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Microbiology (Reading, England) Dec 2000The potential for natural genetic transformation among the seven genomovars (gvs) of Pseudomonas stutzeri was investigated. Of the 12 strains originating from a variety...
The potential for natural genetic transformation among the seven genomovars (gvs) of Pseudomonas stutzeri was investigated. Of the 12 strains originating from a variety of environments, six strains (50%) from five gvs were competent for DNA uptake (Rif(R) marker). The transformation frequencies varied over more than three orders of magnitude. With three highly transformable strains (ATCC 17587, ATCC 17641, JM300) from two gvs and all other strains as DNA donors, sexual isolation from other pseudomonad species (Pseudomonas alcaligenes, Pseudomonas mendocina) and also from other P. stutzeri gvs was observed (i.e. heterogamic transformation was reduced). For ATCC 17587 (gv 2) and ATCC 17641 (gv 8), heterogamic transformation was up to two and three orders of magnitude lower with other P. stutzeri gv and the other species employed, respectively, than in homogamic transformations. Interestingly, whereas with ATCC 17587 and ATCC 17641 heterogamic transformation with donors of the same gv was as high as homogamic transformation, JM300 (gv 8) was sexually isolated from its nearest relative (ATCC 17641). Also, sexual isolation of JM300 from other P. stutzeri gvs was most pronounced among the recipients tested, in some cases reaching the highest levels found with the other species as DNA donors (reduction of heterogamic transformation by 4000-fold). Results obtained here from nucleotide sequence analysis of part (422 nt) of the gene for the RNA polymerase ss subunit (rpoB) from various strains indicated that sexual isolation of ATCC 17641 increased with nucleotide sequence divergence. Implications of the observed great heterogeneity in transformability, competence levels and sexual isolation among strains are discussed with regard to the evolution of P. stutzeri.
Topics: Conjugation, Genetic; DNA, Bacterial; DNA-Directed RNA Polymerases; Gene Transfer, Horizontal; Genotype; Humans; Molecular Sequence Data; Phylogeny; Pseudomonas; Sequence Analysis, DNA; Transformation, Bacterial
PubMed: 11101666
DOI: 10.1099/00221287-146-12-3081 -
Applied Microbiology and Biotechnology Sep 2000Bacterial physiological responses to toluene exposure were investigated in five reference pseudomonad strains that express different toluene degradation pathways:...
Bacterial physiological responses to toluene exposure were investigated in five reference pseudomonad strains that express different toluene degradation pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. The intact phospholipids of these archetypes, grown with and without toluene, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. All strains showed significant changes in phospholipid content and composition as an adaptive response to toluene exposure, as well as considerable diversity in response mechanisms. For example, the phospholipid content of toluene-grown PKO1, F1, and KR1 were 10.9-34.7% of that found in succinate-grown strains, while the phospholipid content of mt-2 and G4 increased by 56% and 94%, respectively, when grown on toluene. In addition, PKO1, F1, and mt-2 responded to the presence of toluene by synthesizing more phosphatidylglycerol, whereas G4 and KR1 synthesized phospholipids with polyunsaturated fatty acids (C18:2) on one or both of the sn-2 positions. These changes in phospholipid composition and concentration probably reflect the sensitivity and degree of tolerance of these strains to toluene, and suggest that different mechanisms are utilized by dissimilar bacteria to maintain optimal lipid ordering in the presence of such environmental pollutants.
Topics: Burkholderia; Burkholderia cepacia; Cell Membrane; Culture Media; Phospholipids; Pseudomonas; Pseudomonas putida; Succinates; Toluene
PubMed: 11030576
DOI: 10.1007/s002530000389 -
Microbiology (Reading, England) Oct 2000Phylogenetic analysis of the genus Pseudomonas: was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas: (a...
Phylogenetic analysis of the genus Pseudomonas: was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas: (a total of 125 strains). Pseudomonas: strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the 'P: aeruginosa complex', which included P: aeruginosa, P: alcaligenes, P: citronellolis, P: mendocina, P: oleovorans and P: pseudoalcaligenes, and the 'P: stutzeri complex', which included P: balearica and P: stutzeri. IGC II was further split into three subclusters that were designated the 'P: putida complex', the 'P: syringae complex' and the 'P: fluorescens complex'. The 'P: putida complex' included P: putida and P: fulva. The 'P: syringae complex' was the cluster of phytopathogens including P: amygdali, P: caricapapayae, P: cichorii, P: ficuserectae, P: viridiflava and the pathovars of P. savastanoi and P. syringae. The 'P. fluorescens complex' was further divided into two subpopulations, the 'P. fluorescens lineage' and the 'P. chlororaphis lineage'. The 'P. fluorescens lineage' contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the 'P. chlororaphis lineage' included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the 'P. fluorescens complex'.
Topics: Base Sequence; DNA Gyrase; DNA Topoisomerases, Type II; DNA-Directed RNA Polymerases; Genes, Bacterial; Molecular Sequence Data; Phylogeny; Polymerase Chain Reaction; Pseudomonas; Sigma Factor
PubMed: 11021915
DOI: 10.1099/00221287-146-10-2385 -
Applied and Environmental Microbiology Sep 2000The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by...
The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.
Topics: Alkanes; Benzene; Biodegradation, Environmental; Chemotaxis; Culture Media; Environmental Pollutants; Hydrocarbons, Aromatic; Hydrocarbons, Chlorinated; Pseudomonas putida; Toluene; Trichloroethylene; Water Pollutants, Chemical
PubMed: 10966434
DOI: 10.1128/AEM.66.9.4098-4104.2000 -
Applied and Environmental Microbiology May 2000Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the...
Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0. 01 to 0.33 micromol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified.
Topics: Alkenes; Cloning, Molecular; Epoxy Compounds; Escherichia coli; Isoenzymes; Kinetics; Oxygenases; Pseudomonas; Recombinant Proteins; Substrate Specificity
PubMed: 10788354
DOI: 10.1128/AEM.66.5.1877-1882.2000 -
FEMS Microbiology Ecology Mar 2000A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species...
A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.
PubMed: 10719200
DOI: 10.1111/j.1574-6941.2000.tb00684.x -
The Biochemical Journal Jun 1999A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield...
A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of approximately 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of approximately 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, pimelic acid-utilizing A) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.
Topics: Acyl Coenzyme A; Adenosine Triphosphate; Amino Acid Sequence; Base Sequence; Chromatography, High Pressure Liquid; Cloning, Molecular; Coenzyme A Ligases; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Molecular Weight; Pimelic Acids; Pseudomonas; Recombinant Proteins; Restriction Mapping; Sequence Analysis; Sequence Homology, Amino Acid; Spectrum Analysis; Substrate Specificity
PubMed: 10359666
DOI: No ID Found -
Applied and Environmental Microbiology Mar 1998This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was...
This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a care painting facility as the inoculum and Solvesso100 as the sole carbon source. The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides. Two significant shifts in the bacterial community structure could be demonstrated. The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria. A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor. The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined. Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor. In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis. Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.
Topics: Bacteria; Biodegradation, Environmental; Bioreactors; Fermentation; In Situ Hybridization, Fluorescence; Oligonucleotide Probes
PubMed: 9501433
DOI: 10.1128/AEM.64.3.930-939.1998 -
Applied and Environmental Microbiology Jan 1998We assayed the tolerance to solvents of three toluene-degrading Pseudomonas putida strains and Pseudomonas mendocina KR1 in liquid and soil systems. P. putida DOT-T1...
We assayed the tolerance to solvents of three toluene-degrading Pseudomonas putida strains and Pseudomonas mendocina KR1 in liquid and soil systems. P. putida DOT-T1 tolerated concentrations of heptane, propylbenzene, octanol, and toluene of at least 10% (vol/vol), while P. putida F1 and EEZ15 grew well in the presence of 1% (vol/vol) propylbenzene or 10% (vol/vol) heptane, but not in the presence of similar concentrations of octanol or toluene. P. mendocina KR1 grew only in the presence of heptane. All three P. putida strains were able to become established in a fluvisol soil from the Granada, Spain, area, whereas P. mendocina KR1 did not survive in this soil. The tolerance to organic solvents of all three P. putida strains was therefore assayed in soil. The addition to soil of 10% (vol/wt) heptane or 10% (vol/wt) propylbenzene did not affect the survival of the three P. putida strains. However, the addition of 10% (vol/wt) toluene led to an immediate decrease of several log units in the number of CFU per gram of soil for all of the strains, although P. putida F1 and DOT-T1 subsequently recovered. This recovery was influenced by the humidity of the soil and the incubation temperature. P. putida DOT-T1 recovered from the shock faster than P. putida F1; this allowed the former strain to become established at higher densities in polluted sites into which both strains had been introduced.
Topics: Benzene Derivatives; Biodegradation, Environmental; Colony Count, Microbial; Culture Media; DNA Transposable Elements; Heptanes; Mutagenesis, Insertional; Octanols; Pseudomonas; Pseudomonas putida; Soil Microbiology; Solvents; Spain; Toluene
PubMed: 9435060
DOI: 10.1128/AEM.64.1.38-42.1998 -
Bioscience, Biotechnology, and... Mar 1997meta-Cleavage dioxygenase genes were isolated from the benzoate-assimilating bacteria Pseudomonas stutzeri A401 and Pseudomonas mendocina KC37. The gene products had the...
meta-Cleavage dioxygenase genes were isolated from the benzoate-assimilating bacteria Pseudomonas stutzeri A401 and Pseudomonas mendocina KC37. The gene products had the enzymatic activity of 2,3-dihydroxy-biphenyl-1,2-dioxygenase. Phylogenetic analysis based on the alignment of amino acid sequences suggests that these enzymes are distantly related to other meta-cleavage enzymes and may be members of a new family of extradiol dioxygenases.
Topics: Amino Acid Sequence; Benzoates; Dioxygenases; Gene Expression Regulation, Enzymologic; Molecular Sequence Data; Oxygenases; Phylogeny; Polymerase Chain Reaction; Pseudomonas; Restriction Mapping; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 9095556
DOI: 10.1271/bbb.61.530