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PubMed1994

Microbial synthesis and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Comamonas acidovorans.

Comamonas acidovorans DS-17 was isolated from activated sludge and found to produce copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) at 30 degrees C under growth-limited conditions. When 1,4-butanediol or 4-hydroxybutyric acid was used as the sole carbon source, a P(4HB) homopolymer was produced. Random copolymers of 3HB and 4HB units were produced on the addition of glucose or 3-hydroxybutyric acid to the culture solution of 4-hydroxybutyric acid. The physical properties of P(3HB-co-4HB) copolyesters with high 4HB fractions (64-100 mol%) were investigated. The copolyester films with high 4HB fractions exhibited the characteristics of a thermoplastic elastomer, and the tensile strength increased from 17 to 104 MPa as the 4HB fraction was increased from 64 to 100 mol%. The biodegradabilities of P(3HB-co-4HB) films were studied in aqueous solutions of extracellular polyhydroxybutyrate (PHB) depolymerase from Alcaligenes faecalis or of lipase from Rhizopus delemer. The erosion rate of P(3HB-co-4HB) films by PHB depolymerase decreased as the 4HB fraction in copolyester was increased from 64 to 100 mol%. In contrast, the erosion rate of films by lipase increased with the 4HB fraction.

Biodegradation, Environmental↗

PubMed1995

Sequence of the bphD gene encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid (HOP/cPDA) hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway in Comamonas testosteroni: evidence suggesting involvement of Ser112 in catalytic activity.

The nucleotide sequence of bphD, encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway of Comamonas testosteroni strain B-356, was determined. Comparison of the deduced amino-acid sequence with published sequences led to the identification of a 'lipase box', containing a consensus pentapeptide sequence GlyXaaSerXaaGly. This suggested that the mechanism of action of this enzyme may involve an Asp-Ser-His catalytic triad similar to that of classical lipases and serine hydrolases. Further biochemical and genetic evidence for the active-site involvement of Ser112 was obtained by showing that a semipurified enzyme was inhibited by PMSF, a classic inhibitor of serine hydrolases, and by site-directed Ser112-->Ala mutagenesis.

Amino Acid Sequence↗

PubMed1995

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.

Amino Acid Sequence↗

PubMed1996

Sequencing of Comamonas testosteroni strain B-356-biphenyl/chlorobiphenyl dioxygenase genes: evolutionary relationships among Gram-negative bacterial biphenyl dioxygenases.

In a previous work, all three components of Comamonas testosteroni B-356 biphenyl (BPH)/chlorobiphenyls (PCBs) dioxygenase (dox) have been purified and characterized. They include an iron-sulphur protein (ISPBPH) which is the terminal oxygenase composed of two subunits (encoded by bphA and bphE), a ferredoxin (FERBPH) encoded by bphF and a reductase (REDBPH) encoded by bphG. bphG Is not located in the neighbourhood of bphAEF in B-356. We are reporting the cloning of B-356-bphG and the sequencing of B-356-BPH dox genes. Comparative analysis of the genes provided genetic evidence showing that two BPH dox lineages have emerged in Gram-negative bacteria. The main features of the lineage that includes B-356 are the location of bphG outside the bph gene cluster and the structure of REDBPH which is very distinct from all other aryl dioxygenase-reductases.

Amino Acid Sequence↗

PubMedDate not supplied

Topology of the anion-selective porin Omp32 from Comamonas acidovorans.

Limited proteolysis experiments were performed with outer membranes from Comamonas acidovorans to probe the topology of its major protein component, the anion-selective porin Omp32. Proteinase K treatment above a critical temperature of 42 degrees C cleaved the surface-exposed regions of the porin, yielding membrane-embedded fragments which were separated by SDS polyacrylamide gel electrophoresis or reversed phase chromatography. The identification of the proteinase K-sensitive sites was performed by microsequencing. This allowed us to determine six surface-exposed sites of the porin, all located in nonconserved primary structure regions. These results along with the previously determined amino acid sequence and in conjunction with some structural constraints applicable to porins allowed us to propose a chain-folding model of the Omp32 porin. The features of our model are compared with the structure of the Rhodobacter capsulatus porin, recently established by X-ray crystallography (Weiss et al., 1991) and they are used to elucidate the structural basis of the anion selectivity.

Amino Acid Sequence↗

PubMed2004

High-level expression and characterization of a highly functional Comamonas acidovorans xanthine dehydrogenase in Pseudomonas aeruginosa.

An improved procedure is described for the high-level expression of Comamonas acidovorans XDH in Pseudomonas aeruginosa PAO1-LAC. The level of functional expression (56 mg protein/L culture) is found to be 7-fold higher than that observed in Escherichia coli and 30-fold higher than that induced in C. acidovorans. Co-expression of the xdhC gene is required for maximal level of functional expression. Comparison of purified preparations of XDH expressed in the absence of xdhC (XDH(AB)) with that expressed in its presence (XDH(ABC)) shows the increased level of activity due to the level of Mo incorporation. The Fe and FAD contents of expressed enzymes are independent of xdhC co-expression. Electron paramagnetic resonance spectroscopy, circular dichroism spectroscopy, metal analysis, and kinetic properties of recombinant purified XDH(ABC) are identical with those exhibited by the native enzyme. This expression system should serve as a valuable tool for further biophysical and mechanistic investigations of xanthine dehydrogenase by site-directed mutagenesis. A method is also described to evaluate the suitability of P. aeruginosa and other organisms as potential expression hosts for five different sources of xdh genes.

Animals↗

PubMed1991

Ocular infections associated with Comamonas acidovorans.

Comamonas acidovorans (Pseudomonas acidovorans) is a ubiquitous gram-negative rod. Although generally considered nonpathogenic, we found C. acidovorans to be associated with six cases of ocular infections. The organism was the only isolate in three cases, whereas an association of other organisms was present in three cases. The multiple resistance patterns of these strains to antibiotic susceptibility testing emphasizes the need for culturing ocular infections. We recommend the identification and susceptibility testing of all ocular gram-negative rod isolates.

Adolescent↗

PubMedDate not supplied

Improved production of poly(4-hydroxybutyrate) by Comamonas acidovorans and its freeze-fracture morphology.

Production of poly(4-hydroxybutyrate) [P(4HB)] by Comamonas acidovorans JCM10181 was studied by introducing additional copies of its PHA synthase gene and the beta-ketothiolase gene. A multi-copy-number broad-host-range plasmid vector, pJRD215, was modified to contain the strong hybrid trc promoter in order to express these genes in the wild-type C. acidovorans. Increased copy-number of genes resulted in significant increase in the activities of corresponding enzymes, which could further be increased by inducing with isopropyl-beta-D-thiogalactopyranoside (IPTG), indicating that the expression is under the transcriptional control of the trc promoter. P(4HB) biosynthesis in the recombinant C. acidovorans increased 2-fold to constitute more than 60 wt% of the dry cell weight. No significant decrease in the number-average molecular weights of P(4HB) in the recombinant strain was observed when compared with that of the wild-type. Freeze-fracture electron microscopy of intracellular P(4HB) granules revealed almost similar fracture morphology to the well-known mushroom-type deformation shown by polyhydroxyalkanoates with medium-chain-length monomers.

Acetyl-CoA C-Acyltransferase↗

PubMedDate not supplied

Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp.

The PHA-degrading isolate, strain P37C, was enriched from residential compost for its ability to hydrolyze the medium chain length PHA, poly(beta-hydroxyoctanoate) (PHO). It was subsequently found to grow on a wide range of PHAs, including both short chain length and medium chain length PHAs. The isolate was identified as belonging to the genus Comamonas. Strain P37C formed clear zones on poly(beta-hydroxybutyrate) (PHB), (PHO) and poly(beta-hydroxyphenylvalerate) (PHPV) overlay plates. PHA clear zone tubes were prepared using seven different kinds of PHAs, ranging from PHB with four-carbon repeating units, to poly(beta-hydroxyoctanoate-co-beta-hydroxyundecanoate) (PHOU) with 8- and 11-carbon repeating units. There was a direct correlation between PHA side chain length and rate of hydrolysis of the PHAs. A series of PHOUs containing varying percentages of unsaturated bonds were used to make a series of epoxidized PHOUs (PHOEs) with varying percentages of epoxy functions. Results of clear zone tube assays showed that these functionalized PHAs were all biodegradable by strain P37C, and there was no apparent correlation between rate of biodegradation and the proportion of functional groups in the PHAs. Biodegradability of these PHAs was verified using respirometry and enzyme assays. Cell-free supernatants containing activity toward PHAs were prepared, and strain P37C was shown to synthesize at least two distinct PHA depolymerases for the hydrolysis of SCL and MCL PHAs.

Carboxylic Ester Hydrolases↗

PubMed1999

Beta-lactamases and outer membrane investigations in beta-lactam-resistant Comamonas acidovorans strains.

Imipenem-induced beta-lactamase (level of expression, specific activity and kinetic parameters (Vmax and Km) in response to nitrocefin) and outer membrane proteins (OMPs) (hydrophobicity, permeability and electrophoretic pattern) were characterized in, one beta-lactam sensitive (PAC-9), one resistant (PAC-1) and two resistant laboratory mutants (PAC-9M, PAC-9M2) of Comamonas acidovorans strains. Beta-lactamases from both mutant strains showed different Vmax values compared to the parental strains. Beta-lactam resistance was found to be associated in PAC-1 with inducible beta-lactamase production and OMP alteration by the appearance of a 102-KDa protein. Moreover, PAC-1 was less permeable to nitrocefin than PAC-9. These data indicate that C. acidovorans resistance to beta-lactam resulted from synergy between beta-lactamase and OMP alterations.

Aminoglycosides↗

PubMed1999

Comamonas acidovorans strain MC1: a new isolate capable of degrading the chiral herbicides dichlorprop and mecoprop and the herbicides 2,4-D and MCPA.

A gram-negative prototrophic bacterial species, strain MC1, was isolated from the vicinity of herbicide-contaminated building rubble and identified by 16S rDNA sequence analysis, its physiological properties, GC content, and fatty acid composition as Comamonas acidovorans. This strain displays activity for the productive degradation of the two enantiomers of dichlorprop [(RS)-2-(2,4-dichlorophenoxy-)propionate; (RS)-2,4-DP] and mecoprop [(RS)-2-(4-chloro-2-methyl-) phenoxypropionate; (RS)-MCPP] in addition phenoxyacetate herbicides, i.e. 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), and various chlorophenols were utilized. Rates amounted to 1.2 mmoles/h g dry mass (2,4-D) and 2.7 mmoles/h g dry mass [(RS)-2,4-DP]. Degradation of (RS)-2,4-DP was not inhibited up to concentrations of 500 mg/l, nor of 2,4-D up to 200 mg/l. The optimum pH value of (RS)-2,4-DP degradation was around 8. The application of respective primers for PCR amplification revealed the presence of tfdB and tfdC genes.

2,4-Dichlorophenoxyacetic Acid↗

PubMedDate not supplied

A new Comamonas testosteroni steroid-inducible gene: cloning and sequence analysis.

Comamonas testosteroni can grow on a variety of steroid compounds as the sole carbon and energy source. In a previous study, we cloned and sequenced the testosterone-inducible betahsd gene from C. testosteroni (Genti-Raimondi, S., Tolmasky, M., Patrito, L., Flury, A. and Actis, L., Molecular cloning and expression of the beta-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni. Gene, 1991, 105, 43-49.). Herein we report the cloning and characterization of another steroid-inducible gene (stdC), located 2400 bp upstream of betahsd. Nucleotide sequencing of a region encompassing the stdC gene revealed an open reading frame 546 bp long including the stop codon TGA with significant similarity to the orf4, orf1 and orf4 of unknown function described in the polyhydroxyalkanoic acid (PHA) cluster of Chromatium vinosum, Rhizobium meliloti and Thiocystis violacea, respectively. The aminoacid sequence deduced from the nucleotide sequence predicts a putative protein of 181 amino acids with a molecular weight of 20715 Da. Northern blot experiments indicate that the stdC gene was transcribed as a monocistronic mRNA with an apparent molecular size of 670 nt. The stdC transcript was abundant in C. testosteroni cells grown with different steroid carbon sources harvested in the exponential phase and was found to be under catabolite repression.

Amino Acid Sequence↗

PubMed2000

Crystal structure of Omp32, the anion-selective porin from Comamonas acidovorans, in complex with a periplasmic peptide at 2.1 A resolution.

BACKGROUND: Porins provide diffusion channels for salts and small organic molecules in the outer membrane of bacteria. In OmpF from Escherichia coli and related porins, an electrostatic field across the channel and a potential, originating from a surplus of negative charges, create moderate cation selectivity. Here, we investigate the strongly anion-selective porin Omp32 from Comamonas acidovorans, which is closely homologous to the porins of pathogenic Bordetella and Neisseria species. RESULTS: The crystal structure of Omp32 was determined to a resolution of 2.1 A using single isomorphous replacement with anomalous scattering (SIRAS). The porin consists of a 16-stranded beta barrel with eight external loops and seven periplasmic turns. Loops 3 and 8, together with a protrusion located within beta-strand 2, narrow the cross-section of the pore considerably. Arginine residues create a charge filter in the constriction zone and a positive surface potential at the external and periplasmic faces. One sulfate ion was bound to Arg38 in the channel constriction zone. A peptide of 5.8 kDa appeared bound to Omp32 in a 1:1 stoichiometry on the periplasmic side close to the symmetry axis of the trimer. Eight amino acids of this peptide could be identified, revealing specific interactions with beta-strand 1 of the porin. CONCLUSIONS: The Omp32 structure explains the strong anion selectivity of this porin. Selectivity is conferred by a positive potential, which is not attenuated by negative charges inside the channel, and by an extremely narrow constriction zone. Moreover, Omp32 represents the anchor molecule for a peptide which is homologous to proteins that link the outer membrane to the cell wall peptidoglycan.

Amino Acid Sequence↗

PubMed1999

Purification and properties of culture-broth-secreted esterase from the polyurethane degrader Comamonas acidovorans TB-35.

The polyester-polyurethane (PUR)-degrading bacterium Comamonas acidovorans TB-35 produces two kinds of esterases, one cell-bound esterase (PUR esterase) and the other secreted in the culture broth (CBS esterase). In this study, the CBS esterase and the two recombinant esterases were purified. Identification of the physical and biochemical properties of the CBS and PUR esterases revealed that they have the same polypeptide from one gene. This finding was supported by the observation that Escherichia coli harboring the PUR esterase gene also produced two kinds of esterases. Though the PUR esterase degraded PUR and poly(diethylene glycol adipate), the soft segment of the PUR, the CBS esterase degraded only poly(diethylene glycol adipate). Furthermore, the hydrophobicity of the CBS esterase was lower than that of the PUR esterase. As the PUR esterase has been previously indicated to possess a PUR-binding domain, it was assumed that structural change around the PUR-binding domain of the CBS esterase was responsible for its inability to degrade PUR.

Journal Article↗

PubMed1995

Characterization of the interaction between PQQ and heme c in the quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni.

Quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni (QH-EDH) contains two cofactors, 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and heme c. Since previous studies on the kinetics of this enzyme suggested that both participate in electron transfer, spectroscopic investigations were performed of the oxidized and reduced holo- and apoenzyme (without PQQ but with heme c) to reveal the nature of the interaction between the two redox centers. From this it appears that the properties of the heme in the enzyme are affected by the presence of PQQ, as judged from the shift of the maxima in the ultraviolet/visible absorption spectra of the heme moiety in both reduced and oxidized QH-EDH and the 60-mV increase of the heme midpoint redox potential caused by PQQ addition. Also 1H-NMR spectroscopy was indicative for interaction since binding of PQQ induced shifts in the resonances of the methyl groups of the porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme. On the other hand, resonance Raman spectra of the heme in the different enzyme forms were nearly similar. These results suggest that a major effect of PQQ binding to apo-QH-EDH is a rotation of the methionine ligand of heme c. Since no intermediate 1H-NMR spectra were observed upon titration of apoenzyme with PQQ, apparently no exchange occurs of PQQ between (oxidized) holo- and apoenzyme at the NMR time scale and at that of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗

PubMed1995

Insights into the catalytic mechanism and active-site environment of Comamonas testosteroni delta 5-3-ketosteroid isomerase as revealed by site-directed mutagenesis of the catalytic base aspartate-38.

Delta 5-3-Ketosteroid isomerase (KSI) of Comamonas testosteroni catalyzes the isomerization of a wide variety of delta 5(6) and delta 5(10) steroids through the formation of an enzyme bound dienol(ate) intermediate. Asp-38 has been strongly implicated in catalysis, apparently serving as a proton shuttle. In this paper the results of a detailed kinetic characterization of the KSI mutants D38E and D38H are presented. Both mutants retain significant activity, with kcat and kcat/Km values 10(3)-10(4) times greater than the D38N mutant. The results allow for a qualitative assessment of the sensitivity of the enzymes catalytic capability to the positioning and chemical nature of the catalytic base. The near identity of the ratios of kcat5-AND/kcat5,10-EST is most easily explained by a mechanism in which the second chemical step, reketonization of the intermediate dienol(ate), is not significantly rate determining. The pH dependence of the rate constants for the D38E and D38H mutants is found to be consistent with earlier proposals that an as yet unidentified titrating functional group is present in the active site and indicates that the electrostatic environment of residue 38 is hydrophobic and positively charged.

Androstenedione↗

PubMed1994

Extent of proton transfer in the transition states of the reaction catalyzed by the delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: site-specific replacement of the active site base, aspartate 38, by the weaker base alanine-3-sulfinate.

Previous studies of the mechanism of the steroid isomerase of Comamonas (Pseudomonas) testosteroni have identified aspartate 38 as the proton porter which transfers the substrate's 4 beta proton to the 6 beta position of the product. Consequently, aspartate 38 functions as a base in the deprotonation of the substrate to form a dienol or dienolate intermediate, which then undergoes reprotonation from protonated aspartate 38 at C-6 beta to give the product. We have tried to characterize the transition states for the proton transfers by altering the pKa' of aspartate 38 and then determining the effect of the alteration on the kinetics of the enzyme. Alteration of the pKa' was accomplished by replacement of the carboxyl carbon of aspartate 38 by sulfur, a change which converts the carboxylate group to the much less basic sulfinate group. Employing Brønsted catalysis theory as applied to the individual steps of the isomerase mechanism, we find that in the enolization step of the reaction proton transfer to aspartate 38 is well advanced in the transition state. In the subsequent ketonization step, proton transfer from aspartate 38 has barely started when that transition state is reached. A series of mutant KSIs with alternative bases at position 38 have been constructed using a combination of site-directed mutagenesis and chemical modification: Asp-38 to Glu (D38E), His (D38H), and S-(carboxymethyl)cysteine (D38CMC). While the D38H and D38E mutants both retain significant isomerase activity, D38CMC is essentially inert. From the results of kinetic experiments it is possible to get a qualitative idea of the sensitivity of the enzyme's catalytic ability to the location of the base responsible for proton transfer.

Aspartic Acid↗

PubMedDate not supplied

Cloning and sequence analysis of a catechol 2,3-dioxygenase gene from the nitrobenzene-degrading strain Comamonas sp JS765.

Comamonas sp strain JS765 utilizes nitrobenzene as a carbon and nitrogen source. The initial attack on nitrobenzene is carried out by nitrobenzene 1,2-dioxygenase, which converts nitrobenzene to an unstable nitrohydrodiol that spontaneously decomposes to form catechol and nitrite. Catechol is then degraded via a meta cleavage pathway. We now report the cloning of a DNA fragment carrying a catechol 2,3-dioxygenase gene from JS765. Nucleotide sequence analysis revealed three open reading frames (ORFs) predicted to encode proteins of 33.6, 13.0, and 35.0 kDa. Homology searches of the deduced amino acid sequences of three proteins suggested that ORF1 encodes a LysR-type transcriptional regulator, ORF2 encodes a XylT-type ferredoxin, and ORF3 encodes a catechol 2,3-dioxygenase. The putative regulatory gene, designated cdoR, is divergently transcribed from the ferredoxin and catechol dioxygenase genes, cdoT and cdoE, respectively. The catechol 2,3-dioxygenase is most similar in amino acid sequence to the 1.2.C subfamily of extradiol dioxygenases which include 3-methylcatechol 2,3-dioxygenase from the aniline- and toluidine-degrading Pseudomonas putida UCC2, TbuE from the toluene monooxygenase pathway of Pseudomonas pickettii PKO1 and catechol 2,3-dioxygenase II from the TOL plasmid pWW15. The substrate range of the catechol 2,3-dioxygenase produced by the recombinant E. coli strains was very similar to that of the enzyme present in nitrobenzene-grown JS765, suggesting that we have cloned the catechol 2,3-dioxygenase gene required for nitrobenzene degradation.

Amino Acid Sequence↗