Science.gov

Sample records for alcaligenes eutrophus ch34

  1. Introduction of Pseudomonas aeruginosa mutator phage D3112 into Alcaligenes eutrophus strain CH34.

    PubMed

    Krylov, V; Merlin, C; Toussaint, A

    1995-01-01

    We have investigated the possibility of growing mutator phages from Pseudomonas aeruginosa on various isolates of Alcaligenes eutrophus. Although none out of 10 A. eutrophus strains were susceptible to infection with any of the phages tested, phage D3112 could be readily transferred in our model strain CH34 by means of an RP4::D3112 plasmid. CH34/RP4::D3112 lysogens were stable and produced phages. However, neither mitomycin C nor UV treatment increased the phage yield.

  2. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals

    SciTech Connect

    Mergeay, M.; Nies, D.; Schlegel, H.G.; Gerits, J.; Charles, P.; Van Gijsegem, F.

    1985-04-01

    Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrocarbon, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic/sup +/, 2.5; Nic/sup -/, 0.6; Cob/sup +/A, 5.0; Cob/sup +/B, 20.0; Cob/sup -/, < 0.07; Zin/sup +/, 12.0; Zin/sup -/, 0.6; Cad/sup +/, 2.5; and Cad/sup -/, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).

  3. Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34.

    PubMed Central

    Liesegang, H; Lemke, K; Siddiqui, R A; Schlegel, H G

    1993-01-01

    From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 [ORF]; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression. ORF0ab and ORF1 exhibit a codon usage that is not typical for A. eutrophus. The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis. Among 72 insertion mutants, the majority were nickel sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30% similarity

  4. [Introduction of mutator phage D3112 of Pseudomonas aeruginosa into Alcaligenes eutrophus var. metallotollerans (Strain CH34)].

    PubMed

    Krylov, V N

    1996-01-01

    It is demonstrated that the intact genome of a D3112 tranposable phage (TP) of Pseudomonas aeruginosa, integrated into a recombinant plasmid RP4 :: D3112, can be transferred by means of conjugation from P. putida PpG1 (RP4npt :: D3112) donor cells into Alcaligenes eutrophus var. metallotollerans cells. P. aeruginosa strains are unacceptable as donors because they have a bactericidal effect on A. eutrophus. RP4npt :: D3112 plasmid is stably inherited by A. eutrophus with D3112 being expressed and successfully reproduced. However, TP loses the induction ability after UV irradiation or mitomycin C treatment. It is suggested that D3112 TP and its miniderivatives could be used in manipulations with A. eutrophus var. metallotolerans.

  5. Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor.

    PubMed Central

    Gilis, A; Khan, M A; Cornelis, P; Meyer, J M; Mergeay, M; van der Lelie, D

    1996-01-01

    Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus. PMID:8808942

  6. High-Level Nickel Resistance in Alcaligenes xylosoxydans 31A and Alcaligenes eutrophus KTO2

    PubMed Central

    Schmidt, Thomas; Stoppel, Ralf-D.; Schlegel, Hans G.

    1991-01-01

    Two new nickel-resistant strains of Alcaligenes species were selected from a large number (about 400) of strains isolated from ecosystems polluted by heavy metals and were studied on the physiological and molecular level. Alcaligenes xylosoxydans 31A is a heterotrophic bacterium, and Alcaligenes eutrophus KTO2 is an autotrophic aerobic hydrogen-oxidizing bacterium. Both strains carry—among other plasmids—a megaplasmid determining resistance to 20 to 50 mM NiCl2 and 20 mM CoCl2 (when growing in defined Tris-buffered media). Megaplasmids pTOM8, pTOM9 from strain 31A, and pGOE2 from strain KTO2 confer nickel resistance to the same degree to transconjugants of all strains of A. eutrophus tested but were not transferred to Escherichia coli. However, DNA fragments carrying the nickel resistance genes, cloned into broad-hostrange vector pVDZ'2, confer resistance to A. eutrophus derivatives as well as E. coli. The DNA fragments of both bacteria, TBA8, TBA9, and GBA (14.5-kb BamHI fragments), appear to be identical. They share equal size, restriction maps, and strong DNA homology but are largely different from fragment HKI of nickel-cobalt resistance plasmid pMOL28 of A. eutrophus CH34. Images PMID:16348590

  7. Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus.

    PubMed Central

    Nies, D H; Silver, S

    1989-01-01

    In Alcaligenes eutrophus CH34, resistance to chromate is plasmid determined, inducible, and based on decreased net accumulation of the metal anion. Plasmid-encoded resistances to zinc, cadmium, cobalt, and nickel are resulting from inducible, energy-dependent cation efflux systems. PMID:2914875

  8. Dense autotrophic cultures of Alcaligenes eutrophus.

    PubMed Central

    Repaske, R; Mayer, R

    1976-01-01

    Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere. It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting. As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h. Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h. The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter. PMID:10840

  9. Molecular and genetic characterization of an Alcaligenes eutrophus insertion element.

    PubMed Central

    Kung, S S; Chen, J; Chow, W Y

    1992-01-01

    An insertion element, ISAE1, was discovered during the molecular analysis of mutants defective in the autotrophic growth (Aut-) of Alcaligenes eutrophus H1-4, a mitomycin C-generated derivative of strain H1. ISAE1 is 1,313 bp long, has 12-bp nearly perfect inverted terminal repeats, and contains an open reading frame that has a coding capacity of 408 amino acids. Direct repeats of 8 bp were generated by insertion of ISAE1 into chromosomes or plasmids. Most insertion were found in the AT-rich target sites. The distribution of ISAE1 is limited to A. eutrophus H1 (ATCC 17698) and H16 (ATCC 17699). Variants with newly transposed copies of ISAE1 could be isolated at an elevated frequency by changing the growth conditions. Images PMID:1334068

  10. Quantitative requirements for exponential growth of Alcaligenes eutrophus.

    PubMed Central

    Repaske, R; Repaske, A C

    1976-01-01

    Quantitative nutrient requirements for unrestricted autotrophic growth of Alcaligenes eutrophus were determined. Minimum saturating concentrations of Mg2+, SO42-, PO43-, Fe3+, and Na2+ for an optical density increase of 2 were 10(-4) M 8 X10(-5) M, 5 X 10(-4) to 6 X 10(-4) M, 10(-5) M, and 10(-7) to 2 X 10(-7) M, respectively. Trace metal requirements for cobalt, chromium, and copper were also demonstrated, but minimum concentrations could not be determined because other reagents contributed a high background of these metals. Under certain conditions an apparent response to zinc was observed, although other experiments suggest the zinc salt contained another metal that was required for growth. Poly-beta-hydroxybutyrate biosynthesis was shown to be initiated by a magnesium or sulfate deficiency as well as by a nitrogen or phosphate deficiency. PMID:984831

  11. Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp.

    PubMed Central

    Siddiqui, R A; Benthin, K; Schlegel, H G

    1989-01-01

    The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A. eutrophus H16 and subjected to cloning procedures. After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated. HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions. Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19. Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance. The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34. The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A. eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance. In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly. The hybrid plasmid metal resistance was not expressed in Escherichia coli. DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant. Images PMID:2549012

  12. Purification, crystallization and preliminary X-ray studies of two isoforms of Rubisco from Alcaligenes eutrophus.

    PubMed

    Hansen, S; Hough, E; Andersen, K

    1999-01-01

    Two different isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Alcaligenes eutrophus have been purified and crystallized. Both isoforms crystallize in space group P43212. Crystals of isoform I (unit-cell dimensions a = 112.0 and c = 402.7 A) diffract to 2.7 A, whereas isoform II (unit-cell dimensions a = 111.8 and c = 400.0 A) presently diffract to 3.2 A, using synchrotron radiation in both cases.

  13. A cytochrome cd1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus.

    PubMed

    Sann, R; Kostka, S; Friedrich, B

    1994-01-01

    Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd1-type nitrite reductase. It appeared to be a dimer of kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d1. The isoelectric point of 8.6 was considerably higher than the pI determined for cd1 nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.

  14. Inducible and constitutive expression of pMOL28-encoded nickel resistance in Alcaligenes eutrophus N9A.

    PubMed Central

    Siddiqui, R A; Schlegel, H G; Meyer, M

    1988-01-01

    The nickel and cobalt resistance plasmid pMOL28 was transferred by conjugation from its natural host Alcaligenes eutrophus CH34 to the susceptible A. eutrophus N9A. Strain N9A and its pMOL28-containing transconjugant M220 were studied in detail. At a concentration of 3.0 mM NiCl2, the wild-type N9A did not grow, while M220 started to grow at its maximum exponential growth rate after a lag of 12 to 24 h. When grown in the presence of subinhibitory concentrations (0.5 mM) of nickel salt, M220 grew actively at 3 mM NiCl2 without a lag, indicating that nickel resistance is an inducible property. Expression of nickel resistance required active growth in the presence of nickel salts at a concentration higher than 0.05 mM. Two mutants of M220 were isolated which expressed nickel resistance constitutively. When the plasmids, pMOL28.1 and pMOL28.2, carried by the mutants were transferred to strains H16 and CH34, the transconjugants expressed constitutive nickel resistance. This indicates that the mutation is plasmid located. Both mutants expressed constitutive resistance to nickel and cobalt. Physiological studies revealed the following differences between strain N9A and its pMOL28.1-harboring mutant derivatives. (i) The uptake of 63NiCl2 occurred more rapidly in the susceptible strain and reached a 30- to 60-fold-higher amount that in the pMOL28.1-harboring mutant; (ii) in intact cells of the susceptible strain N9A, the cytoplasmic hydrogenase was inhibited by 1 to 5 nM NiCl2, whereas 10 mM Ni2+ was needed to inhibit the hydrogenase of mutant cells; (iii) the minimal concentration of nickel chloride for the derepressed synthesis of cytoplasmic hydrogenase was lower in strain N9A (1 to 3 microM) than in the constitutive mutant (8 to 10 microM). PMID:3410828

  15. Trichloroethylene degradation by two independent aromatic-degrading pathways in Alcaligenes eutrophus JMP134.

    PubMed Central

    Harker, A R; Kim, Y

    1990-01-01

    The bacterium Alcaligenes eutrophus JMP134(pJP4) degrades trichloroethylene (TCE) by a chromosomal phenol-dependent pathway and by the plasmid-encoded 2,4-dichlorophenoxyacetic acid pathway. The two pathways were independent and exhibited different rates of removal and capacities for quantity of TCE removed. The phenol-dependent pathway was more rapid (0.2 versus 0.06 nmol of TCE removed per min per mg of protein) and consumed all detectable TCE. The 2,4-dichlorophenoxyacetic acid-dependent pathway removed 40 to 60% of detectable TCE. PMID:2339875

  16. Construction and characterization of heavy metal-resistant haloaromatic-degrading Alcaligenes eutrophus strains.

    PubMed Central

    Springael, D; Diels, L; Hooyberghs, L; Kreps, S; Mergeay, M

    1993-01-01

    Alcaligenes eutrophus strains exhibiting both plasmid-borne heavy metal resistance and haloaromatic-degrading functions were obtained by intraspecific conjugation. The strains which we constructed expressed catabolic and resistance markers together. Degradation of various polychlorinated biphenyl isomers and 2,4-D (2,4-dichlorophenoxyacetic acid) was observed in the presence of 1 mM nickel or 2 mM zinc, provided that the metal resistance determinant was present in the catabolizing strain. Such strains may be useful for decontamination of sites that are polluted with both organic compounds and heavy metals. Images PMID:8439161

  17. Construction and characterization of heavy metal-resistant haloaromatic-degrading Alcaligenes eutrophus strains

    SciTech Connect

    Springael, D.; Diels, L.; Hooyberghs, L.; Kreps, S.; Mergeay, M. )

    1993-01-01

    The use of specialized bacterial strains that are able to degrade organic pollutants, such as polycholorinated biphenyls (PCBs) and pesticides, is becoming increasingly important for decontaminating polluted soils, sludges, and groundwaters. However, bioremediation of sites polluted with organic compounds might be impaired by the presence of heavy metals. The researchers used Alcaligenes eutrophus strains to test responses to heavy metals by evaluating their capacity to degrade organic xenobiotic compounds. The constructed strains were able to grow in the presence of nickel and zinc and exhibited unimpaired biodegradation activities. Both properties are important in considering microorganisms for decontamination of areas with organic xenobiotic and heavy metal pollution. 36 refs., 1 fig., 6 tabs.

  18. Cloning and expression of plasmid genes encoding resistances to chromate and cobalt in Alcaligenes eutrophus.

    PubMed Central

    Nies, A; Nies, D H; Silver, S

    1989-01-01

    Resistances to chromate and cobalt were cloned on a 30-kilobase-pair (kb) DNA region from the large Alcaligenes eutrophus plasmid pMOL28 into the broad-host-range mobilizable cosmid vector pVK102. A restriction nuclease map of the 30-kb region was generated. The resistances expressed from the hybrid plasmids after transfer back into A. eutrophus were inducible and conferred the same degree of resistance as the parent plasmid pMOL28. Resistances were expressed in metal-sensitive Alcaligenes strains and related bacteria but not in Escherichia coli. Resistance to chromate was further localized on a 2.6-kb EcoRI fragment, and resistance to cobalt was localized on an adjoining 8.5-kb PstI-EcoRI fragment. When the 2.6-kb EcoRI fragment was expressed in E. coli under the control of a bacteriophage T7 promoter, three polypeptides with molecular masses of 31,500, 21,000, and 14,500 daltons were visible on autoradiograms. The 31,500- and 21,000-dalton polypeptides were membrane bound; the 14,500-dalton polypeptide was soluble. Images PMID:2549011

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  1. Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase.

    PubMed Central

    Fukumori, F; Hausinger, R P

    1993-01-01

    The Alcaligenes eutrophus JMP134 tfdA gene, encoding the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation, was overexpressed in Escherichia coli, and several enzymatic properties of the partially purified gene product were examined. Although the tfdA-encoded enzyme is typically referred to as 2,4-D monooxygenase, we were unable to observe any reductant-dependent activity. Rather, we demonstrate that this enzyme is a ferrous ion-dependent dioxygenase that uses alpha-ketoglutarate as a cosubstrate. The alpha-ketoglutarate is converted to succinate concomitant with 2,4-D conversion to 2,4-dichlorophenol. By using [1-14C]alpha-ketoglutarate, we established that carbon dioxide is the second product derived from alpha-ketoglutarate. Finally, we verified the proposal that glyoxylate is the second product derived from 2,4-D. PMID:8458850

  2. Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus.

    PubMed Central

    Eberz, G; Friedrich, B

    1991-01-01

    Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes. Images PMID:2001989

  3. A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.

    PubMed Central

    Kortlüke, C; Horstmann, K; Schwartz, E; Rohde, M; Binsack, R; Friedrich, B

    1992-01-01

    One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units. Images PMID:1383192

  4. The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation.

    PubMed Central

    Lenz, O.; Schwartz, E.; Dernedde, J.; Eitinger, M.; Friedrich, B.

    1994-01-01

    Nucleotide sequence analysis revealed a 1,791-bp open reading frame in the hox gene cluster of the gram-negative chemolithotroph Alcaligenes eutrophus H16. In order to investigate the biological role of this open reading frame, we generated an in-frame deletion allele via a gene replacement strategy. The resulting mutant grew significantly more slowly than the wild type under lithoautotrophic conditions (6.1 versus 4.2 h doubling time). A reduction in the level of the soluble NAD-reducing hydrogenase (60% of the wild-type activity) was shown to be the cause of the slow lithoautotrophic growth. We used plasmid-borne gene fusions to monitor the expression of the operons encoding the soluble and membrane-bound hydrogenases. The expression of both operons was lower in the mutant than in the wild-type strain. These results suggest that the newly identified gene, designated hoxX, encodes a regulatory component which, in conjunction with the transcriptional activator HoxA, controls hydrogenase synthesis. Images PMID:8021224

  5. Crystal structure of the flavohemoglobin from Alcaligenes eutrophus at 1.75 A resolution.

    PubMed Central

    Ermler, U; Siddiqui, R A; Cramm, R; Friedrich, B

    1995-01-01

    The molecular structure of the flavohemoglobin from Alcaligenes eutrophus has been determined to a resolution of 1.75 A and refined to an R-factor of 19.6%. The protein comprises two fused modules: a heme binding module, which belongs to the globin family, and an FAD binding oxidoreductase module, which adopts a fold like ferredoxin reductase. The most striking deviation of the bacterial globin structure from those of other species is the movement of helix E in a way to provide more space in the vicinity of the distal heme binding site. A comparison with other members of the ferredoxin reductase family shows similar tertiary structures for the individual FAD and NAD binding domains but largely different interdomain orientations. The heme and FAD molecules approach each other to a minimal distance of 6.3 A and adopt an interplanar angle of 80 degrees. The electron transfer from FAD to heme occurs in a predominantly polar environment and may occur directly or be mediated by a water molecule. Images PMID:8557026

  6. Identification of a catabolic transposon, Tn4371, carrying biphenyl and 4-chlorobiphenyl degradation genes in Alcaligenes eutrophus A5.

    PubMed Central

    Springael, D; Kreps, S; Mergeay, M

    1993-01-01

    Alcaligenes eutrophus A5 catabolizes biphenyl to CO2 via benzoate and 4-chlorobiphenyl to 4-chlorobenzoate. In curing and conjugation experiments, the A5 endogenous 51-kb IncP1 plasmid pSS50 was found to be dispensable for biphenyl and 4-chlorobiphenyl catabolism. Transfer of the biphenyl- and 4-chlorobiphenyl-degrading phenotype by means of pSS50 was observed at a frequency of 10(-5) per transferred plasmid in matings of A5 with other A. eutrophus strains. Transconjugants harbor enlarged pSS50 derivatives which contain additional genetic information governing the oxidation of biphenyl and 4-chlorobiphenyl to benzoate and 4-chlorobenzoate and originating from the chromosome of strain A5. The following observations indicate that the catabolic genes reside on a 59-kb large transposon (Tn4371) for which a restriction map is presented. (i) Tn4371 transposes between different replicons and at different locations of the same replicon. (ii) Transposition was observed in a Rec- strain of A. eutrophus. (iii) Tn4371 transposes as a single, contiguous piece of DNA. Although an RP4::Tn4371 plasmid was stably maintained in different hosts, the plasmid conferred growth on biphenyl only when present in strains of A. eutrophus and in an Acinetobacter sp. strain. Images PMID:8383664

  7. Sequence similarities in the genes encoding polychlorinated biphenyl degradation by pseudomonas strain LB400 and alcaligenes eutrophus H850

    SciTech Connect

    Yates, J.R.; Mondello, F.J.

    1989-01-01

    DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains. Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to LB400 in PCB-degrading capability. These two organisms showed a strong conservation of restriction sites in the region of DNA encoding PCB metabolism. No other sequence similarities were detected in the two genomes. DNA from the other PCB-degrading strains showed no hybridization to the probe, which demonstrated the existence of at least two distinct classes of genes encoding PCB degradation.

  8. [High-level production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by feb-batch culture of Alcaligenes eutrophus].

    PubMed

    Cai, Y B; Liu, M Q; Yi, Z H; Chen, Q; Weng, W Q

    2001-09-01

    Fermentation strategies for production P (3HB-co-3HV) from glucose and propionic (or valeric) acid by Alcaligenes eutrophus were studied. During the culture, we controlled pH of the broth by feeding precusors of 3HV- propionic or valeric acid after Ammonia feeding stopped. When propionic acid were used as the precusor, for 50 hours, we obtained a cell dry weight, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content and a 3HV fraction of 149.9 g/L, 124.9 g/L, 83.3% and 12.4 mol%, respectively, with a PHA productivity of 2.50 g h-1 L-1. When valeric acid were used as the precusor, for 45 hours, we obtained a cell dry weight, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content and a 3HV fraction of 160.2 g/L, 119.0 g/L, 74.2% and 17.7 mol%, respectively, with a PHA productivity of 2.64 g h-1 L-1. Prior to this study, it hasn't been reported to obtain such high level productivity and 3HV fraction at the same time by Alcaligenes eutrophus.

  9. Characterization of the duplicate ribulose-1,5-bisphosphate carboxylase genes and cbb promoters of Alcaligenes eutrophus.

    PubMed

    Kusian, B; Bednarski, R; Husemann, M; Bowien, B

    1995-08-01

    Autotrophic CO2 fixation via the Calvin carbon reduction cycle in Alcaligenes eutrophus H16 is genetically determined by two highly homologous cbb operons, one of which is located on the chromosome and the other on megaplasmid pHG1 of the organism. An activator gene, cbbR, lies in divergent orientation only 167 bp upstream of the chromosomal operon and controls the expression of both cbb operons. The two 5'-terminal genes of the operons, cbbLS, coding for ribulose-1,5-bisphosphate carboxylase/oxygenase, were sequenced. Mapping of the 5' termini of the 2.1-kb cbbLS transcripts by primer extension and by nuclease S1 treatment revealed a single transcriptional start point at the same relative position for the chromosomal and plasmid-borne cbb operons. The derived cbb operon promoter showed similarity to sigma 70-dependent promoters of Escherichia coli. For the 1.4-kb transcripts of cbbR, the transcriptional start points were different in autotrophic and heterotrophic cells. The two corresponding cbbR promoters overlapped the cbb operon promoter and also displayed similarities to sigma 70-dependent promoters. The deficient cbbR gene located on pHG1 was transcribed as well. A newly constructed double operon fusion vector was used to determine the activities of the cbb promoters. Fusions with fragments carrying the cbb intergenic control regions demonstrated that the cbb operon promoters were strongly regulated in response to autotrophic versus heterotrophic growth conditions. In contrast, the cbbR promoters displayed low constitutive activities. The data suggest that the chromosomal and plasmid-borne cbb promoters of A. eutrophus H16 are functionally equivalent despite minor structural differences.

  10. Cytochromes c-552 from two strains of the hydrogenotrophic bacterium Alcaligenes eutrophus are sequence homologs of the cytochromes c8 from the denitrifying pseudomonads.

    PubMed

    Klarskov, K; Bartsch, R G; Meyer, T E; Cusanovich, M A; Van Beeumen, J J

    1997-12-05

    Soluble cytochromes c-552 were purified from two strains of the hydrogenothrophic species Alcaligenes eutrophus and their amino acid sequences determined. The two cytochromes were found to have 5 differences out of a total of 89 residues. The proteins are clearly related to the cytochromes c8 (formerly called Pseudomonas cytochromes c-551), but require a single residue insertion after the methionine sixth heme ligand relative to the Pseudomonas aeruginosa protein. The consensus residues Trp56 and Trp77, characteristic for the c8 family, are also present in the Alcaligenes proteins. Overall, the Alcaligenes cytochromes are only 43% identical to the Pseudomonas proteins which average 68% identity to one another. They are also only 45% identical to cytochrome c8 from Hydrogenobacter thermophilus, another hydrogenothrophic species, which indicates that the hydrogen utilizing bacteria are not more closely related to one another than they are to other species. The finding of cytochrome c8 in Alcaligenes eutrophus completes the recent characterization of a cytochrome cd1-nitrite reductase from this bacterial species and suggests the existence of the same denitrification pathway as in Pseudomonas where these two proteins are reaction partners.

  11. Degradation of Chlorophenols by Alcaligenes eutrophus JMP134(pJP4) in Bleached Kraft Mill Effluent

    PubMed Central

    Valenzuela, J.; Bumann, U.; Cespedes, R.; Padilla, L.; Gonzalez, B.

    1997-01-01

    The ability of Alcaligenes eutrophus JMP134(pJP4) to degrade 2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenol, and other chlorophenols in a bleached kraft mill effluent was studied. The efficiency of degradation and the survival of strain JMP134 and indigenous microorganisms in short-term batch or long-term semicontinuous incubations performed in microcosms were assessed. After 6 days of incubation, 2,4-dichlorophenoxyacetate (400 ppm) or 2,4,6-trichlorophenol (40 to 100 ppm) were extensively degraded (70 to 100%). In short-term batch incubations, indigenous microorganisms were unable to degrade such of compounds. Degradation of 2,4,6-trichlorophenol by strain JMP134 was significantly lower at 200 to 400 ppm of compound. This strain was also able to degrade 2,4-dichlorophenoxyacetate, 2,4,6-trichlorophenol, 4-chlorophenol, and 2,4,5-trichlorophenol when bleached Kraft mill effluent was amended with mixtures of these compounds. On the other hand, the chlorophenol concentration and the indigenous microorganisms inhibited the growth and survival of the strain in short-term incubations. In long-term (>1-month) incubations, strain JMP134 was unable to maintain a large, stable population, although extensive 2,4,6-trichlorophenol degradation was still observed. The latter is probably due to acclimation of the indigenous microorganisms to degrade 2,4,6-trichlorophenol. Acclimation was observed only in long-term, semicontinuous microcosms. PMID:16535488

  12. An rpoN-like gene of Alcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxidation.

    PubMed Central

    Römermann, D; Warrelmann, J; Bender, R A; Friedrich, B

    1989-01-01

    Pleiotropic mutants of Alcaligenes eutrophus with the phenotype Hno- have been characterized previously. They are deficient in several diverse metabolic activities, including hydrogen oxidation, nitrate and urea assimilation, denitrification, and various substrate transport systems. Phenotypically similar mutants were identified among hydrogenase-deficient strains of Pseudomonas facilis. The Tn5-labeled hno gene was cloned from a genomic DNA library of A. eutrophus and used to identify the corresponding unimpaired wild-type DNA sequence. The recombinant plasmid pCH148 contained an insert of 12.3 kilobase pairs and was shown to restore the Hno+ phenotype to mutants of A. eutrophus and P. facilis. A cosmid isolated from a DNA library of P. facilis also exhibited intergeneric Hno-complementing activity. The cloned hno loci from both organisms showed DNA homology by Southern blot hybridization. A subclone of pCH148 which contained a 6.5-kilobase-pair insert was constructed. The resulting hybrid, pCH170, not only was able to complement Hno- mutants but also relieved glutamine auxotrophy in NtrA- mutants of enteric bacteria. This suggests that the hno gene product from A. eutrophus is functionally similar to the NtrA protein, which has been identified as a novel sigma factor (sigma 54) of RNA polymerase. Images PMID:2536672

  13. Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

    PubMed Central

    Eberz, G; Eitinger, T; Friedrich, B

    1989-01-01

    Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium. PMID:2646280

  14. Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism.

    PubMed Central

    Priefert, H; Hein, S; Krüger, N; Zeh, K; Schmidt, B; Steinbüchel, A

    1991-01-01

    Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude extracts of acetoin-grown cells of Alcaligenes eutrophus. Ao:DCPIP OR consisted of alpha and beta subunits (Mrs, 35,500 and 36,000, respectively), and a tetrameric alpha 2 beta 2 structure was most likely for the native protein. The molecular weight of FMP subunits was 39,000. The N-terminal amino acid sequences of the three proteins were determined, and oligonucleotides were synthesized on the basis of the codon usage of A. eutrophus. With these, the structural genes for the alpha and beta subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, acoB, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, and the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified. Together with the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading frame, these genes are probably organized in one single operon (acoXABC). The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial sigma 54-dependent promoter consensus sequence. The amino acid sequences deduced from acoA and acoB for the alpha subunit (Mr, 35,243) and the beta subunit (Mr, 35,788) exhibited significant homologies to the primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whereas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltransferase of Escherichia coli. The occurrence of a new enzyme type for the degradation of acetoin is discussed. Images

  15. Gene escape model: Transfer of heavy metal resistance genes from Escherichia coli to Alcaligenes eutrophus on agar plates and in soil samples

    SciTech Connect

    Top, E. Studiecentrum voor Kernenergie, Mol ); Mergeay, M.; Springael, D. ); Verstraete, W. )

    1990-08-01

    Conjugal transfer from Escherichia coli to Alcaligenes eutrophus of the A. eutrophus genes coding for plasmid-borne resistance to cadmium, cobalt, and zinc (czc genes) was investigated on agar plates and in soil samples. This czc fragment is not expressed in the donor strain, E. coli, but it is expressed in the recipient strain, A. eutrophus. Hence, expression of heavy metal resistance by cells plated on a medium containing heavy metals represents escape of the czc genes. The two plasmids into which this DNA fragment has been cloned previously and which were used in these experiments are the nonconjugative, mobilizable plasmid pDN705 and the nonconjugative, nonmobilizable plasmid pMOL149. The results demonstrate that even genes incorporated into nonmobilizable plasmids can be exchanged between two different genera and that the presence of broad-host-range plasmids in putative recipients among soil bacteria could increase the risk of gene dissemination in case of release of genetically engineered microorganisms. The results also reveal that in certain soils, environmental conditions and particularly nutrient levels are conducive to gene transfer.

  16. Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16.

    PubMed Central

    Tran-Betcke, A; Warnecke, U; Böcker, C; Zaborosch, C; Friedrich, B

    1990-01-01

    The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides alpha, gamma, delta, and beta of the NAD-reducing hydrogenase (HoxS) of Alcaligenes eutrophus H16, were cloned, expressed in Pseudomonas facilis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (alpha and gamma subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) gamma subunit contains two cysteine clusters which may participate in the coordination of two [4F3-4S]centers. The genes hoxY and hoxH code for the small 22.9-kDa delta subunit and the nickel-containing 54.8-kDa beta subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the delta and beta subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide. Images PMID:2188945

  17. Production of poly(D-3-hydroxybutyrate) from CO(2), H(2), and O(2) by high cell density autotrophic cultivation of Alcaligenes eutrophus.

    PubMed

    Tanaka, K; Ishizaki, A; Kanamaru, T; Kawano, T

    1995-02-05

    Hydrogen-oxidizing bacterium, Alcaligenes eutrophus autotrophically produces biodegradable plastic material, poly(D-3-hydroxybutyrate), P(3HB), from carbon dioxide, hydrogen, and oxygen. In autotrophic cultivation of the microorganism, it is essential to eliminate possible occurrence of gas explosions from the fermentation process. We developed a bench-plant scale, recycled-gas, closed-circuit culture system equipped with several safety features to perform autotrophic cultivation of A. eutrophus by maintaining the oxygen concentration in the substrate gas phase below the lower limit for a gas explosion (6.9%). The culture vessel utilized a baskettype agitator, resulting in a K(L) a value of 2970 h(-1). Oxygen gas was also directly fed to the fermentor separately from the other gases. As a result, 91.3 g . dm(-3) of the cells and 61.9 g . dm(-3) of P(3HB) were obtained after 40 h of cultivation under this oxygen-limited condition. The results compared favorably with those reported for mass production of P(3HB) by heterotrophic fermentation. (c) 1995 John Wiley & Sons, Inc.

  18. Aerobic degradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5.

    PubMed Central

    Nadeau, L J; Menn, F M; Breen, A; Sayler, G S

    1994-01-01

    Biotransformation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5 was demonstrated by analysis of ethyl acetate-extracted products from resting cell cultures. Gas chromatography-mass spectrometry characterization of the neutral extracts revealed two hydroxy-DDT intermediates (m/z = 370) with retention times at 19.55 and 19.80 min that shared identical mass spectra. This result suggested that the hydroxylations occurred at the ortho and meta positions on the aromatic ring. UV-visible spectrum spectrophotometric analysis of a yellow metabolite in the culture supernatant showed a maximum A402 with, under acidic and basic conditions, spectrophotometric characteristics similar to those of the aromatic ring meta-cleavage products. 4-Chlorobenzoic acid was detected by thin-layer chromatography radiochemical scanning in samples from mineralization experiments by comparison of Rf values of [14C]DDT intermediates with that of an authentic standard. These results were further confirmed by gas chromatography-mass spectrometry analysis. This study indicates that DDT appears to be oxidized by a dioxygenase in A. eutrophus A5 and that the products of this oxidation are subsequently subjected to ring fission to eventually yield 4-chlorobenzoic acid as a major stable intermediate. PMID:8117093

  19. Identification of acoR, a regulatory gene for the expression of genes essential for acetoin catabolism in Alcaligenes eutrophus H16.

    PubMed Central

    Krüger, N; Steinbüchel, A

    1992-01-01

    Two hundred thirty-nine base pairs upstream from acoXABC, which encodes the Alcaligenes eutrophus H16 structural genes essential for cleavage of acetoin, the 2,004-bp acoR gene was identified. acoR encodes a protein of 668 amino acids with a molecular mass of 72.9 kDa. The amino acid sequence deduced from acoR exhibited homologies to the primary structures of transcriptional activators such as NifA of Azotobacter vinelandii, NtrC of Klebsiella pneumoniae, and HoxA of A. eutrophus. Striking similarities to the central domain of these proteins and the presence of a typical nucleotide-binding site (GETGSGK) as well as of a C-terminal helix-turn-helix motif as a DNA-binding site were revealed. Between acoR and acoXABC, two different types of sequences with dual rotational symmetry [CAC-(N11 to N18)-GTG and TGT-(N10 to N14)-ACA] were found; these sequences are similar to NtrC and NifA upstream activator sequences, respectively. Determination of the N-terminal amino acid sequence of an acoR'-'lacZ gene fusion identified the translational start of acoR. S1 nuclease protection assay identified the transcriptional start site 109 bp upstream of acoR. The promoter region (TTGCGC-N18-TACATT) resembled the sigma 70 consensus sequence of Escherichia coli. Analysis of an acoR'-'lacZ fusion and primer extension studies revealed that acoR was expressed at a low level under all culture conditions, whereas acoXABC was expressed only in acetoin-grown cells. The insertions of Tn5 in six transposon-induced acetoin-negative mutants of A. eutrophus were mapped within acoR. On the basis of these studies, it is probable that AcoR represents a regulatory protein which is required for sigma 54-dependent transcription of acoXABC. Images PMID:1378052

  20. Measurement of Growth at Very Low Rates ((mu) >= 0), an Approach To Study the Energy Requirement for the Survival of Alcaligenes eutrophus JMP 134.

    PubMed

    Muller, R H; Babel, W

    1996-01-01

    Alcaligenes eutrophus JMP 134 was grown in a recycling-mode fermenter with 100% biomass retention on 2,4-dichlorophenoxyacetic acid (2,4-D), phenol, and fructose. The growth pattern obtained given a constant supply of substrates exhibited three phases of linear growth on all three substrates. The transition from phase 1 to phase 2, considered to correspond to the onset of stringent (growth) control as indicated by a significant increase in guanosine 5(prm1)-bisphosphate 3(prm1)-bisphosphate (ppGpp), took place at 0.016 h(sup-1) with 2,4-D and at about 0.02 h(sup-1) with phenol and fructose. In the final phase, phase 4, which was achieved after the growth rate on the respective substrates fell below 0.003 to 0.001 h(sup-1), a constant level of biomass was obtained irrespective of further feeding of substrate at the same rate. The yield coefficients decreased by 70 to 80% from phase 1 to phase 3 and were 0 in phase 4. The stationary substrate concentrations s(infmin) in phase 4, calculated from the kinetic constants of the strain, were 1.23, 0.34, and 0.23 (mu)M for 2,4-D, phenol, and fructose, respectively. These figures characterize the minimum stationary substrate concentrations required in a dynamic system to keep A. eutrophus alive. This is caused by a substrate flux which enables growth at a rate >=0 due to the provision of energy to an extent at least satisfying maintenance requirements. According to the constant feed rates of the substrates and the final and stable biomass concentrations, this maintenance energy amounts to 14.4, 4.0, and 2.4 (mu)mol of ATP (middot) mg of dry mass(sup-1) h(sup-1) for 2,4-D, phenol, and fructose, respectively, after correction for the fraction of living cells. The increased energy expenditure in the case of 2,4-D is discussed with respect to uncoupling.

  1. Effects of rheological change by addition of carboxymethylcellulose in culture media of an air-lift fermentor on poly-D-3-hydroxybutyric acid productivity in autotrophic culture of hydrogen-oxidizing bacterium, Alcaligenes eutrophus.

    PubMed

    Taga, N; Tanaka, K; Ishizaki, A

    1997-03-05

    The effects of rheological change by addition of sodium carboxymethylcellulose (CMC) to culture medium in an air-lift-type fermentor on autotrophic production of poly-(D-3-hydroxybutyric acid) [P(3HB)] by two-stage culture of Alcaligenes eutrophus is investigated. Addition of 0.05% CMC increased P(3HB) production rate during the P(3HB) accumulation phase to twice that of the control culture. It was thought that addition of a small amount of CMC was beneficial for production of P(3HB) employing the air-lift fermentor under safe autotrophic culture conditions in wich oxygen concentration was maintained below 6.9% (v/v). the volumetric mass transfer coefficient (K(L)a) observed in the presence of CMC is shown to correlated with the P(3HB) production rate obtained. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 529-533, 1997.

  2. Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase.

    PubMed

    Zumft, W G; Dreusch, A; Löchelt, S; Cuypers, H; Friedrich, B; Schneider, B

    1992-08-15

    The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri [Viebrock, A. & Zumft, W. G. (1988) J. Bacteriol. 170, 4658-4668] and the subunit II of cytochrome-c oxidase (COII). Both types of enzymes possess the CuA binding site. The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22-kb PstI fragment (NS220) of nosZ from P. stutzeri. The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A. eutrophus) and 70695 Da (P. aeruginosa). The N-terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport. A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences. Only three out of nine cysteine residues of the NosZ protein (P. stutzeri) are invariant. Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus. Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes. The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences. Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination. Of 23 histidine residues in NosZ (P. stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences. Conserved tryptophan residues are located close to several potential copper ligands. Trp615 may contribute to the observed quenching of

  3. The chlorocatechol-catabolic transposon Tn5707 of Alcaligenes eutrophus NH9, carrying a gene cluster highly homologous to that in the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, confers the ability to grow on 3-chlorobenzoate.

    PubMed

    Ogawa, N; Miyashita, K

    1999-02-01

    Alcaligenes eutrophus (Ralstonia eutropha) NH9, isolated in Japan, utilizes 3-chlorobenzoate as its sole source of carbon and energy. Sequencing of the relevant region of plasmid pENH91 from strain NH9 revealed that the genes for the catabolic enzymes were homologous to the genes of the modified ortho-cleavage pathway. The genes from strain NH9 (cbnR-ABCD) showed the highest homology (89 to 100% identity at the nucleotide level) to the tcbR-CDEF genes on plasmid pP51 of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, which was isolated in The Netherlands. The structure of the operon, including the lengths of open reading frames and intervening sequences, was completely conserved between the cbn and tcb genes. Most nucleotide substitutions were localized within and proximal to the cbnB (tcbD) gene. The difference in the chloroaromatics that the two strains could use as growth substrates seemed to be due to differences in enzymes that convert substrates to chlorocatechols. The restriction map of plasmid pENH91 was clearly different from that of pP51 except in the regions that contained the cbnR-ABCD and tcbR-CDEF genes, respectively, suggesting that the chlorocatechol gene clusters might have been transferred as units. Two homologous sequences, present as direct repeats in both flanking regions of the cbnR-ABCD genes on pENH91, were found to be identical insertion sequences (ISs), designated IS1600, which formed a composite transposon designated Tn5707. Although the tcbR-CDEF genes were not associated with similar ISs, a DNA fragment homologous to IS1600 was cloned from the chromosome of strain P51. The sequence of the fragment suggested that it might be a remnant of an IS. The two sequences, together with IS1326 and nmoT, formed a distinct cluster on a phylogenetic tree of the IS21 family. The diversity of the sources of these IS or IS-like elements suggests the prevalence of ISs of this type.

  4. The chlorocatechol-catabolic transposon Tn5707 of Alcaligenes eutrophus NH9, carrying a gene cluster highly homologous to that in the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, confers the ability to grow on 3-chlorobenzoate

    SciTech Connect

    Ogawa, Naoto; Miyashita, Kiyotaka

    1999-02-01

    Alcaligenes eutrophus (Ralstonia eutropha) NH9, isolated in Japan, utilizes 3-chlorobenzoate as its sole source of carbon and energy. Sequencing of the relevant region of plasmid pENH91 from strain NH9 revealed that the genes for the catabolic enzymes were homologous to the genes of the modified ortho-cleavage pathway. The genes from strain NH9 (cbnR-ABCD) showed the highest homology to the tcbR-CDEF genes on plasmid pP51 of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, which was isolated in The Netherlands. The structure of the operon, including the lengths of open reading frames and intervening sequences, was completely conserved between the cbn and tcb genes. Most nucleotide substitutions were localized within and proximal to the cbnB (tcbD) gene. The difference in the chloroaromatics that the two strains could use as growth substrates seemed to be due to differences in enzymes that convert substrates to chlorocatechols. The restriction map of plasmid pENH91 was clearly different from that of pP51 except in the regions that contained the cbnR-ABCD and tcbR-CDEF genes, respectively, suggesting that the chlorocatechol gene clusters might have been transferred as units. Two homologous sequences, present as direct repeats in both flanking regions of the cbnR-ABCD genes on pENH91, were found to be identical insertion sequences (ISs), designated IS1600, which formed a composite transposon designated Tn5707. Although the tcbR-CDEF genes were not associated with similar ISs, a DNA fragment homologous to IS/1600 was cloned from the chromosome of strain P51. The sequence of the fragment suggested that it might be a remnant of an IS. The two sequences, together with IS1326 and nmoT, formed a distinct cluster on a phylogenetic tree of the IS21 family. The diversity of the sources of these IS or IS-like elements suggests the prevalence of ISs of this type.

  5. Chemical Characterization, Crossfeeding and Uptake Studies on Hydroxamate Siderophore of Alcaligenes faecalis.

    PubMed

    Sayyed, R Z; Chincholkar, S B; Meyer, J M; Kale, S P

    2011-06-01

    We report the production of two types of siderophores namely catecholate and hydroxamate in modified succinic acid medium (SM) from Alcaligenes faecalis. Two fractions of siderophores were purified on amberlite XAD, major fraction was hydroxamate type having a λ(max) at 224 nm and minor fraction appeared as catecholate with a λ(max) of 264 nm. The recovery yield obtained from major and minor fractions was 297 and 50 mg ml(-1) respectively. The IEF pattern of XAD-4 purified siderophore suggested the pI value of 6.5. Cross feeding studies revealed that A. faecalis accepts heterologous as well as self (hydroxamate) siderophore in both free and iron complexed forms however; the rate of siderophore uptake was more in case of siderophores complexed to iron. Siderophore iron uptake studies indicated the differences between hydroxamate siderophore of A. faecalis and Alc E, a siderophore of Alcaligenes eutrophus.

  6. The response of Cupriavidus metallidurans CH34 to spaceflight in the international space station.

    PubMed

    Leys, Natalie; Baatout, Sarah; Rosier, Caroline; Dams, Annik; s'Heeren, Catherine; Wattiez, Ruddy; Mergeay, Max

    2009-08-01

    The survival and behavior of Cupriavidus metallidurans strain CH34 were tested in space. In three spaceflight experiments, during three separate visits to the 'International Space Station' (ISS), strain CH34 was grown for 10-12 days at ambient temperature on mineral agar medium. Space- and earth-grown cells were compared post-flight by flow cytometry and using 2D-gel protein analysis. Pre-, in- and post-flight incubation conditions and experiment design had a significant impact on the survival and growth of CH34 in space. In the CH34 cells returning from spaceflight, 16 proteins were identified which were present in higher concentration in cells developed in spaceflight conditions. These proteins were involved in a specific response of CH34 to carbon limitation and oxidative stress, and included an acetone carboxylase subunit, fructose biphosphate aldolase, a DNA protection during starvation protein, chaperone protein, universal stress protein, and alkyl hydroperoxide reductase. The reproducible observation of the over-expression of these same proteins in multiple flight experiments, indicated that the CH34 cells could experience a substrate limitation and oxidative stress in spaceflight where cells and substrates are exposed to lower levels of gravity and higher doses of ionizing radiation. Bacterium C. metallidurans CH34 was able to grow normally under spaceflight conditions with very minor to no effects on cell physiology, but nevertheless specifically altered the expression of a few proteins in response to the environmental changes.

  7. Alcaligenes Eutrophus as a Source of Hydrogenase: An Evaluation of Techniques for Its Large Scale Production

    DTIC Science & Technology

    1990-08-01

    the Azotobacter and the Cynobacteria. K evolution by these micro-organisms are ATP-dependent and is catalyzed by nitrogenase but a hydrogenase Is...of Biochemistry and Biolhysics, Vol. 237, pp. 504- 512, 1985. 33 45. Seefeldt, Lance C. and Daniel J. Arp. Purification to homogeneity of Azotobacter

  8. Soil solid phases effects on the proteomic analysis of Cupriavidus metallidurans CH34

    SciTech Connect

    Giagnoni L.; Taghavi S.; Magherini, F.; Landi, L.; van der Lelie, D.; Puglia, M.; Bianchi, L.; Bini, L.; Nannipieri, P.; Renella, G.; Modesti, A.

    2012-05-01

    Cupriavidus metallidurans CH34 is a completely sequenced soil-borne beta-proteobacterium with known genome and proteome. Comparative 2-D electrophoresis and protein mass spectrometry were used to compare the proteome of C. metallidurans CH34 from liquid culture and after incubation for 1, 3, and 12 days in microcosms containing quartz sand, kaolinite, montmorillonite, or an artificial soil. Results showed that proteome from liquid culture was similar to CH34 proteins extracted from sand and kaolinite, whereas the proteins extracted from artificial soil differed significantly and no proteins were detected from C. metallidurans CH34 incubated in the montmorillonite microcosms. Protein recovery decreased on prolonging incubation time in all microcosms. Mass spectrometry identification showed that the trend of lower recovery upon incubation time was independent on the putative function of protein. These results suggest that the soil solid phase influences the protein recovery and soil proteomic analysis and that distinction between protein recovery and protein expression in soil will be a challenging for soil proteomic researchers.

  9. Alcaligenes infection in cystic fibrosis.

    PubMed

    Tan, Kenneth; Conway, Steven P; Brownlee, Keith G; Etherington, Christine; Peckham, Daniel G

    2002-08-01

    The aim of this study was to investigate the effect of chronic Alcaligenes species infection of the respiratory tract on the clinical status of patients with cystic fibrosis. We conducted a retrospective case-controlled study. The microbiological records of all patients attending the Leeds Regional Pediatric and Adult Cystic Fibrosis Units from 1992-1999 were examined. Chronic Alcaligenes infection was defined as a positive sputum culture on at least three occasions over a 6-month period. These patients were compared with controls matched for age, gender, respiratory function, and Pseudomonas aeruginosa infection status. Respiratory function tests, anthropometric data, Shwachman-Kulczycki score, Northern chest x-ray score, intravenous and nebulized antibiotic treatment, and corticosteroid treatment were compared from 2 years before to 2 years after Alcaligenes infection. From a clinic population of 557, 13 (2.3%) fulfilled the criteria for chronic infection. The median age at acquisition of infection was 17.2 years (range, 6.5-33.6). There was no significant difference in the changes of percentage predicted values for FEV(1), FVC, FEF(25-75), or Shwachman-Kulczycki and Northern chest x-ray scores, or in weight, height, and body mass index z-scores between Alcaligenes-infected cases and controls. There was also no significant difference in the use of antibiotics (intravenous and nebulized) or corticosteroids (inhaled and oral). We conclude that in our clinic, chronic infection with Alcaligenes species was uncommon. Chronically infected patients showed no excess deterioration in clinical or pulmonary function status from 2 years before to 2 years after primary acquisition.

  10. The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity

    NASA Astrophysics Data System (ADS)

    van Houdt, Rob; de Boever, Patrick; Coninx, Ilse; Janssen, Ann; Benotmane, Rafi; Leys, Natalie; Mergeay, Max

    The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity R. Van Houdt, P. De Boever, I. Coninx, A. Janssen, M.A. Benotmane, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. We have studied the response of Cupriavidus (formerly Ralstonia) metallidurans CH34 to simulated microgravity by culturing in a Rotating Wall Vessel (RWV) bioreactor. This bioreactor technology generates a unique Low-Shear Modeled Microgravity (LSMMG) environment and is exploited as analogue for in vivo medical and space environments. Cupriavidus and Ralstonia species are relevant model bacteria since they are often isolated from the floor, air and surfaces of spacecraft assembly rooms and not only contaminate the clean rooms but have also been found prior-to-flight on surfaces of space robots such as the Mars Odyssey Orbiter and even in-flight in ISS cooling water and Shuttle drinking water. In addition, C. metallidurans CH34 is also being used in fundamental space flight experiments aimed to gain a better insight in the bacterial adaptation to space. The first objective was to elucidate the stress response of C. metallidurans CH34 grown in LSMMG compared to a normal gravity control. Transcriptomic analysis revealed that a significant part of the heat shock response was induced in LSMMG. Transcription of d naK, encoding the major heat-shock protein and a prokaryotic homologue of the eukaryotic Hsp70 protein, was induced 6.4 fold in LSMMG. DnaK is assisted by partner chaperones DnaJ and GrpE for which transcription respectively were induced 2.0 and 2.6 fold. Transcription of other chaperones known to belong to the heat shock response was also induced in LSMMG: hslV and hsl U, encoding the HslVU protease, were induced respectively 5.5 and 3.4 fold; htpG, encoding a Hsp90 family chaperone, was induced 4.6 fold

  11. Heavy metal resistance in Cupriavidus metallidurans CH34 is governed by an intricate transcriptional network.

    PubMed

    Monsieurs, Pieter; Moors, Hugo; Van Houdt, Rob; Janssen, Paul J; Janssen, Ann; Coninx, Ilse; Mergeay, Max; Leys, Natalie

    2011-12-01

    The soil bacterium Cupriavidus metallidurans CH34 contains a high number of heavy metal resistance genes making it an interesting model organism to study microbial responses to heavy metals. In this study the transcriptional response of strain CH34 was measured when challenged to sub-lethal concentrations of various essential or toxic metals. Based on the global transcriptional responses for each challenge and the overlap in upregulated genes between different metal responses, the sixteen metals were clustered in three groups. In addition, the transcriptional response of already known metal resistance genes was assessed, and new metal response gene clusters were identified. The majority of the studied metal response loci showed similar expression profiles when cells were exposed to different metals, suggesting complex interplay at transcriptional level between the different metal responses. The pronounced redundancy of these metal resistant regions-as illustrated by the large number of paralogous genes-combined with the phylogenetic distribution of these metal response regions within either evolutionary related or other metal resistant bacteria, provides important insights on the recent evolutionary forces shaping this naturally soil-dwelling bacterium into a highly metal-resistant strain well adapted to harsh and anthropogenic environments.

  12. Sodium Ions Affect the pH behavior of the Soluble Hydrogenase of Alcaligenes Eutrophus H16

    DTIC Science & Technology

    1993-01-01

    were relatively ineffective as inhibitors. Similar results were observed with MgCI2, 5 CaC2, and BaCl2 , possibly suggesting a common binding site on...Inhibition at pH 8.5 at pH 7.0 100 mM LiCI 50.2 4.7 300 mM KCI 50.1 4.4 30 mM MgCl2 43.8 0 30 mM CaCI2 54.6 3.4 40 mM BaCQ2 65.3 7.3 10 FIGURE LEGENDS

  13. Uranium interaction with two multi-resistant environmental bacteria: Cupriavidus metallidurans CH34 and Rhodopseudomonas palustris.

    PubMed

    Llorens, Isabelle; Untereiner, Guillaume; Jaillard, Danielle; Gouget, Barbara; Chapon, Virginie; Carriere, Marie

    2012-01-01

    Depending on speciation, U environmental contamination may be spread through the environment or inversely restrained to a limited area. Induction of U precipitation via biogenic or non-biogenic processes would reduce the dissemination of U contamination. To this aim U oxidation/reduction processes triggered by bacteria are presently intensively studied. Using X-ray absorption analysis, we describe in the present article the ability of Cupriavidus metallidurans CH34 and Rhodopseudomonas palustris, highly resistant to a variety of metals and metalloids or to organic pollutants, to withstand high concentrations of U and to immobilize it either through biosorption or through reduction to non-uraninite U(IV)-phosphate or U(IV)-carboxylate compounds. These bacterial strains are thus good candidates for U bioremediation strategies, particularly in the context of multi-pollutant or mixed-waste contaminations.

  14. Uranium Interaction with Two Multi-Resistant Environmental Bacteria: Cupriavidus metallidurans CH34 and Rhodopseudomonas palustris

    PubMed Central

    Llorens, Isabelle; Untereiner, Guillaume; Jaillard, Danielle; Gouget, Barbara; Chapon, Virginie; Carriere, Marie

    2012-01-01

    Depending on speciation, U environmental contamination may be spread through the environment or inversely restrained to a limited area. Induction of U precipitation via biogenic or non-biogenic processes would reduce the dissemination of U contamination. To this aim U oxidation/reduction processes triggered by bacteria are presently intensively studied. Using X-ray absorption analysis, we describe in the present article the ability of Cupriavidus metallidurans CH34 and Rhodopseudomonas palustris, highly resistant to a variety of metals and metalloids or to organic pollutants, to withstand high concentrations of U and to immobilize it either through biosorption or through reduction to non-uraninite U(IV)-phosphate or U(IV)-carboxylate compounds. These bacterial strains are thus good candidates for U bioremediation strategies, particularly in the context of multi-pollutant or mixed-waste contaminations. PMID:23251623

  15. CzcE from Cupriavidus metallidurans CH34 is a copper-binding protein

    SciTech Connect

    Zoropogui, Anthony; Gambarelli, Serge; Coves, Jacques

    2008-01-25

    CzcE is encoded by the most distal gene of the czc determinant that allows Cupriavidus metallidurans CH34 to modulate its internal concentrations of cobalt, zinc and cadmium by regulation of the expression of the efflux pump CzcCBA. We have overproduced and purified CzcE. CzcE is a periplasm-located dimeric protein able to bind specifically 4 Cu-equivalent per dimer. Spectrophotometry and EPR are indicative of type II copper with typical d-d transitions. Re-oxidation of fully reduced CzcE led to the formation of an air stable semi-reduced form binding both 2 Cu(I) and 2 Cu(II) ions. The spectroscopic characteristics of the semi-reduced form are different of those of the oxidized one, suggesting a change in the environment of Cu(II)

  16. Proteomic responses to gold(iii)-toxicity in the bacterium Cupriavidus metallidurans CH34.

    PubMed

    Zammit, Carla M; Weiland, Florian; Brugger, Joël; Wade, Benjamin; Winderbaum, Lyron Juan; Nies, Dietrich H; Southam, Gordon; Hoffmann, Peter; Reith, Frank

    2016-11-09

    The metal-resistant β-proteobacterium Cupriavidus metallidurans drives gold (Au) biomineralisation and the (trans)formation of Au nuggets largely via unknown biochemical processes, ultimately leading to the reductive precipitation of mobile, toxic Au(i/iii)-complexes. In this study proteomic responses of C. metallidurans CH34 to mobile, toxic Au(iii)-chloride are investigated. Cells were grown in the presence of 10 and 50 μM Au(iii)-chloride, 50 μM Cu(ii)-chloride and without additional metals. Differentially expressed proteins were detected by difference gel electrophoresis and identified by liquid chromatography coupled mass spectrometry. Proteins that were more abundant in the presence of Au(iii)-chloride are involved in a range of important cellular functions, e.g., metabolic activities, transcriptional regulation, efflux and metal transport. To identify Au-binding proteins, protein extracts were separated by native 2D gel electrophoresis and Au in protein spots was detected by laser absorption inductively coupled plasma mass spectrometry. A chaperon protein commonly understood to bind copper (Cu), CupC, was identified and shown to bind Au. This indicates that it forms part of a multi-metal detoxification system and suggests that similar/shared detoxification pathways for Au and Cu exist. Overall, this means that C. metallidurans CH34 is able to mollify the toxic effects of cytoplasmic Au(iii) by sequestering this Au-species. This effect may in the future be used to develop CupC-based biosensing capabilities for the in-field detection of Au in exploration samples.

  17. Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation.

    PubMed

    Biondo, Ronaldo; da Silva, Felipe Almeida; Vicente, Elisabete José; Souza Sarkis, Jorge Eduardo; Schenberg, Ana Clara Guerrini

    2012-08-07

    This work describes the effects of the cell surface display of a synthetic phytochelatin in the highly metal tolerant bacterium Cupriavidus metallidurans CH34. The EC20sp synthetic phytochelatin gene was fused between the coding sequences of the signal peptide (SS) and of the autotransporter β-domain of the Neisseria gonorrhoeae IgA protease precursor (IgAβ), which successfully targeted the hybrid protein toward the C. metallidurans outer membrane. The expression of the SS-EC20sp-IgAβ gene fusion was driven by a modified version of the Bacillus subtilis mrgA promoter showing high level basal gene expression that is further enhanced by metal presence in C. metallidurans. The recombinant strain showed increased ability to immobilize Pb(2+), Zn(2+), Cu(2+), Cd(2+), Mn(2+), and Ni(2+) ions from the external medium when compared to the control strain. To ensure plasmid stability and biological containment, the MOB region of the plasmid was replaced by the E. coli hok/sok coding sequence.

  18. Response of the bacterium Cupriavidus metallidurans CH34 to space flight conditions.

    NASA Astrophysics Data System (ADS)

    Leys, N.; Wattiez, R.; Rosier, C.; de Boever, P.; Baatout, S.; Mergeay, M.

    Background When man goes to space inevitably microbes hitchhike along some needed others unwanted Knowledge is required to understand the behaviour of bacteria in spaceflight conditions Aim The aim of this work was to investigate the physiological and metabolic response and adaptation of the environmental model bacterium Cupriavidus metallidurans CH34 to space flight conditions The strain was grown in the International Space Station ISS during 2 separated Soyuz missions MESSAGE 1 2 experiments and in the Rotating Wall Vessel RWV mimicking microgravity on ground Results It was clear that pre- in- and post-flight incubation conditions are critical in spaceflight experiments and should be controlled monitored and taken into account as much as possible when comparing space flight with ground grown cells Distinct changes in physiology and metabolism were observed in the cell cultures grown in space flight when compared to correct ground control cultures A total of 12 proteins over-produced in space conditions were identified and divided in functional groups One group are proteins that protect the cell against physical damage such as heat-shock GrpE UspA and oxidative agents AhpC TrxB DpsA Another group of proteins is probably involved in a metabolic pathway to produce the energy-rich Acetyl-CoA Ald ExaC LpsJ CaiA with the help of a de carboxylase AcxABC Higher concentrations of this group of proteins were also detected in cells grown with acetone or 2-propanol as

  19. The Complete Genome Sequence of Cupriavidus metallidurans Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments

    PubMed Central

    Janssen, Paul J.; Van Houdt, Rob; Moors, Hugo; Monsieurs, Pieter; Morin, Nicolas; Michaux, Arlette; Benotmane, Mohammed A.; Leys, Natalie; Vallaeys, Tatiana; Lapidus, Alla; Monchy, Sébastien; Médigue, Claudine; Taghavi, Safiyh; McCorkle, Sean; Dunn, John; van der Lelie, Daniël; Mergeay, Max

    2010-01-01

    Many bacteria in the environment have adapted to the presence of toxic heavy metals. Over the last 30 years, this heavy metal tolerance was the subject of extensive research. The bacterium Cupriavidus metallidurans strain CH34, originally isolated by us in 1976 from a metal processing factory, is considered a major model organism in this field because it withstands milli-molar range concentrations of over 20 different heavy metal ions. This tolerance is mostly achieved by rapid ion efflux but also by metal-complexation and -reduction. We present here the full genome sequence of strain CH34 and the manual annotation of all its genes. The genome of C. metallidurans CH34 is composed of two large circular chromosomes CHR1 and CHR2 of, respectively, 3,928,089 bp and 2,580,084 bp, and two megaplasmids pMOL28 and pMOL30 of, respectively, 171,459 bp and 233,720 bp in size. At least 25 loci for heavy-metal resistance (HMR) are distributed over the four replicons. Approximately 67% of the 6,717 coding sequences (CDSs) present in the CH34 genome could be assigned a putative function, and 9.1% (611 genes) appear to be unique to this strain. One out of five proteins is associated with either transport or transcription while the relay of environmental stimuli is governed by more than 600 signal transduction systems. The CH34 genome is most similar to the genomes of other Cupriavidus strains by correspondence between the respective CHR1 replicons but also displays similarity to the genomes of more distantly related species as a result of gene transfer and through the presence of large genomic islands. The presence of at least 57 IS elements and 19 transposons and the ability to take in and express foreign genes indicates a very dynamic and complex genome shaped by evolutionary forces. The genome data show that C. metallidurans CH34 is particularly well equipped to live in extreme conditions and anthropogenic environments that are rich in metals. PMID:20463976

  20. The Complete Genome Sequence of Cupriavidus metallidurans Strain CH34, a Master Survivalist in Harsh and Antropogenic Environments

    SciTech Connect

    Janssen, P.J.; van der Lelie, D.; Van Houdt, R.; Moors, H.; Monsieurs, P.; Morin, N.; Michaux, A.; Benotmane, M. A.; Leys, N.; Vallaeys, T.; Lapidus, A.; Monchy, S.; Medique, C.; Taghavi, S.; McCorkle, S.; Dunn, J.; Mergeay, M.

    2010-05-01

    Many bacteria in the environment have adapted to the presence of toxic heavy metals. Over the last 30 years, this heavy metal tolerance was the subject of extensive research. The bacterium Cupriavidus metallidurans strain CH34, originally isolated by us in 1976 from a metal processing factory, is considered a major model organism in this field because it withstands milli-molar range concentrations of over 20 different heavy metal ions. This tolerance is mostly achieved by rapid ion efflux but also by metal-complexation and -reduction. We present here the full genome sequence of strain CH34 and the manual annotation of all its genes. The genome of C. metallidurans CH34 is composed of two large circular chromosomes CHR1 and CHR2 of, respectively, 3,928,089 bp and 2,580,084 bp, and two megaplasmids pMOL28 and pMOL30 of, respectively, 171,459 bp and 233,720 bp in size. At least 25 loci for heavy-metal resistance (HMR) are distributed over the four replicons. Approximately 67% of the 6,717 coding sequences (CDSs) present in the CH34 genome could be assigned a putative function, and 9.1% (611 genes) appear to be unique to this strain. One out of five proteins is associated with either transport or transcription while the relay of environmental stimuli is governed by more than 600 signal transduction systems. The CH34 genome is most similar to the genomes of other Cupriavidus strains by correspondence between the respective CHR1 replicons but also displays similarity to the genomes of more distantly related species as a result of gene transfer and through the presence of large genomic islands. The presence of at least 57 IS elements and 19 transposons and the ability to take in and express foreign genes indicates a very dynamic and complex genome shaped by evolutionary forces. The genome data show that C. metallidurans CH34 is particularly well equipped to live in extreme conditions and anthropogenic environments that are rich in metals.

  1. Overproduction, purification and preliminary X-ray diffraction analysis of CzcE from Cupriavidus metallidurans CH34

    SciTech Connect

    Pompidor, Guillaume; Zoropogui, Anthony; Kahn, Richard; Covès, Jacques

    2007-10-01

    Crystals of the mature form of CzcE from C. metallidurans CH34 were obtained which diffracted synchrotron radiation to 1.96 Å. CzcE is encoded by the czc determinant that allows Cupriavidus metallidurans CH34 to modulate its internal concentrations of cobalt, zinc and cadmium. This periplasmic protein was overproduced in its mature form in Escherichia coli and purified in two steps. After preliminary screening of crystallization conditions using a robot, well diffracting crystals were obtained using the hanging-drop vapour-diffusion method. Crystals diffracted to 1.96 Å using synchrotron radiation. They belonged to the monoclinic space group C2, with unit-cell parameters a = 105.54, b = 29.68, c = 71.10 Å. The asymmetric unit is expected to contain a dimer, in agreement with the quaternary structure deduced from gel-filtration experiments.

  2. The interactions of the bacterium Cupriavidus metallidurans CH34 with basalt rock, on Earth and in Space

    NASA Astrophysics Data System (ADS)

    Byloos, Bo; Van Houdt, Rob; Leys, Natalie; Ilyin, Vyacheslav; Nicholson, Natasha; Childers, Delma; Cockell, Charles; Boon, Nico

    2016-07-01

    Microbe-mineral interactions have become of interest for space exploration as microorganisms can biomine elements from extra-terrestrial materials, which could be used as nutrients in a life support system. This research is aimed at identifying the molecular mechanisms behind the interaction of Cupriavidus metallidurans CH34 with basalt, a lunar-type rock, and determining the influence of space flight conditions on this interaction. Survival and physiology of CH34 was monitored, with and without basalt, in mineral water over several months by flow cytometry, plate counts, ICP-MS, microscopy and proteomics. To study the influence of space conditions, a flight experiment on board the Russian FOTON-M4 capsule was performed. The results obtained from from water survival experiments on ground showed that CH34 was able to survive in mineral water, in the absence and presence of basalt, for several months. The total cell concentration remained stable but the cultivable fraction dropped to 10%, indicating a transition to a more dormant state. In the presence of basalt, this transition was less pronounced and cultivability was enhanced. In addition, with basalt, CH34 attached to the rock surface and formed a biofilm. The space flight experiment indicated more viable and cultivable cells compared to the ground experiment, both in the absence and presence of basalt, indicating a positive effect of space flight on survival. Chemical analysis indicated that basalt leaches out elements which may contribute to a positive effect of basalt on survival. Basalt may thus enhance survival and viability of CH34 both in ground and space flight experimental conditions. This study hopefully can contribute to a better understanding of microbe-mineral interactions, opening the door to future applications, in space, and on Earth. Acknowledgments: This work is supported by the European Space Agency (ESA-PRODEX) and the Belgian Science Policy (Belspo) through the BIOROCK project. We thank Kai

  3. Determinants encoding resistance to several heavy metals in newly isolated copper-resistant bacteria

    SciTech Connect

    Dressler, C.; Kues, U.; Nies, D.H.; Friedrich, B. )

    1991-11-01

    Three copper-resistant, gram-negative bacteria were isolated and characterized. Of the three strains, Alcaligenes dentrificans AH tolerated the highest copper concentration (MIC = 4 mM CuSO{sub 4}). All three strains showed various levels of resistance to other metal ions. A. denitrificans AH contains sequences which cross-hybridized with the mer (mercury resistance) determinant of Tn21 and the czc (cobalt, zinc, and cadmium resistance), cnr (cobalt and nickel resistance), and chr (chromate resistance) determinants of A. eutrophus CH34. DNA-DNA hybridization with probes prepared from A. eutrophus CH34 and Tn21 revealed the presence of chr-, cnr-, and mer-like sequences on the 200-kb plasmid pHG27 and of czc, cnr, and mer homologs located on the chromosomes. The second strain, classified as Alcaligenes sp. strain PW, carries czc, cnr, and mer homologs on the 240-kb plasmid pHG29-c and chr determinant on the 290-kb plasmid pHG29-a; a third plasmid, the 260-kb large plasmid pHG29-b, is cryptic. In contrast to the Alcaligenes strains, which were isolated from metal-contaminated water, Pseudomonas paucimobilis CD was isolated from the air. This strain harbors two cryptic plasmids: the 210-kb large plasmid pHG28-a and the 40-kb plasmid pHG28-b. Southern analysis revealed no homology between the metal ion resistance determinants of A. eutrophus CH34 and P. paucimonilis CD.

  4. Differentiation of Alcaligenes-like bacteria of avian origin and comparison with Alcaligenes spp. reference strains.

    PubMed

    Berkhoff, H A; Riddle, G D

    1984-04-01

    Although standard biochemical tests used for the identification of Alcaligenes spp. revealed only minor differences, the oxidative low-peptone technique clearly differentiated between Alcaligenes-like bacteria of avian origin and Alcaligenes spp. reference strains. Based on their colonial morphology, biochemical profiles, and hemagglutination, the Alcaligenes-like bacteria of avian origin were further divided into two subgroups, C1-T1 and C2-T2. Colonies of subgroup C1-T1 were nondescript, round, raised, glistening, translucent, greyish, and about 2 mm in diameter. Colonies of subgroup C2-T2 were off-white, flat, dry and wrinkled, generally round, and resembled tiny lily pads. Biochemical profiles by the oxidative low-peptone method showed the C1-T1 subgroup alkalinizing only three substrates (citrate, acetate, and succinate), whereas the C2-T2 subgroup alkalinized eight substrates (citrate, acetate, butyrate, itaconate, malonate, saccharate, succinate, and M-tartrate). Subgroup C1-T1 agglutinated human, chicken, and turkey erythrocytes, whereas subgroup C2-T2 did not. The recognition of these two subgroups within the Alcaligenes-like bacteria of avian origin is important, since it may explain the differences seen in pathogenicity among isolates.

  5. Differentiation of Alcaligenes-like bacteria of avian origin and comparison with Alcaligenes spp. reference strains.

    PubMed Central

    Berkhoff, H A; Riddle, G D

    1984-01-01

    Although standard biochemical tests used for the identification of Alcaligenes spp. revealed only minor differences, the oxidative low-peptone technique clearly differentiated between Alcaligenes-like bacteria of avian origin and Alcaligenes spp. reference strains. Based on their colonial morphology, biochemical profiles, and hemagglutination, the Alcaligenes-like bacteria of avian origin were further divided into two subgroups, C1-T1 and C2-T2. Colonies of subgroup C1-T1 were nondescript, round, raised, glistening, translucent, greyish, and about 2 mm in diameter. Colonies of subgroup C2-T2 were off-white, flat, dry and wrinkled, generally round, and resembled tiny lily pads. Biochemical profiles by the oxidative low-peptone method showed the C1-T1 subgroup alkalinizing only three substrates (citrate, acetate, and succinate), whereas the C2-T2 subgroup alkalinized eight substrates (citrate, acetate, butyrate, itaconate, malonate, saccharate, succinate, and M-tartrate). Subgroup C1-T1 agglutinated human, chicken, and turkey erythrocytes, whereas subgroup C2-T2 did not. The recognition of these two subgroups within the Alcaligenes-like bacteria of avian origin is important, since it may explain the differences seen in pathogenicity among isolates. Images PMID:6715517

  6. New mobile genetic elements in Cupriavidus metallidurans CH34, their possible roles and occurrence in other bacteria.

    PubMed

    Van Houdt, Rob; Monchy, Sébastien; Leys, Natalie; Mergeay, Max

    2009-08-01

    Cupriavidus metallidurans strain CH34 is a beta-Proteobacterium that thrives in low concentrations of heavy metals. The genetic determinants of resistance to heavy metals are located on its two chromosomes, and are particularly abundant in the two megaplasmids, pMOL28 and pMOL30. We explored the involvement of mobile genetic elements in acquiring these and others traits that might be advantageous in this strain using genome comparison of Cupriavidus/Ralstonia strains and related beta-Proteobacteria. At least eleven genomic islands were identified on the main replicon, three on pMOL28 and two on pMOL30. Multiple islands contained genes for heavy metal resistance or other genetic determinants putatively responding to harsh environmental conditions. However, cryptic elements also were noted. New mobile genetic elements (or variations of known ones) were identified through synteny analysis, allowing the detection of mobile genetic elements outside the bias of a selectable marker. Tn4371-like conjugative transposons involved in chemolithotrophy and degradation of aromatic compounds were identified in strain CH34, while similar elements involved in heavy metal resistance were found in Delftia acidovorans SPH-1 and Bordetella petrii DSM12804. We defined new transposons, viz., Tn6048 putatively involved in the response to heavy metals and Tn6050 carrying accessory genes not classically associated with transposons. Syntenic analysis also revealed new transposons carrying metal response genes in Burkholderia xenovorans LB400, and other bacteria. Finally, other putative mobile elements, which were previously unnoticed but apparently common in several bacteria, were also revealed. This was the case for triads of tyrosine-based site-specific recombinases and for an int gene paired with a putative repressor and associated with chromate resistance.

  7. Chemical forms of selenium in the metal-resistant bacterium Ralstonia metallidurans CH34 exposed to selenite and selenate.

    PubMed

    Sarret, Géraldine; Avoscan, Laure; Carrière, Marie; Collins, Richard; Geoffroy, Nicolas; Carrot, Francine; Covès, Jacques; Gouget, Barbara

    2005-05-01

    Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se(0)). We have studied the kinetics of selenite (Se(IV)) and selenate (Se(VI)) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se(0) and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se(0). Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se(0) was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se(IV) was detected as a transient species in the first 12 h after selenate introduction, Se(0) also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.

  8. Lead(II) resistance in Cupriavidus metallidurans CH34: interplay between plasmid and chromosomally-located functions

    SciTech Connect

    Taghavi, S.; van der Lelie, D.; Lesaulnier, C.; Monchy, S.; Wattier, R.; Mergeay, M.

    2009-08-01

    Proteome and transcriptome analysis, combined with mutagenesis, were used to better understand the response of Cupriavidus metallidurans CH34 against lead(II). Structural Pb(II)-resistance genes of the pMOL30-encoded pbrUTRABCD operon formed the major line of defense against Pb(II). However, several general stress response mechanisms under the control of alternative sigma factors such as {sigma}24/rpoK, {sigma}32/rpoH and {sigma}28/fliA were also induced. In addition, the expression of the pbrR 2 cadA pbrC 2 operon of the CMGI-1 region and the chromosomally encoded zntA were clearly induced in the presence of Pb(II), although their respective gene products were not detected via proteomics. After inactivation of the pbrA, pbrB or pbrD genes, the expression of the pbrR 2 cadA pbrC 2 operon went up considerably. This points towards synergistic interactions between pbrUTRABCD and pbrR 2 cadA pbrC 2 to maintain a low intracellular Pb(II) concentration, where pbrR 2 cadA pbrC 2 gene functions can complement and compensate for the mutations in the pbrA and pbrD genes. This role of zntA and cadA to complement for the loss of pbrA was further confirmed by mutation analysis. The pbrB:Tn(Km2) mutation resulted in the most significant decrease of Pb(II) resistance, indicating that Pb(II) sequestration, avoiding re-entry of this toxic metal ion, forms a critical step in the pbr-encoded Pb(II) resistance mechanism.

  9. Lead(II) resistance in Cupriavidus metallidurans CH34: interplay between plasmid and chromosomally-located functions.

    PubMed

    Taghavi, Safiyh; Lesaulnier, Celine; Monchy, Sebastien; Wattiez, Ruddy; Mergeay, Max; van der Lelie, Daniel

    2009-08-01

    Proteome and transcriptome analysis, combined with mutagenesis, were used to better understand the response of Cupriavidus metallidurans CH34 against lead(II). Structural Pb(II)-resistance genes of the pMOL30-encoded pbrUTRABCD operon formed the major line of defense against Pb(II). However, several general stress response mechanisms under the control of alternative sigma factors such as sigma24/rpoK, sigma32/rpoH and sigma28/fliA were also induced. In addition, the expression of the pbrR(2) cadA pbrC(2) operon of the CMGI-1 region and the chromosomally encoded zntA were clearly induced in the presence of Pb(II), although their respective gene products were not detected via proteomics. After inactivation of the pbrA, pbrB or pbrD genes, the expression of the pbrR(2) cadA pbrC(2) operon went up considerably. This points towards synergistic interactions between pbrUTRABCD and pbrR(2) cadA pbrC(2) to maintain a low intracellular Pb(II) concentration, where pbrR(2) cadA pbrC(2) gene functions can complement and compensate for the mutations in the pbrA and pbrD genes. This role of zntA and cadA to complement for the loss of pbrA was further confirmed by mutation analysis. The pbrB:: colonsTn(Km2) mutation resulted in the most significant decrease of Pb(II) resistance, indicating that Pb(II) sequestration, avoiding re-entry of this toxic metal ion, forms a critical step in the pbr-encoded Pb(II) resistance mechanism.

  10. Fluoranthene degradation in Pseudomonas alcaligenes PA-10.

    PubMed

    Gordon, L; Dobson, A D

    2001-01-01

    Pseudomonas alcaligenes strain PA-10 degrades the four-ring polycyclic aromatic hydrocarbon fluoranthene, co-metabolically. HPLC analysis of the growth medium identified four intermediates, 9-fluorenone-1-carboxylic acid; 9-hydroxy-1-fluorene carboxylic acid; 9-fluorenone and 9-fluorenol, formed during fluoranthene degradation. Pre-exposure of PA-10 to 9-fluorenone-1-carboxylic acid and 9-hydroxy-1-fluorene-carboxylic acid resulted in increases in fluoranthene removal, while pre-exposure to 9-fluorenone and 9-fluorenol resulted in a decrease in fluoranthene degradation. The rate of indole transformation was similarly affected by pre-exposure to these metabolic intermediates, indicating a link between fluoranthene degradation and indigo formation in this strain.

  11. Pancreatic abscess secondary to Alcaligenes faecalis.

    PubMed

    Ashwath, Mahi Lakshmi; Katner, Harold P

    2005-01-01

    We report a patient with pancreatic abscesses and necrosis secondary to Alcaligenes faecalis infection. He initially presented with alcohol-induced acute pancreatitis. Twenty days after the initial presentation, he re-presented with increasing pain and was found to have pancreatic necrosis and abscesses. Treatment was initiated with meropenem. Because of persistent fevers, computed tomography-guided drainage was performed. The fluid grew A faecalis resistant to meropenem and the patient continued to be febrile. He recovered only after adequate surgical intervention and appropriate antibiotic coverage. Although this is the first case of A faecalis reported to cause pancreatic abscess, we believe selection of this organism occurred because surgical drainage was delayed while the patient was on the recommended treatment with meropenem. This case emphasizes the need for early surgical drainage of pancreatic abscesses to avoid the selection of such resistant pathogens.

  12. Antibiotic aerosolization for treatment of alcaligenes rhinotracheitis.

    PubMed

    Ficken, M D

    1983-01-01

    Aerosolization of oxytetracycline hydrochloride for treatment and control of rhinotracheitis caused by Alcaligenes faecalis was performed in complete confinement on two turkey flocks immediately preceding vaccination with Newcastle disease virus (NDV). Tracheal culture results indicated populations of A. faecalis decreased following treatment. Clinical observations showed the treated turkeys had greater activity and more alertness compared with untreated A. faecalis-infected flocks, and respiratory sounds were diminished overall. Increased food and water consumption and no stunting was observed in treated turkeys. Treated flocks had a substantial decrease in mortality during the 4-week post-NDV vaccination period compared with untreated flocks. These results, although inadequately controlled, suggest a promising treatment program for A. faecalis infection in turkeys.

  13. [Comparative susceptibility of Ochrobactrum anthropi, Agrobacterium tumefaciens, Alcaligenes faecalis, Alcaligenes denitrificans subsp. denitrificans, Alcaligenes denitrificans subsp. xylosidans and Bordetella bronchiseptica against 35 antibiotics including 17 beta-lactams].

    PubMed

    Bizet, C; Bizet, J

    1995-04-01

    Ochrobactrum anthropi, formerly known as "Achromobacter sp." or CDC group Vd has been isolated from water, hospital environment (antiseptic solutions, dialysis fluids ... ). O. anthropi is a Gram negative, motile, strictly aerobic, oxydase positive and non-fermentative bacteria with a strong urease activity. The susceptibility of 13 strains of O. anthropi was determined by agar diffusion method and compared to those of type strains of Agrobacterium tumefaciens, Alcaligenes faecalis, Alcaligenes denitrificans subsp. denitrificans, Alcaligenes denitrificans subsp. xylosoxydans and Bordetella bronchiseptica. The MICs of 20 antimicrobial agents confirmed the distinct phenotype susceptibility of O. anthropi. All the strains of O. anthropi are sensitive to imipenem, amikacin, gentamicin, netilmicin, nalidixic acid, pefloxacin, ciprofloxacin, tetracyclin, colistin, sulphonamides and rifampicin and resistant to ampicillin, amoxycillin + clavulanic acid, ticarcillin, mezlocillin, cefuroxime, cefamandol, cefoxitin, cefotaxime, cefoperazon, ceftazidime, cefsulodin, aztreonam, streptomycin, kanamycin, pipemidic acid, chloramphenicol, erythromicin, pristinamycin, trimethoprim and fosfomycin. O. anthropi is implicated in nosocomial infections. O. anthropi was the species with the greatest resistance to beta-lactamins.

  14. Alcaligenes endophyticus sp. nov., isolated from roots of Ammodendron bifolium.

    PubMed

    Lu, Chun-Yan; Li, Yu-Qian; Tian, Ye; Han, Ming-Xian; Narsing Rao, Manik Prabhu; Li, Yan-Ru; Zhu, Zhi-Nan; Wei, Da-Qiao; An, Deng-Di; Li, Wen-Jun

    2016-12-12

    A Gram-staining negative, rod-shaped, motile bacterium, designated AER10T, was isolated from roots of Ammodendron bifolium collected from Takeermohuer desert in Xinjiang Uygur Autonomous Region, north western China. Growth was found to occur from 10°C to 45°C, at pH 5.0-9.0 and could tolerate NaCl up to 10 % (w/v). 16S rRNA gene sequence result indicated that the strain AER10T belongs to the genus Alcaligenes and was closely related to Alcaligenes aquatilis (98.4 %), Alcaligenes faecalis subsp. parafaecalis (98.4 %), Alcaligenes faecalis subsp. faecalis (98.1 %) and Alcaligenes faecalis subsp. phenolicus (97.9 %). However, the DNA-DNA hybridization values between the strain AER10T and the above strains were less than the threshold value (below 70 %) for the delineation of genomic species. The DNA G+C content was 53.3 mol %. Ubiquinone-8 (Q-8) was the only quinone system present. The major fatty acid were summed feature 8 (C18:1ω7c, 25 %), C16:0 (24.2 %), summed feature 3 (C16:1ω7c /C16:1ω6c, 19.3 %) and cyclo-C17:0 (10.5 %). The polar lipid profile of the strain AER10T consist diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), two unidentified aminolipid (AL) and five unknown polar lipids (UL). On the basis of the evidence presented in this study, strain AER10T is a representative of a novel species in the genus Alcaligenes, for which the name Alcaligenes endophyticus sp. nov. is proposed. The type strain is AER10T (=DSM 100498T=KCTC 42688T).

  15. Size of diffusion pore of Alcaligenes faecalis.

    PubMed Central

    Ishii, J; Nakae, T

    1988-01-01

    The diffusion pore of the outer membrane of Alcaligenes faecalis was shown to be substantially smaller than the Escherichia coli porin pore. In experiments with intact cells, pentoses and hexoses penetrated into the NaCl-expanded periplasm, whereas saccharides of Mr greater than 342 did not. Cells treated with 0.5 M saccharides of Mr greater than 342 weighed 33 to 38% less than cells treated with isotonic solution, suggesting that these saccharides do not permeate through the outer membrane. The diffusion rates of various solutes through the liposome membranes reconstituted from the Mr-43,000 outer membrane protein showed the following characteristics. (i) The relative diffusion rates of pentoses, hexoses, and methylhexoses appeared to be about 1.0, 0.6, and negligibly small, respectively. (ii) The diffusion rate of glucose appeared to be about 1/10th that with the E. coli B porin. (iii) The diffusion rate of gluconic acid was five to seven times higher than that of glucose. (iv) The diffusion rates of beta-lactam antibiotics appeared to be 40 to less than 10% of those with the E. coli B porin. Images PMID:2835003

  16. The cnrY gene, a tool to monitor DNA rearrangements by IS translocation in Cupriavidus metallidurans CH34 in response to space flight

    NASA Astrophysics Data System (ADS)

    Leys, N.; Monchy, S.; Vallaeys, T.; Dams, A.; Mergeay, M.

    Background The beta -Proteobacterium Cupriavidus metallidurans CH34 carries a chromosome 3 9 Mb a megaplasmid 2 6 Mb and many different Mobile Genetic Elements MGEs including 2 large plasmids 234 kb and 170 kb and at least 1 genomic island 7 transposons and 13 IS elements Mobility and rearrangements of these MGEs could play a direct part in genome adaptation and evolution in response to environmental stresses such as space flight conditions Aim In this study a new tool was developed and tested to detect the mobility and functionality of the IS elements in response to environmental stresses such as space flight Method The cnrYXHCBAT gene cluster on the pMOL28 plasmid of CH34 Tibazarwa et al 2000 governs the efficient efflux of Co 2 and Ni 2 and a slight unspecific efflux of Zn 2 Mutations inactivating the cnrY gene 300 bp encoding an antisigma repressor protein allow a constitutive over-expression of nickel cobalt resistance Collard et al 1993 Such cnrY mutants can be positively selected when the medium is supplemented with 0 6mM Zn 2 ZnR mutants As functional test 35 independent cultures of CH34 were incubated on agar containing 0 6mM Zn 2 during 10 days in the International Space Station ISS and on corresponding control plates at the ground From these cultures in total ca 600 ZnR mutants were selected and the promoter- cnrY fragment was amplified and sequenced Result This study revealed that the

  17. Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34.

    PubMed

    Bersch, Beate; Derfoufi, Kheiro-Mouna; De Angelis, Fabien; Auquier, Vanessa; Ekendé, Elisabeth Ngonlong; Mergeay, Max; Ruysschaert, Jean-Marie; Vandenbussche, Guy

    2011-03-29

    Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34.

  18. The Impact of Space Flight on Survival and Interaction of Cupriavidus metallidurans CH34 with Basalt, a Volcanic Moon Analog Rock

    PubMed Central

    Byloos, Bo; Coninx, Ilse; Van Hoey, Olivier; Cockell, Charles; Nicholson, Natasha; Ilyin, Vyacheslav; Van Houdt, Rob; Boon, Nico; Leys, Natalie

    2017-01-01

    Microbe-mineral interactions have become of interest for space exploration as microorganisms could be used to biomine from extra-terrestrial material and extract elements useful as micronutrients in life support systems. This research aimed to identify the impact of space flight on the long-term survival of Cupriavidus metallidurans CH34 in mineral water and the interaction with basalt, a lunar-type rock in preparation for the ESA spaceflight experiment, BIOROCK. Therefore, C. metallidurans CH34 cells were suspended in mineral water supplemented with or without crushed basalt and send for 3 months on board the Russian FOTON-M4 capsule. Long-term storage had a significant impact on cell physiology and energy status (by flow cytometry analysis, plate count and intracellular ATP measurements) as 60% of cells stored on ground lost their cell membrane potential, only 17% were still active, average ATP levels per cell were significantly lower and cultivability dropped to 1%. The cells stored in the presence of basalt and exposed to space flight conditions during storage however showed less dramatic changes in physiology, with only 16% of the cells lost their cell membrane potential and 24% were still active, leading to a higher cultivability (50%) and indicating a general positive effect of basalt and space flight on survival. Microbe-mineral interactions and biofilm formation was altered by spaceflight as less biofilm was formed on the basalt during flight conditions. Leaching from basalt also changed (measured with ICP-OES), showing that cells release more copper from basalt and the presence of cells also impacted iron and magnesium concentration irrespective of the presence of basalt. The flight conditions thus could counteract some of the detrimental effects observed after the 3 month storage conditions. PMID:28503167

  19. The Impact of Space Flight on Survival and Interaction of Cupriavidus metallidurans CH34 with Basalt, a Volcanic Moon Analog Rock.

    PubMed

    Byloos, Bo; Coninx, Ilse; Van Hoey, Olivier; Cockell, Charles; Nicholson, Natasha; Ilyin, Vyacheslav; Van Houdt, Rob; Boon, Nico; Leys, Natalie

    2017-01-01

    Microbe-mineral interactions have become of interest for space exploration as microorganisms could be used to biomine from extra-terrestrial material and extract elements useful as micronutrients in life support systems. This research aimed to identify the impact of space flight on the long-term survival of Cupriavidus metallidurans CH34 in mineral water and the interaction with basalt, a lunar-type rock in preparation for the ESA spaceflight experiment, BIOROCK. Therefore, C. metallidurans CH34 cells were suspended in mineral water supplemented with or without crushed basalt and send for 3 months on board the Russian FOTON-M4 capsule. Long-term storage had a significant impact on cell physiology and energy status (by flow cytometry analysis, plate count and intracellular ATP measurements) as 60% of cells stored on ground lost their cell membrane potential, only 17% were still active, average ATP levels per cell were significantly lower and cultivability dropped to 1%. The cells stored in the presence of basalt and exposed to space flight conditions during storage however showed less dramatic changes in physiology, with only 16% of the cells lost their cell membrane potential and 24% were still active, leading to a higher cultivability (50%) and indicating a general positive effect of basalt and space flight on survival. Microbe-mineral interactions and biofilm formation was altered by spaceflight as less biofilm was formed on the basalt during flight conditions. Leaching from basalt also changed (measured with ICP-OES), showing that cells release more copper from basalt and the presence of cells also impacted iron and magnesium concentration irrespective of the presence of basalt. The flight conditions thus could counteract some of the detrimental effects observed after the 3 month storage conditions.

  20. Biodegradation of phenol at high initial concentration by Alcaligenes faecalis.

    PubMed

    Jiang, Yan; Wen, Jianping; Bai, Jing; Jia, Xiaoqiang; Hu, Zongding

    2007-08-17

    Strain Alcaligenes faecalis was isolated and identified as a member of the genus Alcaligenes by using BIOLOG and 16S rDNA sequence analysis. The phenol biodegradation tests showed that the phenol-degrading potential of A. faecalis related greatly to the different physiological phases of inoculum. The maximum phenol degradation occurred at the late phase of the exponential growth stages, where 1600 mg L(-1) phenol was completely degraded within 76 h. A. faecalis secreted and accumulated a vast quantity of phenol hydroxylase in this physiological phase, which ensured that the cells could quickly utilize phenol as a sole carbon and energy source. In addition, the kinetic behavior of A. faecalis in batch cultures was also investigated over a wide range of initial phenol concentrations (0-1600 mg L(-1)) by using Haldane model. It was clear that the Haldane kinetic model adequately described the dynamic behavior of the phenol biodegradation by the strain of A. faecalis.

  1. Purification of siderophores of Alcaligenes faecalis on Amberlite XAD.

    PubMed

    Sayyed, R Z; Chincholkar, S B

    2006-05-01

    Studies on siderophore production using Alcaligenes faecalis BCCM ID 2374 revealed hydroxamate and catecholate type of siderophores at 347 microg mL-1. These fractions were purified on Amberlite XAD-4 column, which resulted in the separation of two bands having absorption maxima at 264 and 224 nm. The amount of pure siderophore obtained in powdered form from first and second fraction was 297 and 50 microg mL-1 respectively.

  2. Isolation and identification of dexamethasone sodium phosphate degrading Pseudomonas alcaligenes.

    PubMed

    Yi, Wang; Zhibang, Yang; Lili, Zhu; Zhongquan, Shi; Lianju, Ma; Ziwei, Tang; Renju, Jiang

    2015-02-01

    Glucocorticosteroids such as dexamethasone have polluted hospital wastewater, urban sewage, and river water in varying degrees. However, dexamethasone degradation by bioremediation technology is less understood. This study aims to isolate bacteria that could degrade dexamethasone and to identify their degradation characteristics. Hospital wastewater contaminated by dexamethasone was collected. After culturing in inorganic salt medium and in carbon deficient medium containing dexamethasone sodium phosphate, a bacterial strain with dexamethasone sodium phosphate as the sole carbon and energy source was enriched and isolated from the contaminated wastewater. The strain was identified as Pseudomonas alcaligenes by morphology, Gram staining, biochemical test, and 16S rDNA sequencing. Isolated bacteria were domesticated. Then its degradation characteristic was determined by high-performance liquid chromatography method. The degradation rate of P. alcaligenes on dexamethasone sodium phosphate was 50.86%. Of the degraded dexamethasone sodium phosphate, 75.23% of dexamethasone sodium phosphate was degraded to dexamethasone and 23.63% was degraded to other metabolites. In conclusion, the isolated P. alcaligenes in this study would provide experimental evidence for further research on the bioremediation technology to treat dexamethasone sodium phosphate and dexamethasone polluted water and further for the elimination of side effects of dexamethasone.

  3. Metabolic energy from arsenite oxidation in Alcaligenes faecalis

    NASA Astrophysics Data System (ADS)

    Anderson, G. L.; Love, M.; Zeider, B. K.

    2003-05-01

    The aerobic soil bacterium, Alcaligenes faecalis, survives in cultures containing greater than 10 g/L of aqueous arsenic. Toleration of arsenite occurs by the enzymatic oxidation of arsenite (As^III), to the less toxic arsenate (As^V). In defined media, the bacterium grows faster in the presence of arsenite than in its absence. This suggests that the bacterium uses the redox potential of arsenite oxidation as metabolic energy. The oxidation occurs via periplasmic arsenite oxidase, azurin, and cytochrome c [11] which presumably pass electron equivalents through an electron transport chain involving cytochrome c oxidase aud oxygen as the terminal electron acceptor. The associated proton translocation would allow synthesis of ATP and provide a useful means of harnessing the redox potential of arsenite oxidation. Arsenite and arsenate assays of the media during bacterial growth indicate that arsenite is depleted during the exponential growth phase and occurs concomitantly with the expression of arsenite oxidase. These results suggest that arsenite is detoxified to arsenate during bacterial growth and are inconsistent with previous reported interpretations of growth data. Alcaligenes faecalis is dependent on organic carbon sources and is therefore not chemolithoautotrophic. The relationship between succinate and arsenite utilisation provides evidence for the use of arsenite as a supplemental energy source. Because Alcaligenes faecalis not only tolerates, but thrives, in very high concentrations of arsenic has important implications in bioremediation of environments contaminated by aqueous arsenic.

  4. Potential of Cupriavidus metallidurans CH34 for in situ resource utilization from basalt by determining the molecular micro-mineral interactions

    NASA Astrophysics Data System (ADS)

    Byloos, Bo; Van Houdt, Rob; Boon, Nico; Leys, Natalie

    In order to maintain a persistent human presence in space, materials must either be provided from Earth or generated from material already present in space, in a process referred to as 'in situ resource utilization (ISRU)'. Microorganisms can biomine useful elements from extra-terrestrial materials for use as nutrients in a life support system or to aid in the creation of soil. To effectively use bacteria in an ISRU process more needs to be known about the molecular mechanisms behind microbe-mineral interaction and the influence of microgravity and radiation that affect bioleaching. The aim of this research project is to define the microbe-mineral interactions on basalt, which is a major constituent of Lunar or Martian regolith, the mechanisms that are important in bioleaching and how this process will be altered by space flight conditions. In particular, the research will be focussed on the determination of the genes and proteins involved in the biosynthesis of metallophores and exopolysaccharides (EPS), and biofilm formation. Iron, copper and phosphate uptake mechanisms are investigated in detail because these have been shown to be essential for life and bacteria are faced with limitation of these nutrients in the environment. In this study the bacterium Cupriavidus metallidurans CH34 is used to study these interactions. C. metallidurans CH34 is a soil bacterium which is resistant to up to 20 different heavy metal ions. Its behaviour in space has already been determined with earlier flight experiments to the ISS. It was recently discovered that C. metallidurans forms a biofilm and is capable of leaching calcium, magnesium and iron from basalt to sustain its growth First, C. metallidurans was grown in conditions with and without basalt, iron, copper and phosphate and the production of EPS and metallophores was examined. The iron, copper and phosphate concentrations which are limiting and optimal to allow C. metallidurans cell proliferation could be determined as

  5. Genome Sequence of a Strain of the Human Pathogenic Bacterium Pseudomonas alcaligenes That Caused Bloodstream Infection.

    PubMed

    Suzuki, Masato; Suzuki, Satowa; Matsui, Mari; Hiraki, Yoichi; Kawano, Fumio; Shibayama, Keigo

    2013-10-31

    Pseudomonas alcaligenes, a Gram-negative aerobic bacterium, is a rare opportunistic human pathogen. Here, we report the whole-genome sequence of P. alcaligenes strain MRY13-0052, which was isolated from a bloodstream infection in a medical institution in Japan and is resistant to antimicrobial agents, including broad-spectrum cephalosporins and monobactams.

  6. Draft Genome Sequence of Alcaligenes faecalis Strain IITR89, an Indole-Oxidizing Bacterium.

    PubMed

    Regar, Raj Kumar; Gaur, Vivek Kumar; Mishra, Gayatri; Jadhao, Sudhir; Kamthan, Mohan; Manickam, Natesan

    2016-03-03

    We report the draft genome sequence of Alcaligenes faecalis strain IITR89, a bacterium able to form indigo by utilizing indole as the sole carbon source. The Alcaligenes species is increasingly reported for biodegradation of diverse toxicants and thus complete sequencing may provide insight into biodegradation capabilities and other phenotypes.

  7. Differential proteomic analysis using isotope-coded protein-labeling strategies: comparison, improvements and application to simulated microgravity effect on Cupriavidus metallidurans CH34.

    PubMed

    Leroy, Baptiste; Rosier, Caroline; Erculisse, Vanessa; Leys, Natalie; Mergeay, Max; Wattiez, Ruddy

    2010-06-01

    Among differential proteomic methods based on stable isotopic labeling, isotope-coded protein labeling (ICPL) is a recent non-isobaric technique devised to label primary amines found in proteins. ICPL overcomes some of the disadvantages found in other chemical-labeling techniques, such as iTRAQ or ICAT. However, previous analyses revealed that more than 30% of the proteins identified in regular ICPL generally remain unquantified. In this study, we describe a modified version of ICPL, named Post-digest ICPL, that makes it possible to label and thus to quantify all the peptides in a sample (bottom-up approach). Optimization and validation of this Post-digest ICPL approach were performed using a standard protein mixture and complex protein samples. Using this strategy, the number of proteins that were identified and quantified was greatly increased in comparison with regular ICPL and cICAT approaches. The pros and cons of this improvement are discussed. This complementary approach to traditional ICPL was applied to the analysis of modification of protein abundances in the model bacterium Cupriavidus metallidurans CH34 after cultivation under simulated microgravity. In this context, two different systems - a 2-D clinorotation and 3-D random positioning device - were used and the results were compared and discussed.

  8. Detoxification of lantana hepatotoxin, lantadene A, using Alcaligenes faecalis.

    PubMed

    Singh, A; Sharma, O P; Kurade, N P; Ojha, S

    2001-01-01

    Detoxification of lantadene A (LA), the hepatotoxin from Lantana camara var. aculeata, by the bacterial strain Alcaligenes faecalis has been investigated. Lantadene A induced hepatotoxicity concomitant with increases in plasma bilirubin, blood plasma enzymes and histopathological lesions that typify lantana toxicity. The extract of fermentation broth in which LA was incubated with A. faecalis did not elicit any alterations in blood enzyme prolife or liver histopathology, which were comparable with the control group. It is concluded that A. faecalis detoxified LA and no noxious product was formed on incubation of LA with A. faecalis.

  9. Specific and sensitive detection of Alcaligenes species from an agricultural environment.

    PubMed

    Nakano, Miyo; Niwa, Masumi; Nishimura, Norihiro

    2013-03-01

    A quantitative real-time PCR assay to specifically detect and quantify the genus Alcaligenes in samples from the agricultural environment, such as vegetables and farming soils, was developed. The minimum detection sensitivity was 106 fg of pure culture DNA, corresponding to DNA extracted from two cells of Alcaligenes faecalis. To evaluate the detection limit of A. faecalis, serially diluted genomic DNA from this organism was mixed with DNA extracted from soil and vegetables and then a standard curve was constructed. It was found that Alcaligenes species are present in the plant phytosphere at levels 10(2)-10(4) times lower than those in soil. The approach presented here will be useful for tracking or quantifying species of the genus Alcaligenes in the agricultural environment.

  10. Unusual causes of peritonitis in a peritoneal dialysis patient: Alcaligenes faecalis and Pantoea agglomerans.

    PubMed

    Kahveci, Arzu; Asicioglu, Ebru; Tigen, Elif; Ari, Elif; Arikan, Hakki; Odabasi, Zekaver; Ozener, Cetin

    2011-04-10

    An 87 -year-old female who was undergoing peritoneal dialysis presented with peritonitis caused by Alcaligenes faecalis and Pantoea agglomerans in consecutive years. With the following report we discuss the importance of these unusual microorganisms in peritoneal dialysis patients.

  11. Alcaligenes xylosoxidans endophthalmitis following phacoemulsification and intraocular lens implantation.

    PubMed

    Robert, Pierre-Yves; Chainier, Delphine; Garnier, Fabien; Ploy, Marie-Cécile; Parneix, Pierre; Adenis, Jean-Paul; Martin, Christian

    2008-01-01

    Five consecutive cases of endophthalmitis that developed after cataract extraction by a single surgeon using the same operating room during one morning session are described. Following preoperative topical administration of ciprofloxacin, surgery consisted of phacoemulsification with peristaltic pump and fluid venting, polymethylmethacrylate intraocular lens implantation, and corneal suture. No complications occurred during surgery. All five patients developed endophthalmitis caused by infection with Alcaligenes xylosoxidans in less than 24 hours. Pulsed-field gel electrophoresis was used to prove similarity between strains. Bacterial inquiry on contamination of the operating room environment revealed massive colonization of phacoemulsifier irrigation channels by Pseudomonas fluorescens bacteria from an unestablished source. Four of the five patients ultimately recovered visual acuity better than 20/60.

  12. Development of a lipase fermentation process that uses a recombinant Pseudomonas alcaligenes strain.

    PubMed

    Gerritse, G; Hommes, R W; Quax, W J

    1998-07-01

    Pseudomonas alcaligenes M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions. A fed-batch fermentation process based on the secretion of the alkaline lipase from P. alcaligenes was developed. Due to the inability of P. alcaligenes to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively. The gene encoding the high-alkaline lipase from P. alcaligenes was isolated and characterized. Amplification of lipase gene copies in P. alcaligenes with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number. It was found that overexpression of the lipase helper gene, lipB, produced a stimulating effect in strains with high copy numbers (> 20) of the lipase structural gene, lipA. In strains with lipA on a low-copy-number vector, the lipB gene did not show any effect, suggesting that LipB is required in a low ratio to LipA only. During scaling up of the fermentation process to 100 m3, severe losses in lipase productivity were observed. Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses. A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.

  13. Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans

    SciTech Connect

    Ingvorsen, K.; Hojer-Pederson, B.; Godtfredsen, S.E. )

    1991-06-01

    A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp. denitrificans. Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H{sub 2}O {r arrow} HCOOH + NH{sub 3}) without forming formamide as a free intermediate. The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media. Cyanate (OCN{sup {minus}}) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A. xylosoxidans subsp. denitrificans DF3. Strain DF3 hydrolyzed cyanide with great efficacy. Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h. Enzyme purification established that cyanide hydrolysis by A. xylosoxidans subsp. denitrificans DF3 was due to a single intracellular enzyme. The molecular mass of the active enzyme (purity, {gt}97% as determined by amino acid sequencing) was estimated to be {gt}300,000 Da. The cyanide-hydrolyzing enzyme of A. xylosoxidans subsp. denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles.

  14. Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans.

    PubMed Central

    Ingvorsen, K; Højer-Pedersen, B; Godtfredsen, S E

    1991-01-01

    A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp. denitrificans. Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H2O----HCOOH + NH3) without forming formamide as a free intermediate. The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media. Cyanate (OCN-) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A. xylosoxidans subsp. denitrificans DF3. Strain DF3 hydrolyzed cyanide with great efficacy. Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h. Enzyme purification established that cyanide hydrolysis by A. xylosoxidans subsp. denitrificans DF3 was due to a single intracellular enzyme. The soluble enzyme was purified approximately 160-fold, and the first 25 NH2-terminal amino acids were determined by automated Edman degradation. The molecular mass of the active enzyme (purity, greater than 97% as determined by amino acid sequencing) was estimated to be greater than 300,000 Da. The cyanide-hydrolyzing enzyme of A. xylosoxidans subsp. denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles. Images PMID:1872607

  15. Comparison of aspartate transcarbamoylase regulation in Pseudomonas alcaligenes and Pseudomonas mendocina.

    PubMed

    Santiago, Manuel F; West, Thomas P

    2003-01-01

    The regulation of aspartate transcarbamoylase activity in cell extracts of Pseudomonas alcaligenes ATCC 14909 and Pseudomonas mendocina ATCC 25411 was compared. Under saturating substrate concentrations, pyrophosphate, CTP, UDP and ADP were highly inhibitory of the P. alcaligenes transcarbamoylase activity while pyrophosphate, UDP, ADP, ATP and GTP were the most effective inhibitors of the P. mendocina transcarbamoylase. By examining transcarbamoylase inhibition by ribonucleotide triphosphates, it was possible to differentiate these species assigned to different DNA homology groups and such an analysis might prove useful in the reclassification of Pseudomonas species.

  16. DEGRADATION OF ERGOTHIONEINE BY CELL-FREE EXTRACTS OF ALCALIGENES FAECALIS II.

    PubMed Central

    Booth, James S.; Appleman, Milo D.

    1963-01-01

    Booth, James S. (University of Southern California, Los Angeles) and Milo D. Appleman. Degradation of ergothioneine by cell-free extracts of Alcaligenes faecalis. II. Production of glutamic acid. J. Bacteriol. 85:654–657. 1963.—On the basis of oxidation and paper chromatographic procedures, glutamic acid was identified as the end product of ergothioneine degradation by cell-free extracts of Alcaligenes faecalis. Hydrogen sulfide and ammonia yields were determined. Several differences between the metabolism of whole cells and cell-free extracts were noted. Cleavage of the imidazole ring by cell-free extracts appeared to be hydrolytic rather than oxidative. PMID:14042946

  17. Nickel-Resistant Bacteria from Anthropogenically Nickel-Polluted and Naturally Nickel-Percolated Ecosystems

    PubMed Central

    Stoppel, R.; Schlegel, H. G.

    1995-01-01

    DNA fragments harboring the nickel resistance determinants from bacteria isolated from anthropogenically polluted ecosystems in Europe and Zaire were compared with those harboring the nickel resistance determinants from bacteria isolated from naturally nickel-percolated soils from New Caledonia by DNA-DNA hybridization. The biotinylated DNA probes were derived from the previously described Alcaligenes eutrophus CH34, Alcaligenes xylosoxidans 31A, Alcaligenes denitrificans 4a-2, and Klebsiella oxytoca CCUG 15788 and four new nickel resistance-determining fragments cloned from strains isolated from soils under nickel-hyperaccumulating trees. Nine probes were hybridized with endonuclease-cleaved plasmid and total DNA samples from 56 nickel-resistant strains. Some of the New Caledonian strains were tentatively identified as Acinetobacter, Pseudomonas mendocina, Comamonas, Hafnia alvei, Burkholderia, Arthrobacter aurescens, and Arthrobacter ramosus strains. The DNA of most strains showed homologies to one or several of the following nickel resistance determinants: the cnr and ncc operons of the strains A. eutrophus CH34 and A. xylosoxidans 31A, respectively, the nre operon of strain 31A, and the nickel resistance determinants of K. oxytoca. On the basis of their hybridization reactions the nickel resistance determinants of the strains could be assigned to four groups: (i) cnr/ncc type, (ii) cnr/ncc/nre type, (iii) K. oxytoca type, and (iv) others. The majority of the strains were assigned to the known groups. Among the strains from Belgium and Zaire, exclusively the cnr/ncc and the cnr/ncc/nre types were found. Among the New Caledonian strains all four types were represented. Homologies to the nre operon were found only in combination with the cnr/ncc operon. The homologies to the cnr/ncc operon were the most abundant and were detected alone or together with homologies to the nre operon. Only the DNA of the strains isolated from soil in Scotland and the United States

  18. Metabolism of Cyclohexane Carboxylic Acid by Alcaligenes Strain W1

    PubMed Central

    Taylor, David G.; Trudgill, Peter W.

    1978-01-01

    Thirty-three microorganisms capable of growth with cyclohexane carboxylate as the sole source of carbon were isolated from mud, water, and soil samples from the Aberystwyth area. Preliminary screening and whole-cell oxidation studies suggested that, with one exception, all of the strains metabolized the growth substrate by beta-oxidation of the coenzyme A ester. This single distinctive strain, able to oxidize rapidly trans-4-hydroxycyclohexane carboxylate, 4-ketocyclohexane carboxylate, p-hydroxybenzoate, and protocatechuate when grown with cyclohexane carboxylate, was classified as a strain of Alcaligenes and given the number W1. Enzymes capable of converting cyclohexane carboxylate to p-hydroxybenzoate were induced by growth with the alicyclic acid and included the first unambiguous specimen of a cyclohexane carboxylate hydroxylase. Because it is a very fragile protein, attempts to stabilize the cyclohexane carboxylate hydroxylase so that a purification procedure could be developed have consistently failed. In limited studies with crude cell extracts, we found that hydroxylation occurred at the 4 position, probably yielding the trans isomer of 4-hydroxycyclohexane carboxylate. Simultaneous measurement of oxygen consumption and reduced nicotinamide adenine dinucleotide oxidation, coupled with an assessment of reactant stoichiometry, showed the enzyme to be a mixed-function oxygenase. Mass spectral analysis enabled the conversion of cyclohexane carboxylate to p-hydroxybenzoate by cell extracts to be established unequivocally, and all of our data were consistent with the pathway: cyclohexane carboxylate → trans-4-hydroxycyclohexane carboxylate → 4-ketocyclohexane carboxylate → p-hydroxybenzoate. The further metabolism of p-hydroxybenzoate proceeded by meta fission and by the oxidative branch of the 2-hydroxy-4-carboxymuconic semialde-hyde-cleaving pathway. PMID:207665

  19. Degradation of dexamethasone by acclimated strain of Pseudomonas Alcaligenes.

    PubMed

    Zhu, Lili; Yang, Zhibang; Yang, Qian; Tu, Zeng; Ma, Lianju; Shi, Zhongquan; Li, Xiaoyu

    2015-01-01

    This study is to investigate the use of microbial remediation technology for degradation of dexamethasone in polluted water. A strain of Pseudomonas Alcaligenes with the ability of dexamethasone degradation was isolated from hospital polluted water. This strain was further acclimated into a bacterial strain that could highly degrade dexamethasone. Domesticated bacterial proteins were separated by osmotic shock method and were analyzed using SDS-PAGE. Enzyme activity of dexamethasone degradation was detected by high performance liquid chromatography. Protein bands with different molecular weight were found in all regions of the bacteria and a band with molecular weight of about 100 kDa was most obvious. In intracellular and periplasmic liquid, there was a band with molecular weight of about 41 kDa. Enzyme activity mainly existed in intracellular liquid. The 41 kDa protease was purified using ammonium sulfate precipitation, DEAE-52 ion exchange column and Sephadex G-100 column. Dexamethasone and dexamethasone sodium phosphate degrading rates of the purified enzyme were 36% and 95%, respectively. The 100 kDa protein had a 19% coverage rate to TonB receptor dependent protein, with 11 peptides matching. The 41 kDa protein had a 56% coverage rate to isovaleryl coenzyme A dehydrogenase, with 5 peptides matching. The 41 kDa protein had good degradation between the temperature of 25-40°C and PH value of 6.5-8.5. The enzyme kinetics equation was Ct = C0 e(-0.1769t), in accordance with the first-order kinetic equation. This study laid the foundation for further preparation of bioremediation agents for clearance of dexamethasone pollution in water.

  20. Investigation of cadmium resistance in an Alcaligenes sp.

    PubMed Central

    McEntee, J D; Woodrow, J R; Quirk, A V

    1986-01-01

    The mechanisms of metal resistance of a cadmium-resistant Alcaligenes sp. were studied. Growth in a defined medium was unaffected by cadmium at concentrations up to 0.1 mM, while at concentrations up to 2.5 mM, growth occurred after an extended lag phase. The increase in length of the lag phase was abolished by repeated subculturing at these higher concentrations. However, subculture in the absence of cadmium reversed the adaptation process. Plasmid DNA was not detected in adapted cells, suggesting that adaptation is not plasmid mediated. Increased sulfide production in response to cadmium was observed, although the levels were too low to account fully for cadmium resistance. Adaptation of cells to cadmium resulted in the appearance of a major new membrane protein (molecular weight, 34,500) whose presence was not dependent upon the method of membrane preparation. This protein was induced at cadmium concentrations of 0.1 mM and above, but below this level the protein was absent. The onset of growth at concentrations above 0.1 mM was coincident with the appearance of this protein, which was also induced by zinc (0.4 mM) but not by manganese or nickel. The protein was only solubilized by a sodium dodecyl sulfate-2-mercaptoethanol mixture. Similar solubility properties were shown by a second major membrane protein (molecular weight, 33,000). These two proteins proved to be similar by peptide-mapping experiments and amino acid analysis. The appearance of the 34,500-molecular-weight protein and its possible role in cadmium resistance are discussed. Images PMID:3963810

  1. Isolation and characterization of the pesticide-degrading plasmid pJP1 from Alcaligenes paradoxus.

    PubMed Central

    Fisher, P R; Appleton, J; Pemberton, J M

    1978-01-01

    A strain of Alcaligenes paradoxus, unable to degrade phenoxyacetic acid, was shown to degrade two synthetic derivatives of this molecule, the herbicides 2,4-dichlorophenoxyacetic acid and 2-methyl-4-chlorophenoxyacetic acid. The ability to degrade these pesticides is encoded by a 58-megadalton conjugal plasmid, pJP1. PMID:690076

  2. Draft Genome Sequence of Alcaligenes faecalis subsp. faecalis NCIB 8687 (CCUG 2071)

    PubMed Central

    Phung, Le T.; Trimble, William L.; Meyer, Folker

    2012-01-01

    Alcaligenes faecalis subsp. faecalis NCIB 8687, the betaproteobacterium from which arsenite oxidase had its structure solved and the first “arsenate gene island” identified, provided a draft genome of 3.9 Mb in 186 contigs (with the largest 15 comprising 90% of the total) for this opportunistic pathogen species. PMID:22933773

  3. Unusual causes of peritonitis in a peritoneal dialysis patient: Alcaligenes faecalis and Pantoea agglomerans

    PubMed Central

    2011-01-01

    An 87 -year-old female who was undergoing peritoneal dialysis presented with peritonitis caused by Alcaligenes faecalis and Pantoea agglomerans in consecutive years. With the following report we discuss the importance of these unusual microorganisms in peritoneal dialysis patients. PMID:21477370

  4. Treatment of dairy wastewater using a selected bacterial isolate, Alcaligenes sp. MMRR7.

    PubMed

    Rajeshkumar, K; Jayachandran, K

    2004-01-01

    Physicochemical and biologic analysis of dairy wastewater showed that the effluent had a high organic load (chemical oxygen demand [COD]: 5095 mg/L), an acidic pH (6.4), and a high probability of coliforms (most probable number [MPN] >1100). The various bacterial strains isolated and purified were identified as Sporolactobacillus sp., Citrobacter sp., Pseudomonas sp., Alcaligenes sp., Bacillus sp., Staphylococcus sp., and Proteus sp., as per the Bergey's manual of systematic bacteriology. Among the five selected bacterial strains, the strain designated as MMRR7 and identified as Alcaligenes sp. was found to give a maximum reduction in COD (62%) in 5 d of incubation. Chemical coagulation using alum at a concentration of 0.5 g/100 mL was found to be effective in the primary treatment of the effluent. Studies on free-cell treatment of the coagulated effluent with the selected bacterial strain Alcaligenes sp. MMRR7 gave a maximum COD reduction of 91% in 120 h. This study clearly indicates the possibility of using Alcaligenes sp. MMRR7 for the effective treatment of dairy wastewater.

  5. Draft genome sequence of Alcaligenes faecalis subsp. faecalis NCIB 8687 (CCUG 2071).

    PubMed

    Phung, Le T; Trimble, William L; Meyer, Folker; Gilbert, Jack A; Silver, Simon

    2012-09-01

    Alcaligenes faecalis subsp. faecalis NCIB 8687, the betaproteobacterium from which arsenite oxidase had its structure solved and the first "arsenate gene island" identified, provided a draft genome of 3.9 Mb in 186 contigs (with the largest 15 comprising 90% of the total) for this opportunistic pathogen species.

  6. A Newly Sequenced Alcaligenes faecalis Strain: Implications for Novel Temporal Symbiotic Relationships.

    PubMed

    Hernández-Mendoza, Armando; Lozano-Aguirre Beltrán, Luis Fernando; Martínez-Ocampo, Fernando; Quiroz-Castañeda, Rosa Estela; Dantán-González, Edgar

    2014-12-24

    We report here the draft genome sequence of Alcaligenes faecalis strain MOR02, a bacterium that is able to colonize nematodes in a temporary fashion and kill insects for their own benefit. The availability of the genome should enable us to explain these phenotypes.

  7. The characterisation of Bordetella/Alcaligenes-like organisms and their effects on turkey poults and chicks.

    PubMed

    Varley, J

    1986-01-01

    Eight isolates of the Bordetella or Alcaligenes-like organisms associated with turkey rhino-tracheitis were examined. Five of these isolates had been recovered from the United Kingdom and three were foreign isolates. Four of the UK isolates came from flocks with mild respiratory disease. The fifth isolate came from birds with no respiratory signs and this appears to be the first report of the recovery of Bordetella/Alcaligenes from apparently normal turkeys. The field isolates and type strains Alcaligenes faecalis NCTC 415 and Bordetella bronchiseptica NCTC 452 were characterised by biochemical tests, but these did not include any electrophoresis or nucleic acid studies. Cluster analysis using the group average method and the similarly coefficient of Sokal and Sneath indicated that all the strains were distinct from Alcaligenes faecalis but were quite closely related to Bordetella bronchiseptica. Each field isolate was used to infect separate groups of day-old turkey poults and chicks, and each group contained birds which were experimentally infected and others which were in-contact. Observations were made over a 32-day period. In turkey poults, some of the isolates induced severe respiratory disease and mortality, and others very little or none. The UK isolates were less pathogenic than the foreign isolates. It was not possible to correlate the pathogenicity of the isolates for turkey poults with their biochemical characteristics. Chicks infected with two of the eight isolates showed slight respiratory signs, but there was no significant mortality.

  8. Alcaligenes faecalis subsp. parafaecalis subsp. nov., a bacterium accumulating poly-beta-hydroxybutyrate from acetone-butanol bioprocess residues.

    PubMed

    Schroll, G; Busse, H J; Parrer, G; Rölleke, S; Lubitz, W; Denner, E B

    2001-04-01

    The authors have previously isolated a solvent tolerant bacterium, strain G(T), (T = type strain) capable to convert acetone-butanol bioprocess residues into poly-beta-hydroxybutyrate. Strain G(T) was initially identified as Alcaligenes spp by standard bacteriological tests. In this study the taxonomic position of the bacterium was investigated in detail. The 165 rDNA sequence analysis, the G + C content of DNA (56 mol%) and the presence of ubiquinone Q-8 confirmed strain G(T) as a representative of the genus Alcaligenes. In the polyamine pattern of the bacterium putrescine and cadaverine were detected, but only trace amounts of 2-hydroxyputrescine. The extremely low content of 2-hydroxyputrescine is remarkable, since this unique diamine is a common marker for beta-proteobacteria. Phylogenetic analyses of 16S rDNA demonstrated that Alcaligenes sp. G(T) is most closely related to the species Alcaligenes faecalis (99.6% sequence similarity to A. faecalis HR4 and 98.7% sequence similarity to A. faecalis [ATCC 8750T = DSM 30030T]. On the basis of DNA-DNA relatedness (56% similarity), the unique polyamine pattern, the physiological and biochemical differences strain G(T) could be distinguished from the species A. faecalis. Therefore, a new subspecies for the species Alcaligenes faecalis is proposed; Alcaligenes faecalis subsp. parafaecalis subsp. nov.

  9. Meningitis caused by Alcaligenes xylosoxidans in a patient with HIV/AIDS.

    PubMed

    Espinoza-Gómez, F; Newton-Sánchez, O A; Melnikov, V; Virgen-González, O; Unrau, J

    2007-12-01

    The purpose of the present work was to inform about the first case of meningitis associated to the bacteria Alcaligenes xylosoxidans in a patient with HIV/AIDS. The patient was a 46-year-old male, with the antecedent of have been diagnosed for HIV/ AIDS, who attended in the Hospital Universitario de Colima, Mexico, with fever, shock and meningismus. The study of the cerebrospinal fluid showed pleocytosis, elevated protein levels and hypoglycorrhachia. The culture yielded the presence of Alcaligenes xylosoxidans with sensitivity to ciprofloxacin. After 14 days of treatment with this antibiotic, the patient showed neurologic improvement and was able to continue with his outpatient antiretroviral treatment. The present case shows the importance of the inclusion of this bacterium in the differential diagnosis of the neurological infections in HIV/AIDS patients and emphasizes the importance of considering the bacterial meningitis in this population.

  10. Effect of surfactants on fluoranthene degradation by Pseudomonas alcaligenes PA-10.

    PubMed

    Hickey, Anne Marie; Gordon, Linda; Dobson, Alan D W; Kelly, Catherine T; Doyle, Evelyn M

    2007-03-01

    Two surfactants, Tween 80 and JBR, were investigated for their effect on fluoranthene degradation by a Pseudomonad. Both surfactants enhanced fluoranthene degradation by Pseudomonas alcaligenes PA-10 in shake flask culture. This bacterium was capable of utilising the synthetic surfactant and the biosurfactant as growth substrates and the critical micelle concentration of neither compound inhibited bacterial growth. The biosurfactant JBR significantly increased polycyclic aromatic hydrocarbon (PAH) desorption from soil. Inoculation of fluoranthene-contaminated soil microcosms with P. alcaligenes PA-10 resulted in the removal of significant amounts (45 +/- 5%) of the PAH after 28 days compared to an uninoculated control. Addition of the biosurfactant increased the initial rate of fluoranthene degradation in the inoculated microcosm. The presence of a lower molecular weight PAH, phenanthrene, had a similar effect on the rate of fluoranthene removal.

  11. [Case report: respiratory infection due to Alcaligenes xylosoxidans in a patient with Mounier-Kuhn syndrome].

    PubMed

    Arroyo-Cózar, Marta; Ruiz-García, Montserrat; Merlos, Eva M; Vielba, David; Macías, Enrique

    2012-10-01

    Mounier-Kuhn syndrome is a rare entity characterized by abnormal dilatation of the trachea and main bronchi (tracheobronchomegaly). Alcaligenes xylosoxidans is a non fermenting gram-negative pathogen common in extra-and intra-hospital environment, which may be related to immunosuppression states. We describe the case of a 75 years old male, ex-smoker with moderate functional obstruction, chronic respiratory failure and chronic colonization by Pseudomonas aeuriginosa. He had an infectious exacerbation of his disease, reason that previously required several hospital admissions. The patient was treated with antibiotics and his evolution was favourable with negativization in cultures of the pathogen. This is the first description of the isolation of Alcaligenes xylosoxidans as a cause of respiratory infection in a patient with Mounier-Kuhn syndrome.

  12. Effect of carbon source on pyrimidine biosynthesis in Pseudomonas alcaligenes ATCC 14909.

    PubMed

    Santiago, Manuel F; West, Thomas P

    2003-01-01

    The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in Pseudomonas alcaligenes ATCC 14909 was investigated. The de novo pyrimidine biosynthetic enzymes were measured in extracts of P. alcaligenes ATCC 14909 cells and of cells from an auxotroph deficient for orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic enzyme activities in ATCC 14909 were influenced by pyrimidine supplementation to the culture medium but not by the carbon source present. Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be controlled at the transcriptional level by a pyrimidine-related compound. Its regulation seemed to be subject to less transcriptional control by a pyrimidine-related compound than what was observed in the closely related species Pseudomonas pseudoalcaligenes.

  13. Immobilization of Alcaligenes faecalis penicillin G acylase on epoxy-type supports.

    PubMed

    Sun, J; Zhou, Y; Yuan, Z; Xu, G

    2009-01-01

    Alcaligenes faecalis penicillin G acylase has several desired features over other penicillin G acylases and its use in industry requires immobilization. In this work, two novel supports ZH-EP (epoxy-type) and ZH-HA (epoxy-amino type) were used to immobilize Alcaligenes faecalis penicillin G acylase (AfPGA) with Eupergit C as reference. The saturation of immobilized protein on ZH-EP (269 mg/g, 116 h) and ZH-HA (296 mg/g, 15 h) was obtained more rapidly than Eupergit C (197 mg/g, 260 h). And the activity of immobilized AfPGA on ZH-EP (520 U/g) and ZH-HA (2200 U/g) was higher than that on Eupergit C (310 U/g). The properties of three immobilized enzymes were compared and no obvious difference was observed, which indicated that ZH-EP and ZH-HA were promised in industry.

  14. Lipase expression in Pseudomonas alcaligenes is under the control of a two-component regulatory system.

    PubMed

    Krzeslak, Joanna; Gerritse, Gijs; van Merkerk, Ronald; Cool, Robbert H; Quax, Wim J

    2008-03-01

    Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.

  15. Arsenic methylation and volatilization by arsenite S-adenosylmethionine methyltransferase in Pseudomonas alcaligenes NBRC14159.

    PubMed

    Zhang, Jun; Cao, Tingting; Tang, Zhu; Shen, Qirong; Rosen, Barry P; Zhao, Fang-Jie

    2015-04-01

    Inorganic arsenic (As) is highly toxic and ubiquitous in the environment. Inorganic As can be transformed by microbial methylation, which constitutes an important part of the As biogeochemical cycle. In this study, we investigated As biotransformation by Pseudomonas alcaligenes NBRC14159. P. alcaligenes was able to methylate arsenite [As(III)] rapidly to dimethylarsenate and small amounts of trimethylarsenic oxide. An arsenite S-adenosylmethionine methyltransferase, PaArsM, was identified and functionally characterized. PaArsM shares low similarities with other reported ArsM enzymes (<55%). When P. alcaligenes arsM gene (PaarsM) was disrupted, the mutant lost As methylation ability and became more sensitive to As(III). PaarsM was expressed in the absence of As(III) and the expression was further enhanced by As(III) exposure. Heterologous expression of PaarsM in an As-hypersensitive strain of Escherichia coli conferred As(III) resistance. Purified PaArsM protein methylated As(III) to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases. We propose that PaArsM plays a role in As methylation and detoxification of As(III) and could be exploited in bioremediation of As-contaminated environments.

  16. Identification and antimicrobial susceptibility of Alcaligenes xylosoxidans isolated from patients with cystic fibrosis.

    PubMed

    Saiman, L; Chen, Y; Tabibi, S; San Gabriel, P; Zhou, J; Liu, Z; Lai, L; Whittier, S

    2001-11-01

    In the past decade, potential pathogens, including Alcaligenes species, have been increasingly recovered from cystic fibrosis (CF) patients. Accurate identification of multiply antibiotic-resistant gram-negative bacilli is critical to understanding the epidemiology and clinical implications of emerging pathogens in CF. We examined the frequency of correct identification of Alcaligenes spp. by microbiology laboratories affiliated with American CF patient care centers. Selective media, an exotoxin A probe for Pseudomonas aeruginosa, and a commercial identification assay, API 20 NE, were used for identification. The activity of antimicrobial agents against these clinical isolates was determined. A total of 106 strains from 78 patients from 49 CF centers in 22 states were studied. Most (89%) were correctly identified by the referring laboratories as Alcaligenes xylosoxidans. However, 12 (11%) strains were misidentified; these were found to be P. aeruginosa (n = 10), Stenotrophomonas maltophilia (n = 1), and Burkholderia cepacia (n = 1). Minocycline, imipenem, meropenem, piperacillin, and piperacillin-tazobactam were the most active since 51, 59, 51, 50, and 55% of strains, respectively, were inhibited. High concentrations of colistin (100 and 200 microg/ml) inhibited 92% of strains. Chloramphenicol paired with minocycline and ciprofloxacin paired with either imipenem or meropenem were the most active combinations and inhibited 40 and 32%, respectively, of strains. Selective media and biochemical identification proved to be useful strategies for distinguishing A. xylosoxidans from other CF pathogens. Standards for processing CF specimens should be developed, and the optimal method for antimicrobial susceptibility testing of A. xylosoxidans should be determined.

  17. Isolation and characterization of group II introns from Pseudomonas alcaligenes and Pseudomonas putida.

    PubMed

    Yeo, C C; Yiin, S; Tan, B H; Poh, C L

    2001-05-01

    Group II introns isolated from Pseudomonas alcaligenes NCIB 9867, Pseudomonas putida NCIB 9869, and P. putida KT2440 were closely related with nucleotide sequence identities of between 87 and 96%. The genome of P. alcaligenes also harbored a truncated group II intron of 682 bp that lacks the gene for the intron-encoded protein (IEP). Unlike most bacterial group II introns, the Pseudomonas introns were found to lack the Zn domains in their IEPs, did not appear to interrupt any genes, and were located downstream of open reading frames which were adjacent to hairpin loop structures that resemble rho-independent terminators. These structures also contain the intron binding sites 1 and 2 (IBS1 and IBS2 sequences) that were required for intron target site recognition in transposition. One of the group II introns found in P. alcaligenes, Xln3, was shown to have transposed from the chromosome to the endogenous pRA2 plasmid at a site adjacent to IBS1- and IBS2-like sequences.

  18. Isolation of Indole Utilizing Bacteria Arthrobacter sp. and Alcaligenes sp. From Livestock Waste.

    PubMed

    Kim, Minsu; Lee, Jin-Hyung; Kim, Eonmi; Choi, Hyukjae; Kim, Younghoon; Lee, Jintae

    2016-06-01

    Indole is an interspecies and interkingdom signaling molecule widespread in different environmental compartment. Although multifaceted roles of indole in different biological systems have been established, little information is available on the microbial utilization of indole in the context of combating odor emissions from different types of waste. The present study was aimed at identifying novel bacteria capable of utilizing indole as the sole carbon and energy source. From the selective enrichment of swine waste and cattle feces, we identified Gram-positive and Gram-negative bacteria belonging to the genera Arthrobacter and Alcaligenes. Bacteria belonging to the genus Alcaligenes showed higher rates of indole utilization than Arthrobacter. Indole at 1.0 mM for growth was completely utilized by Alcaligenes sp. in 16 h. Both strains produced two intermediates, anthranilic acid and isatin, during aerobic indole metabolism. These isolates were also able to grow on several indole derivatives. Interestingly, an adaptive response in terms of a decrease in cell size was observed in both strains in the presence of indole. The present study will help to explain the degradation of indole by different bacteria and also the pathways through which it is catabolized. Furthermore, these novel bacterial isolates could be potentially useful for the in situ attenuation of odorant indole and its derivatives emitted from different types of livestock waste.

  19. Identification of Strains of Alcaligenes and Agrobacterium by a Polyphasic Approach

    PubMed Central

    Clermont, Dominique; Harmant, Christine; Bizet, Chantal

    2001-01-01

    The number of stable discriminant biochemical characters is limited in the genera Alcaligenes and Agrobacterium, whose species are consequently difficult to distinguish from one another by conventional tests. Moreover, genomic studies have recently drastically modified the nomenclature of these genera; for example, Alcaligenes xylosoxidans was transferred to the genus Achromobacter in 1998. Twenty-five strains of Achromobacter xylosoxidans, three strains of an Agrobacterium sp., five strains of an Alcaligenes sp., and four unnamed strains belonging to the Centers for Disease Control and Prevention group IVc-2 were examined. These strains were characterized by conventional tests, including biochemical tests. The assimilation of 99 carbohydrates, organic acids, and amino acids was studied by using Biotype-100 strips, and rRNA gene restriction patterns were obtained with the automated Riboprinter microbial characterization system after cleavage of total DNA with EcoRI or PstI restriction endonuclease. This polyphasic approach allowed the two subspecies of A. xylosoxidans to be clearly separated. Relationships between five strains and the Ralstonia paucula type strain were demonstrated. Likewise, three strains were found to be related to the Ochrobactrum anthropi type strain. We showed that substrate assimilation tests and automated ribotyping provide a simple, rapid, and reliable means of identifying A. xylosoxidans subspecies and that these two methods can be used as alternative methods to characterize unidentified strains rapidly when discriminant biochemical characters are missing. PMID:11526136

  20. Development of a PCR-based method for monitoring the status of Alcaligenes species in the agricultural environment.

    PubMed

    Nakano, Miyo; Niwa, Masumi; Nishimura, Norihiro

    2014-01-01

    To analyze the status of the genus Alcaligenes in the agricultural environment, we developed a PCR method for detection of these species from vegetables and farming soil. The selected PCR primers amplified a 107-bp fragment of the 16S rRNA gene in a specific PCR assay with a detection limit of 1.06 pg of pure culture DNA, corresponding to DNA extracted from approximately 23 cells of Alcaligenes faecalis. Meanwhile, PCR primers generated a detectable amount of the amplicon from 2.2×10(2) CFU/ml cell suspensions from the soil. Analysis of vegetable phylloepiphytic and farming soil microbes showed that bacterial species belonging to the genus Alcaligenes were present in the range from 0.9×10(0) CFU per gram (or cm(2)) (Japanese radish: Raphanus sativus var. longipinnatus) to more than 1.1×10(4) CFU/g (broccoli flowers: Brassica oleracea var. italic), while 2.4×10(2) to 4.4×10(3) CFU/g were detected from all soil samples. These results indicated that Alcaligenes species are present in the phytosphere at levels 10-1000 times lower than those in soil. Our approach may be useful for tracking or quantifying species of the genus Alcaligenes in the agricultural environment.

  1. Septic arthritis caused by a gram-negative bacterium representing a new species related to the Bordetella-Alcaligenes complex.

    PubMed

    Kronvall, G; Hanson, H S; von Stedingk, L V; Törnqvist, E; Falsen, E

    2000-03-01

    A knee-joint exudate culture yielded on two occasions a gram-negative bacterium. Regular methods for speciation did not provide an identification. The infection was successfully treated with ciprofloxacin. The unknown isolate, CCUG 36768, was subjected to further investigation, including 16S rDNA sequencing, protein profiling, cellular fatty acid analysis, and various biochemical tests, in order to produce a species identification. The 1469 bp-long 16S rDNA sequence did not reveal identity with any known species sequence. CCUG 36768 clustered in a group of species, including Alcaligenes defragrans, Denitrobacter permanens, Taylorella equigenitalis, Alcaligenes faecalis, and four strains of Alcaligenes species without a specific species name. Bordetella species also showed a high degree of similarity with CCUG 36768. Protein profiling, cellular fatty acid analysis and computer-assisted analysis of biochemical profiles indicated similarity with Bordetella-Alcaligenes species, often close to B. holmesii and B. avium. API 20 NE indicated the profile of Moraxella species of poor identity. It is concluded that CCUG 36768 represents a new bacterial species of pathogenic potential in humans. It is related to the Bordetella-Alcaligenes group. Powerful new methods for speciation are available and it is recommended that unknown isolates from normally sterile sites be submitted for further analysis. Several isolates are required for the definition of new species.

  2. Cell growth and accumulation of polyhydroxyalkanoates from CO2 and H2 of a hydrogen-oxidizing bacterium, Cupriavidus eutrophus B-10646.

    PubMed

    Volova, Tatiana G; Kiselev, Evgeniy G; Shishatskaya, Ekaterina I; Zhila, Natalia O; Boyandin, Anatoly N; Syrvacheva, Daria A; Vinogradova, Olga N; Kalacheva, Galina S; Vasiliev, Alexander D; Peterson, Ivan V

    2013-10-01

    Synthesis of polyhydroxyalkanoates (PHAs) by a new strain of Cupriavidus - Cupriavidus eutrophus B-10646 - was investigated under autotrophic growth conditions. Under chemostat, at the specific flow rate D=0.1h(-1), on sole carbon substrate (CO2), with nitrogen, sulfur, phosphorus, potassium, and manganese used as growth limiting elements, the highest poly(3-hydroxybutyrate) [P(3HB)] yields were obtained under nitrogen deficiency. In batch autotrophic culture, in the fermenter with oxygen mass transfer coefficient 0.460 h(-1), P(3HB) yields reached 85% of dry cell weight (DCW) and DCW reached 50 g/l. Concentrations of supplementary PHA precursor substrates (valerate, hexanoate, γ-butyrolactone) and culture conditions were varied to produce, for the first time under autotrophic growth conditions, PHA ter- and tetra-polymers with widely varying major fractions of 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate, and 3-hydroxyhexanoate monomer units. Investigation of the high-purity PHA specimens showed significant differences in their physicochemical and physicomechanical properties. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867.

    PubMed

    Zhao, Bing; Yeo, Chew Chieng; Lee, Chee Chow; Geng, Anli; Chew, Fook Tim; Poh, Chit Laa

    2004-07-01

    Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions. One set of the enzymes is constitutive whereas the other is strictly inducible. To date, only the gene encoding the constitutively-expressed gentisate dioxygenase had been cloned and characterized. A mutant strain of P25X, designated G56, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate. The proteome profiles of P. alcaligenes P25X and mutant G56 cells grown in the presence and absence of gentisate were compared after two-dimensional polyacrylamide gel electrophoresis. Eight distinctive protein spots (designated M1-M8) which were observed only in induced cells of strain G56 but absent in noninduced cells were further analyzed by matrix-assisted laser desorption/ionization-time of flight, quadrupole-TOF and N-terminal sequencing. Of the 15 proteins (including seven up-regulated) examined, 13 showed sequence similarities to proteins with assigned functions in other microorganisms. The identification of protein M5 which showed high homology to a gentisate dioxygenase from Ralstonia sp. U2 indicated the putative function of this protein being consistent with the inducible gentisate 1,2-dioxygenase in P. alcaligenes. In addition, the induction of stress proteins and other adaptation phenomena were also observed.

  4. Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer.

    PubMed

    Kwong, S M; Yeo, C C; Suwanto, A; Poh, C L

    2000-01-01

    The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.

  5. Growth kinetics of Pseudomonas alcaligenes C-0 relative to inoculation and 3-chlorobenzoate metabolism in soil.

    PubMed Central

    Focht, D D; Shelton, D

    1987-01-01

    Pseudomonas alcaligenes C-0 was isolated from activated sewage sludge by enrichment with 3-chlorobenzoate (3CB) as the sole carbon source. The carbon balance from [14C]3CB in pure culture could be accounted for in substrate, biomass, and CO2 from all sampling periods and inoculum densities (0.012, 0.092, 0.20, and 0.92 micrograms of dry cells X ml-1), and inorganic chloride was produced stoichiometrically. Monod parameters as determined in culture were compared with the kinetics of 3CB metabolism in soil with decreasing inoculum densities (1.9 X 10(-1), 1.9 X 10(-3), and 1.9 X 10(-5) micrograms of cells X g-1). 3CB was refractile to attack in soil by indigenous microflora, but it was completely metabolized upon inoculation with P. alcaligenes C-0. The saturation constant KS was much higher in soil than in culture, but the yield coefficient Y and the growth rate constant were the same in both systems: mu max = 0.32 h-1; Y = 34 micrograms cells X mumol-1; KS = 0.18 mM in culture and 6.0 mM in soil solution (1.1 mumol X g-1 of soil). The parameter estimates obtained from the highest inoculum density could be used for the lower inoculum densities with reasonable agreement between predicted and observed 3CB concentrations in soil, although the residual sum of squares was progressively higher. Since the growth rate of P. alcaligenes C-0 in soil was comparable to its growth rate in culture, inoculation should be a viable strategy for biodegradation of 3CB in soil if indigenous microflora are unable to exploit this metabolic niche. PMID:3662518

  6. Genomic Characterization of VIM metallo-β-lactamase-producing Alcaligenes faecalis from GAZA, Palestine.

    PubMed

    Al Laham, Nahed; Chavda, Kalyan D; Cienfuegos-Gallet, Astrid V; Kreiswirth, Barry N; Chen, Liang

    2017-08-28

    Carbapenemase-producing Gram-negative bacteria (CP-GNB) have increasingly spread worldwide, and different families of carbapenemases have been identified in various bacterial species. Here we report the identification of five VIM metallo-β-lactamase-producing Alcaligenes faecalis, associated with a small outbreak in a large hospital in GAZA, Palestine. Next generation sequencing analysis showed blaVIM-2 is harbored by a chromosomal genomic island among three strains, while blaVIM-4 is carried by a novel plasmid in two strains. Copyright © 2017 American Society for Microbiology.

  7. Modification of cell surface properties of Pseudomonas alcaligenes S22 during hydrocarbon biodegradation.

    PubMed

    Kaczorek, Ewa; Moszyńska, Sylwia; Olszanowski, Andrzej

    2011-04-01

    Biodegradation of water insoluble hydrocarbons can be significantly increased by the addition of natural surfactants one. Very promising option is the use of saponins. The obtained results indicated that in this system, after 21 days, 92% biodegradation of diesel oil could be achieved using Pseudomonas alcaligenes. No positive effect on the biodegradation process was observed using synthetic surfactant Triton X-100. The kind of carbon source influences the cell surface properties of microorganisms. Modification of the surface cell could be observed by control of the sedimentation profile. This analytical method is a new approach in microbiological analysis.

  8. Bioreactor cultivation of Escherichia coli for production of recombinant penicillin G amidase from Alcaligenes faecalis.

    PubMed

    Deak, Peter M; Lutz-Wahl, Sabine; Bothe, Harald; Fischer, Lutz

    2003-03-01

    The penicillin G amidase (PGA) from Alcaligenes faecalis, which has interesting properties for use in combinatorial biochemistry, was produced by recombinant expression in Escherichia coli. The corresponding gene was cloned into a multicopy vector under the strict regulatory control of the rhamnose inducible promoter. Cells were grown in a synthetic minimal medium in a bioreactor (5 l working vol.), and production of PGA was induced by repeated addition of the inducer rhamnose, that served also as a carbon source. The fermentation yield was about 4500 units PGA activity per liter of culture medium.

  9. Structure-based stabilization of an enzyme: the case of penicillin acylase from Alcaligenes faecalis.

    PubMed

    Wang, Tianwen; Zhu, Hu; Ma, Xingyuan; Ma, Yushu; Wei, Dongzhi

    2006-01-01

    The modeled structure of penicillin acylase from Alcaligenes faecali (AFPGA) was constructed by comparative modeling with the Modeller program. Candidate positions that could be replaced with cysteine were estimated by scanning the modeled structure of AFPGA with the program MODIP (modeling disulfide bond in protein). The mutant Q3C/P751C had a higher optimum temperature by three degrees than that of the wild type AFPGA. The half life of the double mutant Q3C/P751C at 55 degrees C was increased by 50%. To our knowledge, this was the first structure-based genetic modification of AFPGA.

  10. The complete genome sequence of Alcaligenes faecalis ZD02, a novel potential bionematocide.

    PubMed

    Ju, Shouyong; Zheng, Jinshui; Lin, Jian; Geng, Ce; Zhu, Lei; Guan, Ziyu; Zheng, Ziqiang; Sun, Ming

    2016-01-20

    Root-knot nematodes (RKNs) can infect almost all crops, and result in huge economic losses in agriculture. There is no effective and environmentally safe means available to control RKNs. Alcaligenes faecalis ZD02 isolated from free living nematode Caenorhabditis elegans cadavers shows toxicity against RKN Meloidogyne incognita, that makes this strain to be a good bionematicide candidate for controlling of RKNs. Here, we firstly report the complete genome of A. faecalis ZD02 and describe its features. Additionally, we found two potential virulence factors in this genome, which play important roles for the nematocidal activity of A. faecalis ZD02.

  11. Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions

    SciTech Connect

    Otte, S.; Grobben, N.G.; Robertson, L.A.; Jetten, M.S.M.; Kuenen, J.G.

    1996-07-01

    Nitrous oxide production contributes to both greenhouse effect and ozone depletion in the stratosphere. A significant part of the global N2O emission can be attributed to microbial processes, especially nitrification and denitrification, used in biological wastewater treatment systems. This study looks at the efficiency of denitrification and the enzymes involved, with the emphasis on N2O production during the transient phase from aerobic to anaerobic conditions and vice versa. The effect of repetitive changing aerobic-anaerobic conditions on N2O was also studied. Alcaligenes faecalis was used as the model denitrofing organism. 35 refs., 3 figs., 1 tab.

  12. IS1491 from Pseudomonas alcaligenes NCIB 9867: characterization and distribution among Pseudomonas species.

    PubMed

    Yeo, C C; Wong, D T; Poh, C L

    1998-01-01

    A new insertion sequence, IS1491, has been cloned and sequenced. The 2489-bp IS1491 was isolated from a Pseudomonas alcaligenes NCIB 9867 (strain P25X) 4.8-kb PstI chromosomal fragment. IS1491 is flanked by an imperfect inverted repeat of 23 bp and carries two overlapping open reading frames, ORF1 and ORF2. Both ORF1 and ORF2 displayed homology to the IstA-like and IstB-like transposases encoded by the IS21 family of insertion sequences, which include two IS elements previously isolated from P. alcaligenes P25X, IS1474, and IS1475 (Yeo, C. C., and Poh, C. L. (1997). FEMS Microbiol. Lett. 149, 257-263). Transposition assays showed that IS1491 transposed at a frequency of approximately 1.4 x 10(-6). Transposition of IS1491 into the target pRK415 replicon was observed but when ORF2 was disrupted, a fusion between the donor and target replicons was detected. IS1491-like sequences were detected in total DNA of Pseudomonas putida NCIB 9869 (strain P35X), Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas mendocina, Comomonas acidovorans, and Comomonas testosteroni by hybridization with IS1491 DNA.

  13. [Extraction, Purification and Identification of a Dexamethasone-degrading Enzymes Generated by Pseudomonas Alcaligenes].

    PubMed

    Zhu, Lili; Yang, Zhibang; Yang, Qian; Shi, Zhongquan; Deng, Xichuan

    2015-10-01

    In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.

  14. Preliminary observations on the application of the multiple inocula (replicator) method for carbon substrate utilization studies of Alcaligenes faecalis.

    PubMed Central

    Riley, P S; Rarick, H R; Utter, G

    1977-01-01

    The use of the replicator technique in evaluating carbon source utilization by 55 cultures of Alcaligenes faecalis was examined. Six of the 20 substrates tested were utilized by the bacterial cultures. In this study, the reproducibility of the technique was 100%. Images PMID:323288

  15. The phenotype enhancement method identifies the Xcp outer membrane secretion machinery from Pseudomonas alcaligenes as a bottleneck for lipase production.

    PubMed

    Gerritse, G; Ure, R; Bizoullier, F; Quax, W J

    1998-09-17

    Pseudomonas alcaligenes M-1 has been selected from an intensive screening for micro-organisms that can naturally produce a lipase active in detergent formulations. The lipase expression has been increased to allow high level secretion from Pseudomonas alcaligenes, via the introduction of multi-copy plasmids. In order to improve the lipase yield further, the phenotype enhancement method has been developed. This idea comprises the reintroduction of a cosmid library with random chromosomal fragments in a P. alcaligenes strain with already high lipase productivity. One of the strains which showed an enhanced lipase production appeared to contain a cosmid encoding the outer membrane secretion genes. These xcp-genes are clustered in two divergently transcribed operons similar to the situation in Pseudomonas aeruginosa. Remarkably and dissimilar to P. aeruginosa, in between the two xcp gene clusters, two reading frames of unknown function--OrfV and OrfX--are present. For OrfX no equivalent can be found in the known protein data bases. On the other hand, OrfV shows homology to the regulatory proteins MalT and AcoK. Some evidence is provided that suggests that OrfV acts as a regulator of the xcp operons. A model is proposed for the regulation of the secretion system from P. alcaligenes.

  16. Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition.

    PubMed

    de Groot, A; Koster, M; Gérard-Vincent, M; Gerritse, G; Lazdunski, A; Tommassen, J; Filloux, A

    2001-02-01

    Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

  17. Fluorescence in situ hybridization for rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis recovered from cystic fibrosis patients.

    PubMed

    Wellinghausen, Nele; Wirths, Beate; Poppert, Sven

    2006-09-01

    Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients.

  18. The Genome Sequence of Alcaligenes faecalis NBIB-017 Contains Genes with Potentially High Activities against Erwinia carotovora.

    PubMed

    Liu, Xiaoyan; Huang, Daye; Wu, Jinping; Yu, Cui; Zhou, Ronghua; Liu, Cuijun; Zhang, Wei; Yao, Jingwu; Cheng, Meng; Guo, Suxia

    2016-04-07

    Alcaligenes faecalisNBIB-017, a Gram-negative bacterium, was isolated from soil in China. Here, we provide the complete genome sequence of this bacterium, which possesses a high number of genes encoding antibacterial factors, including proteins and small molecular peptides.

  19. The Genome Sequence of Alcaligenes faecalis NBIB-017 Contains Genes with Potentially High Activities against Erwinia carotovora

    PubMed Central

    Liu, Xiaoyan; Huang, Daye; Wu, Jinping; Yu, Cui; Zhou, Ronghua; Liu, Cuijun; Zhang, Wei; Yao, Jingwu; Cheng, Meng

    2016-01-01

    Alcaligenes faecalis NBIB-017, a Gram-negative bacterium, was isolated from soil in China. Here, we provide the complete genome sequence of this bacterium, which possesses a high number of genes encoding antibacterial factors, including proteins and small molecular peptides. PMID:27056227

  20. Castellaniella gen. nov., to accommodate the phylogenetic lineage of Alcaligenes defragrans, and proposal of Castellaniella defragrans gen. nov., comb. nov. and Castellaniella denitrificans sp. nov.

    PubMed

    Kämpfer, P; Denger, K; Cook, A M; Lee, S-T; Jäckel, U; Denner, E B M; Busse, H-J

    2006-04-01

    Comparative 16S rRNA gene sequence analysis indicates that two distinct sublineages exist within the genus Alcaligenes: the Alcaligenes faecalis lineage, comprising Alcaligenes aquatilis and A. faecalis (with the three subspecies A. faecalis subsp. faecalis, A. faecalis subsp. parafaecalis and A. faecalis subsp. phenolicus), and the Alcaligenes defragrans lineage, comprising A. defragrans. This phylogenetic discrimination is supported by phenotypic and chemotaxonomic differences. It is proposed that the A. defragrans lineage constitutes a distinct genus, for which the name Castellaniella gen. nov. is proposed. The type strain for Castellaniella defragrans gen. nov., comb. nov. is 54PinT (=CCUG 39790T = CIP 105602T = DSM 12141T). Finally, on the basis of data from the literature and new DNA-DNA hybridization and phenotypic data, the novel species Castellaniella denitrificans sp. nov. (type strain NKNTAUT = DSM 11046T = CCUG 39541T) is proposed for two strains previously identified as strains of A. defragrans.

  1. Cysteine coordination of Pb(II) is involved in the PbrR-dependent activation of the lead-resistance promoter, PpbrA, from Cupriavidus metallidurans CH34.

    PubMed

    Hobman, Jon L; Julian, Daniel J; Brown, Nigel L

    2012-06-18

    The pbr resistance operon from Cupriavidus metallidurans CH34 plasmid pMOL30 confers resistance to Pb(II) salts, and is regulated by the Pb(II) responsive regulator PbrR, which is a MerR family activator. In other metal sensing MerR family regulators, such as MerR, CueR, and ZntR the cognate regulator binds to a promoter with an unusually long spacer between the -35 and -10 sequences, and activates transcription of resistance genes as a consequence of binding the appropriate metal. Cysteine residues in these regulators are essential for metal ion coordination and activation of expression from their cognate promoter. In this study we investigated the interaction of PbrR with the promoter for the structural pbr resistance genes, PpbrA, effects on transcriptional activation of altering the DNA sequence of PpbrA, and effects on Pb(II)-induced activation of PpbrA when cysteine residues in PbrR were mutated to serine. Gel retardation and footprinting assays using purified PbrR show that it binds to, and protects from DNase I digestion, the PpbrA promoter, which has a 19 bp spacer between its -35 and -10 sites. Using β-galactosidase assays in C. metallidurans, we show that when PpbrA is changed to an 18 bp spacer, there is an increase in transcriptional activation both in the presence and absence of Pb(II) salts up to a maximum induction equivalent to that seen in the fully-induced wild-type promoter. Changes to the -10 sequence of PpbrA from TTAAAT to the consensus E. coli -10 sequence (TATAAT) increased transcriptional activation from PpbrA, whilst changing the -10 sequence to that of the Tn501 mer promoter (TAAGGT) also increased the transcriptional response, but only in the presence of Pb(II). Individual PbrR mutants C14S, C55S, C79S, C114S, C123S, C132S and C134S, and a double mutant C132S/C134S, were tested for Pb(II) response from PpbrA, using β-galactosidase assays in C. metallidurans. The PbrR C14S, C79S, C134S, and C132S/C134S mutants were defective in Pb

  2. Cysteine coordination of Pb(II) is involved in the PbrR-dependent activation of the lead-resistance promoter, PpbrA, from Cupriavidus metallidurans CH34

    PubMed Central

    2012-01-01

    Background The pbr resistance operon from Cupriavidus metallidurans CH34 plasmid pMOL30 confers resistance to Pb(II) salts, and is regulated by the Pb(II) responsive regulator PbrR, which is a MerR family activator. In other metal sensing MerR family regulators, such as MerR, CueR, and ZntR the cognate regulator binds to a promoter with an unusually long spacer between the −35 and −10 sequences, and activates transcription of resistance genes as a consequence of binding the appropriate metal. Cysteine residues in these regulators are essential for metal ion coordination and activation of expression from their cognate promoter. In this study we investigated the interaction of PbrR with the promoter for the structural pbr resistance genes, PpbrA, effects on transcriptional activation of altering the DNA sequence of PpbrA, and effects on Pb(II)-induced activation of PpbrA when cysteine residues in PbrR were mutated to serine. Results Gel retardation and footprinting assays using purified PbrR show that it binds to, and protects from DNase I digestion, the PpbrA promoter, which has a 19 bp spacer between its −35 and −10 sites. Using β-galactosidase assays in C. metallidurans, we show that when PpbrA is changed to an 18 bp spacer, there is an increase in transcriptional activation both in the presence and absence of Pb(II) salts up to a maximum induction equivalent to that seen in the fully-induced wild-type promoter. Changes to the −10 sequence of PpbrA from TTAAAT to the consensus E. coli −10 sequence (TATAAT) increased transcriptional activation from PpbrA, whilst changing the −10 sequence to that of the Tn501 mer promoter (TAAGGT) also increased the transcriptional response, but only in the presence of Pb(II). Individual PbrR mutants C14S, C55S, C79S, C114S, C123S, C132S and C134S, and a double mutant C132S/C134S, were tested for Pb(II) response from PpbrA, using β-galactosidase assays in C. metallidurans. The PbrR C14S, C79S, C134S, and C132S

  3. Production of R-(-)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750.

    PubMed Central

    Yamamoto, K; Oishi, K; Fujimatsu, I; Komatsu, K

    1991-01-01

    R-(-)-Mandelic acid was produced from racemic mandelonitrile by Alcaligenes faecalis ATCC 8750. Ammonium acetate or L-glutamic acid as the carbon source and n-butyronitrile as the inducer in the culture medium were effective for bacterial growth and the induction of R-(-)-mandelic acid-producing activity. The R-(-)-mandelic acid formed from mandelonitrile by resting cells was present in a 100% enantiomeric excess. A. faecalis ATCC 8750 has an R-enantioselective nitrilase for mandelonitrile and an amidase for mandelamide. As R-(-)-mandelic acid was produced from racemic mandelonitrile in a yield of 91%, whereas no S-mandelonitrile was left, the S-mandelonitrile remaining in the reaction is spontaneously racemized because of the chemical equilibrium and is used as the substrate. Consequently, almost all the mandelonitrile is consumed and converted to R-(-)-mandelic acid. R-(-)-Mandelic acid was also produced when benzaldehyde plus HCN was used as the substrate. PMID:1660699

  4. The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri.

    PubMed

    Vermeiren, H; Willems, A; Schoofs, G; de Mot, R; Keijers, V; Hai, W; Vanderleyden, J

    1999-05-01

    The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.

  5. Soil bacteria Pseudomonas putida and Alcaligenes xylosoxidans subsp. denitrificans inactivate triclosan in liquid and solid substrates.

    PubMed

    Meade, M J; Waddell, R L; Callahan, T M

    2001-10-16

    Triclosan is a broad-spectrum antimicrobial agent that has been incorporated into many household and medical products. Bacteria with high levels of triclosan resistance were isolated from compost, water, and soil samples. Two of these bacteria, Pseudomonas putida TriRY and Alcaligenes xylosoxidans subsp. denitrificans TR1, were able to use triclosan as a sole carbon source and clear particulate triclosan from agar. A decrease in triclosan concentration was measured by HPLC within 6 h of inoculation with strain TriRY and 24 h with strain TR1. Bioassays demonstrated that triclosan was inactivated in liquid cultures and/or embedded in plastic by the growth of strain TriRY and strain TR1, permitting the growth of triclosan-sensitive bacteria.

  6. The purification and chemical composition of the lipopolysaccharide of Pseudomonas alcaligenes

    PubMed Central

    Key, B. A.; Gray, G. W.; Wilkinson, S. G.

    1970-01-01

    1. A method for obtaining lipopolysaccharide free from glycosaminopeptide from isolated cell walls of Pseudomonas alcaligenes is discussed. 2. About 70–75% of the lipopolysaccharide and 86–90% of the isolated lipid A have been accounted for in terms of identifiable components. 3. Hydrolysates of lipid A contain mainly inorganic phosphate, glucosamine, O-phosphorylglucosamine and fatty acids (dodecanoic acid, dodec-2-enoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid), of which the last is the main N-acylating acid of the glucosamine backbone. 4. Material corresponding to the polysaccharide moiety of the lipopolysaccharide is extensively degraded. 5. Solubilization of the lipopolysaccharide by using sodium deoxycholate appreciably affects the chemical composition of the material. PMID:5499968

  7. Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis No. 4.

    PubMed

    Joo, Hung-Soo; Hirai, Mitsuyo; Shoda, Makoto

    2005-08-01

    Alcaligenes faecalis no. 4 has heterotrophic nitrification and aerobic denitrification abilities. By taking the nitrogen balance under different culture conditions, 40-50% of removed NH4+-N was denitrified and about one-half of removed NH4+-N was converted to intracellular nitrogen. The maximum ammonium removal rate of no. 4 (28.9 mg-N/l/h) and its denitrification rate at high-strength NH4+-N of about 1200 ppm in aerated batch experiments at a C/N ratio of 10 were 5-40 times higher than those of other bacteria with the same ability. Only a few percent of the removed ammonium was converted to nitrite, and the main denitrification process was speculated to be via hydroxylamine which was produced by ammonium oxidation.

  8. Production of bioemulsifier by Bacillus subtilis, Alcaligenes faecalis and Enterobacter species in liquid culture.

    PubMed

    Toledo, F L; Gonzalez-Lopez, J; Calvo, C

    2008-11-01

    Three bacterial strains isolated from waste crude oil were selected due to their capacity of growing in the presence of hydrocarbons and production of bioemulsifier. The genetic identification (PCR of the 16S rDNA gene using fD1 and rD1 primers) of these strains showed their affiliation to Bacillus subtilis, Alcaligenes faecalis and Enterobacter sp. These strains were able to emulsify n-octane, toluene, xylene, mineral oils and crude oil, look promising for bioremediation application. Finally, chemical composition, emulsifying activity and surfactant activity of the biopolymers produced by the selected strains were studies under different culture conditions. Our results showed that chemical and functional properties of the bioemulsifiers were affected by the carbon source added to the growth media.

  9. Alcaligenes faecalis: an unusual cause of skin and soft tissue infection.

    PubMed

    Tena, Daniel; Fernández, Cristina; Lago, María R

    2015-01-01

    Skin and soft tissue infection (SSTI) due to Alcaligenes faecalis is very rare and has never been studied. The aim of the present study was to investigate the clinical and microbiological characteristics of this infection. We conducted a retrospective review of 5 cases that occurred at our institution over a period of 6 years. All patients had underlying diseases, and infection was secondary to vascular disease or recent surgery in 4 of them. The most common clinical presentations were vascular ulcer infection and surgical site infection. The clinical outcome was uniformly good after treatment, except in 1 patient. In conclusion, A. faecalis should be considered a potential pathogen of SSTI, particularly in patients with vascular diseases or after surgery. The history of contact with water or aqueous solutions should be investigated in all cases. The clinical outcome is usually good, but treatment can be difficult in some cases due to the high level of resistance to commonly used antibiotics.

  10. Growth and siderophores production in Alcaligenes faecalis is regulated by metal ions.

    PubMed

    Sayyed, R Z; Chincholkar, S B

    2010-06-01

    During stationary phase of growth under low stress of iron in succinic acid medium, Alcaligenes feacalis BCCM ID 2374 produced microbial iron chelators. Increase in iron concentration supported bacterial growth but suppressed siderophores production, 1 μM and 2 μM of iron was optimum for maximum siderophore yield, i.e. 354 and 360 μg/ml in untreated and deferrated medium, respectively. Threshold level of iron, which suppressed siderophores production in A. feacalis BCCM ID 2374, was 20 μM. Ten micromoles and above concentration of CuCl(2) and CoCl(2), and 20 μM of MgCl(2), MgSO(4), ZnCl(2) and ZnSO(4) severely affected siderophores production.

  11. Tributyltin chloride (TBTCl)-enhanced exopolysaccharide and siderophore production in an estuarine Alcaligenes faecalis strain.

    PubMed

    Khanolkar, Dnyanada; Dubey, S K; Naik, Milind Mohan

    2015-05-01

    Tributyltin chloride (TBTCl) has been used extensively as an antifouling agent in ship paints, which results in the contamination of aquatic sites. These contaminated sites serve as enrichment areas for TBTCl-resistant bacterial strains. One TBTCl-resistant bacterial strain was isolated from the sediments of Zuari estuary, Goa, India, which is a major hub of various ship-building activities. Based on biochemical characteristics and 16S rDNA sequence analysis, this bacterial strain was identified as Alcaligenes faecalis and designated as strain SD5. It could degrade ≥3 mM TBTCl by using it as a sole carbon source and transform it into the less toxic dibutyltin chloride, which was confirmed by nuclear magnetic resonance and mass spectroscopy. Interestingly, this bacterial strain also showed enhanced exopolysaccharide and siderophore production when cells were exposed to toxic levels of TBTCl, suggesting their involvement in conferring resistance to this antifouling biocide as well as degradative capability respectively.

  12. A novel synthesis of iminodiacetic acid: biocatalysis by whole Alcaligenes faecalis ZJB-09133 cells from iminodiacetonitrile.

    PubMed

    Liu, Zhi-Qiang; Li, Fei-Fei; Cheng, Feng; Zhang, Tao; You, Zhong-Yu; Xu, Jian-Miao; Xue, Ya-Ping; Zheng, Yu-Guo; Shen, Yin-Chu

    2011-01-01

    Iminodiacetic acid (IDA) has been widely used as an important intermediate in the fine chemical industry. In this study, a novel synthesis route of IDA from iminodiacetonitrile by whole microorganisms was investigated. A strain with the capability of producing nitrilase, ZJB-09133, was isolated and identified, and later named Alcaligenes faecalis ZJB-09133. In addition, the detailed biocatalysis of iminodiacetonitrile to produce IDA using ZJB-09133 was investigated. The results showed that the conversion reached 65.3% in Na(2)HPO(4)-NaH(2)PO(4) buffer of pH 8.0 under the following conditions: cells in the amount of 0.075-g DCW/L, 1.5% substrate, conversion time of 8 h, and a reaction temperature of 35°C. To the best of our knowledge, this is the first time that the production of IDA using a biocatalysis method has been reported.

  13. Hypochlorite digestion method for efficient recovery of PHB from Alcaligenes faecalis.

    PubMed

    Sayyed, R Z; Gangurde, N S; Chincholkar, S B

    2009-09-01

    We reported the optimum amount of PHB accumulated by Alcaligenes faecalis during its 24 h growth under nitrogen deficient conditions. After 24 h incubation decrease in the amount of PHB was recorded. Hypochlorite digestion of biomass of organism followed by extraction with a solvent system consisting of 1:1 mixture of ethanol and acetone resulted in efficient recovery of PHB vis-à-vis earlier methods. This solvent system gave a high recovery yield, i.e. 5.6 gL(-1) vis-à-vis earlier reported yield, 1.34 gL(-1) (by same method), 0.63 gL(-1) (by chloroform extraction method) and 1.1 gL(-1) (by dispersion method).

  14. Effect of nitrogen source on curdlan production by Alcaligenes faecalis ATCC 31749.

    PubMed

    Jiang, Longfa

    2013-01-01

    This study aims to investigate the effect of nitrogen source on curdlan production by Alcaligenes faecalis ATCC 31749. Curdlan production fell when excess nitrogen source was present, while biomass accumulation increased as the level of nitrogen source raised. Curdlan production and biomass accumulation were greater with urea compared with those with other nitrogen sources. The highest production of curdlan and biomass accumulation by A. faecalis ATCC 31749 was 28.16 g L(-1) and 9.58 g L(-1), respectively, with urea, whereas those with NH(4)Cl were 15.17 g L(-1) and 6.25 g L(-1), respectively. The optimum fermentation time for curdlan production was also affected by the nitrogen source in the medium.

  15. Piggery wastewater treatment using Alcaligenes faecalis strain No. 4 with heterotrophic nitrification and aerobic denitrification.

    PubMed

    Joo, Hung-Soo; Hirai, Mitsuyo; Shoda, Makoto

    2006-09-01

    Alcaligenes faecalis strain No. 4, which has heterotrophic nitrification and aerobic denitrification abilities, was used to treat actual piggery wastewater containing high-strength ammonium under aerobic conditions. In a continuous experiment using a solids-free wastewater (SFW) mixed with feces, almost all of the 2000 NH4+ -N mg/L and 12,000 COD mg/L in the wastewater was removed and the ammonium removal rate was approximately 30 mg-N/L/h, which was 5-10 times higher than the rates achieved by other bacteria with the same abilities. The denitrification ratio was more than 65% of removed NH4+ -N, indicating that strain No. 4 exhibited its heterotrophic nitrification and aerobic denitrification abilities in the piggery wastewater.

  16. N2O and N2 production during heterotrophic nitrification by Alcaligenes faecalis strain NR.

    PubMed

    Zhao, Bin; An, Qiang; He, Yi Liang; Guo, Jin Song

    2012-07-01

    A heterotrophic nitrifier, strain NR, was isolated from a membrane bioreactor. Strain NR was identified as Alcaligenes faecalis by Auto-Microbic system and 16S rRNA gene sequence analysis. A. faecalis strain NR shows a capability of heterotrophic nitrification and N(2)O and N(2) production as well under the aerobic condition. Further tests demonstrated that neither nitrite nor nitrate could be denitrified aerobically by strain NR. However, when hydroxylamine was used as the sole nitrogen source, nitrogenous gases were detected. With an enzyme assay, a 0.063 U activity of hydroxylamine oxidase was observed, while nitrate reductase and nitrite reductase were undetectable. Thus, nitrogenous gas was speculated to be produced via hydroxylamine. Therefore, two different metabolic pathways might exist in A. faecalis NR. One is heterotrophic nitrification by oxidizing ammonium to nitrite and nitrate. The other is oxidizing ammonium to nitrogenous gas directly via hydroxylamine.

  17. Alcaligenes faecalis Cellulitis After a Dog Bite: Case Report and Literature Review.

    PubMed

    Chu, Andrew S; Harkness, Julia

    2016-01-12

    Alcaligenes faecalis is a gram-negative organism that is commonly found in the environment and may also be a part of normal fecal flora in humans. Although various infections with this bacteria have been described in the pediatric population, it has not been previously identified in infections as the pathogen after a dog bite. A case of a 19-month-old boy is presented with a cellulitis secondary to a dog bite, which failed oral antibiotic therapy, and progressed to worsening fever and swelling. The patient ultimately required hospitalization, intravenous antibiotics, and incision and drainage. The wound culture grew A. faecalis, whose identity was confirmed through recombinant DNA sequence analysis. Although it has been identified in cat bite wounds, A. faecalis has not been cited in the literature before in an infection after a dog bite.

  18. Nitrification and denitrification in high-strength ammonium by Alcaligenes faecalis.

    PubMed

    Joo, Hung-Soo; Hirai, Mitsuyo; Shoda, Makoto

    2005-06-01

    Alcaligenes faecalis sp. No. 4, that has the ability of heterotrophic nitrification and aerobic denitrification in high-strength ammonium at about 1200 mg-N/l, converted about one-half of removed NH4+-N to intracellular nitrogen and nitrified only 3% of the removed NH4+. From the nitrogen balance, 40-50% of removed NH4+-N was estimated to be denitrified. Production of N2 was confirmed by GC-MS and 90% of denitrified products was N2. The maximum ammonium removal rate, 29 mg-N/l h and its denitrification rate in aerated batch experiments, were 5-40 times higher than those of other bacteria with the same ability.

  19. Potential application of Alcaligenes faecalis strain No. 4 in mitigating ammonia emissions from dairy wastewater.

    PubMed

    Neerackal, George M; Ndegwa, Pius M; Joo, Hung-Soo; Wang, Xiang; Frear, Craig S; Harrison, Joseph H; Beutel, Marc W

    2016-04-01

    This research examined the potential mitigation of NH3 emissions from dairy manure via an enhanced aerobic bio-treatment with bacterium Alcaligenes faecalis strain No. 4. The studies were conducted in aerated batch reactors using air and pure oxygen. Aeration with air and oxygen removed approximately 40% and 100% total ammoniacal nitrogen (TAN), respectively. Intermittent oxygenation (every 2 or 4 h) reduced oxygen consumption by 95%, while attaining nearly identical TAN removal to continuous aeration. The results revealed that adequate oxygen supply and supplementing dairy wastewater with carbon are essential for this bioprocess. Based on the nitrogen mass balance, only 4% of TAN was released as NH3 gas, while the majority was retained in either the microbial biomass (58%) or converted to nitrogen gas (36%). The mass balance results reveal high potential for environmentally friendly bio-treatment of dairy wastewater using A. faecalis strain No. 4 with respect to NH3 emissions.

  20. Metal induced changes in trivalent chromium resistant Alcaligenes faecalis VITSIM2.

    PubMed

    Matilda, Shiny C; Shanthi, Chittibabu

    2017-02-20

    The changes induced in bacterial strains under stress conditions provide an insight into metal resistance strategies. Trivalent chromium resistant bacterium were isolated and identified by 16S rRNA gene sequencing and designated as Alcaligenes faecalis VITSIM2. The growth pattern was monitored. The organism also showed resistance to copper, cadmium, and certain antibiotics. The differentially expressed proteins in SDS PAGE were identified by mass spectrometry as flagellin and 50S ribosomal L36 protein. The morphological changes were identified by scanning electron microscopy. The changes in the cell wall content were estimated by peptidoglycan analysis and transformation of phosphates was detected by (31) P NMR. Flow cytometry was employed to measure the membrane integrity, esterase activity and intracellular pH. In conclusion spectrum of proteomic, physiological, and morphological alterations was observed that aid the organism to overcome chromium stress.

  1. Anaerobic taurine oxidation: a novel reaction by a nitrate-reducing Alcaligenes sp.

    PubMed

    Denger, K; Laue, H; Cook, A M

    1997-06-01

    Enrichment cultures were prepared under strictly anoxic conditions in medium representing fresh water and containing an organosulfonate as electron donor and carbon source, and nitrate as electron acceptor. The inoculum was from the anaerobic digestor of two communal sewage works. The natural organosulfonates 2-aminoethanesulfonate (taurine), DL-2-amino-3-sulfopropionate (cysteate) and 2-hydroxyethanesulfonate (isethionate) all gave positive enrichments, whereas unsubstituted alkanesulfonates, such as methanesulfonate and arenesulfonates, gave no enrichment. Two representative enrichments were used to obtain pure cultures, and strains NKNTAU (utilizing taurine) and NKNIS (utilizing isethionate) were isolated. Strain NKNTAU was examined in detail. Out of 18 tested organosulfonates, it utilized only one, taurine, and was identified as a novel Alcaligenes sp., a facultatively anaerobic bacterium. Carbon from taurine was converted to cell material and carbon dioxide. The amino group was released as ammonium ion and the sulfonate moiety was recovered as sulfate. Nitrate was reduced to nitrogen gas.

  2. Strain of alcaligenes latus bacteria used for the decomposition of polychlorinated biphenyls

    DOEpatents

    Dyadischev, Nikolai Romanovich; Zharikov, Gennady Alekseevich; Kapranov, Vladimir Vladimirovich

    2001-09-11

    Alcaligenes latus bacterial strain TXD-13 VKPM B 75-05 is capable of degrading polychlorinated biphenyls (PCBs). The strain may be employed to detoxicate environment media and PCB-containing industrial waste. To produce biomass, the strain is incubated on media which contain carbon sources, nitrogen sources and mineral salts. The strain is cultivated by a subsurface method up to a titer from 6.0.multidot.10.sup.8 to 2.0.times.10.sup.9 cells per cu cm. The produced biomass is used for degrading PCBs in concentrations from 10.sup.7 to 10.sup.8 cells per cu cm. The strain ensures from 35 to 50% reduction in PCB content in soil and water.

  3. Molecular characterization of extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase genes from Pseudomonas alcaligenes strains.

    PubMed

    Kim, Do Young; Kim, Hyun Chul; Kim, Sun Young; Rhee, Young Ha

    2005-06-01

    A bacterial strain M4-7 capable of degrading various polyesters, such as poly(epsilon-caprolactone), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyoctanoate), and poly(3-hydroxy-5-phenylvalerate), was isolated from a marine environment and identified as Pseudomonas alcaligenes. The relative molecular mass of a purified extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase (PhaZ(PalM4-7)) from P. alcaligenes M4-7 was 28.0 kDa, as determined by SDS-PAGE. The PhaZ(PalM4-7) was most active in 50 mM glycine-NaOH buffer (pH 9.0) at 35 degrees C. It was insensitive to dithiothreitol, sodium azide, and iodoacetamide, but susceptible to p-hydroxymercuribenzoic acid, N-bromosuccinimide, acetic anhydride, EDTA, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, Tween 80, and Triton X-100. In this study, the genes encoding MCL-PHA depolymerase were cloned, sequenced, and characterized from a soil bacterium, P. alcaligenes LB19 (Kim et al., 2002, Biomacromolecules 3, 291-296) as well as P. alcaligenes M4-7. The structural gene (phaZ(PalLB19)) of MCL-PHA depolymerase of P. alcaligenes LB19 consisted of an 837 bp open reading frame (ORF) encoding a protein of 278 amino acids with a deduced M((r)) of 30,188 Da. However, the MCL-PHA depolymerase gene (phaZ(PalM4-7)) of P. alcaligenes M4-7 was composed of an 834 bp ORF encoding a protein of 277 amino acids with a deduced Mr of 30,323 Da. Amino acid sequence analyses showed that, in the two different polypeptides, a substrate-binding domain and a catalytic domain are located in the N-terminus and in the C-terminus, respectively. The PhaZ(PalLB19) and the PhaZ(PalM4-7) commonly share the lipase box, GISSG, in their catalytic domains, and utilize 111Asn and 110Ser residues, respectively, as oxyanions that play an important role in transition-state stabilization of hydrolytic reactions.

  4. Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1.

    PubMed

    Sallam, Ahmed; Kalkandzhiev, Dimitar; Steinbüchel, Alexander

    2011-11-07

    Pseudomonas alcaligenes DIP1 produces an extracellular cyanophycinase (CphEal). The corresponding gene (cphEal) was identified from subclones of a genomic DNA gene library by heterologously expressing the functionally active enzyme in Escherichia coli. The nucleotide sequence of the gene (1260 base pairs) was determined indicating a theoretical mass of 43.6 kDa (mature CphEal) plus a leader peptide of 2,6 kDa which corresponds well to the apparent molecular mass of 45 kDa as revealed by SDS-PAGE. The enzyme exhibited a high sequence identity of 91% with the extracellular cyanophycinase from P. anguilliseptica strain BI and carried an N-terminal Sec secretion signal peptide. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the serine motif GXSXG plus a histidine and a glutamate residue, suggesting a catalytic mechanism similar to serine-type proteases. The cyanophycinase (CphEal) was heterologously produced in two different E. coli strains (Top10 and BL21(DE3)) from two plasmid vectors (pBBR1MCS-4 and pET-23a(+)). The signal peptide of CphEal was cleaved in E. coli, suggesting active export of the protein at least to the periplasm. Substantial enzyme activity was also present in the culture supernatants. The extracellular cyanophycinase activities in E. coli were higher than activities in the wild type P. alcaligenes DIP1 in complex LB medium. Highest extracellular enzyme production was achieved with E. coli BL21(DE3) expressing CphEal from pBBR1MCS-4. Using M9 minimal medium was less effective, but the relatively low cost of mineral salt media makes these results important for the industrial-scale production of dipeptides from cyanophycin.

  5. Purification and characterization of a novel caffeine oxidase from Alcaligenes species.

    PubMed

    Mohapatra, B R; Harris, N; Nordin, R; Mazumder, A

    2006-09-18

    Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.

  6. Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1

    PubMed Central

    2011-01-01

    Pseudomonas alcaligenes DIP1 produces an extracellular cyanophycinase (CphEal). The corresponding gene (cphEal) was identified from subclones of a genomic DNA gene library by heterologously expressing the functionally active enzyme in Escherichia coli. The nucleotide sequence of the gene (1260 base pairs) was determined indicating a theoretical mass of 43.6 kDa (mature CphEal) plus a leader peptide of 2,6 kDa which corresponds well to the apparent molecular mass of 45 kDa as revealed by SDS-PAGE. The enzyme exhibited a high sequence identity of 91% with the extracellular cyanophycinase from P. anguilliseptica strain BI and carried an N-terminal Sec secretion signal peptide. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the serine motif GXSXG plus a histidine and a glutamate residue, suggesting a catalytic mechanism similar to serine-type proteases. The cyanophycinase (CphEal) was heterologously produced in two different E. coli strains (Top10 and BL21(DE3)) from two plasmid vectors (pBBR1MCS-4 and pET-23a(+)). The signal peptide of CphEal was cleaved in E. coli, suggesting active export of the protein at least to the periplasm. Substantial enzyme activity was also present in the culture supernatants. The extracellular cyanophycinase activities in E. coli were higher than activities in the wild type P. alcaligenes DIP1 in complex LB medium. Highest extracellular enzyme production was achieved with E. coli BL21(DE3) expressing CphEal from pBBR1MCS-4. Using M9 minimal medium was less effective, but the relatively low cost of mineral salt media makes these results important for the industrial-scale production of dipeptides from cyanophycin. PMID:22060187

  7. Kerstersia gyiorum gen. nov., sp. nov., a novel Alcaligenes faecalis-like organism isolated from human clinical samples, and reclassification of Alcaligenes denitrificans Rüger and Tan 1983 as Achromobacter denitrificans comb. nov.

    PubMed

    Coenye, Tom; Vancanneyt, Marc; Cnockaert, Margo C; Falsen, Enevold; Swings, Jean; Vandamme, Peter

    2003-11-01

    A polyphasic taxonomic study was performed on nine isolates recovered from various human clinical samples. Phenotypically, these isolates resembled Alcaligenes faecalis. Whole-cell protein analysis distinguished two different species, and this was confirmed by DNA-DNA hybridizations. Cellular fatty acid analysis and 16S rDNA sequence analysis indicated that these isolates were related to the genera Alcaligenes, Bordetella, Achromobacter and Pigmentiphaga and belonged to the family Alcaligenaceae. On the basis of the results of this study, the organisms were classified in a novel genus, Kerstersia gen. nov. This genus comprises one species, Kerstersia gyiorum sp. nov. (type strain LMG 5906(T)=API 184-2-84(T)=CCUG 47000(T)), and several unnamed isolates. The DNA G+C content of members of the genus Kerstersia is between 61.5 and 62.9 mol%. On the basis of previously published DNA-DNA hybridization results and data from chemotaxonomic studies, it is proposed that Alcaligenes denitrificans Rüger and Tan 1983 be reclassified as Achromobacter denitrificans comb. nov.

  8. Delivery of a genetically marked Alcaligenes sp. to the glassy-winged sharpshooter for use in a paratransgenic control strategy.

    PubMed

    Bextine, Blake; Lauzon, Carol; Potter, Sarah; Lampe, David; Miller, Thomas A

    2004-05-01

    An artificial feeding system was designed for the glassy-winged sharpshooter (GWSS), Homalodisca coagulata Say (Hemiptera: Cicadellidae). The system, unlike previous systems, provided enough nutrients to GWSS to survive for 48 h. A system like this is a prerequisite to examining the potential use of paratransgenesis to interrupt transmission of Xylella fastidiosa, the bacterial pathogen causing Pierce's disease of grape, by insect vectors. We developed a system for short-term feeding of GWSS that allows for the introduction of bacteria in liquid medium, and we have demonstrated the ability of Alcaligenes xylosoxidans denitrificans, expressing a red fluorescent protein (dsRed), to colonize the cibarial region of the GWSS foregut for up to 5 weeks post-exposure. Alcaligenes xylosoxidans denitrificans thus occupies the same region in the foregut as the pathogen, Xylella fastidiosa.

  9. HbzF catalyzes direct hydrolysis of maleylpyruvate in the gentisate pathway of Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Liu, Kun; Liu, Ting-Ting; Zhou, Ning-Yi

    2013-02-01

    HbzF from Pseudomonas alcaligenes NCIMB 9867 was purified to homogeneity as a His-tagged protein and likely a dimer by SDS-PAGE and gel filtration. This protein was demonstrated to be a novel maleylpyruvate hydrolase, catalyzing direct hydrolysis of maleylpyruvate to maleate and pyruvate, and belongs to the fumarylacetoacetate hydrolase superfamily. This study reveals the genetic determinate for the direct maleylpyruvate hydrolysis in the gentisate pathway, complementary to the well-studied maleylpyruvate isomerization route.

  10. Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor.

    PubMed

    Rehfuss, Marc; Urban, James

    2005-07-01

    A Gram (-) coccobacillary bacterium, J(T), was isolated from a graywater bioprocessor. 16S rRNA and biochemical analysis has revealed strain J(T) closely resembles Alcaligenes faecalis ATCC 8750T and A. faecalis subsp. parafaecalis DSM 13975T, but is a distinct, previously uncharacterized isolate. Strain J(T), along with the type strain of A. faecalis and its previously described subspecies share the ability to aerobically degrade phenol. The degradation rates of phenol for strain J(T) and reference phenol degrading bacteria were determined by photometrically measuring the change in optical density when grown on 0.1% phenol as the sole carbon source, followed by addition of Gibb's reagent to measure depletion of substrate. The phenol degradation rates of strain J(T) was found to exceed that of the phenol hydroxylase group III bacterium Pseudomonas pseudoalcaligenes, with isolate J(T) exhibiting a doubling time of 4.5 h. The presence of the large subunit of the multicomponent phenol hydroxylase gene in strain J(T) was confirmed by PCR. The presence of the nirK nitrite reductase gene as demonstrated by PCR as well as results obtained from nitrite media indicated denitrification at least to N2O. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA DNA hybridization, we propose assigning a novel subspecies of Alcaligenes faecalis, to be named Alcaligenes faecalis subsp. phenolicus with the type strain J(T) (= DSM 16503) (= NRRL B-41076).

  11. Structure of the 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP

    SciTech Connect

    Keegan, R.; Lebedev, A.; Erskine, P.; Guo, J.; Wood, S. P.; Hopper, D. J.; Rigby, S. E. J.; Cooper, J. B.

    2014-09-01

    The first X-ray structure of a 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP at a resolution of 2.2 Å is reported. This structure establishes that the enzyme adopts the cupin-fold, forming compact dimers with a pronounced hydrophobic interface between the monomers. Each monomer possesses a catalytic ferrous iron that is coordinated by three histidines (76, 78 and 114) and an additional ligand which has been putatively assigned as a carbonate, although formate and acetate are possibilities. The enzyme 2, 4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2, 4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in

  12. Alcaligenes aquatilis sp. nov., a novel bacterium from sediments of the Weser Estuary, Germany, and a salt marsh on Shem Creek in Charleston Harbor, USA.

    PubMed

    Van Trappen, Stefanie; Tan, Tjhing-Lok; Samyn, Emly; Vandamme, Peter

    2005-11-01

    Four nitrite-dissimilating strains, isolated from Weser Estuary sediments, were investigated using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belong to the 'Betaproteobacteria' and are related to the genus Alcaligenes. The highest level of sequence similarity (100 %) was found with strain M3A (=ATCC 700596), a dimethyl sulfide-producing marine isolate that was included in this study. DNA-DNA hybridizations between the five strains and related Alcaligenes faecalis strains confirmed that the former belong to a single and novel species within the genus Alcaligenes. The isolates are Gram-negative, motile, rod-shaped cells with a DNA G+C content of about 56 mol%. The whole-cell fatty acid profiles of the isolates were very similar and included C(16 : 0), C(17 : 0) cyclo, C(18 : 1)omega7c, summed feature 2 (comprising any combination of C(12 : 0) aldehyde, an unknown fatty acid of equivalent chain length 10.928, C(16 : 1) iso I and C(14 : 0) 3-OH) and summed feature 3 (C(15 : 0) iso 2-OH and/or C(16 : 1)omega7c) as the major fatty acid components. On the basis of their phylogenetic, genomic and phenotypic properties, the five novel strains can be assigned to the genus Alcaligenes as a novel species, for which the name Alcaligenes aquatilis sp. nov. is proposed. The type strain is LMG 22996T (=CCUG 50924T).

  13. Improved welan gum production by Alcaligenes sp. ATCC31555 from pretreated cane molasses.

    PubMed

    Ai, Hongxia; Liu, Min; Yu, Pingru; Zhang, Shaozhi; Suo, Yukai; Luo, Ping; Li, Shuang; Wang, Jufang

    2015-09-20

    Welan gum production by Alcaligenes sp. ATCC31555 from cane molasses was studied in batch fermentation to reduce production costs and enhance gum production. The pretreatment of cane molasses, agitation speed and the addition of supplements were investigated to optimize the process. Sulfuric acid hydrolysis was found to be the optimal pretreatment, resulting in a maximum gum concentration of 33.5 g/L, which is 50.0% higher than those obtained from the molasses' mother liquor. Agitation at 600 rpm at 30°C and addition of 10% n-dodecane following fermentation for 36 h increased the maximum gum production up to 41.0 ± 1.41 g/L, which is 49.1% higher than the greatest welan gum concentration in the literature so far. The welan gum product showed an acceptable molecular weight, similar rheological properties and better thermal stability to that obtained from glucose. These results indicate that cane molasses may be a suitable and inexpensive substrate for cost-effective industrial-scale welan gum production.

  14. Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil

    PubMed Central

    Castignetti, Domenic; Hollocher, Thomas C.

    1982-01-01

    Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (Vmax) of 0.066 and 0.003 μmol of N × min−1 × mg of protein−1, respectively, at 22°C. Km values were 15 and 42 μM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion. PMID:16346117

  15. The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.

    PubMed

    Anderson, G L; Williams, J; Hille, R

    1992-11-25

    The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.

  16. Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.

    PubMed Central

    Verhaert, R M; Riemens, A M; van der Laan, J M; van Duin, J; Quax, W J

    1997-01-01

    Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge. PMID:9292993

  17. Efficient enzymatic synthesis of ampicillin by mutant Alcaligenes faecalis penicillin G acylase.

    PubMed

    Deng, Senwen; Su, Erzheng; Ma, Xiaoqiang; Yang, Shengli; Wei, Dongzhi

    2015-04-10

    Semi-synthetic β-lactam antibiotics (SSBAs) are one of the most important antibiotic families in the world market. Their enzymatic synthesis can be catalyzed by penicillin G acylases (PGAs). In this study, to improve enzymatic synthesis of ampicillin, site-saturating mutagenesis was performed on three conserved amino acid residues: βF24, αR146, and αF147 of thermo-stable penicillin G acylase from Alcaligenes faecalis (Af PGA). Four mutants βF24G, βF24A, βF24S, and βF24P were recovered by screening the mutant bank. Kinetic analysis of them showed up to 800-fold increased kcat/Km value for activated acyl donor D-phenylglycine methyl ester (D-PGME). When βF24G was used for ampicillin synthesis under kinetic control at industrially relevant conditions, 95% of nucleophile 6-aminopenicillanic acid (6-APA) was converted to ampicillin in aqueous medium at room temperature while 12% process time is needed to reach maximum product accumulation at 25% enzyme concentration compared with the wild-type Af PGA. Consequently, process productivity of enzymatic synthesis of ampicillin catalyzed by Af PGA was improved by more than 130 times, which indicated an enzyme viable for efficient SSBAs synthesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene.

    PubMed

    Cox, M; Gerritse, G; Dankmeyer, L; Quax, W J

    2001-03-09

    Pseudomonas alcaligenes secretes a lipase with a high pH optimum, which has interesting properties for application in detergents. The expression of the lipase is strongly dependent on the presence of lipids in the growth medium such as soybean oil. The promoter of the gene was characterized and found to have resemblance to sigma54 controlled promoters, which are known to be tightly regulated. The transcription start was mapped precisely downstream of a sequence with close similarity to the -12/-24 consensus sequence of sigma54 controlled promoters. Interestingly, a hyperproducer mutant strain was isolated and found to have a C to T mutation in the -12/-24 promoter consensus region. In addition an Upstream Activating Sequence (UAS) with homology to sigma54 UAS consensus sequences was identified. It was demonstrated that an increase of the distance from the UAS to the transcription start or the deletion of the UAS results in significantly lower expression levels of lipase. A systematic mutational analysis of the UAS sequence has resulted in a variant with an increased lipase expression.

  19. Characterization of hbzE-encoded gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Yeo, Chew Chieng; Tan, Chew Ling; Gao, Xiaoli; Zhao, Bing; Poh, Chit Laa

    2007-09-01

    Pseudomonas alcaligenes NCIMB 9867 (strain P25X) is known to synthesize two isofunctional gentisate 1,2-dioxygenases (GDO; EC 1.13.11.4) as well as other enzymes involved in the degradation of xylenols and cresols via the gentisate pathway. The hbzE gene encoding what is possibly the strictly inducible gentisate 1,2-dioxygenase II (GDO-II) was cloned, overexpressed and purified as a hexahistidine fusion protein from Escherichia coli. Active recombinant GDO-II had an estimated molecular mass of 150kDa and is likely a tetrameric protein with a subunit mass of approximately 40kDa, similar to the previously characterized gentisate 1,2-dioxygenase I (GDO-I) encoded by xlnE. However, GDO-II was unable to utilize gentisate that is substituted at the carbon-4 position, unlike GDO-I which had broader substrate specificity. GDO-II also possessed different kinetic characteristics when compared to GDO-I. The hbzE-encoded GDO-II shared higher sequence identities (53%) with GDOs from Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2, compared with only 35% identity with the xlnE-encoded GDO-I. The hbzE gene was found to be part of a cluster of nine genes including the putative regulatory gene designated hbzR, which encodes an LysR-type regulator and is divergently transcribed from the other genes of the hbzHIJKLFED cluster.

  20. Wheat Bran Enhances the Cytotoxicity of Immobilized Alcaligenes aquatilis F8 against Microcystis aeruginosa

    PubMed Central

    Sun, Pengfei; Lin, Hui; Wang, Guan; Zhang, Ximing; Zhang, Qichun; Zhao, Yuhua

    2015-01-01

    Algicidal bacteria offer a promising option for killing cyanobacteria. Therefore, a new Alcaligenes aquatilis strain F8 was isolated to control Microcystis aeruginosa in this study. The algicidal activity of strain F8 was dependent on the cell density of M. aeruginosa, and the maximal algicidal rate of the free bacterium reached 88.45% within 72 h. With a view to its application to the control of M. aeruginosa in the natural environment, strain F8 was immobilized in sodium alginate beads, but immobilization of the strain decreased its algicidal rate compared to that of the free bacterium. However, addition of wheat bran to the sodium alginate matrix used to immobilize strain F8 not only eliminated the adverse effects of immobilization on the bacteria but also resulted in an 8.83% higher algicidal rate of the immobilized than free bacteria. Exclusion and recovery methods were used to identify key ingredients of wheat bran and gain insight into the mechanism underlying the observed enhancement of algicidal activity. This analysis indicated that certain factors in wheat bran, including vitamins B1, B2, B9, and E were responsible for promoting bacterial growth and thereby improving the algicidal rate of immobilized strain F8. Our findings indicate that wheat bran is able to improve the algicidal efficiency of A. aquatilis strain F8 for killing M. aeruginosa and is a good source of not only carbon and nitrogen but also vitamins for bacteria. PMID:26295573

  1. Characterization of Alcaligenes faecalis strain AD15 indicating biocontrol activity against plant pathogens.

    PubMed

    Yokoyama, Shin-ichiro; Adachi, Yoshitomi; Asakura, Shuichi; Kohyama, Erina

    2013-01-01

    Bacterial strain possessing both bacteriostatic and fungistatic activity (biocontrol activity) against pathogens of cyclamen (Cyclamen sp.) was isolated from the soil in Gifu Prefecture, Japan, and characterized with respect to its taxonomic and biocontrol properties. The sequence of its 16S rRNA gene, morphology, biochemistry, and fatty acid composition demonstrated that it is a strain most closely related to Alcaligenes faecalis subsp. faecalis LMG 1229(T). The isolate was named A. faecalis strain AD15. A. faecalis AD15 produced hydroxylamine at maximum yields of 33.3±1.7 mg/L after 16 h cultivation in LB medium and 19.0±0.44 mg/L after 19 h cultivation in synthetic medium. Moreover, minimum inhibitory concentrations of hydroxylamine against the cyclamen pathogens Pantoea agglomerans and Colletotrichum gloeosporioides were 4.20±0.98 and 16.5±0.67 mg/L. These results indicated that the biocontrol activity of strain AD15 might be attributed to hydroxylamine, a metabolite in the culture medium, and it had the potential for biopesticide application.

  2. [Influence of pH control on the production of curdlan by Alcaligenes faecalis strain].

    PubMed

    Wang, Lei; Zhan, Xiao-Bei; Zhu, Yi-Hui; Li, Zhen-Yu; Yang, Ye

    2002-09-01

    A two-stage pH control method was employed in batch fermentation of curdlan by Alcaligenes faecalis WX-C12 where cell-growth stage was constantly controlled at pH 7.0 and stationary stage was controlled at a constant pH as well. The influence of pH control on the curdlan production was investigated. The optimal pH control of batch process for curdlan production was obtained when cell-growth stage was controlled at pH 7.0 and stationary stage was constantly controlled at pH 5.6. Production and productivity of curdlan, QP and YP/S reached 28.19 g/L, 291 mg/(L.h), 132.27 mg/(L.h.g) and 0.659, an improvement of 20.4%, 38.1%, 38.1% and 29.5% compared to a pH uncontrolled operation respectively.

  3. Biodegradation of nicosulfuron by a novel Alcaligenes faecalis strain ZWS11.

    PubMed

    Zhao, Weisong; Wang, Chen; Xu, Li; Zhao, Chunqing; Liang, Hongwu; Qiu, Lihong

    2015-09-01

    A bacterial strain ZWS11 was isolated from sulfonylurea herbicide-contaminated farmland soil and identified as a potential nicosulfuron-degrading bacterium. Based on morphological and physicochemical characterization of the bacterium and phylogenetic analysis of the 16S rRNA sequence, strain ZWS11 was identified as Alcaligenes faecalis. The effects of the initial concentration of nicosulfuron, inoculation volume, and medium pH on degradation of nicosulfuron were investigated. Strain ZWS11 could degrade 80.56% of the initial nicosulfuron supplemented at 500.0mg/L under the conditions of pH7.0, 180r/min and 30°C after incubation for 6days. Strain ZWS11 was also capable of degrading rimsulfuron, tribenuron-methyl and thifensulfuron-methyl. Four metabolites from biodegradation of nicosulfuron were identified, which were 2-aminosulfonyl-N, N-dimethylnicotinamide (M1), 4, 6-dihydroxypyrimidine (M2), 2-amino-4, 6-dimethoxypyrimidine (M3) and 2-(1-(4,6-dimethoxy-pyrimidin-2-yl)-ureido)-N,N-dimethyl-nicotinamide (M4). Among the metabolites detected, M2 was reported for the first time. Possible biodegradation pathways of nicosulfuron by strain ZWS11 were proposed. The degradation proceeded mainly via cleavage of the sulfonylurea bridge, O-dealkylation, and contraction of the sulfonylurea bridge by elimination of a sulfur dioxide group. The results provide valuable information for degradation of nicosulfuron in contaminated environments.

  4. The crystal structure of cobalt-substituted pseudoazurin from Alcaligenes faecalis.

    PubMed

    Gessmann, Renate; Kyvelidou, Christiana; Papadovasilaki, Maria; Petratos, Kyriacos

    2011-03-01

    The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo-protein, followed by addition of an aqueous solution of CoCl(2). Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P6(5), with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X-ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co-substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86-Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40-Nδ(1), Cys78-Sγ, His81-Nδ(1), and 86Met-Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)-substituted pseudoazurin.

  5. Mutant AFM 2 of Alcaligenes faecalis for phenol biodegradation using He-Ne laser irradiation.

    PubMed

    Jiang, Yan; Wen, Jianping; Caiyin, Qinggele; Lin, Liangcai; Hu, Zongding

    2006-11-01

    He-Ne laser technology was utilized in this study to investigate the response of Alcaligenes faecalis to laser stimulation. The irradiation experiments were conducted by the adjustment of the output power from 5 to 25 mW and the exposure time from 5 to 25 min. The results showed that the survival rate changed regularly with the variety of irradiation dose, and high positive mutation frequency was determined by both the energy density and the output power. The mutant strain AFM 2 was obtained. Phenol biodegradation assay demonstrated that AFM 2 possessed a more prominent phenol-degrading potential than its parent strain, which presumably attributed to the improvements of phenol hydroxylase and catechol 1,2-dioxygenase activities. The phenol of 2000 mgl(-1) was completely degraded by AFM 2 within 85.5h at 30 degrees C. In addition, the cell growth and phenol degradation kinetics of the mutant strain AFM 2 and its parent strain in batch cultures were also investigated at the wide initial phenol concentration ranging from 0 to 2000 mgl(-1) by Haldane model. The results of these experiments further demonstrated that the mutant strain AFM 2 possessed a higher capacity to resist phenol.

  6. Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis.

    PubMed

    Varshney, Nishant Kumar; Kumar, R Suresh; Ignatova, Zoya; Prabhune, Asmita; Pundle, Archana; Dodson, Eleanor; Suresh, C G

    2012-03-01

    The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C222(1), with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 , and P4(1)2(1)2, with unit-cell parameters a = b = 85.6, c = 298.8 . Data were collected at 293 and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G cylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme.

  7. Alcaligenes faecalis kw-a biofilm for denitrification of nitrate-rich effluent.

    PubMed

    Jadhav, T S; Faldu, N J; Patel, P; Narolkar, S N; Nerurkar, A S

    2005-06-01

    Alcaligenes faecalis kw-A selected for possessing good denitrification efficiency was used for biofilm development. The biofilm could be developed on a glass surface within 12 hr when 5%, Ix 10(8) cells/ml was used as inoculum. The microcolonies were seen in 6 hr and glycocalyx in 9 hr stage. At 24 hr the biofilm was developed fully and hence was visualised as dense mass. The biofilm protein content showed 48.5% increase in shake flask than in static condition. The exopoplymer is produced in larger amounts in biofilm as compared to the suspended cells. Also, its amount was more by 43% in the biofilm produced in shake flask condition than in static condition. The biofilm could remove 95% nitrate from nitrate-rich effluent in a bench-scale process in 36 hr. The attached growth technique demonstrated here can be utilised to study the effect of favourable as well as adverse conditions on the denitrification efficiency of a culture. The ultimate application of a denitrifying biofilm would be in attached growth or biofilm reactor.

  8. Colonization of Alcaligenes faecalis strain JBW4 in natural soils and its detoxification of endosulfan.

    PubMed

    Kong, Lingfen; Zhu, Shaoyuan; Zhu, Lusheng; Xie, Hui; Wei, Kai; Yan, Tongxiang; Wang, Jun; Wang, Jinhua; Wang, Fenghua; Sun, Fengxia

    2014-02-01

    Alcaligenes faecalis strain JBW4, a strain of bacteria that is capable of degrading endosulfan, was inoculated into sterilized and natural soils spiked with endosulfan. JBW4 degraded 75.8 and 87.0 % of α-endosulfan and 58.5 and 69.5 % of β-endosulfan in sterilized and natural soils, respectively, after 77 days. Endosulfan ether and endosulfan lactone were the major metabolites that were detected by gas chromatography-mass spectrometry. This result suggested that A. faecalis strain JBW4 degrades endosulfan using a non-oxidative pathway in soils. The ability of strain JBW4 to colonize endosulfan-contaminated soils was confirmed by polymerase chain reaction-denaturing gradient gel electrophoresis. This result suggested that strain JBW4 competed with the original inhabitants in the soil to establish a balance and successfully colonize the soils. In addition, the detoxification of endosulfan by strain JBW4 was evaluated using single-cell gel electrophoresis and by determining the soil microbial biomass carbon and enzymatic activities. The results showed that the genotoxicity and ecotoxicity of endosulfan in soil were reduced after degradation. The natural degradation of endosulfan in soil is inadequate; therefore, JBW4 shows potential for the bioremediation of industrial soils that are contaminated with endosulfan residues.

  9. Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor.

    PubMed

    Ju, Shouyong; Lin, Jian; Zheng, Jinshui; Wang, Shaoying; Zhou, Hongying; Sun, Ming

    2016-01-29

    Root knot nematodes (RKNs) are the world's most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs.

  10. Enzymatic properties of immobilized Alcaligenes faecalis cells with cell-associated beta-glucosidase activity

    SciTech Connect

    Wheatly, M.A.; Phillips, C.R.

    1984-06-01

    Enzymatic properties of Alcaligenes faecalis cells immobilized in polyacrylamide were characterized and compared with those reported for the extracted enzyme, and with those measured for free cells. Many of the properties reflected those of the extracted enzyme rather than those measured in the free whole cells prior to immobilization, suggesting cell disruption during immobilization. These properties included the pH activity profile, a slightly broader pH stability profile, and the activation energy. Electron micrographs showed evidence of cell debris among the polymer matrix. The immobilized cells were not viable, and did not consume glucose. Thermal stability was less after immobilization with a half-line of 16 h at 45 degrees C, and 3.5 h at 50 degrees C. The immobilized preparation was more stable when stored lyophilized rather than in buffer, losing 23 and 52% activity, respectively, after six months. The enzyme was irreversibly inhibited by both acetate and citrate buffers. If the immobilized enzyme is to be used in conjunction with cellulases from Trichoderma reesei for cellulase saccharification, the optimal conditions would be pH 5.5 and 45 degrees C in a buffer containing no carboxylic acid groups.

  11. Aspartate beta-decarboxylase from Alcaligenes faecalis: carbon-13 kinetic isotope effect and deuterium exchange experiments

    SciTech Connect

    Rosenberg, R.M.; O'Leary, M.H.

    1985-03-26

    The authors have measured the /sup 13/C kinetic isotope effect at pH 4.0, 5.0, 6.0, and 6.5 and in D/sub 2/O at pH 5.0 and the rate of D-H exchange of the alpha and beta protons of aspartic acid in D/sub 2/O at pH 5.0 for the reaction catalyzed by the enzyme aspartate beta-decarboxylase from Alcaligenes faecalis. The /sup 13/C kinetic isotope effect, with a value of 1.0099 +/- 0.0002 at pH 5.0, is less than the intrinsic isotope effect for the decarboxylation step, indicating that the decarboxylation step is not entirely rate limiting. The authors have been able to estimate probable values of the relative free energies of the transition states of the enzymatic reaction up to and including the decarboxylation step from the /sup 13/C kinetic isotope effect and the rate of D-H exchange of alpha-H. The pH dependence of the kinetic isotope effect reflects the pKa of the pyridine nitrogen of the coenzyme pyridoxal 5'-phosphate but not that of the imine nitrogen. A mechanism is proposed for the exchange of aspartate beta-H that is consistent with the stereochemistry suggested earlier.

  12. Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study.

    PubMed

    Singh, Renu; Banerjee, Anirban; Kaul, Praveen; Barse, Brajesh; Banerjee, U C

    2005-10-01

    Nitrilases constitute an important class of hydrolases, however, cheap and ready availability of enzyme sources limit their practical synthetic applications. The present investigation was directed to compare the applicability of various physical cell disintegration methods namely, solid shear, liquid shear and sonication, for the release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126. Different parameters associated with each method were optimized in order to ensure maximal release of active nitrilase. The methods were also compared under optimal conditions for their efficiency of nitrilase release and extent of cell disruption, and enzyme release were visualized under a differential interference contrast microscope (DIC) and SDS-PAGE, respectively. Maximum release of the enzyme protein from the cells was observed in case of liquid shear method employing high-pressure homogenization, however, the specific activity of nitrilase was highest in cell-free extract (CFE) generated by sonication. Both the solid shear and liquid shear proved to be equally effective for maximum release of intracellular enzymes, however, from the specific activity point of view, sonication was found to be a better one compared to other two methodologies. The generated cell-free extract can be further employed for the production of enantiopure chiral carboxylic acids, which are important chiral building blocks.

  13. Fragments of pro-peptide activate mature penicillin amidase of Alcaligenes faecalis.

    PubMed

    Kasche, Volker; Galunsky, Boris; Ignatova, Zoya

    2003-12-01

    Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (kcat/Km) for the hydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin amidase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number (kcat).

  14. Kinetic characteristics and modelling of growth and substrate removal by Alcaligenes faecalis strain NR.

    PubMed

    Chen, Jie; Zhao, Bin; An, Qiang; Wang, Xia; Zhang, Yi Xin

    2016-04-01

    Alcaligenes faecalis strain NR has the capability of simultaneous ammonium and organic carbon removal under sole aerobic conditions. The growth and substrate removal characteristics of A. faecalis strain NR were studied and appropriate kinetic models were developed. The maximum substrate removal rate of NH4 (+)-N and TOC were determined as 2.27 mg NH4 (+)-N/L/h and 30.00 mg TOC/L/h, respectively with initial NH4 (+)-N = 80 mg/L and TOC = 800 mg/L. Single-substrate models and double-substrate models based on Monod, Contois, Moser and Teissier were employed to describe the bioprocess kinetic coefficients. As a result, two double-substrate models, Teissier-Contois and Contois-Contois, were considered to be appropriate to model growth kinetics with both NH4 (+)-N and TOC as limiting substrates. The kinetic constants of maximum growth rate (μ max) and half-saturation constant (K S and B S) were obtained by solving multiple equations with regression. This work can be used to further understand and predict the performance of heterotrophic nitrifiers, and thus provides specific guidance of these functional strains in practical wastewater treatment process.

  15. Purification and characterization of Alcaligenes faecalis penicillin G acylase expressed in Bacillus subtilis.

    PubMed

    Zhou, Zheng; Zhou, Li-Ping; Chen, Mei-Juan; Zhang, Yan-Liang; Li, Ren-Bao; Yang, Sheng; Yuan, Zhong-Yi

    2003-05-01

    The Alcaligenes faecalis PGA gene encoding heterodimeric penicillin G acylase (PGA) was cloned and successfully expressed in Escherichia coli and Bacillus subtilis respectively. In contrast to E.coli hosts where the enzymes were retained in the periplasm, B. subtilis cell secreted the recombinant enzyme into the medium. Contrary to limited expression yield of E. coli (pETAPGA), PGA extracellularly expressed by B. subtilis (pBAPGA) and B. subtilis (pMAPGA) reached the highest yield of 653 u/L. This yield increased 109-fold higher than the native expression of A. faecalis CICC AS1.767. The enzyme was fractionated with (NH(4))(2)SO(4) and purified by DEAE-Sepharose CL-6B with a yield of 81%. The purified enzyme had a specific activity of 1.469 u/mg. Furthermore, some enzyme characteristics, such as the pH and temperature optimum, the stability against organic solvent and the ratio of cepholexin synthesis to hydrolysis were determined. By overexpressing A. faecalis PGA in B. subtilis and purifying secreted enzyme from culture medium one could readily obtain a large amount of an alternative source of PGA.

  16. Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis

    PubMed Central

    Varshney, Nishant Kumar; Suresh Kumar, R.; Ignatova, Zoya; Prabhune, Asmita; Pundle, Archana; Dodson, Eleanor; Suresh, C. G.

    2012-01-01

    The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C2221, with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 Å, and P41212, with unit-cell parameters a = b = 85.6, c = 298.8 Å. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-­terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme. PMID:22442220

  17. Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor

    PubMed Central

    Ju, Shouyong; Lin, Jian; Zheng, Jinshui; Wang, Shaoying; Zhou, Hongying

    2016-01-01

    Root knot nematodes (RKNs) are the world's most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs. PMID:26826227

  18. Biodegradation of Picolinic Acid by a Newly Isolated Bacterium Alcaligenes faecalis Strain JQ135.

    PubMed

    Qiu, Jiguo; Zhang, Junjie; Zhang, Yanting; Wang, Yuhong; Tong, Lu; Hong, Qing; He, Jian

    2017-04-01

    We isolated a bacterial strain JQ135 from municipal wastewater, which was capable of efficiently degrading picolinic acid (PA). Based on the physico-biochemical characteristics and 16S rDNA analysis, strain JQ135 was identified as Alcaligenes faecalis. In addition, strain JQ135 produced an orange pigment when cultured in the Luria-Bertani medium, which is different from the previously reported strains of A. faecalis. During the degradation of PA by the resting strain JQ135 cells, only one intermediate, 6-hydroxypicolinic acid (6HPA), was detected by ultraviolet spectrophotometry, high-pressure liquid chromatography, and liquid chromatography-mass spectrometry. A random transposon mutagenesis library of strain JQ135 was constructed. One mutant, Mut-G31, could convert PA into 6HPA without further degradation. The disrupted gene (orf2) was amplified from Mut-G31, and its product showed 32% identity to the 3-deoxy-D-manno-octulosonic acid kinase (KdkA) from Haemophilus influenzae. Results from complementation analysis confirmed that GTG was the initiation codon of the kdkA-like orf2, and that it was essential for PA biodegradation by strain JQ135. This study provides the first genetic evidence for the bacterial degradation of PA.

  19. Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes.

    PubMed

    Meyners, Christian; Baud, Matthias G J; Fuchter, Matthew J; Meyer-Almes, Franz-Josef

    2014-03-01

    Thermodynamic studies on ligand-protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4-7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH(0)/ΔG(0). The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design.

  20. Antimicrobial activity of Alcaligenes sp. HPC 1271 against multidrug resistant bacteria.

    PubMed

    Kapley, Atya; Tanksale, Himgouri; Sagarkar, Sneha; Prasad, A R; Kumar, Rathod Aravind; Sharma, Nandita; Qureshi, Asifa; Purohit, Hemant J

    2016-01-01

    Alcaligenes sp. HPC 1271 demonstrated antibacterial activity against multidrug resistant bacteria, Enterobacter sp., resistant to sulfamethoxazole, ampicillin, azithromycin, and tetracycline, as well as against Serratia sp. GMX1, resistant to the same antibiotics with the addition of netilmicin. The cell-free culture supernatant was analyzed for possible antibacterials by HPLC, and the active fraction was further identified by LC-MS. Results suggest the production of tunicamycin, a nucleoside antibiotic. The draft genome of this bacterial isolate was analyzed, and the 4.2 Mb sequence data revealed six secondary metabolite-producing clusters, identified using antiSMASH platform as ectoine, butyrolactone, phosphonate, terpene, polyketides, and nonribosomal peptide synthase (NRPS). Additionally, the draft genome demonstrated homology to the tunicamycin-producing gene cluster and also defined 30 ORFs linked to protein secretion that could also play a role in the antibacterial activity observed. Gene expression analysis demonstrated that both NRPS and dTDP-glucose 4,6-dehydratase gene clusters are functional and could be involved in antibacterial biosynthesis.

  1. Purification and properties of the membrane-bound hydrogenase from N2-fixing Alcaligenes latus.

    PubMed

    Pinkwart, M; Schneider, K; Schlegel, H G

    1983-06-29

    The nitrogen-fixing, aerobic hydrogen-oxidizing bacterium Alcaligenes latus forms hydrogenase when growing lithoautotrophically with hydrogen as electron donor and carbon dioxide as sole carbon source or when growing heterotrophically with N2 as sole nitrogen source. The hydrogenase is membrane-bound and relatively oxygen-sensitive. The enzymes formed under both conditions are identical on the basis of the following criteria: molecular mass, mobility in polyacrylamide gel electrophoresis, Km value for hydrogen (methylene blue reduction), stability properties, localization, and cross-reactivity to antibodies raised against the 'autotrophic' hydrogenase. The hydrogenase was solubilized by Triton X-100 and deoxycholate treatment and purified by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose C1-4B, DEAE-Sephacel and Matrix Gel Red A under hydrogen to homogeneity to a specific activity of 113 mumol H2 oxidized/min per mg protein (methylene blue reduction). SDS gel electrophoresis revealed two nonidentical subunits of molecular weights of 67 000 and 34 000, corresponding to a total molecular weight of 101 000. The pure enzyme was able to reduce FAD, FMN, riboflavin, flavodoxin isolated from Megasphaera elsdenii, menadione and horse heart cytochrome c as well as various artificial electron acceptors. The reversibility of the hydrogenase function was demonstrated by H2 evolution from reduced methyl viologen.

  2. Alcaligenes defragrans sp. nov., description of four strains isolated on alkenoic monoterpenes ((+)-menthene, alpha-pinene, 2-carene, and alpha-phellandrene) and nitrate.

    PubMed

    Foss, S; Heyen, U; Harder, J

    1998-06-01

    Four pseudomonad strains 51Men, 54Pin, 62Car and 65Phen were recently isolated on the monoterpenes (+)-menthene, alpha-pinene, 2-carene and alpha-phellandrene as sole carbon source and nitrate as electron acceptor. These bacteria were characterised. The motile, mesophilic, Gram-negative rods had a strictly respiratory metabolism. Monoterpenes as carbon sources were completely mineralised to carbon dioxide. The physiology of all strains was very similar, but displayed an individual utilisation preference for the isolation substrate. The fatty acid composition of whole cells showed a high degree of similarity to that of Alcaligenes faecalis. Comparative 16S rDNA data analysis placed the isolates into the beta-subclass of Proteobacteria in a common offshoot together with Alcaligenes and Bordetella species. On the basis of these characteristics, the strains are described as a new species belonging to the genus Alcaligenes, A. defragrans sp. nov., with strain 54Pin (DSM 12141T) as type strain.

  3. Maintenance and induction of naphthalene degradation activity in Pseudomonas putida and an Alcaligenes sp. under different culture conditions.

    PubMed

    Guerin, W F; Boyd, S A

    1995-11-01

    The expression of xenobiotic-degradative genes in indigenous bacteria or in bacteria introduced into an ecosystem is essential for the successful bioremediation of contaminated environments. The maintenance of naphthalene utilization activity is studied in Pseudomonas putida (ATCC 17484) and an Alcaligenes sp. (strain NP-Alk) under different batch culture conditions. Levels of activity decreased exponentially in stationary phase with half-lives of 43 and 13 h for strains ATCC 17484 and NP-Alk, respectively. Activity half-lives were 2.7 and 5.3 times longer, respectively, in starved cultures than in stationary-phase cultures following growth on naphthalene. The treatment of starved cultures with chloramphenicol caused a loss of activity more rapid than that measured in untreated starved cultures, suggesting a continued enzyme synthesis in starved cultures in the absence of a substrate. Following growth in nutrient medium, activity decreased to undetectable levels in the Alcaligenes sp. but remained at measurable levels in the pseudomonad even after 9 months. The induction of naphthalene degradation activities in these cultures, when followed by radiorespirometry with 14C-labeled naphthalene as the substrate, was consistent with activity maintenance data. In the pseudomonad, naphthalene degradation activity was present constitutively at low levels under all growth conditions and was rapidly (in approximately 15 min) induced to high levels upon exposure to naphthalene. Adaptation in the uninduced Alcaligenes sp. occurred after many hours of exposure to naphthalene. In vivo labeling with 35S, to monitor the extent of de novo enzyme synthesis by naphthalene-challenged cells, provided an independent confirmation of the results.

  4. Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Zhao, Bing; Yeo, Chew Chieng; Poh, Chit Laa

    2005-05-01

    Pseudomonas alcaligenes NCIMB 9867 (strain P25X) utilizes the gentisate pathway for the degradation of aromatic hydrocarbons. The gene encoding the alternative sigma (sigma) factor sigma(54), rpoN, was cloned from strain P25X and a rpoN knock-out strain, designated G54, was constructed by insertional inactivation with a kanamycin resistance gene cassette. The role of sigma(54) in the physiological response of P. alcaligenes P25X to gentisate induction was assessed by comparing the global protein expression profiles of the wild-type P25X with the rpoN mutant strain G54. Analysis of two-dimensional polyacrylamide gel electrophoresis gels showed that 39 out of 355 prominent protein spots exhibited differential expression as a result of the insertional inactivation of rpoN. Identification of the protein spots by matrix-assisted laser desorption/ionization-time of flight/time of flight revealed a wide diversity of proteins that are affected by the sigma(54) mutation, the largest group being proteins that are involved in carbon metabolism. The strictly inducible gentisate 1,2-dioxygenase, one of two isofunctional copies of the key enzyme in the gentisate pathway, and enzymes of the TCA cycle, pyruvate metabolism and gluconeogenesis were part of this group. Other proteins that are part of the sigma(54) regulon include enzymes implicated in nitrogen metabolism, transport proteins, stress-response proteins and proteins involved in cell motility. The results of this study showed that sigma(54) plays a global regulatory role in the expression of a wide variety of genes in P. alcaligenes, including the wild-type response to the presence of the aromatic inducer, gentisate.

  5. Maintenance and induction of naphthalene degradation activity in Pseudomonas putida and an Alcaligenes sp. under different culture conditions

    SciTech Connect

    Guerin, W.F.; Boyd, S.A.

    1995-11-01

    The expression of xenobiotic-degradative genes in indigenous bacteria or in bacteria introduced into an ecosystem is essential for the successful bioremediation of contaminated environments. The maintenance of naphthalene utilization activity is studied in Pseudomonas putida (ATCC 17484) and an Alcaligenes sp. (strain NP-Alk) under different batch culture conditions. Levels of activity decreased exponentially in stationary phase with half-lives of 43 and 13 h for strains ATCC 17484 nad NP-Alk, respectively. Activity half-lives were 2.7 and 5.3 times longer, respectively, in starved cultures than in stationary-phase cultures following growth on naphthalene. The treatment of starved cultures with chloramphenicol caused a loss of activity more rapid than that measured in untreated starved cultures, suggesting a continued enzyme synthesis in starved cultures in the absence of a substrate. Following growth in nutrient medium, activity decreased to undetectable levels in the Alcaligenes sp. but remained at measureable levels int he pseudomonad even after 9 months. The induction of naphthalene degradation activities in these cultures, when followed by radiorespirometry with {sup 14}C-labeled naphthalene as the substrate, was consistent with activity maintenance data. In the pseudomonad, naphthalene degradation activity was present constitutively at low levels under all growth conditions and was rapidly (in approximately 15 min) induced to high levels upon exposure to naphthalene. Adaptation in the uninduced Alcaligenes sp. occurred after many hours of exposure to naphthalene. In vivo labeling with {sup 35}S, to monitor the extent of de novo enzyme synthesis by naphthalene-challenged cells, provided an independent confirmation of the results. 43 refs., 9 figs., 1 tab.

  6. Plasmid pCBI carries genes for anaerobic benzoate catabolism in Alcaligenes xylosoxidans subsp. denitrificans PN-1.

    PubMed Central

    Blake, C K; Hegeman, G D

    1987-01-01

    Pseudomonas sp. strain PN-1 is reclassified as Alcaligenes xylosoxidans subsp. denitrificans PN-1. Strain PN-1 is a gram-negative, rod-shaped organism, is motile by means of lateral flagella, is oxidase positive, and does not ferment sugars. Plasmid pCBI, carrying genes for the anaerobic degradation of benzoate in strain PN-1, is 17.4 kilobase pairs in length and is transmissible to a number of denitrifying Pseudomonas aeruginosa and Pseudomonas stutzeri strains. A restriction endonuclease map was constructed. PMID:2822651

  7. Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov.

    PubMed

    Coenye, T; Falsen, E; Vancanneyt, M; Hoste, B; Govan, J R; Kersters, K; Vandamme, P

    1999-04-01

    A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analysis, whole-cell protein and fatty acid analyses, AFLP (amplified fragment length polymorphism) fingerprinting and an extensive biochemical characterization was performed on 10 strains provisionally identified as Alcaligenes faecalis-like bacteria. The six environmental and four human isolates belonged to the genus Ralstonia and were assigned to a new species for which the name Ralstonia gilardii sp. nov. is proposed. The type strain is LMG 5886T.

  8. [Constitutive expression and purification of Alcaligenes faecalis penicillin G acylase in Escherichia coli].

    PubMed

    Yang, Zhi-Jian; Cai, Jin; Sun, Jian; Yuan, Zhong-Yi

    2004-09-01

    Considering Alcaligenes faecalis pencillin G acylase(AfPGA), which possesses the attractive characteristics for beta-lactam antibiotics conversions, the gene of PGA was cloned into an expressing vector pKKFPGA. The recombinant plasmid contained multicopy replicon(COLE 1), trc promoter, AfPGA gene, rrnB transcript terminator and ampicillin marker transformed Escherichia coli DH5alpha. As both the recombinant plasmid and the host DH5alpha had no laclq gene, the trc promoter was always active and the AfPGA could be constitutively expressed without IPTG induction in the host DH5alpha. In the shaking flask, the recombinant cell was inoculated into the fermentation medium (tryptone 10g/L, yeast extract 5g/L, MgSO4 x 7 H2O 1g, KH2 PO4 2g/L, K2HPO4 x 3H2O 5g/L, Na2HPO4 x 12H2O 7g/L, (NH4)2SO4 1.2g/L, NH4Cl 0.2 g/L, NaCl 0.1g/L, dextrin 30g/L) and cultured at 28 degrees C for 20h. The production of AfPGA reached 2,590u/L(NIPAB method), with a cell-density-specific activity of more than 300(u/L)/A600, this yield increased 432 fold higher than the native expression of Alcaligenes faecalis . Without ammonium sulphate fractionation and dialysis, the supernatant of crude extract was directly loaded on DEAE-Sepharose CL 6B column equilibrated by phosphate buffer (50mmol/L, pH7.8), and the enzyme fraction was not absorbed on the column but impurities were absorbed. Subsequently the effluent was added ammonium sulphate to 1mol/L and loaded on Butyl-Sepharose CL 4B column equilibrated by 50mmol/L phosphate buffer pH7.8-1mol/L ammonium sulphate. The enzyme was eluted as concentration of ammonium sulphate in phosphate buffer decreased to 0, PGA was eluted. After these two column chromatography, the enzyme was enriched 20 times with a 91% activity recovery. The purified enzyme had a specific activity of 68.6u/mg protein. However, the overproduction of PGA was often limited by translocation and/or periplasmic processing steps, subsequently resulted in intracellular accumulation of

  9. Inhibition of Serratia marcescens Smj-11 biofilm formation by Alcaligenes faecalis STN17 crude extract

    SciTech Connect

    Lutfi, Zainal; Ahmad, Asmat; Usup, Gires

    2014-09-03

    Serratia marcescens biofilms are formed when they are bound to surfaces in aqueous environments. S. marcescens utilizes N-acylhomoserine lactone (AHL) as its quorum sensing signal molecule. The accumulation of AHL indicates the bacteria to produce matrices to form biofilms. Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosin), which causes red pigmentation in the colonies, are also produced when the AHL reaches a certain threshold. The Alcaligenes faecalis STN17 crude extract is believed to inhibit quorum sensing in the S. marcescens Smj-11 and, thus, impedes its biofilm formation ability. A. faecalis STN17 was grown in marine broth, and ethyl acetate extraction was carried out. The crude compound of A. faecalis STN17 was diluted at high concentration (0.2-6.4 mg/mL) and was taken to confirm anti-biofilm activity through the crystal violet method in 96-wells plate. Then, the crude extract underwent purification using simple solvents partitioning test to discern the respective compounds that had the anti-biofilm activity under the crystal violet method. The crystal violet test showed that the crude did have anti-biofilm activity on S. marcescens Smj-11, but did not kill the cells. This finding signifies that the suppression of biofilm formation in S. marcescens by A. faecalis STN17 has a strong correlation. The partitioning test showed that A. faecalis STN17 crude extract has several compounds and only the compound(s) in chloroform showed activities. In conclusion, the crude extract of A. faecalis STN17 has the ability to inhibit S. marcescens Smj-11 biofilm formation.

  10. Inhibition of Serratia marcescens Smj-11 biofilm formation by Alcaligenes faecalis STN17 crude extract

    NASA Astrophysics Data System (ADS)

    Lutfi, Zainal; Usup, Gires; Ahmad, Asmat

    2014-09-01

    Serratia marcescens biofilms are formed when they are bound to surfaces in aqueous environments. S. marcescens utilizes N-acylhomoserine lactone (AHL) as its quorum sensing signal molecule. The accumulation of AHL indicates the bacteria to produce matrices to form biofilms. Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosin), which causes red pigmentation in the colonies, are also produced when the AHL reaches a certain threshold. The Alcaligenes faecalis STN17 crude extract is believed to inhibit quorum sensing in the S. marcescens Smj-11 and, thus, impedes its biofilm formation ability. A. faecalis STN17 was grown in marine broth, and ethyl acetate extraction was carried out. The crude compound of A. faecalis STN17 was diluted at high concentration (0.2-6.4 mg/mL) and was taken to confirm anti-biofilm activity through the crystal violet method in 96-wells plate. Then, the crude extract underwent purification using simple solvents partitioning test to discern the respective compounds that had the anti-biofilm activity under the crystal violet method. The crystal violet test showed that the crude did have anti-biofilm activity on S. marcescens Smj-11, but did not kill the cells. This finding signifies that the suppression of biofilm formation in S. marcescens by A. faecalis STN17 has a strong correlation. The partitioning test showed that A. faecalis STN17 crude extract has several compounds and only the compound(s) in chloroform showed activities. In conclusion, the crude extract of A. faecalis STN17 has the ability to inhibit S. marcescens Smj-11 biofilm formation.

  11. Structure of the 2,4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP

    PubMed Central

    Keegan, R.; Lebedev, A.; Erskine, P.; Guo, J.; Wood, S. P.; Hopper, D. J.; Rigby, S. E. J.; Cooper, J. B.

    2014-01-01

    The enzyme 2,4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in the predominantly hydrophobic active-site pocket where it undergoes peroxide radical-mediated heterolysis. PMID:25195757

  12. Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions.

    PubMed Central

    Otte, S; Grobben, N G; Robertson, L A; Jetten, M S; Kuenen, J G

    1996-01-01

    Nitrous oxide can be a harmful by-product in nitrogen removal from wastewater. Since wastewater treatment systems operate under different aeration regimens, the influence of different oxygen concentrations and oxygen fluctuations on denitrification was studied. Continuous cultures of Alcaligenes faecalis TUD produced N2O under anaerobic as well as aerobic conditions. Below a dissolved oxygen concentration of 5% air saturation, the relatively highest N2O production was observed. Under these conditions, significant activities of nitrite reductase could be measured. After transition from aerobic to anaerobic conditions, there was insufficient nitrite reductase present to sustain growth and the culture began to wash out. After 20 h, nitrite reductase became detectable and the culture started to recover. Nitrous oxide reductase became measurable only after 27 h, suggesting sequential induction of the denitrification reductases, causing the transient accumulation of N2O. After transition from anaerobic conditions to aerobic conditions, nitrite reduction continued (at a lower rate) for several hours. N2O reduction appeared to stop immediately after the switch, indicating inhibition of nitrous oxide reductase, resulting in high N2O emissions (maximum, 1.4 mmol liter-1 h-1). The nitrite reductase was not inactivated by oxygen, but its synthesis was repressed. A half-life of 16 to 22 h for nitrite reductase under these conditions was calculated. In a dynamic aerobic-anaerobic culture of A. faecalis, a semisteady state in which most of the N2O production took place after the transition from anaerobic to aerobic conditions was obtained. The nitrite consumption rate in this culture was equal to that in an anaerobic culture (0.95 and 0.92 mmol liter-1 h-1, respectively), but the production of N2O was higher in the dynamic culture (28 and 26% of nitrite consumption, respectively). PMID:8779582

  13. Immunostimulatory activities of a decapeptide derived from Alcaligenes faecalis FY-3 to crucian carp.

    PubMed

    Wang, G-X; Li, F-Y; Cui, J; Wang, Y; Liu, Y-T; Han, J; Lei, Y

    2011-07-01

    A strain was isolated from a soil sample collected from Weihe river in Shaanxi province (108°03'E 34°14'N), which was identified as Alcaligenes faecalis by 16S rRNA analysis. A compound M showing potent immune activity was isolated from secondary metabolites of the strain through bioassay-guided isolation techniques. The structure of the compound M was elucidated using FT-IR, EI-MS, 1H NMR and 13C NMR spectra and identified as cyclo-(L-Pro-Gly)5 which was first time reported as a natural product. We evaluated the immune effects of the cyclo-(L-Pro-Gly)5 on the basis of serum lysozyme activity, bacterial agglutination titre assay, superoxide anion production and phagocytic activity assay, and they were found to be significantly increased by cyclo-(L-Pro-Gly)5. The effects of cyclo-(L-Pro-Gly)5 on immune-related gene expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 6β (IL-6β) and inducible nitric oxide synthase 1β (iNOS-1β) mRNA in the blood leucocytes have been augmented by cyclo-(L-Pro-Gly)5. The challenge experiment showed that crucian carp injected the cyclo-(L-Pro-Gly)5 had significantly (P < 0.05) lower cumulative mortality (13.0%) compared with the control (45.4%) after infection with live Aeromonas hydrophila. These results suggested that cyclo-(L-Pro-Gly)5 is a possible immunostimulant and may strengthen the immune response and protect the heath status of crucian carp against A. hydrophila.

  14. Enhanced Alcaligenes faecalis Denitrification Rate with Electrodes as the Electron Donor.

    PubMed

    Wang, Xin; Yu, Ping; Zeng, Cuiping; Ding, Hongrui; Li, Yan; Wang, Changqiu; Lu, Anhuai

    2015-08-15

    The utilization by Alcaligenes faecalis of electrodes as the electron donor for denitrification was investigated in this study. The denitrification rate of A. faecalis with a poised potential was greatly enhanced compared with that of the controls without poised potentials. For nitrate reduction, although A. faecalis could not reduce nitrate, at three poised potentials of +0.06, -0.06, and -0.15 V (versus normal hydrogen electrode [NHE]), the nitrate was partially reduced with -0.15- and -0.06-V potentials at rates of 17.3 and 28.5 mg/liter/day, respectively. The percentages of reduction for -0.15 and -0.06 V were 52.4 and 30.4%, respectively. Meanwhile, for nitrite reduction, the poised potentials greatly enhanced the nitrite reduction. The nitrite reduction rates for three poised potentials (-0.06, -0.15, and -0.30 V) were 1.98, 4.37, and 3.91 mg/liter/h, respectively. When the potentials were cut off, the nitrite reduction rate was maintained for 1.5 h (from 2.3 to 2.25 mg/liter/h) and then greatly decreased, and the reduction rate (0.38 mg/liter/h) was about 1/6 compared with the rate (2.3 mg/liter/h) when potential was on. Then the potentials resumed, but the reduction rate did not resume and was only 2 times higher than the rate when the potential was off.

  15. Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis.

    PubMed

    Shahi, Puja; Kumar, Ish; Sharma, Ritu; Sanger, Shefali; Jolly, Ravinder S

    2006-06-01

    A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.

  16. Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1.

    PubMed

    Hsu, Cheng-Sheng; Lai, Wen-Lin; Chang, Wei-Wei; Liaw, Shwu-Huey; Tsai, Ying-Chieh

    2002-11-01

    D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the zinc-assistant hydrolysis of N-acyl-D-amino acids. We report here the cloning, expression, and structural-based mutation of the D-aminoacylase from Alcaligenes faecalis DA1. A 1,007-bp PCR product amplified with degenerate primers, was used to isolate a 4-kb genomic fragment, encoding a 484-residue D-aminoacylase. The enzyme amino-terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D-PSSM to be similar to urease and dihydroorotase, which have grouped into a novel alpha/beta-barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His-67, His-69, and His-250, putative metal ligands in D-aminoacylase, have been mutated previously, the remaining histidine (His-220) and aspartate (Asp-366) Asp-65, and four cysteines were then characterized. Substitution of Asp-65, Cys-96, His-220, and Asp-366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc-binding ability, revealing that Cys-96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino-terminal segment including Asp-65 may be responsible for structural stabilization.

  17. Production of poly-beta-hydroxybutyrate (PHB) by Alcaligenes latus from maple sap.

    PubMed

    Yezza, Abdessalem; Halasz, Annamaria; Levadoux, Wayne; Hawari, Jalal

    2007-11-01

    Maple sap, an abundant natural product especially in Canada, is rich in sucrose and thus may represent an ideal renewable feedstock for the production of a wide variety of value-added products. In the present study, maple sap or sucrose was employed as a carbon source to Alcaligenes latus for the production of poly-beta-hydroxybutyrate (PHB). In shake flasks, the biomass obtained from both the sap and sucrose were 4.4 +/- 0.5 and 2.9 +/- 0.3 g/L, and the PHB contents were 77.6 +/- 1.5 and 74.1 +/- 2.0%, respectively. Subsequent batch fermentation (10 L sap) resulted in the formation of 4.2 +/- 0.3 g/L biomass and a PHB content of 77.0 +/- 2.6%. The number average molecular weights of the PHB produced by A. latus from maple sap and pure sucrose media were 300 +/- 66 x 10(3) and 313 +/- 104 x 10(3) g/mol, respectively. Near-infrared, (1)H magnetic resonance imaging (MRI), and (13)C-MRI spectra of the microbially produced PHB completely matched those obtained with a reference material of poly[(R)-3-hydroxybutyric acid]. The polymer was found to be optically active with [alpha](25) (D) equaled to -7.87 in chloroform. The melting point (177.0 degrees C) and enthalpy of fusion (77.2 J/g) of the polymer were also in line with those reported, i.e., 177 degrees C and 81 J/g, respectively.

  18. Factors affecting biodegradation of 2-chlorophenol by Alcaligenes sp. in aerobic reactors.

    PubMed

    Gallego, A; Gómez, C E; Fortunato, M S; Cenzano, L; Rossi, S; Paglilla, M; Goméz Hermida, D G; Higa, L E; Korol, S E

    2001-01-01

    The influence of variations in carbon source concentration, cell inocula, pH, presence of other substrates, and other organisms on the biodegradation of 2-chlorophenol (2-CP) was studied for Alcaligenes sp. isolated from natural sources. Assays of biodegradation were performed in batch and continuous-flow fluidized-bed aerobic reactors. Evaluation of biodegradation was performed by determining total phenols, chemical oxygen demand (COD), and 2-CP by ultraviolet (UV) spectrophotometry. Measurement of microbial growth was carried out by the plate count method. Bioassays of acute toxicity were performed to evaluate detoxification by using Daphnia magna. Results obtained show that under batch conditions with initial inocula of 10(6) cells/mL the strain grew exponentially with 100, 200, and 300 mg/L of 2-CP within 48 hr. A lag period was observed with low cell density inocula (10(5) cells/mL). The strain showed marked delay in the biodegradation of 2-CP at pH 5. Removal of target substrate from mixtures containing other carbon sources demonstrated the possibility of concurrent growth. Mineralization of 2-CP was assessed by gas chromatography carried out at the end of the batch assays and at the exit of the continuous-flow reactor. The presence of other organisms (bacteria, rotifers, ciliate, and algae) that developed in the fluidized-bed reactor did not affect the efficacy of the biodegradation of 2-CP. The removal of 2-CP in the two assayed systems was over 97% in all cases. Toxicity was not detected at the exit of the continuous reactor.

  19. Enhanced Alcaligenes faecalis Denitrification Rate with Electrodes as the Electron Donor

    PubMed Central

    Wang, Xin; Yu, Ping; Zeng, Cuiping; Ding, Hongrui; Wang, Changqiu

    2015-01-01

    The utilization by Alcaligenes faecalis of electrodes as the electron donor for denitrification was investigated in this study. The denitrification rate of A. faecalis with a poised potential was greatly enhanced compared with that of the controls without poised potentials. For nitrate reduction, although A. faecalis could not reduce nitrate, at three poised potentials of +0.06, −0.06, and −0.15 V (versus normal hydrogen electrode [NHE]), the nitrate was partially reduced with −0.15- and −0.06-V potentials at rates of 17.3 and 28.5 mg/liter/day, respectively. The percentages of reduction for −0.15 and −0.06 V were 52.4 and 30.4%, respectively. Meanwhile, for nitrite reduction, the poised potentials greatly enhanced the nitrite reduction. The nitrite reduction rates for three poised potentials (−0.06, −0.15, and −0.30 V) were 1.98, 4.37, and 3.91 mg/liter/h, respectively. When the potentials were cut off, the nitrite reduction rate was maintained for 1.5 h (from 2.3 to 2.25 mg/liter/h) and then greatly decreased, and the reduction rate (0.38 mg/liter/h) was about 1/6 compared with the rate (2.3 mg/liter/h) when potential was on. Then the potentials resumed, but the reduction rate did not resume and was only 2 times higher than the rate when the potential was off. PMID:26048940

  20. [Influence of yeast extract on the fermentation of glucose by the demulsifying strain Alcaligenes sp. S-XJ-1].

    PubMed

    Huang, Xiang-Feng; Wang, Kai; Li, Ming-Xia; Wang, Cai-Lin; Lu, Li-Jun; Liu, Jia

    2013-04-01

    The demulsifying strain Alcaligenes sp. S-XJ-1, isolated from oil contaminated soil, was cultivated with glucose as the carbon source. The influences of yeast extract on the growth, demulsifying ability and the element composition of the strain were investigated. The results showed that the yeast extract could increase the biomass and enhance the glucose utilization of Alcaligenes sp. S-XJ-1. When the concentration of the yeast extract was 5 g x L(-1), the biomass was increased up to 3.0 g x L(-1), and the glucose utilization achieved 58%. The demulsifying ability of the strain was improved with increasing yeast extract concentration. When the concentration of the yeast extract was 10 g x L(-1), the demulsification ratio of the obtained cell was 76%. While the C/N ratio of the cells decreased with the increasing concentration of yeast extract. The proteins of cells were extracted and measured. The results showed that the proteins of the obtained cell increased with the increasing concentration of yeast extract, in accordance with the increased concentrations of proteins on the surface of the cells as measured by FTIR. It is estimated that the increase of the proteins leads to the improvement of the demulsifying ability of the demulsifying strain and theses proteins play essential roles in the demulsifying process.

  1. Thermostable Alkaline Phytase from Alcaligenes sp. in Improving Bioavailability of Phosphorus in Animal Feed: In Vitro Analysis

    PubMed Central

    Vijayaraghavan, Ponnuswamy; Primiya, R. Raja; Prakash Vincent, Samuel Gnana

    2013-01-01

    A bacterial isolate, Alcaligenes sp. secreting phytase (EC 3.1.3.8), was isolated and characterized. The optimum conditions for the production of phytase included a fermentation period of 96 h, pH 8.0, and the addition of 1% (w/v) maltose and 1% (w/v) beef extract to the culture medium. This enzyme was purified to homogeneity and had an apparent molecular mass of 41 kDa. The optimum pH range and temperature for the activity of phytase were found to be 7.0-8.0 and 60°C, respectively. This enzyme was strongly inhibited by 0.005 M of Mn2+, Mg2+, and Zn2+. In vitro studies revealed that the phytase from Alcaligenes sp. released inorganic phosphate from plant phytates. Phytase released 1930 ± 28, 1740 ± 13, 1050 ± 31, 845 ± 7, 1935 ± 32, and 1655 ± 21 mg inorganic phosphate/kg plant phytates, namely, chick pea, corn, green pea, groundnut, pearl pea, and chick feed, respectively. PMID:25969790

  2. Arsenite oxidation by Alcaligenes sp. strain RS-19 isolated from arsenic-contaminated mines in the Republic of Korea.

    PubMed

    Yoon, In-Ho; Chang, Jin-Soo; Lee, Ji-Hoon; Kim, Kyoung-Woong

    2009-02-01

    Arsenite [As(III)]-oxidizing bacteria play important roles in reducing arsenic [As] toxicity and mobility in As-contaminated areas. As-resistant bacteria were isolated from the soils of two abandoned mines in the Republic of Korea. The isolated bacteria showed relatively high resistances to As(III) up to 26 mM. The PCR-based 16S rRNA analysis revealed that the isolated As-resistant bacteria were close relatives to Serratia marcescensa, Pseudomonas putida, Pantoea agglomerans, and Alcaligenes sp. Among the five As-resistant bacterial isolates, Alcaligenes sp. strain RS-19 showed the highest As(III)-oxidizing activity in batch tests, completely oxidizing 1 mM of As(III) to As(V) within 40 h during heterotrophic growth. This study suggests that the indigenous bacteria have evolved to retain the ability to resist toxic As in the As-contaminated environments and moreover to convert the species to a less toxic form [e.g., from As(III) to As(V)] and also contribute the biogeochemical cycling of As by being involved in speciation of As.

  3. [Enhanced production of curdlan by Alcaligenes faecalis by selective feeding with ammonia water during the cell growth phase of fermentation].

    PubMed

    Wu, Jianrong; Zhan, Xiaobei; Liu, Hui; Zheng, Zhiyong

    2008-06-01

    Curdlan is a water insoluble exopolysaccharide produced by Alcaligenes faecalis under nitrogen-limiting conditions. After excretion, the polysaccharide is attached the cell wall. Thus enhancement of biomass production during the cell growth phase is important to curdlan production. A strategy of increasing nitrogen source to improve biomass production was adopted for curdlan production by Alcaligenes faecalis (ATCC 31749). In the batch fermentation of curdlan, a relatively higher NH4Cl level of 3.6 g/L with continuous glucose feeding increased the cell density leading to improvement of curdlan production. However, excessive NH4Cl would inhibit curdlan production and biomass production was not improved significantly. In addition, feeding of ammonia water at the initial phase replaced NaOH solution to control pH at 7.0. Subsequently, feeding of NaOH solution was resumed to control pH at 5.6 for curdlan production after ammonia was consumed. As a result, biomass production and curdlan yield were both enhanced remarkably. Feeding of ammonia water during the first 24 h led to biomass production of 18.8 g/L. However, higher cell density did not lead to increase in curdlan production. The maximum curdlan production (72 g/L) was obtained by feeding ammonia water for the first 14 h, during which the cell density was about 11.9 g/L.

  4. Rhizosphere colonization and arsenic translocation in sunflower (Helianthus annuus L.) by arsenate reducing Alcaligenes sp. strain Dhal-L.

    PubMed

    Cavalca, Lucia; Corsini, Anna; Bachate, Sachin Prabhakar; Andreoni, Vincenza

    2013-10-01

    In the present study, six arsenic-resistant strains previously isolated were tested for their plant growth promoting characteristics and heavy metal resistance, in order to choose one model strain as an inoculum for sunflower plants in pot experiments. The aim was to investigate the effect of arsenic-resistant strain on sunflower growth and on arsenic uptake from arsenic contaminated soil. Based on plant growth promoting characteristics and heavy metal resistance, Alcaligenes sp. strain Dhal-L was chosen as an inoculum. Beside the ability to reduce arsenate to arsenite via an Ars operon, the strain exhibited 1-amino-cyclopropane-1-carboxylic acid deaminase activity and it was also able to produce siderophore and indole acetic acid. Pot experiments were conducted with an agricultural soil contaminated with arsenic (214 mg kg⁻¹). A real time PCR method was set up based on the quantification of ACR3(2) type of arsenite efflux pump carried by Alcaligenes sp. strain Dhal-L, in order to monitor presence and colonisation of the strain in the bulk and rhizospheric soil. As a result of strain inoculation, arsenic uptake by plants was increased by 53 %, whereas ACR3(2) gene copy number in rhizospheric soil was 100 times higher in inoculated than in control pots, indicating the colonisation of strain. The results indicated that the presence of arsenate reducing strains in the rhizosphere of sunflower influences arsenic mobilization and promotes arsenic uptake by plant.

  5. Co-immobilization of Pseudomonas stutzeri YHA-13 and Alcaligenes sp. ZGED-12 with polyvinyl alcohol-alginate for removal of nitrogen and phosphorus from synthetic wastewater.

    PubMed

    Han, Yonghe; Zhang, Wenxian; Lu, Wenxian; Zhou, Zhihua; Zhuang, Zhigang; Li, Min

    2014-01-01

    Nitrogen (N) and phosphorus (P) are the two main factors causing water eutrophication. Immobilized micro-organisms have been widely studied in N and P removal. However, the effects of various immobilizing conditions on the removal efficiency of N and P using immobilized micro-organism beads (IMOBs) remain unclear. Polyvinyl alcohol (PVA) and alginate, as the two frequently immobilizing-used matrixes, were used for co-immobilizing Pseudomonas stutzeri YHA-13 and Alcaligenes sp. ZGED-12. PVA, alginate and CaCl₂contents, immobilization time and different wet biomass ratios of P. stutzeri to Alcaligenes sp. were conducted to elucidate their roles in and influences on the removal efficiency of N and P from synthetic wastewater. The application potential of IMOBs was estimated as well. Results showed that IMOBs prepared by cross-link of 4% PVA and 2-3% alginate with 5% CaCl₂and saturated boric acid solution for 10-15 min are the best ones in removal of N and P. Though IMOBs containing P. stutzeri and/or Alcaligenes sp. were capable of removal of the two nutrients, the highest removal efficiency was observed when the wet biomass ratio of P. stutzeri to Alcaligenes sp. was adjusted to 2:2. In addition, the IMOBs were of good ability to remove chemical oxygen demand (COD), NO(3)(-), NO(2)(-), NH(4)(+)- N, total nitrogen (TN) and total phosphorus (TP) from artificial wastewater. Of which, micro-organisms immobilized in matrixes were mainly responsible for NO(3)(-) and TP removal. Therefore, P. stutzeri YHA-13 and Alcaligenes sp. ZGED-12 are reliable bioresources to remove N and P from wastewater.

  6. Exogenous cofactors for the improvement of bioremoval and biotransformation of sulfamethoxazole by Alcaligenes faecalis.

    PubMed

    Zhang, Yi-Bi; Zhou, Jiao; Xu, Qiu-Man; Cheng, Jing-Sheng; Luo, Yu-Lu; Yuan, Ying-Jin

    2016-09-15

    Sulfamethoxazole (SMX), an extensively prescribed or administered antibiotic pharmaceutical product, is usually detected in aquatic environments, because of its incomplete metabolism and elimination. This study investigated the effects of exogenous cofactors on the bioremoval and biotransformation of SMX by Alcaligenes faecalis. High concentration (100mg·L(-1)) of exogenous vitamin C (VC), vitamin B6 (VB6) and oxidized glutathione (GSSG) enhanced SMX bioremoval, while the additions of vitamin B2 (VB2) and vitamin B12 (VB12) did not significantly alter the SMX removal efficiency. Globally, cellular growth of A. faecalis and SMX removal both initially increased and then gradually decreased, indicating that SMX bioremoval is likely dependent on the primary biomass activity of A. faecalis. The decreases in the SMX removal efficiency indicated that some metabolites of SMX might be transformed into parent compound at the last stage of incubation. Two transformation products of SMX, N-hydroxy sulfamethoxazole (HO-SMX) and N4-acetyl sulfamethoxazole (Ac-SMX), were identified by a high-performance liquid chromatograph coupled with mass spectrometer. High concentrations of VC, nicotinamide adenine dinucleotide hydrogen (NADH, 7.1mg·L(-1)), and nicotinamide adenine dinucleotide (NAD(+), 6.6mg·L(-1)), and low concentrations of reduced glutathione (GSH, 0.1 and 10mg·L(-1)) and VB2 (1mg·L(-1)) remarkably increased the formation of HO-SMX, while VB12 showed opposite effects on HO-SMX formation. In addition, low concentrations of GSH and NADH enhanced Ac-SMX formation by the addition of A. faecalis, whereas cofactors (VC, VB2, VB12, NAD(+), and GSSG) had no obvious impact on the formation of Ac-SMX compared with the controls. The levels of Ac-SMX were stable when biomass of A. faecalis gradually decreased, indicating the direct effect of biomass on the formation of Ac-SMX by A. faecalis. In sum, these results help us understand the roles played by exogenous cofactors in

  7. Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria.

    PubMed Central

    Schlömann, M; Schmidt, E; Knackmuss, H J

    1990-01-01

    Of various benzoate-utilizing bacteria tested, Alcaligenes eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, A. eutrophus JMP134, Alcaligenes strain A7, and Pseudomonas cepacia were able to grow with 4-fluorobenzoate as the sole source of carbon and energy. P. cepacia also utilizes 3-fluorobenzoate. Except for A. eutrophus JMP134, which is known to grow with 2,4-dichlorophenoxyacetate and 3-chlorobenzoate (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981), the strains were unable to grow at the expense of these compounds or 4-chlorobenzoate. Assays of cell extracts revealed that all strains express dienelactone hydrolase and maleylacetate reductase activities in addition to enzymes of the catechol branch of the 3-oxoadipate pathway when growing with 4-fluorobenzoate. Induction of dienelactone hydrolase and maleylacetate reductase apparently is not necessarily connected to synthesis of catechol 1,2-dioxygenase type II and chloromuconate cycloisomerase activities, which are indispensable for the degradation of chlorocatechols. Substrate specificities of the dienelactone hydrolases provisionally differentiate among three types of this activity. (i) Extracts of A. eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide (trans-dienelactone) much faster than the cis-isomer (type I). (ii) The enzyme present in P. cepacia shows the opposite preference for the isomeric substrates (type II). (iii) Cell extracts of A. eutrophus JMP134, as well as purified dienelactone hydrolase from Pseudomonas strain B13 (E. Schmidt and H.-J. Knackmuss, Biochem. J. 192:339-347, 1980), hydrolyze both dienelactones at rates that are of the same order of magnitude (type III). This classification implies that A. eutrophus JMP134 possesses at least two different dienelactone hydrolases, one of type III encoded by the plasmid pJP4 and one of type I, which is also present in the cured strain JMP222. PMID

  8. Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867.

    PubMed

    Chua, C H; Feng, Y; Yeo, C C; Khoo, H E; Poh, C L

    2001-10-16

    Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product. Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding. A mutant enzyme with increased catalytic activities was also characterized.

  9. Tn5563, a transposon encoding putative mercuric ion transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes.

    PubMed

    Yeo, C C; Tham, J M; Kwong, S M; Yiin, S; Poh, C L

    1998-08-15

    Sequence analysis of pRA2, an endogenous 33-kb plasmid from Pseudomonas alcaligenes NCIB 9867 (strain P25X), revealed the presence of a 6256-bp transposon of the Tn3 family, designated Tn5563. Tn5563, which is flanked by two 39-bp inverted repeats, encodes a transposase, a resolvase, and two open reading frames which share amino acid sequence similarities with the mercuric ion transport proteins MerT and MerP encoded by several mer operons. However, no other mer operon genes were found on Tn5563. Sequencing of a RP4::XIn hybrid plasmid indicates possible interactions between pRA2 and the P25X chromosome mediated by Tn5563.

  10. Improvement in ammonium removal efficiency in wastewater treatment by mixed culture of Alcaligenes faecalis no. 4 and L1.

    PubMed

    Joo, Hung-Soo; Hirai, Mitsuyo; Shoda, Makoto

    2007-01-01

    To improve ammonium removal efficiency in wastewater treatment, a mixed culture of Alcaligenes faecalis no. 4 and its mutant L1, both of which have heterotrophic nitrification and aerobic denitrification abilities, was performed. In a batch culture, no. 4 has a higher denitrification ability than L1, but its ammonium removal rate was lower. In a mixed continuous culture in the ammonium loading range of 750 to 3500 mg-N/l/d, the average ammonium removal rate and the average denitrification ratio were 61 mg-N/l/h and 31%, respectively. In the mixed culture, the ammonium removal rate was twofold higher than that in a single culture of no. 4, the rate was similar to that in a single culture of L1, and the denitrification ratio was very high compared with that in the single culture of L1.

  11. The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue copper protein from Escherichia coli cells.

    PubMed Central

    Yamamoto, K; Uozumi, T; Beppu, T

    1987-01-01

    The gene encoding a blue copper protein (a member of the pseudoazurins) of 123 amino acid residues, containing a single type I Cu2+ ion, was cloned from Alcaligenes faecalis S-6. The nucleotide sequence of the coding region, as well as the 5'- and 3'-flanking regions, was determined. The deduced amino acid sequence after Glu-24 coincided with the reported sequence of the blue protein, and its NH2-terminal sequence of 23 residues resembled a typical signal peptide. The cloned gene was expressed under the control of the tac promoter in Escherichia coli, and the correctly processed blue protein was secreted into the periplasm. The blue protein produced in E. coli possessed the activity to transfer electrons to the copper-containing nitrite reductase of A. faecalis S-6 in vitro. Images PMID:2824441

  12. Amphoteric surfactant N-oleoyl-N-methyltaurine utilized by Pseudomonas alcaligenes with excretion of N-methyltaurine.

    PubMed

    Denger, Karin; Mayer, Jutta; Hollemeyer, Klaus; Cook, Alasdair M

    2008-11-01

    The amphoteric surfactant N-oleoyl-N-methyltaurine, which is in use in skin-care products, was utilized by aerobic bacteria as the sole source of carbon or of nitrogen in enrichment cultures. One isolate, which was identified as Pseudomonas alcaligenes, grew with the xenobiotic compound as the sole source of carbon and energy. The sulfonate moiety, N-methyltaurine, was excreted quantitatively during growth, while the fatty acid was dissimilated. The initial degradative reaction was shown to be hydrolytic and inducible. This amidase reaction could be demonstrated with crude cell extracts. The excreted N-methyltaurine could be utilized by other bacteria in cocultures. Complete degradation of similar natural compounds in bacterial communities seems likely.

  13. Biochemical and histochemical analyses revealing endophytic Alcaligenes faecalis mediated suppression of oxidative stress in Abelmoschus esculentus challenged with Sclerotium rolfsii.

    PubMed

    Ray, Shatrupa; Singh, Vivek; Singh, Surendra; Sarma, Birinchi Kumar; Singh, Harikesh Bahadur

    2016-12-01

    Sclerotium rolfsii is a highly aggressive pathogen that causes huge economic losses, especially in temperate climates. Alcaligenes faecalis, particularly in endophytic form, has rarely been used to control this fungus. In this study, endophytic Alcaligenes sp. strain BHU 12, BHU 16 (isolated from Abelmoschus esculentus leaf) and BHU M7 (isolated from Andrographis paniculata leaf) were reported to trigger a wide range of host defenses in Okra plant against the collar-rot pathogen S. rolfsii. Endophytic colonization of the strains in ten days old plants was assessed through re-isolation of the rif-tagged strains on rifampicin augmented nutrient agar media. The ability of the endophytic strains to induce systemic defense responses in above-ground organs was assessed by collecting leaf tissues of the Okra plants grown under non-gnotobiotic conditions at different time intervals post seedling bacterization with the endophytic biocontrol agents. The pathogen challenged unprimed plants exhibited flaccidity of the stem and leaves at 48 h post infection (hpi) in contrast to the bioprimed and challenged plants. Biochemical and histochemical analyses explained the above phenomenon as activation of phyto-peroxidases leading to an increased metabolism of the reactive oxygen species (ROS), accompanied by activation of the phenylpropanoid network and a subsequent enhancement in plant phenolics. Interestingly, though the maximum increase in the defense pathways was observed in treatments with native endophytes of Okra plant, yet the enhancement in antioxidant pathway due to A. paniculata borne endophytes was also quite significant. Thus, this work clearly demonstrates how Okra plants respond to the "non-hostile" colonization of bacterial endophytes and how induced defense response can contribute to the biocontrol activity of the endophytic strains.

  14. Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Pseudomonas alcaligenes LB19.

    PubMed

    Kim, Do Young; Nam, Jin Sik; Rhee, Young Ha

    2002-01-01

    An extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase from an isolate, Pseudomonas alcaligenes LB19, was purified to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the enzyme, which consisted of a single polypeptide chain, was approximately 27.6 kDa. The pI value of the enzyme was estimated to be 5.7, and its maximum activity was observed at pH 9.0 and 45 degreesC. The enzyme was significantly inactivated by EDTA and phenylmethylsulfonyl fluoride (PMSF) but insensitive to dithiothreitol. It was also markedly inhibited by 0.1% Tween 80 and 0.05% Triton X-100. The purified enzyme could hydrolyze various types of bacterial aliphatic and aromatic MCL-PHAs but not poly(3-hydroxybutyrate), polycaprolactone, and poly(L-lactide). Biodegradation rates of the aromatic MCL-PHAs were significantly lower than those of the aliphatic MCL-PHAs, regardless of the compositions and types of aromatic substituents. It was able to hydrolyze medium-chain-length p-nitrophenylalkanoates more efficiently than the shorter-chain forms. The main hydrolysis products of poly(3-hydroxynonanoate) were identified as monomer units. The results demonstrated in this study suggest that the MCL-PHA depolymerase from P. alcaligenes LB19 is a distinct enzyme, which are different from those of other MCL-PHA degrading bacteria in its quaternary structure, pI value, sensitivity to EDTA and PMSF, and hydrolysis products of MCL-PHA.

  15. Cloning, sequencing and mutagenesis of the genes for aromatic amine dehydrogenase from Alcaligenes faecalis and evolution of amine dehydrogenases.

    PubMed

    Chistoserdov, A Y

    2001-08-01

    The nucleotide sequence of the aromatic amine utilization (aau) gene region from Alcaligenes faecalis contained nine genes (orf-1, aauBEDA, orf-2, orf-3, orf-4 and hemE) transcribed in the same direction. The aauB and aauA genes encode the periplasmic aromatic amine dehydrogenase (AADH) large and small subunit polypeptides, respectively, and were homologous to mauB and mauA, the genes for the large and small subunits of methylamine dehydrogenase (MADH). aauE and aauD are homologous to mauE and mauD and apparently carry out the same function of transport and folding of the small subunit polypeptide in the periplasm. No analogues of the mauF, mauG, mauL, mauM and mauN genes responsible for biosynthesis of tryptophan tryptophylquinone (the prosthetic group of amine dehydrogenases) were found in the aau cluster. orf-2 was predicted to encode a small periplasmic monohaem c-type cytochrome. No biological function can be assigned to polypeptides encoded by orf-1, orf-3 and orf-4 and mutations in these genes appeared to be lethal. Mutants generated by insertions into mauD were not able to use phenylethylamine, tyramine and tryptamine as a source of carbon and phenylethylamine, 3'-hydroxytyramine (dopamine) and tyramine as a source of nitrogen, indicating that AADH is the only enzyme involved in utilization of primary amines in A. faecalis. AADH genes are present in Alcaligenes xylosoxydans subsp. xylosoxydans, but not in other beta- and gamma-proteobacteria. Phylogenetic analysis of amine dehydrogenases (MADH and AADH) indicated that AADH and MADH evolutionarily diverged before separation of proteobacteria into existing subclasses.

  16. Synergistic activities of macrolide antibiotics against Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans isolated from patients with cystic fibrosis.

    PubMed

    Saiman, Lisa; Chen, Yunhua; Gabriel, Pablo San; Knirsch, Charles

    2002-04-01

    Azithromycin and clarithromycin were paired with other antibiotics to test synergistic activity against 300 multidrug-resistant pathogens isolated from cystic fibrosis (CF) patients. Clarithromycin-tobramycin was most active against Pseudomonas aeruginosa and inhibited 58% of strains. Azithromycin-trimethoprim-sulfamethoxazole, azithromycin-ceftazidime, and azithromycin-doxycycline or azithromycin-trimethoprim-sulfamethoxazole inhibited 40, 20, and 22% of Stenotrophomonas maltophilia, Burkholderia cepacia complex, and Achromobacter (Alcaligenes) xylosoxidans strains, respectively.

  17. Biocatalytic synthesis of (R)-(-)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of Alcaligenes faecalis ECU0401.

    PubMed

    He, Yu-Cai; Zhang, Zhi-Jun; Xu, Jian-He; Liu, You-Yan

    2010-07-01

    The nitrilase from Alcaligenes faecalis ECU0401 belongs to the category of arylacetonitrilase, which could hydrolyze 2-chloromandelonitrile, 3,4-dimethoxyphenylacetonitrile, mandelonitrile, and phenylacetonitrile into the corresponding arylacetic acids. To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Alcaligenes faecalis ECU0401 in relation to nitrilase activity was optimized by using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. The nitrilase activity from Alcaligenes faecalis ECU0401 increased 4.5-fold when the cells were permeabilized with 0.3% (w/v) CTAB for 20 min at 25 degrees C and pH 6.5. Consequently, almost all the mandelonitrile was consumed and converted to (R)-(-)-mandelic acid with greater than 99.9% enantiomeric excess (e.e.) by the CTAB-permeabilized cells. The permeability barrier has been significantly reduced in the hydrolysis of mandelonitrile by using CTAB-permeabilized cells and a dynamic resolution was successfully achieved, giving a 100% theoretical yield of (R)-(-)-mandelic acid. Efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 3.82 g (R)-(-)-mandelic acid g(-1) dry cell weight (dcw) cell after 20 cycles of repeated use.

  18. Fate of a metal-resistant inoculum in contaminated and pristine soils assessed by denaturing gradient gel electrophoresis

    SciTech Connect

    Stephen, J.R.; Chang, Y.J.; MacNaughton, S.J.; Leung, K.T.; Flemming, C.A. . Center for Environmental Biotechnology); Whitaker, S.L.; Hicks, C.L. ); White, D.C. . Center for Environmental Biotechnology Oak Ridge National Lab., TN . Environmental Sciences Div.)

    1999-06-01

    Cesium, cadmium, cobalt, and strontium are four contaminants frequently found in soils at biotoxic levels. Introduction of certain nongenetically modified bacteria has been frequently suggested as a method for the immobilization of heavy metal contaminants in soil, thereby reducing runoff and bioavailability. In this study, the authors have used the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to track the survival of the five bacterial species added to soil microcosms with and without the addition of a mixture of these metals. The PCR primers targeted conserved regions of the 165 rDNA molecular present in all bacteria. The reaction products were shown to reflect the relative abundance of the bacteria both in mixtures of pure cultures and against a background of all the eubacterial species present in the soil following inoculation. Three of the species (Pseudomonas aeruginosa FRD-1, Shewanella putrifaciens 200, and Desulfovibrio vulgaris Hildenborough) decreased rapidly following inoculation into both soils. The proportion of Alcaligenes eutrophus CH34 remained at a constant level throughout the 8-week experiment in both soil treatments. Sphingomonas aromaticivorans B0695 showed toxic metal-dependent survival in that its relative abundance dropped rapidly in pristine soil but remained at approximately inoculation levels throughout the experiment in contaminated microcosms.

  19. Fed batch bioconversion of 2-propanol by a solvent tolerant strain of Alcaligenes faecalis entrapped in Ca-alginate gel.

    PubMed

    Mohammad, Balsam T; Bustard, Mark T

    2008-07-01

    A gram-negative, rod-shaped, aerobe, capable of converting 2-propanol (isopropanol, IPA) to acetone was isolated from an oil/sump, and identified by 16 S rDNA analysis as Alcaligenes faecalis. Investigations showed this strain to be extremely solvent-tolerant and it was subsequently named ST1. In this study, A. faecalis ST1 cells were immobilized by entrapment in Ca-alginate beads (3 mm in diameter), and used in the bioconversion of high concentration IPA. The biodegradation rates and the corresponding microbial growth inside the beads were measured at four different IPA concentration ranges from 2 to 15 g l(-1). The maximum cell concentration obtained was 9.59 g dry cell weight (DCW) l(-1) medium which equated to 66 g DCW l(-1) gel, at an initial IPA concentration of 15 g l(-1) after 216 h of incubation. A maximum biodegradation rate of 0.067 g IPA g cells(-1) h(-1) was achieved for 5 g l(-1) IPA where an increase in IPA concentration to 38 g l(-1) caused reduction in bead integrity. A modified growth medium was developed which allowed repeated use of the beads for more than 42 days without any loss of integrity and continued bioconversion activity.

  20. Upstream process optimization of polyhydroxybutyrate (PHB) by Alcaligenes latus using two-stage batch and fed-batch fermentation strategies.

    PubMed

    Wang, Bingqing; Sharma-Shivappa, Ratna R; Olson, Jonathan W; Khan, Saad A

    2012-11-01

    This research focused on optimizing the upstream process time for production of polyhydroxybutyrate (PHB) from sucrose by two-stage batch and fed-batch fermentation with Alcaligenes latus ATCC 29714. The study included selection of strain, two-stage batch fermentations with different time points for switching to nitrogen limited media (14, 16 or 18 h) and fed-batch fermentations with varied time points (similar to two stage) for introducing nitrogen limited media. The optimal strain to produce PHB using sucrose as carbon source was A. latus ATCC 29714 with maximum-specific growth rate of 0.38 ± 0.01 h(-1) and doubling time of 1.80 ± 0.05 h. Inducing nitrogen limitation at 16 h and ending second stage at 26 h gave optimal performance for PHB production, resulting in a PHB content of 46.7 ± 12.2 % (g PHB per g dry cell weight) at the end of fermentation. This was significantly higher (P ≤ 0.05) (approximately 7 %) than the corresponding fed batch run in which nitrogen limitation was initiated at 16 h.

  1. Production and characterization of poly(3-hydroxybutyrate) generated by Alcaligenes latus using lactose and whey after acid protein precipitation process.

    PubMed

    Berwig, Karina Hammel; Baldasso, Camila; Dettmer, Aline

    2016-10-01

    Whey after acid protein precipitation was used as substrate for PHB production in orbital shaker using Alcaligenes latus. Statistical analysis determined the most appropriate hydroxide for pH neutralization of whey after protein precipitation among NH4OH, KOH and NaOH 10%w/v. The results were compared to those of commercial lactose. A scale-up test in a 4L bioreactor was done at 35°C, 750rpm, 7L/min air flow, and 6.5 pH. The PHB was characterized through Fourier Transform Infrared Spectroscopy, thermogravimetry and differential scanning calorimetry. NH4OH provided the best results for productivity (p), 0.11g/L.h, and for polymer yield, (YP/S), 1.08g/g. The bioreactor experiment resulted in lower p and YP/S. PHB showed maximum degradation temperature (291°C), melting temperature (169°C), and chemical properties similar to those of standard PHB. The use of whey as a substrate for PHB production did not affect significantly the final product quality.

  2. Efficient cascade synthesis of ampicillin from penicillin G potassium salt using wild and mutant penicillin G acylase from Alcaligenes faecalis.

    PubMed

    Deng, Senwen; Ma, Xiaoqiang; Su, Erzheng; Wei, Dongzhi

    2016-02-10

    To avoid isolation and purification of the intermediate 6-aminopenicillanic acid (6-APA), a two-enzyme two-step cascade synthesis of ampicillin from penicillin G was established. In purely aqueous medium, penicillin G hydrolysis and ampicillin synthesis were catalyzed by immobilized wild-type and mutagenized penicillin G acylases from Alcaligenes faecalis (Af PGA), respectively (Fig. 1). The βF24 G mutant Af PGA (the 24th Phenylalanine of the β-subunit was replaced by Glycine) was employed for its superior performance in enzymatic synthesis of ampicillin. By optimizing the reaction conditions, including enzyme loading, temperature, initial pH and D-PGME/6-APA ratio, the conversion of the second step of ampicillin synthesis reached approximately 90% in 240 min and less than 1.7 mole D-PGME were required to produce 1 mole ampicillin. Overall, in a 285 min continuous two-step procedure, an ampicillin yield of 87% was achieved, demonstrating the possibility of improving the cascade synthesis of ampicillin by mutagenized PGA, providing an economically efficient and environmentally benign procedure for semi-synthetic penicillins antibiotics synthesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Application of waste frying oils in the biosynthesis of biodemulsifier by a demulsifying strain Alcaligenes sp. S-XJ-1.

    PubMed

    Liu, Jia; Peng, Kaiming; Huang, Xiangfeng; Lu, Lijun; Cheng, Hang; Yang, Dianhai; Zhou, Qi; Deng, Huiping

    2011-01-01

    Exploration of biodemulsifiers has become a new research aspect. Using waste frying oils (WFOs) as carbon source to synthesize biodemulsifiers has a potential prospect to decrease production cost and to improve the application of biodemulsifiers in the oilfield. In this study, a demulsifying strain, Alcaligenes sp. S-XJ-1, was investigated to synthesize a biodemulsifier using waste frying oils as carbon source. It was found that the increase of initial pH of culture medium could increase the biodemulsifier yield but decrease the demulsification ratio compared to that using paraffin as carbon source. In addition, a biodemulsifier produced by waste frying oils and paraffin as mixed carbon source had a lower demulsification capability compared with that produced by paraffin or waste frying oil as sole carbon source. Fed-batch fermentation of biodemulsifier using waste frying oils as supplementary carbon source was found to be a suitable method. Mechanism of waste frying oils utilization was studied by using tripalmitin, olein and tristearin as sole carbon sources to synthesize biodemulsifier. The results showed saturated long-chain fatty acid was difficult for S-XJ-1 to utilize but could effectively enhance the demulsification ability of the produced biodemulsifier. Moreover, FT-IR result showed that the demulsification capability of biodemulsifiers was associated with the content of C=O group and nitrogen element.

  4. Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase.

    PubMed

    Iwakiri, Ryo; Yoshihira, Kunichika; Ngadiman; Futagami, Taiki; Goto, Masatoshi; Furukawa, Kensuke

    2004-06-01

    We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.

  5. Genetic diversity and horizontal transfer of genes involved in oxidation of reduced phosphorus compounds by Alcaligenes faecalis WM2072.

    PubMed

    Wilson, Marlena M; Metcalf, William W

    2005-01-01

    Enrichment was performed to isolate organisms that could utilize reduced phosphorus compounds as their sole phosphorus sources. One isolate that grew well with either hypophosphite or phosphite was identified by 16S rRNA gene analysis as a strain of Alcaligenes faecalis. The genes required for oxidation of hypophosphite and phosphite by this organism were identified by using transposon mutagenesis and include homologs of the ptxD and htxA genes of Pseudomonas stutzeri WM88, which encode an NAD-dependent phosphite dehydrogenase (PtxD) and 2-oxoglutarate-dependent hypophosphite dioxygenase (HtxA). This organism also has the htxB, htxC, and htxD genes that comprise an ABC-type transporter, presumably for hypophosphite and phosphite transport. The role of these genes in reduced phosphorus metabolism was confirmed by analyzing the growth of mutants in which these genes were deleted. Sequencing data showed that htxA, htxB, htxC, and htxD are virtually identical to their homologs in P. stutzeri at the DNA level, indicating that horizontal gene transfer occurred. However, A. faecalis ptxD is very different from its P. stutzeri homolog and represents a new ptxD lineage. Therefore, this gene has ancient evolutionary roots in bacteria. These data suggest that there is strong evolutionary selection for the ability of microorganisms to oxidize hypophosphite and phosphite.

  6. A combination of site-directed mutagenesis and chemical modification to improve diastereopreference of Pseudomonas alcaligenes lipase.

    PubMed

    Chen, Hui; Wu, Jianping; Yang, Lirong; Xu, Gang

    2013-12-01

    A combination of site-directed mutagenesis and chemical modification was employed to alter protein structure with the objective of improving diastereopreference over that achieved by simple site-directed mutagenesis. Conformational analysis using molecular dynamic (MD) simulation of Pseudomonas alcaligenes lipase (PAL) indicated that stronger steric exclusion and structural rigidity facilitated diastereopreference. A cysteine (Cys) residue was introduced using site-directed mutagenesis to construct variant A272C. The modifier 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was then reacted with the introduced Cys residue to provide stronger steric exclusion and structural rigidity. The modification was verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Diastereopreference was improved significantly. The diastereomeric excess (dep) of l-menthol increased from 35% with wild type PAL to 90% with A272C-DTNB modified PAL when the conversion ratio of l-menthyl propionate was nearly 100%. Conformation and kinetic parameter analysis showed that A272C-DTNB modified PAL exhibited stronger steric exclusion and increased structural rigidity around the modification site that inhibited the hydrolysis of non-targeted substrates. The combination of site-directed mutagenesis and chemical modification could be an effective method to alter protein properties and enhance diastereopreference through the combined effect of steric exclusion and structural rigidity.

  7. Metabolism of 2-, 3- and 4-hydroxybenzoates by soil isolates Alcaligenes sp. strain PPH and Pseudomonas sp. strain PPD.

    PubMed

    Deveryshetty, Jaigeeth; Suvekbala, V; Varadamshetty, Gautham; Phale, Prashant S

    2007-03-01

    Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment culture technique utilize 2-, 3- and 4-hydroxybenzoates as the sole source of carbon and energy. The degradation pathways were elucidated by performing whole-cell O(2) uptake, enzyme activity and induction studies. Depending on the mixture of carbon source and the preculture condition, strain PPH was found to degrade 2-hydroxybenzoate either via the catechol or gentisate route and has both salicylate 1-hydroxylase and salicylate 5-hydroxylase. Strain PPD utilizes 2-hydroxybenzoate via gentisate. Both strains degrade 3- and 4-hydroxybenzoate via gentisate and protocatechuate, respectively. Enzymes were induced by respective hydroxybenzoate. Growth pattern, O(2) uptake and enzyme activity profiles on the mixture of three hydroxybenzoates as a carbon source suggest coutilization by both strains. When 3- or 4-hydroxybenzoate grown culture was used as an inoculum, strain PPH failed to utilize 2-hydroxybenzoate via catechol, indicating the modulation of the metabolic pathways, thus generating metabolic diversity.

  8. Enantioselective acylation of β-phenylalanine acid and its derivatives catalyzed by penicillin G acylase from Alcaligenes faecalis.

    PubMed

    Li, Dengchao; Ji, Lilian; Wang, Xinfeng; Wei, Dongzhi

    2013-01-01

    This study developed a simple, efficient method for producing racemic β-phenylalanine acid (BPA) and its derivatives via the enantioselective acylation catalyzed by the penicillin G acylase from Alcaligenes faecalis (Af-PGA). When the reaction was run at 25°C and pH 10 in an aqueous medium containing phenylacetamide and BPA in a molar ratio of 2:1, 8 U/mL enzyme and 0.1 M BPA, the maximum BPA conversion efficiency at 40 min only reached 36.1%, which, however, increased to 42.9% as the pH value and the molar ratio of phenylacetamide to BPA were elevated to 11 and 3:1, respectively. Under the relatively optimum reaction conditions, the maximum conversion efficiencies of BPA derivatives all reached about 50% in a relatively short reaction time (45-90 min). The enantiomeric excess value of product (ee(p)) and enantiomeric excess value of substrate (ee(s)) were all above 98% and 95%, respectively. These results suggest that the method established in this study is practical, effective, and environmentally benign and may be applied to industrial production of enantiomerically pure BPA and its derivatives.

  9. The sulfonated osmolyte N-methyltaurine is dissimilated by Alcaligenes faecalis and by Paracoccus versutus with release of methylamine.

    PubMed

    Weinitschke, Sonja; Denger, Karin; Smits, Theo H M; Hollemeyer, Klaus; Cook, Alasdair M

    2006-04-01

    Selective enrichments yielded bacterial cultures able to utilize the osmolyte N-methyltaurine as sole source of carbon and energy or as sole source of fixed nitrogen for aerobic growth. Strain MT1, which degraded N-methyltaurine as a sole source of carbon concomitantly with growth, was identified as a strain of Alcaligenes faecalis. Stoichiometric amounts of methylamine, whose identity was confirmed by matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry, and of sulfate were released during growth. Inducible N-methyltaurine dehydrogenase, sulfoacetaldehyde acetyltransferase (Xsc) and a sulfite dehydrogenase could be detected. Taurine dehydrogenase was also present and it was hypothesized that taurine dehydrogenase has a substrate range that includes N-methyltaurine. Partial sequences of a tauY-like gene (encoding the putative large component of taurine dehydrogenase) and an xsc gene were obtained by PCR with degenerate primers. Strain N-MT utilized N-methyltaurine as a sole source of fixed nitrogen for growth and could also utilize the compound as sole source of carbon. This bacterium was identified as a strain of Paracoccus versutus. This organism also expressed inducible (N-methyl)taurine dehydrogenase, Xsc and a sulfite dehydrogenase. The presence of a gene cluster with high identity to a larger cluster from Paracoccus pantotrophus NKNCYSA, which is now known to dissimilate N-methyltaurine via Xsc, allowed most of the overall pathway, including transport and excretion, to be defined. N-Methyltaurine is thus another compound whose catabolism is channelled directly through sulfoacetaldehyde.

  10. Purification and characterization of chitinase from Alcaligenes faecalis AU02 by utilizing marine wastes and its antioxidant activity.

    PubMed

    Annamalai, Neelamegam; Veeramuthu Rajeswari, Mayavan; Vijayalakshmi, Shanmugam; Balasubramanian, Thangavel

    2011-12-01

    Marine waste is an abundant renewable source for the recovery of several value added metabolites with potential industrial applications. This study describes the production of chitinase on marine waste, with the subsequent use of the same marine waste for the extraction of antioxidants. A chitinase-producing bacterium isolated from seafood effluent was identified as Alcaligenes faecalis AU02. Optimal chitinase production was obtained in culture conditions of 37°C for 72 h in 100 ml medium containing 1% shrimp and crab shell powder (1:1) (w/v), 0.1% K(2)HPO(4), and 0.05% MgSO(4)·7H(2)O. The molecular weight of chitinase was determined by SDS-PAGE to be 36 kDa. The optimum pH, temperature, pH stability, and thermal stability of chitinase were about 8, 37°C, 5-12, and 40-80°C, respectively. The antioxidant activity of A. faecalis AU02 culture supernatant was determined through scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) as 84%, and the antioxidant compound was characterized by TLC and its FT-IR spectrum. The present study proposed that marine wastes can be utilized to generate a high-value-added product and that pharmacological studies can extend its use to the field of medicine.

  11. Heterotrophic nitrification and aerobic denitrification of high-strength ammonium in anaerobically digested sludge by Alcaligenes faecalis strain No. 4.

    PubMed

    Shoda, Makoto; Ishikawa, Yoichi

    2014-06-01

    Alcaligenes faecalis strain No. 4 which is capable of heterogeneous nitrification and aerobic denitrification, was used to remove high-strength ammonium (approximately 1 g NH4(+)-N/l) from digested sludge, the product of an anaerobic digestion reactor, in which methane was produced from excess municipal sewage sludge. Repeated batch operations were conducted at 20°C and 30°C for 550 h, using a jar fermentor. The removal ratios of high-strength ammonium reached 90-100% within 24 h, and the average ammonium removal rate was 2.9 kg-N/m(3)/day, more than 200 times higher than that in conventional nitrification-denitrification processes. During these operations, the cell density was maintained at 10(8)-10(9) cells of A. faecalis strain No. 4/ml. At 3% NaCl in the digested sludge, strain No. 4 exhibited an ammonium removal rate of 3 kg-N/m(3)/day.

  12. Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity.

    PubMed

    Nageshwar, Y V D; Sheelu, Gurrala; Shambhu, Rekha Rao; Muluka, Hemalatha; Mehdi, Nooreen; Malik, M Shaheer; Kamal, Ahmed

    2011-06-01

    Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.

  13. Identification of a new Alcaligenes faecalis strain MOR02 and assessment of its toxicity and pathogenicity to insects.

    PubMed

    Quiroz-Castañeda, Rosa Estela; Mendoza-Mejía, Ared; Obregón-Barboza, Verónica; Martínez-Ocampo, Fernando; Hernández-Mendoza, Armando; Martínez-Garduño, Felipe; Guillén-Solís, Gabriel; Sánchez-Rodríguez, Federico; Peña-Chora, Guadalupe; Ortíz-Hernández, Laura; Gaytán-Colín, Paul; Dantán-González, Edgar

    2015-01-01

    We report the isolation of a bacterium from Galleria mellonella larva and its identification using genome sequencing and phylogenomic analysis. This bacterium was named Alcaligenes faecalis strain MOR02. Microscopic analyses revealed that the bacteria are located in the esophagus and intestine of the nematodes Steinernema feltiae, S. carpocapsae, and H. bacteriophora. Using G. mellonella larvae as a model, when the larvae were injected with 24,000 CFU in their hemocoel, more than 96% mortality was achieved after 24 h. Additionally, toxicity assays determined that 1 μg of supernatant extract from A. faecalis MOR02 killed more than 70% G. mellonella larvae 96 h after injection. A correlation of experimental data with sequence genome analyses was also performed. We discovered genes that encode proteins and enzymes that are related to pathogenicity, toxicity, and host/environment interactions that may be responsible for the observed phenotypic characteristics. Our data demonstrates that the bacteria are able to use different strategies to colonize nematodes and kill insects to their own benefit. However, there remains an extensive group of unidentified microorganisms that could be participating in the infection process. Additionally, a nematode-bacterium association could be established probably as a strategy of dispersion and colonization.

  14. New molecular packing in a crystal of pseudoazurin from Alcaligenes faecalis: a double-helical arrangement of blue copper.

    PubMed

    Fukuda, Yohta; Mizohata, Eiichi; Inoue, Tsuyoshi

    2017-03-01

    Pseudoazurin from the denitrifying bacterium Alcaligenes faecalis (AfPAz) is a blue copper protein and functions as an electron donor to copper-containing nitrite reductase (CuNIR). Conventionally, AfPAz has been crystallized using highly concentrated ammonium sulfate as a precipitant. Here, a needle-like crystal of AfPAz grown in a solution containing a macromolecular precipitant, polyethylene glycol 8000 (PEG 8000), is reported. The crystal belonged to space group P61, with unit-cell parameters a = b = 68.7, c = 94.2 Å. The structure has been determined and refined at 2.6 Å resolution. The asymmetric unit contained two AfPAz molecules contacting each other on negatively charged surfaces. The molecular packing of the crystal showed a right-handed double-helical arrangement of AfPAz molecules and hence of blue copper sites. This structure provides insight into the excluded-volume effect of PEG and the manner of assembly of AfPAz.

  15. [Evaluation of occurrence of Alcaligenes faecalis in clinical samples of patients of the university hospital in Bydgoszcz].

    PubMed

    Jachna-Sawicka, Katarzyna; Gospodarek, Eugenia

    2009-01-01

    Alcaligenes faecalis is an aerobic Gram-negative, non-fermentative rod. It's saprophyte of water and soil. It may be recovered from wet places of hospital environment. It is considered as an opportunistic pathogen. The aim of this review was evaluation of occurrence in clinical samples and susceptibility to antibiotics of 72 A. faecalis strains isolated in years 2003-2008. Over 30% of strains were isolated from patients in surgical ward, 19.6% from patients in outpatient clinic and almost 14% from patients in Department of Dermatology. 70.8% of strains were isolated from purulent material samples, whereas from urine--16.7% of strains. Nearly 88% out of examined strains were grown in mixed culture together with one (26.4%), two (32.0%), three (23.6%) or four (5.6%) microorganisms. All out of strains were sensitive to piperacyline, piperacyline/tazobactam and carbapenems. Sensitivity to aztreonam was observed at 22.2% of strains and to co-trimoxazole at 57.1% of strains.

  16. Arsenite oxidation by immobilized cells of Alcaligenes faecalis strain O1201 in a fluidized-bed reactor.

    PubMed

    Wang, Yi-Tin; Suttigarn, Arthon; Dastidar, Aniruddha

    2009-02-01

    Arsenic(III) oxidation was evaluated in a continuous-flow fluidized-bed reactor (FBR) with Alcaligenes faecalis strain 01201 immobilized in gel beads. The FBR was operated under 300 mg/L citrate and a range of influent As(III) concentrations (75 to 3000 mg/L) at short hydraulic retention times (1.06 to 3.17 hours). The pH and temperature in the FBR were maintained at optimal growth conditions for strain O1201 (pH 7 and 30 degrees C) throughout the study. A total of 10 quasi-steady-state operating conditions were obtained after 54 days of operation under an As(III) concentration of 441 mg/L (10 000 mg/L/d loading rate), with As (III) removal efficiency ranging from 76% to near complete. Material balance analysis over the FBR revealed that the difference between the cumulative influent As (III) and the sum of cumulative effluent As(III) and As(V) was insignificant. The major mechanism of As(III) removal from the FBR is biological oxidation to As(V).

  17. Effect of Alcaligenes faecalis on nitrous oxide emission and nitrogen removal in three phase fluidized bed process.

    PubMed

    Kim, Jeong-Sook; Kim, Shi-Jun; Lee, Byung-Hun

    2004-01-01

    Nitrous oxide (N2O), one of the greenhouse effect gases, has not been known that how much N2O is produced from municipal wastewater treatment and what its management should be. In this study, for controlling nitrous oxide emission and removing nitrogen from municipal wastewater, we experimented the three phase fluidized bed process equipped with draft tube along with immobilized Alcaligenes faecalis, a typical heterotrophic nitrifer and a predominant genus. Also we evaluated the optimum treatment condition of the three phase fluidized bed process for emitting nitrous oxide. The results of this study showed that the three phase fluidized bed process was more effective than the activated sludge process for controlling nitrous oxide emission and removing nitrogen. Increasing amount of A. faecalis in reactor should be encouraged for controlling nitrous oxide emission and removing nitrogen. In addition, the activated sludge process using immobilized A. faecalis as a carrier had more nitrogen removal efficiency than conventional activated sludge process. The accumulation of NO2-N, NO3-N resulted in high N2O emission. Therefore, we suggested that it is necessary to reduce NO2-N and NO3-N for both reducing N2O emission and improving nitrogen removal.

  18. Genetic Diversity and Horizontal Transfer of Genes Involved in Oxidation of Reduced Phosphorus Compounds by Alcaligenes faecalis WM2072

    PubMed Central

    Wilson, Marlena M.; Metcalf, William W.

    2005-01-01

    Enrichment was performed to isolate organisms that could utilize reduced phosphorus compounds as their sole phosphorus sources. One isolate that grew well with either hypophosphite or phosphite was identified by 16S rRNA gene analysis as a strain of Alcaligenes faecalis. The genes required for oxidation of hypophosphite and phosphite by this organism were identified by using transposon mutagenesis and include homologs of the ptxD and htxA genes of Pseudomonas stutzeri WM88, which encode an NAD-dependent phosphite dehydrogenase (PtxD) and 2-oxoglutarate-dependent hypophosphite dioxygenase (HtxA). This organism also has the htxB, htxC, and htxD genes that comprise an ABC-type transporter, presumably for hypophosphite and phosphite transport. The role of these genes in reduced phosphorus metabolism was confirmed by analyzing the growth of mutants in which these genes were deleted. Sequencing data showed that htxA, htxB, htxC, and htxD are virtually identical to their homologs in P. stutzeri at the DNA level, indicating that horizontal gene transfer occurred. However, A. faecalis ptxD is very different from its P. stutzeri homolog and represents a new ptxD lineage. Therefore, this gene has ancient evolutionary roots in bacteria. These data suggest that there is strong evolutionary selection for the ability of microorganisms to oxidize hypophosphite and phosphite. PMID:15640200

  19. Antimicrobial activity and determination of bioactive components from marine Alcaligenes faecalis extract against a sulfate-reducing bacteria

    NASA Astrophysics Data System (ADS)

    AbdSharad, Ali; Usup, Gires; Sahrani, Fathul Karim; Ahmad, Asmat

    2016-11-01

    Biogenic souring and microbial-influenced corrosion is a common scenario in petroleum reservoir. The serious threat normally comes from sulfate-reducing bacteria (SRB). Alcaligenes faecalis was tested in this study for the ability to inhibit the growth of SRB. Ethyl acetate extraction of A. faecalis grown in marine broth was carried out to produce crude ethyl acetate of A. faecalis (CEAF). CEAF was diluted at concentrations 0.2-12.8 mg/mL and was tested for anti-microbial activity by microdilution susceptibility tests in 96-wells plate. CEAF was then analyzed by Gas Chromatography Mass Spectrometry (GC-MS). The microdilution susceptibility tests showed that the crude have anti- microbial activities on SRB. CEAF showed immediate killing effect against SRB in liquid medium which suggest the presence of active chemical compounds with antimicrobial activity. The GC-MS analysis showed the presence of 20 different chemical compounds in CEAF, The major components in CEAF can be related to antimicrobial, antifungal, antioxidant, pesticide, metabolism, toxicity, anticancer and corrosion inhibition activities. In conclusion, crude ethyl acetate extract of A. faecalis has the ability to inhibit SRB growth.

  20. An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid.

    PubMed

    Bhatia, S K; Mehta, P K; Bhatia, R K; Bhalla, T C

    2013-05-01

    Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V max and K m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 μmol mg(-1) min(-1) and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml (-) resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.

  1. Replacement of tyrosine 181 by phenylalanine in gentisate 1,2-dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 enhances catalytic activities.

    PubMed

    Tan, Chew Ling; Yeo, Chew Chieng; Khoo, Hoon Eng; Poh, Chit Laa

    2005-11-01

    xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k(cat)/Km) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.

  2. Curdlan, a (1----3)-beta-D-glucan from Alcaligenes faecalis var. myxogenes IFO13140, activates the alternative complement pathway by heat treatment.

    PubMed

    Matsushita, M

    1990-10-01

    From the results of consumption experiments of guinea pig complement, Curdlan, a (1----3)-beta-D-glucan obtained from Alcaligenes faecalis var. myxogenes IFO13140, has been found to lack the ability to activate complement when unheated or preheated at either 40 degrees C or 50 degrees C. However, Curdlan heated at or above 60 degrees C increased complement consumption. This activation, dependent on the temperature of the Curdlan, was via the alternative complement pathway as assessed by cleavage of factor B into Ba and Bb fragments. These results suggest a substantial change must occur in Curdlan with heat treatment for alternative pathway activation.

  3. Molecular Characterization of a Novel ortho-Nitrophenol Catabolic Gene Cluster in Alcaligenes sp. Strain NyZ215▿

    PubMed Central

    Xiao, Yi; Zhang, Jun-Jie; Liu, Hong; Zhou, Ning-Yi

    2007-01-01

    Alcaligenes sp. strain NyZ215 was isolated for its ability to grow on ortho-nitrophenol (ONP) as the sole source of carbon, nitrogen, and energy and was shown to degrade ONP via a catechol ortho-cleavage pathway. A 10,152-bp DNA fragment extending from a conserved region of the catechol 1,2-dioxygenase gene was obtained by genome walking. Of seven complete open reading frames deduced from this fragment, three (onpABC) have been shown to encode the enzymes involved in the initial reactions of ONP catabolism in this strain. OnpA, which shares 26% identity with salicylate 1-monooxygenase of Pseudomonas stutzeri AN10, is an ONP 2-monooxygenase (EC 1.14.13.31) which converts ONP to catechol in the presence of NADPH, with concomitant nitrite release. OnpC is a catechol 1,2-dioxygenase catalyzing the oxidation of catechol to cis,cis-muconic acid. OnpB exhibits 54% identity with the reductase subunit of vanillate O-demethylase in Pseudomonas fluorescens BF13. OnpAB (but not OnpA alone) conferred on the catechol utilizer Pseudomonas putida PaW340 the ability to grow on ONP. This suggests that OnpB may also be involved in ONP degradation in vivo as an o-benzoquinone reductase converting o-benzoquinone to catechol. This is analogous to the reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone by a tetrachlorobenzoquinone reductase (PcpD, 38% identity with OnpB) in the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723. PMID:17616586

  4. Class 1 integrons and tetracycline resistance genes in alcaligenes, arthrobacter, and Pseudomonas spp. isolated from pigsties and manured soil.

    PubMed

    Agersø, Yvonne; Sandvang, Dorthe

    2005-12-01

    The presence of tetracycline resistance (Tc(r)) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc(r) gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli, Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc(r) genes tet(A), tet(B), and tet(C), and all (n = 72) gram-positive isolates were screened for tet(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet(B). All gram-positive isolates with in-1 also contained tet(33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc(r) and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc(r) but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.

  5. Molecular characterization of an inducible gentisate 1,2-dioxygenase gene, xlnE, from Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Yeo, Chew Chieng; Wong, Mark Vee-Meng; Feng, Yongmei; Song, Keang Peng; Poh, Chit Laa

    2003-07-17

    Pseudomonas alcaligenes NCIMB 9867 (strain P25X) produces isofunctional enzymes of the gentisate pathway that enables the degradation of xylenols and cresols via gentisate. Previous reports had indicated that one set of enzymes is constitutively expressed whereas the other set is strictly inducible by aromatic hydrocarbon substrates. The gene encoding gentisate 1,2-dioxygenase (GDO), the enzyme that catalyzes the cleavage of the gentisate aromatic ring, was cloned from strain P25X. The GDO gene, designated xlnE, is 1,044 bp, and is part of a 5.4 kb operon which consists of six genes, xlnEFGHID. Transcription of this operon was driven by a sigma 70-type promoter, PxlnE, located 123 bp upstream of the xlnE start codon. Primer extension analysis showed that the xlnE transcription start point is located at the -87 adenine residue. In a P25X xlnE knockout mutant, GDO activity could only be detected when cells were grown in the presence of aromatic substrates, suggesting that xlnE encodes for the constitutive copy of GDO. This was verified by constructing a P25X strain with xlnE transcriptionally fused to a promoterless catechol 2,3-dioxygenase gene. In this strain, catechol 2,3-dioxygenase activity was detected in cells that were grown in the absence of aromatic inducers. However, catechol 2,3-dioxygenase activity increased up to four fold when these cells were grown in the presence of aromatic substrates, in particular 3-hydroxybenzoate. Thus, xlnE is in fact, inducible and the constitutive activity observed under non-inducing conditions was due to its relatively high basal levels of expression.

  6. Molecular characterization of a novel ortho-nitrophenol catabolic gene cluster in Alcaligenes sp. strain NyZ215.

    PubMed

    Xiao, Yi; Zhang, Jun-Jie; Liu, Hong; Zhou, Ning-Yi

    2007-09-01

    Alcaligenes sp. strain NyZ215 was isolated for its ability to grow on ortho-nitrophenol (ONP) as the sole source of carbon, nitrogen, and energy and was shown to degrade ONP via a catechol ortho-cleavage pathway. A 10,152-bp DNA fragment extending from a conserved region of the catechol 1,2-dioxygenase gene was obtained by genome walking. Of seven complete open reading frames deduced from this fragment, three (onpABC) have been shown to encode the enzymes involved in the initial reactions of ONP catabolism in this strain. OnpA, which shares 26% identity with salicylate 1-monooxygenase of Pseudomonas stutzeri AN10, is an ONP 2-monooxygenase (EC 1.14.13.31) which converts ONP to catechol in the presence of NADPH, with concomitant nitrite release. OnpC is a catechol 1,2-dioxygenase catalyzing the oxidation of catechol to cis,cis-muconic acid. OnpB exhibits 54% identity with the reductase subunit of vanillate O-demethylase in Pseudomonas fluorescens BF13. OnpAB (but not OnpA alone) conferred on the catechol utilizer Pseudomonas putida PaW340 the ability to grow on ONP. This suggests that OnpB may also be involved in ONP degradation in vivo as an o-benzoquinone reductase converting o-benzoquinone to catechol. This is analogous to the reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone by a tetrachlorobenzoquinone reductase (PcpD, 38% identity with OnpB) in the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723.

  7. Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

    PubMed Central

    Okazaki, Fumiyoshi; Tamaru, Yutaka; Hashikawa, Shinnosuke; Li, Yu-Teh; Araki, Toshiyoshi

    2002-01-01

    A β-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the Kd and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan. PMID:11948152

  8. Microbial degradation of alkylbenzenesulphonates. Metabolism of homologues of short alkyl-chain length by an Alcaligenes sp

    PubMed Central

    Bird, J. Anthony; Cain, Ronald B.

    1974-01-01

    1. An organism isolated from sewage and identified as an Alcaligenes sp. utilized benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate as sole source of carbon and energy for growth. Higher alkylbenzenesulphonate homologues and the hydrocarbons, benzene, toluene, phenylethane and 1-phenyldodecane were not utilized. 2. 2-Phenylpropanesulphonate was metabolized to 4-isopropylcatechol. 3. 1-Phenylpropanesulphonate was metabolized to an ortho-diol, which was tentatively identified, in the absence of an authentic specimen, as 4-n-propylcatechol. 4. In the presence of 4-isopropylcatechol, which inhibited catechol 2,3-dioxygenase, 4-ethylcatechol accumulated in cultures growing on phenylethane-p-sulphonate. 5. Authentic samples of catechol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol and 3-isopropylcatechol were oxidized by heat-treated extracts to the corresponding 2-hydroxyalkylmuconic semialdehydes. Ring cleavage occurred between C-2 and C-3. 6. The catechol derived from 1-phenylpropanesulphonate was oxygenated by catechol 2,3-dioxygenase to a compound with all the properties of a 2-hydroxyalkylmuconic semialdehyde, but it was not rigorously identified. 7. The catechol 2,3-dioxygenase induced by growth on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate showed a constant ratio of specific activities with catechol, 3-methylcatechol, 4-methylcatechol and 4-ethylcatechol that was independent of the growth substrate. At 60°C, activity towards these substrates declined at an identical first-order rate. 8. Enzymes of the `ortho' pathway of catechol metabolism were present in small amounts in cells grown on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate. 9. The catechol 1,2-dioxygenase oxidized the alkylcatechols, but the rates and the total extents of oxidation were less than for catechol itself. The oxidation products of these alkylcatechols were not further metabolized. PMID:4375955

  9. Separation and characterization of effective demulsifying substances from surface of Alcaligenes sp. S-XJ-1 and its application in water-in-kerosene emulsion.

    PubMed

    Huang, Xiangfeng; Peng, Kaiming; Feng, Yi; Liu, Jia; Lu, Lijun

    2013-07-01

    The main goal of this work was to analyze the effect of surface substances on demulsifying capability of the demulsifying strain Alcaligenes sp. S-XJ-1. The demulsifying substances were successfully separated from the cell surface with dichloromethane-alkali treatment, and exhibited 67.5% of the demulsification ratio for water-in-kerosene emulsions at a dosage of 356mg/L. FT-IR, TLC and ESI-MS analysis confirmed the presence of a carbohydrate-protein-lipid complex in the demulsifying substances with the major molecular ions from mass-to-charge ratio (m/z) 165 to 814. After the substances separated, the cell morphology changed from aggregated to dispersed, and the concentration of cell surface functional groups decreased. Cell surface hydrophobicity and the ability of cell adhesion to hydrophobic surface of the treated cells was also reduced compared with original cell. It was proved that the demulsifying substances had a significant effect on cell surface properties and accordingly with demulsifying capability of Alcaligenes sp. S-XJ-1. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Efficacy of polysaccharide from Alcaligenes xylosoxidans MSA3 administration as protection against γ-radiation in female rats.

    PubMed

    Hassan, Amal I; Ghoneim, Mona A M; Mahmoud, Manal G; Asker, Mohsen M S; Mohamed, Saher S

    2016-03-01

    Damage to normal tissues is a consequence of both therapeutic and accidental exposures to ionizing radiation. A water-soluble heteropolysaccharide called AXEPS, composed of glucose, galactose, rhamnose and glucouronic acid in a molar ratio of nearly 1.0:1.6:0.4:2.3, respectively, was isolated from culture medium of strain Alcaligenes xylosoxidans MSA3 by ethanol precipitation followed by freeze-drying. Chemical analysis, Fourier-transform infrared (FTIR) and chromatographic studies revealed that the molecular weight was 1.6 × 10(4) g mol(-1). This study was designed to investigate the radioprotective and biological effects of AXEPS in alleviating the toxicity of ionizing radiation in female albino rats. A total of 32 female albino rats were divided into four groups. In the control group, rats were administered vehicle by tube for four weeks. The second group was administered AXEPS (100 mg/kg) orally by gavage for four weeks. Animals in the third group were exposed to whole-body γ-rays (5 Gy) and remained for 2 weeks without treatment. The fourth group received AXEPS (100 mg/kg) orally by gavage for two weeks before being exposed to whole-body γ-rays (5 Gy), then 24 h post γ-rays, they received AXEPS (100 mg/kg) in a treatment continuing till the end of the experiment (15 days after the whole-body γ-irradiation). Oral administration of AXEPS (100 mg/kg) significantly reversed the oxidative stress effects of radiation, as evidenced by the decrease in DNA damage in the bone marrow. Assessment of apoptosis and cell proliferation markers revealed that caspase-3 significantly increased in the irradiated group. Moreover, a significant decrease in the hematological constituents of peripheral blood, the chemotactic index and CD8+ T cells were observed in animals in the irradiation-only group, whereas an increase in the lymphocyte index was observed in animals in that group. In contrast, AXEPS treatment prevented these alterations. From our results, we conclude that

  11. Efficacy of polysaccharide from Alcaligenes xylosoxidans MSA3 administration as protection against γ-radiation in female rats

    PubMed Central

    Hassan, Amal I.; Ghoneim, Mona A. M.; Mahmoud, Manal G.; Asker, Mohsen M. S.; Mohamed, Saher S.

    2016-01-01

    Damage to normal tissues is a consequence of both therapeutic and accidental exposures to ionizing radiation. A water-soluble heteropolysaccharide called AXEPS, composed of glucose, galactose, rhamnose and glucouronic acid in a molar ratio of nearly 1.0:1.6:0.4:2.3, respectively, was isolated from culture medium of strain Alcaligenes xylosoxidans MSA3 by ethanol precipitation followed by freeze-drying. Chemical analysis, Fourier-transform infrared (FTIR) and chromatographic studies revealed that the molecular weight was 1.6 × 104 g mol−1. This study was designed to investigate the radioprotective and biological effects of AXEPS in alleviating the toxicity of ionizing radiation in female albino rats. A total of 32 female albino rats were divided into four groups. In the control group, rats were administered vehicle by tube for four weeks. The second group was administered AXEPS (100 mg/kg) orally by gavage for four weeks. Animals in the third group were exposed to whole-body γ-rays (5 Gy) and remained for 2 weeks without treatment. The fourth group received AXEPS (100 mg/kg) orally by gavage for two weeks before being exposed to whole-body γ-rays (5 Gy), then 24 h post γ-rays, they received AXEPS (100 mg/kg) in a treatment continuing till the end of the experiment (15 days after the whole–body γ-irradiation). Oral administration of AXEPS (100 mg/kg) significantly reversed the oxidative stress effects of radiation, as evidenced by the decrease in DNA damage in the bone marrow. Assessment of apoptosis and cell proliferation markers revealed that caspase-3 significantly increased in the irradiated group. Moreover, a significant decrease in the hematological constituents of peripheral blood, the chemotactic index and CD8+ T cells were observed in animals in the irradiation-only group, whereas an increase in the lymphocyte index was observed in animals in that group. In contrast, AXEPS treatment prevented these alterations. From our results, we conclude that

  12. Purification and characterization of gentisate 1,2-dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869.

    PubMed

    Feng, Y; Khoo, H E; Poh, C L

    1999-03-01

    Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from Pseudomonas alcaligenes NCIB 9867 (P25X) and Pseudomonas putida NCIB 9869 (P35X), respectively. The estimated molecular mass of the purified P25X gentisate 1, 2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa. Its structure is deduced to be a tetramer. The pI of this enzyme was established to be 4.8 to 5.0. The subunit mass of P35X gentisate 1, 2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa. The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8. Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent Kms of 92 and 143 microM for gentisate, respectively. Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs. Both enzymes had similar kinetic turnover characteristics for gentisate, with kcat/Km values of 44.08 x 10(4) s-1 M-1 for the P25X enzyme and 39.34 x 10(4) s-1 M-1 for the P35X enzyme. Higher kcat/Km values were expressed by both enzymes against the substituted gentisates. Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases. The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0. The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0. The optimal temperature for both P25X and P35X gentisate 1, 2-dioxygenases was around 50 degrees C, but the P35X enzyme was more heat stable than that from P25X. Both enzymes were strongly stimulated by 0.1 mM Fe2+ but were completely inhibited by the presence of 5 mM Cu2+. Partial inhibition of both enzymes was also observed with 5 mM Mn2+, Zn2+, and EDTA.

  13. Nickel(II)-substituted azurin I from Alcaligenes xylosoxidans as characterized by resonance Raman spectroscopy at cryogenic temperature.

    PubMed

    Fitzpatrick, Marzena B; Czernuszewicz, Roman S

    2009-05-01

    Metal-substituted blue copper proteins (cupredoxins) have been successfully used to study the effect of metal-ion identity on their active-site properties, specifically the coordination geometry and metal-ligand bond strengths. In this work, low-temperature (77 K) resonance Raman (RR) spectra of the blue copper protein Alcaligenes xylosoxidans azurin I and its Ni(II) derivative are reported. A detailed analysis of all observed bands is presented and responsiveness to metal substitution is discussed in terms of structural and bonding changes. The native cupric site exhibits a RR spectrum characteristic of a primarily trigonal planar (type 1) coordination geometry, identified by the nu(Cu-S)(Cys) markers at 373, 399, 409, and 430 cm(-1). Replacement of Cu(II) with Ni(II) results in optical and RR spectra that reveal (1) a large hypsochromic shift in the main (Cys)S --> M(II) charge-transfer absorption from 622 to 440 nm, (2) greatly reduced metal-thiolate bonding interaction, indicated by substantially lower nu(Ni-S)(Cys) stretching frequencies, (3) elevation of the cysteine nu(C( beta )-S) stretching, amide III, and rho (s)(C( beta )H(2)) scissors vibrational modes, and (4) primarily four-coordinated, trigonally distorted tetrahedral geometry of the Ni(II) site that is marked by characteristic nu(Ni-S)(Cys) stretching RR bands at 347, 364, and 391 cm(-1). Comparisons of the electronic and vibrational properties between A. xylosoxidans azurin I and its closely structurally related azurin from Pseudomonas aeruginosa are made and discussed. For cupric azurins, the intensity-weighted average M(II)-S(Cys) stretching frequencies are calculated to be nu(Cu-S)(iwa) = 406.3 and 407.6 cm(-1), respectively. These values decreased to nu(Ni-S)(iwa) = 359.3 and 365.5 cm(-1), respectively, after Ni(II) --> Cu(II) exchange, suggesting that the metal-thiolate interactions are similar in the two native proteins but are much less alike in their Ni(II)-substituted forms.

  14. Uptake of benzoic acid and chloro-substituted benzoic acids by alcaligenes denitrificans BRI 3010 and BRI 6011

    SciTech Connect

    Miguez, C.B.; Ingram, J.M.; MacLeod, R.A.

    1995-12-01

    The mechanism of uptake of benzoic and 2,4-dichlorobenzoic acid (2,4-DCBA) by Alcaligenes denitrificans BRI 3010 and BRI 6011 and Pseudomonas sp. strain B13, three organisms capable of degrading isomers of chlorinated benzoic acids, was investigated. In all three organisms, uptake of benzoic acid was inducible. For benzoic acid uptake into BRI 3010, monophasic saturation kinetics with apparent K{sub m} and V{sub max} values of 1.4 {mu}M and 3.2 nmol/min/mg of cell dry weight, respectively, were obtained. For BRI 6011, biphasic saturation kinetics were observed, suggesting presence of two uptake systems for benzoic acid with distinct K{sub m} (0.72 and 5.3 {mu}M) and V{sub max} (3.3 and 4.6 nmol/min/mg of cell dry weight) values. BRI 3010 and BRI 6011 accumulated benzoic acid against a concentration gradient by a factor of 8 and 10, respectively. A wide range of structural analogs, at 50-fold excess concentrations, inhibited benzoic acid uptake by BRI 3010 and BRI 6011, whereas with B13, only 3-chlorobenzoic acid was an effective inhibitor. For BRI 3010 and BRI 6011, the inhibition by the structural analogs was not of a competitive nature. Uptake of benzoic acid by BRI 3010 and BRI 6011 was inhibited by KCN, by the protonophore 3,5,3`, 4`-tetrachlorosalicylanilide (TCS), and, for BRI 6011, by anaerobiosis unless nitrate was present, thus indicating that energy was required for the uptake process. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation uptake kinetics were not observed. Uptake of 2,4-DCBA by BRI 6011 was inhibited by KCN, TCS, and anaerobiosis even if nitrate was present, but the compound was not accumulated intracellularly against a concentration gradient. Uptake of 2,4-DCBA by BRI 6011 appears to occur by passive diffusion into the cell down its concentration gradient, which is maintained by the intracellular metabolism of the compound. This process could play an important role in the degradation of xenobiotic compounds by microorganisms.

  15. DEGRADATION OF THE CHLORINATED PHENOXYACETATE HERBICIDES 2,4-DICHLOROPHENOXYACETIC ACID AND 2,4,5- TRICHLOROPHENOXYACETIC BY PURE AND MIXED BACTERIAL CULTURES

    EPA Science Inventory

    Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compo...

  16. Final Treatability Study in Support of Remediation by Natural Attenuation Site FT-1 at Fairchild Air Force Base, Spokane, Washington

    DTIC Science & Technology

    1997-10-01

    Altenschmidt, U. and Fuchs, G., 1991, Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: Indication for toluene methyihydroxylation and...Washington. July. * Harker, A.R., and Kim, Y., 1990, Trichloroethylene degradation by two independent aromatic-degrading pathways in Alcaligenes eutrophus

  17. DEGRADATION OF THE CHLORINATED PHENOXYACETATE HERBICIDES 2,4-DICHLOROPHENOXYACETIC ACID AND 2,4,5- TRICHLOROPHENOXYACETIC BY PURE AND MIXED BACTERIAL CULTURES

    EPA Science Inventory

    Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compo...

  18. Treatability Study in Support of the Intrinsic Remediation Option at the Christmas Tree Fire Training Area at Westover Air Reserve Base Chicopee, Massachusetts

    DTIC Science & Technology

    1997-01-01

    Summary. Brooks Air Force Base, p. 8. Altenschmidt, U. and Fuchs, G., 1991, Anaerobic degradation of toluene in denitrifying Pseudomonas sp...Trichloroethylene degradation by two independent aromatic-degrading pathways in Alcaligenes eutrophus JMP134: Appl. Environ. Microbiol., vol. 56, no. 4, p. 1179

  19. Determination of the active sites serine of the poly (3-hydroxybutyrate) depolymerases of Pseudomonas lemoignei (PhaZ5) and of Alcaligenes faecalis.

    PubMed

    Shinohe, T; Nojiri, M; Saito, T; Stanislawski, T; Jendrossek, D

    1996-07-15

    Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S138 (P. lemoignei) and S139 (A. faecalis) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase box of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S195 (P. lemoignei) and S196 (A. faecalis), resulted in mutant proteins with almost full activity and proved that S195 and S196 are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.

  20. Lipase A gene transcription in Pseudomonas alcaligenes is under control of RNA polymerase σ54 and response regulator LipR.

    PubMed

    Krzeslak, Joanna; Papaioannou, Evelina; van Merkerk, Ronald; Paal, Krisztina A; Bischoff, Rainer; Cool, Robbert H; Quax, Wim J

    2012-04-01

    Initial analysis has shown that the transcription of the Pseudomonas alcaligenes lipA gene, which encodes an extracellular lipase, is governed by the LipQR two-component system consisting of sensor kinase LipQ and DNA-binding regulator LipR. This study further analyzes lipA gene expression and demonstrates that the RNA polymerase σ54 is involved in the transcription. Purified LipR has an ATPase activity that is stimulated by the presence of lipA promoter DNA. Surface plasmon resonance measurements with purified and in vitro phosphorylated LipR reveal that phosphorylation of LipR is required for specific binding to the upstream activating sequence of the lipA promoter. Furthermore, mass spectrometric analysis combined with mutagenesis demonstrates that Asp52 is the phosphorylated aspartate. This analysis exposes LipR as a prominent member of the growing family of bacterial enhancer-binding proteins.

  1. Biocontrol Potential of Siderophore Producing Heavy Metal Resistant Alcaligenes sp. and Pseudomonas aeruginosa RZS3 vis-à-vis Organophosphorus Fungicide.

    PubMed

    Sayyed, R Z; Patel, P R

    2011-07-01

    In present study in vitro phytopathogen suppression activity of siderophoregenic preparations of Ni and Mn resistant Alcaligenes sp. STC1 and Pseudomonas aeruginosa RZS3 SH-94B isolated from soil were found superior over the chemical pesticide. Siderophore rich culture broth and siderophore rich supernatant exerted antifungal activity against Aspergillus niger NCIM 1025, Aspergillus flavus NCIM 650, Fusarium oxysporum NCIM 1281, Alternaria alternata ARI 715, Cercospora arachichola, Metarhizium anisopliae NCIM 1311 and Pseudomonas solanacerum NCIM 5103. Siderophore rich broth and supernatant exhibited potent antifungal activity vis-à-vis oraganophosphorus chemical fungicide; kitazine. The minimum fungicidal concentration required was 25 μl for Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, Cercospora arachichola, Metarhizium anisopliae, Pseudomonas solanacerum and 75 μl for A. alternata.

  2. Cloning and functional analysis by gene disruption of a novel gene involved in indigo production and fluoranthene metabolism in Pseudomonas alcaligenes PA-10.

    PubMed

    Alemayehu, D; Gordon, L M; O'Mahony, M M; O'Leary, N D; Dobson, A D W

    2004-10-15

    A novel indole dioxygenase (idoA) gene has been cloned from Pseudomonas alcaligenes PA-10, based on its ability to convert indole to indigo. The chromosomally encoded idoA gene exhibits no similarity to previously cloned naphthalene dioxygenases or to aromatic oxygenases from other species at the nucleotide level. Phylogenetic analysis indicates that the idoA gene product is most similar to an acyl-CoA dehydrogenase from Novosphingobium aromaticivorans. The enzyme encoded by the idoA gene is essential for the metabolism of fluoranthene, since a mutant in which the idoA gene has been disrupted looses the ability to degrade this compound. The idoA gene appears to be constitutively expressed in PA-10, but its expression is also subject to regulation following prior exposure to salicylate and to fluoranthene degradative intermediates.

  3. Heterotrophic nitrification by Alcaligenes faecalis: NO sub 2 sup minus , NO sub 3 sup minus , N sub 2 O, and NO production in exponentially growing cultures

    SciTech Connect

    Papen, H.; von Berg, R.; Hinkel, I.; Thoene, B.; Rennenberg, H. )

    1989-08-01

    Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO{sub 2}{sup {minus}}, NO{sub 3}{sup {minus}}, NO, and N{sub 2}O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO{sub 2}{sup {minus}}, NO{sub 3}{sup {minus}}, NO, and N{sub 2}O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO{sub 2}{sup {minus}} and NO{sub 3}{sup {minus}} production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.

  4. Pathway of oxidation of pyruvic oxime by a heterotrophic nitrifier of the genus Alcaligenes: evidence against hydrolysis to pyruvate and hydroxylamine

    SciTech Connect

    Castignetti, D.; Petithory, J.R.; Hollocher, T.C.

    1983-07-15

    A heterotrophic nitrifier of the genus Alcaligenes, which grows vigorously on pyruvic oxime, was tested by several methods for possible differences or similarities in metabolic performance between pyruvic oxime and its hydrolysis products, pyruvate and hydroxylamine. Major differences were observed between pyruvic oxime and one or both of the other reductants with regard to growth yield, rates of reductant uptake, rates of oxygen uptake, sensitivity of their oxidations to inhibition by thiocyanate, and performance in reductant pulse experiments. Other oximes, some of which are structural analogs of pyruvic oxime and all of which are potential sources of hydroxylamine, were not metabolized by cells or cell-free extract. Collectively the results indicate a pathway of oxidation of pyruvic oxime to nitrite and CO/sub 2/ that does not involve its initial hydrolysis, but probably involves the oxidation of N and/or C before C-N bond breakage.

  5. A comparison of NO and N2O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis.

    PubMed Central

    Anderson, I C; Poth, M; Homstead, J; Burdige, D

    1993-01-01

    Soil microorganisms are important sources of the nitrogen trace gases NO and N2O for the atmosphere. Present evidence suggests that autotrophic nitrifiers such as Nitrosomonas europaea are the primary producers of NO and N2O in aerobic soils, whereas denitrifiers such as Pseudomonas spp. or Alcaligenes spp. are responsible for most of the NO and N2O emissions from anaerobic soils. It has been shown that Alcaligenes faecalis, a bacterium common in both soil and water, is capable of concomitant heterotrophic nitrification and denitrification. This study was undertaken to determine whether heterotrophic nitrification might be as important a source of NO and N2O as autotrophic nitrification. We compared the responses of N. europaea and A. faecalis to changes in partial O2 pressure (pO2) and to the presence of typical nitrification inhibitors. Maximal production of NO and N2O occurred at low pO2 values in cultures of both N. europaea (pO2, 0.3 kPa) and A. faecalis (pO2, 2 to 4 kPa). With N. europaea most of the NH4+ oxidized was converted to NO2-, with NO and N2O accounting for 2.6 and 1% of the end product, respectively. With A. faecalis maximal production of NO occurred at a pO2 of 2 kPa, and maximal production of N2O occurred at a pO2 of 4 kPa. At these low pO2 values there was net nitrite consumption. Aerobically, A. faecalis produced approximately the same amount of NO but 10-fold more N2O per cell than N. europaea did. Typical nitrification inhibitors were far less effective for reducing emissions of NO and N2O by A. faecalis than for reducing emissions of NO and N2O by N. europaea.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8285659

  6. The structures of Alcaligenes faecalis D-3-hydroxybutyrate dehydrogenase before and after NAD+ and acetate binding suggest a dynamical reaction mechanism as a member of the SDR family.

    PubMed

    Hoque, Md Mominul; Shimizu, Satoru; Hossain, Md Tofazzal; Yamamoto, Tamotsu; Imamura, Shigeyuki; Suzuki, Kaoru; Tsunoda, Masaru; Amano, Hitoshi; Sekiguchi, Takeshi; Takénaka, Akio

    2008-05-01

    D-3-Hydroxybutyrate dehydrogenase, which catalyzes the reversible reaction between D-3-hydroxybutyrate and acetoacetate, has been classified into the short-chain dehydrogenase/reductase family and is a useful marker in the assay of diabetes mellitus and/or ketoacidosis. The enzyme from Alcaligenes faecalis was crystallized in the apo form and in the holo form with acetate as a substrate analogue. The crystal structures of both forms were determined at 2.2 angstroms resolution. The enzyme is a tetramer composed of four subunits assembled with noncrystallographic 222 point symmetry. Each subunit has two domains. The principal domain adopts the Rossmann fold essential for nucleotide binding, which is a common feature of the SDR family. NAD+ is bound in a large cleft in the domain. The pyrophosphate group of NAD+ is covered by the small additional domain, which is supported by two extended arms allowing domain movement. In the catalytic site, a water molecule is trapped by the catalytic Tyr155 and Ser142 residues in the vicinity of the bound NAD+ and acetate. The substrate analogue acetate is bound above the nicotinamide plane. A substrate (D-3-hydroxybutylate) bound model can reasonably be constructed by adding two C atoms into the void space between the water O atom and the methyl group of the acetate, suggesting a substrate-bound state before enzymatic reaction occurs. Based on these structural features, a reaction mechanism has been proposed.

  7. Molecular Cloning, Sequencing, and Expression in Escherichia coli of the Gene Encoding a Novel 5-Oxoprolinase without ATP-Hydrolyzing Activity from Alcaligenes faecalis N-38A

    PubMed Central

    Nishimura, Atsuhisa; Oyama, Hiroshi; Hamada, Takatoshi; Nobuoka, Katsunori; Shin, Takashi; Murao, Sawao; Oda, Kohei

    2000-01-01

    The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-β-d-thiogalactopyranoside-inducible tac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited by p-chloromercuribenzoic acid, EDTA, o-phenanthroline, HgCl2, and CuSO4. The EDTA-inactivated enzyme was completely restored by the addition of Zn2+ or Co2+. In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme. PMID:10919770

  8. Pyruvic oxime dioxygenase from heterotrophic nitrifier Alcaligenes faecalis is a nonheme Fe(II)-dependent enzyme homologous to class II aldolase

    PubMed Central

    Tsujino, Shuhei; Uematsu, Chisato; Dohra, Hideo; Fujiwara, Taketomo

    2017-01-01

    Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn(II)-dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe(II). Affinity and catalytic constants were estimated at 470 μM and 4.69 sec−1, respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe(II), not Zn(II), is essential for POD activity and that Mn(II) could partially fulfill the function of Fe(II). A mutant POD with replacement of His183, corresponding to one of three Zn(II)-binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe(II) as a catalytic center instead of Zn(II). A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe(II)-dependent dioxygenase. PMID:28059164

  9. Effect of bacterial inoculation of strains of Pseudomonas aeruginosa, Alcaligenes feacalis and Bacillus subtilis on germination, growth and heavy metal (Cd, Cr, and Ni) uptake of Brassica juncea.

    PubMed

    Ndeddy Aka, Robinson Junior; Babalola, Olubukola Oluranti

    2016-01-01

    Bacterial inoculation may influence Brassica juncea growth and heavy metal (Ni, Cr, and Cd) accumulation. Three metal tolerant bacterial isolates (BCr3, BCd33, and BNi11) recovered from mine tailings, identified as Pseudomonas aeruginosa KP717554, Alcaligenes feacalis KP717561, and Bacillus subtilis KP717559 were used. The isolates exhibited multiple plant growth beneficial characteristics including the production of indole-3-acetic acid, hydrogen cyanide, ammonia, insoluble phosphate solubilization together with the potential to protect plants against fungal pathogens. Bacterial inoculation improved seeds germination of B. juncea plant in the presence of 0.1 mM Cr, Cd, and Ni, as compared to the control treatment. Compared with control treatment, soil inoculation with bacterial isolates significantly increased the amount of soluble heavy metals in soil by 51% (Cr), 50% (Cd), and 44% (Ni) respectively. Pot experiment of B. juncea grown in soil spiked with 100 mg kg(-1) of NiCl2, 100 mg kg(-1) of CdCl2, and 150 mg kg(-1) of K2Cr2O7, revealed that inoculation with metal tolerant bacteria not only protected plants against the toxic effects of heavy metals, but also increased growth and metal accumulation of plants significantly. These findings suggest that such metal tolerant, plant growth promoting bacteria are valuable tools which could be used to develop bio-inoculants for enhancing the efficiency of phytoextraction.

  10. Anaerobic and aerobic degradation of cyanophycin by the denitrifying bacterium Pseudomonas alcaligenes strain DIP1 and role of three other coisolates in a mixed bacterial consortium.

    PubMed

    Sallam, Ahmed; Steinbüchel, Alexander

    2008-06-01

    Four bacterial strains were isolated from a cyanophycin granule polypeptide (CGP)-degrading anaerobic consortium, identified by 16S rRNA gene sequencing, and assigned to species of the genera Pseudomonas, Enterococcus, Clostridium, and Paenibacillus. The consortium member responsible for CGP degradation was assigned as Pseudomonas alcaligenes strain DIP1. The growth of and CGP degradation by strain DIP1 under anaerobic conditions were enhanced but not dependent on the presence of nitrate as an electron acceptor. CGP was hydrolyzed to its constituting beta-Asp-Arg dipeptides, which were then completely utilized within 25 and 4 days under anaerobic and aerobic conditions, respectively. The end products of CGP degradation by strain DIP1 were alanine, succinate, and ornithine as determined by high-performance liquid chromatography analysis. The facultative anaerobic Enterococcus casseliflavus strain ELS3 and the strictly anaerobic Clostridium sulfidogenes strain SGB2 were coisolates and utilized the beta-linked isodipeptides from the common pool available to the mixed consortium, while the fourth isolate, Paenibacillus odorifer strain PNF4, did not play a direct role in the biodegradation of CGP. Several syntrophic interactions affecting CGP degradation, such as substrate utilization, the reduction of electron acceptors, and aeration, were elucidated. This study demonstrates the first investigation of CGP degradation under both anaerobic and aerobic conditions by one bacterial strain, with regard to the physiological role of other bacteria in a mixed consortium.

  11. Proteome analysis of heat shock protein expression in Pseudomonas alcaligenes NCIMB 9867 in response to gentisate exposure and elevated growth temperature.

    PubMed

    Zhao, Bing; Yeo, Chew Chieng; Tan, Chew Ling; Poh, Chit Laa

    2007-06-15

    Pseudomonas alcaligenes NCIMB 9867 (strain P25X) degrades xylenols and cresols via the gentisate pathway. P25X expresses two isofunctional gentisate 1,2-dioxygenases (GDO I and GDO II). The expression of both GDOs was not detected when P25X cells were grown at 42 degrees C, even in the presence of gentisate. A total of 19 heat shock proteins (Hsps) belonging to the Hsp100, Hsp90, Hsp70, Hsp60, Hsp45, and small heat shock protein (sHsp) families were identified among the protein spots that were either newly detected or were expressed at levels of at least twofold higher when P25X cells were cultured at 32 or 42 degrees C in the presence and absence of gentisate. Among these, 16 Hsps were commonly expressed at 42 degrees C. Two additional Hsps (H5 and H13) from the Hsp90 and Hsp60 families, respectively, were expressed only when P25X cells were grown at 42 degrees C and in the presence of gentisate. A protein of the sHsp (H16) family was expressed only in the presence of gentisate at 32 degrees C but not at 42 degrees C. The GroEL chaperonins of the Hsp60 family comprised the largest group of Hsps identified and exhibited high level of expression at 42 degrees C following gentisate exposure.

  12. Improving the bioremoval of sulfamethoxazole and alleviating cytotoxicity of its biotransformation by laccase producing system under coculture of Pycnoporus sanguineus and Alcaligenes faecalis.

    PubMed

    Li, Xin; Xu, Qiu-Man; Cheng, Jing-Sheng; Yuan, Ying-Jin

    2016-11-01

    The occurrence of sulfamethoxazole (SMX) in aquatic environment is a health concern. The presence of SMX significantly inhibited the laccase activity of Pycnoporus sanguineus with a lower removal efficiency of SMX. Although a laccase system with 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) eliminated 100% SMX within 6h, ABTS might cause an environmental issue. An alternative to SMX elimination is the coculture of Alcaligenes faecalis and P. sanguineus. The SMX removal efficiency at 48h under the coculture with vitamins was higher than that under their pure culture alone, indicating that a coculture was more efficient in eliminating SMX than a pure culture. Only 1% SMX was detected in mycelia, indicating that SMX elimination is achieved primarily through biotransformation rather than adsorption. Laccase production by the coculture effectively inhibited the accumulations of N4-acetyl-SMX and N-hydroxy-SMX and alleviated the cytotoxicity of SMX transformation products. The mixture of SMX and sulfadiazine inhibited their removal efficiency.

  13. Pyruvic oxime dioxygenase from heterotrophic nitrifier Alcaligenes faecalis is a nonheme Fe((II))-dependent enzyme homologous to class II aldolase.

    PubMed

    Tsujino, Shuhei; Uematsu, Chisato; Dohra, Hideo; Fujiwara, Taketomo

    2017-01-06

    Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn((II))-dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe((II)). Affinity and catalytic constants were estimated at 470 μM and 4.69 sec(-1), respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe((II)), not Zn((II)), is essential for POD activity and that Mn((II)) could partially fulfill the function of Fe((II)). A mutant POD with replacement of His(183), corresponding to one of three Zn((II))-binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe((II)) as a catalytic center instead of Zn((II)). A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe((II))-dependent dioxygenase.

  14. Identification of two vicinal operons for the degradation of 2-aminobenzenesulfonate encoded on plasmid pSAH in Alcaligenes sp. strain O-1.

    PubMed

    Ruff, Jürgen; Smits, Theo H M; Cook, Alasdair M; Schleheck, David

    2010-05-30

    Alcaligenes sp. strain O-1 inducibly deaminates 2-aminobenzenesulfonate (ABS) via dioxygenation to 3-sulfocatechol, which is desulfonated during meta ring-cleavage to yield 2-hydroxymuconate. This intermediate is transformed through the oxalocrotonate-branch of the sulfocatechol meta-pathway (Scm). The complete pathway is encoded on the 180-kb plasmid pSAH, 20kb of which was sequenced. Twenty open reading frames (ORFs) were detected. Two clusters (abs and scm) with degradative genes were surrounded by several transposon-related ORFs. The six genes of the abs cluster were shown to be co-transcribed, and contained the genes for two characterised subunits of the oxygenase component of the ABS-dioxygenase system, and genes putatively encoding ABS-transport functions with similarities to (a) an ABC-type transporter system and (b) a putative major facilitator superfamily transporter. No gene encoding the reductase for the oxygenase system was present in the abs gene cluster, but a candidate gene was found in the scm cluster. The seven-gene scm cluster was also transcribed as single polycistronic message. Functions could be attributed to the gene products, but one enzyme, which was shown to be present, 2-hydroxymuconate isomerase, was not encoded in the scm cluster. No transcriptional regulator was found. This genetic information on the degradation of ABS in strain O-1 provides another example of both split operons and dispersed pathway genes.

  15. The importance of Asn47 for structure and reactivity of azurin from Alcaligenes denitrificans as studied by site-directed mutagenesis and spectroscopy.

    PubMed

    Hoitink, C W; Canters, G W

    1992-07-15

    To study the importance of a rigid copper site for the structure and function of azurin, a mutant with a reduced number of internal hydrogen bonds around the copper has been prepared and characterized. To this purpose, the previously cloned azu gene from Alcaligenes denitrificans (Hoitink, C. W. G., Woudt, L. P., Turenhout, J. C. M., Van de Kamp, M., and Canters, G. W. (1990) Gene (Amst.) 90, 15-20) was expressed in Escherichia coli and an isolation and purification procedure for the azurin was developed. The azurin obtained after heterologous expression in E. coli appears spectroscopically indistinguishable from azurin derived from A. denitrificans. The hydrogen bonding network around the copper site was altered by replacing Asn47 by a leucine by means of site-directed mutagenesis. Asn47 is a conserved residue in all blue copper proteins of which the primary structure has been reported. Characterization of the mutant protein with UV-visible, electron spin resonance, and NMR spectroscopy, and comparison with the wild type azurin revealed that the structure of the copper site as well as the overall structure of the protein have been largely retained. The redox activity as measured by the electron self-exchange rate appears not to have changed either. However, the mutant differs from the wild type azurin with respect to stability and midpoint potential. Midpoint potentials of mutant and wild type azurin amount to 396 and 286 mV, respectively. The difference is due to sizable entropic and enthalpic contributions which to a large extent cancel. Possible explanations for the outcome of these experiments are discussed.

  16. Long-term analysis of diesel fuel consumption in a co-culture of Acinetobacter venetianus, Pseudomonas putida and Alcaligenes faecalis.

    PubMed

    Pepi, Milva; Minacci, Andrea; Di Cello, Francescopaolo; Baldi, Franco; Fani, Renato

    2003-01-01

    The dynamics of a microbial population isolated from superficial waters of Venice Lagoon and the ability to utilise diesel fuel (n-alkanes mixture C12-C28) as the sole carbon and energy source were studied in a long-term reconstruction experiment. The reconstructed microbial population consisted of three bacterial strains belonging to the species Acinetobacter venetianus, Pseudomonas putida, and Alcaligenes faecalis, which were able to oxidise n-alkanes to alkanoates, n-alkanols to alkanoates, or only n-alkanoates, respectively. Three different approaches: plate counting, cell counting by epifluorescence microscopy with DAPI staining, and by fluorescence in situ hybridisation (FISH) by using a probe conjugate with fluoresceine isothiocyanate specifically targeted towards the 16S rRNA of bacteria belonging to the genus Acinetobacter were used to monitor the growth of the bacterial population. The growth of A. venetianus was stimulated by the presence of other strains, suggesting a beneficial interaction. After the first week of growth A. venetianus cells formed aggregates, as confirmed by confocal microscopy (CLSM), which allowed them to be distinguished from free cells. A relationship between cell number and measured areas (microm2) per aggregate was found. Each cell presented an average surface of 1.21 microm2. Each aggregate was formed by a cellular monolayer biofilm consisting of up to several thousands of cells. The A. venetianus aggregates increased in number and size over time, but after two weeks fragmentation events, which had a beneficial effect on the growth of P. putida and A. faecalis, occurred.

  17. Relative rates of nitric oxide and nitrous oxide production by nitrifiers, denitrifiers, and nitrate respirers. [Pseudomonas fluorescens; Serratia marcescens; Alcaligenes faecalis

    SciTech Connect

    Anderson, I.C.; Levine, J.S.

    1986-05-01

    The authors investigated the effect of the partial pressure of oxygen (pO/sub 2/) on the production of NO and N/sub 2/O by a wide variety of common soil nitrifying, denitrifying, and nitrate-respiring bacteria under laboratory conditions. The production of NO per cell was highest by autotrophic nitrifiers and was independent of pO/sub 2/ in the range tested (0.5 to 10%), whereas N/sub 2/O production was inversely proportional to pO/sub 2/. Nitrous oxide production was highest in the denitrifier Pseudomonas fluorescens, but only under anaerobic conditions. The molar ratio of NO/N/sub 2/O produced was usually greater than unity for nitrifiers and much less than unity for denitrifiers. Chemodenitrification was the major source of both the NO and N/sub 2/O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of NO and N/sub 2/O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of No and N/sub 2/O in nitrifier cultures but only when high concentrations of nitrite had accumulated or were added to the medium. Although most of the denitrifiers produced NO and N/sub 2/O only under anaerobic conditions, chemostat cultures of Alcaligenes faecalis continued to emit these gases even when the cultures were sprayed with air. Based upon these results, we predict that aerobic soils are primary sources of NO and that N/sub 2/O is produced only when there is sufficient soil moisture to provide the anaerobic microsites necessary for denitrification by either denitrifiers or nitrifiers.

  18. Cyanophycinase CphE from P. alcaligenes produced in different compartments of N. benthamiana degrades high amounts of cyanophycin in plant extracts.

    PubMed

    Nausch, Henrik; Broer, Inge

    2017-03-01

    One of the major constraints in pig and poultry farming is the supply of protein-rich forage, containing sufficient amounts of key amino acids such as arginine (Ufaz and Galili 2008). Since these are underrepresented in plant proteins, the usage of plants as feed is limited. The heterologous production of the cyanobacterial storage polymer cyanophycin granule polypeptide (CGP) in plastids increases the amount of arginine substantially (Huhns et al. 2008; Huhns et al. 2009; Nausch et al. 2016a). CGP degradation releases arginine-aspartate dipeptides. CGP is stable in plants because its degradation is exclusively restricted to bacterial cyanophycinases (CGPases; Law et al. 2009). Since animals are also unable to digest CGP, CGPases need to be co-delivered with CGP-containing plant feed in order to release the dipeptides in the gastrointestinal tract of animals during digestion. Therefore, an extracellular CGPase, CphE from Pseudomonas alcaligenes DIP-1, was targeted to the cytosol, ER, and apoplasm of Nicotiana benthamiana. Translocation to the chloroplast was not successful. Although CphE accumulated in high amounts in the cytosol, only moderate levels were present in the ER, while the enzyme was nearly undetectable in the apoplasm. This correlates with the higher instability of post-translationally modified CphE in crude plant extracts. In addition, the production in the ER led to an increased number and size of necroses compared with cytosolic expression and might therefore interfere with the endogenous metabolism in the ER. Due to the high and robust enzyme activity, even moderate CphE concentrations were sufficient to degrade CGP in plant extracts.

  19. The crystal structure of D-threonine aldolase from Alcaligenes xylosoxidans provides insight into a metal ion assisted PLP-dependent mechanism.

    PubMed

    Uhl, Michael K; Oberdorfer, Gustav; Steinkellner, Georg; Riegler-Berket, Lina; Mink, Daniel; van Assema, Friso; Schürmann, Martin; Gruber, Karl

    2015-01-01

    Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

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

    PubMed

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

    2012-08-01

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

  1. Metabolism of acrylate to {beta}-hydroxypropionate and its role in dimethylsulfoniopropionate lyase induction by a salt marsh sediment bacterium, Alcaligenes faecalis M3A

    SciTech Connect

    Ansede, J.H.; Pellechia, P.J.; Yoch, D.C.

    1999-11-01

    Dimethylsulfoniopropionate (DMSP) is degraded to dimethylsulfide (DMS) and acrylate by the enzyme DMSP lyase. DMS or acrylate can serve as a carbon source for both free-living and endophytic bacteria in the marine environment. In this study, the authors report on the mechanism of DMSP-acrylate metabolism by Alcaligenes faecalis M3A. Suspensions of citrate-grown cells expressed a low level of DMSP lyase activity that could be induced to much higher levels in the presence of DMSP, acrylate, and its metabolic product, {beta}-hydroxypropionate. DMSP was degraded outside the cell, resulting in an extracellular accumulation of acrylate, which in suspensions of citrate-grown cells was then metabolized at a low endogenous rate. The inducible nature of acrylate metabolism was evidenced by both an increase in the rate of its degradation over time and the ability of acrylate-grown cells to metabolize this molecule at about an eight times higher rate than citrate-grown cells. Therefore, acrylate induces both its production (from DMSP) and its degradation by an acrylase enzyme. {sup 1}H and {sup 13}C nuclear magnetic resonance analyses were used to identify the products resulting from [1-{sup 13}C]acrylate metabolism. The results indicated that A.faecalis first metabolized acrylate to {beta}-hydroxypropionate outside the cell, which was followed by its intracellular accumulation and subsequent induction of DMSP lyase activity. In summary, the mechanism of DMSP degradation to acrylate and the subsequent degradation of acrylate to {beta}-hydroxypropionate in the aerobic {beta}-Proteobacterium A.faecalis has been described.

  2. Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine.

    PubMed

    Boulanger, Martin J; Murphy, Michael E P

    2003-02-01

    Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], I257T[NO(2)(-)], I257M[NO(2)(-)] and I257G[NO(2)(-)] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)

  3. Improvement of Alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of (R)-(-)-mandelic acid.

    PubMed

    Liu, Zhi-Qiang; Zhang, Xin-Hong; Xue, Ya-Ping; Xu, Ming; Zheng, Yu-Guo

    2014-05-21

    Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(-)-mandelic acid after 7.5 h of conversion reached 693 mM with an enantiomeric excess of 99%, and the space-time productivity of Mut3 was 21.50-fold higher than that of wild-type nitrilase. The Km, Vmax, and k(cat) of wild-type and Mut3 for mandelonitrile were 20.64 mM, 33.74 μmol mg(-1) min(-1), 24.45 s(-1), and 9.24 mM, 47.68 μmol mg(-1) min(-1), and 34.55 s(-1), respectively. A homology modeling and molecular docking study showed that the diameter of the catalytic tunnel of Mut3 became longer and that the tunnel volume was smaller. These structural changes are proposed to improve the hydrolytic activity and pH stability of Mut3. Mut3 has the potential for industrial applications in the upscale production of (R)-(-)-mandelic acid.

  4. An asynchronous unfolding among molecular different regions of lobster D-glyceraldehyde-3-phosphate dehydrogenase and maltotetraose-forming amylase from an Alcaligenes sp. during guanidine denaturation.

    PubMed

    He, R Q; Zhao, K Y; Yan, Z Z; Li, M

    1993-06-04

    Changes in ultraviolet absorbance and intrinsic protein fluorescence of 1,4-alpha-D-glucan maltotetrahydrolase (EC 3.2.1.60) from an Alcaligenes sp. (Gram-negative bacteria 537.1) and D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) have been compared with their inactivation during denaturation in guanidinium-Cl solutions. The two enzymes were completely inactivated at GuHCl concentrations less than 0.6 M and this was accompanied by marked absorbance and intrinsic fluorescence changes suggesting exposure of aromatic residues. The changes of the intrinsic fluorescence of the amylase have a relatively constant plateau in emission intensities and maxima at GuHCl concentrations from 0.8-2.0 M, similar to that of muscle GAPDH. The relative activity of the enzyme increased markedly in dilute GuHCl solutions accompanied by very little change of its intrinsic fluorescence at 8 degrees C. The kinetic decrease in emission intensities, excited respectively by 230 nm and 292 nm, was different for the two enzymes. The inactivation was a biphasic process with a fast phase faster than the unfolding rate as measured by fluorescence changes in 0.5 M GuHCl solution. Similar to the inactivation process, changes in intensity of 410 nm NAD fluorescent derivative of GAPDH which is in situ at the active site is also a biphasic process under the same condition. It appears that there may be an unfolding intermediate state of the enzymes and an asynchronous unfolding process among the different regions in the molecules during GuHCl denaturation, this may be due to differences in their flexibility.

  5. Characterization of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid]-degrading Alcaligenes sp.CS1 and Ralstonia sp. CS2 isolated from agricultural soils.

    PubMed

    Smejkal, C W; Vallaeys, T; Seymour, F A; Burton, S K; Lappin-Scott, H M

    2001-04-01

    The herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] is widely applied to corn fields in order to control broad-leaved weeds. However, it is often detected in groundwater where it can be a persistent contaminant. Two mecoprop-degrading bacterial strains were isolated from agricultural soils through their capability to degrade (R/S)-mecoprop rapidly. 16S rDNA sequencing of the isolates demonstrated that one was closely related to the genera Alcaligenes sp. (designated CS1) and the other to Ralstonia sp. (designated CS2). Additionally, these isolates demonstrated ability to grow on other related herbicides, including 2,4-D (2,4-dichlorophenoxyacetic acid), MCPA [4-chloro-2-methyl phenoxy acetic acid] and (R/S)-2,4-DP [2-(2,4-dichlorophenoxy)propionic acid] as sole carbon sources. tfdABC gene-specific probes derived from the 2,4-D-degrading Variovorax paradoxus TV1 were used in hybridization analyses to establish whether tfd-like genes are present in mecoprop-degrading bacteria. Hybridization analysis demonstrated that both Alcaligenes sp. CS1 and Ralstonia sp. CS2 harboured tfdA, tfdB and tfdC genes on plasmids that have approximately > 60% sequence similarity to the tfdA, tfdB and tfdC genes of V. paradoxus. It is therefore likely that tfd-like genes may be involved in the degradation of mecoprop, and we are currently investigating this further.

  6. Keratinase production and biodegradation of polluted secondary chicken feather wastes by a newly isolated multi heavy metal tolerant bacterium-Alcaligenes sp. AQ05-001.

    PubMed

    Yusuf, Ibrahim; Ahmad, Siti Aqlima; Phang, Lai Yee; Syed, Mohd Arif; Shamaan, Nor Aripin; Abdul Khalil, Khalilah; Dahalan, Farrah Aini; Shukor, Mohd Yunus

    2016-12-01

    Biodegradation of agricultural wastes, generated annually from poultry farms and slaughterhouses, can solve the pollution problem and at the same time yield valuable degradation products. But these wastes also constitute environmental nuisance, especially in Malaysia where their illegal disposal on heavy metal contaminated soils poses a serious biodegradation issue as feather tends to accumulate heavy metals from the surrounding environment. Further, continuous use of feather wastes as cheap biosorbent material for the removal of heavy metals from effluents has contributed to the rising amount of polluted feathers, which has necessitated the search for heavy metal-tolerant feather degrading strains. Isolation, characterization and application of a novel heavy metal-tolerant feather-degrading bacterium, identified by 16S RNA sequencing as Alcaligenes sp. AQ05-001 in degradation of heavy metal polluted recalcitrant agricultural wastes, have been reported. Physico-cultural conditions influencing its activities were studied using one-factor-at-a-time and a statistical optimisation approach. Complete degradation of 5 g/L feather was achieved with pH 8, 2% inoculum at 27 °C and incubation period of 36 h. The medium optimisation after the response surface methodology (RSM) resulted in a 10-fold increase in keratinase production (88.4 U/mL) over the initial 8.85 U/mL when supplemented with 0.5% (w/v) sucrose, 0.15% (w/v) ammonium bicarbonate, 0.3% (w/v) skim milk, and 0.01% (w/v) urea. Under optimum conditions, the bacterium was able to degrade heavy metal polluted feathers completely and produced valuable keratinase and protein-rich hydrolysates. About 83% of the feathers polluted with a mixture of highly toxic metals were degraded with high keratinase activities. The heavy metal tolerance ability of this bacterium can be harnessed not only in keratinase production but also in the bioremediation of heavy metal-polluted feather wastes. Copyright © 2016. Published by

  7. Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp. C34 and Alcaligenes faecalis T1.

    PubMed

    Sun, Jian; Matsumoto, Ken'ichiro; Tabata, Yuta; Kadoya, Ryosuke; Ooi, Toshihiko; Abe, Hideki; Taguchi, Seiichi

    2015-11-01

    Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZVs) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(D-LA-co-D-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZVs for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZAf) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZVs generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZVs cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZVs but also PhaZAf hydrolyzed all of these substrates, namely PhaZAf also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZVs exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZAf. Therefore, the cleaving capability of PhaZs used here toward the D-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.

  8. Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis. Cofactor assembly and catalytic properties of recombinant enzyme expressed in Paracoccus denitrificans.

    PubMed

    Hothi, Parvinder; Khadra, Khalid Abu; Combe, Jonathan P; Leys, David; Scrutton, Nigel S

    2005-11-01

    The heterologous expression of tryptophan trytophylquinone (TTQ)-dependent aromatic amine dehydrogenase (AADH) has been achieved in Paracoccus denitrificans. The aauBEDA genes and orf-2 from the aromatic amine utilization (aau) gene cluster of Alcaligenes faecalis were placed under the regulatory control of the mauF promoter from P. denitrificans and introduced into P. denitrificans using a broad-host-range vector. The physical, spectroscopic and kinetic properties of the recombinant AADH were indistinguishable from those of the native enzyme isolated from A. faecalis. TTQ biogenesis in recombinant AADH is functional despite the lack of analogues in the cloned aau gene cluster for mauF, mauG, mauL, mauM and mauN that are found in the methylamine utilization (mau) gene cluster of a number of methylotrophic organisms. Steady-state reaction profiles for recombinant AADH as a function of substrate concentration differed between 'fast' (tryptamine) and 'slow' (benzylamine) substrates, owing to a lack of inhibition by benzylamine at high substrate concentrations. A deflated and temperature-dependent kinetic isotope effect indicated that C-H/C-D bond breakage is only partially rate-limiting in steady-state reactions with benzylamine. Stopped-flow studies of the reductive half-reaction of recombinant AADH with benzylamine demonstrated that the KIE is elevated over the value observed in steady-state turnover and is independent of temperature, consistent with (a) previously reported studies with native AADH and (b) breakage of the substrate C-H bond by quantum mechanical tunnelling. The limiting rate constant (k(lim)) for TTQ reduction is controlled by a single ionization with pK(a) value of 6.0, with maximum activity realized in the alkaline region. Two kinetically influential ionizations were identified in plots of k(lim)/K(d) of pK(a) values 7.1 and 9.3, again with the maximum value realized in the alkaline region. The potential origin of these kinetically influential

  9. Influence of Copper Resistance Determinants on Gold Transformation by Cupriavidus metallidurans Strain CH34

    PubMed Central

    Wiesemann, Nicole; Mohr, Juliane; Grosse, Cornelia; Herzberg, Martin; Hause, Gerd; Reith, Frank

    2013-01-01

    Cupriavidus metallidurans is associated with gold grains and may be involved in their formation. Gold(III) complexes influence the transcriptome of C. metallidurans (F. Reith et al., Proc. Natl. Acad. Sci. U. S. A. 106:17757–17762, 2009), leading to the upregulation of genes involved in the detoxification of reactive oxygen species and metal ions. In a systematic study, the involvement of these systems in gold transformation was investigated. Treatment of C. metallidurans cells with Au(I) complexes, which occur in this organism's natural environment, led to the upregulation of genes similar to those observed for treatment with Au(III) complexes. The two indigenous plasmids of C. metallidurans, which harbor several transition metal resistance determinants, were not involved in resistance to Au(I/III) complexes nor in their transformation to metallic nanoparticles. Upregulation of a cupA-lacZ fusion by the MerR-type regulator CupR with increasing Au(III) concentrations indicated the presence of gold ions in the cytoplasm. A hypothesis stating that the Gig system detoxifies gold complexes by the uptake and reduction of Au(III) to Au(I) or Au(0) reminiscent to detoxification of Hg(II) was disproven. ZupT and other secondary uptake systems for transition metal cations influenced Au(III) resistance but not the upregulation of the cupA-lacZ fusion. The two copper-exporting P-type ATPases CupA and CopF were also not essential for gold resistance. The copABCD determinant on chromosome 2, which encodes periplasmic proteins involved in copper resistance, was required for full gold resistance in C. metallidurans. In conclusion, biomineralization of gold particles via the reduction of mobile Au(I/III) complexes in C. metallidurans appears to primarily occur in the periplasmic space via copper-handling systems. PMID:23475973

  10. Two quick methods for isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase.

    PubMed

    Jakob, R

    1988-01-01

    Methods are described which allow the isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (rubisco) in a very short time. Source of the material was highly impure commercial enzyme in the case of spinach rubisco or bacteria grown from a fermentor in the case of Alcaligenes eutrophus rubisco. Purity of the enzymes is demonstrated by gel electrophoreses. Enzyme isolated from fresh cells gave crystals of excellent diffraction, suitable for X-ray structure analyisis.

  11. Chemical and Physical Characterization of the Activation of Ribulosebiphosphate Carboxylase/Oxygenase

    DOE R&D Accomplishments Database

    Donnelly, M. I.; Ramakrishnan, V.; Hartman, F. C.

    1983-08-01

    Molecular structure of ribulosebiphosphate carboxylase/oxygenase isolated from Rhodospirillium was compared with the enzyme isolated from Alcaligens eutrophus. Peptides derived from the active center of the bacterial enzyme were highly homologous with those isolated from spinach. Molecular shapes of the carboxylases were estimated using neutron scattering data. These studies suggested that the enzyme as isolated from R. rubrum is a solid prolate ellipsoid or cylinder, while the spinach enzyme resembles a hollow sphere.

  12. Molecular Ecology of Bacterial Populations in Environmental Hazardous Chemical Control

    DTIC Science & Technology

    1994-01-14

    Pseudomonas fluorescens HK44 for naphthalene and salicylate catabolism was used for analysis of naphthalene and bioavailability in aqueous extracts of soils...reporter bacterium, Pseudomonas fluorescens HK44, was developed for continuous, rapid on-line monitoring of naphthalene and salicylate bioavailablity...G.S. Sayler (1993). Degradation of DDT by Alcaligenes eutrophus. Appl. Environ. Microbiol. 60:51-55. Sanseverino, J., B.M. Applegate, J.M. Henry King

  13. Molecular and Cellular Fundamentals of Aerobic Cometabolism

    DTIC Science & Technology

    1998-10-01

    1 99 1 ) Butane Pseudomonas butane monooxygenase (Hamamura, et al . , butanovorars (BMO) 1 997) 2,4-Dichloro Alcaligenes eutrophus...Leadbetter and Foster, 1 960). These studies initially revealed that the methane-utilizing bacterium Pseudomonas (Methylomonas) methanica could not grow...enzyme is required for each insertion. Pseudomonas mendocina KR 1 toluene-4-monooxygenase (T4MO) produces p-cresol ; Pseudomonas picketii toluene

  14. Molecular Ecology of Bacterial Population in Environmental Hazardous Chemical Control

    DTIC Science & Technology

    1993-01-14

    Pseudomonas putida F1 to measure toluene driven co-metabolic oxidation of TCE. (2) Demonstration of a new pathway for aerobic biodegradation of DDT...mediated by Alcaligenes eutrophus strain A5 previously shown competent for biodegradation of chlorobiphenyl congeners. (3) Confirmation that...the dynamics in microbial population density and activity during environmental biodegradation processes. Metabolism of PAHs. Pseudomonas £luorescens 5RL

  15. Chemical and physical characterization of the activation of ribulosebiphosphate carboxylase/oxygenase

    SciTech Connect

    Donnelly, M.I.; Ramakrishnan, V.; Hartman, F.C.

    1983-01-01

    Molecular structure of ribulosebiphosphate carboxylase/oxygenase isolated from Rhodospirillium was compared with the enzyme isolated from Alcaligens eutrophus. Peptides derived from the active center of the bacterial enzyme were highly homologous with those isolated from spinach. Molecular shapes of the carboxylases were estimated using neutron scattering data. These studies suggested that the enzyme as isolated from R. rubrum is a solid prolate ellipsoid or cylinder, while the spinach enzyme resembles a hollow sphere. 1 drawing.

  16. Reclassification of Alcaligenes latus strains IAM 12599T and IAM 12664 and Pseudomonas saccharophila as Azohydromonas lata gen. nov., comb. nov., Azohydromonas australica sp. nov. and Pelomonas saccharophila gen. nov., comb. nov., respectively.

    PubMed

    Xie, Cheng-Hui; Yokota, Akira

    2005-11-01

    The aim of this study was to clarify the taxonomic position of the nitrogen-fixing and hydrogen-oxidizing bacteria Alcaligenes latus strains IAM 12599T, IAM 12664 and IAM 12665 and Pseudomonas saccharophila IAM 14368T. It was found that the type strain of Alcaligenes latus, IAM 12599T, showed 99 x 9 and 96 x 1 % 16S rRNA gene sequence similarity to strains IAM 12665 and IAM 12664, respectively. A comparison using DNA-DNA hybridization suggested that strains IAM 12599T and IAM 12665 belong to a single species (89 x 7 %) and that strain IAM 12664 (35 x 1 %) forms a separate species. The phenotypic characteristics also support the conclusion that these bacteria should be identified as two species of a new genus: Azohydromonas lata gen. nov., comb. nov. (type strain IAM 12599T=DSM 1122T=LMG 3321T=ATCC 29712T; reference strain IAM 12665=DSM 1123=LMG 3325=ATCC 29714) and Azohydromonas australica sp. nov. (type strain IAM 12664T=DSM 1124T=LMG 3324T=ATCC 29713T). Pseudomonas saccharophila IAM 14368T was found to be closely related to the phototrophic bacterium Roseateles depolymerans, with 96 x 8 % 16S rRNA gene sequence similarity, but the two bacteria are quite different with respect to their metabolism and some significant phenotypic characteristics, suggesting that they cannot be included in a single genus. Further studies on their nifH gene sequences, G+C content of the DNA and cellular fatty acid composition confirm that Pseudomonas saccharophila should be reclassified: the name Pelomonas saccharophila gen. nov., comb. nov. is proposed, with the type strain IAM 14368T (=LMG 2256T=ATCC 15946T).

  17. Monitoring populations of 4-chlorobiphenyl-degrading bacterial in soil and lake water microcosms using colony hybridization

    SciTech Connect

    Packard, J.; Breen, A.; Sayler, G.S. ); Palumbo, A.V. )

    1989-01-01

    Population dynamics and maintenance of a 4-chlorobiphenyl (4CB)- degrading bacteria, Alcaligenes eutrophus, added to field lysimeters and lake water microcosms were monitored using colony hybridization. Six field lysimeters (55 gal steel drums) were set up and monitored for one year. Each lysimeter contained 60 kg of soil from a PCB-contaminated floodplain and all but one were watered and stirred during the experiment. At the start of the field lysimeter experiment, half of the lysimeters received an inoculation of Alcaligenes eutrophus. The lysimeters differed in the treatments administered over the year; some received inorganic nutrients and yeast extract and one also received biphenyl. Four 20 l microcosms were set up with water from Fort Loudon Reservoir. They were supplemented with mineral salts and glucose and stirred and monitored for two months. Alcaligenes eutrophus was inoculated into two of the microcosms, one of which was maintained with selective pressure by the addition of 4CB. One of the uninoculated microcosms also had selective pressure for 4CB-degrading organisms. 18 refs., 2 figs., 1 tab.

  18. Molecular and biochemical characterization of the xlnD-encoded 3-hydroxybenzoate 6-hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Gao, Xiaoli; Tan, Chew Ling; Yeo, Chew Chieng; Poh, Chit Laa

    2005-11-01

    The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.

  19. Raman Spectroscopic Analysis of Cupriavidus metallidurans LMG 1195 (CH34) Cultured in Low-shear Microgravity Conditions

    NASA Astrophysics Data System (ADS)

    De Gelder, Joke; Vandenabeele, Peter; De Boever, Patrick; Mergeay, Max; Moens, Luc; De Vos, Paul

    2009-07-01

    In this study, the effect of low-shear microgravity on the metabolism of Cupriavidus metallidurans LMG 1195 was studied with Raman spectroscopy. Therefore, the strain was cultured for 24 or 48 h in a rotating wall vessel to simulate microgravity (SMG) and in a control setup. The differences in Raman spectra recorded from both setups after 24 h of culturing were small. The most prominent features in a difference spectrum, calculated between the mean spectra from the microgravity and the control setup separately, could be assigned to the presence of poly-β-hydroxybutyrate (PHB). SMG seems to yield a higher PHB production after 24 h of culturing. Additional processing of the spectra suggested that SMG induced also other changes in the carbon-metabolism. After 48 h, similar results were found for the carbon metabolism, while PHB concentrations were reduced in SMG compared to the control. However, these differences could also be caused by interfering effects that may occur in the bioreactors after a prolonged incubation time.

  20. Contributions of Five Secondary Metal Uptake Systems to Metal Homeostasis of Cupriavidus metallidurans CH34▿†

    PubMed Central

    Kirsten, Andreas; Herzberg, Martin; Voigt, Alexander; Seravalli, Javier; Grass, Gregor; Scherer, Judith; Nies, Dietrich H.

    2011-01-01

    Cupriavidus metallidurans is adapted to high concentrations of transition metal cations and is a model system for studying metal homeostasis in difficult environments. The elemental composition of C. metallidurans cells cultivated under various conditions was determined, revealing the ability of the bacterium to shield homeostasis of one essential metal from the toxic action of another. The contribution of metal uptake systems to this ability was studied. C. metallidurans contains three CorA members of the metal inorganic transport (MIT) protein family of putative magnesium uptake systems, ZupT of the ZRT/IRT protein, or ZIP, family, and PitA, which imports metal phosphate complexes. Expression of the genes for all these transporters was regulated by zinc availability, as shown by reporter gene fusions. While expression of zupT was upregulated under conditions of zinc starvation, expression of the other genes was downregulated at high zinc concentrations. Only corA1 expression was influenced by magnesium starvation. Deletion mutants were constructed to characterize the contribution of each system to transition metal import. This identified ZupT as the main zinc uptake system under conditions of low zinc availability, CorA1 as the main secondary magnesium uptake system, and CorA2 and CorA3 as backup systems for metal cation import. PitA may function as a cation-phosphate uptake system, the main supplier of divalent metal cations and phosphate in phosphate-rich environments. Thus, metal homeostasis in C. metallidurans is achieved by highly redundant metal uptake systems, which have only minimal cation selectivity and are in combination with efflux systems that “worry later” about surplus cations. PMID:21742896

  1. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid

    PubMed Central

    Mishra, Pradeep; Kaur, Suneet; Sharma, Amar Nath; Jolly, Ravinder S.

    2016-01-01

    Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S)-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R)-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S)-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline)-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S)-amide to (S)-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH). IaaH is known to catalyse conversion of indole-3-acetamide (IAM) to indole-3-acetic acid (IAA), which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To the best of

  2. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid.

    PubMed

    Mishra, Pradeep; Kaur, Suneet; Sharma, Amar Nath; Jolly, Ravinder S

    2016-01-01

    Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S)-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R)-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S)-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline)-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S)-amide to (S)-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH). IaaH is known to catalyse conversion of indole-3-acetamide (IAM) to indole-3-acetic acid (IAA), which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To the best of

  3. Determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with Rhodococcus sp. Strain DN22

    DTIC Science & Technology

    2002-01-01

    directly through the decomposition of the hydroxylated product III or via one of its ring cleavage intermediates possibly formamide (NH2CHO). Pseudomonas ...4. Babu, G. R. V., O. K. Vijaya, V. L. Ross, J. H. Wolfram, and K. D. Cahpat- wala. 1996. Cell free extract(s) of Pseudomonas putida catalyzes the...dinitrophenol by Alcaligenes eutrophus JMP 134 and JMP 222. Arch. Microbiol. 158:149–154. 9. Haas, R., I. Schreiber, E. von Löw, and G. Stork. 1990. Conception

  4. Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids.

    PubMed Central

    Ramsay, B A; Lomaliza, K; Chavarie, C; Dubé, B; Bataille, P; Ramsay, J A

    1990-01-01

    Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid. PMID:2117877

  5. Acetylene is an active-site-directed, slow-binding, reversible inhibitor of Azotobacter vinelandii hydrogenase

    SciTech Connect

    Hyman, M.R.; Arp, D.J.

    1987-10-06

    The inhibition of purified and membrane-bound hydrogenase from Azotobacter vinelandii by dihydrogen-free acetylene was investigated. The inhibition was a time-dependent process which exhibited first-order kinetics. Both H/sub 2/ and CO protected against the inhibition by acetylene. K/sub protect(app)/ values of 0.41 and 24 ..mu..M were derived for these gases, respectively. Both H/sub 2/-oxidizing activity and the tritium exchange capacity of the purified enzyme were inhibited at the same rate by acetylene. Removal of acetylene reversed the inhibition for both the purified and the membrane-associated form of the enzyme. The purified hydrogenases from both Rhizobium japonicum and Alcaligenes eutrophus H16 were also inhibited by acetylene in a time-dependent fashion. These findings suggest that acetylene is an active-site-directed, slow-binding, reversible inhibitor of some membrane-bound hydrogenases from aerobic bacteria.

  6. Degradation of poly(3-hydroxyoctanoic acid) [P(3HO)] by bacteria: Purification and properties of a P(3HO) depolymerase from Pseudomonas fluorescens GK13

    SciTech Connect

    Schirmer, A.; Jendrossek, D.; Schlegel, H.G. )

    1993-04-01

    Poly(3-hydroxyoctanoic acid)[P(3HO)] and other poly(hydroxyalkanoic acids) PHA are widespread bacterial storage compounds of carbon and reducing power. They are biodegradable to carbon dioxide and water, and both aerobic and anaerobic P(3HB)-degradable bacteria are widely distributed in various ecosytems: soil, activated sludge, lake water and air, sea water, estuarine sediment, and anaerobic sewage sludge. This study describes the isolation and characterization of P(3HO) degrading bacteria: Alcaligenes eutrophus, Comamonas violaceum, Pseudomonas citronellolis, and P. fluorescenes (2 strains). The authors also describe purified P(3HO) depolymerase and compared it to PHB and PHA deploymerases. P(3HO) depolymerase activity was found not only in the sulture supernatant but also in the soluble fraction and membrane fractions of P(3HO) grown cells.39 refs.,5 figs.,3 tabs.

  7. A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha

    PubMed Central

    Srinivasan, Sriram; Barnard, Gavin C.; Gerngross, Tillman U.

    2002-01-01

    We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli. PMID:12450812

  8. Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules.

    PubMed

    Pieper-Fürst, U; Madkour, M H; Mayer, F; Steinbüchel, A

    1995-05-01

    The function of the polyhydroxyalkanoic acid (PHA) granule-associated GA14 protein of Rhodococcus ruber was investigated in Escherichia coli XL1-Blue, which coexpressed this protein with the polyhydroxybutyric acid (PHB) biosynthesis operon of Alcaligenes eutrophus. The GA14 protein had no influence on the biosynthesis rate of PHB in E. coli XL1-Blue(pSKCO7), but this recombinant E. coli strain formed smaller PHB granules than were formed by an E. coli strain that expressed only the PHB operon. Immunoelectron microscopy with GA14-specific antibodies demonstrated the binding of GA14 protein to these mini granules. In a previous study, two hydrophobic domains close to the C terminus of the GA14 protein were analyzed, and a working hypothesis that suggested an anchoring of the GA14 protein in the phospholipid monolayer surrounding the PHA granule core by these hydrophobic domains was developed (U. Pieper-Fürst, M. H. Madkour, F. Mayer, and A. Steinbüchel, J. Bacteriol. 176:4328-4337, 1994). This hypothesis was confirmed by the construction of C-terminally truncated variants of the GA14 protein lacking the second or both hydrophobic domains and by the demonstration of their inability to bind to PHB granules. Further confirmation of the hypothesis was obtained by the construction of a fusion protein composed of the acetaldehyde dehydrogenase II of A. eutrophus and the C terminus of the GA14 protein containing both hydrophobic domains and by its affinity to native and artificial PHB granules.

  9. Production of NO and N(inf2)O by Pure Cultures of Nitrifying and Denitrifying Bacteria during Changes in Aeration

    PubMed Central

    Kester, R. A.; De Boer, W.; Laanbroek, H. J.

    1997-01-01

    Peak emissions of NO and N(inf2)O are often observed after wetting of soil. The reactions to sudden changes in the aeration of cultures of nitrifying and denitrifying bacteria with respect to NO and N(inf2)O emissions were compared to obtain more information about the microbiological aspects of peak emissions. In continuous culture, the nitrifier Nitrosomonas europaea and the denitrifiers Alcaligenes eutrophus and Pseudomonas stutzeri were cultured at different levels of aeration (80 to 0% air saturation) and subjected to changes in aeration. The relative production of NO and N(inf2)O by N. europaea, as a percentage of the ammonium conversion, increased from 0.87 and 0.17%, respectively, at 80% air saturation to 2.32 and 0.78%, respectively, at 1% air saturation. At 0% air saturation, ammonium oxidation and N(inf2)O production ceased but NO production was enhanced. Coculturing of N. europaea with the nitrite oxidizer Nitrobacter winogradskyi strongly reduced the relative levels of NO and N(inf2)O production, probably as an effect of the lowered nitrite concentration. After lowering the aeration, N. europaea produced large short-lasting peaks of NO and N(inf2)O emissions in the presence but not in the absence of nitrite. A. eutrophus and P. stutzeri began to denitrify below 1% air saturation, with the former accumulating nitrite and N(inf2)O and the latter reducing nitrate almost completely to N(inf2). Transition of A. eutrophus and P. stutzeri from 80 to 0% air saturation resulted in transient maxima of denitrification intermediates. Such transient maxima were not observed after transition from 1 to 0%. Reduction of nitrate by A. eutrophus continued 48 h after the onset of the aeration, whereas N(inf2)O emission by P. stutzeri increased for only a short period. It was concluded that only in the presence of nitrite are nitrifiers able to dominate the NO and N(inf2)O emissions of soils shortly after a rainfall event. PMID:16535707

  10. Cloning, Molecular Analysis, and Expression of the Polyhydroxyalkanoic Acid Synthase (phaC) Gene from Chromobacterium violaceum

    PubMed Central

    Kolibachuk, Dana; Miller, Andrea; Dennis, Douglas

    1999-01-01

    The polyhydroxyalkanoic acid synthase gene from Chromobacterium violaceum (phaCCv) was cloned and characterized. A 6.3-kb BamHI fragment was found to contain both phaCCv and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaACv). Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While C. violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on a fatty acid carbon source, Klebsiella aerogenes and Ralstonia eutropha (formerly Alcaligenes eutrophus), harboring phaCCv, accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the C. violaceum PHA synthase. Neither recombinant E. coli nor recombinant Pseudomonas putida harboring phaCCv accumulated significant levels of PHA. Sequence analysis of the phaCCv product shows homology with several PHA synthases, most notably a 48% identity with that of Alcaligenes latus (GenBank accession no. AAD10274). PMID:10427049

  11. Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4-dichlorophenoxyacetic acid.

    PubMed Central

    Amy, P S; Schulke, J W; Frazier, L M; Seidler, R J

    1985-01-01

    Water samples from rivers, streams, ponds, and activated sewage were tested for the presence of bacteria which utilize 2,4-dichlorophenoxyacetic acid (2,4-D) as a sole source of carbon. Seventy percent of the attempted enrichments yielded pure cultures of 2,4-D-metabolizing bacteria. All but 1 of the 30 isolates were gram-negative rods, all but 2 were motile, and all were nonfermentative and oxidase and catalase positive. Nine isolates had DNA guanine-plus-cytosine values of 61.1 to 65 mol%. One isolate had a 67 mol% guanine-plus-cytosine value. The results suggest that these 2,4-D-metabolizing bacteria belong to the genus Alcaligenes. Fourteen of 23 isolates contained one or more detectable plasmids of about 20, 60, or 100 megadaltons. HindIII restriction fragment patterns showed these plasmids to be different from each other with one exception. Very similar restriction fragment patterns were revealed with a plasmid isolated from an Alcaligenes eutrophus strain obtained from Australia (pJMP397) and in an Alcaligenes sp. isolated in Oregon (pEML159). These two plasmids were about 56 megadaltons, had the same guanine-plus-cytosine value, were transmissable, and coded for 2,4-D metabolism and resistance to HgCl2. Hybridization of these two plasmids was demonstrated by using nick-translated 32P-labeled pJMP397. The vector pBR325 was used to clone HindIII fragments from pEML159. One cloned fragment of 14.8 megaldaltons expressed in Escherichia coli the ability to release 14CO2 from 2,4-D labeled in the acetate portion. Images PMID:3923930

  12. Plasmid Transfer between Spatially Separated Donor and Recipient Bacteria in Earthworm-Containing Soil Microcosms

    PubMed Central

    Daane, L. L.; Molina, J.; Sadowsky, M. J.

    1997-01-01

    Most gene transfer studies have been performed with relatively homogeneous soil systems in the absence of soil macrobiota, including invertebrates. In this study we examined the influence of earthworm activity (burrowing, casting, and feeding) on transfer of plasmid pJP4 between spatially separated donor (Alcaligenes eutrophus) and recipient (Pseudomonas fluorescens) bacteria in nonsterile soil columns. A model system was designed such that the activity of earthworms would act to mediate cell contact and gene transfer. Three different earthworm species (Aporrectodea trapezoides, Lumbricus rubellus, and Lumbricus terrestris), representing each of the major ecological categories (endogeic, epigeic, and anecic), were evaluated. Inoculated soil microcosms, with and without added earthworms, were analyzed for donor, recipient, and transconjugant bacteria at 5-cm-depth intervals by using selective plating techniques. Transconjugants were confirmed by colony hybridization with a mer gene probe. The presence of earthworms significantly increased dispersal of the donor and recipient strains. In situ gene transfer of plasmid pJP4 from A. eutrophus to P. fluorescens was detected only in earthworm-containing microcosms, at a frequency of (symbl)10(sup2) transconjugants per g of soil. The depth of recovery was dependent on the burrowing behavior of each earthworm species; however, there was no significant difference in the total number of transconjugants among the earthworm species. Donor and recipient bacteria were recovered from earthworm feces (casts) of all three earthworm species, with numbers up to 10(sup6) and 10(sup4) bacteria per g of cast, respectively. A. trapezoides egg capsules (cocoons) formed in the inoculated soil microcosms contained up to 10(sup7) donor and 10(sup6) recipient bacteria per g of cocoon. No transconjugant bacteria, however, were recovered from these microhabitats. To our knowledge, this is the first report of gene transfer between physically

  13. Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules.

    PubMed Central

    Pieper-Fürst, U; Madkour, M H; Mayer, F; Steinbüchel, A

    1995-01-01

    The function of the polyhydroxyalkanoic acid (PHA) granule-associated GA14 protein of Rhodococcus ruber was investigated in Escherichia coli XL1-Blue, which coexpressed this protein with the polyhydroxybutyric acid (PHB) biosynthesis operon of Alcaligenes eutrophus. The GA14 protein had no influence on the biosynthesis rate of PHB in E. coli XL1-Blue(pSKCO7), but this recombinant E. coli strain formed smaller PHB granules than were formed by an E. coli strain that expressed only the PHB operon. Immunoelectron microscopy with GA14-specific antibodies demonstrated the binding of GA14 protein to these mini granules. In a previous study, two hydrophobic domains close to the C terminus of the GA14 protein were analyzed, and a working hypothesis that suggested an anchoring of the GA14 protein in the phospholipid monolayer surrounding the PHA granule core by these hydrophobic domains was developed (U. Pieper-Fürst, M. H. Madkour, F. Mayer, and A. Steinbüchel, J. Bacteriol. 176:4328-4337, 1994). This hypothesis was confirmed by the construction of C-terminally truncated variants of the GA14 protein lacking the second or both hydrophobic domains and by the demonstration of their inability to bind to PHB granules. Further confirmation of the hypothesis was obtained by the construction of a fusion protein composed of the acetaldehyde dehydrogenase II of A. eutrophus and the C terminus of the GA14 protein containing both hydrophobic domains and by its affinity to native and artificial PHB granules. PMID:7730285

  14. Paleomicrobiology to investigate copper resistance in bacteria: isolation and description of Cupriavidus necator B9 in the soil of a medieval foundry.

    PubMed

    Gillan, David C; Van Camp, Camille; Mergeay, Max; Provoost, Ann; Thomas, Nicolas; Vermard, Laurent; Billon, Gabriel; Wattiez, Ruddy

    2017-02-01

    Remains of a medieval foundry were excavated by archaeologists in 2013 in Verdun (France). Ancient workshops specialized in brass and copper alloys were found with an activity between 13th to 16th c. Levels of Cu, Zn and Pb reached 20000, 7000 and 6000 mg kg(-1) (dw), respectively, in several soil horizons. The objective of the present work was to examine the microbial community in this contaminated site. A total of 8-22 10(6) reads were obtained by shotgun metagenomics in four soil horizons. Bioinformatic analyses suggest the presence of complex bacterial communities dominated by Proteobacteria. The structure of the community was not affected by metals, contrary to the set of metal-resistance genes. Using selective media, a novel strain of Cupriavidus necator (eutrophus), strain B9, was isolated. Its genome was sequenced and a novel metal resistance gene cluster with Hg resistance genes (merRTPCA) followed by 24 copper-resistance genes (actP, cusCBAF, silP, copK1, copH4QLOFGJH3IDCBARS, copH2H1, copK2) was found. This cluster is partly homologous to the cop genes of Cupriavidus gilardii CR3 and C. metallidurans CH34. Proteomics indicated that the four copH genes were differentially expressed: CopH1 and CopH2 were mostly induced by Cd while CopH4 was highly expressed by Cu.

  15. Integrated systems for biopolymers and bioenergy production from organic waste and by-products: a review of microbial processes.

    PubMed

    Pagliano, Giorgia; Ventorino, Valeria; Panico, Antonio; Pepe, Olimpia

    2017-01-01

    Recently, issues concerning the sustainable and harmless disposal of organic solid waste have generated interest in microbial biotechnologies aimed at converting waste materials into bioenergy and biomaterials, thus contributing to a reduction in economic dependence on fossil fuels. To valorize biomass, waste materials derived from agriculture, food processing factories, and municipal organic waste can be used to produce biopolymers, such as biohydrogen and biogas, through different microbial processes. In fact, different bacterial strains can synthesize biopolymers to convert waste materials into valuable intracellular (e.g., polyhydroxyalkanoates) and extracellular (e.g., exopolysaccharides) bioproducts, which are useful for biochemical production. In particular, large numbers of bacteria, including Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinelandii, Azotobacter chroococcum, Azotobacter beijerincki, methylotrophs, Pseudomonas spp., Bacillus spp., Rhizobium spp., Nocardia spp., and recombinant Escherichia coli, have been successfully used to produce polyhydroxyalkanoates on an industrial scale from different types of organic by-products. Therefore, the development of high-performance microbial strains and the use of by-products and waste as substrates could reasonably make the production costs of biodegradable polymers comparable to those required by petrochemical-derived plastics and promote their use. Many studies have reported use of the same organic substrates as alternative energy sources to produce biogas and biohydrogen through anaerobic digestion as well as dark and photofermentation processes under anaerobic conditions. Therefore, concurrently obtaining bioenergy and biopolymers at a reasonable cost through an integrated system is becoming feasible using by-products and waste as organic carbon sources. An overview of the suitable substrates and microbial strains used in low-cost polyhydroxyalkanoates for biohydrogen and biogas production is

  16. Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M.

    PubMed

    Saito, T; Saegusa, H; Miyata, Y; Fukui, T

    1992-12-01

    Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.

  17. Molecular systematics of the genus Zoogloea and emendation of the genus.

    PubMed

    Shin, Y K; Hiraishi, A; Sugiyama, J

    1993-10-01

    Phylogenetic relationships among strains of Zoogloea and related taxa were determined by 16S rDNA sequencing and genomic DNA hybridization techniques. The 16S rRNA gene was amplified by the polymerase chain reaction with a pair of eubacterial consensus primers and sequenced directly by using an automated fluorescent DNA sequencer. Sequence comparisons and distance matrix tree analysis revealed that Zoogloea ramigera IAM 12136 (= N. C. Dondero 106, type strain) and Zoogloea sp. ATCC 19324 formed a lineage with Rhodocyclus purpureus in the beta subclass of Proteobacteria. Z. ramigera IAM 12670 (= P. R. Dugan 115) was shown to belong to another cluster with Alcaligenes eutrophus and Pseudomonas cepacia in the beta subclass. In contrast, Z. ramigera IAM 12669 (= K. Crabtree I-16-M) proved to be a member of the alpha subclass of the Proteobacteria, closely related to Agrobacterium tumefaciens. Genomic DNA hybridization studies also showed that there is genetic diversity among the strains currently designated Z. ramigera, but typical Zoogloea strains, characterized by their production of rhodoquinones, are highly related to each other and can be regarded as a single species. On the basis of the molecular data, together with the early phenotypic and chemotaxonomic information, we have emended the generic description of Zoogloea.

  18. A physical genome map of the Burkholderia cepacia type strain.

    PubMed

    Rodley, P D; Römling, U; Tümmler, B

    1995-07-01

    Burkholderia cepacia (basonym Pseudomonas cepacia), the type species of the new genus Burkholderia, is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil-borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type-strain ATCC 25416 was constructed by applying two-dimensional pulsed-field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 SpeI, 11 SwaI, 11 PacI, 11 PmeI and six I-CeuI sites, resulting in an average resolution of 105 kbp. Random single-hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase-size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia. The phylogenetically related species Burkholderia glumae, Burkholderia pickettii, Burkholderia solanacearum, Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase-size replicon.

  19. Oxygen-controlled regulation of the flavohemoglobin gene in Bacillus subtilis.

    PubMed Central

    LaCelle, M; Kumano, M; Kurita, K; Yamane, K; Zuber, P; Nakano, M M

    1996-01-01

    A gene, hmp, which encodes a ubiquitous protein homologous to hemoglobin was isolated among genes from Bacillus subtilis that are induced under anaerobic conditions. The hmp protein belongs to the family of two-domain flavohemoproteins, homologs of which have been isolated from various organisms such as Escherichia coli, Alcaligenes eutrophus, and Saccharomyces cerevisiae. These proteins consist of an amino-terminal hemoglobin domain and a carboxy-terminal redox active site domain with potential binding sites for NAD(P)H and flavin adenine dinucleotide. The expression of hmp is strongly induced upon oxygen limitation, and the induction is dependent on a two-component regulatory pair, ResD and ResE, an anaerobic regulator, FNR, and respiratory nitrate reductase, NarGHJI. The requirement of FNR and NarGHJI for hmp expression is completely bypassed by the addition of nitrite in the culture medium, indicating that fnr is required for transcriptional activation of narGHJI, which produces nitrite, leading to induction of hmp expression. In contrast, induction of hmp was still dependent on resDE in the presence of nitrite. A defect in hmp in B. subtilis has no significant effect on anaerobic growth. PMID:8682784

  20. Conversion of food industrial wastes into bioplastics.

    PubMed

    Yu, P H; Chua, H; Huang, A L; Lo, W; Chen, G Q

    1998-01-01

    The usage of plastics in packaging and disposable products, and the generation of plastic waste, have been increasing drastically. Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. In the authors' laboratories, various carbohydrates in the growth media, including sucrose, lactic acid, butyric acid, valeric acid, and various combinations of butyric and valeric acids, were utilized as the carbon (c) sources for the production of bioplastics by Alcaligenes eutrophus. As the first step in pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesize bioplastics, the authors investigated the usage of malt wastes from a beer brewery plant as the C sources for the production of bioplastics by microorganisms. Specific polymer production yield by A. Latus DSM 1124 increased to 70% polymer/cell (g/g) and 32 g/L cell dry wt, using malt wastes as the C source. The results of these experiments indicated that, with the use of different types of food wastes as the C source, different polyhydroxyalkanoate copolymers could be produced with distinct polymer properties.

  1. Direct Production of Propene from the Thermolysis of Poly(..beta..-hydroxybutyrate)

    SciTech Connect

    Mittal, Ashutosh; Pilath, Heidi M.; Johnson, David K.

    2015-03-22

    To transform biomass components into hydrocarbon fuels it is clear that there are two main transformations that need to occur, i.e., deoxygenation and carbon chain extension. The potential routes for decreasing the oxygen content of biomass intermediates include dehydration, hydrodeoxygenation and decarboxylation. One route that is examined here is the conversion of polyhydroxyalkanoates (PHA) to alkenes that would be intermediates to hydrocarbon fuels.Thermal breakdown of PHA proceeds via an intermediate carboxylic acid, which can then be decarboxylated to an alkene. Oligomerization of alkenes by well-known commercial technologies would permit production of a range of hydrocarbon fuels from a carbohydrate derived intermediate. Moreover, polyhydroxybutyrate (PHB) can be produced in Cupriavidus necator (formerly known as Ralstonia eutropha) and Alcaligenes eutrophus on a variety of carbon sources including glucose, fructose and glycerol with PHB accumulation reaching 75 percent of dry cell mass. We conducted thermal conversion of PHB and pure crotonic acid (CA), the intermediate carboxylic acid produced by thermal depolymerization of PHB, in a flow-through reactor. The results of initial experiments on the thermal conversion of CA showed that up to 75 mole percent yields of propene could be achieved by optimizing the residence time and temperature of the reactor. Further experiments are being investigated to optimize the reactor parameters and enhance propene yields via thermal conversion of PHB.

  2. Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions.

    PubMed

    Kuhn, M; Steinbüchel, A; Schlegel, H G

    1984-08-01

    When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.

  3. Polyhydroxybutyrate: plastic made and degraded by microorganisms.

    PubMed

    Hankermeyer, C R; Tjeerdema, R S

    1999-01-01

    Polyhydroxybutyrate (PHB) offers many advantages over traditional petrochemically derived plastics. In addition to its complete biodegradability, PHB is formed from renewable resources. It possesses better physical properties than polypropylene for food packaging applications and is completely nontoxic. The poor low-impact strength of PHB is solved by incorporation of hydroxyvalerate monomers into the polymer to produce polyhydroxybutyrate-co-valerate (PHBV), which is commercially marketed under the trade name Biopol. Like PHB, PHBV completely degrades into carbon dioxide and water under aerobic conditions. Microbial synthesis of PHB is the best method for industrial production because it ensures the proper stereochemistry for biodegradation. Microorganisms synthesize and store PHB under nutrient-limited conditions and degrade and metabolize it when the limitation is removed. Current production employs Alcaligenes eutrophus because it grows efficiently on glucose as a carbon source, accumulates PHB up to 80% of its dry weight, and is able to synthesize PHBV when propionic acid is added to the feedstock. PHBV is currently 16 times the price of polypropylene. However, the development of transgenic PHA-producing organisms is expected to greatly reduce its cost. Benefits of using transgenic systems include lack of a depolymerase system, ability to use faster-growing organisms, production of highly purified polymers, and ability to utilize inexpensive carbon sources. Because transgenic plants may someday result in the evolution of plastic crops that could lower the price of PHA to a competitive level, future research will surely focus on such recombinant DNA techniques.

  4. Microbial remediation of soils co-contaminated with 2,4-dichlorophenoxyacetic acid and cadmium

    SciTech Connect

    Roane, T.M.; Pepper, I.L.

    1997-12-31

    One-third of organically-polluted sites are also contaminated with metals; however, the bioremediation potential of such sites is not clear. While metals are thought to inhibit the abilities of microbial communities to degrade organic pollutants, several microbial-metal resistance mechanisms are known to exist. This study utilizes cadmium-resistant soil microorganisms to enhance the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of cadmium. Cadmium-resistant bacteria were isolated from both a 40-year-old metal-contaminated soil and an uncontaminated soil. During growth experiments, it was found that the uncontaminated soil had a greater number of resistant isolates at low concentrations of cadmium, while the cadmium-contaminated soil exhibited higher microbial resistance with increased cadmium concentrations. ERIC PCR fingerprints discriminated among the cadmium-resistant isolates identified by BIOLOG as Bacillus, Corynecbacterium, Pseudomonas, and Xanthomonas spp. These isolates were resistant to concentrations ranging from 5 to 275 ppm soluble cadmium. In conventional degradation studies, two resistant isolates, a Bacillus and an unidentified Gram positive rod, supported the degradation of 500 ppm 2,4-D by the cadmium-sensitive 2,4-D degrader Alcaligenes eutrophus JMP134 in the presence of 20 and 40 ppm soluble cadmium, respectively.

  5. Gene cloning, expression, and substrate specificity of an imidase from the strain Pseudomonas putida YZ-26.

    PubMed

    Shi, Ya-wei; Cui, Li-fang; Yuan, Jing-ming

    2007-07-01

    A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni(2+)-NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated.

  6. Enzyme-catalyzed synthesis of poly[(R)-(-)-3-hydroxybutyrate]: Formation of macroscopic granules in vitro

    SciTech Connect

    Gerngross, T.U.; Martin, D.P.

    1995-07-03

    A combined chemical and enzymatic procedure has been developed to synthesize macroscopic poly[(R)-(-)-3-hydroxybutyrate] (PHB) granules in vitro. The granules form in a matter of minutes when purified polyhydroxyalkanoate (PHA) synthase from Alcaligenes eutrophus is exposed to synthetically prepared (R)-3-hydroxybutyryl coenzyme A, thereby establishing the minimal requirements for PHB granule formation. The artificial granules are spherical with diameters of up to 3 {mu}m and significantly larger than their native counterparts (0.5 {mu}m). The isolated PHB was characterized by {sup 1}H and {sup 13}C NMR, gel-permeation chromatography, and chemical analysis. The in vitro polymerization system yields PHB with a molecular mass > 10 x 10{sup 6} Da, exceeding by an order of magnitude the mass of PHAs typically extracted from microorganisms. We also demonstrate that the molecular mass of the polymer can be controlled by the initial PHA synthase concentration. Preliminary kinetic analysis of de novo granule formation confirms earlier findings of a lag time for the enzyme but suggests the involvement of an additional granule assembly step. Minimal requirements for substrate recognition were investigated. Since substrate analogs lacking the adenosine 3{prime}, 5{prime}-bisphosphate moiety of (R)-3-hydroxybutyryl coenzyme A were not accepted by the PHA synthase, we provide evidence that this structural element of the substrate is essential for catalysis. PHAs provide a range of natural, renewable, biodegradable thermoplastics with a broad range of useful material properties. 33 refs., 6 figs., 1 tab.

  7. Bioavailability of zinc in runoff water from roofing materials.

    PubMed

    Heijerick, D G; Janssen, C R; Karlèn, C; Wallinder, I Odnevall; Leygraf, C

    2002-06-01

    Corrosion and runoff from zinc-coated materials and outdoor structures is an important source for the dispersion of zinc in the environment. Being part of a large inter-disciplinary research project, this study presents the bioavailability of zinc in runoff water immediately after release from the surface of 15 different commercially available zinc-based materials exposed to the urban environment of Stockholm, Sweden. Runoff water was analysed chemically and evaluated for its possible environmental impact, using both a biosensor test with the bacteria Alcaligenes eutrophus (Biomet) and the conventional 72 h growth inhibition test with the green alga Raphidocelis subcapitata. Chemical speciation modelling revealed that most zinc (94.3-99.9%) was present as the free Zn ion, the most bioavailable speciation form. These findings were confirmed by the results of the biosensor test (Biomet) which indicated that all zinc was indeed bioavailable. Analysis of the ecotoxicity data also suggested that the observed toxic effects were due to the presence of Zn2+ ions. Finally, regression analysis showed that, for this type of runoff samples, the rapid screening biosensor was capable of predicting (a) the total amount of zinc present in the runoff samples (R2 of 0.93-0.98; p < 0.05) and (b) the observed 72 h-EbC50s (R2 of 0.69-0.97; p < 0.05).

  8. Microbial degradation of 2,4-dichlorophenoxyacetic acid: Insight into the enzymes and catabolic genes involved, their regulation and biotechnological implications.

    PubMed

    Kumar, Ajit; Trefault, Nicole; Olaniran, Ademola Olufolahan

    2016-01-01

    A considerable progress has been made to understand the mechanisms of biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D biodegradation pathway has been elucidated in many microorganisms including Cupriavidus necator JMP134 (previously known as Wautersia eutropha, Ralstonia eutropha and Alcaligenes eutrophus) and Pseudomonas strains. It generally involves the side chain removal of 2,4-D by α-ketoglutarate-dependent 2,4-D dioxygenase (tfdA) to form 2,4-dichlorophenol (2,4-DCP); hydroxylation of 2,4-DCP by 2,4-DCP hydroxylase (tfdB) to form dichlorocatechol; ortho or meta cleavage of dichlorocatechol by chlorocatechol 1,2-dioxygenase (tfdC) to form 2,4-dichloro-cis,cis-muconate; conversion of 2,4-dichloro-cis,cis-muconate to 2-chlorodienelactone by chloromuconate cycloisomerase (tfdD); conversion of 2-chlorodienelactone to 2-chloromaleylacetate by chlorodienelactone hydrolase (tfdE) and, finally, conversion of 2-chloromaleylacetate to 3-oxoadepate via maleylacetate by chloromaleylacetate reductase and maleylacetate reductase (tfdF), respectively, which is funnelled to the tricarboxylic acid cycle. The latest review on microbial breakdown of 2,4-D, other halogenated aromatic pesticides, and related compounds was compiled by Haggblom, however, a considerable progress has been made in this area of research since then. Thus, this review focuses on the recent advancement on 2,4-D biodegradation, the enzymes, and genes involved and their biotechlogical implications.

  9. Substrate diversity and expression of the 2,4,5-trichlorophenoxyacetic acid oxygenase from Burkholderia cepacia AC1100.

    PubMed Central

    Danganan, C E; Shankar, S; Ye, R W; Chakrabarty, A M

    1995-01-01

    Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid as a sole source of carbon and energy. The genes encoding the proteins involved in the first step (tftA and tftB [previously designated tftA1 and tftA2, respectively]) have been cloned and sequenced. The oxygenase, TftAB, is capable of converting not only 2,4,5-trichlorophenoxyacetic acid to 2,4,5-trichlorophenol but also a wide range of chlorinated aromatic phenoxyacetates to their corresponding phenolic derivatives, as shown by whole-cell and cell-free assays. The rate of substrate utilization by TftAB depends upon the extent of chlorination of the substrate, the positions of the chlorines, and the phenoxy group. These results indicate a mechanistic similarity between TftAB and the 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate-dependent dioxygenase, TfdA, from Alcaligenes eutrophus JMP134. The promoter of the oxygenase genes was localized by promoter-probe analysis, and the transcriptional start site was identified by primer extension. The beta-galactosidase activity of the construct containing the promoter region cloned upstream of the beta-galactosidase gene in the promoter-probe vector pKRZ-1 showed that this construct is constitutively expressed in Escherichia coli and in AC1100. The -35 and -10 regions of the oxygenase genes show significant sequence identity to typical Escherichia coli sigma 70 promoters. PMID:8534119

  10. Specific binding of Thiobacillus ferrooxidans RbcR to the intergenic sequence between the rbc operon and the rbcR gene.

    PubMed Central

    Kusano, T; Sugawara, K

    1993-01-01

    The presence of two sets (rbcL1-rbcS1 and rbcL2-rbcS2) of rbc operons has been demonstrated in Thiobacillus ferrooxidans Fe1 (T. Kusano, T. Takeshima, C. Inoue, and K. Sugawara, J. Bacteriol. 173:7313-7323, 1991). A possible regulatory gene, rbcR, 930 bp long and possibly translated into a 309-amino-acid protein, was found upstream from the rbcL1 gene as a single copy. The gene is located divergently to rbcL1 with a 144-bp intergenic sequence. As in the cases of the Chromatium vinosum RbcR and Alcaligenes eutrophus CfxR, T. ferrooxidans RbcR is thought to be a new member of the LysR family, and these proteins share 46.5 and 42.8% identity, respectively. Gel mobility shift assays showed that T. ferrooxidans RbcR, produced in Escherichia coli, binds specifically to the intergenic sequence between rbcL1 and rbcR. Footprinting and site-directed mutagenesis experiments further demonstrated that RbcR binds to overlapping promoter elements of the rbcR and rbcL1 genes. The above data strongly support the participation of RbcR in regulation of the rbcL1-rbcS1 operon and the rbcR gene in T. ferrooxidans. Images PMID:8432695

  11. Cloning of the amphibolic Calvin cycle/OPPP enzyme D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects.

    PubMed

    Nowitzki, U; Wyrich, R; Westhoff, P; Henze, K; Schnarrenberger, C; Martin, W

    1995-12-01

    Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme D-ribulose-5-phosphate 3-epimerase (R5P3E)(EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.

  12. A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha.

    PubMed

    Solaiman, Daniel K Y; Swingle, Bryan M; Ashby, Richard D

    2010-08-01

    Ralstonia eutropha (formerly Alcaligenes eutrophus) is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and molecular engineering of this organism. We report here a versatile gene expression method for the genetic engineering of R. eutropha. This method, based on a simplified electroporation protocol, uses a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). Using this method, we successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB(-)4, with various pBS29-P2-based recombinants. A transformation frequency as high as 4x10(3) Km-resistance colonies/mug DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor alpha-galactosidase in R. eutropha.

  13. Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil.

    PubMed

    Neilson, J W; Josephson, K L; Pepper, I L; Arnold, R B; Di Giovanni, G D; Sinclair, N A

    1994-11-01

    Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment. Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms. The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site. Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation. A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil. Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4. In addition, we confirmed the transfer through PCR amplifications of the tfdB gene. Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses. An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.

  14. Spontaneous liberation of intracellular polyhydroxybutyrate granules in Escherichia coli.

    PubMed

    Jung, Il Lae; Phyo, Ki Heon; Kim, Kug Chan; Park, Hyo Kook; Kim, In Gyu

    2005-09-01

    Despite receiving much attention as a biodegradable substitute for conventional non-biodegradable plastics, the commercial use of polyhydroxybutyrate (PHB) remains limited because of its high production cost. In order to reduce the recovery/purification cost, which forms over half of the total production cost, we have developed a new cultivation method which enables spontaneous liberation of PHB by modulation of the initial inoculum size and the medium composition in recombinant Escherichia coli harboring Alcaligenes eutrophus phbCAB genes. In flask cultivation using a low cell inoculum and 2x LB medium containing 21% glucose, autolysis of 80.2% as well as yields of 85.2 g/l of PHB and a PHB content of 99.0% (w/w) were obtained. The glucose conversion rate was 0.43. The strategies developed in this study can minimize complex and unfavorable efforts required for efficient recovery/purification processes, thereby enabling biodegradable plastic to be produced by the recombinant E. coli so as to compete with conventional non-biodegradable plastic.

  15. Crystal structure of an electron transfer complex between aromatic amine dehydrogenase and azurin from Alcaligenes faecalis.

    PubMed

    Sukumar, Narayanasami; Chen, Zhi-wei; Ferrari, Davide; Merli, Angelo; Rossi, Gian Luigi; Bellamy, Henry D; Chistoserdov, Andrei; Davidson, Victor L; Mathews, F Scott

    2006-11-14

    The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of the TTQ-containing methylamine dehydrogenase (MADH) and the cupredoxin amicyanin. Despite significant similarities between the two quinoproteins and the two cupredoxins, each is specific for its respective partner and the ionic strength dependence and magnitude of the binding constant for each complex are quite different. The AADH-azurin interface is largely hydrophobic, covering approximately 500 A(2) of surface on each molecule, with one direct hydrogen bond linking them. The closest distance from TTQ to copper is 12.6 A compared with a distance of 9.3 A in the MADH-amicyanin complex. When the MADH-amicyanin complex is aligned with the AADH-azurin complex, the amicyanin lies on top of the azurin but is oriented quite differently. Although the copper atoms differ in position by approximately 4.7 A, the amicyanin bound to MADH appears to be rotated approximately 90 degrees from its aligned position with azurin. Comparison of the structures of the two complexes identifies features of the interface that dictate the specificity of the protein-protein interaction and determine the rate of interprotein electron transfer.

  16. Biodegradation of organochlorine pesticide endosulfan by bacterial strain Alcaligenes faecalis JBW4.

    PubMed

    Kong, Lingfen; Zhu, Shaoyuan; Zhu, Lusheng; Xie, Hui; Su, Kunchang; Yan, Tongxiang; Wang, Jun; Wang, Jinhua; Wang, Fenghua; Sun, Fengxia

    2013-11-01

    The recently discovered endosulfan-degrading bacterial strain Alcaligenesfaecalis JBW4 was isolated from activated sludge. This strain is able to use endosulfan as a carbon and energy source. The optimal conditions for the growth of strain JBW4 and for biodegradation by this strain were identified, and the metabolic products of endosulfan degradation were studied in detail. The maximum level of endosulfan biodegradation by strain JBW4 was obtained using broth at an initial pH of 7.0, an incubation temperature of 40 degreeC and an endosulfan concentration of 100 mg/L. The concentration of endosulfan was determined by gas chromatography. Strain JBW4 was able to degrade 87.5% of alpha-endosulfan and 83.9% of beta-endosulfan within 5 days. These degradation rates are much higher than the previously reported bacterial strains. Endosulfan diol and endosulfan lactone were the major metabolites detected by gas chromatography-mass spectrometry; endosulfan sulfate, which is a persistent and toxic metabolite, was not detected. These results suggested that A. faecalis JBW4 degrades endosulfan via a non-oxidative pathway. The biodegradation of endosulfan by A. faecalis is reported for the first time. Additionally, the present study indicates that strain JBW4 may have potential for the biodegradation of endosulfan residues.

  17. [Influence of nitrogen source NH4 Cl concentration on curdlan production in Alcaligenes faecalis].

    PubMed

    Sun, Yong-Sheng; Wang, Lei; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Chen, Yuan-Zhi

    2005-03-01

    The effect of initial ammonium chloride level on production of curdlan in Alcaligenesfaecalis was investigated. It was found that ammonium chloride was the limiting substrate for cell growth during the batch fermentation process. However, the cell growth and curdlan production could not be enhanced by solely increasing the initial ammonium chloride level. The pH drop in the broth due to the consumption of ammonium chloride also effected the cell growth and curdlan production. By simultaneously increasing the initial ammonium chloride concentration and implementing an optimal pH control strategy, which is to control pH at 7.0 in the growth phase, and then shift to 5.6 in the production phase, the biomass and curdlan production in batch fermentation were increased markedly. If the initial ammonium chloride concentration was increased from 1.1 g/L to 3.6 g/L, biomass concentration of 7.2 g/L was obtained, and the final curdlan concentration reached 30.5 g/L, which was 51.7% higher than that of the former case. As the cell growth was improved due to the increase of the initial ammonium chloride concentration, the agitation speed and aeration rates must be enhanced to suit the higher oxygen uptake requirement. However, as curdlan molecules is subject to the structural breakage due to the high shear stress at higher agitation speed, an overall optimal condition for both productivity and quality of curdlan should be considered comprehensively.

  18. Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10.

    PubMed

    Liu, Zhi-Qiang; Dong, Li-Zhu; Cheng, Feng; Xue, Ya-Ping; Wang, Yuan-Shan; Ding, Jie-Nv; Zheng, Yu-Guo; Shen, Yin-Chu

    2011-11-09

    Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 °C, respectively. Mg(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+), Ni(2+), Ag(+), and Hg(2+) showed a strong inhibitory effect. The K(m) and v(max) for mandelonitrile were 4.74 mM and 15.85 μmol min(-1) mg(-1) protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.

  19. Effect of Tween 80 on the production of curdlan by Alcaligenes faecalis ATCC 31749.

    PubMed

    Xia, Zhenqiang

    2013-10-15

    This study aims to investigate the effects of Tween 80 on curdlan production, cell growth, and glucosyltransferase activity. The addition of Tween 80 to the culture medium increased curdlan production. However, curdlan production did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. The addition of Tween 80 to the culture medium did not affect cell growth. The glucosyltransferase activity involved in the curdlan synthesis increased with the increase of Tween 80 concentration. The glucosyltransferase activity did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. Maximum curdlan was observed at day 5 and then levelled off. The biomass continued to increase until the end of the experimental period (6d). Maximum glucosyltransferase activity was also observed at day 5 and decreased thereafter. The results indicate that the enhanced curdlan production by Tween 80 is highly correlated with glucosyltransferase activity.

  20. Tracheal mucus transport rate in normal turkeys and in turkeys infected with Bordetella avium (Alcaligenes faecalis).

    PubMed

    Ficken, M D; Edwards, J F; Lay, J C; Tveter, D E

    1986-01-01

    Using the radiopharmaceutical 99mtechnetium-sulfur colloid, the tracheal mucus transport rate (TMTR) was measured in healthy unanesthetized turkeys and in turkeys infected with Bordetella avium. The TMTR of uninfected turkeys was 35.6 +/- 14.4 cm/min. The TMTR of B. avium-infected turkeys was normal on days 0 through 14 postexposure (PE), despite heavy bacterial colonization of the tracheal epithelium. On day 21 PE, the TMTR of B. avium-infected turkeys was significantly depressed (P less than or equal to 0.01) compared with that of control turkeys. Depressed transport was associated with extensive loss of ciliated epithelium from the tracheal mucosa and replacement of the normal mucosa by immature nonciliated epithelium or metaplastic squamous epithelium.

  1. Pristine environments harbor a new group of oligotrophic 2,4-dichlorophenoxyacetic acid-degrading bacteria.

    PubMed Central

    Kamagata, Y; Fulthorpe, R R; Tamura, K; Takami, H; Forney, L J; Tiedje, J M

    1997-01-01

    2,4-Dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated from pristine environments which had no history of 2,4-D exposure. By using 2,4-D dye indicator medium or 14C-labeled 2,4-D medium, six strains were isolated from eight enrichment cultures capable of degrading 2,4-D. Phylogenetic analyses based on 16S ribosomal DNA (rDNA) sequencing and physiological properties revealed that one isolate from Hawaiian volcanic soil could be classified in the genus Variovorax (a member of the beta subdivision of the class Proteobacteria) and that the other five isolates from Hawaiian volcanic soils, Saskatchewan forest soil, and Chilean forest soil have 16S rDNAs with high degrees of similarity to those of the Bradyrhizobium group (a member of the alpha subdivision of the class Proteobacteria). All the isolates grow slowly on either nutrient media (0.1 x Bacto Peptone-tryptone-yeast extract-glucose [PTYG] or 0.1 x Luria broth [LB] medium) or 2,4-D medium, with mean generation times of 16 to 30 h, which are significantly slower than previously known 2,4-D degraders. Nutrient-rich media such as full-strength PTYG and LB medium did not allow their growth. PCR amplification using internal consensus sequences of tfdA (a gene encoding an enzyme for the first step of 2,4-D mineralization, found in pJP4 of Alcaligenes eutrophus JMP134 and some other 2,4-D-degrading bacteria) as primers and Southern hybridization with pJP4-tfdA as a probe revealed that the isolate belonging to the genus Variovorax carried the tfdA gene. This gene was transmissible to A. eutrophus JMP228 carrying a plasmid with a mutant tfdA gene. The other five isolates did not appear to carry tfdA, and 2,4-D-specific alpha-ketoglutarate-dependent dioxygenase activity could not be detected in cell lysates. These results indicate that 2,4-D-degrading bacteria in pristine environments are slow-growing bacteria and that most of their phylogenies and catabolic genes differ from those of 2,4-D degraders

  2. Poly-ß-hydroxybutyrate content and dose of the bacterial carrier for Artemia enrichment determine the performance of giant freshwater prawn larvae.

    PubMed

    Thai, Truong Quoc; Wille, Mathieu; Garcia-Gonzalez, Linsey; Sorgeloos, Patrick; Bossier, Peter; De Schryver, Peter

    2014-06-01

    The beneficial effects of poly-β-hydroxybutyrate (PHB) for aquaculture animals have been shown in several studies. The strategy of applying PHB contained in a bacterial carrier has, however, hardly been considered. The effect of administering PHB-accumulated Alcaligenes eutrophus H16 containing 10 or 80 % PHB on dry weight, named A10 and A80, respectively, through the live feed Artemia was investigated on the culture performance of larvae of the giant freshwater prawn (Macrobrachium rosenbergii). Feeding larvae with Artemia nauplii enriched in a medium containing 100 and 1,000 mg L(-1) A80 significantly increased the survival with about 15 % and the development of the larvae with a larval stage index of about 1 as compared to feeding non-enriched Artemia. The survival of the larvae also significantly increased with about 35 % in case of a challenge with Vibrio harveyi. The efficiency of these treatments was equal to a control treatment of Artemia enriched in an 800 mg L(-1) PHB powder suspension, while Artemia enriched in 10 mg L(-1) A80, 100 mg L(-1) A10, and 1,000 mg L(-1) A10 did not bring similar effects. From our results, it can be concluded that PHB supplemented in a bacterial carrier (i.e., amorphous PHB) can increase the larviculture efficiency of giant freshwater prawn similar to supplementation of PHB in powdered form (i.e., crystalline PHB). When the level of PHB in the bacterial carrier is high, similar beneficial effects can be achieved as crystalline PHB, but at a lower live food enrichment concentration expressed on PHB basis.

  3. Production of bioplastics and hydrogen gas by photosynthetic microorganisms

    NASA Astrophysics Data System (ADS)

    Yasuo, Asada; Masato, Miyake; Jun, Miyake

    1998-03-01

    Our efforts have been aimed at the technological basis of photosynthetic-microbial production of materials and an energy carrier. We report here accumulation of poly-(3-hydroxybutyrate) (PHB), a raw material of biodegradable plastics and for production of hydrogen gas, and a renewable energy carrier by photosynthetic microorganisms (tentatively defined as cyanobacteria plus photosynthetic bateria, in this report). A thermophilic cyanobacterium, Synechococcus sp. MA19 that accumulates PHB at more than 20% of cell dry wt under nitrogen-starved conditions was isolated and microbiologically identified. The mechanism of PHB accumulation was studied. A mesophilic Synechococcus PCC7942 was transformed with the genes encoding PHB-synthesizing enzymes from Alcaligenes eutrophus. The transformant accumulated PHB under nitrogen-starved conditions. The optimal conditions for PHB accumulation by a photosynthetic bacterium grown on acetate were studied. Hydrogen production by photosynthetic microorganisms was studied. Cyanobacteria can produce hydrogen gas by nitrogenase or hydrogenase. Hydrogen production mediated by native hydrogenase in cyanobacteria was revealed to be in the dark anaerobic degradation of intracellular glycogen. A new system for light-dependent hydrogen production was targeted. In vitro and in vivo coupling of cyanobacterial ferredoxin with a heterologous hydrogenase was shown to produce hydrogen under light conditions. A trial for genetic trasformation of Synechococcus PCC7942 with the hydrogenase gene from Clostridium pasteurianum is going on. The strong hydrogen producers among photosynthetic bacteria were isolated and characterized. Co-culture of Rhodobacter and Clostriumdium was applied to produce hydrogen from glucose. Conversely in the case of cyanobacteria, genetic regulation of photosynthetic proteins was intended to improve conversion efficiency in hydrogen production by the photosynthetic bacterium, Rhodobacter sphaeroides RV. A mutant acquired by

  4. Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic

    PubMed Central

    Madison, Lara L.; Huisman, Gjalt W.

    1999-01-01

    Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of

  5. Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp. strain RASC.

    PubMed Central

    Suwa, Y; Wright, A D; Fukimori, F; Nummy, K A; Hausinger, R P; Holben, W E; Forney, L J

    1996-01-01

    The findings of previous studies indicate that the genes required for metabolism of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) are typically encoded on broad-host-range plasmids. However, characterization of plasmid-cured strains of Burkholderia sp. strain RASC, as well as mutants obtained by transposon mutagenesis, suggested that the 2,4-D catabolic genes were located on the chromosome of this strain. Mutants of Burkholderia strain RASC unable to degrade 2,4-D (2,4-D- strains) were obtained by insertional inactivation with Tn5. One such mutant (d1) was shown to have Tn5 inserted in tfdARASC, which encodes 2,4-D/alpha-ketoglutarate dioxygenase. This is the first reported example of a chromosomally encoded tfdA. The tfdARASC gene was cloned from a library of wild-type Burkholderia strain RASC DNA and shown to express 2,4-D/alpha-ketoglutarate dioxygenase activity in Escherichia coli. The DNA sequence of the gene was determined and shown to be similar, although not identical, to those of isofunctional genes from other bacteria. Moreover, the gene product (TfdARASC) was purified and shown to be similar in molecular weight, amino-terminal sequence, and reaction mechanism to the canonical TfdA of Alcaligenes eutrophus JMP134. The data presented here indicate that tfdA genes can be found on the chromosome of some bacterial species and suggest that these catabolic genes are rather mobile and may be transferred by means other than conjugation. PMID:8779585

  6. Expressed genes for plant-type ribulose 1,5-bisphosphate carboxylase/oxygenase in the photosynthetic bacterium Chromatium vinosum, which possesses two complete sets of the genes.

    PubMed Central

    Viale, A M; Kobayashi, H; Akazawa, T

    1989-01-01

    Two sets of genes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were detected in the photosynthetic purple sulfur bacterium Chromatium vinosum by hybridization analysis with RuBisCO gene probes, cloned by using the lambda Fix vector, and designated rbcL-rbcS and rbcA-rbcB. rbcL and rbcA encode the large subunits, and rbcS and rbcB encode the small subunits. rbcL-rbcS was the same as that reported previously (A. M. Viale, H. Kobayashi, T. Takabe, and T. Akazawa, FEBS Lett. 192:283-288, 1985). A DNA fragment bearing rbcA-rbcB was subcloned in plasmid vectors and sequenced. We found that rbcB was located 177 base pairs downstream of the rbcA coding region, and both genes were preceded by plausible procaryotic ribosome-binding sites. rbcA and rbcD encoded polypeptides of 472 and 118 amino acids, respectively. Edman degradation analysis of the subunits of RuBisCO isolated from C. vinosum showed that rbcA-rbcB encoded the enzyme present in this bacterium. The large- and small-subunit polypeptides were posttranslationally processed to remove 2 and 1 amino acid residues from their N-termini, respectively. Among hetero-oligomeric RuBisCOs, the C. vinosum large subunit exhibited higher homology to that from cyanobacteria, eucaryotic algae, and higher plants (71.6 to 74.2%) than to that from the chemolithotrophic bacterium Alcaligenes eutrophus (56.6%). A similar situation has been observed for the C. vinosum small subunit, although the homology among small subunits from different organisms was lower than that among the large subunits. Images PMID:2708310

  7. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates.

    PubMed Central

    Anderson, A J; Dawes, E A

    1990-01-01

    Polyhydroxyalkanoates (PHAs), of which polyhydroxybutyrate (PHB) is the most abundant, are bacterial carbon and energy reserve materials of widespread occurrence. They are composed of 3-hydroxyacid monomer units and exist as a small number of cytoplasmic granules per cell. The properties of the C4 homopolymer PHB as a biodegradable thermoplastic first attracted industrial attention more than 20 years ago. Copolymers of C4 (3-hydroxybutyrate [3HB]) and C5 (3-hydroxyvalerate [3HV]) monomer units have modified physical properties; e.g., the plastic is less brittle than PHB, whereas PHAs containing C8 to C12 monomers behave as elastomers. This family of materials is the centre of considerable commercial interest, and 3HB-co-3HV copolymers have been marketed by ICI plc as Biopol. The known polymers exist as 2(1) helices with the fiber repeat decreasing from 0.596 nm for PHB to about 0.45 nm for C8 to C10 polymers. Novel copolymers with a backbone of 3HB and 4HB have been obtained. The native granules contain noncrystalline polymer, and water may possibly act as a plasticizer. Although the biosynthesis and regulation of PHB are generally well understood, the corresponding information for the synthesis of long-side-chain PHAs from alkanes, alcohols, and organic acids is still incomplete. The precise mechanisms of action of the polymerizing and depolymerizing enzymes also remain to be established. The structural genes for the three key enzymes of PHB synthesis from acetyl coenzyme A in Alcaligenes eutrophus have been cloned, sequenced, and expressed in Escherichia coli. Polymer molecular weights appear to be species specific. The factors influencing the commercial choice of organism, substrate, and isolation process are discussed. The physiological functions of PHB as a reserve material and in symbiotic nitrogen fixation and its presence in bacterial plasma membranes and putative role in transformability and calcium signaling are also considered. PMID:2087222

  8. Mechanisms of bacterial resistance to chromium compounds.

    PubMed

    Ramírez-Díaz, Martha I; Díaz-Pérez, César; Vargas, Eréndira; Riveros-Rosas, Héctor; Campos-García, Jesús; Cervantes, Carlos

    2008-06-01

    Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species, the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species. At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported. The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidus metallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of iron and sulfur.

  9. Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii.

    PubMed Central

    Menon, A L; Mortenson, L E; Robson, R L

    1992-01-01

    Azotobacter vinelandii contains a heterodimeric, membrane-bound [NiFe]hydrogenase capable of catalyzing the reversible oxidation of H2. The beta and alpha subunits of the enzyme are encoded by the structural genes hoxK and hoxG, respectively, which appear to form part of an operon that contains at least one further potential gene (open reading frame 3 [ORF3]). In this study, determination of the nucleotide sequence of a region of 2,344 bp downstream of ORF3 revealed four additional closely spaced or overlapping ORFs. These ORFs, ORF4 through ORF7, potentially encode polypeptides with predicted masses of 22.8, 11.4, 16.3, and 31 kDa, respectively. Mutagenesis of the chromosome of A. vinelandii in the area sequenced was carried out by introduction of antibiotic resistance gene cassettes. Disruption of hoxK and hoxG by a kanamycin resistance gene abolished whole-cell hydrogenase activity coupled to O2 and led to loss of the hydrogenase alpha subunit. Insertional mutagenesis of ORF3 through ORF7 with a promoterless lacZ-Kmr cassette established that the region is transcriptionally active and involved in H2 oxidation. We propose to call ORF3 through ORF7 hoxZ, hoxM, hoxL, hoxO, and hoxQ, respectively. The predicted hox gene products resemble those encoded by genes from hydrogenase-related operons in other bacteria, including Escherichia coli and Alcaligenes eutrophus. Images PMID:1624446

  10. Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90.

    PubMed Central

    Bhat, M A; Tsuda, M; Horiike, K; Nozaki, M; Vaidyanathan, C S; Nakazawa, T

    1994-01-01

    Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine. PMID:7509586

  11. Effect of nonionic surfactant on the degradation of glass-sorbed PCB congeners by integrated chemical-biological treatment

    SciTech Connect

    Aronstein, B.N.; Paterek, J.R.

    1995-05-01

    A study was conducted on the effect of a nonionic surfactant on the efficiency of the integrated chemical-biological treatment (ICBT) of polychlorinated biphenyls (PCBs) sorbed to the glass walls of the experimental vessels. The addition of surfactant associated with the chemical oxidation was proposed to overcome two limitations of biodegradation of hydrophobic compounds: adsorption to surfaces,a nd lack of solubility and thus availability to the microorganisms. Two {sup 14}C-labeled PCB congeners, 2-chlorobiphenyl and 2,2{prime},4,4{prime}-tetrachlorobiphenyl, were used as the test compounds. The integrated chemical-biological treatment consisted of sequential applications of Fenton`s reagent (5% H{sub 2}O{sub 2}, 1 mM FeSO{sub 4}) at pH 4.0 (with and without non-ionic surfactant Novel 2 1412-60) and the inoculation with a mixed culture of Pseudomonas sp., strain LB400, and Alcaligenes eutrophus, strain H850, at pH 8.0. The chemical treatment in the presence of surfactant affected the removal of the congeners bound to the glass surfaces of the experimental test systems. As indicated by high-pressure liquid chromatography analysis, these PCBs were converted to unidentified soluble products that are hypothesized to be partial oxidation products. No parent test compounds were detectable in the system after chemical treatment with or without surfactant. The addition of the mixed culture of PCB-degrading bacteria after completion of chemical treatment in the presence of surfactant resulted in the removal of approximately 45% of 2-chlorobiphenyl and 32% of 2,2{prime},4,4{prime}-tetrachlorobiphenyl in the form of {sup 14}CO{sub 2}. This compared to 29% and 23% without the addition of surfactant, respectively. The authors suggest that the combination of surfactant-induced desorption and chemical oxidation would increase the availability of hydrophobic pollutants for microbial degradation.

  12. Effect of alcohol feeding mode on the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

    SciTech Connect

    Park, C.H.; Damodaran, V.K. )

    1994-12-01

    An alcohol utilizing Alcaligenes eutrophus produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer under phosphate limitation. Fermentation was performed for 42--46 h in a computer-controlled 5-L working volume fed-batch fermentor using ethanol and propanol as carbon sources. The culture experienced phosphate limitation in approximately 19 h. When propanol was used as a sole carbon source, 24 g/L of copolymer with 36.5 mol % of hydroxyvalerate (HV) was produced at a polymer yield of 0.41 g polymer/g alcohol (g/g) and an average polymer production rate of 0.08 g polymer/g residual biomass-h (g/g-h). Two experiments switching alcohols after phosphate exhaustion resulted in better polymer production (g/L), polymer yield (g/g) on alcohol, HV yield (g/g) on propanol, and average polymer production rate (g/g-h) as compared to propanol run without alcohol switching. One switching experiment was from a mixture of 50% ethanol and 50% propanol to 100% propanol and the other experiment was from 100% ethanol to a mixture of 65% ethanol and 35% propanol. Polymer yield for these two experiments was 0.51 g/g and 0.46 g/g, respectively. However, HV mol % in the copolymer for these two runs was lower compared to propanol run without alcohol switching. Direct switch from ethanol to propanol did not support cell growth and polymer production. Polymer production rate and polymer yield changed with time, and the pattern was dependent upon the alcohol feeding mode.

  13. Biodegradation and detoxification of melanoidin from distillery effluent using an aerobic bacterial strain SAG5 of Alcaligenes faecalis.

    PubMed

    Santal, Anita Rani; Singh, N P; Saharan, Baljeet Singh

    2011-10-15

    Distillery effluent retains very dark brown color even after anaerobic treatment due to presence of various water soluble, recalcitrant and coloring compounds mainly melanoidins. In laboratory conditions, melanoidin decolorizing bacteria was isolated and optimized the cultural conditions at various incubation temperatures, pH, carbon sources, nitrogen sources and combined effect of both carbon and nitrogen sources. The optimum decolorization (72.6 ± 0.56%) of melanoidins was achieved at pH 7.5 and temperature 37 °C on 5th day of cultivation. The toxicity evaluation with mung bean (Vigna radiata) revealed that the raw distillery effluent was environmentally highly toxic as compared to biologically treated distillery effluent, which indicated that the effluent after bacterial treatment is environmentally safe. This proves to be novel biological treatment technique for biodegradation and detoxification of melanoidin from distillery effluent using the bacterial strain SAG(5).

  14. Feasibility study on the utilization of rubber latex effluent for producing bacterial biopolymers.

    PubMed

    Tang, S N; Fakhru'l-Razi, A; Hassan, M A; Karim, M I

    1999-01-01

    Rubber latex effluent is a polluting source that has a high biochemical oxygen demand (BOD). It is estimated that about 100 million liters of effluent are discharged daily from rubber processing factories. Utilization of this effluent such as the use of a coupled system not only can reduce the cost of treatment but also yield a fermentation feedstock for the production of bioplastic. This study initially was carried out to increase the production of organic acids by anaerobic treatment of rubber latex effluent. It was found that through anaerobic treatment the concentration of organic acids did not increase. Consequently, separation of organic acids from rubber latex effluent by anion exchange resin was examined as a preliminary study of recovering acetic and propionic acids. However, the suspended solids (SS) content in the raw effluent was rather high which partially blocked the ion-exchange columns. Lime was used to remove the SS in the rubber latex effluent. After the lime precipitation process, organic acids were found to adsorb strongly onto the anion exchange resin. Less adsorption of organic acids onto the resin was observed before the lime precipitation. This was probably due to more sites being occupied by colloidal particles on the resin thus inhibiting the adsorption of organic acids. The initial concentration of organic acids in the raw effluent was 3.9 g/L. After ion exchange, the concentration of the organic acids increased to 27 g/L, which could be utilized for production of polyhydroxyalkanoates (PHA). For PHA accumulation stage, concentrated rubber latex effluent obtained from ion exchange resins and synthetic acetic acid were used as the carbon source. Quantitative analyses from fed batch culture via HPLC showed that the accumulation of PHA in Alcaligenes eutrophus was maximum with a concentration of 1.182 g/L when cultivated on synthetic acetic acid, corresponding to a yield of 87% based on its cell dry weight. The dry cell weight increased from

  15. Widespread occurrence of the tfd-II genes in soil bacteria revealed by nucleotide sequence analysis of 2,4-dichlorophenoxyacetic acid degradative plasmids pDB1 and p712.

    PubMed

    Kim, Dong-Uk; Kim, Min-Sun; Lim, Jong-Sung; Ka, Jong-Ok

    2013-05-01

    Variovorax sp. strain DB1 and Pseudomonas pickettii strain 712 are 2,4-dicholorophenoxy-acetic acid (2,4-D)-degrading bacteria, which were isolated from agricultural soils in Republic of Korea and USA, respectively. Each strain harbors a 2,4-D degradative plasmid and is able to utilize 2,4-D as the sole source of carbon for its growth. The 2,4-D degradative plasmid pDB1 of strain DB1 consisted of a 65,269-bp circular molecule with a G+C content of 66.23% and had 68 ORFs. The 2,4-D degradative plasmid p712 of strain 712 was composed of a 62,798-bp circular molecule with a 62.11% G+C content and had 62 ORFs. The plasmids pDB1 and p712 share significantly homologous 2,4-D degradative genes with high similarity to the tfdR, tfdB-II, tfdC-II, tfdD-II, tfdE-II, tfdF-II, tfdK and tfdA genes of plasmid pJP4 of Alcaligenes eutrophus isolated from Australia. In a phylogenetic analysis with trfA, traL, and trbA genes, pDB1 belonged to IncP-1β with pJP4, while p712 belonged to IncP-1ε with pKJK5 and pEMT3. The results indicated that, in spite of the differences in their backbone regions, the 2,4-D catabolic genes of the two plasmids were closely related and also related to the well-known 2,4-D degradative plasmid pJP4 even though all were isolated from different geographic regions. Other similarities in the genetic organization and the presence of IS1071 suggested that these catabolic genes may be on a transposable element, leading to widespread occurrence in soil bacteria.

  16. Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis.

    PubMed

    Knaebel, D B; Crawford, R L

    1995-10-01

    We investigated the use of multiplex polymerase chain reaction (PCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWW0), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWW0, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10(0) to 10(6) cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 microL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradative cells, at a lower detection limit of approximately 10 cells per gram of highly contaminated

  17. Sequence analysis of the Pseudomonas sp. strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenases for chlorinated substrates.

    PubMed Central

    van der Meer, J R; Eggen, R I; Zehnder, A J; de Vos, W M

    1991-01-01

    Pseudomonas sp. strain P51 contains two gene clusters located on catabolic plasmid pP51 that encode the degradation of chlorinated benzenes. The nucleotide sequence of a 5,499-bp region containing the chlorocatechol-oxidative gene cluster tcbCDEF was determined. The sequence contained five large open reading frames, which were all colinear. The functionality of these open reading frames was studied with various Escherichia coli expression systems and by analysis of enzyme activities. The first gene, tcbC, encodes a 27.5-kDa protein with chlorocatechol 1,2-dioxygenase activity. The tcbC gene is followed by tcbD, which encodes cycloisomerase II (39.5 kDa); a large open reading frame (ORF3) with an unknown function; tcbE, which encodes hydrolase II (25.8 kDa); and tcbF, which encodes a putative trans-dienelactone isomerase (37.5 kDa). The tcbCDEF gene cluster showed strong DNA homology (between 57.6 and 72.1% identity) and an organization similar to that of other known plasmid-encoded operons for chlorocatechol metabolism, e.g., clcABD of Pseudomonas putida and tfdCDEF of Alcaligenes eutrophus JMP134. The identity between amino acid sequences of functionally related enzymes of the three operons varied between 50.6 and 75.7%, with the tcbCDEF and tfdCDEF pair being the least similar of the three. Measurements of the specific activities of chlorocatechol 1,2-dioxygenases encoded by tcbC, clcA, and tfdC suggested that a specialization among type II enzymes has taken place. TcbC preferentially converts 3,4-dichlorocatechol relative to other chlorinated catechols, whereas TfdC has a higher activity toward 3,5-dichlorocatechol. ClcA takes an intermediate position, with the highest activity level for 3-chlorocatechol and the second-highest level for 3,5-dichlorocatechol. Images PMID:2013566

  18. Detection of a variant metallo-beta-lactamase, IMP-10, from two unrelated strains of Pseudomonas aeruginosa and an alcaligenes xylosoxidans strain.

    PubMed

    Iyobe, Shizuko; Kusadokoro, Haruko; Takahashi, Ayako; Yomoda, Sachie; Okubo, Toyoji; Nakamura, Akio; O'Hara, Koji

    2002-06-01

    The gene bla(IMP-10) of a variant metallo-beta-lactamase, IMP-10, had a single base replacement of G by T at nucleotide 145, which led to an amino acid alteration of Val49 to Phe compared to the IMP-1 enzyme, indicating that IMP-10 was a point mutation derivative of IMP-1. Highly purified enzymes revealed that IMP-10 was different from IMP-1 in its extremely low hydrolyzing activities for penicillins, such as benzylpenicillin, ampicillin, and piperacillin.

  19. New Biocatalysts: Essential Tools for a Sustainable 21st Century Chemical Industry

    DTIC Science & Technology

    2005-01-01

    sp. Candida parapsilosis Fusarium oxysporum Pseudomonas putida Saccharomyces sake Alcaligenes faecalis Enterobacter aerogenes Erwinia carotovora...Rhodococcus rhodochrous Serratia marcescens Xanthobacter agilis Nocardia corallina Beauveria bassiana Rhodococcus erythropolis Alcaligenes sp., Pseudomonas

  20. Marine Science Initiative at South Carolina State College: An Investigation of the Biosensing Parameters Regulating Bacterial and Larval Attachment on Substrata

    DTIC Science & Technology

    1993-08-12

    haemolytica/ P. ureae MSA Yellow........................... 0001.......... Pseudomonas speciesl Alcaligenes faecalis/ Pasteurella ureaef Moraxella specie sI...hydrophila 52 OSL: Colony Color ID Value Identity EMB Purple-Creamy ...................... 0031 .............. Alcaligenes faccalisi Pseudomonas species...Group 4E Alcaligenes -likef Group M-4f MORAXELLA-LIKE EMB Dark Blue ................................... 2171 .............. Pseudomonas aeruginosa/ P

  1. Detection of Staphylococcus Aureus Enterotoxin A and B Genes with PCR-EIA and a Hand-Held Electrochemical Sensor

    DTIC Science & Technology

    2004-06-11

    that included genomic and cloned S. aureus DNA, and genomic DNA from Alcaligens , Bacillus, Bacteroides, Bordetella, Borkholderia, Clostridium...studies, each with 81 samples that included genomic and cloned S. aureus DNA, and genomic DNA from Alcaligens , Bacillus, Bacteroides, Bordetella...Organism Strain/ isolate Alcaligens xylosoxidans 19606 Pseudomonas aeruginosa 17933D Bacillus anthracis Ames Salmonella choleraesuis 9150D Bacillus anthracis

  2. Polyhydroxyalkanoate copolymers from forest biomass.

    PubMed

    Keenan, Thomas M; Nakas, James P; Tanenbaum, Stuart W

    2006-07-01

    /w) PHA contents, and 4-67 mol% 3HV compositions. These data are comparable to copolymer yields and cellular contents reported for hexose plus levulinic acid-based shake-flask cultures, as reported using Alcaligenes eutrophus and Pseudomonas putida. However, our findings presage a conceivable alternative, forestry-based biorefinery approach for the production of value-added biodegradable PHA polymers. Specifically, this review describes the current and potential utilization of lignocellulosic process streams as platform precursors to PHA polymers including hemicellulosic hydrolysates, residual cellulose-derived levulinic acid, tall oil fatty acids (Kraft pulping residual), and lignin-derived aromatics.

  3. JPRS Report, Science & Technology, USSR: Life Sciences

    DTIC Science & Technology

    1988-04-05

    Biodegradation of Aniline by Alcaligenes Sp. Isolates [T.P. Chekhovskaya, et ai; MIKROBIOLOGICHESKIY ZHURNAL, Nov-Dec 87] 10 Hydrophobicity of Paintwork...Microbiology Biodegradation of Aniline by Alcaligenes Sp. 18400192a Kiev M1KROBIOLOGICHESKIY ZHURNAL in Russian Vol 49, No 6, Nov-Dec 87...Chemistry, Ukrainian SSR Academy of Sciences, Kiev] [Abstract] An Alcaligenes sp. isolated from the aniline- polluted soil of the Dneproderzhinsk "Azof

  4. 21 CFR 172.809 - Curdlan.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... from the nonpathogenic and nontoxicogenic bacterium Alcaligenes faecalis var. myxogenes. (b) Curdlan... per square centimeter. (10) Aerobic plate count, not more than 103 per gram. (11) Coliform bacteria...

  5. Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6.

    PubMed

    Kukimoto, M; Nishiyama, M; Tanokura, M; Murphy, M E; Adman, E T; Horinouchi, S

    1996-09-23

    Kinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Akaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR.

  6. Oil and Hydrocarbon Spill Bioremediation Product and Application Technology

    DTIC Science & Technology

    1993-05-01

    Degradation Concentration Concentration Source South Louisiana 1.0%, 5.0% Aeromonas, Crude Oil and Alcaligenes , Motor Oil’ Pseudomonas , Vibrio Mixed...important found in marine and soil environments are Achromobacter, Acinetobacter, Alcaligenes , Arthrobacter, Bacillus, Flavobacterium, Nocardia, and... Pseudomonas (Leahy and Colwell, 1990). Among the fungi listed, Aureobasidium, Candida, 2 Table 1 -Major Genera of Oil-Degrading Bacteria and Fungi Bacteria

  7. Antimicrobial Susceptibility as a Diagnostic Aid in the Identification of Nonfermenting Gram-Negative Bacteria

    PubMed Central

    Gilardi, G. L.

    1971-01-01

    Antimicrobial susceptibility data regarding nonfermentative, gram-negative bacteria (Pseudomonas, Alcaligenes, Acinetobacter, Moraxella, Flavobacterium) are presented showing that the antibiograms of most species examined can be used as an important auxillary aid in their differentiation. PMID:5132093

  8. Effects of Microbial Trophic Interactions on the Fate and Mobility of Soil Contaminants.

    DTIC Science & Technology

    2007-11-02

    characterized for their feeding and I growth on the bacterial isolates obtained. Stable consortia of bacteria and protista’ in biphenyl cultures will be...eutrophus H850 in soil samples with and without protists will also begin. The effects of nutrient limited growth and predation pressure as pre...morphological features for light microscopy, and characterized for their feeding and growth on the bacterial isolates obtained. Stable consortia of

  9. AGARD Flight Test Techniques Series. Volume 14. Introduction to Flight Test Engineering (Introduction a la Technique d’essais en vol)

    DTIC Science & Technology

    1995-09-01

    W.B. Clarks, 1897. Gast, R. de, "Pax River: the Naval Air Test Center", US Naval Institute Proceedings, Dec. 1969. Gibbs -Smith, C.H., "The Aeroplane...An Historical Survey of Its Origin and Development", London, HMSO, 1960. Gibbs -Smith, C.H., "The Invention of the Aeroplane 1799-1909", New York...145302. 16-6. Dobrzynski, W., "The Effect of Ground Reflections on the Propeller Aircraft Noise Measures LAmax and EPNL", Paper 8th Symposium FASE 󈨝

  10. ADA Implementation of an Object Data Repository

    DTIC Science & Technology

    1992-12-01

    AtUNtute Inheorwtne ConneciOn AeText NGdeoup /no" Group Ihhwodinc Iiiedlce Ydoe Polyon Owdip Dl~Int Polygn Polygo Ui-sod U-emprh Figure 39. Inheritance Group...Inheritance Monnecton) (onci Aggregation vncs Cnet (Ch&#34ss (Cba) (Cam,) (croass)Mb" Polyon Point Sardli (Canvs) C onnects connects Wudw Conne as Con nects More

  11. 38 CFR 21.7050 - Ending dates of eligibility.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... assistance under 38 U.S.C. ch. 34 through service on active duty which— (i) Commenced within 12 months of.... 3031(c); Pub. L. 98-525) (h) Time limitation for a spouse eligible for transferred entitlement. (1... (Montgomery GI Bill-Active Duty) Eligibility § 21.7050 Ending dates of eligibility. The ending date...

  12. Effects of Cyclic Loading on the Deformation and Elastic-Plastic Fracture Behavior of a Cast Stainless Steel

    DTIC Science & Technology

    1991-10-01

    USNRC report NUREG /CR- 5585. INTRODUCTION Tests conducted in Japan as part of the High Level Vibration Test (HLVT) program for reactor piping systems...Cuerri, J.R., and Hofmayer, C.H., "The High Level Vibration Test Program Final Report," USNRC NUREG /CR-5585, 1991, U.S. Nuclear Regulatory Commission

  13. Mildenhall RAF, United Kingdom Revised Uniform Summary of Surface Weather Observations (RUSSWO). Parts A-F.

    DTIC Science & Technology

    1984-04-01

    was not reported prior to 1946 . Relative humidity usually was nct reported prior to 1949, nor subsequent to June 1958; and was computed by machine...nAL CLTMAT3L^,Y ? AsCH ,.&#34 , :- c EXTREMEVAUS i T .AT"FR SERvICE/-AC E 44XIMJM TEMPERATRE (FROM DAILY OBIEVATIONS) STATION STATION NAME YEARS ,H^L- OE

  14. DddY, a periplasmic dimethylsulfoniopropionate lyase found in taxonomically diverse species of Proteobacteria

    PubMed Central

    Curson, Andrew R J; Sullivan, Matthew J; Todd, Jonathan D; Johnston, Andrew W B

    2011-01-01

    The abundant compatible solute dimethylsulfoniopropionate (DMSP) is made by many marine algae. Different marine bacteria catabolise DMSP by various mechanisms, some of which liberate the environmentally important gas dimethyl sulfide (DMS). We describe an enzyme, DddY, which cleaves DMSP into DMS plus acrylate and is located in the bacterial periplasm, unlike other DMSP lyases that catalyse this reaction. There are dddY-like genes in strains of Alcaligenes, Arcobacter and Shewanella, in the β-, ɛ- and γ-proteobacteria, respectively. In Alcaligenes, dddY is in a cluster of ddd and acu genes that resemble, but also have significant differences to, those in other bacteria that catabolise both DMSP and acrylate. Although production of DMS and transcription of Alcaligenes dddY are both apparently inducible by pre-growth of cells with DMSP, this substrate must be catabolised to form acrylate, the bona fide coinducer. PMID:21248856

  15. DddY, a periplasmic dimethylsulfoniopropionate lyase found in taxonomically diverse species of Proteobacteria.

    PubMed

    Curson, Andrew R J; Sullivan, Matthew J; Todd, Jonathan D; Johnston, Andrew W B

    2011-07-01

    The abundant compatible solute dimethylsulfoniopropionate (DMSP) is made by many marine algae. Different marine bacteria catabolise DMSP by various mechanisms, some of which liberate the environmentally important gas dimethyl sulfide (DMS). We describe an enzyme, DddY, which cleaves DMSP into DMS plus acrylate and is located in the bacterial periplasm, unlike other DMSP lyases that catalyse this reaction. There are dddY-like genes in strains of Alcaligenes, Arcobacter and Shewanella, in the β-, ɛ- and γ-proteobacteria, respectively. In Alcaligenes, dddY is in a cluster of ddd and acu genes that resemble, but also have significant differences to, those in other bacteria that catabolise both DMSP and acrylate. Although production of DMS and transcription of Alcaligenes dddY are both apparently inducible by pre-growth of cells with DMSP, this substrate must be catabolised to form acrylate, the bona fide coinducer.

  16. Evaluation of the rapid NFT system for identification of gram-negative, nonfermenting rods.

    PubMed Central

    Appelbaum, P C; Leathers, D J

    1984-01-01

    This study evaluated the ability of the Rapid NFT system (API System SA, Montalieu-Vercieu, France) to accurately identify 262 clinically isolated, gram-negative, nonfermentative rods without additional tests. Identifications were classified as correct; low discrimination, with a spectrum of two or more possibilities (additional tests necessary for accurate identification); and incorrect. Correct identification rates were analyzed in two categories: (i) correct to species or biotype for all organism groups except Alcaligenes faecalis-odorans, Moraxella, Pseudomonas testosteroni-alcaligenes-pseudoalcaligenes, and Acinetobacter calcoaceticus biotype haemolyticus-alcaligenes (in this category, the latter four genus-biotype group identifications were taken as correct) and (ii) correct to species or biotype in all cases, including the above four groups. In category i, 87.4% of the strains were correctly identified, with 4.2% low discrimination and 8.4% incorrect. When the criteria of category ii were used, 71.8% of the strains were correctly identified, with 19.9% low discrimination. The Rapid NFT system provided excellent species identification of Pseudomonas and Flavobacterium spp., Bordetella bronchiseptica, and Achromobacter xylosoxidans strains. Within Acinetobacter calcoaceticus, differentiation between biotypes anitratus and lwoffi was satisfactory, but the system did not differentiate between biotypes haemolyticus and alcaligenes. Species resolution within the genera Moraxella and Alcaligenes was incomplete. All Alcaligenes faecalis strains were misidentified and accounted for 50% of misidentifications with the Rapid NFT system; however, these results may reflect taxonomic differences rather than true misidentifications. The Rapid NFT system is easy to inoculate and interpret and represents a worthwhile advance in the identification of gram-negative, nonfermentative rods. PMID:6490857

  17. Aerobic Degradation of Dinitrotoluenes and Pathway for Bacterial Degradation of 2,6- Dinitrotoluene.

    DTIC Science & Technology

    2000-01-01

    of 3M4NC, 9.8 t±mol of sodium phosphate (pH 7.0), and cell extract (0.1 been designated Alcaligenes sp. They are represented by strain to 0.5 mg of...protein) in a final volume of 1 ml. The molar extinction coefficients JS867 ( Alcaligenes denitrificans Biolog cluster) and strain JS871 of compounds X...cepacia Pseudomonas sp. 1; 1992 2,4 - + + PR7 Burkholderia cepacia Pseudomonas sp. 1; 1992 2,4 - + + JS850 Burkholderia cepacia None 3; 1995 2,6

  18. A Glucose-Utilizing Strain, Cupriavidus euthrophus B-10646: Growth Kinetics, Characterization and Synthesis of Multicomponent PHAs

    PubMed Central

    Volova, Tatiana; Kiselev, Evgeniy; Vinogradova, Olga; Nikolaeva, Elena; Chistyakov, Anton; Sukovatiy, Aleksey; Shishatskaya, Ekaterina

    2014-01-01

    This study investigates kinetic and production parameters of a glucose-utilizing bacterial strain, C. eutrophus B-10646, and its ability to synthesize PHA terpolymers. Optimization of a number of parameters of bacterial culture (cell concentration in the inoculum, physiological activity of the inoculum, determined by the initial intracellular polymer content, and glucose concentration in the culture medium during cultivation) provided cell concentrations and PHA yields reaching 110 g/L and 80%, respectively, under two-stage batch culture conditions. Addition of precursor substrates (valerate, hexanoate, propionate, γ-butyrolactone) to the culture medium enabled synthesis of PHA terpolymers, P(3HB/3HV/4HB) and P(3HB/3HV/3HHx), with different composition and different molar fractions of 3HB, 3HV, 4HB, and 3HHx. Different types of PHA terpolymers synthesized by C. eutrophus B-10646 were used to prepare films, whose physicochemical and physical-mechanical properties were investigated. The properties of PHA terpolymers were significantly different from those of the P3HB homopolymer: they had much lower degrees of crystallinity and lower melting points and thermal decomposition temperatures, with the difference between these temperatures remaining practically unchanged. Films prepared from all PHA terpolymers had higher mechanical strength and elasticity than P3HB films. In spite of dissimilar surface structures, all films prepared from PHA terpolymers facilitated attachment and proliferation of mouse fibroblast NIH 3T3 cells more effectively than polystyrene and the highly crystalline P3HB. PMID:24586280

  19. A glucose-utilizing strain, Cupriavidus euthrophus B-10646: growth kinetics, characterization and synthesis of multicomponent PHAs.

    PubMed

    Volova, Tatiana; Kiselev, Evgeniy; Vinogradova, Olga; Nikolaeva, Elena; Chistyakov, Anton; Sukovatiy, Aleksey; Shishatskaya, Ekaterina

    2014-01-01

    This study investigates kinetic and production parameters of a glucose-utilizing bacterial strain, C. eutrophus B-10646, and its ability to synthesize PHA terpolymers. Optimization of a number of parameters of bacterial culture (cell concentration in the inoculum, physiological activity of the inoculum, determined by the initial intracellular polymer content, and glucose concentration in the culture medium during cultivation) provided cell concentrations and PHA yields reaching 110 g/L and 80%, respectively, under two-stage batch culture conditions. Addition of precursor substrates (valerate, hexanoate, propionate, γ-butyrolactone) to the culture medium enabled synthesis of PHA terpolymers, P(3HB/3HV/4HB) and P(3HB/3HV/3HHx), with different composition and different molar fractions of 3HB, 3HV, 4HB, and 3HHx. Different types of PHA terpolymers synthesized by C. eutrophus B-10646 were used to prepare films, whose physicochemical and physical-mechanical properties were investigated. The properties of PHA terpolymers were significantly different from those of the P3HB homopolymer: they had much lower degrees of crystallinity and lower melting points and thermal decomposition temperatures, with the difference between these temperatures remaining practically unchanged. Films prepared from all PHA terpolymers had higher mechanical strength and elasticity than P3HB films. In spite of dissimilar surface structures, all films prepared from PHA terpolymers facilitated attachment and proliferation of mouse fibroblast NIH 3T3 cells more effectively than polystyrene and the highly crystalline P3HB.

  20. Draft Genome Sequence of Achromobacter piechaudii Strain HLE

    PubMed Central

    Trimble, William L.; Phung, Le T.; Meyer, Folker

    2012-01-01

    Achromobacter piechaudii strain HLE is a betaproteobacterium (previously known as Alcaligenes faecalis) that was an early isolate with arsenite oxidase activity. This draft genome of 6.89 Mb is the second available genome for this species in the opportunistic pathogen Alcaligenaceae family. PMID:23105084

  1. Degradation of polychlorinated biphenyls by microorganisms

    SciTech Connect

    Yagi, O.; Sudo, R.

    1980-05-01

    The biodegradation of PCB's by microorganisms and the degradation pathway of PCB's are investigated. Experimental methods and materials are described. Only several strains of bacteria, Achromobacter sp., Alcaligenes sp., Acinetobacter sp., Pseudomonas sp., and soil microorganisms were able to decompose PCB's. A possible relationships between the structure and biodegradability of related biphenyl compounds was examined. (5 diagrams, 11 graphs, 18 references, 1 table)

  2. Impact Of Landfill Closure Designs On Long-Term Natural Attenuation Of Chlorinated Hydrocarbons

    DTIC Science & Technology

    2002-03-01

    and W. Francke. 1991. Degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene by Pseudomonas strains. Applied Environmental Microbiology...Neerven, P.J. Colberg, and A.J.B. Zehnder. 1986. Degradation of 1,2-dichlorobenzene by Alcaligene sp. Strain A175. Applied Environmental Microbiology

  3. Poly(Beta-Hydroxybutyrate) Stereoisomers: A Model Study of the Effects of Stereochemical and Morphological Variables on Polymer Biological Degradability

    DTIC Science & Technology

    1992-01-01

    sole source of carbon. The bacterial ybutyrate), (R)-PHB, which is a highly crystalline bio- strains Pseudomonas ?remoignei (ATCC 17989)15-18 and...degradable thermoplastic.8 Alcaligenes faecalis T1,1s9A isolated from soil and activated Several researchers have studied the depolymerization sewage sludge

  4. Strain-specific virulence of Bordetella hinzii in turkeys

    USDA-ARS?s Scientific Manuscript database

    Bordetella hinzii is commonly acquired from the respiratory tract of diseased poultry but regarded as nonpathogenic in avian hosts. Recently, it was recognized that some previously used isolates were misidentified at the time of their acquisition as B. avium, B. avium-like or Alcaligenes faecalis ty...

  5. Method for treating waste water

    SciTech Connect

    Masaki, Y.; Odawara, Y.; Shimizu, N.

    1982-10-26

    The invention relates to an improvement of the floc-formation property of activated sludge contained in waste water. A waste water treatment process comprises steps culturing a novel strain-alcaligenes faecalis hrl-1-and adding the cultured cells to to-be-treated waste water.

  6. Draft genome sequence of Achromobacter piechaudii strain HLE.

    PubMed

    Trimble, William L; Phung, Le T; Meyer, Folker; Silver, Simon; Gilbert, Jack A

    2012-11-01

    Achromobacter piechaudii strain HLE is a betaproteobacterium (previously known as Alcaligenes faecalis) that was an early isolate with arsenite oxidase activity. This draft genome of 6.89 Mb is the second available genome for this species in the opportunistic pathogen Alcaligenaceae family.

  7. Development of eco-friendly bioplastic like PHB by distillery effluent microorganisms.

    PubMed

    Gangurde, Nilesh S; Sayyed, Riyaz Z; Kiran, Shashi; Gulati, Arvind

    2013-01-01

    During screening for poly-β-hydroxybutyrate (PHB) producing bacteria from distillery effluent sample, six out of 30 isolates comprising of three strains of Alcaligenes sp., two strains of Bacillus sp., and one strain of Pseudomonas sp. were found to accumulate varying levels of intracellular PHB. Amongst the various isolates, Alcaligenes sp. RZS4 was found as the potent PHB-producing organism, accumulating higher amounts of PHB. PHB productivity was further enhanced in the presence of oxygen, nitrogen-limiting conditions, and cloning of PHB synthesizing genes of Alcaligenes sp. RZS 4 into Escherichia coli. A twofold increase in PHB yield was obtained from recombinant E. coli vis-à-vis Alcaligenes sp.; the recombinant E. coli accumulated more PHB in NDMM, produced good amount of PHB in a single-stage cultivation process under both nutrient-rich and nutrient-deficient conditions. Extraction of PHB with acetone-alcohol (1:1) was found as suitable method for optimum extraction of PHB as this mixture selectively extracted PHB without affecting the non-PHB cell mass. PHB extract from recombinant E. coli showed the presence of C-H, =O stretching, =C-H deformation, =C-H, =CH, and =C-O functional groups characteristic of PHB.

  8. Evaluation of Hyperbaric Oxygen Therapy in the Treatment of Spinal Cord Injury Using the Rabbit Spinal Stroke Model

    DTIC Science & Technology

    1990-11-30

    Oxygen Therapy, Springer- Verlag, 1988 2. Peirce , E.C., Jacobson C.H., "Cerebral Edema" in Hyperbaric Oxygen Therapy, Eds: Davis, J.C., Hunt, T.K., pp. 287...al. (eds.) Fifth International Hyperbaric Congress (Proceedings), Simon Fraser University, Burnaby, B.C., Canada, pp. 350 - 361, 1974 14.Marsala, M...consistant with the theory that ischemia- reperfusion is accompanied by oxygen free-radical production in the CNS and that the vasculature is a target for free

  9. Variation in genomic islands contribute to genome plasticity in cupriavidus metallidurans

    PubMed Central

    2012-01-01

    Background Different Cupriavidus metallidurans strains isolated from metal-contaminated and other anthropogenic environments were genotypically and phenotypically compared with C. metallidurans type strain CH34. The latter is well-studied for its resistance to a wide range of metals, which is carried for a substantial part by its two megaplasmids pMOL28 and pMOL30. Results Comparative genomic hybridization (CGH) indicated that the extensive arsenal of determinants involved in metal resistance was well conserved among the different C. metallidurans strains. Contrary, the mobile genetic elements identified in type strain CH34 were not present in all strains but clearly showed a pattern, although, not directly related to a particular biotope nor location (geographical). One group of strains carried almost all mobile genetic elements, while these were much less abundant in the second group. This occurrence was also reflected in their ability to degrade toluene and grow autotrophically on hydrogen gas and carbon dioxide, which are two traits linked to separate genomic islands of the Tn4371-family. In addition, the clear pattern of genomic islands distribution allowed to identify new putative genomic islands on chromosome 1 and 2 of C. metallidurans CH34. Conclusions Metal resistance determinants are shared by all C. metallidurans strains and their occurrence is apparently irrespective of the strain's isolation type and place. Cupriavidus metallidurans strains do display substantial differences in the diversity and size of their mobile gene pool, which may be extensive in some (including the type strain) while marginal in others. PMID:22443515

  10. Size-, composition- and shape-dependent toxicological impact of metal oxide nanoparticles and carbon nanotubes toward bacteria.

    PubMed

    Simon-Deckers, Angélique; Loo, Sylvain; Mayne-L'hermite, Martine; Herlin-Boime, Nathalie; Menguy, Nicolas; Reynaud, Cécile; Gouget, Barbara; Carrière, Marie

    2009-11-01

    Ecotoxicological effects of nanoparticles (NP) are still poorly documented while their commercialization for industrial and household applications increases. The aim of this study was to evaluate the influence of physicochemical characteristics on metal oxide NP and carbon nanotubes toxicological effects toward bacteria. Two strains of bacteria, Cupriavidus metallidurans CH34 and Escherichia coli MG1655 were exposed to TiO(2) or Al(2)O(3) NP or to multiwalled-carbon nanotubes (MWCNT). Particular attention was paid on optimizing NP dispersion to obtain nonagglomerated suspensions. Our results show that NP toxicity depends on their chemical composition, size, surface charge, and shape but not on their crystalline phase. MWCNT toxicity does not depend on their purity. Toxicity also depends on the bacterial strain: E. coli MG1655 is sensitive to NP, whereas C. metallidurans CH34 is not. Interestingly, NP are accumulated in both bacterial strains, and association between NP and bacteria is necessary for bacterial death to occur. NP may then represent a danger for the environment, causing the disappearance of some sensitive bacterial strains such as E. coli MG1655, but also being mobilized by nonsensitive strains such as C. metallidurans CH34 and transported through the whole ecosystem.

  11. Characterization of the Metabolically Modified Heavy Metal-Resistant Cupriavidus metallidurans Strain MSR33 Generated for Mercury Bioremediation

    PubMed Central

    Rojas, Luis A.; Yáñez, Carolina; González, Myriam; Lobos, Soledad; Smalla, Kornelia; Seeger, Michael

    2011-01-01

    Background Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds. Methodology/Principal Findings To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel merB, merG genes and additional other mer genes was introduced into the bacterium by biparental mating. The transconjugant Cupriavidus metallidurans strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg2+. The minimum inhibitory concentrations (mM) for strain MSR33 were: Hg2+, 0.12 and CH3Hg+, 0.08. The addition of Hg2+ (0.04 mM) at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg2+ addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg2+ no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg2+ showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg2+ (0.10 and 0.15 mM) was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM) after 2 h. Conclusions/Significance A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into C. metallidurans CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel and stable bacterial strain

  12. Mobile genetic elements, a key to microbial adaptation in extreme environments

    NASA Astrophysics Data System (ADS)

    van Houdt, Rob; Mijnendonckx, Kristel; Provoost, Ann; Monsieurs, Pieter; Mergeay, Max; Leys, Natalie

    To ensure well-being of the crew during manned spaceflight, continuous monitoring of different microbial contaminants in air, in water and on surfaces in the spacecraft is vital. Next to microorganisms originating mainly from human activity, strains from the closely related gen-era Cupriavidus and Ralstonia have been identified and isolated during numerous monitoring campaigns from different space-related environments. These strains have been found in the air of the Mars Exploration Rover assembly room, on the surface of the Mars Odyssey Orbiter and in different water sources from the International Space Station, Shuttle and Mir space station. In previous studies, we investigated the response of the model bacterium Cupriavidus metallidurans CH34 when cultured in the international space station (ISS) and space gravity and radiation simulation facilities, to understand it's ways to adapt to space flight conditions. It was also demonstrated that genetic rearrangements due to the movement of IS (insertion sequence) elements, enabled CH34 to adapt to toxic zinc concentrations, in space flight and on ground. In this study, we screened the full genome sequence of C. metallidurans CH34 for the presence of mobile genetic elements (MGEs), with the purpose to identified their putative role in adaptation to the new environments. Eleven genomic islands (GI) were identified in chro-mosome 1, three on the native plasmid pMOL28 and two on the native plasmid pMOL30. On the plasmids pMOL28 and pMOL30, all genes involved in the response to metals were located within GIs. Three of the GIs on chromosome 1 contained genes involved in the response to metals. Three GIs (CMGI-2, -3 and -4) on chromosome 1 belonged to the Tn4371 family, with CMGI-2 containing at least 25 genes involved in the degradation of toluene corresponding to CH34's ability to grow at expense of toluene, benzene or xylene as sole carbon source. CMGI-3 sheltered accessory genes involved in CO2 fixation and

  13. Study on the biodegradation of crude oil by free and immobilized bacterial consortium in marine environment

    PubMed Central

    Li, Jingjing; Liu, Mei; Sun, Huiling; Bao, Mutai

    2017-01-01

    Five strains of bacteria, namely, Exiguobacterium sp. ASW-1, Pseudomonas aeruginosa strain ASW-2, Alcaligenes sp. ASW-3, Alcaligenes sp. ASS-1, and Bacillus sp. ASS-2, were isolated from the Zhejiang coast in China. The mixed flora of the five strains performed well with degrading 75.1% crude oil (1%, w/v) in 7 days. The calcium alginate—activated carbon embedding carrier was used to immobilize bacterial consortium. Immobilized cells performed better than free ones in variations of environmental factors containing incubated temperature, initial pH, salinity of the medium and crude oil concentration. The degradation process of crude oil by immobilized bacteria was accelerated compared with that of the free ones. Bacterial consortium showed better performance on biodegradation of normal alkanes than that of PAHs. Improvement of immobilization on the biodegradation efficiency of normal alkanes (31.9%) was apparently high than that of PAHs (1.9%). PMID:28346510

  14. Compositional studies on succinoglycan-like extracellular water-soluble Rhizobium polysaccharides.

    PubMed

    Ghai, S K

    1981-01-01

    This study reports structural information on extracellular, water-soluble polysaccharides from 5 different strains of Rhizobium, viz. R. trifolii J60, R. meliloti J1017, 202, 204 and 207. All the 5 polysaccharides had glucose and galactose in approximate molar ratio of 7:1. Methylation analysis revealed that the polysaccharides contained (1 leads to 3), (1 leads to 6), (1 leads to 4), (1 leads to 4, 1 leads to 6)-linked D-glucose residues, (1 leads to 3)-linked D-galactose and non-reducing terminal D-glucose attached to pyruvate. This structure was found to be exactly the same as that of succinoglycan, a succinic acid containing water-soluble polysaccharide elaborated by Alcaligenes faecalis var. myxogenes 10C3. The similarity of the structure of polysaccharides of two different Rhizobium species and also to the polysaccharide produced by Alcaligenes are discussed in terms of host specificity.

  15. Degradation of polychlorinated biphenyls by mixed microbial cultures.

    PubMed Central

    Clark, R R; Chian, E S; Griffin, R A

    1979-01-01

    Three different enriched mixed cultures capable of degrading polychlorinated biphenylas were isolated from two soil samples and a river sediment, respectively. The predominant organisms found in all three mixed cultures were Alcaligenes odorans, Alcaligenes dentrificans, and an unidentified bacterium. The polychlorinated biphenyl isomers that were more water soluble and had lower chlorination were not only degraded at a faster rate than those that were less water soluble and had higher chlorination, but were also more completely utilized by these mixed cultures. This resulted in the presence in the environment of polychlorinated biphenyl residues consisting mainly of higher-chlorinated isomers. A form of cometabolism of polychlorinated biphenyls was also found with these cultures in the presence of acetate as the cosubstrate. PMID:110265

  16. MEDIUM FOR DIFFERENTIATING THE GRAM-NEGATIVE, NONFERMENTING BACILLI OF MEDICAL INTEREST

    PubMed Central

    Sellers, Walter

    1964-01-01

    Sellers, Walter (U.S. Air Force School of Aerospace Medicine, Brooks Air Force Base, Tex.). Medium for differentiating the gram-negative, nonfermenting bacilli of medical interest. J. Bacteriol. 87:46–48. 1964.—An agar-slant medium is described for differentiating gram-negative, nonfermenting bacilli of medical interest. Differences in the ability of bacilli to grow anaerobically in the presence of nitrate, to produce pH changes, to produce N2 from nitrite and nitrate (singly and in combination), to fluoresce, and to oxidize a drop of glucose solution to acid in the presence of high peptone concentrations were used in the development of the medium. Organisms differentiated by the medium include Pseudomonas aeruginosa, Bacterium anitratum, Mima polymorpha, and Vibrio alcaligenes or Alcaligenes faecalis. PMID:14102872

  17. Hydrocarbons biodegradation by activated sludge bacteria in the presence of natural and synthetic surfactants.

    PubMed

    Zdarta, Agata; Smułek, Wojciech; Pietraszak, Emilia; Kaczorek, Ewa; Olszanowski, Andrzej

    2016-12-05

    Fuels, such as diesel oil, can have a substantial impact on the microbial equilibrium of activated sludge and have a negative influence on work of wastewater treatment plant. The primary objective of the research was to examine the possibility of using the surfactants to improve pollutants biodegradation by bacteria from activated sludge. The results showed that the addition of rhamnolipids allows to increase the hydrocarbon biodegradation from 47% up to 75% in the cultures inoculated with the consortium. The saponins increased the degradation of diesel oil by the two isolated strains: from 27% to 43% for Alcaligenes sp. and from 44% to 64% for Pseudomonas sp. The addition of surfactants to the cultures growth with diesel oil caused a significant decrease of the surface charge for Alcaligenes strain in the presence of saponins, but not in other cases. The obtained results revealed the potential of natural surfactants to support hydrocarbon biodegradation in wastewater treatment plants.

  18. HYDROCARBON-DEGRADING BACTERIA AND SURFACTANT ACTIVITY

    SciTech Connect

    Brigmon, R; Topher Berry, T; Grazyna A. Plaza, G; jacek Wypych, j

    2006-08-15

    Fate of benzene ethylbenzene toluene xylenes (BTEX) compounds through biodegradation was investigated using two different bacteria, Ralstonia picketti (BP-20) and Alcaligenes piechaudii (CZOR L-1B). These bacteria were isolated from extremely polluted petroleum hydrocarbon contaminated soils. PCR and Fatty Acid Methyl Ester (FAME) were used to identify the isolates. Biodegradation was measured using each organism individually and in combination. Both bacteria were shown to degrade each of the BTEX compounds. Alcaligenes piechaudii biodegraded BTEXs more efficiently while mixed with BP-20 and individually. Biosurfactant production was observed by culture techniques. In addition 3-hydroxy fatty acids, important in biosurfactant production, was observed by FAME analysis. In the all experiments toluene and m+p- xylenes were better growth substrates for both bacteria than the other BTEX compounds. In addition, the test results indicate that the bacteria could contribute to bioremediation of aromatic hydrocarbons (BTEX) pollution increase biodegradation through the action by biosurfactants.

  19. A new biopolymer for high-temperature profile control. Part 1; Laboratory testing

    SciTech Connect

    Storm, E.T.; Paul, J.M.; Phelps, C.H.; Sampath, K. )

    1991-02-01

    This paper reports on an extracellular polysaccharide produced by Alcaligenes bacteria that has been crosslinked to form firm gels that are stable for long periods at elevated temperatures. The biopolymer can be gelled with multivalent ions such as Cr{sup +3}; it can also be gelled at the 2,000-to-4,000-ppm level without Cr{sup 3+} in high-salinity ({approximately}20% total dissolved solids (TDS)) brines. Gel samples have been stable for 2 years at 74 and 90{degrees} C. Gels could be formed over a wide pH range, but best long-term stability was achieved in the pH range of 7 to 8. A flow test of Alcaligenes biopolymer gel in Berea sandstone at 74{degrees} C showed that the gel gave a brine permeability reduction factor of 320.

  20. Differentiation among bacteria isolated from turkeys with coryza (rhinotracheitis).

    PubMed

    Rimler, R B; Simmons, D G

    1983-01-01

    Gram-negative bacteria isolated from turkeys with coryza in the United States, the Federal Republic of Germany, and the Republic of South Africa were compared with known Alcaligenes species and Bordetella bronchiseptica. The turkey isolates were separated into three distinct groups based on biochemical and physiologic tests. Forty of the 68 isolates studied (group I) were different from Alcaligenes sp. and B. bronchiseptica. Isolates in group I produced a heat-labile hemagglutinin and did not grow on Simmons' citrate agar. Isolates in group II (25 isolates) were similar to A. faecalis and A. odorans, grew on Simmons' citrate agar, and did not produce a hemagglutinin. Isolates in group III were B. bronchiseptica. Isolates from groups I and II caused coryza in poults. Group III isolates were not pathogenic.

  1. Study on the biodegradation of crude oil by free and immobilized bacterial consortium in marine environment.

    PubMed

    Chen, Qingguo; Li, Jingjing; Liu, Mei; Sun, Huiling; Bao, Mutai

    2017-01-01

    Five strains of bacteria, namely, Exiguobacterium sp. ASW-1, Pseudomonas aeruginosa strain ASW-2, Alcaligenes sp. ASW-3, Alcaligenes sp. ASS-1, and Bacillus sp. ASS-2, were isolated from the Zhejiang coast in China. The mixed flora of the five strains performed well with degrading 75.1% crude oil (1%, w/v) in 7 days. The calcium alginate-activated carbon embedding carrier was used to immobilize bacterial consortium. Immobilized cells performed better than free ones in variations of environmental factors containing incubated temperature, initial pH, salinity of the medium and crude oil concentration. The degradation process of crude oil by immobilized bacteria was accelerated compared with that of the free ones. Bacterial consortium showed better performance on biodegradation of normal alkanes than that of PAHs. Improvement of immobilization on the biodegradation efficiency of normal alkanes (31.9%) was apparently high than that of PAHs (1.9%).

  2. Genome sequence of the β-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia

    PubMed Central

    Amadou, Claire; Pascal, Géraldine; Mangenot, Sophie; Glew, Michelle; Bontemps, Cyril; Capela, Delphine; Carrère, Sébastien; Cruveiller, Stéphane; Dossat, Carole; Lajus, Aurélie; Marchetti, Marta; Poinsot, Véréna; Rouy, Zoé; Servin, Bertrand; Saad, Maged; Schenowitz, Chantal; Barbe, Valérie; Batut, Jacques; Médigue, Claudine; Masson-Boivin, Catherine

    2008-01-01

    We report the first complete genome sequence of a β-proteobacterial nitrogen-fixing symbiont of legumes, Cupriavidus taiwanensis LMG19424. The genome consists of two chromosomes of size 3.42 Mb and 2.50 Mb, and a large symbiotic plasmid of 0.56 Mb. The C. taiwanensis genome displays an unexpected high similarity with the genome of the saprophytic bacterium C. eutrophus H16, despite being 0.94 Mb smaller. Both organisms harbor two chromosomes with large regions of synteny interspersed by specific regions. In contrast, the two species host highly divergent plasmids, with the consequence that C. taiwanensis is symbiotically proficient and less metabolically versatile. Altogether, specific regions in C. taiwanensis compared with C. eutrophus cover 1.02 Mb and are enriched in genes associated with symbiosis or virulence in other bacteria. C. taiwanensis reveals characteristics of a minimal rhizobium, including the most compact (35-kb) symbiotic island (nod and nif) identified so far in any rhizobium. The atypical phylogenetic position of C. taiwanensis allowed insightful comparative genomics of all available rhizobium genomes. We did not find any gene that was both common and specific to all rhizobia, thus suggesting that a unique shared genetic strategy does not support symbiosis of rhizobia with legumes. Instead, phylodistribution analysis of more than 200 Sinorhizobium meliloti known symbiotic genes indicated large and complex variations of their occurrence in rhizobia and non-rhizobia. This led us to devise an in silico method to extract genes preferentially associated with rhizobia. We discuss how the novel genes we have identified may contribute to symbiotic adaptation. PMID:18490699

  3. Biopolymers production with carbon source from the wastes of a beer brewery industry

    NASA Astrophysics Data System (ADS)

    Wong, Phoeby Ai Ling

    The main purpose of this study was to assess the potential and feasibility of malt wastes, and other food wastes, such as soy wastes, ice-cream wastes, confectionery wastes, vinegar wastes, milk waste and sesame oil, in the induction of biosynthesis of PHA, in the cellular assembly of novel PHA with improved physical and chemical properties, and in the reduction of the cost of PHA production. In the first part of the experiments, a specific culture of Alcaligenes latus DSM 1124 was selected to ferment several types of food wastes as carbon sources into biopolymers. In addition, the biopolymer production, by way of using malt waste, of microorganisms from municipal activated sludge was also investigated. In the second part, the experiments focused on the synthesis of biopolymer with a higher molecular mass via the bacterial strain, which was selected and isolated from sesame oil, identified as Staphylococcus epidermidis . Molecular weight and molecular weight distribution of PHB were studied by GPC. Molecular weight of PHB produced from various types of food wastes by Alcaligenes latus was higher than using synthetic sucrose medium as nutrient, however, it resulted in the reverse by Staphylococcus epidermidis. Thermal properties of biopolymers were studied by DSC and TG. Using malt wastes as nutrients by Alcaligenes latus gave a higher melting temperature. Using sucrose, confectionery and sesame oil as nutrients by Staphylococcus epidermidis gave higher melting temperature. Optimization was carried out for the recovery of microbial PHB from Alcaligenes latus. Results showed that molecular weight can be controlled by changing the hypochlorite concentration, the ratio of chloroform to hypochlorite solution and the extraction time. In addition, the determination of PHB content by thermogravimetric analysis method with wet cell was the first report in our study. (Abstract shortened by UMI.)

  4. Surface active properties of bacterial strains isolated from petroleum hydrocarbon-bioremediated soil.

    PubMed

    Płaza, Grazyna A; Ulfig, Krzysztof; Brigmon, Robin L

    2005-01-01

    Two bacterial strains identified as Ralstonia picketti (BP 20) and Alcaligenes piechaudii (CZOR L-1B) were isolated from petroleum hydrocarbon-contaminated soil following bioremediation treatment. The surface active properties, e.g. surface tension, emulsification and foamability of their culture filtrates were evaluated. Bacterial cell-surface hydrophobicity (BAH) as measured by analyzing cell affinity towards aliphatic and aromatic compounds was also determinated. The bacteria grew in liquid cultures containing 1% (v/v) of crude oil as carbon and energy source at 30 degrees C under aerobic conditions. The surface tensions were reduced to 61 mN/m and 55 mN/m by Ralstonia picketti and Alcaligenes piechaudii, respectively. The emulsification index (EI24) was almost 100% for all tested compounds except diesel oil. The stability of the emulsions was deteminated at 4 degrees C, 45 degrees C and 65 degrees C. The emulsions were stable at 4 degrees C. Ralstonia picketti was better foam inducer (FV = 50 ml) compared to Alcaligenes piechaudii (FV = 10 ml). The BAH measurements revealed higher adhesion of Alcaligenes piechaudii cells towards different hydrocarbons compared to Ralstonia picketti cells. The strains were found to have a surface hydrophobicity in the following order: aliphatic hydrocarbons, BTEX, and PAHs. The ability to adhere to bulk hydrocarbon is mostly a characteristic of hydrocarbon-degrading bacteria. The strains were found to be better emulsifiers than surface tension reducers. They produce water-soluble extracellular bioemulsifiers. Both bacterial isolates have good properties to use them, mainly in the petroleum industry, e.g. in enhanced oil recovery and in bioremediation processes-primarily due to their emulsification property, i.e. emulsion forming and stabilizing capacity.

  5. The bacterial transformation of abietic acid

    PubMed Central

    Cross, B. E.; Myers, P. L.

    1968-01-01

    An Alcaligenes species, which was isolated from soil, can utilize abietic acid as its sole carbon source. During growth, the bacterium transforms abietic acid into 5α-hydroxyabietic acid (I, R=OH), a product considered to be 7β-hydroxy-13-isopropyl-8ξ-podocarp-13-en-15-oic acid (II, R=H) and a compound, C20H28O3, which is believed to be an epoxy-γ-lactone. PMID:5665894

  6. Installation Restoration Program. Environmental Technology Development Task Order - 8. Field Demonstration - Composting of Explosives - Contaminated Sediments at the Louisiana Army Ammunition Plant (LAAP)

    DTIC Science & Technology

    1988-09-30

    acids were added, a 10 mg/L initial TNT concentration was reduced to 0.1 mg/L in 5 days. Naumora et al. (1979) isolated a Pseudomonas denitrificans...removed from Holston Army Ammunition Plant (HAAP) Area B wastes in pilot-plant studies using an activated sludge reactor dominated by Pseudomonas and... Alcaligens species (Green, 1972). Stillwell (1977) presented data indicating that RDX and fIl JX could be biologically removed from pilot-plant units

  7. Hybrid density functional approach to the isotropic and anisotropic hyperfine couplings with 14N and 1H nuclei in the blue copper proteins

    NASA Astrophysics Data System (ADS)

    Jaszewski, Adrian R.; Jezierska, Julia

    2001-08-01

    We report UB1LYP hybrid density functional studies on the hyperfine parameters of wild-type azurin from Pseudomonas aeruginosa and M121Q mutant of azurin from Alcaligenes denitrificans. The large models of the copper site used in the calculations give quantitative insight into the spin density distribution and confirm highly delocalized character of the unpaired electron. Theoretically predicted isotropic and anisotropic hyperfine couplings are compared to the available EPR data and the assignment of the hyperfine splittings is verified.

  8. Evidence for 4-chlorobenzoic acid dehalogenation mediated by plasmids related to pSS50.

    PubMed Central

    Layton, A C; Sanseverino, J; Wallace, W; Corcoran, C; Sayler, G S

    1992-01-01

    The biodegradation of 4-chlorobiphenyl usually proceeds through the intermediate 4-chlorobenzoate. Few bacterial strains can degrade 4-chlorobiphenyl to 4-chlorobenzoate and 4-chlorobenzoate to CO2. This study demonstrates that the 4-chlorobiphenyl-degrading Alcaligenes sp. strain ALP83 can degrade 4-chlorobenzoate to 4-hydroxybenzoate. The dehalogenase activity is correlated with a 10-kb fragment carried on plasmid pSS70. Images PMID:1539985

  9. Importance of Extracellular Processes in the Oxygen Enhancement of Radiation Lethality.

    DTIC Science & Technology

    1982-03-08

    used to demonstrate the presence of an iron superoxide dismutase in Alcaligenes faecalis and of mangane. superoxide dismutases in Streptococcus...superoxide dismutase (SOD). Streptococcus faecalis contains a single manganese-SOD which was isolated and used as an antigen in the rabbit. A...precipitating and inhibiting antibody was obtained and used to suppress the SOD in crude lysates of S. faecalis . It allowed the demonstration that 170 of the

  10. Respirator Filter Efficiency Testing Against Particulate and Biological Aerosols Under Moderate to High Flow Rates

    DTIC Science & Technology

    2006-08-01

    mainly of charge- neutralized dioctyl phthalate (DOP), sodium chloride (NaCl), corn oil, or polystyrene latex (PSL) spheres. The aerosol concentrations...function of particle size for several filtering facepieces and DM, DFM, and HEPA respirator cartridges using corn oil particles over a size range of...globigii, M luteus, and P. alcaligenes, monodisperse urea aerosols (1.5, 3, 5, 7, and 9 um), and a NaCl aerosol (0.6 Vtm MMAD, 2.3 GSD). Bacterial

  11. Identification of dimethyl disulfide-forming bacteria isolated from activated sludge.

    PubMed Central

    Tomita, B; Inoue, H; Chaya, K; Nakamura, A; Hamamura, N; Ueno, K; Watanabe, K; Ose, Y

    1987-01-01

    Twenty-four strains with high dimethyl disulfide (DMDS)-forming ability were isolated from activated sludge and identified to the genus level. These bacteria were classified into four groups (A, B, C, and D) by the API ZYM System (API System S.A., Montalieu, France). Group A (three strains) was identified as genus Lactobacillus by the API 20B System, by the method of Cowan and Steel, and by production of lactic acid as confirmed by gas-liquid chromatography. Group B (eight strains) was identified as genus Corynebacterium by API 20B and the Cowan and Steel method. Group C (one strain) was suggested to belong to genus Corynebacterium by the API 20B System. Group D (12 strains) was identified as genus Pseudomonas or Alcaligenes by the API 20B System, as genus Alcaligenes by the Cowan and Steel method, and as Achromobacter group Vd by the API 20NE System. However, on the basis of guanine-plus-cytosine contents in DNA and form of flagella, these strains were identified as genus Pseudomonas. Formation of DMDS from DL-methionine and S-methyl-L-cysteine was tested. DMDS-forming bacteria isolated from activated sludge formed DMDS from both precursors. In genus Pseudomonas, P. aeruginosa could not form DMDS from either precursor, but P. acidovorans, P. alcaligenes, P. pseudoalcaligenes, and P. testosteroni formed DMDS. In genus Alcaligenes, A. denitrificans subsp. xylosoxydans, A. denitrificans subsp. denitrificans, A. faecalis, and A. odorans formed DMDS from both precursors. Achromobacter group Vd formed DMDS from S-methyl-L-cysteine, but could not from DL-methionine. PMID:3662505

  12. [Species composition and microbial contamination of atmospheric humidity condensate and potable water from the Mir water supply systems during Mir main missions 4 through 27].

    PubMed

    Skuratov, V M; Zagibalova, L B; Pushkin, V P

    2002-01-01

    Analyzed were quantitative and qualitative characteristics, and processes of formation and transformation of microflora in the atmospheric humidity condensate (AHC) and potable water (PW) regenerated from the condensate by the long-operating system (SRV-K) aboard space station Mir. The paper presents data on species composition of microflora of AHC, and PW regenerated by SRV-K in the period from 1989 through to 1999. The following microbial species were identified in AHC: Alcaligenes (14.8%), Bacillus (3.7%), Citrobacter (7.4%), Clostridium (11.1%), Enterobacter (7.4%), Proteus (7.4%), Pseudomonas (18.5%), and Staphylococcus (100%). There were two PW cocks in SRV-K--"hot" and "cold". "Hot" PW microflora included Alcaligenes (15%), Bacillus (5%), Citrobacter (20%), Clostridium (5%), Enterobacter (15%), Proteus (10%), Pseudomonas (15%), and Staphylococcus (75%). "Cold" PW microflora included Aeromonas (14.3%), Alcaligenes (21.4%), Bacillus (7.1%), Citrobacter (7.1%), Clostridium (7.1%), Enterobacter (7.1%), Moraxella (7.1%), Pseudomonas (7.1%), and Staphylococcus (78.5%). Microbial content averaged 8.9 x 10(2) +/- 1.3 (0.01-4) x 10(3) CFU/ml in condensate and 1.5 +/- 0.1 (0-3.0) CFU/ml in regenerated potable water.

  13. Quality changes in squid (Loligo duvaucelli) tubes chilled with dry ice and water ice.

    PubMed

    Jeyasekaran, G; Jeya Shakila, R; Sukumar, D; Ganesan, P; Anandaraj, R

    2010-08-01

    Squid tubes were packed with 100% (w/w of squid) dry ice (PI), 20% dry ice and 50% water ice (PII) and 50% water ice (PIII) in polyethylene bags and store in thermocole boxes at room temperature (32 ± 2°C) for 24 h. Quality changes curing storage were studied. Lowest temperature of -30.3°C was attained in PI while it was 15-16°C in PII and PIII at 1 h of storage. The gas compositions in packages initially were 21% O2, 0.4% CO2 and 78.1% N2 in PI, PII and PIII, respectively. During storage for 24 h highest level of 82.5% CO2 was noticed in PII. Fresh squid tubes had bacterial flora of Hafnia, Pseudomonas, Bacillus, Flavobacterium and Alcaligens. Hafnia constituted 74% of the flora. Alcaligenes (47%), Alteromonas (30%) and Alcaligenes (56%) were dominant in squid tubes stored in 100% dry ice, in the combination package, and in 100% water ice, respectively. Increase in total volatile base nitrogen and trimethylamine nitrogen, no definite trend in free fatty acid values in all packages while increase in pH in PI and PIII and no consistent changes in PI were noticed during storage for 24 h. The PI had lowest bacterial counts and PIII the highest. Squids stored in PI and PII were sensorily acceptable after 24 and 18 h, respectively.

  14. Evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate.

    PubMed Central

    Oltmanns, R H; Müller, R; Otto, M K; Lingens, F

    1989-01-01

    Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolated by selective enrichment from various water and soil samples from the Stuttgart area. According to their responses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes, Pseudomonas, and Aureobacterium. To elucidate the degradation pathway of 4-fluorobenzoate, metabolic intermediates were identified. Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as described in previous studies. In growth experiments, these strains excreted 50 to 90% of the fluoride from fluorobenzoate. Alcaligenes sp. strains RHO21 and RHO22 used all three isomers of monofluorobenzoate. Alcaligenes sp. strain RHO22 also grew on 4-chlorobenzoate. Aureobacterium sp. strain RHO25 transiently excreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometric amounts of fluoride were released. In cell extracts from this strain, the enzymes for the conversion of 4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected. All these enzymes were inducible by 4-fluorobenzoate. These data suggest a new pathway for the degradation of 4-fluorobenzoate by Aureobacterium sp. strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate. PMID:2604392

  15. [Activated Sludge Bacteria Transforming Cyanopyridines and Amides of Pyridinecarboxylic Acids].

    PubMed

    Demakov, V A; Vasil'ev, D M; Maksimova, Yu G; Pavlova, Yu A; Ovechkina, G V; Maksimov, A Yu

    2015-01-01

    Species diversity of bacteria from the activated sludge of Perm biological waste treatment facilities capable of transformation of cyanopyridines and amides of pyridinecarboxylic acids was investigated. Enrichment cultures in mineral media with 3-cyanopyridine as the sole carbon and nitrogen source were used to obtain 32 clones of gram-negative heterotrophic bacteria exhibiting moderate growth on solid and liquid media with 3- and 4-cyanopyridine. Sequencing of the 16S rRNA gene fragments revealed that the clones with homology of at least 99% belonged to the genera Acinetobacte, Alcaligenes, Delftia, Ochrobactrum, Pseudomonas, Stenotrophomonas, and Xanthobacter. PCR analysis showed that 13 out of 32 isolates contained the sequences (-1070 bp) homologous to the nitrilase genes reported previously in Alcaligenes faecalis JM3 (GenBank, D13419.1). Nine clones were capable of nitrile and amide transformation in minimal salt medium. Acinetobacter sp. 11 h and Alcaligenes sp. osv transformed 3-cyanopyridine to nicotinamide, while most of the clones possessed amidase activity (0.5 to 46.3 mmol/(g h) for acetamide and 0.1 to 5.6 mmol/(g h) for nicotinamide). Nicotinamide utilization by strain A. faecalis 2 was shown to result in excretion of a secondary metabolite, which was identified as dodecyl acrylate at 91% probability.

  16. Species-specific functioning of the Pseudomonas XcpQ secretin: role for the C-terminal homology domain and lipopolysaccharide.

    PubMed

    Bitter, Wilbert; van Boxtel, Ria; Groeneweg, Mathijs; Carballo, Patricia Sánchez; Zähringer, Ulrich; Tommassen, Jan; Koster, Margot

    2007-04-01

    Secretins are oligomeric proteins that mediate the export of macromolecules across the bacterial outer membrane. The members of the secretin superfamily possess a C-terminal homology domain that is important for oligomerization and channel formation, while their N-terminal halves are thought to be involved in system-specific interactions. The XcpQ secretin of Pseudomonas spp. is a component of the type II secretion pathway. XcpQ from Pseudomonas alcaligenes is not able to functionally replace the secretin of the closely related species Pseudomonas aeruginosa. By analysis of chimeric XcpQ proteins, a region important for species-specific functioning was mapped between amino acid residues 344 and 478 in the C-terminal homology domain. Two chromosomal suppressor mutations were obtained that resulted in the proper functioning in P. aeruginosa of P. alcaligenes XcpQ and inactive hybrids. These mutations caused a defect in the synthesis of the lipopolysaccharide (LPS) outer core region. Subsequent analysis of different LPS mutants showed that changes in the outer core and not the loss of O antigen caused the suppressor phenotype. High concentrations of divalent cations in the growth medium also allowed P. alcaligenes XcpQ and inactive hybrids to function properly in P. aeruginosa. Since divalent cations are known to affect the structure of LPS, this observation supports the hypothesis that LPS has a role in the functioning of secretins.

  17. Hierarchical meso-macroporous silica grafted with glyoxyl groups: opportunities for covalent immobilization of enzymes.

    PubMed

    Bernal, Claudia; Urrutia, Paulina; Illanes, Andrés; Wilson, Lorena

    2013-06-25

    Hierarchical meso-macroporous silica (average mesopore diameter 20 nm) was synthesized and chemically modified to be used as a support for the immobilization of lipases from Candida antarctica B and Alcaligenes sp. and β-galactosidases from Bacillus circulans and Aspergillus oryzae. Catalytic activities and thermal stabilities of enzymes immobilized by multipoint covalent attachment in silica derivatized with glyoxyl groups were compared with those immobilized in glyoxyl-agarose, assessing biocatalyst performance under non-reactive conditions in aqueous medium. In the case of A. oryzae β-galactosidase and Alcaligenes sp. lipase, an additional step of amination was needed to improve immobilization yield. Specific activities of lipases immobilized in glyoxyl-silica were high (232 and 62 IU per gram, for C. antarctica B and Alcaligenes sp. respectively); thermal stabilities were higher than those immobilized in glyoxyl-agarose. Although in the case of β-galactosidases from B. circulans and A. oryzae, the specific activities (250 and 310 IU per gram, respectively) were lower than the ones obtained with glyoxyl-agarose, expressed activities were similar to values previously reported. Thermal stabilities of both β-galactosidases immobilized in glyoxyl-silica were higher than when glyoxyl-agarose was used as support. Results indicate that hierarchical meso-macroporous silica is a versatile support for the production of robust biocatalysts.

  18. Center for Advanced Electrical and Structural Polymers

    DTIC Science & Technology

    1993-10-15

    I RO OR + HC-C-Ar-C-CH (O’P)YwI"I, CulASX (CH3CHz)NH/HFt BrHC-CH&#34S"C H=CHBr IVa: trans. trans IVb: cistrans RO OR 4C=C-CH=CH I CH=CH-C-C-Ar--h U VI ...SENIOR RESEARCH PERSONNEL V. JUNIOR RESEARCH PERSONNEL VI . EXTRA-MURAL SUBCONTRACTORS VII. ABSTRACT OF ACCOMPLISHMENTS VIII. DESCRIPTION OF RESEARCH...MIT) D-K. Ding (MIT) 5 Peter Schuler (FMI) Bill Slager (FMI) John Larouco (FMI) VI . SUBCONTRACTORS: Profs. W.A. Feld, J.J. Kane; Wright State

  19. Basic Techniques in Environmental Simulation.

    DTIC Science & Technology

    1982-07-01

    PROJECT, TASKAREA & WORK UNIT NUMBERS US Air Force Environmental Technical Applications Center/DNS Scott AFB, Illinois 62225 I. CONTROLLING OFFICE...74. MONITORING AGENCY NAME & ADDRESS(II dilferent from Controlling Office) IS. SECURITY CLASS. (of this repor) Unclassified 15a. DECLASSIFICATION...re e, j onc n0 :ut- ch&#34ange. [n using an anin hiLi Li il weather sequenn" in p1 annin 24or clo rgrn. on.. runs tho risk Qr Ih f."fl(r chcn~ may he too

  20. Classical Combinatorial Sequential Machine Components Implemented with Quantum Devices

    DTIC Science & Technology

    1998-05-01

    1,1994) |4| Bennett. C.H., "Notes on the History of Reversible Computation", 1988 (5] Bose, S., Knight, P, Murao, M .. Plenio , M ., Vedral, V...Engineer f^H^ M , H^. FOR THE DIRECTOR: ROBERT G. POLCE, Acting Chief Rome Operations Office Sensors Directorate If your address has changed or...conventional multi-level logic implementation is shown in Figure 2. Inputs Level 1 Level 2 Level 3 =D—iG A B C D E F H 3> K 4j- Output

  1. The Military Relationship between the United States and Turkey Since 1947.

    DTIC Science & Technology

    1981-06-01

    scientific, technical and cultural areas, and grants of $1.2 billion in long-term Soviet credit for industrial development (13:232). In 1975 Turkey received...Submarines Type 209 (8 torpedo tubes), built in 1974 FR Germany 3 1’ Guppy -class (10 torpedo tubes) built in 1940’s USA 10 2 Destroyers Gearing-class...ex- Dutch 1970 3 Coastal minesweeper (USA) ex-Fre;.ch &#34MSC" type ex-British 1970-71 4 Submarine " Guppy 11 A" USA Completed type 1944 1971 2

  2. The Reactions of C2H2 and CH3C2H on Ag Powder.

    DTIC Science & Technology

    1987-12-10

    Ag microclusters . This powder is then exposed to subsequent pulses of 20 or CH&#34C2H (3.7% in NP). The surface enhanced Raman Scattering (SERS) spectra...pulsing it with NO 2 gas which forms fresh Ag microclusters . This powder is then exposed to subsequent pulses of C2H2 or CH3 C2H (3.7% in N2). The surface...gas. The Ag imbedded in the AgNO migrates to sites where Ag microclusters are formed. These microstructures are a necessary part of the SERS enhancement

  3. The Light Infantry Company and Tactical Mobility: A Step in Which Direction?

    DTIC Science & Technology

    2007-11-02

    estimated to t Tg t er t St9ne. nlUvding teUi oreeinIMUtii.larg ch&#34 in data sorirc; ofhein ndnjnaning the data needed, and completing and...the light force, Operation JUST CAUSE was not a definitive test and did provide food for thought concerning possible future tactical changes and...February 1993). 19 Transportation capabilities for the LID DISCOM (trucks) are extracted from Student Text 101-6, G1/ G4 Battle Book (Fort Leavenworth, KS

  4. Lead resistance in micro-organisms.

    PubMed

    Jarosławiecka, Anna; Piotrowska-Seget, Zofia

    2014-01-01

    Lead (Pb) is an element present in the environment that negatively affects all living organisms. To diminish its high toxicity, micro-organisms have developed several mechanisms that allow them to survive exposure to Pb(II). The main mechanisms of lead resistance involve adsorption by extracellular polysaccharides, cell exclusion, sequestration as insoluble phosphates, and ion efflux to the cell exterior. This review describes the various lead resistance mechanisms, and the regulation of their expression by lead binding regulatory proteins. Special attention is given to the Pbr system from Cupriavidus metallidurans CH34, which involves a unique mechanism combining efflux and lead precipitation.

  5. Poland in NATO? A Case Study of the United States Foreign Policymaking Process

    DTIC Science & Technology

    1994-06-01

    Science Monitor, 22 September 1993: "As East Bloc Drifts West, Russia Frets Over Security" by Daniel Sneider. 17 David Remnick , "Russia Appeals for...Affairs David Wipple, Ch &#34i Department of Systems Management ii ABSTRACT The opportunity for nations such as Poland to enter NATO is of vital concern...November 5, 1991. Reisch, Alfred A., "Central and Eastern Europe’s Quest for NATO Membership", RFE/RL Research Report, Vol.2, No.28, July 9, 1993. Remnick

  6. Screening and identification of polyhydroxyalkanoates producing bacteria and biochemical characterization of their possible application.

    PubMed

    Sangkharak, Kanokphorn; Prasertsan, Poonsuk

    2012-01-01

    Polyhydroxyalkanoates (PHAs) accumulating bacteria were isolated under various selective conditions such as pH, salt concentrations and types of heavy metal. Fifty strains of bacterial isolates were found to belong to Bacillus, Proteus, Pseudomonas, Aeromonas, Alcaligenes and Chromobacterium, based on phenotypical features and genotypic investigation. Only twenty five bacterial isolates were selected and observed for the production of PHAs. Interestingly, bacteria belonging to Firmucutes Bacillus sp. produced a high amount of PHAs. The maximum PHAs were accumulated by B. licheniformis PHA 007 at 68.80% of dry cell weight (DCW). Pseudomonas sp., Aeromonas sp., Alcaligenes sp. and Chromobacterium sp. were recorded to produce a moderate amount of PHAs, varying from 10.00-44.32% of DCW. The enzymatic activity was preliminarily analyzed by the ratio of the clear zone diameter to colony diameter. Bacillus gave the highest ratio of hydrolysis zone which corresponds to the highest hydrolytic enzyme activities. Bacillus licheniformis PHA 007 had the highest lipase and protease activity at 2.1 and 5.1, respectively. However, the highest amylase activity was observed in Bacillus sp. PHA 023 at 1.4. Determination of metabolic characteristics was also investigated to check for their ability to consume a wide range of substrates. Bacillus, Aeromonas sp. and Alcaligenes sp. had great ability to utilize a variety of substrates. To decrease high PHA cost, different sources of cheap substrates were tested for the production of PHAs. Bacillus cereus PHA 008 gave the maximal yield of PHA production (64.09% of DCW) when cultivated in anaerobically treated POME. In addition, the accumulation of PHA copolymers such as 3-hydroxyvalerate and 3-hydroxyhexanoate was also observed in Bacillus and Pseudomomas sp. strain 012 and 045, respectively. Eight of the nine isolates accumulated a significant amount of PHAs when inexpensive carbon sources were used as substrates. Here it varied from 1

  7. Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event

    SciTech Connect

    Kroeckel, L.; Focht, D.D.

    1987-10-01

    Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-0, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absences of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene of 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant-unlike it parent, R5-3-did not grown on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown in 4-methylbenzoate than in the same cells grown on benzene. Benzene was metabolized through the MP pathway in CB1-9, while chlorobenzene was metabolized through the OP pathway.

  8. Achromobacter buckle infection diagnosed by a 16S rDNA clone library analysis: a case report.

    PubMed

    Hotta, Fumika; Eguchi, Hiroshi; Naito, Takeshi; Mitamura, Yoshinori; Kusujima, Kohei; Kuwahara, Tomomi

    2014-11-24

    In clinical settings, bacterial infections are usually diagnosed by isolation of colonies after laboratory cultivation followed by species identification with biochemical tests. However, biochemical tests result in misidentification due to similar phenotypes of closely related species. In such cases, 16S rDNA sequence analysis is useful. Herein, we report the first case of an Achromobacter-associated buckle infection that was diagnosed by 16S rDNA sequence analysis. This report highlights the significance of Achromobacter spp. in device-related ophthalmic infections. A 56-year-old woman, who had received buckling surgery using a silicone solid tire for retinal detachment eighteen years prior to this study, presented purulent eye discharge and conjunctival hyperemia in her right eye. Buckle infection was suspected and the buckle material was removed. Isolates from cultures of preoperative discharge and from deposits on the operatively removed buckle material were initially identified as Alcaligenes and Corynebacterium species. However, sequence analysis of a 16S rDNA clone library using the DNA extracted from the deposits on the buckle material demonstrated that all of the 16S rDNA sequences most closely matched those of Achromobacter spp. We concluded that the initial misdiagnosis of this case as an Alcaligenes buckle infection was due to the unreliability of the biochemical test in discriminating Achromobacter and Alcaligenes species due to their close taxonomic positions and similar phenotypes. Corynebacterium species were found to be contaminants from the ocular surface. Achromobacter spp. should be recognized as causative agents for device-related ophthalmic infections. Molecular species identification by 16S rDNA sequence analysis should be combined with conventional cultivation techniques to investigate the significance of Achromobacter spp. in ophthalmic infections.

  9. Novel approach for the ammonium removal by simultaneous heterotrophic nitrification and denitrification using a novel bacterial species co-culture.

    PubMed

    Angar, Yassmina; Kebbouche-Gana, Salima; Djelali, Nacer-Eddine; Khemili-Talbi, Souad

    2016-03-01

    Agricultural activities lead excessive emission of ammonia nitrogen in the environment and can profoundly interfere the equilibrium of the natural ecosystems leading to their contamination. Actually, the biological purification of wastewaters is the most adopted technique thanks to its several advantages such as high performance and low energy consumption. For this reason, two novel strains of Alcaligenes sp. S84S3 and Proteus sp. S19 genus were isolated from an activated sludge and applied in the treatment of ammonium and nitrite in aqueous solution. Under the optimum operating conditions of temperature (30 °C), pH (7), carbon substrate (2 g/L of glucose) and duration of incubation time (69 h), the strain Alcaligenes sp. S84S3 could oxidize 65% of the ammonium as high as 272.72 mg-NH4(+)/L. Moreover, during 48 h, the nitrate reduction rate performed by the strain Proteus S19 was about 99 % without production of nitrite intermediate (negligible concentration). Moreover, the coculture of the strains Alcaligenes sp. S84S3 and Proteus sp. S19 could eliminate 65.83% of the ammonium ions without production of toxic forms of nitrogen oxides during a short time of incubation (118 h) at the same operational conditions with providing the aeration in the first treatment phase. The coculture of our isolated strains is assumed to have a good potential for nitrification and denitrification reactions applied in the treatment of wastewater containing ammonium, nitrite and nitrate. As a result, we can consider that the mixed culture is a practical method in the treatment of high-strength ammonium wastewater with reducing of sludge production.

  10. [Pathogenic bacteria in cystic fibrosis].

    PubMed

    Mariani-Kurkdjian, P; Bingen, E

    2003-09-01

    Since the CF gene identification in 1989 and despite the improvement of our knowledge in the physiopathology of the disease, bronchopulmonary infection determines the vital prognosis. Following Staphylococcus aureus infection, patients are colonized or colonized by Pseudomonas aeruginosa, greatly involved in the pulmonary deterioration. Other bacteria may be involved Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenes sp. Intensive antibiotic treatment of primocolonisation helps to prevent or delay chronic colonisation. Chronic colonization needs a rational long term antibiotic strategy to prevent the occurrence of multiresistant germs; antibiotic cures are performed every 3 or 4 months before pulmonary exacerbation symptoms.

  11. Multidrug-Resistant Gram-Negative Bacteria Colonization of Healthy US Military Personnel in the US and Afghanistan

    DTIC Science & Technology

    2013-02-05

    1) 0 4 (5) 2 (2) 8 (9) Alcaligenes species 0 1 (1) 0 0 1 (1) 2 (2) 1 (1) 5 (5) Citrobacter species 0 0 0 0 0 0 11 (11) 11 (11) Enterobacter...Klebsiella oxytoca 0 1 (1) 0 1 (1) 0 0 2 (3) 2 (5) Klebsiella pneumoniae 0 1 (1) 1 (1) 2 (2) 0 1 (1) 8 (8) 12 (13) Proteus species 0 0 1 (1) 0 0 1...bacteria or clones of bacteria. ***Other isolates include: Acinetobacter species (2), Acinetobacter haemolyticus (2), Burkholderia cepacia, Burkholderia

  12. BIODEGRADATION OF PETROLEUM-WASTE BY BIOSURFACTANT-PRODUCING BACTERIA

    SciTech Connect

    Brigmon, R; Grazyna A. Plaza, G; Kamlesh Jangid, K; Krystyna Lukasik, K; Grzegorz Nalecz-Jawecki, G; Topher Berry, T

    2007-05-16

    The degradation of petroleum waste by mixed bacterial cultures which produce biosurfactants: Ralstonia pickettii SRS (BP-20), Alcaligenes piechaudii SRS (CZOR L-1B), Bacillus subtilis (1'- 1a), Bacillus sp. (T-1) and Bacillus sp. (T'-1) was investigated. The total petroleum hydrocarbons were degraded substantially (91 %) by the mixed bacterial culture in 30 days (reaching up to 29 % in the first 72 h). Similarly, the toxicity of the biodegraded petroleum waste decreased 3 times after 30 days as compared to raw petroleum waste. Thus, the mixed bacterial strains effectively clean-up the petroleum waste and they can be used in other bioremediation processes.

  13. Isolation and identification of autochthonous microbiota from a granitic aquifer and its variation after the bottling process.

    PubMed

    Urmeneta, J; Navarrete, A; Sancho, J

    2000-12-01

    The autochthonous microbiota from a granitic aquifer in Spain were studied. Several bacterial strains were isolated and identified. The major components of the microbiota were Pseudomonas-like strains, Flavobacterium, Acinetobacter, and Alcaligenes. The variation in the number of microorganisms after the bottling process was studied. The initial bacterial population increased over the first 5 days after bottling. This increase was higher in samples from polyvinyl chloride bottles. Sonication usually increased the total cell counts. As expected, most of the autochthonous microbiota were not detected in the viable cell counts.

  14. Occurrence of Potential Pathogens in Water Containing Ornamental Fishes

    PubMed Central

    Trust, T. J.; Bartlett, K. H.

    1974-01-01

    The bacterial population of the water supplied with ornamental fish purchased from retail outlets was examined qualitatively and quantitatively. As many as 109 viable aerobic organisms per 100 ml were present, with fecal coliform counts as high as 105 per 100 ml. Citrobacter, Escherichia, Pseudomonas, and Vibrio were isolated from 75% or more of the samples, whereas Aeromonas, Alcaligenes, Enterobacter, Flavobacterium, and Streptococcus were isolated from 45 to 65% of the samples. Pseudomonas aeruginosa, Edwardsiella tarda, and Klebsiella pneumoniae were also isolated. PMID:4602309

  15. Incidence of otitis externa in dogs and cats in Japan.

    PubMed

    Baba, E; Fukata, T

    1981-05-02

    The incidence of otitis externa in dogs and cats admitted to the animal hospital of the University of Osaka Prefecture was investigated and the bacteria isolated were tested for antibiotic susceptibility. Of the various breeds examined, the incidence of otitis externa was highest in miniature poodles and cocker spaniels and Himalayan and Persian cats. The organisms most commonly associated with otitis externa were coagulase-negative staphylococci, followed by coagulase-positive staphylococci, streptococci and Escherichia coli. Most staphylococci were susceptible to the antibiotics tested, but 15 per cent of staphylococci were resistant to more than three antibiotics. Pseudomonas and Alcaligenes species were resistant to almost all antibiotics except gentamicin and colistin.

  16. Antimicrobial activity of essential oil from Schinus molle Linn.

    PubMed

    Gundidza, M

    1993-11-01

    The essential oil from the fresh leaves of Schinus molle isolated by hydrodistillation was tested for antibacterial activity using the hole plate diffusion method and for antifungal activity using the mycelium or single cell growth inhibition method. Results obtained showed that the volatile oil exhibited significant activity against the following bacterial species: Klebsiella pneumoniae, Alcaligenes faecalis, Pseudomonas aeruginosa, Leuconostoc cremoris, Enterobacter aerogenes, Proteus vulgaris, Clostridium sporogenes, Acinetobacter calcoacetica, Escherichia coli, Beneckea natriegens, Citrobacter freundii, Serratia marcescens, Bacillus subtilis and Brochothrix thermosphacata. The fungal species Aspergillus ochraceus, Aspergillus parasiticus, Fusarium culmorum and Alternaria alternata exhibited significant sensitivity to the volatile oil.

  17. Complicated intra-abdominal infection caused by extended drug-resistant Achromobacter xylosoxidans.

    PubMed

    Teng, Sing-On; Ou, Tsong-Yih; Hsieh, Yu-Chia; Lee, Wuan-Chan; Lin, Yi-Chun; Lee, Wen-Sen

    2009-04-01

    Achromobacter xylosoxidans (formerly Alcaligenes xylosoxidans) is a rare but important nosocomial pathogen. Antibiotic resistance has been increasing during the past decade. A. xylosoxidans may be confused with Pseudomonas spp. but, unlike Pseudomonas spp., this organism has peritrichous flagella. Complicated intra-abdominal infection with A. xylosoxidans has rarely been reported in the literature. This report is of an immunocompetent patient with acute cholecystitis complicated by an intra-abdominal abscess after surgery. Culture of both blood and ascites yielded extended drug-resistant A. xylosoxidans, which was only sensitive to colistin. The clinical and laboratory characteristics of A. xylosoxidans are presented.

  18. Microbial contamination of a disinfectant-soaked unwoven cleaning cloth.

    PubMed

    Oie, S; Arakawa, J; Furukawa, H; Matsumoto, S; Matsuda, N; Wakamatsu, H

    2012-09-01

    In December 2009, a 76-year-old male patient developed pneumonia due to Burkholderia cepacia whilst in an intensive care unit at a Japanese university hospital. During the subsequent environmental investigation to find the source, B. cepacia with an identical DNA type was found in his denture storage solution. Open packets of unwoven rayon cloths soaked in 0.2% alkyldiaminoethylglycine hydrochloride, used for environmental cleaning, were shown to be contaminated with B. cepacia, Alcaligenes xylosoxidans, Pseudomonas fluorescens and Pseudomonas aeruginosa. B. cepacia of a different DNA type was found in five of 42 samples from sealed packets of cloths.

  19. Microbial oxidation of dimethylnaphthalene isomers

    SciTech Connect

    Miyachi, Nobuya; Tanaka, Tohru; Suzuki, Takaya; Hotta, Yasushi ); Omori, Toshio )

    1993-05-01

    Few studies have focused on the microbial oxidation and metabolism of dimethylnaphthalenes (DMNs), which are supplied from the light oil fraction of crude oil. This report describes the isolation of 2,6-DMN-utilizing bacteria, the identification of the isolates (Alcaligenes sp. strain D-59, Pseudomonas sp. strains D-87 and D-186), the accumulation of 2,6-naphthalene dicarboxylic acid (NDCA) from 2,6-DMN, the identification of some metabolic intermediates of 2,6-DMN, and a proposal of a metabolic pathway of 2,6-DMN. 11 refs., 2 figs.

  20. Characteristics and restriction analysis of the 4-chlorobiphenyl catabolic plasmid, pSS50.

    PubMed Central

    Hooper, S W; Dockendorff, T C; Sayler, G S

    1989-01-01

    The plasmid pSS50 is a 53-kilobase self-transmissible plasmid of broad host range that has been isolated from several Alcaligenes and Acinetobacter species. This plasmid has previously been shown to mediate the mineralization of 4-chlorobiphenyl to carbon dioxide and water. Physical characterization of this plasmid by restriction analysis indicates that most hexanucleotide cleavage sites are clustered in a 5-kilobase region, leaving large regions without restriction sites. The paucity of restriction sites is not due to DNA methylation. PMID:2757383

  1. Biodegradation of Asphalt Cement-20 by Aerobic Bacteria

    PubMed Central

    Pendrys, John P.

    1989-01-01

    Seven gram-negative, aerobic bacteria were isolated from a mixed culture enriched for asphalt-degrading bacteria. The predominant genera of these isolates were Pseudomonas, Acinetobacter, Alcaligenes, Flavimonas, and Flavobacterium. The mixed culture preferentially degraded the saturate and naphthene aromatic fractions of asphalt cement-20. A residue remained on the surface which was resistant to biodegradation and protected the underlying asphalt from biodegradation. The most potent asphalt-degrading bacterium, Acinetobacter calcoaceticus NAV2, excretes an emulsifier which is capable of emulsifying the saturate and naphthene aromatic fractions of asphalt cement-20. This emulsifier is not denatured by phenol. PMID:16347928

  2. Growth suppressing activity for endothelial cells induced from macrophages by carboxymethylated curdlan.

    PubMed

    Usui, S; Matsunaga, T; Ukai, S; Kiho, T

    1997-11-01

    A carboxymethylated derivative of a linear (1-->3)-beta-D-glucan (CMCD) from Alcaligenes faecalis var. myxogenes acted directly on mouse peritoneal macrophages and mouse lymphoma P388D1 cells, and induced a growth suppressing activity for bovine artery endothelial cells (BAEs) from themselves at a concentration of 100 micrograms/ml. The suppressing activity was also detected in the mouse serum administered as an i.p. injection of CMCD at a dose of 100 mg/kg, suggesting that the growth suppressing activity was induced from macrophages potentiated by CMCD in vivo.

  3. Degradation of the Herbicide Mecoprop [2-(2-Methyl-4-Chlorophenoxy)Propionic Acid] by a Synergistic Microbial Community

    PubMed Central

    Lappin, Hilary M.; Greaves, Michael P.; Slater, J. Howard

    1985-01-01

    A microbial community isolated from wheat root systems was capable of growth on mecoprop as the sole carbon and energy source. When exposed to fresh herbicide additions, the community was able to shorten the lag phase from 30 days to less than 24 h. The community comprised two Pseudomonas species, an Alcaligenes species, a Flavobacterium species, and Acinetobacter calcoaceticus. None of the pure cultures was capable of growing on mecoprop. Certain combinations of two or more community constituents were required before growth commenced. The mecoprop-degrading community could also degrade 2,4-dichlorophenoxyacetic acid and 2-methyl-4-chlorophenoxyacetic acid but not 2,4,5-trichlorophenoxyacetic acid. PMID:16346731

  4. Marine biosurfactants, I. Screening for biosurfactants among crude oil degrading marine microorganisms from the North Sea.

    PubMed

    Schulz, D; Passeri, A; Schmidt, M; Lang, S; Wagner, F; Wray, V; Gunkel, W

    1991-01-01

    Three bacterial strains of marine origin were isolated during a screening for biosurfactants among n-alkane degrading microorganisms. One strain-identified as Alcaligenes sp. MM1-produced a novel glucose lipid. In the case of Arthrobacter sp. EK 1 the well-known trehalose tetraester was found as major component. From another pure culture classified as Arthrobacter sp. SI 1, extracellular emulsifying agents with properties indicating high molecular weight substances were detected. Furthermore trehalose corynomycolates were found at up to 2 g/l. The isolated biosurfactants showed good interfacial and emulsifying properties.

  5. Antibiotic-resistant heterotrophic plate count bacteria and amoeba-resistant bacteria in aquifers of the Mooi River, North West province, South Africa.

    PubMed

    Carstens, Alewyn; Bartie, Catheleen; Dennis, Rainier; Bezuidenhout, Carlos

    2014-12-01

    Groundwater in the Mooi River catchment is prone to mining, agricultural, municipal and septic tank pollution. In this study physico-chemical and microbiological parameters were determined using appropriate methods. Bacterial isolates were identified by 16S rRNA sequencing (heterotrophic plate count (HPC) bacteria and amoeba-resistant bacteria (ARB)) and multiplex polymerase chain reaction (Escherichia coli). Antibiotic resistance tests were also performed. Physico-chemical parameters were generally within target water quality ranges for drinking water. HPC bacteria ranged between 10(5) and 10(7) colony-forming units (cfu)/ml. E. coli were enumerated from Trimpark, School and Cemetery. The Blaauwbank borehole was negative for faecal streptococci. Pseudomonas spp. were most abundant in the bulk water. Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying patterns of antibiotic resistance were observed. Most HPC bacterial isolates were resistant to cephalothin and/or amoxicillin and a few were resistant to erythromycin and streptomycin. Pseudomonas spp. was also the most abundant ARB. Other ARBs included Alcaligenes faecalis, Ochrobactrum sp. and Achromobacter sp. ARBs were resistant to streptomycin, chloramphenicol, cephalothin, and/or amoxicillin compared to HPCs. The presence of E. coli and ARB in these groundwater sources indicates potential human health risks. These risks should be further investigated and quantified, and groundwater should be treated before use.

  6. Involvement of a chlorobenzoate-catabolic transposon, Tn5271, in community adaptation to chlorobiphenyl, chloroaniline, and 2,4-dichlorophenoxyacetic acid in a freshwater ecosystem

    SciTech Connect

    Fulthorpe, R.R.; Wyndham, R.C. )

    1992-01-01

    A chlorobenzoate-catabolic transposon (Tn5271) was introduced on a conjugative plasmid (pBRC60) in the natural host, Alcaligenes sp. strain BR60, into lake water and sediment flowthrough microcosms. Experimental microcosms were exposed to micromolar levels of 3-chlorobenzoate, 4-chloroaniline, 2,4-dichlorophenoxyacetate, or 3-chlorobiphenyl. The populations of the host, BR60, and organisms carrying Tn5271 were monitored over a 100-day period by use of selective plate counts and the most-probable-number-DNA hybridization method. Populations of Tn5271-carrying bacteria were significantly higher in microcosms dosed with 3-chlorobenzoate, 4-chloroaniline, and 3-chlorobiphenyl than in the control microcosms, indicating that each of these chemicals exerts a selective force on this particular genotype in natural systems. The rates of 3-chlorobenzoate uptake and respiration correlated with Tn5271-carrying populations, as did the rates of 4-chloroaniline uptake and respiration. Plasmid transfer in the 3-chlorobenzoate- and 3-chlorobiphenyl-dosed microcosms resulted in the selection of three phenotypic clusters of chlorobenzoate degraders, only one of which was closely related to the original pBRC60 (Tn5271) donor, Alcaligenes sp. strain BR60. Bacteria dominating 3-chloroaniline-dosed microcosms carried IS1071, the class II insertion sequence that brackets Tn5271, on a plasmid unrelated to pBRC60. The importance of plasmid transfer and transposition during chemical adaptation is discussed.

  7. Enrichment, isolation and identification of sulfur-oxidizing bacteria from sulfide removing bioreactor.

    PubMed

    Luo, Jianfei; Tian, Guoliang; Lin, Weitie

    2013-07-01

    Sulfur-oxidizing bacteria (SOB) are the main microorganisms that participate in the natural sulfur cycle. To obtain SOB with high sulfur-oxidizing ability under aerobic or anaerobic conditions, aerobic and anaerobic enrichments were carried out. Denaturing gradient gel electrophoresis (DGGE) profiles showed that the microbial community changed according to the thiosulfate utilization during enrichments, and Rhodopseudomonas and Halothiobacillus were the predominant bacteria in anaerobic enrichment and aerobic enrichment, respectively, which mainly contributed to the thiosulfate oxidization in the enrichments. Based on the enriched cultures, six isolates were isolated from the aerobic enrichment and four isolates were obtained from the anaerobic enrichment. Phylogenetic analysis suggested the 16S rRNA gene of isolates belonged to the genus Acinetobacter, Rhodopseudomonas, Pseudomonas, Halothiobacillus, Ochrobactrum, Paracoccus, Thiobacillus, and Alcaligenes, respectively. The tests suggested isolates related to Halothiobacillus and Rhodopseudomonas had the highest thiosulfate oxidizing ability under aerobic or anaerobic conditions, respectively; Paracoccus and Alcaligenes could aerobically and anaerobically oxidize thiosulfate. Based on the DGGE and thiosulfate oxidizing ability analysis, Rhodopseudomonas and Halothiobacillus were found to be the main SOB in the sulfide-removing reactor, and were responsible for the sulfur-oxidizing in the treatment system.

  8. Systematic investigation and microbial community profile of indole degradation processes in two aerobic activated sludge systems

    PubMed Central

    Ma, Qiao; Qu, Yuanyuan; Zhang, Xuwang; Liu, Ziyan; Li, Huijie; Zhang, Zhaojing; Wang, Jingwei; Shen, Wenli; Zhou, Jiti

    2015-01-01

    Indole is widely spread in various environmental matrices. Indole degradation by bacteria has been reported previously, whereas its degradation processes driven by aerobic microbial community were as-yet unexplored. Herein, eight sequencing batch bioreactors fed with municipal and coking activated sludges were constructed for aerobic treatment of indole. The whole operation processes contained three stages, i.e. stage I, glucose and indole as carbon sources; stage II, indole as carbon source; and stage III, indole as carbon and nitrogen source. Indole could be completely removed in both systems. Illumina sequencing revealed that alpha diversity was reduced after indole treatment and microbial communities were significantly distinct among the three stages. At genus level, Azorcus and Thauera were dominant species in stage I in both systems, while Alcaligenes, Comamonas and Pseudomonas were the core genera in stage II and III in municipal sludge system, Alcaligenes and Burkholderia in coking sludge system. In addition, four strains belonged to genera Comamonas, Burkholderia and Xenophilus were isolated using indole as sole carbon source. Burkholderia sp. IDO3 could remove 100 mg/L indole completely within 14 h, the highest degradation rate to date. These findings provide novel information and enrich our understanding of indole aerobic degradation processes. PMID:26657581

  9. Nonspecific toxicites in the mouse assay test for botulinum toxin.

    PubMed

    Segner, W P; Schmidt, C F

    1968-08-01

    In inoculated pack experiments on Clostridium botulinum type E, unirradiated and 0.1-Mrad irradiated haddock fillets often gave nonspecific toxicities by the mouse assay test for botulinum toxin. Samples given 0.2-Mrad radiation failed to produce nonspecific reactions. Nonspecific deaths sometimes occurred within 24 hr after injection, although deaths between 24 and 48 hr were more common. The symptoms and the pattern of these deaths suggested a septicemia. Heart-blood cultured from mice showing nonspecific symptoms indicated an infectious process. Among 23 isolates from the blood, eight were identified as Proteus vulgaris, two P. morganii, one P. rettgeri, one Providence subgroup B, two Aerobacter aerogenes, one Actinobacillus, three enterococci, one Alcaligenes marshalli, and four Erysipelothrix insidiosa. The E. insidiosa, Aerobacter, Providence group, and most of the Proteus isolates were infectious for mice when injected by the intraperitoneal route. But the enterococci, Alcaligenes, and Actinobacillus isolates were not infectious and probably represent secondary invaders. The cultural characteristics of the E. insidiosa isolates conform to those described in the literature, with the exception that the four strains grew in the temperature range 50 F (10 C) to 40 F (4.4 C). Nonspecific toxicities were avoided in assays for botulinum toxin by the protection of mice with chloramphenicol and oxytetracycline.

  10. Survival of added bacterial species and metabolism of toxic compounds in natural environments

    SciTech Connect

    King, V.M.

    1987-01-01

    Bacteria able to degrade either 2,4-dichlorophenol (DCP) or phenanthrene (PHEN) were isolated from polluted freshwater environments. Two isolates able to degrade each compound were tested for mineralization with a sensitive /sup 14/C assay and for survival in lake water and sewage using a selective medium. One DCP isolate was identified as Alcaligenes paradoxus and the other as Alcaligenes sp. One PHEN isolate was identified as Pseudomonas fluorescens and the other as Pseudomonas sp. All four isolates survived and grew in sterile environments which indicated that starvation would not be a factor in survival of these strains. The number of organisms declined immediately in number in nonsterile lake water. However, they did survive or even grow in nonsterile sewage for a short period before declining in number. Biotic factors appeared to be influential for survival and mineralization of target compounds in many environments. The removal of protozoa, which prey on bacteria, improved survival of the added cells, but had no influence on the mineralization of 10 ..mu..g DCP/L. In comparison, degradation of 10 and 25 mg DCP/L stopped after a few days. Yeast nitrogen base appeared to overcome the lack of nutrient regeneration, a function attributed to protozoa. The additional nutrients increased toxicant mineralization, especially when seeded with appropriate species. Thus, protozoa may limit growth of added cells but appear to be needed for mineralization of higher concentrations of DCP.

  11. Plasmid-mediated mineralization of 4-chlorobiphenyl.

    PubMed Central

    Shields, M S; Hooper, S W; Sayler, G S

    1985-01-01

    Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 X 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB. Images PMID:2993249

  12. Molecular Analysis of Surfactant-Driven Microbial Population Shifts in Hydrocarbon-Contaminated Soil†

    PubMed Central

    Colores, Gregory M.; Macur, Richard E.; Ward, David M.; Inskeep, William P.

    2000-01-01

    We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC′) (determined to be 13 mg g−1). Addition of the surfactant at a concentration below the CMC′ (2 mg g−1) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC′ (10 mg g−1), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC′ (40 mg g−1) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulated Rhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenes populations at elevated surfactant levels. Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas and Alcaligenes populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization. PMID:10877792

  13. Efficacy of root-associated fungi and PGPR on the growth of Pisum sativum (cv. Arkil) and reproduction of the root-knot nematode Meloidogyne incognita.

    PubMed

    Akhtar, Mohd Sayeed; Panwar, Jitendra

    2013-04-01

    The effects of root-associated fungi (Aspergillus awamori and Glomus mosseae) and plant growth promoting rhizobacteria (PGPR) (Pseudomonas putida, Pseudomonas alcaligenes and Paenibacillus polymyxa) were studied alone and in combination in glasshouse experiments on the growth of pea, enzyme activity (peroxidase and catalase) and reproduction of root-knot nematode Meloidogyne incognita. Application of A. awamori, G. mosseae and PGPR caused a significant increase in pea growth and enzyme activities of both nematode inoculated and uninoculated plants. A. awamori was more effective in reducing galling and improving the growth of nematode inoculated plants than P. alcaligenes or P. polymyxa. The greatest increase in growth, enzyme activities of nematode-inoculated plants and reduction in galling and nematode multiplication was observed when A. awamori was used with P. putida or G. mosseae as compared to the other combinations tested. Percentage root colonization was higher when AM fungus inoculated plants were treated with P. putida both in presence and absence of nematode. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Simple whole-cell biodetection and bioremediation of heavy metals based on an engineered lead-specific operon.

    PubMed

    Wei, Wei; Liu, Xiangzhi; Sun, Peiqing; Wang, Xin; Zhu, Hong; Hong, Mei; Mao, Zong-Wan; Zhao, Jing

    2014-03-18

    A lead-specific binding protein, PbrR, and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 was incorporated into E. coli in conjunction with an engineered downstream RFP (red fluorescence protein), which allowed for highly sensitive and selective whole-cell detection of lead ions. The subsequent display of PbrR on the E. coli cell surface permitted selective adsorption of lead ions from solution containing various heavy metal ions. The surface-engineered E. coli bacteria effectively protected Arabidopsis thaliana seed germination from the toxicity of lead ions at high concentrations. Engineering the E. coli bacteria harboring these lead-specific elements from the pbr operon may potentially be a valuable general strategy for biodetection and bioremediation of toxic heavy metal ions in the environment.

  15. Isolation and identification of cobalt- and caesium-resistant bacteria from a nuclear fuel storage pond.

    PubMed

    Dekker, Linda; Osborne, Thomas H; Santini, Joanne M

    2014-10-01

    One of the issues facing the nuclear power industry is how to store spent nuclear fuel which is contaminated with radionuclides produced during nuclear fission, including caesium ((134)Cs(+), (135)Cs(+) and (137)Cs(+)) and cobalt ((60)Co(2+)). In this study, we have isolated Co(2+)- and Cs(+)-resistant bacteria from water collected from a nuclear fuel storage pond. The most resistant Cs(+) and Co(2+) isolates grew in the presence of 500 mM CsCl and 3 mM CoCl2. Strain Cs67-2 is resistant to fourfold more Cs(+) than Cupriavidus metallidurans str. CH34 making it the most Cs(+)-resistant strain identified to date. The Cs(+)-resistant isolates were closely related to bacteria in the Serratia and Yersinia genera, while the Co(2+)-resistant isolates were closely related to the Curvibacter and Tardiphaga genera. These new isolates could be used for bioremediation.

  16. Proton-detected solid-state NMR spectroscopy of a zinc diffusion facilitator protein in native nanodiscs

    PubMed Central

    Bersch, Beate; Dörr, Jonas M.; Hessel, Audrey; Killian, J. Antoinette; Schanda, Paul

    2017-01-01

    The structure, dynamics and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane, and reconstituted in a suitable membrane-mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co-polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes, forming nanosize discoidal proteolipid particles, also referred to as native nanodiscs. Here we show, for the first time, that high-resolution solid-state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified with the 2 x 34 kDa-large bacterial cation diffusion facilitator CzcD from Cupriavidus metallidurans CH34. PMID:28128538

  17. Precipitin response of potato processing workers to work-related microbial allergens.

    PubMed

    Dutkiewicz, Jacek; Skórska, Czesława; Krysińska-Traczyk, Ewa; Cholewa, Grazyna; Sitkowska, Jolanta; Milanowski, Janusz; Góra, Anna

    2002-01-01

    Serum samples from 61 potato processing workers and 30 urban dwellers not exposed to organic dusts (as a reference group) were examined in agar-gel precipitation test performed by Ouchterlony double diffusion method with the antigens of 12 microorganisms associated with organic dusts. Each serum was tested twice: not concentrated, and three-fold concentrated, for the detection of low levels of precipitins. The antibody response of workers to the antigen of coryneform bacterium Agromyces ramosus was high, at both not concentrated and 3-fold concentrated sera (respectively 29.5% and 45.9%)--significantly greater than in reference group (p < 0.001). Workers' response to the antigens of Gram-negative bacterium Alcaligenes faecalis and thermophilic actinomycete Thermoactinomyces vulgaris was lower (respectively 13.1% and 13.1% at not concentrated sera, 24.6% and 29.5% at 3-fold concentrated sera) but in all cases significantly greater than in reference group (p < 0.05 at not concentrated sera, p < 0.01 and p < 0.001 at 3-fold concentrated sera). The frequency of positive precipitin reactions of potato workers to antigen of Penicillium citrinum was high only at 3-fold concentrated sera (55.7%)--significantly higher compared to reference group (p < 0.001). The antibody response of potato workers to other antigens was either unspecific or low, showing no significant difference compared to reference group. Twenty eight out of 61 examined potato processing workers (45.9%) reported the occurrence of the work-related pulmonary symptoms. The frequency of positive precipitin reactions to Agromyces ramosus, Alcaligenes faecalis, Thermoactinomyces vulgaris, Penicillium citrinum and Acinetobacter calcoaceticus was significantly greater in the subgroup of 28 workers reporting work-related pulmonary symptoms compared to 33 asymptomatic workers (p < 0.05). Study results suggest that antigens of Agromyces ramosus, Alcaligenes faecalis, Thermoactinomyces vulgaris and Penicillium

  18. Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

    PubMed Central

    Mazzola, Priscila G; Martins, Alzira MS; Penna, Thereza CV

    2006-01-01

    Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite

  19. The adsorption of substrate-binding domain of PHB depolymerases to the surface of poly(3-hydroxybutyric acid).

    PubMed

    Shinomiya, M; Iwata, T; Doi, Y

    1998-04-01

    The binding characteristic of PHB depolymerase has been studied by using glutathione S-transferase (GST) fusion proteins with substrate-binding domain of three bacterial PHB depolymerases, Alcaligenes faecalis, Comamonas acidovorans and Comamonas testosteroni. Analysis using immuno-gold labeling technique and transmission electron microscopy indicated that a novel GST fusion protein derived from A. Faecalis enzyme adsorbed to the surface of poly(3-hydroxybutyric acid) (P(3HB)) single crystals like other fusion proteins. Comparison of inhibiting degree of P(3HB) powder hydrolysis activity of PHB depolymerase by fusion proteins indicated that three fusion proteins bind to P(3HB) powder in the same degree. The measurement of the surface hydrophobicity of proteins suggests that the interaction of the substrate-binding domain with insoluble P(3HB) may include not only a hydrophobic effect but also molecule-specific contacts.

  20. Dynamism of PGPR in bioremediation and plant growth promotion in heavy metal contaminated soil.

    PubMed

    Patel, P R; Shaikh, S S; Sayyed, R Z

    2016-04-01

    Heavy metal contamination, particularly of cultivable lands, is a matter of concern. Bioremediation helps in reversing such contamination to certain extent. Here, we report isolation, polyphasic identification and the role of siderophore producing rhizobacteria Alcaligenes feacalis RZS2 and Pseudomonas aeruginosa RZS3 in bioremediation of heavy metal contaminated soil and plant growth promotion activity in such contaminated soil. Siderophore produced by A. feacalis RZS2 and P. aeruginosa RZS3 strains chelated various heavy metal ions like MnCl₂.4H₂O, NiCl₂.6H₂O, ZnCl₂, CuCl₂ and CoCl₂ other than FeCl₃.6H2O at batch scale. Their bioremediation potential was superior over the chemical ion chelators like EDTA and citric acid. These isolates also promoted growth of wheat and peanut seeds sown in heavy metal contaminated soil. Effective root colonizing ability of these isolates was observed in wheat and peanut plants.

  1. Light controlled reversible inversion of nanophosphor-stabilized Pickering emulsions for biphasic enantioselective biocatalysis.

    PubMed

    Chen, Zhaowei; Zhou, Li; Bing, Wei; Zhang, Zhijun; Li, Zhenhua; Ren, Jinsong; Qu, Xiaogang

    2014-05-21

    In this work, by utilizing photochromic spiropyrans conjugated upconversion nanophosphors, we have successfully prepared NIR/visible light tuned interfacially active nanoparticles for the formulation of Pickering emulsions with reversible inversion properties. By loading a model enantioselective biocatalytic active bacteria Alcaligenes faecalis ATCC 8750 in the aqueous phase, we demonstrated for the first time that the multifunctional Pickering emulsion not only highly enhanced its catalytic performance but also relieved the substrate inhibition effect. In addition, product recovery, and biocatalysts and colloid emulsifiers recycling could be easily realized based on the inversion ability of the Pickering emulsion. Most importantly, the utilization of NIR/visible light to perform the reversible inversion without any chemical auxiliaries or temperature variation showed little damage toward the biocatalysts, which was highlighted by the high catalytic efficiency and high enantioselectivity even after 10 cycles. The NIR/visible light controlled Pickering emulsion showed promising potential as a powerful technique for biocatalysis in biphasic systems.

  2. Characterization of bacterial isolates from rubber dump site and their use in biodegradation of isoprene in batch and continuous bioreactors.

    PubMed

    Srivastva, Navnita; Shukla, Awadhesh Kumar; Singh, Ram Sharan; Upadhyay, Siddh Nath; Dubey, Suresh Kumar

    2015-01-01

    Bacterial isolates from contaminated soil of a waste rubber dumping site were isolated and characterized using biochemical and molecular approaches. Isoprene degradation kinetics in batch mode (isoprene concentration: 100-1000 ppm) revealed the degradation efficiency of isolates as: Pseudomonas sp. (83%)>Alcaligenes sp. (70%)>Klebsiella sp. (68.5%). The most efficient isolate Pseudomonas sp. was finally inoculated in a specifically designed bioreactor system comprising a bioscrubber and a biofilter packed with polyurethane foam connected in series. The bioscrubber and biofilter units when operated in a series showed more than 90% removal efficiency up to the inlet loading rate (IL) of 371.1g/m(3)/h. Maximum elimination capacity (EC) of biofilter was found to be an order of magnitude greater than that for bioscrubber. Oxidative cleavage of the double bond of isoprene has been revealed through IR spectra of the leachate. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Reduction of petroleum hydrocarbons and toxicity in refinery wastewater by bioremediation.

    PubMed

    Płaza, Grazyna A; Jangid, Kamlesh; Lukasik, Krystyna; Nałecz-Jawecki, Grzegorz; Berry, Christopher J; Brigmon, Robin L

    2008-10-01

    The aim of the study was to investigate petroleum waste remediation and toxicity reduction by five bacterial strains: Ralstonia picketti SRS (BP-20), Alcaligenes piechaudii SRS (CZOR L-1B), Bacillus subtilis (I'-1a), Bacillus sp. (T-1), and Bacillus sp. (T'-1), previously isolated from petroleum-contaminated soils. Petroleum hydrocarbons were significantly degraded (91%) by the mixed bacterial cultures in 30 days (reaching up to 29% in the first 72 h). Similarly, the toxicity of the biodegraded petroleum waste decreased 3-fold after 30 days. This work shows the influence of bacteria on hydrocarbon degradation and associated toxicity, and its dependence on the specific microorganisms present. The ability of these mixed cultures to degrade hydrocarbons and reduce toxicity makes them candidates for environmental restoration applications at other hydrocarbon-contaminated environments.

  4. Enumeration and Characterization of Bacterial Colonists of a Submersed Aquatic Plant, Eurasian Watermilfoil (Myriophyllum spicatum L.)

    PubMed Central

    Chand, T.; Harris, R. F.; Andrews, J. H.

    1992-01-01

    A simple procedure for enumerating and grouping the bacterial colonists of Eurasian watermilfoil (Myriophyllum spicatum L.) is described. Colony characteristics of bacteria associated with M. spicatum were better defined and more stable on nutrient-poor, diluted nutrient broth agar than on high-nutrient media. Acinetobacter, Cytophaga, Flavobacterium, Moraxella, Pseudomonas and/or Alcaligenes, and Vibrio/Aeromonas spp., as well as two highly fastidious unidentified bacterial groups (gram-negative rods and gram-negative cocci), were associated with cultured watermilfoil during January, February, May, June, July, and August 1988. In Lake Wingra (Madison, Wis.), Micrococcus spp. and enterobacters were also associated with Eurasian watermilfoil during July, August, and October 1987. PMID:16348792

  5. Aerobic biotransformation and mineralization of 2,4,6-trinitrotoluene

    SciTech Connect

    Bae, B.H.; Autenrieth, R.L.; Bonner, J.S.

    1995-12-31

    Respirometric mineralization studies of 2,4,6-trinitrotoluene (TNT) were conducted with microorganisms isolated from a site contaminated with munitions waste in Illinois. Nine aerobic bacterial species were isolated under a carbon- and nitrogen-limited condition and tentatively identified as: one Pseudomonas species; one Enterobacter species; and seven Alcaligenes species. Experiments were performed using each of the nine organisms individually and with a consortium of all nine bacterial species. The aerobic microorganisms were cultured in a sterile nutrient solution with glucose and 20 mg/L TNT. Mineralization was determined using uniformly ring-labeled {sup 14}C-TNT in a respirometer that trapped the evolved CO{sub 2}. Biodegradation behavior was characterized based on oxygen consumption, distribution of {sup 14}C activity, and high-performance liquid chromatography (HPLC) analysis of TNT and its transformation products.

  6. Aerobic granulation of aggregating consortium X9 isolated from aerobic granules and role of cyclic di-GMP.

    PubMed

    Wan, Chunli; Yang, Xue; Lee, Duu-Jong; Wang, Xin-Yue; Yang, Qiaoli; Pan, Xiangliang

    2014-01-01

    This study monitored the granulation process of an aggregating functional consortium X9 that was consisted of Pseudomonas putida X-1, Acinetobacter sp. X-2, Alcaligenes sp. X-3 and Comamonas testosteroni X-4 in shaken reactors. The growth curve of X9 was fit using logistic model as follows y=1.49/(1+21.3*exp(-0.33x)), the maximum specific cell growth rate for X9 was 0.33 h(-1). Initially X9 consumed polysaccharides (PS) and secreted proteins (PN) to trigger granulation. Then X9 grew in biomass and formed numerous micro-granules, driven by increasing hydrophobicity of cell membranes and of accumulated extracellular polymeric substances (EPS). In later stage the intracellular cyclic diguanylate (c-di-GMP) was at high levels for inhibiting bacteria swarming motility, thereby promotion formation of large aerobic granules. The findings reported herein advise the way to accelerate granule formation and to stabilize operation in aerobic granular reactors.

  7. [Computational fluid dynamics simulation of different impeller combinations in high viscosity fermentation and its application].

    PubMed

    Dong, Shuhao; Zhu, Ping; Xu, Xiaoying; Li, Sha; Jiang, Yongxiang; Xu, Hong

    2015-07-01

    Agitator is one of the essential factors to realize high efficient fermentation for high aerobic and viscous microorganisms, and the influence of different impeller combination on the fermentation process is very important. Welan gum is a microbial exopolysaccharide produced by Alcaligenes sp. under high aerobic and high viscos conditions. Computational fluid dynamics (CFD) numerical simulation was used for analyzing the distribution of velocity, shear rate and gas holdup in the welan fermentation reactor under six different impeller combinations. The best three combinations of impellers were applied to the fermentation of welan. By analyzing the fermentation performance, the MB-4-6 combination had better effect on dissolved oxygen and velocity. The content of welan was increased by 13%. Furthermore, the viscosity of production were also increased.

  8. An adsorption-release-biodegradation system for simultaneous biodegradation of phenol and ammonium in phenol-rich wastewater.

    PubMed

    Wang, Ying; Chen, Hu; Liu, Yu-Xiang; Ren, Rui-Peng; Lv, Yong-Kang

    2016-07-01

    The feasibility of simultaneous biodegradation of phenol and ammonium in phenol-rich wastewater was evaluated in a reusable system, which contained macroporous adsorption resin and Alcaligenes faecalis strain WY-01. In the system, up to 6000mg/L phenol could be completely degraded by WY-01; meanwhile, 99.03±3.95% of ammonium was removed from the initial concentration of 384mg/L. This is the first study to show the capability of single strain in simultaneous removal of ammonium and phenol in wastewater containing such high concentrations of phenol. Moreover, the resin was regenerated during the biodegradation process without any additional manipulations, indicating the system was reusable. Furthermore, enzyme assay, gene expression patterns, HPLC-MS and gas chromatography analysis confirmed that phenol biodegradation accompanied with aerobic nitrifier denitrification process. Results imply that the reusable system provides a novel strategy for more efficient biodegradation of phenol and ammonium contained in some particular industrial wastewater.

  9. A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry.

    PubMed

    He, Yu-Cai; Ma, Cui-Luan; Xu, Jian-He; Zhou, Li

    2011-02-01

    Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.

  10. Improvement of efficiency in the enzymatic synthesis of lactulose palmitate.

    PubMed

    Bernal, Claudia; Illanes, Andres; Wilson, Lorena

    2015-04-15

    Sugar esters are considered as surfactants due to its amphiphilic balance that can lower the surface tension in oil/water mixtures. Enzymatic syntheses of these compounds are interesting both from economic and environmental considerations. A study was carried out to evaluate the effect of four solvents, temperature, substrate molar ratio, biocatalyst source, and immobilization methodology on the yield and specific productivity of lactulose palmitate monoester synthesis. Lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), immobilized in porous silica functionalized with octyl groups (adsorption immobilization, OS) and with glyoxyl-octyl groups (both adsorption and covalent immobilization, OGS), were used. The highest lactulose palmitate yields were obtained at 47 °C in acetone, for all biocatalysts, while the best lactulose:palmitic acid molar ratio differed according to the immobilization methodology, being 1:1 for AsL-OGS biocatalyst (20.7 ± 3%) and 1:3 for the others (30-50%).

  11. Exploiting metagenomic diversity for novel polyhydroxyalkanoate synthases: production of a terpolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) with a recombinant Pseudomonas putida strain.

    PubMed

    Cheema, Simrita; Bassas-Galia, Monica; Sarma, Priyangshu M; Lal, Banwari; Arias, Sagrario

    2012-01-01

    A metagenomic library of 2.1×10(6) clones was constructed using oil-contaminated soil from Gujarat (India). One of the fosmid clones, 40N22, encodes a polyhydroxyalkanoate synthase showing 76% identity with an Alcaligenes sp. synthase. The corresponding gene was expressed in Pseudomonas putida KT2440 ΔphaC1 which is impaired in PHA production. The gene conferred the recombinant strain PpKT-40N22 with the ability to produce copolymers with up to 21% in medium-chain-length content. Thus, 37% and 45% of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate), respectively were obtained when using sodium heptanoate and oleic acid as carbon sources. These 3-hydroxybutyrate-(3HB)-based polymers are of interest since they incorporate the properties of medium chain length polymers and thus increase the range of applications of PHAs.

  12. Characterization of Pseudomonas species isolated from the rhizosphere of plants grown in serozem soil, semi arid region of Uzbekistan.

    PubMed

    Egamberdiyeva, Dilfuza

    2005-07-08

    Collections of native Pseudomonas spp. are kept at the NCAM of Uzbekistan. Some of those organisms were isolated from the rhizosphere of cotton, wheat, corn, melon, alfalfa, and tomato grown in field locations within a semi-arid region of Uzbekistan. Strains used for this study were Pseudomonas alcaligenes, P. aurantiaca, P. aureofaciens, P. denitrificans, P. mendocina, P. rathonis, and P. stutzeri. Some of the pseudomonads have been characterized in this report. These strains produced enzymes, phytohormone auxin (IAA), and were antagonist against plant pathogenic fungi in in vitro experiments. Most of the strains were salt tolerant and temperature resistant. Some of the Pseudomonas spp. isolated in this study have been found to increase the growth of wheat, corn, and tomato in pot experiments.

  13. Predacious bacteria, Bdellovibrio with potential for biocontrol.

    PubMed

    Markelova, Natalia Y

    2010-11-01

    Bacteria of the genus of Bdellovibrio are highly motile Gram-negative predators of other Gram-negative bacteria causing lysis of their prey. Here we report results of studies on the interactions of Bdellovibrio with species of Alcaligenes, Campylobacter, Erwinia, Escherichia, Helicobacter, Pseudomonas, Legionella, and Shigella in agar lower, liquid media and cells attached to a surface. Helicobacter pylori was studied employing both actively growing and viable but nonculturable (VBNC) cells. The majority of the bacterial strains tested were found to be susceptible to Bdellovibrio. A significant observation was that Bdellovibrio attacked both actively growing and VBNC H. pylori, that phenomenon has never been reported. The results indicate that bdellovibrios have potential as biocontrol agents.

  14. Whole genome sequences of a free-living Pseudomonas sp. strain ML96 isolated from a freshwater Maar Lake.

    PubMed

    Li, Xiuling; Blom, Jochen; Zeng, Yonghui

    2015-12-01

    A free living freshwater Pseudomonas strain ML96 was isolated from the Huguangyan Maar Lake in southern China. Genome sequencing of strain ML96 revealed a 4.7 Mb long draft genome consisting of 47 contigs with a G+C content of 64.8%. Its 16S and 23S rRNA gene sequences were 99.8% and 99.3% identical to those of its closest relative, Pseudomonas alcaligenes NBRC 14159, respectively. ML96's genome shared 73% orthologous CDS (3256/4457) with the genome of NBRC 14159. Comparative genomics analysis provide further insight into the diversity and evolution of aquatic Pseudomonas species, which may help enhance our understanding of this both environmentally and medically important group of bacteria.

  15. Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N sub 2 O reductase

    SciTech Connect

    Lee, H.S.; Ingraham, J.L. ); Hancock, R.E.W. )

    1989-04-01

    A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N{sub 2}O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Line N{sub 2}O reductase, NosA contained copper and was only made anaerobically. In contrast to N{sub 2}O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N{sub 2}O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.

  16. Bacteriological and Shelf-Life Characteristics of Canned, Pasteurized Crab Cake Mix

    PubMed Central

    Loaharanu, Paisan; Lopez, Anthony

    1970-01-01

    The bacteriological spoilage characteristics of a canned, pasteurized crab cake mix product stored at various temperatures were investigated. A large number of bacteria, both mesophilic and psychrophilic, survived the pasteurization process. Bacillus and Micrococcus were found to predominate when the product was stored at 30 C (86 F) and 18 C (64 F), whereas Alcaligenes predominated at 2 C (36 F). The product was found to be free of Escherichia coli. Bacterial counts, trimethylamine nitrogen, volatile reducing substances, and ammonia determinations were evaluated as indices of quality for the product. Close correlation was observed between bacterial counts, volatile reducing substance values, and organoleptic tests when the product was stored at 30 C (86 F). The shelf-life of the product was approximately 6 months at 2 C (36 F), 4 days at 18 C (64 F), and 27 hr at 30 C (86 F). PMID:5422305

  17. Screening and Improving the Recombinant Nitrilases and Application in Biotransformation of Iminodiacetonitrile to Iminodiacetic Acid

    PubMed Central

    Liu, Zhi-Qiang; Baker, Peter James; Cheng, Feng; Xue, Ya-Ping; Zheng, Yu-Guo; Shen, Yin-Chu

    2013-01-01

    In this study, several nitrilase genes from phylogenetically distinct organisms were expressed and purified in E. coli in order to study their ability to mediate the biotransformation of nitriles. We identified three nitrilases: Acidovorax facilis nitrilase (AcN); Alcaligenes fecalis nitrilase (AkN); and Rhodococcus rhodochrous nitrilase (RkN), which catalyzed iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA). AcN demonstrated 8.8-fold higher activity for IDAN degradation as compared to AkN and RkN. Based on homology modeling and previously described ‘hot spot’ mutations, several AcN mutants were screened for improved activity. One mutant M3 (F168V/L201N/S192F) was identified, which demonstrates a 41% enhancement in the conversion as well as a 2.4-fold higher catalytic efficiency towards IDAN as compared to wild-type AcN. PMID:23826231

  18. Differentiation of Gram-Negative, Nonfermentative Bacteria Isolated from Biofilters on the Basis of Fatty Acid Composition, Quinone System, and Physiological Reaction Profiles

    PubMed Central

    Lipski, André; Klatte, Stefan; Bendinger, Bernd; Altendorf, Karlheinz

    1992-01-01

    Gram-negative, nonfermentative bacteria isolated from biofilters for off-gas treatment of animal-rendering-plant emissions were differentiated by whole-cell fatty acid analysis, quinone analysis, and numerical taxonomy based on their physiological reaction profiles. The last system consisted of 60 physiological tests and was arranged as a microtest system on microtitration plates. Based on fatty acid analyses, 31 isolates were separated into six clusters and five single-member clusters. The isolates of two clusters were identified as Alcaligenes faecalis and Pseudomonas diminuta. The remaining nine clusters were characterized by their fatty acid profiles, quinone systems, and physiological reaction profiles. Clusters resulting from fatty acid analyses were compared with those resulting from physiological reaction profiles. Six clusters could be confirmed this way. The efficiency of the physiological test system was increased by the prearrangement of the isolates according to their quinone type. PMID:16348724

  19. Scanning electron microscopy studies of bacterial cultures

    NASA Astrophysics Data System (ADS)

    Swinger, Tracy; Blust, Brittni; Calabrese, Joseph; Tzolov, Marian

    2012-02-01

    Scanning electron microscopy is a powerful tool to study the morphology of bacteria. We have used conventional scanning electron microscope to follow the modification of the bacterial morphology over the course of the bacterial growth cycle. The bacteria were fixed in vapors of Glutaraldehyde and ruthenium oxide applied in sequence. A gold film of about 5 nm was deposited on top of the samples to avoid charging and to enhance the contrast. We have selected two types of bacteria Alcaligenes faecalis and Kocuria rhizophila. Their development was carefully monitored and samples were taken for imaging in equal time intervals during their cultivation. These studies are supporting our efforts to develop an optical method for identification of the Gram-type of bacterial cultures.

  20. Tropospheric sources of NO(x) - Lightning and biology

    NASA Astrophysics Data System (ADS)

    Levine, J. S.; Augustsson, T. R.; Anderson, I. C.; Hoell, J. M., Jr.; Brewer, D. A.

    Laboratory tests were performed to quantify the expected NO(x) production by lightning and biological processes. Attention was focused on energy deposition by lightning and the oxygen partial pressure of soil, and one-dimensional photochemical models were defined for the tropospheric distributions of NO and HNO3 for various NO source strengths. The Lightning Facility data were compared with air samples of N2O production gathered during over 2 yr of flights through storms. Soil NO(x) productions were studied in terms of nitrification processes involving Nitrosomonas europaea and Alcaligenes faecalis bacteria, which change ammonium to nitrite and release NO and N2O as byproducts. The results indicate that atmospheric NO(x) is generated at two tropospheric levels, with lightning and soil bacteria being significant contributors.