living, immotile bacteria, and isolated flagella were observed. Hence, methylobacteria that live in a biofilm, transferred into aqueous environments, assemble a flagellum that is lost when cell density has reached a maximum. This swimming motility, which appeared during ontogenetic development within growing microbial populations, may be a means to colonize the moist outer surfaces of leaves.

Aerobic methanol-oxidizing bacteria in soil.

PubMed

Kolb, Steffen

2009-11-01

Methanol is an atmospheric compound that is primarily released from plant polymers and impacts ozone formation. The global methanol emission rate from terrestrial ecosystems is of the same order of magnitude (4.9 x 10(12) mol year(-1)) as that of methane (10 x 10(12) mol year(-1)). The major proportion of the annual plant-released methanol does not enter the atmosphere, but may be reoxidized by biological methanol oxidation, which is catalyzed by methanol-oxidizing prokaryotes. Fifty-six aerobic methanol-oxidizing species have been isolated from soils. These methylotrophs belong to the Alpha-, Beta-, and Gammaproteobacteria, Verrucomicrobia, Firmicutes, and Actinobacteria. Their ecological niches are determined by oxygen and methanol concentration, temperature, pH, the capability to utilize nitrate as an electron acceptor, and the spectrum of nitrogen sources and utilizable multicarbon substrates. Recently discovered interactions with eukaryotes indicate that their ecological niches may not solely be defined by physicochemical parameters. Nonetheless, there are still gaps in knowledge; based on global methanol budgets, methanol oxidation in soil is important, but has not been addressed adequately by biogeochemical studies. Ratios of above-ground and soil-internal methanol oxidation are not known. The contribution to methanol-oxidation by aerobic and anaerobic methylotrophs in situ also needs further research.

Methane assimilation and trophic interactions with marine Methylomicrobium in deep-water coral reef sediment off the coast of Norway.

PubMed

Jensen, Sigmund; Neufeld, Josh D; Birkeland, Nils-Kåre; Hovland, Martin; Murrell, John Colin

2008-11-01

Deep-water coral reefs are seafloor environments with diverse biological communities surrounded by cold permanent darkness. Sources of energy and carbon for the nourishment of these reefs are presently unclear. We investigated one aspect of the food web using DNA stable-isotope probing (DNA-SIP). Sediment from beneath a Lophelia pertusa reef off the coast of Norway was incubated until assimilation of 5 micromol 13CH4 g(-1) wet weight occurred. Extracted DNA was separated into 'light' and 'heavy' fractions for analysis of labelling. Bacterial community fingerprinting of PCR-amplified 16S rRNA gene fragments revealed two predominant 13C-specific bands. Sequencing of these bands indicated that carbon from 13CH4 had been assimilated by a Methylomicrobium and an uncultivated member of the Gammaproteobacteria. Cloning and sequencing of 16S rRNA genes from the heavy DNA, in addition to genes encoding particulate methane monooxygenase and methanol dehydrogenase, all linked Methylomicrobium with methane metabolism. Putative cross-feeders were affiliated with Methylophaga (Gammaproteobacteria), Hyphomicrobium (Alphaproteobacteria) and previously unrecognized methylotrophs of the Gammaproteobacteria, Alphaproteobacteria, Deferribacteres and Bacteroidetes. This first marine methane SIP study provides evidence for the presence of methylotrophs that participate in sediment food webs associated with deep-water coral reefs.

Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress.

PubMed

Müller, Jonas E N; Meyer, Fabian; Litsanov, Boris; Kiefer, Patrick; Vorholt, Julia A

2015-12-01

Bacillus methanolicus MGA3 is a model facultative methylotroph of interest for fundamental research and biotechnological applications. Previous research uncovered a number of pathways potentially involved in one-carbon substrate utilization. Here, we applied dynamic (13) C labeling to elucidate which of these pathways operate during growth on methanol and to uncover potentially new ones. B. methanolicus MGA3 uses the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycles for conversion of the central but toxic intermediate formaldehyde. Additionally, the operation of two cofactor-dependent formaldehyde oxidation pathways with distinct roles was revealed. One is dependent on tri- and tetraglutamylated tetrahydrofolate (THF) and is involved in formaldehyde oxidation during growth on methanol. A second pathway was discovered that is dependent on bacillithiol, a thiol cofactor present also in other Bacilli where it is known to function in redox-homeostasis. We show that bacillithiol-dependent formaldehyde oxidation is activated upon an upshift in formaldehyde induced by a substrate switch from mannitol to methanol. The genes and the corresponding enzymes involved in the biosynthesis of bacillithiol were identified by heterologous production of bacillithiol in Escherichia coli. The presented results indicate metabolic plasticity of the methylotroph allowing acclimation to fluctuating intracellular formaldehyde concentrations. © 2015 John Wiley & Sons Ltd.

Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity production from methanol.

PubMed

Müller, Jonas E N; Heggeset, Tonje M B; Wendisch, Volker F; Vorholt, Julia A; Brautaset, Trygve

2015-01-01

Using methanol as an alternative non-food feedstock for biotechnological production offers several advantages in line with a methanol-based bioeconomy. The Gram-positive, facultative methylotrophic and thermophilic bacterium Bacillus methanolicus is one of the few described microbial candidates with a potential for the conversion of methanol to value-added products. Its capabilities of producing and secreting the commercially important amino acids L-glutamate and L-lysine to high concentrations at 50 °C have been demonstrated and make B. methanolicus a promising target to develop cell factories for industrial-scale production processes. B. methanolicus uses the ribulose monophosphate cycle for methanol assimilation and represents the first example of plasmid-dependent methylotrophy. Recent genome sequencing of two physiologically different wild-type B. methanolicus strains, MGA3 and PB1, accompanied with transcriptome and proteome analyses has generated fundamental new insight into the metabolism of the species. In addition, multiple key enzymes representing methylotrophic and biosynthetic pathways have been biochemically characterized. All this, together with establishment of improved tools for gene expression, has opened opportunities for systems-level metabolic engineering of B. methanolicus. Here, we summarize the current status of its metabolism and biochemistry, available genetic tools, and its potential use in respect to overproduction of amino acids.

Haloacetonitriles are low K1 inhibitors of bacterial dichloromethane dehalogenases.

PubMed

Logan, M S; Blocki, F A; Stimpfl, K J; Wackett, L P

1993-12-15

Distinct dichloromethane dehalogenases from Methylobacterium sp. strain DM4 and Methylophilus DM11 were inhibited by low concentrations of haloacetonitriles. Chloroacetonitrile (ClCH2CN) showed maximal inhibition at a stoichiometry of 1 mol inhibitor:1 mol holoenzyme for both enzymes. This stoichiometry is suggestive of one active site per holoenzyme or extreme negative cooperativity amongst the subunits. Radiolabelled ClCH2CN dissociated completely or partially from the two dehalogenases, respectively, during chromatography. This suggested ClCH2CN was bound non-covalently.

[Analysis of DNA-DNA homologies in obligate methylotrophic bacteria].

PubMed

Doronina, N V; Govorukhina, N I; Lysenko, A M; Trotsenko, Iu A

1988-01-01

The genotypic affinity of 19 bacterial strains obligately dependent on methanol or methylamine as carbon and energy sources was studied by techniques of molecular DNA hybridization. The high homology level (35-88%) between motile strain Methylophilus methanolovorus V-1447D and nonmotile strain Methylobacillus sp. VSB-792 as well as other motile strains (Pseudomonas methanolica ATCC 21704, Methylomonas methanolica NRRL 5458, Pseudomonas sp. W6, strain A3) indicates that all of them belong to one genus. Rather high level of homology (62-63%) was found between Methylobacillus glycogenes ATCC 29475 and Pseudomonas insueta ATCC 21276 and strain G-10. The motile strain Methylophilus methylotrophus NCIB 10515 has a low homology (below 20%) to other of the studied obligate methylobacteria. Therefore, at least two genetically different genera of obligate methylobacteria can be distinguished, namely Methylophilus and Methylobacillus, the latter being represented by both motile and nonmotile forms.

Biosorption for the separation of radionuclides from drainage and process waters of the uranium mining industry

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

Glombitza, F.; Eckardt, L.; Hummel, A.

1995-12-31

Biosorption means the storage of substances at the cell envelope. Different microbial biomasses were tested for the separation of radionuclides from mining waters. Results of a pilot plant demonstrate the ability of these techniques for water cleaning processes. An effluent concentration of lower than 1 mg/l (in most cases 0.1 mg/1) could be realized in a pilot plant by using pure cells of a methylotrophic strain of bacteria as well as using of a fungal mycelia.

Biosynthesis of pyrroloquinoline quinone. 1. Identification of biosynthetic precursors using /sup 13/C labeling and NMR spectroscopy

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

Houck, D.R.; Hanners, J.L.; Unkefer, C.J.

The biosynthesis of pyrroloquinoline quinone (PQQ) in the methylotropic bacterium methylobacterium AM1 has been investigated using /sup 13/C-labelling of the products and NMR spectroscopy. The data indicated that the quinoline portion of PQQ is formed by a novel condensation of N-1, C-2, -3, and -4 of glutamate with a symmetrical six-carbon ring derived from the shikimate pathway. It is postulated that tyrosine is the shikimate-derived percursor, since pyrrole could be formed by the internal cyclization of the amino acid backbone. 18 references, 2 figures, 2 tables.

Archaeal Diversity Associated with Deep Sea Whalefalls

NASA Astrophysics Data System (ADS)

Wilpiszeski, R.; Goffredi, S.; Turk, K.; Vrijenhoek, R.; House, C. H.; Orphan, V.

2005-12-01

Deep sea whale fall sites support a diverse population of organisms in an otherwise sparsely populated environment. While the macro- and megafauna of these ecosystems have been investigated in some detail, less is known about the nature of associated microbial populations. 16S rRNA gene surveys were used to evaluate the diversity of Archaea in the sediment below one such whale fall at 2800 m water depth and at a nearby control site. A variety of Archaea were identified, including diverse uncultured marine crenarchaeota, phylotypes related to hydrogenotrophic methanogens (Methanogenium spp.), and methylotrophic methanogens associated with the Methanococcoides. No methanogens were discovered at the control site, while hydrogenotrophic methanogens accounted for approximately 20% of the samples from surface sediments below the whale and 35% of the Archaea identified from 12.5 to 15 cm below the whale; the single methylotrophic methanogen was identified within the 12.5 to 15 cm depth sample. The presence of methanogenic phylotypes associated with the whale fall corroborates geochemical observations of elevated methane concentrations observed in the shallow sediments directly beneath the whale fall. This combined geochemical and microbiological evidence suggests that near surface organic matter remineralization is occurring via a methanogenic pathway within this deep sea whale fall habitat rather than the typical sulfidogenic dominated diagenesis commonly observed at other whale fall locations and within shallow marine sediments worldwide.

Overexpression of the genes PDC1 and ADH1 activates glycerol conversion to ethanol in the thermotolerant yeast Ogataea (Hansenula) polymorpha.

PubMed

Kata, Iwona; Semkiv, Marta V; Ruchala, Justyna; Dmytruk, Kostyantyn V; Sibirny, Andriy A

2016-08-01

Conversion of byproduct from biodiesel production glycerol to high-value compounds is of great importance. Ethanol is considered a promising product of glycerol bioconversion. The methylotrophic thermotolerant yeast Ogataea (Hansenula) polymorpha is of great interest for this purpose as the glycerol byproduct contains methanol and heavy metals as contaminants, and this yeast utilizes methanol and is relatively resistant to heavy metals. Besides, O. polymorpha shows robust growth on glycerol and produces ethanol from various carbon sources. The thermotolerance of this yeast is an additional advantage, allowing increased fermentation temperature to 45-48 °C, leading to increased rate of the fermentation process and a fall in the cost of distillation. The wild-type strain of O. polymorpha produces insignificant amounts of ethanol from glycerol (0.8 g/l). Overexpression of PDC1 coding for pyruvate decarboxylase enhanced ethanol production up to 3.1 g/l, whereas simultaneous overexpression of PDC1 and ADH1 (coding for alcohol dehydrogenase) led to further increase in ethanol production from glycerol. Moreover, the increased temperature of fermentation up to 45 °C stimulated the production of ethanol from glycerol used as the only carbon source up to 5.0 g/l, which exceeds the data obtained by methylotrophic yeast strains reported so far. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

DOEpatents

Young, Travis; Schultz, Peter G

2013-12-17

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

DOEpatents

Young, Travis; Schultz, Peter G

2014-01-28

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

DOEpatents

Young, Travis; Schultz, Peter G.

2015-08-18

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

MONITORING FOR METHYLOBACTERIUM IN WATER SYSTEMS - Letter to the Editor

EPA Science Inventory

Methylobacteria are slow growing pink-pigmented organisms that have been reported to be opportunistic pathogens in immunocompromised patients. Methylthylobacterium mesophilicum and M. zatmanii have been the two most commonly reported species isolated in clinical samples. Tap wate...

Diversity of endophytic fungal and bacterial communities in Ilex paraguariensis grown under field conditions.

PubMed

Pérez, María Laura; Collavino, Mónica Mariana; Sansberro, Pedro Alfonso; Mroginski, Luis Amado; Galdeano, Ernestina

2016-04-01

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.

Microbial oxidation of gaseous hydrocarbons: production of methylketones from corresponding n-alkanes by methane-utilizing bacteria

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

Patel, R.N.; Hou, C.T.; Laskin, A.I.

Cell suspensions of methane-utilizing bacteria grown on methane oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding methylketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). The product methylketones accumulated extracellularly. The rate of production of methylketones varied with the organism used for oxidation; however, the average rate of acetone, 2-butanone, 2-pentanone, and 2-hexanone production was 1.2, 1.0, 0.15, and 0.025 ..mu..mol/h per 5.0 mg of protein in cell suspensions. Primary alcohols and aldehydes were also detected in low amounts as products of n-alkane (propane and butane) oxidation, but were rapidly metabolized further by cell suspensions. The optimal conditions for in vivo methylketone formationmore » from n-alkanes were compared in Methylococcus capsulatus (Texas strain), Methylosinus sp. (CRL-15), and Methylobacterium sp. (CRL-26). The rate of acetone and 2-butanone production was linear for the first 60 min of incubation and directly increased with cell concentration up to 10 mg of protein per ml for all three cultures tested. The optimal temperatures for the production of acetone and 2-butanone were 35/sup 0/C for Methylosinus trichosporium sp. (CRL-15) and Methylobacterium sp. (CRL-26) and 40/sup 0/C for Methylococcus capsulatus (Texas). Metal-chelating agents inhibited the production of methylketones, suggesting the involvement of a metal-containing enzymatic system in the oxidation of n-alkanes to the corresponding methylketones. The soluble crude extracts derived from methane-utilizing bacteria contained an oxidized nicotinamide adenine dinucleotide-dependent dehydrogenase which catalyzed the oxidation of secondary alcohols.« less

Effects of growth stage and fulvic acid on the diversity and dynamics of endophytic bacterial community in Stevia rebaudiana Bertoni leaves

PubMed Central

Yu, Xuejian; Yang, Jinshui; Wang, Entao; Li, Baozhen; Yuan, Hongli

2015-01-01

The aim of this study was to learn the interactions among the endophytic bacteria, the plant growth, the foliar spray of fulvic acid, and the accumulation of steviol glycosides in the leaves of Stevia rebaudiana. Metagenomic DNA was extracted from the Stevia leaves at different growth stages with or without the fulvic acid treatment; and the diversity of endophytic bacteria in Stevia leaves was estimated by pyrosequencing of 16S rRNA genes. As results, Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were found to be the dominant phyla despite the growth stages and fulvic acid application. Stevia growth stages strongly regulated composition of endophytic community. The genera Agrobacterium (12.3%) and Erwinia (7.2%) dominated in seedling stage were apparently declined in the vegetable and initial flowering stages, while Sphingomonas and Methylobacterium increased in mature leaves at harvest time, which showed that the mature leaves of Stevia preferred to accumulate some certain endophytic bacteria. Sphingomonas and Methylobacterium constituted an important part of the core endophytic community and were positively correlated with the stevioside content and UGT74G1 gene expression, respectively; while Erwinia, Agrobacterium, and Bacillus were negatively correlated with the stevioside accumulation. Fulvic acid treatment accelerated the variation of endophytes along the growth stages and increased the steviol glycosides content. This is the first study to reveal the community composition of endophytic bacteria in the Stevia leaves, to evidence the strong effects of growth stage and fulvic acid application on the endophytes of Stevia, and to demonstrate the correlation between the endophytic bacteria and the steviol glycosides accumulation. PMID:26379644

Molecular tools to track bacteria responsible for fuel deterioration and microbiologically influenced corrosion.

PubMed

Suflita, Joseph M; Aktas, Deniz F; Oldham, Athenia L; Perez-Ibarra, Beatriz Monica; Duncan, Kathleen

2012-01-01

Investigating the susceptibility of various fuels to anaerobic biodegradation has become complicated with the recognition that the fuels themselves are not sterile. Bacterial DNA could be obtained when various fuels were filtered through a hydrophobic teflon (0.22 μm) membrane filter. Bacterial 16S rRNA genes from these preparations were PCR amplified, cloned, and the resulting libraries sequenced to identify the fuel-borne bacterial communities. The most common sequence, found in algal- and camelina-based biofuels as well as in ultra-low sulfur diesel (ULSD) and F76 diesel, was similar to that of a Tumebacillus. The next most common sequence was similar to Methylobacterium and was found in the biofuels and ULSD. Higher level phylogenetic groups included representatives of the Firmicutes (Bacillus, Lactobacillus and Streptococcus), several Actinobacteria, Deinococcus-Thermus, Chloroflexi, Cyanobacteria, Bacteroidetes, Alphaproteobacteria (Methylobacterium and Sphingomonadales), Betaproteobacteria (Oxalobacteraceae and Burkholderiales) and Deltaproteobacteria. All of the fuel-associated bacterial sequences, except those obtained from a few facultative microorganisms, were from aerobes and only remotely affiliated with sequences that resulted from anaerobic successional events evident when ULSD was incubated with a coastal seawater and sediment inoculum. Thus, both traditional and alternate fuel formulations harbor a characteristic microflora, but these microorganisms contributed little to the successional patterns that ultimately resulted in fuel decomposition, sulfide formation and metal biocorrosion. The findings illustrate the value of molecular approaches to track the fate of bacteria that might come in contact with fuels and potentially contribute to corrosion problems throughout the energy value chain.

[Phosphate-solubilizing activity of aerobic methylobacteria].

PubMed

Agafonova, N V; Kaparullina, E N; Doronina, N V; Trotsenko, Iu A

2014-01-01

Phosphate-solubilizing activity was found in 14 strains of plant-associated aerobic methylobacteria belonging to the genera Methylophilus, Methylobacillus, Methylovorus, Methylopila, Methylobacterium, Delftia, and Ancyclobacter. The growth of methylobacteria on medium with methanol as the carbon and energy source and insoluble tricalcium phosphate as the phosphorus source was accompanied by a decrease in pH due to the accumulation of up to 7 mM formic acid as a methanol oxidation intermediate and by release of 120-280 μM phosphate ions, which can be used by both bacteria and plants. Phosphate-solubilizing activity is a newly revealed role of methylobacteria in phytosymbiosis.

The Methanol Dehydrogenase Gene, mxaF, as a Functional and Phylogenetic Marker for Proteobacterial Methanotrophs in Natural Environments

PubMed Central

Lau, Evan; Fisher, Meredith C.; Steudler, Paul A.; Cavanaugh, Colleen M.

2013-01-01

The mxaF gene, coding for the large (α) subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the Alpha-, Beta- and Gammaproteobacteria. It is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoA and mmoX genes, which are absent in some methanotrophic proteobacterial genera. This study examined the potential for using the mxaF gene as a functional and phylogenetic marker for methanotrophs. mxaF and 16S rRNA gene phylogenies were constructed based on over 100 database sequences of known proteobacterial methanotrophs and other methylotrophs to assess their evolutionary histories. Topology tests revealed that mxaF and 16S rDNA genes of methanotrophs do not show congruent evolutionary histories, with incongruencies in methanotrophic taxa in the Methylococcaceae, Methylocystaceae, and Beijerinckiacea. However, known methanotrophs generally formed coherent clades based on mxaF gene sequences, allowing for phylogenetic discrimination of major taxa. This feature highlights the mxaF gene’s usefulness as a biomarker in studying the molecular diversity of proteobacterial methanotrophs in nature. To verify this, PCR-directed assays targeting this gene were used to detect novel methanotrophs from diverse environments including soil, peatland, hydrothermal vent mussel tissues, and methanotroph isolates. The placement of the majority of environmental mxaF gene sequences in distinct methanotroph-specific clades (Methylocystaceae and Methylococcaceae) detected in this study supports the use of mxaF as a biomarker for methanotrophic proteobacteria. PMID:23451130

Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate.

PubMed

Leßmeier, Lennart; Pfeifenschneider, Johannes; Carnicer, Marc; Heux, Stephanie; Portais, Jean-Charles; Wendisch, Volker F

2015-12-01

Methanol, a one-carbon compound, can be utilized by a variety of bacteria and other organisms as carbon and energy source and is regarded as a promising substrate for biotechnological production. In this study, a strain of non-methylotrophic Corynebacterium glutamicum, which was able to produce the polyamide building block cadaverine as non-native product, was engineered for co-utilization of methanol. Expression of the gene encoding NAD+-dependent methanol dehydrogenase (Mdh) from the natural methylotroph Bacillus methanolicus increased methanol oxidation. Deletion of the endogenous aldehyde dehydrogenase genes ald and fadH prevented methanol oxidation to carbon dioxide and formaldehyde detoxification via the linear formaldehyde dissimilation pathway. Heterologous expression of genes for the key enzymes hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase of the ribulose monophosphate (RuMP) pathway in this strain restored growth in the presence of methanol or formaldehyde, which suggested efficient formaldehyde detoxification involving RuMP key enzymes. While growth with methanol as sole carbon source was not observed, the fate of 13C-methanol added as co-substrate to sugars was followed and the isotopologue distribution indicated incorporation into central metabolites and in vivo activity of the RuMP pathway. In addition, 13C-label from methanol was traced to the secreted product cadaverine. Thus, this synthetic biology approach led to a C. glutamicum strain that converted the non-natural carbon substrate methanol at least partially to the non-native product cadaverine.

Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha

PubMed Central

2012-01-01

Background Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study’s goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system. Results The development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved. Conclusion The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent. PMID:23253823

Applications of methylotrophs: can single carbon be harnessed for biotechnology?

PubMed

Chistoserdova, Ludmila

2018-04-01

This review summarizes developments in the field of applied research involving microbial conversion of single carbon compounds (methane, methanol, CO 2 ). The potential of the microorganisms involved in biotechnological applications could be realized via engineering native C1 utilizers toward higher output of value-added compounds, including biofuels, or via production of value chemicals as parts of novel, heterologously expressed biochemical pathways. Alternatively, C1 metabolism could be implemented in traditional industrial platforms (Escherichia coli, yeast), via introduction of specific metabolic modules. Most recent research spanning both approaches is covered. The potential of C1 utilizers in biomining of rare Earth elements, as well as the potential of C1 consuming microbial consortia in industrial applications are discussed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Protection of Arabidopsis thaliana against Leaf-Pathogenic Pseudomonas syringae by Sphingomonas Strains in a Controlled Model System ▿ †

PubMed Central

Innerebner, Gerd; Knief, Claudia; Vorholt, Julia A.

2011-01-01

Diverse bacterial taxa live in association with plants without causing deleterious effects. Previous analyses of phyllosphere communities revealed the predominance of few bacterial genera on healthy dicotyl plants, provoking the question of whether these commensals play a particular role in plant protection. Here, we tested two of them, Methylobacterium and Sphingomonas, with respect to their ability to diminish disease symptom formation and the proliferation of the foliar plant pathogen Pseudomonas syringae pv. tomato DC3000 on Arabidopsis thaliana. Plants were grown under gnotobiotic conditions in the absence or presence of the potential antagonists and then challenged with the pathogen. No effect of Methylobacterium strains on disease development was observed. However, members of the genus Sphingomonas showed a striking plant-protective effect by suppressing disease symptoms and diminishing pathogen growth. A survey of different Sphingomonas strains revealed that most plant isolates protected A. thaliana plants from developing severe disease symptoms. This was not true for Sphingomonas strains isolated from air, dust, or water, even when they reached cell densities in the phyllosphere comparable to those of the plant isolates. This suggests that plant protection is common among plant-colonizing Sphingomonas spp. but is not a general trait conserved within the genus Sphingomonas. The carbon source profiling of representative isolates revealed differences between protecting and nonprotecting strains, suggesting that substrate competition plays a role in plant protection by Sphingomonas. However, other mechanisms cannot be excluded at this time. In conclusion, the ability to protect plants as shown here in a model system may be an unexplored, common trait of indigenous Sphingomonas spp. and may be of relevance under natural conditions. PMID:21421777

Bacterial communities in the phylloplane of Prunus species.

PubMed

Jo, Yeonhwa; Cho, Jin Kyong; Choi, Hoseong; Chu, Hyosub; Lian, Sen; Cho, Won Kyong

2015-04-01

Bacterial populations in the phylloplane of four different Prunus species were investigated by 16 S rRNA pyrosequencing. Bioinformatic analysis identified an average of 510 operational taxonomic units belonging to 159 genera in 76 families. The two genera, Sphingomonas and Methylobacterium, were dominant in the phylloplane of four Prunus species. Twenty three genera were commonly identified in the four Prunus species, indicating a high level of bacterial diversity dependent on the plant species. Our study based on 16 S rRNA sequencing reveals the complexity of bacterial diversity in the phylloplane of Prunus species in detail. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biodegradation of Metal-EDTA Complexes by an Enriched Microbial Population

PubMed Central

Thomas, Russell A. P.; Lawlor, Kirsten; Bailey, Mark; Macaskie, Lynne E.

1998-01-01

A mixed culture utilizing EDTA as the sole carbon source was isolated from a mixed inoculum of water from the River Mersey (United Kingdom) and sludge from an industrial effluent treatment plant. Fourteen component organisms were isolated from the culture, including representatives of the genera Methylobacterium, Variovorax, Enterobacter, Aureobacterium, and Bacillus. The mixed culture biodegraded metal-EDTA complexes slowly; the biodegradability was in the order Fe>Cu>Co>Ni>Cd. By incorporation of inorganic phosphate into the medium as a precipitant ligand, heavy metals were removed in parallel to EDTA degradation. The mixed culture also utilized a number of possible EDTA degradation intermediates as carbon sources. PMID:9546167

Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

PubMed

Tran, Anh-Minh; Nguyen, Thanh-Thao; Nguyen, Cong-Thuan; Huynh-Thi, Xuan-Mai; Nguyen, Cao-Tri; Trinh, Minh-Thuong; Tran, Linh-Thuoc; Cartwright, Stephanie P; Bill, Roslyn M; Tran-Van, Hieu

2017-04-04

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.

Recent advances in the production of recombinant subunit vaccines in Pichia pastoris

PubMed Central

Wang, Man; Jiang, Shuai; Wang, Yefu

2016-01-01

ABSTRACT Recombinant protein subunit vaccines are formulated using defined protein antigens that can be produced in heterologous expression systems. The methylotrophic yeast Pichia pastoris has become an important host system for the production of recombinant subunit vaccines. Although many basic elements of P. pastoris expression system are now well developed, there is still room for further optimization of protein production. Codon bias, gene dosage, endoplasmic reticulum protein folding and culture condition are important considerations for improved production of recombinant vaccine antigens. Here we comment on current advances in the application of P. pastoris for the synthesis of recombinant subunit vaccines. PMID:27246656

Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications.

PubMed

Kang, Zhen; Huang, Hao; Zhang, Yunfeng; Du, Guocheng; Chen, Jian

2017-01-01

Pichia pastoris: (reclassified as Komagataella phaffii), a methylotrophic yeast strain has been widely used for heterologous protein production because of its unique advantages, such as readily achievable high-density fermentation, tractable genetic modifications and typical eukaryotic post-translational modifications. More recently, P. pastoris as a metabolic pathway engineering platform has also gained much attention. In this mini-review, we addressed recent advances of molecular toolboxes, including synthetic promoters, signal peptides, and genome engineering tools that established for P. pastoris. Furthermore, the applications of P. pastoris towards synthetic biology were also discussed and prospected especially in the context of genome-scale metabolic pathway analysis.

[Biosynthesis of the bioprotectant ectoin by aerobic methylotrophic bacteria from methanol].

PubMed

Doronina, N V; Ezhov, V A; Beschastnyĭ, A P; Trotsenko, Iu A

2010-01-01

It is shown that neutrophilic methylobacteria Methylophaga thalassica and M. marina have higher rates of growth and ectoin accumulation compared to the haloalkaliphilic species M. alcalica and M. natronia and methanotrophs Methylomicrobium alcaliphilum and M. kenyense. The conditions of M. thalassica cultivation in methanol-containing medium were optimized. The yield of this process reached 60 g/l of absolutely dry biomass containing 15-19% (9-11 g/l) ectoine. The scheme of ectoin isolation from the biomass by extraction and subsequent purification, which allowed obtaining preparations of different degree of purity, was developed.

[Receptor elements for biosensors in two ways of methylotrophic yeast immobilization].

PubMed

Zaĭtsev, M G; Arliapov, V A; Alferov, V A; Reshetilov, A N

2012-01-01

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 In yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05-1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2-1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzochinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.

Purification and characterization of the amine dehydrogenase from a facultative methylotroph.

PubMed

Coleman, J P; Perry, J J

1984-01-01

Strain RA-6 is a pink-pigmented organism which can grow on a variety of substrates including methylamine. It can utilize methylamine as sole source of carbon via an isocitrate lyase negative serine pathway. Methylamine grown cells contain an inducible primary amine dehydrogenase [primary amine: (acceptor) oxidoreductase (deaminating)] which is not present in succinate grown cells. The amine dehydrogenase was purified to over 90% homogeneity. It is an acidic protein (isoelectric point of 5.37) with a molecular weight of 118,000 containing subunits with approximate molecular weights of 16,500 and 46,000. It is active on an array of primary terminal amines and is strongly inhibited by carbonyl reagents. Cytochrome c or artificial electron acceptors are required for activity; neither NAD nor NADP can serve as primary electron acceptor.

Biodegradation of haloacetic acids by bacterial isolates and enrichment cultures from drinking water systems.

PubMed

Zhang, Ping; Lapara, Timothy M; Goslan, Emma H; Xie, Yuefeng; Parsons, Simon A; Hozalski, Raymond M

2009-05-01

Biodegradation is a potentially important loss process for haloacetic acids (HAAs), a class of chlorination byproducts, in water treatment and distribution systems, but little is known about the organisms involved (i.e., identity, substrate range, biodegradation kinetics). In this research, 10 biomass samples (i.e., tap water, distribution system biofilms, and prechlorinated granular activated carbon filters) from nine drinking water systems were used to inoculate a total of thirty enrichment cultures fed monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), or trichloroacetic (TCAA) as sole carbon and energy source. HAA degraders were successfully enriched from the biofilm samples (GAC and distribution system) but rarely from tap water. Half of the MCAA and DCAA enrichment cultures were positive, whereas only one TCAA culture was positive (two were inconclusive). Eight unique HAA-degrading isolates were obtained including several Afipia spp. and a Methylobacterium sp.; all isolates were members of the phylum Proteobacteria. MCAA, monobromoacetic acid (MBAA), and monoiodoacetic acid (MIAA) were rapidly degraded by all isolates, and DCAA and tribromoacetic (TBAA) were also relatively labile. TCAA and dibromoacetic acid (DBAA)were degraded by only three isolates and degradation lagged behind the other HAAs. Detailed DCAA biodegradation kinetics were obtained for two selected isolates and two enrichment cultures. The maximum biomass-normalized degradation rates (Vm) were 0.27 and 0.97 microg DCAA/ microg protein/h for Methylobacterium fujisawaense strain PAWDI and Afipia felis strain EMD2, respectively, which were comparable to the values obtained for the enrichment cultures from which those organisms were isolated (0.39 and 1.37 microg DCAN/microg protein/h, respectively). The half-saturation constant (Km) values ranged from 4.38 to 77.91 microg DCAA/L and the cell yields ranged from 14.4 to 36.1 mg protein/g DCAA.

Microbial diversity and methanogenic activity of Antrim Shale formation waters from recently fractured wells

PubMed Central

Wuchter, Cornelia; Banning, Erin; Mincer, Tracy J.; Drenzek, Nicholas J.; Coolen, Marco J. L.

2013-01-01

The Antrim Shale in the Michigan Basin is one of the most productive shale gas formations in the U.S., but optimal resource recovery strategies must rely on a thorough understanding of the complex biogeochemical, microbial, and physical interdependencies in this and similar systems. We used Illumina MiSeq 16S rDNA sequencing to analyze the diversity and relative abundance of prokaryotic communities present in Antrim shale formation water of three closely spaced recently fractured gas-producing wells. In addition, the well waters were incubated with a suite of fermentative and methanogenic substrates in an effort to stimulate microbial methane generation. The three wells exhibited substantial differences in their community structure that may arise from their different drilling and fracturing histories. Bacterial sequences greatly outnumbered those of archaea and shared highest similarity to previously described cultures of mesophiles and moderate halophiles within the Firmicutes, Bacteroidetes, and δ- and ε-Proteobacteria. The majority of archaeal sequences shared highest sequence similarity to uncultured euryarchaeotal environmental clones. Some sequences closely related to cultured methylotrophic and hydrogenotrophic methanogens were also present in the initial well water. Incubation with methanol and trimethylamine stimulated methylotrophic methanogens and resulted in the largest increase in methane production in the formation waters, while fermentation triggered by the addition of yeast extract and formate indirectly stimulated hydrogenotrophic methanogens. The addition of sterile powdered shale as a complex natural substrate stimulated the rate of methane production without affecting total methane yields. Depletion of methane indicative of anaerobic methane oxidation (AMO) was observed over the course of incubation with some substrates. This process could constitute a substantial loss of methane in the shale formation. PMID:24367357

Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis.

PubMed

Tsuji, Daisuke; Akeboshi, Hiromi; Matsuoka, Kazuhiko; Yasuoka, Hiroko; Miyasaki, Eri; Kasahara, Yoshiko; Kawashima, Ikuo; Chiba, Yasunori; Jigami, Yoshifumi; Taki, Takao; Sakuraba, Hitoshi; Itoh, Kohji

2011-04-01

Novel recombinant human lysosomal β-hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency. A recombinant human HexA (Om4HexA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb⁻/⁻ mice) at different doses (0.5-2.5 mg/kg), and then the replacement and therapeutic effects were examined. The Om4HexA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (< 63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases. Copyright © 2010 American Neurological Association.

An Examination of the Carbon Isotope Effects Associated with Amino Acid Biosynthesis

NASA Astrophysics Data System (ADS)

Scott, James H.; O'Brien, Diane M.; Emerson, David; Sun, Henry; McDonald, Gene D.; Salgado, Antonio; Fogel, Marilyn L.

2006-12-01

Stable carbon isotope ratios (δ13C) were determined for alanine, proline, phenylalanine, valine, leucine, isoleucine, aspartate (aspartic acid and asparagine), glutamate (glutamic acid and glutamine), lysine, serine, glycine, and threonine from metabolically diverse microorganisms. The microorganisms examined included fermenting bacteria, organotrophic, chemolithotrophic, phototrophic, methylotrophic, methanogenic, acetogenic, acetotrophic, and naturally occurring cryptoendolithic communities from the Dry Valleys of Antarctica. Here we demonstrated that reactions involved in amino acid biosynthesis can be used to distinguish amino acids formed by life from those formed by nonbiological processes. The unique patterns of δ13C imprinted by life on amino acids produced a biological bias. We also showed that, by applying discriminant function analysis to the δ13C value of a pool of amino acids formed by biological activity, it was possible to identify key aspects of intermediary carbon metabolism in the microbial world. In fact, microorganisms examined in this study could be placed within one of three metabolic groups: (1) heterotrophs that grow by oxidizing compounds containing three or more carbon-to-carbon bonds (fermenters and organotrophs), (2) autotrophs that grow by taking up carbon dioxide (chemolitotrophs and phototrophs), and (3) acetoclastic microbes that grow by assimilation of formaldehyde or acetate (methylotrophs, methanogens, acetogens, and acetotrophs). Furthermore, we demonstrated that cryptoendolithic communities from Antarctica grouped most closely with the autotrophs, which indicates that the dominant metabolic pathways in these communities are likely those utilized for CO2 fixation. We propose that this technique can be used to determine the dominant metabolic types in a community and reveal the overall flow of carbon in a complex ecosystem.

Microbial consortia controlling biogenic gas formation in the Qaidam Basin of western China

NASA Astrophysics Data System (ADS)

Shuai, Yanhua; Zhang, Shuichang; Grasby, Stephen E.; Hou, Weiguo; Chen, Zhuoheng; Huang, Ling; Kui, Mingqing; Xu, Yirui; Wang, Yang

2016-08-01

Knowledge of what controls the activity of subsurface microbial communities is critical for assessing and managing biogenic methane resources. In this study, 19 formation waters and five gas samples were collected at depths of 800 to 1900 m from Quaternary biogenic gas fields of the Qaidam Basin, China. The formation waters were brines with chloride (Cl) concentrations from 1200 to 2700 mM. Bacterial 16S rRNA gene copies ranged from 3.75 × 104 to 2.23 × 106 copies mL-1 of water, and those of archaea ranged from 2.44 × 103 to 4.66 × 107 copies mL-1 of water. Both bacterial and archaea 16 s rRNA gene copies were negatively correlated with Cl concentration. The microbial community structure differed significantly depending on Cl concentrations. At high Cl waters (>1800 mM), the microbial community showed a halophilic signature made up of several abundant taxonomic groups within Firmicules, γ-Proteobacteria, and methylotrophic Methanosarcinales. At low Cl, Firmicules and hydrogenotrophic methanogens were dominant members. The proportion of inferred hydrogenotrophic methanogens decreased from 89% to 14% of total archaeal reads with increasing Cl concentration; in contrast, methylotrophic species increased from 11% to 85%. Given that the proportion of hydrogenotrophic species was positively correlated with the archaeal gene abundances, we suggest that Cl concentrations primarily constrain the activity of archaea catalyzing H2 reduction of CO2. Our results show that dilution of formation waters is critical in the process of biogenic gas formation, suggesting that an engineered decrease in Cl concentrations may induce methanogenesis as a potential method to increase gas reserves in such areas in the future.

Effects of nickel and cobalt on kinetics of methanol conversion by methanogenic sludge as assessed by on-line CH4 monitoring.

PubMed

Gonzalez-Gil, G; Kleerebezem, R; Lettinga, G

1999-04-01

When metals were added in a pulse mode to methylotrophic-methanogenic biomass, three methane production rate phases were recognized. Increased concentrations of Ni and Co accelerated the initial exponential and final arithmetic increases in the methane production rate and reduced the temporary decrease in the rate. When Ni and Co were added continuously, the temporary decrease phase was eliminated and the exponential production rate increased. We hypothesize that the temporary decrease in the methane production rate and the final arithmetic increase in the methane production rate were due to micronutrient limitations and that the precipitation-dissolution kinetics of metal sulfides may play a key role in the biovailability of these compounds.

Energy Metabolism during Anaerobic Methane Oxidation in ANME Archaea

PubMed Central

McGlynn, Shawn E.

2017-01-01

Anaerobic methane oxidation in archaea is often presented to operate via a pathway of “reverse methanogenesis”. However, if the cumulative reactions of a methanogen are run in reverse there is no apparent way to conserve energy. Recent findings suggest that chemiosmotic coupling enzymes known from their use in methylotrophic and acetoclastic methanogens—in addition to unique terminal reductases—biochemically facilitate energy conservation during complete CH4 oxidation to CO2. The apparent enzyme modularity of these organisms highlights how microbes can arrange their energy metabolisms to accommodate diverse chemical potentials in various ecological niches, even in the extreme case of utilizing “reverse” thermodynamic potentials. PMID:28321009

[The influence of colonizing methylobacteria on morphogenesis and resistance of sugar beet and white cabbage plants to Erwinia carotovora].

PubMed

Pigoleva, S V; Zakharchenko, N S; Pigolev, A V; Trotsenko, Iu A; Bur'ianov, Ia I

2009-01-01

The influence of colonization of sugar beet (Beta vulgaris var. saccharifera (Alef) Krass) and white cabbage (Brassica oleracea var. capitata L.) plants by methylotrophic bacteria Methylovorus mays on the growth, rooting, and plant resistance to phytopathogen bacteria Erwinia carotovora was investigated. The colonization by methylobacteria led to their steady association with the plants which had increased growth speed, root formation and photosynthetic activity. The colonized plants had increased resistance to Erwinia carotovora phytopathogen and were better adapted to greenhouse conditions. The obtained results showed the perspectives for the practical implementation of methylobacteria in the ecologically clean microbiology substances used as the plant growth stimulators and for the plant protection from pathogens.

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

PubMed Central

Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kèvin; Stellato, Francesco; Liang, Mengning; White, Thomas A.; Seine, Thomas; Messerschmidt, Marc; Chapman, Henry N.; Wilmanns, Matthias

2016-01-01

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined. PMID:27006771

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

DOE PAGES

Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin; ...

2016-03-01

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

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

Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

Assessment of bacterial contamination of lipstick using pyrosequencing.

PubMed

Lee, So Y; Lee, Si Y

As soon as they are exposed to the environment, cosmetics become contaminated with microorganisms, and this contamination accumulates with increased use. In this study, we employed pyrosequencing to investigate the diversity of bacteria found on lipstick. Bacterial DNA was extracted from 20 lipstick samples and mixed in equal ratios for pyrosequencing analysis. As a result, 105 bacterial genera were detected, four of which ( Leifsonia , Methylobacterium , Streptococcus , and Haemophilus ) were predominant in 92% of the 19,863 total sequence reads. Potentially pathogenic genera such as Staphylococcus , Pseudomonas , Escherichia , Salmonella , Corynebacterium , Mycobacterium , and Neisseria accounted for 27.6% of the 105 genera. The most commonly identified oral bacteria belonged to the Streptococcus genus, although other oral genera such as Actinomyces , Fusobacterium , Porphyromonas , and Lactobacillus were also detected.

Two Cases of Bacteremia Due to Roseomonas mucosa.

PubMed

Kim, Yu Kyung; Moon, Jung Suk; Song, Kyung Eun; Lee, Won Kil

2016-07-01

Roseomonas is a genus of pink-pigmented nonfermentative bacilli. These slow-growing, gram-negative cocobacilli form pink-colored colonies on sheep blood agar. They differ from other pink-pigmented nonfermenters, including Methylobacterium, in morphology, biochemical characteristics, and DNA sequence. Roseomonas strains are rarely isolated in clinical laboratories; therefore, we report two cases in order to improve our ability to identify these pathogens. We isolated two strains of Roseomonas mucosa from the venous blood cultures of two patients, an 84-yr-old woman with common bile duct obstruction and a 17-yr-old male with acute myeloid leukemia who had an indwelling central-venous catheter for chemotherapy. The isolated strains were confirmed as R. mucosa by 16S rRNA sequencing.

Two Cases of Bacteremia Due to Roseomonas mucosa

PubMed Central

Kim, Yu Kyung; Moon, Jung Suk; Song, Kyung Eun

2016-01-01

Roseomonas is a genus of pink-pigmented nonfermentative bacilli. These slow-growing, gram-negative cocobacilli form pink-colored colonies on sheep blood agar. They differ from other pink-pigmented nonfermenters, including Methylobacterium, in morphology, biochemical characteristics, and DNA sequence. Roseomonas strains are rarely isolated in clinical laboratories; therefore, we report two cases in order to improve our ability to identify these pathogens. We isolated two strains of Roseomonas mucosa from the venous blood cultures of two patients, an 84-yr-old woman with common bile duct obstruction and a 17-yr-old male with acute myeloid leukemia who had an indwelling central-venous catheter for chemotherapy. The isolated strains were confirmed as R. mucosa by 16S rRNA sequencing. PMID:27139611

Genomics of Nitrogen Cycle in Freshwater Lakes with Focus on Methylotrophic Bacteria

NASA Astrophysics Data System (ADS)

Chistoserdova, L.

2014-12-01

Data will be presented on communities of microbes active in methane oxidation in Lake Washington, Seattle. Metagenomic sequencing of sediment samples reveals dominant presence of Methylobacter, contrary to prior understanding based on cultivation of methanotrophs. Stable isotope probing of microcosms incubated with methane at varying concentrations of oxygen and nitrate uncover a dominant response by Methylobacter species and a correlation between the populations of Methylobacter and Methylotenera, both responding positively to nitrate. We also uncover a propensity of Methylobacter to act in microoxic conditions, in this case transferring carbon down a food chain represented by a variety of bacteria. Functional gene profiling detects upwards shifts in the abundances of nitrogen metabolism genes in response to nitrate, with Methylococcaceae and Methylophilaceae genes being most abundant. We test a hypothesis of cooperative behavior between Methylobacter, Methylotenera and other species using two alternative approaches: a top-down approach in which we incubate native lake sediments under different conditions and observe trajectories of community simplification, and a bottom-up approach in which we construct synthetic communities from pure cultures of bacteria and observe their behavior. We also cultivate Methylobacter as well as multiple species of Methylophilaceae and analyze their genomes. Among the Methylophilaceae, we uncover a remarkable flexibility in terms of both central carbon and nitrogen metabolic pathways. We hypothesize that this diversity may be driven by microniche conditions along the methane and oxygen countergradients, as well as by the availability of nitrogen sources. Our future plans include deciphering the mechanistic details of cooperative behavior in methane oxidation, using Lake Washington communities as a model.

Characterization of a restriction-modification system of the thermotolerant methylotroph Bacillus methanolicus.

PubMed Central

Cue, D; Lam, H; Hanson, R S; Flickinger, M C

1996-01-01

We report the isolation of a restriction endonuclease, BmeTI, an isoschizomer of BclI, that recognizes the DNA sequence 5' TGATCA 3'. We also report that BmeTI sites are modified to TGm6ATCA. These findings provide the basis for devising strategies to prevent BmeTI restriction of any DNA introduced into Bacillus methanolicus. PMID:8975604

In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

DOEpatents

Young, Travis [San Diego, CA; Schultz, Peter G [La Jolla, CA

2014-02-11

The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris are also provided.

Acidotolerant Bacteria and Fungi as a Sink of Methanol-Derived Carbon in a Deciduous Forest Soil

PubMed Central

Morawe, Mareen; Hoeke, Henrike; Wissenbach, Dirk K.; Lentendu, Guillaume; Wubet, Tesfaye; Kröber, Eileen; Kolb, Steffen

2017-01-01

Methanol is an abundant atmospheric volatile organic compound that is released from both living and decaying plant material. In forest and other aerated soils, methanol can be consumed by methanol-utilizing microorganisms that constitute a known terrestrial sink. However, the environmental factors that drive the biodiversity of such methanol-utilizers have been hardly resolved. Soil-derived isolates of methanol-utilizers can also often assimilate multicarbon compounds as alternative substrates. Here, we conducted a comparative DNA stable isotope probing experiment under methylotrophic (only [13C1]-methanol was supplemented) and combined substrate conditions ([12C1]-methanol and alternative multi-carbon [13Cu]-substrates were simultaneously supplemented) to (i) identify methanol-utilizing microorganisms of a deciduous forest soil (European beech dominated temperate forest in Germany), (ii) assess their substrate range in the soil environment, and (iii) evaluate their trophic links to other soil microorganisms. The applied multi-carbon substrates represented typical intermediates of organic matter degradation, such as acetate, plant-derived sugars (xylose and glucose), and a lignin-derived aromatic compound (vanillic acid). An experimentally induced pH shift was associated with substantial changes of the diversity of active methanol-utilizers suggesting that soil pH was a niche-defining factor of these microorganisms. The main bacterial methanol-utilizers were members of the Beijerinckiaceae (Bacteria) that played a central role in a detected methanol-based food web. A clear preference for methanol or multi-carbon substrates as carbon source of different Beijerinckiaceae-affiliated phylotypes was observed suggesting a restricted substrate range of the methylotrophic representatives. Apart from Bacteria, we also identified the yeasts Cryptococcus and Trichosporon as methanol-derived carbon-utilizing fungi suggesting that further research is needed to exclude or prove

Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus.

PubMed

Brautaset, Trygve; Jakobsen M, Øyvind M; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E

2004-03-01

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50 degrees C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

PubMed Central

Brautaset, Trygve; Jakobsen, Øyvind M.; Flickinger, Michael C.; Valla, Svein; Ellingsen, Trond E.

2004-01-01

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50°C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs. PMID:14973041

Experimental Systems to Study Yeast Pexophagy.

PubMed

Yamashita, Shun-Ichi; Oku, Masahide; Sakai, Yasuyoshi; Fujiki, Yukio

2017-01-01

Peroxisome abundance is tightly regulated according to the physiological contexts, through regulations of both proliferation and degradation of the organelles. Here, we describe detailed methods to analyze processes for autophagic degradation of peroxisomes, termed pexophagy, in yeast organisms. The assay systems include a method for biochemical detection of pexophagy completion, and one for microscopic visualization of specialized membrane structures acting in pexophagy. As a model yeast organism utilized in studies of pexophagy, the methylotrophic yeast Komagataella phaffii (Pichia pastoris) is referred to in this chapter and related information on the studies with baker's yeast (Saccharomyces cerevisiae) is also included. The described techniques facilitate elucidation of molecular machineries for pexophagy and understanding of peroxisome-selective autophagic pathways.

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

PubMed

Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

2014-01-01

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

Hansenula polymorpha (Pichia angusta): Biology and Applications

NASA Astrophysics Data System (ADS)

Kunze, Gotthard; Kang, Hyun Ah; Gellissen, Gerd

Hansenula polymorpha (Pichia angusta) belongs to a limited number of methylotrophic yeast species. It is able to assimilate nitrate and can grow on a range of carbon sources. Furthermore, H. polymorpha is a thermo-tolerant microorganism with some strains growing at temperatures up to 50° C and more. These unusual characteristics render H. polymorpha attractive as a model organism to study the development and functions of peroxisomes and the biochemistry of nitrate assimilation. H. polymorpha provides an established platform for heterologous gene expression and is distinguished by an impressive track record as producer of recom-binant proteins that include commercially available pharmaceuticals like hepatitis B vaccine, insulin and the IFN α-2a

Effects of Nickel and Cobalt on Kinetics of Methanol Conversion by Methanogenic Sludge as Assessed by On-Line CH4 Monitoring

PubMed Central

Gonzalez-Gil, Graciela; Kleerebezem, Robbert; Lettinga, Gatze

1999-01-01

When metals were added in a pulse mode to methylotrophic-methanogenic biomass, three methane production rate phases were recognized. Increased concentrations of Ni and Co accelerated the initial exponential and final arithmetic increases in the methane production rate and reduced the temporary decrease in the rate. When Ni and Co were added continuously, the temporary decrease phase was eliminated and the exponential production rate increased. We hypothesize that the temporary decrease in the methane production rate and the final arithmetic increase in the methane production rate were due to micronutrient limitations and that the precipitation-dissolution kinetics of metal sulfides may play a key role in the biovailability of these compounds. PMID:10103284

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

PubMed Central

de Vries, G E; Arfman, N; Terpstra, P; Dijkhuizen, L

1992-01-01

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed. Images PMID:1644761

In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed.

PubMed

Ochsner, Andrea M; Müller, Jonas E N; Mora, Carlos A; Vorholt, Julia A

2014-08-25

In the Gram-positive methylotroph Bacillus methanolicus, methanol oxidation is catalyzed by an NAD-dependent methanol dehydrogenase (Mdh) that belongs to the type III alcohol dehydrogenase (Adh) family. It was previously shown that the in vitro activity of B. methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase. Here we show that this feature is not unique, but more widespread among type III Adhs in combination with Act or other Act-like Nudix hydrolases. In addition, we studied the effect of site directed mutations in the predicted active site of Mdh and two other type III Adhs with regard to activity and activation by Act. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Microbial growth on oxalate by a route not involving glyoxylate carboligase

PubMed Central

Blackmore, Maureen A.; Quayle, J. R.

1970-01-01

1. The metabolism of oxalate by the pink-pigmented organisms, Pseudomonas AM1, Pseudomonas AM2, Protaminobacter ruber and Pseudomonas extorquens has been compared with that of the non-pigmented Pseudomonas oxalaticus. 2. During growth on oxalate, all the organisms contain oxalyl-CoA decarboxylase, formate dehydrogenase and oxalyl-CoA reductase. This is consistent with oxidation of oxalate to carbon dioxide taking place via oxalyl-CoA, formyl-CoA and formate as intermediates, and also reduction of oxalate to glyoxylate taking place via oxalyl-CoA. 3. The pink-pigmented organisms, when grown on oxalate, contain l-serine–glyoxylate aminotransferase and hydroxypyruvate reductase but do not contain glyoxylate carboligase. The converse of this obtains in oxalate-grown Ps. oxalaticus. This indicates that, in contrast with Ps. oxalaticus, synthesis of C3 compounds from oxalate by the pink-pigmented organisms occurs by a variant of the `serine pathway' used by Pseudomonas AM1 during growth on C1 compounds. 4. Evidence in favour of this scheme is provided by the finding that a mutant of Pseudomonas AM1 that lacks hydroxypyruvate reductase is not able to grow on oxalate. PMID:5472155

Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia.

PubMed

Hou, Shaobin; Makarova, Kira S; Saw, Jimmy H W; Senin, Pavel; Ly, Benjamin V; Zhou, Zhemin; Ren, Yan; Wang, Jianmei; Galperin, Michael Y; Omelchenko, Marina V; Wolf, Yuri I; Yutin, Natalya; Koonin, Eugene V; Stott, Matthew B; Mountain, Bruce W; Crowe, Michelle A; Smirnova, Angela V; Dunfield, Peter F; Feng, Lu; Wang, Lei; Alam, Maqsudul

2008-07-01

The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia. We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C1-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift

The Genome Sequence of Methanohalophilus mahii SLP T Reveals Differences in the Energy Metabolism among Members of the Methanosarcinaceae Inhabiting Freshwater and Saline Environments

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

Spring, Stefan; Scheuner, Carmen; Lapidus, Alla

Methanohalophilus mahii is the type species of the genus Methanohalophilus , which currently comprises three distinct species with validly published names. Mhp. mahii represents moderately halophilic methanogenic archaea with a strictly methylotrophic metabolism. The type strain SLP T was isolated from hypersaline sediments collected from the southern arm of Great Salt Lake, Utah. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,012,424 bp genome is a single replicon with 2032 protein-coding and 63 RNA genes and part of the Genomic Encyclopedia of Bacteria and Archaea project. A comparison of the reconstructedmore » energy metabolism in the halophilic species Mhp. mahii with other representatives of the Methanosarcinaceae reveals some interesting differences to freshwater species.« less

The Genome Sequence of Methanohalophilus mahii SLP T Reveals Differences in the Energy Metabolism among Members of the Methanosarcinaceae Inhabiting Freshwater and Saline Environments

DOE PAGES

Spring, Stefan; Scheuner, Carmen; Lapidus, Alla; ...

2010-01-01

Methanohalophilus mahii is the type species of the genus Methanohalophilus , which currently comprises three distinct species with validly published names. Mhp. mahii represents moderately halophilic methanogenic archaea with a strictly methylotrophic metabolism. The type strain SLP T was isolated from hypersaline sediments collected from the southern arm of Great Salt Lake, Utah. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,012,424 bp genome is a single replicon with 2032 protein-coding and 63 RNA genes and part of the Genomic Encyclopedia of Bacteria and Archaea project. A comparison of the reconstructedmore » energy metabolism in the halophilic species Mhp. mahii with other representatives of the Methanosarcinaceae reveals some interesting differences to freshwater species.« less

Methanol Metabolism in Pseudomonad C

PubMed Central

Stieglitz, B.; Mateles, R. I.

1973-01-01

Cell suspensions of pseudomonad C, a bacterium capable of growth on methanol as sole carbon source, were able to oxidize methanol, formaldehyde, and formate, although the rates of oxidation for the latter two compounds were much slower. The latter compounds also could not serve as sole carbon sources. Through the use of labeled compounds, it was shown that in the presence of methanol, formaldehyde, formate, and bicarbonate were incorporated into trichloroacetic acid-precipitable material. Hexose phosphate synthetase activity was found, indicating the assimilation of methanol via an allulose pathway. No hydroxypyruvate reductase activity was found, nor was any complex membrane structure observed. Such a combination of characteristics has been observed in an obligate methylotroph (Pseudomonas W1), but pseudomonad C can utilize a variety of non-methyl substrates. Images PMID:4349032

A novel directly coupled gradostat

NASA Technical Reports Server (NTRS)

Wimpenny, J. W.; Earnshaw, R. G.; Gest, H.; Hayes, J. M.; Favinger, J. L.

1992-01-01

The original bidirectional compound chemostat (gradostat) described by Lovitt and Wimpenny has been simplified by making a more compact apparatus in which chemical gradients are established by diffusion between adjacent culture chambers. The experimental model (diffusion coupled (DC) gradostat) consisted of five chambers whose contents could be agitated by turbines rotating in the horizontal plane on a common shaft. Two biological experiments were designed to reveal the value of the DC gradostat. A methylotroph (Methylophilus methylotrophus) grown in a methanol gradient showed expected changes in cell viability as a function of position in the five vessel array. Cells of two species of photosynthetic bacteria (Rhodobacter capsulata and Rhodopseudomonas marina/agilis) with different salt sensitivities could be mixed and subsequently separated by the DC gradostat operating with a NaCl gradient of 0-3% w/v.

Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

PubMed Central

Stiefel, Philipp; Zambelli, Tomaso

2013-01-01

In their natural environment, bacteria often behave differently than they do under laboratory conditions. To gain insight into the physiology of bacteria in situ, dedicated approaches are required to monitor their adaptations and specific behaviors under environmental conditions. Optical microscopy is crucial for the observation of fundamental characteristics of bacteria, such as cell shape, size, and marker gene expression. Here, fluidic force microscopy (FluidFM) was exploited to isolate optically selected bacteria for subsequent identification and characterization. In this study, bacteriochlorophyll-producing bacteria, which can be visualized due to their characteristic fluorescence in the infrared range, were isolated from leaf washes. Bacterial communities from the phyllosphere were investigated because they harbor genes indicative of aerobic anoxygenic photosynthesis. Our data show that different species of Methylobacterium express their photosystem in planta, and they show a distinct pattern of bacteriochlorophyll production under laboratory conditions that is dependent on supplied carbon sources. PMID:23770907

cDNA cloning, functional expression and antifungal activities of a dimeric plant defensin SPE10 from Pachyrrhizus erosus seeds.

PubMed

Song, Xiaomin; Wang, Jing; Wu, Fang; Li, Xu; Teng, Maikun; Gong, Weimin

2005-01-01

SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal alpha-mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far.

Methanolobus taylorii sp nov, a new methylotropic, estuarine methanogen

USGS Publications Warehouse

Oremland, Ronald S.; Boone, David R.

1994-01-01

Strain GS-16T (T = type strain) is a methylotrophic methanogen that was isolated from estuarine sediments from San Francisco Bay (4) and has been deposited in the Oregon Collection of Methanogens (Oregon Graduate Institute, Portland) as strain OCM 5ST. This strain was isolated by using dimethyl sulfide as the catabolic substrate (4), but it can also grow on methylamines (13) and methanethiol (8, 9) and grew when it was inoculated into MSHA medium (6) supplemented with 20 mM methanol as the sole catabolic substrate. Strain GS-16T cells form methane from methylmercury (12) and dimethylselenide (16), although they cannot grow on these substrates, and form traces of ethane from diethyl sulfide (15). Methanogenesis from trimethylamine is inhibited by methyl fluoride (11) and methyl bromide (14), but not by dimethyl ether (1 1).

Expression of enzymes for the usage in food and feed industry with Pichia pastoris.

PubMed

Spohner, Sebastian C; Müller, Hagen; Quitmann, Hendrich; Czermak, Peter

2015-05-20

The methylotrophic yeast Pichia pastoris is an established protein expression host for the production of industrial enzymes. This yeast can be grown to very high cell densities and produces high titers of recombinant protein, which can be expressed intercellularly or be secreted to the fermentation medium. P. pastoris offers some advantages over other established expression systems especially in protein maturation. In food and feed production many enzymatically catalyzed processes are reported and the demand for new enzymes grows continuously. For instance the unique catalytic properties of enzymes are used to improve resource efficiency, maintain quality, functionalize food, and to prevent off-flavors. This review aims to provide an overview on recent developments in heterologous production of enzymes with P. pastoris and their application within the food sector. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols.

PubMed

Yamamoto, Hiroaki; Kudoh, Masatake

2013-09-01

A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD(+)-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A gene-encoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4-chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99 % optical purity.

Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae.

PubMed

Dai, Zhongxue; Gu, Honglian; Zhang, Shangjie; Xin, Fengxue; Zhang, Wenming; Dong, Weiliang; Ma, Jiangfeng; Jia, Honghua; Jiang, Min

2017-12-01

The aim of this study was to metabolically construct Saccharomyces cerevisiae for achievement of direct methanol utilization and value added product (mainly pyruvate) production. After successful integration of methanol oxidation pathway originated from Pichia pastoris into the chromosome of S. cerevisiae, the recombinant showed 1.04g/L consumption of methanol and 3.13% increase of cell growth (OD 600 ) when using methanol as the sole carbon source. Moreover, 0.26g/L of pyruvate was detected in the fermentation broth. The supplementation of 1g/L yeast extract could further improve cell growth with increase of 11.70% and methanol consumption to 2.35g/L. This represents the first genetically modified non-methylotrophic eukaryotic microbe for direct methanol utilization and would be of great value concerning the development of biotechnological processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Microbial methanogenesis in the sulfate-reducing zone of sediments in the Eckernförde Bay, SW Baltic Sea

NASA Astrophysics Data System (ADS)

Maltby, Johanna; Steinle, Lea; Löscher, Carolin R.; Bange, Hermann W.; Fischer, Martin A.; Schmidt, Mark; Treude, Tina

2018-01-01

Benthic microbial methanogenesis is a known source of methane in marine systems. In most sediments, the majority of methanogenesis is located below the sulfate-reducing zone, as sulfate reducers outcompete methanogens for the major substrates hydrogen and acetate. The coexistence of methanogenesis and sulfate reduction has been shown before and is possible through the usage of noncompetitive substrates by methanogens such as methanol or methylated amines. However, knowledge about the magnitude, seasonality, and environmental controls of this noncompetitive methane production is sparse. In the present study, the presence of methanogenesis within the sulfate reduction zone (SRZ methanogenesis) was investigated in sediments (0-30 cm below seafloor, cm b.s.f.) of the seasonally hypoxic Eckernförde Bay in the southwestern Baltic Sea. Water column parameters such as oxygen, temperature, and salinity together with porewater geochemistry and benthic methanogenesis rates were determined in the sampling area Boknis Eck quarterly from March 2013 to September 2014 to investigate the effect of seasonal environmental changes on the rate and distribution of SRZ methanogenesis, to estimate its potential contribution to benthic methane emissions, and to identify the potential methanogenic groups responsible for SRZ methanogenesis. The metabolic pathway of methanogenesis in the presence or absence of sulfate reducers, which after the addition of a noncompetitive substrate was studied in four experimental setups: (1) unaltered sediment batch incubations (net methanogenesis), (2) 14C-bicarbonate labeling experiments (hydrogenotrophic methanogenesis), (3) manipulated experiments with the addition of either molybdate (sulfate reducer inhibitor), 2-bromoethanesulfonate (methanogen inhibitor), or methanol (noncompetitive substrate, potential methanogenesis), and (4) the addition of 13C-labeled methanol (potential methylotrophic methanogenesis). After incubation with methanol

Growth in liverworts of the Marchantiales is promoted by epiphytic methylobacteria

NASA Astrophysics Data System (ADS)

Kutschera, U.; Koopmann, V.

2005-07-01

Liverworts, the most basal lineage of extant land plants, have been used as model systems in the reconstruction of adaptations to life on land. In this study, we used gemmae (specialized propagules) that were isolated from mature gemma cups of two distantly related species of liverworts, Marchantia polymorpha L. and Lunularia cruciata L. (order Marchantiales). We show that methylobacteria (genus Methylobacterium), microbes that inhabit the surfaces of land plants where they secrete phytohormones (cytokinines), promote the growth of isolated gemmae cultivated on agar plates. As a control, two species of higher plants, maize (Zea mays L.) and sunflower (Helianthus annuus L.) were raised aseptically from sterile seeds (i.e., caryopses, achenes). Inoculation of these propagules with methylobacteria was without effect on growth in the above-ground phytosphere (expansion of stems and leaves). We conclude that normal development in Marchantia and Lunularia is dependent on (and possibly regulated by) epiphytic methylobacteria, whereas representative higher plants grow at optimal rates in the absence of these prokaryotic epiphytes.

Species-specific cell mobility of bacteria-feeding myxamoebae in plasmodial slime molds.

PubMed

Hoppe, Thomas; Kutschera, Ulrich

2015-01-01

On decaying wood or litter in forests, plasmodial slime molds (myxomycetes) represent a large fraction of eukaryotic protists that feed on bacteria. In his seminal book Experimental Physiology of Plants (1865), Julius Sachs referred to the multinucleate plasmodium of myxomycetes, which were considered at that time as primitive plants (or fungi). Today it is well established that myxomycetes are members of the Amoebozoa (Protista). In this study we compare the mobility of myxamoebae of 3 European species, Lycogala epidendrum (order Liceales), Tubulifera arachnoidea, and Trichia decipiens (order Trichiales). Using agar plates, on which 3 separate bacterial species were cultivated as prey organisms (Methylobacterium mesophilicum, Escherichia coli, Agrobacterium tumefaciens), we document large differences in cell motility between the myxomycetes investigated. In addition, we show that the 3 species of myxamoebae can be distinguished based on their average cell size. These data shed light on the mode of co-occurrence via differential substrate utilization in these members of the Amoebozoa.

Species-specific cell mobility of bacteria-feeding myxamoebae in plasmodial slime molds

PubMed Central

Hoppe, Thomas; Kutschera, Ulrich

2015-01-01

On decaying wood or litter in forests, plasmodial slime molds (myxomycetes) represent a large fraction of eukaryotic protists that feed on bacteria. In his seminal book Experimental Physiology of Plants (1865), Julius Sachs referred to the multinucleate plasmodium of myxomycetes, which were considered at that time as primitive plants (or fungi). Today it is well established that myxomycetes are members of the Amoebozoa (Protista). In this study we compare the mobility of myxamoebae of 3 European species, Lycogala epidendrum (order Liceales), Tubulifera arachnoidea, and Trichia decipiens (order Trichiales). Using agar plates, on which 3 separate bacterial species were cultivated as prey organisms (Methylobacterium mesophilicum, Escherichia coli, Agrobacterium tumefaciens), we document large differences in cell motility between the myxomycetes investigated. In addition, we show that the 3 species of myxamoebae can be distinguished based on their average cell size. These data shed light on the mode of co-occurrence via differential substrate utilization in these members of the Amoebozoa. PMID:26357877

Characterization of the gut bacterial community in Manduca sexta and effect of antibiotics on bacterial diversity and nematode reproduction.

PubMed

van der Hoeven, Ransome; Betrabet, Geeta; Forst, Steven

2008-09-01

The tobacco hornworm, Manduca sexta, is a model lepidopteran insect used to study the pathogenic and mutualistic phases of entomopathogenic nematodes (EPNs) and their bacterial symbionts. While intestinal microbial communities could potentially compete with the EPN and its bacterial partner for nutrient resources of the insect, the microbial gut community had not been characterized previously. Here, we show that the midgut of M. sexta raised on an artificial diet contained mostly Gram-positive cocci and coryneforms including Staphylococcus, Pediococcus, Micrococcus and Corynebacterium. Major perturbation in the gut community was observed on addition of antibiotics to the diet. Paenibacillus and several Proteobacteria such as Methylobacterium, Sphingomonas and Acinetobacter were primary genera identified under these conditions. Furthermore, the reproduction of the nematode Steinernema carpocapsae was less efficient, and the level of nematode colonization by its symbiont Xenorhabdus nematophila reduced, in insects reared on a diet containing antibiotics. The effect of antibiotics and perturbation of gut microbiota on nematode reproduction is discussed.

Growth in liverworts of the Marchantiales is promoted by epiphytic methylobacteria.

PubMed

Kutschera, U; Koopmann, V

2005-07-01

Liverworts, the most basal lineage of extant land plants, have been used as model systems in the reconstruction of adaptations to life on land. In this study, we used gemmae (specialized propagules) that were isolated from mature gemma cups of two distantly related species of liverworts, Marchantia polymorpha L. and Lunularia cruciata L. (order Marchantiales). We show that methylobacteria (genus Methylobacterium), microbes that inhabit the surfaces of land plants where they secrete phytohormones (cytokinines), promote the growth of isolated gemmae cultivated on agar plates. As a control, two species of higher plants, maize (Zea mays L.) and sunflower (Helianthus annuus L.) were raised aseptically from sterile seeds (i.e., caryopses, achenes). Inoculation of these propagules with methylobacteria was without effect on growth in the above-ground phytosphere (expansion of stems and leaves). We conclude that normal development in Marchantia and Lunularia is dependent on (and possibly regulated by) epiphytic methylobacteria, whereas representative higher plants grow at optimal rates in the absence of these prokaryotic epiphytes.

Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

PubMed Central

2012-01-01

Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed

Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia

PubMed Central

Hou, Shaobin; Makarova, Kira S; Saw, Jimmy HW; Senin, Pavel; Ly, Benjamin V; Zhou, Zhemin; Ren, Yan; Wang, Jianmei; Galperin, Michael Y; Omelchenko, Marina V; Wolf, Yuri I; Yutin, Natalya; Koonin, Eugene V; Stott, Matthew B; Mountain, Bruce W; Crowe, Michelle A; Smirnova, Angela V; Dunfield, Peter F; Feng, Lu; Wang, Lei; Alam, Maqsudul

2008-01-01

Background The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia. Results We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C1-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a

6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle.

PubMed

Le, Simone Balzer; Heggeset, Tonje Marita Bjerkan; Haugen, Tone; Nærdal, Ingemar; Brautaset, Trygve

2017-05-01

D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The B. methanolicus wild-type strain MGA3 possesses two putative rpe and pfk genes encoded on plasmid pBM19 (rpe1-MGA3 and pfk1-MGA3) and on the chromosome (rpe2-MGA3 and pfk2-MGA3). The wild-type strain PB1 also encodes putative rpe and pfk genes on plasmid pBM20 (rpe1-PB1 and pfk1-PB1*); however, it only harbours a chromosomal pfk gene (pfk2-PB1). Transcription of the plasmid-encoded genes was 10-fold to 15-fold upregulated in cells growing on methanol compared to mannitol, while the chromosomal genes were transcribed at similar levels under both conditions in both strains. All seven gene products were recombinantly produced in Escherichia coli, purified and biochemically characterized. All three RPEs were active as hexamers, catalytically stimulated by Mg 2+ and Mn 2+ and displayed similar K' values (56-75 μM) for ribulose 5-phosphate. Rpe2-MGA3 showed displayed 2-fold lower V max (49 U/mg) and a significantly reduced thermostability compared to the two Rpe1 proteins. Pfk1-PB1* was shown to be non-functional. The PFKs were active both as octamers and as tetramers, were catalytically stimulated by Mg 2+ and Mn 2+ , and displayed similar thermostabilities. The PFKs have similar K m values for fructose 6-phosphate (0.61-0.94 μM) and for ATP (0.38-0.82 μM), while Pfk1-MGA3 had a 2-fold lower V max (6.3 U/mg) compared to the two Pfk2 proteins. Our results demonstrate that MGA3 and PB1 exert alternative solutions to plasmid-dependent methylotrophy, including genetic organization, regulation, and biochemistry of RuMP cycle enzymes.

The interaction of methanol dehydrogenase and cytochrome cL in the acidophilic methylotroph Acetobacter methanolicus.

PubMed Central

Chan, H T; Anthony, C

1991-01-01

The quinoprotein methanol dehydrogenase (MDH) of Acetobacter methanolicus has an alpha 2 beta 2 structure. By contrast with other MDHs, the beta-subunit (approx. 8.5 kDa) does not contain the five lysine residues previously proposed to be involved in ionic interactions with the electron acceptor cytochrome cL. That electrostatic interactions are involved was confirmed by the demonstration that methanol:cytochrome cL oxidoreductase activity was inhibited by high ionic strength (I), the strength of interaction being inversely related to the square root of I. Specific modifiers of arginine residues on MDH inhibited this reaction but not the dye-linked MDH activity. Modification of lysine residues on MDH that altered its charge had no effect on the dye-linked activity but inhibited reaction with cytochrome cL. When the charge was retained on modification of lysine residues, little effect on either activity was observed. Cross-linking experiments confirmed that lysine residues on the alpha-subunit, but not the beta-subunit, are involved in the 'docking' process between the proteins. Images Fig. 4. PMID:1660263

Production of Delta(1)-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.

PubMed

Taura, Futoshi; Dono, Emi; Sirikantaramas, Supaart; Yoshimura, Kohji; Shoyama, Yukihiro; Morimoto, Satoshi

2007-09-28

Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

PubMed Central

Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

2013-01-01

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

Identification of bacteria on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties by 16S rRNA gene sequencing and by microbiological culture

PubMed Central

Dempsey, Kate E; Riggio, Marcello P; Lennon, Alan; Hannah, Victoria E; Ramage, Gordon; Allan, David; Bagg, Jeremy

2007-01-01

It has been postulated that bacteria attached to the surface of prosthetic hip joints can cause localised inflammation, resulting in failure of the replacement joint. However, diagnosis of infection is difficult with traditional microbiological culture methods, and evidence exists that highly fastidious or non-cultivable organisms have a role in implant infections. The purpose of this study was to use culture and culture-independent methods to detect the bacteria present on the surface of prosthetic hip joints removed during revision arthroplasties. Ten consecutive revisions were performed by two surgeons, which were all clinically and radiologically loose. Five of the hip replacement revision surgeries were performed because of clinical infections and five because of aseptic loosening. Preoperative and perioperative specimens were obtained from each patient and subjected to routine microbiological culture. The prostheses removed from each patient were subjected to mild ultrasonication to dislodge adherent bacteria, followed by aerobic and anaerobic microbiological culture. Bacterial DNA was extracted from each sonicate and the 16S rRNA gene was amplified with the universal primer pair 27f/1387r. All 10 specimens were positive for the presence of bacteria by both culture and PCR. PCR products were then cloned, organised into groups by RFLP analysis and one clone from each group was sequenced. Bacteria were identified by comparison of the 16S rRNA gene sequences obtained with those deposited in public access sequence databases. A total of 512 clones were analysed by RFLP analysis, of which 118 were sequenced. Culture methods identified species from the genera Leifsonia (54.3%), Staphylococcus (21.7%), Proteus (8.7%), Brevundimonas (6.5%), Salibacillus (4.3%), Methylobacterium (2.2%) and Zimmermannella (2.2%). Molecular detection methods identified a more diverse microflora. The predominant genus detected was Lysobacter, representing 312 (60.9%) of 512 clones

MxaY regulates the lanthanide-mediated methanol dehydrogenase switch in Methylomicrobium buryatense

DOE PAGES

Chu, Frances; Beck, David A. C.; Lidstrom, Mary E.

2016-09-07

Many methylotrophs, microorganisms that consume carbon compounds lacking carbon–carbon bonds, use two different systems to oxidize methanol for energy production and biomass accumulation. The MxaFI methanol dehydrogenase (MDH) contains calcium in its active site, while the XoxF enzyme contains a lanthanide in its active site. The genes encoding the MDH enzymes are differentially regulated by the presence of lanthanides. In this study, we found that the histidine kinase MxaY controls the lanthanide-mediated switch in Methylomicrobium buryatense 5GB1C. MxaY controls the transcription of genes encoding MxaFI and XoxF at least partially by controlling the transcript levels of the orphan response regulatormore » MxaB. We identify a constitutively active version of MxaY, and identify the mutated residue that may be involved in lanthanide sensing. Finally, we find evidence to suggest that tight control of active MDH production is required for wild-type growth rates.« less

Strain IMB-1, a novel bacterium for the removal of methyl bromide in fumigated agricultural soils

USGS Publications Warehouse

Connell, Hancock T.L.; Costello, A.M.; Lidstrom, M.E.; Oremland, R.S.

1998-01-01

A facultatively methylotrophic bacterium, strain IMB-1, that has been isolated from agricultural soil grows on methyl bromide (MeBr), methyl iodide, methyl chloride, and methylated amines, as well as on glucose, pyruvate, or acetate. Phylogenetic analysis of its 16S rRNA gene sequence indicates that strain IMB-1 classes in the alpha subgroup of the class Proteobacteria and is closely related to members of the genus Rhizobium. The ability of strain IMB-1 to oxidize MeBr to CO2 is constitutive in cells regardless of the growth substrate. Addition of cell suspensions of strain IMB-1 to soils greatly accelerates the oxidation of MeBr, as does pretreatment of soils with low concentrations of methyl iodide. These results suggest that soil treatment strategies can be devised whereby bacteria can effectively consume MeBr during field fumigations, which would diminish or eliminate the outward flux of MeBr to the atmosphere.

Characterization of bacterial diversity associated with deep sea ferromanganese nodules from the South China Sea.

PubMed

Zhang, De-Chao; Liu, Yan-Xia; Li, Xin-Zheng

2015-09-01

Deep sea ferromanganese (FeMn) nodules contain metallic mineral resources and have great economic potential. In this study, a combination of culture-dependent and culture-independent (16S rRNA genes clone library and pyrosequencing) methods was used to investigate the bacterial diversity in FeMn nodules from Jiaolong Seamount, the South China Sea. Eleven bacterial strains including some moderate thermophiles were isolated. The majority of strains belonged to the phylum Proteobacteria; one isolate belonged to the phylum Firmicutes. A total of 259 near full-length bacterial 16S rRNA gene sequences in a clone library and 67,079 valid reads obtained using pyrosequencing indicated that members of the Gammaproteobacteria dominated, with the most abundant bacterial genera being Pseudomonas and Alteromonas. Sequence analysis indicated the presence of many organisms whose closest relatives are known manganese oxidizers, iron reducers, hydrogen-oxidizing bacteria and methylotrophs. This is the first reported investigation of bacterial diversity associated with deep sea FeMn nodules from the South China Sea.

The deep-subsurface sulfate reducer Desulfotomaculum kuznetsovii employs two methanol-degrading pathways.

PubMed

Sousa, Diana Z; Visser, Michael; van Gelder, Antonie H; Boeren, Sjef; Pieterse, Mervin M; Pinkse, Martijn W H; Verhaert, Peter D E M; Vogt, Carsten; Franke, Steffi; Kümmel, Steffen; Stams, Alfons J M

2018-01-16

Methanol is generally metabolized through a pathway initiated by a cobalamine-containing methanol methyltransferase by anaerobic methylotrophs (such as methanogens and acetogens), or through oxidation to formaldehyde using a methanol dehydrogenase by aerobes. Methanol is an important substrate in deep-subsurface environments, where thermophilic sulfate-reducing bacteria of the genus Desulfotomaculum have key roles. Here, we study the methanol metabolism of Desulfotomaculum kuznetsovii strain 17 T , isolated from a 3000-m deep geothermal water reservoir. We use proteomics to analyze cells grown with methanol and sulfate in the presence and absence of cobalt and vitamin B12. The results indicate the presence of two methanol-degrading pathways in D. kuznetsovii, a cobalt-dependent methanol methyltransferase and a cobalt-independent methanol dehydrogenase, which is further confirmed by stable isotope fractionation. This is the first report of a microorganism utilizing two distinct methanol conversion pathways. We hypothesize that this gives D. kuznetsovii a competitive advantage in its natural environment.

Preliminary Evaluation of Glyceric Acid-producing Ability of Acidomonas methanolica NBRC104435 from Glycerol Containing Methanol.

PubMed

Sato, Shun; Kitamoto, Dai; Habe, Hiroshi

2017-06-01

Some acetic acid bacteria produce large amounts of glyceric acid (GA) from glycerol in culture broth. However, methanol, which is a major contaminant of raw glycerol derived from the biodiesel fuel industry, sharply decreases cell growth and GA production [AMB Express, 3, 20, 2013]. Thus, we evaluated the methylotrophic acetic acid bacterium Acidomonas methanolica NBRC104435 for its ability to produce GA from glycerol containing methanol. This strain accumulated GA in its culture broth when 1-3 wt% glycerol was available as a carbon source. We observed improved cell growth and GA accumulation when 1 vol% methanol was added to the 3-5 wt% glycerol medium. The maximum concentration of GA was 12.8 g/L in medium containing 3 wt% glycerol plus 1 vol% methanol. In addition, the enantiomeric excess (ee) of the GA produced was revealed to be 44%, indicating that this strain converted glycerol to d-GA with a lower enantioselectivity than other acetic acid bacteria, which had 70-99% ee.

Methanol metabolism and archaeal community changes in a bioelectrochemical anaerobic digestion sequencing batch reactor with copper-coated graphite cathode.

PubMed

Park, Jungyu; Lee, Beom; Shi, Peng; Kwon, Hyejeong; Jeong, Sang Mun; Jun, Hangbae

2018-07-01

In this study, the metabolism of methanol and changes in an archaeal community were examined in a bioelectrochemical anaerobic digestion sequencing batch reactor with a copper-coated graphite cathode (BEAD-SBR Cu ). Copper-coated graphite cathode produced methanol from food waste. The BEAD-SBR Cu showed higher methanol removal and methane production than those of the anaerobic digestion (AD)-SBR. The methane production and pH of the BEAD-SBR Cu were stable even under a high organic loading rate (OLR). The hydrogenotrophic methanogens increased from 32.2 to 60.0%, and the hydrogen-dependent methylotrophic methanogens increased from 19.5 to 37.7% in the bulk of BEAD-SBR Cu at high OLR. Where methanol was directly injected as a single substrate into the BEAD-SBR Cu , the main metabolism of methane production was hydrogenotrophic methanogenesis using carbon dioxide and hydrogen released by the oxidation of methanol on the anode through bioelectrochemical reactions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Evaluation of the information content of long-term wastewater characteristics data in relation to activated sludge model parameters.

PubMed

Alikhani, Jamal; Takacs, Imre; Al-Omari, Ahmed; Murthy, Sudhir; Massoudieh, Arash

2017-03-01

A parameter estimation framework was used to evaluate the ability of observed data from a full-scale nitrification-denitrification bioreactor to reduce the uncertainty associated with the bio-kinetic and stoichiometric parameters of an activated sludge model (ASM). Samples collected over a period of 150 days from the effluent as well as from the reactor tanks were used. A hybrid genetic algorithm and Bayesian inference were used to perform deterministic and parameter estimations, respectively. The main goal was to assess the ability of the data to obtain reliable parameter estimates for a modified version of the ASM. The modified ASM model includes methylotrophic processes which play the main role in methanol-fed denitrification. Sensitivity analysis was also used to explain the ability of the data to provide information about each of the parameters. The results showed that the uncertainty in the estimates of the most sensitive parameters (including growth rate, decay rate, and yield coefficients) decreased with respect to the prior information.

Regulation of NAD+- and NADP+-linked isocitrate dehydrogenase in the obligate methylotrophic bacterium Pseudomonas W6.

PubMed

Hofmann, K H; Babel, W

1980-01-01

Cell-free extracts of the obligate methanol-utilizing bacterium Pseudomonas W6 catalyze the oxydation of isocitrate to alpha-ketoglutarate in the presence of NAD+ and NADP+. After electro-focusing of the crude extract of Pseudomonas W6 actually two distinct bands each of NAD+-linked isocitrate dehydrogenase (NAD+-IDH) and of NADP+-linked isocitrate dehydrogenase (NADP+-IDH) could be observed. The NAD+-IDH was completely separated from the NADP+-IDH by employing DEAE ion exchange chromatography and further purified by affinity chromatography using Cibacron blue F 3G-A. The NAD+-IDH was inhibited by a high energy charge, whereas the NADP+-IDH was found to be independent of energy charge. Consequently the NAD+-IDH showed the control behaviour of an enzyme of an energy-generating sequence which, however, equally fulfils a catabolic and an anabolic function. With respect to the inhibition by reduced pyridine nucleotides and alpha-ketoglutarate differences between NAD+-IDH and NADP+-IDH were also found. Only the NADP+-linked enzyme exhibited a feedback inhibition by its reaction products alpha-ketoglutarate and NADPH. This control behaviour gives evidence for the biosynthetic function of the NADP+-IDH. These results confer an amphibolic character to the sequence from citrate to alpha-ketoglutarate in the incomplete citric-acid cycle of Pseudomonas W6.

Overexpression of Wild-Type Aspartokinase Increases l-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus▿

PubMed Central

Jakobsen, Øyvind M.; Brautaset, Trygve; Degnes, Kristin F.; Heggeset, Tonje M. B.; Balzer, Simone; Flickinger, Michael C.; Valla, Svein; Ellingsen, Trond E.

2009-01-01

Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar Vmax values (between 47 and 58 μmol/min/mg protein) and Km values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC50], 0.1 mM) and by l-lysine (IC50, 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC50, 4 mM) and by l-lysine (IC50, 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK. PMID:19060158

Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.

PubMed

Jakobsen, Oyvind M; Brautaset, Trygve; Degnes, Kristin F; Heggeset, Tonje M B; Balzer, Simone; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E

2009-02-01

Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V(max) values (between 47 and 58 micromol/min/mg protein) and K(m) values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC(50)], 0.1 mM) and by l-lysine (IC(50), 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC(50), 4 mM) and by l-lysine (IC(50), 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

Oharaeibacter diazotrophicus gen. nov., sp. nov., a diazotrophic and facultatively methylotrophic bacterium, isolated from rice rhizosphere.

PubMed

Lv, Haoxin; Masuda, Sachiko; Fujitani, Yoshiko; Sahin, Nurettin; Tani, Akio

2017-04-01

A novel facultatively methanol-utilizing bacterial strain, SM30T, was isolated from rice rhizosphere. Strain SM30T was Gram-stain-negative, aerobic, motile, short rods, and grew optimally at pH 7 and at 28 °C. It could tolerate 0 to 2 % (w/v) NaCl. Based on 16S rRNA gene sequence comparisons, strain SM30T was most closely related to Pleomorphomonas oryzae DSM 16300T, with a low similarity of 94.17 %. One of the lanthanide metals, lanthanum, could enhance its growth slightly on methanol. Phylogenetic trees, based on the mxaF, xoxF and cpn60 genes of SM30T showed its distinct phylogenetic position with respect to species with validly published names. Polymerase chain reaction (PCR) amplification of the nifH and growth on nitrogen-free medium indicated that strain SM30T is a diazotroph. The major cellular fatty acids were summed feature 8 (containing 18 : 1ω7c and 18 : 1ω6c) and cyclo 19 : 0ω8c. The major quinone was ubiquinone 10. The DNA G+C content was 74.6 mol%. Based on the genotypic and phenotypic characteristics, strain SM30T represents a novel genus and species, for which the name Oharaeibacter diazotrophicus gen. nov., sp. nov. is proposed with the type strain SM30T (=NBRC 111955T=DSM 102969T).

Hansschlegelia beijingensis sp. nov., an aerobic, pink-pigmented, facultatively methylotrophic bacterium isolated from watermelon rhizosphere soil.

PubMed

Zou, Xiao-lin; Li, Xiu-ai; Wang, Xu-Ming; Chen, Qiang; Gao, Miao; Qiu, Tian-lei; Sun, Jian-guang; Gao, Jun-lian

2013-10-01

A novel Gram-stain-negative, aerobic, rod-shaped strain designated PG04(T) was isolated from the rhizosphere of watermelon plants cultivated in Beijing, China. A polyphasic taxonomic study was performed on the new isolate. On the basis of 16S rRNA gene sequence similarity studies, isolate PG04(T) belonged clearly to the genus Hansschlegelia and was most closely related to Hansschlegelia zhihuaiae (97.3 % similarity to the type strain). The predominant respiratory quinone was ubiquinone 10 (Q-10) and the polar lipid profile was composed of the major lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major fatty acids were C18 : 1ω7c (41.3 %), C19 : 0 cyclo ω8c (30.6 %) and C16 : 0 (19.1 %). The G+C content of the DNA was about 64.4 mol%. DNA-DNA hybridization experiments showed 34.4 % relatedness between strain PG04(T) and H. zhihuaiae DSM 18984(T). The results of physiological and biochemical tests and differences in fatty acid profiles allowed clear phenotypic differentiation of strain PG04(T) from the most closely related species in the genus, H. zhihuaiae. Strain PG04(T) therefore represents a novel species within the genus Hansschlegelia, for which the name Hansschlegelia beijingensis sp. nov. is proposed, with the type strain PG04(T) ( = DSM 25481(T) = ACCC 05759(T)).

Factors Influencing Bacterial Diversity and Community Composition in Municipal Drinking Waters in the Ohio River Basin, USA

PubMed Central

Stanish, Lee F.; Hull, Natalie M.; Robertson, Charles E.; Harris, J. Kirk; Stevens, Mark J.; Spear, John R.; Pace, Norman R.

2016-01-01

The composition and metabolic activities of microbes in drinking water distribution systems can affect water quality and distribution system integrity. In order to understand regional variations in drinking water microbiology in the upper Ohio River watershed, the chemical and microbiological constituents of 17 municipal distribution systems were assessed. While sporadic variations were observed, the microbial diversity was generally dominated by fewer than 10 taxa, and was driven by the amount of disinfectant residual in the water. Overall, Mycobacterium spp. (Actinobacteria), MLE1-12 (phylum Cyanobacteria), Methylobacterium spp., and sphingomonads were the dominant taxa. Shifts in community composition from Alphaproteobacteria and Betaproteobacteria to Firmicutes and Gammaproteobacteria were associated with higher residual chlorine. Alpha- and beta-diversity were higher in systems with higher chlorine loads, which may reflect changes in the ecological processes structuring the communities under different levels of oxidative stress. These results expand the assessment of microbial diversity in municipal distribution systems and demonstrate the value of considering ecological theory to understand the processes controlling microbial makeup. Such understanding may inform the management of municipal drinking water resources. PMID:27362708

Effects of glucose on microcystin-LR removal and the bacterial community composition through anoxic biodegradation in drinking water sludge.

PubMed

Ma, Guangxiang; Pei, Haiyan; Hu, Wenrong; Xu, Xiangchao; Ma, Chunxia; Pei, Ruoting

2016-01-01

To enhance the degradation efficiency of microcystin (MC) in drinking water sludge (DWS), the underlying mechanisms between organic carbon (glucose) and the biodegradation of MC-LR under anoxic conditions were investigated by polymerase chain reaction-denaturing gradient gel electrophoresis technology. The addition of glucose reduced the rate of the MC-LR biodegradation indicating the occurrence of inhibition of degradation, and an increased inhibition was observed with increases in glucose concentration (0-10,000 mg/L). In addition, the community analysis indicated that the variety and the number of the microbes increased with the concentration of glucose amended (0 -1000 mg/L), but they decreased substantially with the addition of 10,000 mg/L of glucose. The phyla Firmicutes, Proteobacteria and Chloroflexi were found to be the dominant. Methylobacterium and Sphingomonas were MC-degrading bacteria and used glucose as a prior carbon source instead of MC, resulting in the decrease in the MC-LR biodegradation rate under anoxic conditions. Thus, reducing organic carbon could improve the anoxic biodegradation efficiency of MC in DWS.

Purification and properties of the hydroxylase component of methane monooxygenase

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

Patel, R.N.; Savas, J.C.

Methane monooxygenase from Methylobacterium sp. strain CRL-26 which catalyzes the oxygenation of hydrocarbons was resolved into two components, a hydroxylase and a flavoprotein. An anaerobic procedure was developed for the purification of the hydroxylase to homogeneity. The molecular weight of the hydroxylase as determined by gel filtration was 220,000, and that determined by sedimentation equilibrium analysis was about 225,000. The purified hydroxylase contained three nonidentical subunits with molecular weights of about 55,000, 40,000, and 20,000, in equal amounts as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is an ..cap alpha../sub 2/..beta gamma.. protein. Optical absorption spectra revealedmore » peaks near 408 and 280 nm, and fluorescence spectra revealed emission peaks at 490 and 630 nm. The purified hydroxylase contained 2.8 +/- 0.2 mol of iron and 0.5 +/- 0.1 mol of zinc per mol of protein but negligible amounts of acid-labile sulfide. The antisera prepared against the hydroxylase showed cross-reactivity with hydroxylase components in soluble extracts from other methanotrophs.« less

Protein Oxidation: Key to Bacterial Desiccation Resistance?

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

Fredrickson, Jim K.; Li, Shu-Mei W.; Gaidamakova, E.

For extremely ionizing radiation resistant bacteria, survival has been attributed to protection of proteins from oxidative damage during irradiation, with the result that repair systems survive and function with far greater efficiency during recovery than in sensitive bacteria. Here we examined the relationship between survival of dry-climate soil bacteria and the level of cellular protein oxidation induced by desiccation. Bacteria were isolated from surface soils of the shrub-steppe of the U.S. Department of Energy’s Hanford Site in Washington state. A total of 63 isolates were used for phylogenetic analysis. The majority of isolates were closely related to members of themore » genus Deinococcus, with Chelatococcus, Methylobacterium and Bosea also among the genera identified. Desiccation-resistant isolates accumulated high intracellular manganese and low iron concentrations compared to sensitive bacteria. In vivo, proteins of desiccation-resistant bacteria were protected from oxidative modifications that introduce carbonyl groups in sensitive bacteria during drying. We present the case that survival of bacteria that inhabit dry-climate soils are highly dependent on mechanisms which limit protein oxidation during dehydration.« less

Effect of bio-organomineral fertilizer on the growth of chili (Capsicum annum l.)

NASA Astrophysics Data System (ADS)

Nofiyanto, R. T.; Wati, V. R.; Setiawati, S. R.; Noviandi, W. D.; Kuscahyanti, A.; Fuskhah, E.

2018-01-01

The productivity of chili in 2014-2015 has decreased by 29,411 tons due to conversion of agricultural land. One of the efforts to increase the production of chili is by using latosol acid as a cultivation area. The low fertility of latosol land can be overcome by fertilizing. The purpose of this research was to investigate the role of duckweed and Methylobacterium on chili production which cultivated at latosol soil. The design used was Completely Randomized Design (RAL) with 6 treatments and 4 replications. The treatment consisted of P0 = control, P1 = 50 kg / ha, P2 = 100 kg / ha, P3 = 150 kg / ha, P4 = 200 kg / ha, and P5 = 250 kg / ha. Assamed data were analyzed by using variance analysis (ANOVA), Duncan’s Multiple Range Test (DMRT) with confidence level 95% was used for further analysis. The results showed that fertilizer application can increase the fertility of latosol soil, percentage of chili pepper plant life, height, number of productive branches, number of flower, and amount of fruit.

Leguminosae native nodulating bacteria from a gold mine As-contaminated soil: Multi-resistance to trace elements, and possible role in plant growth and mineral nutrition.

PubMed

Rangel, Wesley de M; de Oliveira Longatti, Silvia M; Ferreira, Paulo A A; Bonaldi, Daiane S; Guimarães, Amanda A; Thijs, Sofie; Weyens, Nele; Vangronsveld, Jaco; Moreira, Fatima M S

2017-10-03

Efficient N 2 -fixing Leguminosae nodulating bacteria resistant to As may facilitate plant growth on As-contaminated sites. In order to identify bacteria holding these features, 24 strains were isolated from nodules of the trap species Crotalaria spectabilis (12) and Stizolobium aterrimum (12) growing on an As-contaminated gold mine site. 16S rRNA gene sequencing revealed that most of the strains belonged to the group of α-Proteobacteria, being representatives of the genera Bradyrhizobium, Rhizobium, Inquilinus, Labrys, Bosea, Starkeya, and Methylobacterium. Strains of the first four genera showed symbiotic efficiency with their original host, and demonstrated in vitro specific plant-growth-promoting (PGP) traits (production of organic acids, indole-3-acetic-acid and siderophores, 1-aminocyclopropane-1-carboxylate deaminase activity, and Ca 3 (PO 4 ) 2 solubilization), and increased resistance to As, Zn, and Cd. In addition, these strains and some type and reference rhizobia strains exhibited a wide resistance spectrum to β-lactam antibiotics. Both intrinsic PGP abilities and multi-element resistance of rhizobia are promising for exploiting the symbiosis with different legume plants on trace-element-contaminated soils.

Understanding the degradation of Congo red and bacterial diversity in an air-cathode microbial fuel cell being evaluated for simultaneous azo dye removal from wastewater and bioelectricity generation.

PubMed

Sun, Jian; Li, Youming; Hu, Yongyou; Hou, Bin; Zhang, Yaping; Li, Sizhe

2013-04-01

We investigated the mechanism of Congo red degradation and bacterial diversity in a single-chambered microbial fuel cell (MFC) incorporating a microfiltration membrane and air-cathode. The MFC was operated continuously for more than 4 months using a mixture of Congo red and glucose as fuel. We demonstrated that the Congo red azo bonds were reduced at the anode to form aromatic amines. This is consistent with the known mechanism of anaerobic biodegradation of azo dyes. The MFC developed a less dense biofilm at the anode in the presence of Congo red compared to its absence indicating that Congo red degradation negatively affected biofilm formation. Denaturing gradient gel electrophoresis and direct 16S ribosomal DNA gene nucleotide sequencing revealed that the microbial communities differed depending on whether Congo red was present in the MFC. Geobacter-like species known to generate electricity were detected in the presence or absence of Congo red. In contrast, Azospirillum, Methylobacterium, Rhodobacter, Desulfovibrio, Trichococcus, and Bacteroides species were only detected in its presence. These species were most likely responsible for degrading Congo red.

Microbial Surveillance of Potable Water Sources of the International Space Station

NASA Technical Reports Server (NTRS)

Bruce, Rebekah J.; Ott, C. Mark; Skuratov, Vladimir M.; Pierson, Duane L.

2005-01-01

To mitigate risk to the crew, the microbial surveillance of the quality of potable water sources of the International Space Station (ISS) has been ongoing since before the arrival of the first permanent crew. These water sources have included stored ground-supplied water, water produced by the shuttle fuel cells during flight, and ISS humidity condensate that is reclaimed and processed. Monitoring was accomplished using a self-contained filter designed to allow bacterial growth and enumeration during flight. Upon return to earth, microbial isolates were identified using 16S ribosomal gene sequencing. While the predominant isolates were common Gramnegative bacteria including Ralstonia eutropha, Methylobacterium fujisawaense, and Spingomonas paucimobilis, opportunistic pathogens such as Stenotrophomonas maltophilia and Pseudomonas aeruginosa were also isolated. Results of in-flight enumeration have indicated a fluctuation of bacterial counts above system design specifications. Additional in-flight monitoring capability for the specific detection of coliforms was added in 2004; no coliforms have been detected from any potable water source. Neither the bacterial concentrations nor the identification of the isolates recovered from these samples has suggested a threat to crew health.

Factors Influencing Bacterial Diversity and Community Composition in Municipal Drinking Waters in the Ohio River Basin, USA.

PubMed

Stanish, Lee F; Hull, Natalie M; Robertson, Charles E; Harris, J Kirk; Stevens, Mark J; Spear, John R; Pace, Norman R

2016-01-01

The composition and metabolic activities of microbes in drinking water distribution systems can affect water quality and distribution system integrity. In order to understand regional variations in drinking water microbiology in the upper Ohio River watershed, the chemical and microbiological constituents of 17 municipal distribution systems were assessed. While sporadic variations were observed, the microbial diversity was generally dominated by fewer than 10 taxa, and was driven by the amount of disinfectant residual in the water. Overall, Mycobacterium spp. (Actinobacteria), MLE1-12 (phylum Cyanobacteria), Methylobacterium spp., and sphingomonads were the dominant taxa. Shifts in community composition from Alphaproteobacteria and Betaproteobacteria to Firmicutes and Gammaproteobacteria were associated with higher residual chlorine. Alpha- and beta-diversity were higher in systems with higher chlorine loads, which may reflect changes in the ecological processes structuring the communities under different levels of oxidative stress. These results expand the assessment of microbial diversity in municipal distribution systems and demonstrate the value of considering ecological theory to understand the processes controlling microbial makeup. Such understanding may inform the management of municipal drinking water resources.

A pink-pigmented, oxidative, nonmotile bacterium as a cause of opportunistic infections.

PubMed

Korvick, J A; Rihs, J D; Gilardi, G L; Yu, V L

1989-06-01

We describe two cases of bacteremia due to a pink-pigmented, oxidative, nonmotile, gram-negative, rod-shaped organism. One case occurred in a febrile neutropenic patient and another in a chronically debilitated patient with pancreatic abscess. The first patient was cured with gentamicin and ticarcillin, but the second patient died while receiving cefamandole therapy. The organisms described here are similar to Methylobacterium mesophilicum (Pseudomonas mesophilica) and the "unnamed taxon" organisms. A major difference from M mesophilicum is the lack of methanol utilization. Further distinctions between our isolates and M mesophilicum are the lack of flagella in our organisms, growth at 42 degrees C, growth on MacConkey's agar, lack of acetamide assimilation, and citrate utilization. The lack of flagella is the principle difference between our isolates and those in the unnamed taxon. Both of the isolates were resistant to the cephalosporins, but susceptible to the aminoglycosides, ticarcillin-clavulanic acid, sulfamethoxazole and trimethoprim, and imipenem. With the growing population of immunocompromised and chronically ill patients, these organisms may emerge as important pathogens.

The chemical biology of methanogenesis

NASA Astrophysics Data System (ADS)

Ferry, James G.

2010-12-01

Two distinct pathways account for most of the CH 4 produced in the majority of the diverse and vast anaerobic environments of Earth's biosphere by microbes that are classified in the Archaea domain of life: conversion of the methyl group of acetate to CH 4 in the aceticlastic pathway and reduction of CO 2 with electrons derived from H 2, formate or CO in the CO 2 reduction pathway. Minor, albeit ecologically important, amounts of CH 4 are produced by conversion of methylotrophic substrates methanol, methylamines and methyl sulfides. Although all pathways have terminal steps in common, they deviate in the initial steps leading to CH 4 and mechanisms for synthesizing ATP for growth. Hydrogen gas is the major reductant for CO 2-reducing methanogens in the deep subsurface, although H 2 is also utilized by CO 2-reducing microbes from the Bacteria domain that produce acetate for the aceticlastic methanogens. This review presents fundamentals of the two major CH 4-producing pathways with a focus on understanding the potential for biologically-produced CH 4 on Mars.

Crystal Structure of Alcohol Oxidase from Pichia pastoris

PubMed Central

Valerius, Oliver; Feussner, Ivo; Ficner, Ralf

2016-01-01

FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring. PMID:26905908

Characterization of an aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea with broad substrate specificity against aromatic alcohols.

PubMed

Tamaru, Yoshiaki; Umezawa, Kiwamu; Yoshida, Makoto

2018-07-01

The aim of the study was to obtain information about the enzymatic properties of aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea (rCcAAO), which is classified into the auxiliary activities family 3 subfamily 2 (AA3_2). The gene encoding AAO from the plant saprophytic basidiomycete Coprinopsis cinerea (CcAAO) was cloned, and the recombinant CcAAO (rCcAAO) was heterologously expressed in the methylotrophic yeast Pichia pastoris. The purified rCcAAO showed significant activity not only against trans,trans-2,4-hexadien-1-ol but also against a broad range of aromatic alcohols including aromatic compounds that were reported to be poor substrates for known AAOs. Moreover, site-directed mutagenesis analysis demonstrated that mutants with substitutions from leucine to phenylalanine and tryptophan at position 416 exhibited decreases of activity for aromatic alcohols but still maintained the activity for trans,trans-2,4-hexadien-1-ol. Leucine 416 in CcAAO contributes to the broad substrate specificity against various aromatic alcohols, which is useful for the production of hydrogen peroxide using this enzyme.

Perchlorate bioreduction linked to methane oxidation in a membrane biofilm reactor: Performance and microbial community structure.

PubMed

Xie, Ting; Yang, Qi; Winkler, Mari K H; Wang, Dongbo; Zhong, Yu; An, Hongxue; Chen, Fei; Yao, Fubin; Wang, Xiaolin; Wu, Jiawei; Li, Xiaoming

2018-06-05

Perchlorate bioreduction coupled to methane oxidation was successfully achieved without the addition of nitrate or nitrite in a membrane biofilm reactor (MBfR) inoculated with a mixture of freshwater sediments and anaerobic digester sludge as well as return activated sludge. The reactor was operated at different methane pressures (60, 40 and 20 Kpa) and influent perchlorate concentrations (1, 5 and 10 mg/L) to evaluate the biochemical process of perchlorate bioreduction coupled to methane oxidation. Perchlorate was completely reduced with a higher removal flux of 92.75 mg/m 2 ·d using methane as the sole carbon source and electron donor, other than hydrogen or other limiting organics. Quantitative real-time PCR showed that bacteria prevailed over archaea and the abundances of mcrA, pMMO, pcrA, and nirS genes were correlated with the influent perchlorate flux. High-throughput sequencing of 16S rRNA genes demonstrated that the functional community consisted of methanotrophs, methylotrophs, perchlorate-reducing bacteria, as well as various denitrifiers. Copyright © 2018 Elsevier B.V. All rights reserved.

Occurrence of carotenoids and sporopollenin in Nanochlorum eucaryotum, a novel marine alga with unusual characteristics.

PubMed

Geisert, M; Rose, T; Bauer, W; Zahn, R K

1987-01-01

Pigment analysis of Nanochlorum eucaryotum on two strains grown under different gaseous conditions was performed. Air-gassed control cultures did not differ qualitatively with respect to the content of chlorophylls a and b, carotenes alpha and beta, lutein, violaxanthin, neoxanthin and cryptoxanthin in comparison with cultures grown under natural gas. The absolute pigment content per cell increased in cultures grown with natural gas. Growth of N. eucaryotum depends on CO2 which is present in concentrations up to 2.0 vol% in natural gas. N. eucaryotum cannot utilize methane and is therefore not methylotrophic. In cultures of N. eucaryotum grown with natural gas and in air-gassed cultures under nitrogen deficient conditions the secondary carotenoids canthaxanthin and astaxanthin could be detected. In air-gassed cultures of strain N. eucaryotum Colona the same secondary carotenoids have been found, while secondary carotenoids were never found in strain N. eucaryotum Mainz. Cell walls of N. eucaryotum always contain sporopollenin as confirmed by isolation, elemental analysis, infrared absorption spectrophotometry, acetolysis-resistance and electron microscopy.

Evolution of microbial community along with increasing solid concentration during high-solids anaerobic digestion of sewage sludge.

PubMed

Liu, Can; Li, Huan; Zhang, Yuyao; Si, Dandan; Chen, Qingwu

2016-09-01

High-solids anaerobic digestion (HSAD), a promising method with smaller reactor and less heating energy consumption, showed relatively lower digestion efficiency sometimes and higher tolerance to some inhibitors. To investigate the phenomena, the archaeal and bacterial communities in four anaerobic digesters treating sewage sludge with total solids (TS) of 10-19% were investigated. Although acetoclastic methanogenesis conducted mainly by genus Methanosarcina was still the main pathway producing methane, the total ratio of acetoclastic methanogens decreased along with the increased TS. In contrary, the relative abundance of hydrogenotrophic methanogens increased from 6.8% at TS 10% to 22.3% at TS 19%, and methylotrophic methanogens from 10.4% to 20.9%. The bacterial community was dominated by five phyla. Acidogenic and acetogenic bacteria affiliated to Firmicutes decreased following the increase of TS; while the proteolysis phylum Bacteroidetes increased, with a tolerant family ST-12K33 notably existing in the digesters at TS 17% and 19%. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

PubMed Central

Staley, Chris A.; Huang, Amy; Nattestad, Maria; Oshiro, Kristin T.; Ray, Laura E.; Mulye, Tejas; Li, Zhiguo Harry; Le, Thu; Stephens, Justin J.; Gomez, Seth R.; Moy, Allison D.; Nguyen, Jackson C.; Franz, Andreas H.; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P.

2012-01-01

Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5′ untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5′UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5′UTR clearly decreased expression of a β-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5′UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5′UTR optimized for a higher level of expression may be challenging. PMID:22285974

Epoxidation of Short-Chain Alkenes by Resting-Cell Suspensions of Propane-Grown Bacteria

PubMed Central

Hou, Ching T.; Patel, Ramesh; Laskin, Allen I.; Barnabe, Nancy; Barist, Irene

1983-01-01

Sixteen new cultures of propane-utilizing bacteria were isolated from lake water from Warinanco Park, Linden, N.J. and from lake and soil samples from Bayway Refinery, Linden, N.J. In addition, 19 known cultures obtained from culture collections were also found to be able to grow on propane as the sole carbon and energy source. In addition to their ability to oxidize n-alkanes, resting-cell suspensions of both new cultures and known cultures grown on propane oxidize short-chain alkenes to their corresponding 1,2-epoxides. Among the substrate alkenes, propylene was oxidized at the highest rate. In contrast to the case with methylotrophic bacteria, the product epoxides are further metabolized. Propane and other gaseous n-alkanes inhibit the epoxidation of propylene. The optimum conditions for in vivo epoxidation are described. Results from inhibition studies indicate that a propane monooxygenase system catalyzes both the epoxidation and hydroxylation reactions. Experiments with cell-free extracts show that both hydroxylation and epoxidation activities are located in the soluble fraction obtained after 80,000 × g centrifugation. PMID:16346338

Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

NASA Technical Reports Server (NTRS)

Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

1999-01-01

A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

Cloning of novel cellulases from cellulolytic fungi: heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris.

PubMed

Salinas, Alejandro; Vega, Marcela; Lienqueo, María Elena; Garcia, Alejandro; Carmona, Rene; Salazar, Oriana

2011-12-10

Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation. Copyright © 2011 Elsevier Inc. All rights reserved.

Enzymatic transformation of hydrocarbons by methanotrophic organisms

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

Patel, R.N.; Hou, C.T.

Soluble methane monooxygenase from a facultative methane-utilizing organism, Methylobacterium sp. CRL-26 or R6, catalyzed the NAD(P)H-dependent epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branch-chain alkenes, alkanes (C1-C8), substituted alkanes, branch-chain alkanes, carbon monoxide, ether, cyclic and aromatic compounds. The NAD -linked dehydrogenases such as formate dehydrogenase or secondary alcohol dehydrogenase in the presence of formate or secondary alcohol, respectively, regenerated NAD/NADH required for the methane monooxygenase in a coupled enzymes reactions. Oxidation of secondary alcohols to the corresponding methylketones in methanotrophs is catalyzed by an NAD -dependent, zinc-containing, secondary alcohol hydrogenase. Primary alcohols weremore » oxidized to the corresponding aldehydes by a phenazine methosulfate-dependent, pyrollo quinoline quinone (methoxatin or PQQ) containing, methanol dehydrogenase. Oxidation of aldehydes (C1 to C10) to the corresponding carboxylic acids is catalyzed by a heme-containing aldehyde dehydrogenase. Methanotrophs have been considered potentially useful for single cell protein (SCP), amino acids, and biopolymer production at the expense of growth on cheap and readily available C1 compounds. 80 references, 1 figure, 6 tables.« less

Methanotrophs and Methanogens in Masonry

PubMed Central

Kussmaul, Martin; Wilimzig, Markus; Bock, Eberhard

1998-01-01

Methanotrophs were present in 48 of 225 stone samples which were removed from 19 historical buildings in Germany and Italy. The average cell number of methanotrophs was 20 CFU per g of stone, and their activities ranged between 11 and 42 pmol of CH4 g of stone−1 day−1. Twelve strains of methane-oxidizing bacteria were isolated. They belonged to the type II methanotrophs of the genera Methylocystis, Methylosinus, and Methylobacterium. In masonry, growth substrates like methane or methanol are available in very low concentrations. To determine if methane could be produced by the stone at rates sufficient to support growth of methanotrophs, methane production by stone samples under nonoxic conditions was examined. Methane production of 0.07 to 215 nmol of CH4 g of stone−1 day−1 was detected in 23 of 47 stone samples examined. This indicated the presence of the so-called “mini-methane”-producing bacteria and/or methanogenic archaea. Methanotrophs occurred in nearly all samples which showed methane production. This finding indicated that methanotrophs depend on biogenic methane production in or on stone surfaces of historical buildings. PMID:9797318

Methanotrophs and methanogens in masonry

PubMed

Kussmaul; Wilimzig; Bock

1998-11-01

Methanotrophs were present in 48 of 225 stone samples which were removed from 19 historical buildings in Germany and Italy. The average cell number of methanotrophs was 20 CFU per g of stone, and their activities ranged between 11 and 42 pmol of CH4 g of stone-1 day-1. Twelve strains of methane-oxidizing bacteria were isolated. They belonged to the type II methanotrophs of the genera Methylocystis, Methylosinus, and Methylobacterium. In masonry, growth substrates like methane or methanol are available in very low concentrations. To determine if methane could be produced by the stone at rates sufficient to support growth of methanotrophs, methane production by stone samples under nonoxic conditions was examined. Methane production of 0.07 to 215 nmol of CH4 g of stone-1 day-1 was detected in 23 of 47 stone samples examined. This indicated the presence of the so-called "mini-methane"-producing bacteria and/or methanogenic archaea. Methanotrophs occurred in nearly all samples which showed methane production. This finding indicated that methanotrophs depend on biogenic methane production in or on stone surfaces of historical buildings.

Bacterial community structures in air conditioners installed in Japanese residential buildings.

PubMed

Hatayama, Kouta; Oikawa, Yurika; Ito, Hiroyuki

2018-01-01

The bacterial community structures in four Japanese split-type air conditioners were analyzed using a next-generation sequencer. A variety of bacteria were detected in the air filter of an air conditioner installed on the first floor. In the evaporator of this air conditioner, bacteria belonging to the genus Methylobacterium, or the family of Sphingomonadaceae, were predominantly detected. On the other hand, the majority of bacteria detected in the air filters and evaporators of air conditioners installed on the fifth and twelfth floors belonged to the family Enterobacteriaceae. The source of bacteria belonging to the family Enterobacteriaceae may have been aerosols generated by toilet flushing in the buildings. Our results suggested the possibility that the bacterial contamination in the air conditioners was affected by the floor level on which they were installed. The air conditioner installed on the lower floor, near the ground, may have been contaminated by a variety of outdoor bacteria, whereas the air conditioners installed on floors more distant from the ground may have been less contaminated by outdoor bacteria. However, these suppositions may apply only to the specific split-type air conditioners that we analyzed, because our sample size was small.

[Inactivation of the chlorine-resistant bacteria isolated from the drinking water distribution system].

PubMed

Chen, Yu-Qiao; Duan, Xiao-Di; Lu, Pin-Pin; Wang, Qian; Zhang, Xiao-Jian; Chen, Chao

2012-01-01

Inactivation experiments of seven strains of chlorine-resistant bacteria, isolated from a drinking water distribution system, were conducted with four kinds of disinfectants. All the bacteria showed high resistance to chlorine, especially for Mycobacterium mucogenicum. The CT value of 99.9% inactivation for M. mucogenicum, Sphingomonas sanguinis and Methylobacterium were 120 mg x (L x min)(-1), 7 mg x (L x min)(-1) and 4 mg x (L x min)(-1), respectively. The results of inactivation experiments showed that chlorine dioxide and potassium monopersulfate could inactive 5 lg of M. mucogenicum within 30 min, which showed significantly higher efficiency than free chlorine and monochloramine. Free chlorine was less effective because the disinfectant decayed very quickly. Chloramination needed higher concentration to meet the disinfection requirements. The verified dosage of disinfectants, which could effectively inactivate 99.9% of the highly chlorine-resistant M. mucogenicum within 1 h, were 3.0 mg/L monochloramine, 1.0 mg/L chlorine dioxide (as Cl2), and 1.0 mg/L potassium monopersulfate (as Cl2). It was suggested that the water treatment plants increase the concentration of monochloramine or apply chlorine dioxide intermittently to control the disinfectant-resistant bacteria.

Plant-associated methylobacteria as co-evolved phytosymbionts: a hypothesis.

PubMed

Kutschera, Ulrich

2007-03-01

Due to their wall-associated pectin metabolism, growing plant cells emit significant amounts of the one-carbon alcohol methanol. Pink-pigmented microbes of the genus Methylobacterium that colonize the surfaces of leaves (epiphytes) are capable of growth on this volatile C1-compound as sole source of carbon and energy. In this article the results of experiments with germ-free (gnotobiotic) sporophytes of angiosperms (sunflower, maize) and gametophytes of bryophytes (a moss and two liverwort species) are summarized. The data show that methylobacteria do not stimulate the growth of these angiosperms, but organ development in moss protonemata and in thalli of liverworts is considerably enhanced. Since methylobacteria produce and secrete cytokinins and auxin, a model of plant-microbe-interaction (symbiosis) is proposed in which the methanol-consuming bacteria are viewed as coevolved partners of the gametophyte that determine its growth, survival and reproduction (fitness). This symbiosis is restricted to the haploid cells of moisture-dependent "living fossil" plants; it does not apply to the diploid sporophytes of higher embryophytes, which are fully adapted to life on land and apparently produce sufficient amounts of endogenous phytohormones.

Microbial Characterization During the Early Habitation of the International Space Station

NASA Technical Reports Server (NTRS)

Castro, V. A.; Thrasher, A. N.; Healy, M.; Ott, C. M.; Pierson, D. L.

2004-01-01

An evaluation of the microbiota from air, water, and surface samples provided a baseline of microbial characterization onboard the International Space Station (ISS) to gain insight into bacterial and fungal contamination during the initial stages of construction and habitation. Using 16S genetic sequencing and rep-PCR, 63 bacterial strains were isolated for identification and fingerprinted for microbial tracking. Of the bacterial strains that were isolated and fingerprinted, 19 displayed similarity to each other. The use of these molecular tools allowed for the identification of bacteria not previously identified using automated biochemical analysis and provided a clear indication of the source of several ISS contaminants. Strains of Bradyrhizobium and Sphingomonas unable to be identified using sequencing were identified by comparison of rep-PCR DNA fingerprints. Distinct DNA fingerprints for several strains of Methylobacterium provided a clear indication of the source of an ISS water supply contaminant. Fungal and bacterial data acquired during monitoring do not suggest there is a current microbial hazard to the spacecraft, nor does any trend indicate a potential health risk. Previous spacecraft environmental analysis indicated that microbial contamination will increase with time and will require continued surveillance. Copyright 2004 Springer-Verlag.

Prevalence of pathogenic bacteria in Ixodes ricinus ticks in Central Bohemia.

PubMed

Klubal, Radek; Kopecky, Jan; Nesvorna, Marta; Sparagano, Olivier A E; Thomayerova, Jana; Hubert, Jan

2016-01-01

Bacteria associated with the tick Ixodes ricinus were assessed in specimens unattached or attached to the skin of cats, dogs and humans, collected in the Czech Republic. The bacteria were detected by PCR in 97 of 142 pooled samples including 204 ticks, i.e. 1-7 ticks per sample, collected at the same time from one host. A fragment of the bacterial 16S rRNA gene was amplified, cloned and sequenced from 32 randomly selected samples. The most frequent sequences were those related to Candidatus Midichloria midichlori (71% of cloned sequences), followed by Diplorickettsia (13%), Spiroplasma (3%), Rickettsia (3%), Pasteurella (3%), Morganella (3%), Pseudomonas (2%), Bacillus (1%), Methylobacterium (1%) and Phyllobacterium (1%). The phylogenetic analysis of Spiroplasma 16S rRNA gene sequences showed two groups related to Spiroplasma eriocheiris and Spiroplasma melliferum, respectively. Using group-specific primers, the following potentially pathogenic bacteria were detected: Borellia (in 20% of the 142 samples), Rickettsia (12%), Spiroplasma (5%), Diplorickettsia (5%) and Anaplasma (2%). In total, 68% of I. ricinus samples (97/142) contained detectable bacteria and 13% contained two or more putative pathogenic groups. The prevalence of tick-borne bacteria was similar to the observations in other European countries.

Plant-Associated Methylobacteria as Co-Evolved Phytosymbionts

PubMed Central

2007-01-01

Due to their wall-associated pectin metabolism, growing plant cells emit significant amounts of the one-carbon alcohol methanol. Pink-pigmented microbes of the genus Methylobacterium that colonize the surfaces of leaves (epiphytes) are capable of growth on this volatile C1-compound as sole source of carbon and energy. In this article the results of experiments with germ-free (gnotobiotic) sporophytes of angiosperms (sunflower, maize) and gametophytes of bryophytes (a moss and two liverwort species) are summarized. The data show that methylobacteria do not stimulate the growth of these angiosperms, but organ development in moss protonemata and in thalli of liverworts is considerably enhanced. Since methylobacteria produce and secrete cytokinins and auxin, a model of plant-microbe-interaction (symbiosis) is proposed in which the methanol-consuming bacteria are viewed as coevolved partners of the gametophyte that determine its growth, survival and reproduction (fitness). This symbiosis is restricted to the haploid cells of moisture-dependent “living fossil” plants; it does not apply to the diploid sporophytes of higher embryophytes, which are fully adapted to life on land and apparently produce sufficient amounts of endogenous phytohormones. PMID:19516971

Microbial methane from in situ biodegradation of coal and shale: A review and reevaluation of hydrogen and carbon isotope signatures

USGS Publications Warehouse

Vinson, David S.; Blair, Neal E.; Martini, Anna M.; Larter, Steve; Orem, William H.; McIntosh, Jennifer C.

2017-01-01

Stable carbon and hydrogen isotope signatures of methane, water, and inorganic carbon are widely utilized in natural gas systems for distinguishing microbial and thermogenic methane and for delineating methanogenic pathways (acetoclastic, hydrogenotrophic, and/or methylotrophic methanogenesis). Recent studies of coal and shale gas systems have characterized in situ microbial communities and provided stable isotope data (δD-CH4, δD-H2O, δ13C-CH4, and δ13C-CO2) from a wider range of environments than available previously. Here we review the principal biogenic methane-yielding pathways in coal beds and shales and the isotope effects imparted on methane, document the uncertainties and inconsistencies in established isotopic fingerprinting techniques, and identify the knowledge gaps in understanding the subsurface processes that govern H and C isotope signatures of biogenic methane. We also compare established isotopic interpretations with recent microbial community characterization techniques, which reveal additional inconsistencies in the interpretation of microbial metabolic pathways in coal beds and shales. Collectively, the re-assessed data show that widely-utilized isotopic fingerprinting techniques neglect important complications in coal beds and shales.Isotopic fingerprinting techniques that combine δ13C-CH4 with δD-CH4 and/or δ13C-CO2have significant limitations: (1) The consistent ~ 160‰ offset between δD-H2O and δD-CH4 could imply that hydrogenotrophic methanogenesis is the dominant metabolic pathway in microbial gas systems. However, hydrogen isotopes can equilibrate between methane precursors and coexisting water, yielding a similar apparent H isotope signal as hydrogenotrophic methanogenesis, regardless of the actual methane formation pathway. (2) Non-methanogenic processes such as sulfate reduction, Fe oxide reduction, inputs of thermogenic methane, anaerobic methane oxidation, and/or formation water interaction can cause the apparent carbon

Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus.

PubMed

Romkina, Anastasia Y; Kiriukhin, Michael Y

2017-01-01

The isocitrate dehydrogenase (MfIDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E. coli as a fused protein. This enzyme is homodimeric,-with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD+ and 63 U mg -1 with NADP+. The MfIDH activity was dependent on divalent cations and Mn2+ enhanced the activity the most effectively. MfIDH exhibited a cofactor-dependent pH-activity profile. The optimum pH values were 8.5 (NAD+) and 6.0 (NADP+).The Km values for NAD+ and NADP+ were 113 μM and 184 μM respectively, while the Km values for DL-isocitrate were 9.0 μM (NAD+), 8.0 μM (NADP+). The MfIDH specificity (kcat/Km) was only 5-times higher for NAD+ than for NADP+. The purified MfIDH displayed maximal activity at 60°C. Heat-inactivation studies showed that the MfIDH was remarkably thermostable, retaining full activity at 50°C and losting ca. 50% of its activity after one hour of incubation at 75°C. The enzyme was insensitive to the presence of intermediate metabolites, with the exception of 2 mM ATP, which caused 50% inhibition of NADP+-linked activity. The indispensability of the N6 amino group of NAD(P)+ in its binding to MfIDH was demonstrated. MfIDH showed high sequence similarity with bacterial NAD(P)+-dependent type I isocitrate dehydrogenases (IDHs) rather than with eukaryotic NAD+-dependent IDHs. The unique double coenzyme specificity of MfIDH potentially resulted from the Lys340, Ile341 and Ala347 residues in the coenzyme-binding site of the enzyme. The discovery of a type I IDH with double coenzyme specificity elucidates the evolution of this subfamily IDHs and may provide fundamental information for engineering enzymes with desired properties.

Strain-level genetic diversity of Methylophaga nitratireducenticrescens confers plasticity to denitrification capacity in a methylotrophic marine denitrifying biofilm.

PubMed

Geoffroy, Valérie; Payette, Geneviève; Mauffrey, Florian; Lestin, Livie; Constant, Philippe; Villemur, Richard

2018-01-01

The biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO[Formula: see text]) reduction to N 2 , whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59. The denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated on Methylophaga specific medium to isolate denitrifying Methylophaga isolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO[Formula: see text], nitrite (NO[Formula: see text]) and N 2 O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures. During batch cultures of the biofilm, we observed the disappearance of H. nitrativorans NL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, like H. nitrativorans NL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2-3 orders of magnitude higher than M. nitratireducenticrescens JAM1 and H. nitrativorans NL23. Genome analyses revealed that strain GP59 belongs to the species M. nitratireducenticrescens . The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO[Formula: see text] under oxic culture conditions, and has a 24-h lag time before growth and NO[Formula: see text] reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO[Formula: see text] as it carries a gene encoding a NirK-type NO[Formula: see text] reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process. These results reinforce the unique trait of the species M. nitratireducenticrescens among the Methylophaga genus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor.

Strain-level genetic diversity of Methylophaga nitratireducenticrescens confers plasticity to denitrification capacity in a methylotrophic marine denitrifying biofilm

PubMed Central

Geoffroy, Valérie; Payette, Geneviève; Mauffrey, Florian; Lestin, Livie; Constant, Philippe

2018-01-01

Background The biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${}_{3}^{-}$\\end{document}3−) reduction to N2, whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59. Methods The denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated on Methylophaga specific medium to isolate denitrifying Methylophaga isolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${}_{3}^{-}$\\end{document}3−, nitrite (NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${}_{2}^{-}$\\end{document}2−) and N2O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures. Results During batch cultures of the biofilm, we observed the disappearance of H. nitrativorans NL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, like H. nitrativorans NL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2–3 orders of magnitude higher than M. nitratireducenticrescens JAM1 and H. nitrativorans NL23. Genome analyses revealed that strain GP59 belongs to the species M. nitratireducenticrescens. The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${}_{3}^{-}$\\end{document}3− under oxic culture conditions, and has a 24-h lag time before growth and NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${}_{3}^{-}$\\end{document}3− reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${}_{2}^{-}$\\end{document}2− as it carries a gene encoding a NirK-type NO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${}_{2}^{-}$\\end{document}2− reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process. Discussion These results reinforce the unique trait of the species M. nitratireducenticrescens among the Methylophaga genus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor. PMID:29707436

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter.

PubMed

Mariz, F C; Coimbra, E C; Jesus, A L S; Nascimento, L M; Torres, F A G; Freitas, A C

2015-01-01

The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.

An Evolutionary Perspective on Yeast Mating-Type Switching

PubMed Central

Hanson, Sara J.; Wolfe, Kenneth H.

2017-01-01

Cell differentiation in yeast species is controlled by a reversible, programmed DNA-rearrangement process called mating-type switching. Switching is achieved by two functionally similar but structurally distinct processes in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In both species, haploid cells possess one active and two silent copies of the mating-type locus (a three-cassette structure), the active locus is cleaved, and synthesis-dependent strand annealing is used to replace it with a copy of a silent locus encoding the opposite mating-type information. Each species has its own set of components responsible for regulating these processes. In this review, we summarize knowledge about the function and evolution of mating-type switching components in these species, including mechanisms of heterochromatin formation, MAT locus cleavage, donor bias, lineage tracking, and environmental regulation of switching. We compare switching in these well-studied species to others such as Kluyveromyces lactis and the methylotrophic yeasts Ogataea polymorpha and Komagataella phaffii. We focus on some key questions: Which cells switch mating type? What molecular apparatus is required for switching? Where did it come from? And what is the evolutionary purpose of switching? PMID:28476860

Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode.

PubMed

Stasyuk, Nataliya; Smutok, Oleh; Gayda, Galina; Vus, Bohdan; Koval'chuk, Yevgen; Gonchar, Mykhailo

2012-01-01

A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of human ATP-binding cassette protein subfamily D reconstituted into proteoliposomes.

PubMed

Okamoto, Takumi; Kawaguchi, Kosuke; Watanabe, Shiro; Agustina, Rina; Ikejima, Toshiki; Ikeda, Keisuke; Nakano, Minoru; Morita, Masashi; Imanaka, Tsuneo

2018-02-19

In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1‒3 are located on peroxisomal membrane and play an important role in the transportation of various fatty acid-CoA derivatives, including very long chain fatty acid-CoA, into peroxisomes. ABCD4 is located on lysosomal membrane and is suggested to be involved in the transport of vitamin B 12 from lysosomes to the cytosol. However, the precise transport mechanism by which these ABC transporters facilitate the import or export of substrate has yet to be well elucidated. In this study, the overexpression of human ABCD1‒4 in the methylotrophic yeast Pichia pastoris and a purification procedure were developed. The detergent-solubilized proteins were reconstituted into liposomes. ABCD1‒4 displayed stable ATPase activity, which was inhibited by AlF 3 . Furthermore, ABCD1‒4 were found to possess an equal levels of acyl-CoA thioesterase activity. Proteoliposomes is expected to be an aid in the further biochemical characterization of ABCD transporters. Copyright © 2018 Elsevier Inc. All rights reserved.

Yeast nitrogen utilization in the phyllosphere during plant lifespan under regulation of autophagy

PubMed Central

Shiraishi, Kosuke; Oku, Masahide; Kawaguchi, Kosuke; Uchida, Daichi; Yurimoto, Hiroya; Sakai, Yasuyoshi

2015-01-01

Recently, microbe-plant interactions at the above-ground parts have attracted great attention. Here we describe nitrogen metabolism and regulation of autophagy in the methylotrophic yeast Candida boidinii, proliferating and surviving on the leaves of Arabidopsis thaliana. After quantitative analyses of yeast growth on the leaves of A. thaliana with the wild-type and several mutant yeast strains, we showed that on young leaves, nitrate reductase (Ynr1) was necessary for yeast proliferation, and the yeast utilized nitrate as nitrogen source. On the other hand, a newly developed methylamine sensor revealed appearance of methylamine on older leaves, and methylamine metabolism was induced in C. boidinii, and Ynr1 was subjected to degradation. Biochemical and microscopic analysis of Ynr1 in vitro during a shift of nitrogen source from nitrate to methylamine revealed that Ynr1 was transported to the vacuole being the cargo for biosynthetic cytoplasm-to-vacuole targeting (Cvt) pathway, and degraded. Our results reveal changes in the nitrogen source composition for phyllospheric yeasts during plant aging, and subsequent adaptation of the yeasts to this environmental change mediated by regulation of autophagy. PMID:25900611

The transcriptome of Bathymodiolus azoricus gill reveals expression of genes from endosymbionts and free-living deep-sea bacteria.

PubMed

Egas, Conceição; Pinheiro, Miguel; Gomes, Paula; Barroso, Cristina; Bettencourt, Raul

2012-08-01

Deep-sea environments are largely unexplored habitats where a surprising number of species may be found in large communities, thriving regardless of the darkness, extreme cold, and high pressure. Their unique geochemical features result in reducing environments rich in methane and sulfides, sustaining complex chemosynthetic ecosystems that represent one of the most surprising findings in oceans in the last 40 years. The deep-sea Lucky Strike hydrothermal vent field, located in the Mid Atlantic Ridge, is home to large vent mussel communities where Bathymodiolus azoricus represents the dominant faunal biomass, owing its survival to symbiotic associations with methylotrophic or methanotrophic and thiotrophic bacteria. The recent transcriptome sequencing and analysis of gill tissues from B. azoricus revealed a number of genes of bacterial origin, hereby analyzed to provide a functional insight into the gill microbial community. The transcripts supported a metabolically active microbiome and a variety of mechanisms and pathways, evidencing also the sulfur and methane metabolisms. Taxonomic affiliation of transcripts and 16S rRNA community profiling revealed a microbial community dominated by thiotrophic and methanotrophic endosymbionts of B. azoricus and the presence of a Sulfurovum-like epsilonbacterium.

Field assessment of bacterial communities and total trihalomethanes: Implications for drinking water networks.

PubMed

Montoya-Pachongo, Carolina; Douterelo, Isabel; Noakes, Catherine; Camargo-Valero, Miller Alonso; Sleigh, Andrew; Escobar-Rivera, Juan-Carlos; Torres-Lozada, Patricia

2018-03-01

Operation and maintenance (O&M) of drinking water distribution networks (DWDNs) in tropical countries simultaneously face the control of acute and chronic risks due to the presence of microorganisms and disinfection by-products, respectively. In this study, results from a detailed field characterization of microbiological, chemical and infrastructural parameters of a tropical-climate DWDN are presented. Water physicochemical parameters and the characteristics of the network were assessed to evaluate the relationship between abiotic and microbiological factors and their association with the presence of total trihalomethanes (TTHMs). Illumina sequencing of the bacterial 16s rRNA gene revealed significant differences in the composition of biofilm and planktonic communities. The highly diverse biofilm communities showed the presence of methylotrophic bacteria, which suggest the presence of methyl radicals such as THMs within this habitat. Microbiological parameters correlated with water age, pH, temperature and free residual chlorine. The results from this study are necessary to increase the awareness of O&M practices in DWDNs required to reduce biofilm formation and maintain appropriate microbiological and chemical water quality, in relation to biofilm detachment and DBP formation. Copyright © 2017 Elsevier B.V. All rights reserved.

A thermostable GH26 endo-β-mannanase from Myceliophthora thermophila capable of enhancing lignocellulose degradation.

PubMed

Katsimpouras, Constantinos; Dimarogona, Maria; Petropoulos, Pericles; Christakopoulos, Paul; Topakas, Evangelos

2016-10-01

The endomannanase gene em26a from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 26, was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative endomannanase, dubbed MtMan26A, was purified to homogeneity (60 kDa) and subsequently characterized. The optimum pH and temperature for the enzymatic activity of MtMan26A were 6.0 and 60 °C, respectively. MtMan26A showed high specific activity against konjac glucomannan and carob galactomannan, while it also exhibited high thermal stability with a half-life of 14.4 h at 60 °C. Thermostability is of great importance, especially in industrial processes where harsh conditions are employed. With the aim of better understanding its structure-function relationships, a homology model of MtMan26A was constructed, based on the crystallographic structure of a close homologue. Finally, the addition of MtMan26A as a supplement to the commercial enzyme mixture Celluclast® 1.5 L and Novozyme® 188 resulted in enhanced enzymatic hydrolysis of pretreated beechwood sawdust, improving the release of total reducing sugars and glucose by 13 and 12 %, respectively.

Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris.

PubMed

Aloulou, Ahmed; Grandval, Philippe; De Caro, Josiane; De Caro, Alain; Carrière, Frédéric

2006-06-01

High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.

Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production

PubMed Central

Catlett, Jennie L.; Ortiz, Alicia M.

2015-01-01

Methanogens are anaerobic archaea that grow by producing methane, a gas that is both an efficient renewable fuel and a potent greenhouse gas. We observed that overexpression of the cytoplasmic heterodisulfide reductase enzyme HdrABC increased the rate of methane production from methanol by 30% without affecting the growth rate relative to the parent strain. Hdr enzymes are essential in all known methane-producing archaea. They function as the terminal oxidases in the methanogen electron transport system by reducing the coenzyme M (2-mercaptoethane sulfonate) and coenzyme B (7-mercaptoheptanoylthreonine sulfonate) heterodisulfide, CoM-S-S-CoB, to regenerate the thiol-coenzymes for reuse. In Methanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates. When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates. These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells. PMID:26162885

Archaeal populations in hypersaline sediments underlying orange microbial mats in the Napoli mud volcano.

PubMed

Lazar, Cassandre Sara; L'haridon, Stéphane; Pignet, Patricia; Toffin, Laurent

2011-05-01

Microbial mats in marine cold seeps are known to be associated with ascending sulfide- and methane-rich fluids. Hence, they could be visible indicators of anaerobic oxidation of methane (AOM) and methane cycling processes in underlying sediments. The Napoli mud volcano is situated in the Olimpi Area that lies on saline deposits; from there, brine fluids migrate upward to the seafloor. Sediments associated with a brine pool and microbial orange mats of the Napoli mud volcano were recovered during the Medeco cruise. Based on analysis of RNA-derived sequences, the "active" archaeal community was composed of many uncultured lineages, such as rice cluster V or marine benthic group D. Function methyl coenzyme M reductase (mcrA) genes were affiliated with the anaerobic methanotrophic Archaea (ANME) of the ANME-1, ANME-2a, and ANME-2c groups, suggesting that AOM occurred in these sediment layers. Enrichment cultures showed the presence of viable marine methylotrophic Methanococcoides in shallow sediment layers. Thus, the archaeal community diversity seems to show that active methane cycling took place in the hypersaline microbial mat-associated sediments of the Napoli mud volcano.

Archaeal Populations in Hypersaline Sediments Underlying Orange Microbial Mats in the Napoli Mud Volcano▿†

PubMed Central

Lazar, Cassandre Sara; L'Haridon, Stéphane; Pignet, Patricia; Toffin, Laurent

2011-01-01

Microbial mats in marine cold seeps are known to be associated with ascending sulfide- and methane-rich fluids. Hence, they could be visible indicators of anaerobic oxidation of methane (AOM) and methane cycling processes in underlying sediments. The Napoli mud volcano is situated in the Olimpi Area that lies on saline deposits; from there, brine fluids migrate upward to the seafloor. Sediments associated with a brine pool and microbial orange mats of the Napoli mud volcano were recovered during the Medeco cruise. Based on analysis of RNA-derived sequences, the “active” archaeal community was composed of many uncultured lineages, such as rice cluster V or marine benthic group D. Function methyl coenzyme M reductase (mcrA) genes were affiliated with the anaerobic methanotrophic Archaea (ANME) of the ANME-1, ANME-2a, and ANME-2c groups, suggesting that AOM occurred in these sediment layers. Enrichment cultures showed the presence of viable marine methylotrophic Methanococcoides in shallow sediment layers. Thus, the archaeal community diversity seems to show that active methane cycling took place in the hypersaline microbial mat-associated sediments of the Napoli mud volcano. PMID:21335391