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Grazing Pressure by a Bacterivorous Flagellate Reverses the Relative Abundance of Comamonas acidovorans PX54 and Vibrio Strain CB5 in Chemostat Cocultures

The response of the bacterial strains Comamonas acidovorans PX54 (beta subclass of the class Proteobacteria) and Vibrio strain CB5 (gamma subclass of the class Proteobacteria) to grazing by the bacterivorous flagellate Ochromonas sp. was examined in one-stage chemostat experiments under conditions of low growth rates with a complex carbon source. The two bacterial strains were cultured together; they were cultured without flagellates in the first phase of the experiments and in the presence of the flagellates in the second phase. Monoclonal and polyclonal antibodies were used to determine the numbers and sizes of C. acidovorans PX54 and Vibrio strain CB5 cells. The flagellates caused strong changes in total bacterial cell numbers, in the relative abundances of the individual bacterial strains, and in bacterial cell size distribution. Vibrio strain CB5 dominated the total bacterial cell numbers during the flagellate-free phase of the experiments with a relative abundance of 93%, but this declined to 33% after inoculation with the flagellate. In contrast to Vibrio strain CB5, C. acidovorans PX54 responded to grazing with a strong expansion of cell length distribution toward large, filamentous cells. These changes in cell morphology resulted in a high percentage of inedible cells in the C. acidovorans PX54 population but not in the Vibrio strain CB5 population, which caused the observed change in the relative abundances of the strains. Batch culture experiments without the flagellate demonstrated that the elongation of C. acidovorans PX54 cells was dependent on their growth rate. This indicates that the occurrence of filamentous C. acidovorans PX54 cells is not a direct response to chemical stimuli released by the flagellates but rather a response to increased growth rates due to flagellate grazing.

Journal Article↗

Genetic analysis of Comamonas acidovorans polyhydroxyalkanoate synthase and factors affecting the incorporation of 4-hydroxybutyrate monomer.

The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaCCa) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp beta-ketothiolase gene (phaACa). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaACa. The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB-4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism's PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.

Acetyl-CoA C-Acyltransferase↗

4-Toluene sulfonate methyl-monooxygenase from Comamonas testosteroni T-2: purification and some properties of the oxygenase component.

Comamonas testosteroni T-2 synthesizes an inducible enzyme system that oxygenates 4-toluene sulfontate (TS) to 4-sulfobenzyl alcohol when grown in TS-salts medium. We purified this TS methyl-monooxygenase system (TSMOS) and found it to consist of two components. A monomeric, iron-sulfur flavoprotein (component B), which has been shown to act as a reductase in the 4-sulfobenzoate dioxygenase system of this organism (H. H. Locher, T. Leisinger, and A. M. Cook, Biochem. J. 274:833-842, 1991), carried electrons from NADH to component M, an oxygenase. This oxygenase had the UV-visible spectral characteristics of an iron-sulfur protein. Mrs of about 152,000 for the native oxygenase and of 43,000 under denaturing conditions indicated a homotri- or homotetrameric enzyme, whose N-terminal amino acids and amino acid composition were determined. The activity of the purified enzyme was enhanced about fivefold by the addition of Fe2+. In the presence of O2 and NADH, components B and M together catalyzed the stoichiometric transformation of TS or p-toluate to the corresponding alcohol. The reaction was confirmed as oxygenation of the methyl group by observation of an oxygen atom from 18O2 in carboxybenzyl alcohol. The substrate range of TSMOS included carboxylated analogs of TS (p- and m-toluates and 4-ethylbenzoate), whereas p-xylene, toluene, and p-cresol were not substrates. TSMOS also catalyzed demethylation; 4-methoxybenzoate was transformed to 4-hydroxybenzoate and formaldehyde.

Amino Acids↗

Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins.

Amino Acid Sequence↗

Uptake of 4-toluene sulfonate by Comamonas testosteroni T-2.

The mechanism of transport of the xenobiotic 4-toluene sulfonate (TS) in Comamonas testosteroni T-2 was investigated. Rapid uptake of TS was observed only in cells grown with TS or 4-methylbenzoate as a carbon and energy source. Initial uptake rates under aerobic conditions showed substrate saturation kinetics, with an apparent affinity constant (Kt) of 88 microM and a maximal velocity (Vmax) of 26.5 nmol/min/mg of protein. Uptake of TS was inhibited completely by uncouplers and only marginally by ATPase inhibitors and the phosphate analogs arsenate and vanadate. TS uptake was also studied under anaerobic conditions, which prevented intracellular TS metabolism. TS was accumulated under anaerobic conditions in TS-grown cells upon imposition of an artificial transmembrane pH gradient (delta pH, inside alkaline). Uptake of TS was inhibited by structurally related methylated and chlorinated benzenesulfonates and benzoates. The results provide evidence that the first step in the degradation of TS by C. testosteroni T-2 is uptake by an inducible secondary proton symport system.

Aerobiosis↗

Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2: purification and some properties of the oxygenase component.

Comamonas testosteroni T-2, grown in terephthalate (TER)-salts medium, synthesizes inducible enzymes that convert TER to (1R,2S)-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD) and protocatechuate (PC). Anion-exchange chromatography of cell extracts yielded two sets of fractions, R and Z, that were necessary for oxygenation of TER to DCD; we termed this activity the TER dioxygenase system (TERDOS). An NAD(+)-dependent DCD dehydrogenase, which converted DCD to PC, overlapped all fractions R. No significant purification from fraction R, which contained an NADH-dependent reductase function(s) of TERDOS, was attained. Fraction Z, at the end of the gradient, contained essentially one protein, which was further purified by hydrophobic interaction chromatography. This component, Z, had the UV-visible spectrum and electron paramagnetic resonance characteristics of a Rieske [2Fe-2S] protein and was considered to be the oxygenase. M(r)s of about 126,000 for oxygenase Z under native conditions were observed. Oxygenase Z consisted of two subunits, alpha and beta, with M(r)s of 49,000 and 18,000, respectively, under denaturing conditions. We presume that this oxygenase has an alpha 2 beta 2 structure. The sequences of the N-terminal amino acids of each subunit were determined. The activity of the purified enzyme was enhanced about fivefold by addition of Fe2+. In the presence of O2, NADH, and fraction R, component Z catalyzed the stoichiometric transformation of TER to PC, with the intermediate formation of DCD. The reaction was confirmed as a dioxygenation when we observed incorporation of two oxygen atoms from 18O2 into PC. The substrate range of TERDOS appeared to be narrow; apart from TER, only 2,5-dicarboxypyridine and 1,4-dicarboxynaphthalene (of 11 compounds tested) were converted to a product.

Biodegradation, Environmental↗

Purification and characterization of the Comamonas testosteroni B-356 biphenyl dioxygenase components.

In this report, we describe some of the characteristics of the Comamonas testosteroni B-356 biphenyl (BPH)-chlorobiphenyl dioxygenase system, which includes the terminal oxygenase, an iron-sulfur protein (ISPBPH) made up of an alpha subunit (51 kDa) and a beta subunit (22 kDa) encoded by bphA and bphE, respectively; a ferredoxin (FERBPH; 12 kDa) encoded by bphF; and a ferredoxin reductase (REDBPH; 43 kDa) encoded by bphG. ISPBPH subunits were purified from B-356 cells grown on BPH. Since highly purified FERBPH and REDBPH were difficult to obtain from strain B-356, these two components were purified from recombinant Escherichia coli strains by using the His tag purification system. These His-tagged fusion proteins were shown to support BPH 2,3-dioxygenase activity in vitro when added to preparations of ISPBPH in the presence of NADH. FERBPH and REDBPH are thought to pass electrons from NADH to ISPBPH, which then activates molecular oxygen for insertion into the aromatic substrate. The reductase was found to contain approximately 1 mol of flavin adenine dinucleotide per mol of protein and was specific for NADH as an electron donor. The ferredoxin was found to contain a Rieske-type [2Fe-2S] center (epsilon 460, 7,455 M-1 cm-1) which was readily lost from the protein during purification and storage. In the presence of REDBPH and FERBPH, ISPBPH was able to convert BPH into both 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dihydroxybiphenyl. The significance of this observation is discussed.

Amino Acid Sequence↗

Comamonas testosteroni 3-ketosteroid-delta 4(5 alpha)-dehydrogenase: gene and protein characterization.

Comamonas testosteroni delta 4(5 alpha)- and delta1-dehydrogenases [delta4(5alpha)- and delta1DH] are key enzymes in the degradation of steroids having an A:B ring fusion in a trans configuration. We previously reported the isolation of the delta1dh gene (P. Plesiat, M. Grandguillot, S. Harayama, S. Vragar, and Y. Michel Briand, J. Bacteriol. 173:7219-7227, 1991). In this study, the gene encoding delta 4(5 alpha)DH was cloned in Escherichia coli on a 16-kbp BamHI fragment by screening a genomic bank of C. testosteroni ATCC 17410 with a probe derived from delta1dh. Subcloning experiments in plasmid pUC19 mapped delta 4(5 alpha)dh immediately downstream of delta1dh. The enzyme was overexpressed 18-fold in cells of E. coli JM109 carrying a 2.5-kbp cloned fragment (plasmid pXE25). However, much higher levels of enzymatic activity (264-fold) were obtained in Pseudomonas putida KT2440, using pMMB208 as an expression vector. Studies with crude lysates of KT2440 showed that delta4(5alpha)DH exhibits higher specificity and higher activity toward delta l-androstene-3,17-dione than toward the saturated derivative 5 alpha-androstane-3,17-dione. The reaction was found to be irreversible and to use efficiently typical flavoprotein electron acceptors; optimal conditions for the enzyme activity were pH 8 and 40 degrees C. Analysis of the nucleotide sequence of the insert of pXE25 revealed an open reading frame of 1,593 bp preceded by a putative ribosome-binding site and followed by a potential transcription terminator. The amino acid sequence of the deduced peptide showed a typical flavin adenine dinucleotide-binding site in its N-terminal region, confirming the flavoproteinic structure of delta 4(5 alpha)DH. The predicted molecular mass was consistent with that of the enzyme expressed in a T7 polymerase system (60 kDa). Alignment between delta 4(5 alpha)dh and delta1dh indicated that both genes, though coding for functionally related enzymes, do not derive from a common ancestor.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

Testosterone-regulated expression of enzymes involved in steroid and aromatic hydrocarbon catabolism in Comamonas testosteroni.

The effect of testosterone as the sole carbon source on protein expression was analyzed in Comamonas testosteroni. Testosterone simultaneously induced the expression of steroid- and aromatic hydrocarbon-catabolizing enzymes and repressed one amino acid-degrading enzyme. It is suggested that steroids play a regulative role in catabolic enzyme synthesis during adaptive growth of C. testosteroni.

Amino Acid Sequence↗

Characterization of the p-toluenesulfonate operon tsaMBCD and tsaR in Comamonas testosteroni T-2.

Comamonas testosteroni T-2 uses a standard, if seldom examined, attack on an aromatic compound and oxygenates the side chain of p-toluenesulfonate (TS) (or p-toluenecarboxylate) to p-sulfobenzoate (or terephthalate) prior to complete oxidation. The expression of the first three catabolic enzymes in the pathway, the TS methyl-monooxygenase system (comprising reductase B and oxygenase M; TsaMB), p-sulfobenzyl alcohol dehydrogenase (TsaC), and p-sulfobenzaldehyde dehydrogenase (TsaD), is coregulated as regulatory unit R1 (H. R. Schlafli Oppenberg, G. Chen, T. Leisinger, and A. M. Cook, Microbiology [Reading] 141:1891-1899, 1995). The components of the oxygenase system were repurified, and the N-terminal amino acid sequences were confirmed and extended. An internal sequence of TsaM was obtained, and the identity of the [2Fe-2S] Rieske center was confirmed by electron paramagnetic resonance spectroscopy. We purified both dehydrogenases (TsaC and TsaD) and determined their molecular weights and N-terminal amino acid sequences. Oligonucleotides derived from the partial sequences of TsaM were used to identify cloned DNA from strain T-2, and about 6 kb of contiguous cloned DNA was sequenced. Regulatory unit R1 was presumed to represent a four-gene operon (tsaMBCD) which was regulated by the LysR-type regulator, TsaR, encoded by a deduced one-gene transcriptional unit. The genes for the inducible TS transport system were not at this locus. The oxygenase system was confirmed to be a class IA mononuclear iron oxygenase, and class IA can now be seen to have two evolutionary groups, the monooxygenases and the dioxygenases, though the divergence is limited to the oxygenase components. The alcohol dehydrogenase TsaC was confirmed to belong to the short-chain, zinc-independent dehydrogenases, and the aldehyde dehydrogenase TsaD was found to resemble several other aldehyde dehydrogenases. The operon and its putative regulator are compared with units of the TOL plasmid.

Alcohol Dehydrogenase↗

The regulated outer membrane protein Omp21 from Comamonas acidovorans is identified as a member of a new family of eight-stranded beta-sheet proteins by its sequence and properties.

Omp21, a minor outer membrane protein of the soil bacterium Comamonas acidovorans, was purified from a spontaneous mutant lacking a surface layer and long-chain lipopolysaccharide. Omp21 synthesis is enhanced by oxygen depletion, and the protein has a variable electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to its heat-modifiable behavior. The structural gene omp21 encodes a precursor of 204 amino acids with a putative signal peptide of 21 amino acids. Mature Omp21 is a typical outer membrane protein with a high content of beta structure as determined by infrared spectroscopy. Sequence comparisons show that it belongs to a new outer membrane protein family, characterized by eight amphipathic beta strands, which includes virulence proteins, such as the neisserial opacity proteins, Salmonella typhimurium Rck, and Yersinia enterocolitica Ail, as well as the major outer membrane proteins OmpA from Escherichia coli and OprF from Pseudomonas aeruginosa.

Amino Acid Sequence↗

Endocarditis associated with Comamonas acidovorans.

A case of endocarditis caused by Comamonas acidovorans (Pseudomonas acidovorans) in a 42-year-old intravenous-drug abuser is described. This article appears to be the first detailed report of the isolation of this organism from a systemic clinical infection and its identification as a pathogen.

Adult↗

Evidence for a periplasmic location in Comamonas terrigena of the inducible tyrosine sulfate sulfohydrolase.

The location of the inducible and constitutive forms of tyrosine sulfate sulfohydrolase in Comamonas terrigena was investigated by subjecting resting cells to osmotic shock and to treatment with lysozyme in the presence of EDTA. The bulk of this enzyme present in induced cells was released by these procedures, suggesting that the induced form is cell wall associated. The constitutive form present in noninduced cells was not released under these conditions. Evidence was also presented which suggests that SO4(2-)release by intact cells during exposure to tyrosine sulfate was primarily due to the action of the inducible form of the enzyme.

Cytoplasm↗

Identification and mapping of the gene translation products involved in the first steps of the Comamonas testosteroni B-356 biphenyl/chlorobiphenyl biodegradation pathway.

In this study, we have mapped Comamonas testosteroni B-356 genes encoding enzymes for the conversion of biphenyl and 4-chlorobiphenyl into the corresponding meta-cleavage compounds onto a 6.3-kb DNA fragment, and we have determined the subunit composition of the enzymes involved in this pathway. The various proteins encoded by this 6.3-kb DNA fragment and by subclones derived from it were overexpressed and selectively labelled using the T7 polymerase promoter system in Escherichia coli. They were then analyzed using SDS-PAGE, which allowed the encoding locus of each polypeptide to be mapped. Despite apparent dissimilarity in the congener selectivity patterns of the initial oxygenase of strain B-356 with those of Pseudomonas sp. strain LB400, the number and sizes of the polypeptides involved in the enzymatic conversion of biphenyl or 4-chlorobiphenyl into the meta-cleavage product appear to be similar in the two strains. In both strains, the bph operon encodes the following: the large (51-kDa polypeptide encoded by bphA) and the small (22-kDa polypeptide encoded by bphE) subunits of the iron sulphur protein, which is thought to interact directly with the substrate to introduce the oxygen molecule; the ferredoxin (12-kDa polypeptide encoded by bphF) involved in electron transfer from the reduced ferredoxin reductase to the oxidized iron sulphur protein; the 29-kDa polypeptide of the 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase encoded by bphB; and the 32-kDa polypeptide of the 2,3-dihydroxybiphenyl-1,2-dioxygenase encoded by bphC, which catalyzes meta-1,2 fission of the aromatic ring. A major difference between strain B-356 and strain LB400 is that the bphG gene encoding biphenyl dioxygenase ferredoxin reductase is located outside the bph gene cluster in strain B-356. Several lines of evidence indicate that bphG is absent in clones carrying the bph operon from strain B-356.

Base Sequence↗

Characterization by arbitrary primer polymerase chain reaction of polychlorinated biphenyl (PCB)-degrading strains of Comamonas testosteroni isolated from PCB-contaminated soil.

In this study, we isolated and characterized biphenyl (BP) and polychlorinated biphenyl (PCB) degrading bacterial strains found in PCB-contaminated soil from an auto manufacturing plant located in Syracuse, New York. Twenty-one BP and PCB-degrading bacteria were randomly selected to form a representative sample of the bacterial population present at the site. Of the 21 bacteria, 13 were identified as Comamonas testosteroni, constituting about 60% of the bacterial population examined. Other PCB degraders identified were Acidovorax facilis, Alcaligenes xylosoxydans, Bacillus sphericus, Hydrogenophaga pseudoflava, Pseudomonas avanae, and Rhodococcus fascians. Owing to the abundance of C. testosteroni at this site, only these isolates were further characterized for their PCB congener degradation profile, 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and genetic relatedness by polymerase chain reaction (PCR) analysis. The PCB congener degradation pattern revealed a high degree of variability among the C. testosteroni isolates. The majority of the C. testosteroni isolates tested could degrade more than 95% of the PCB congeners up to pentachlorinated biphenyl. Only four isolates could degrade more than 80% of hexachlorobiphenyl. All 12 isolates of C. testosteroni tested were able to attack 2,3,4,5,6,3',4'-heptachlorobiphenyl, indicating involvement of biphenyl 2,3-dioxygenase, while 2,3,5,6,2',3',6'-heptachlorobiphenyl was attacked by 6 strains, suggesting an oxidation reaction mediated by 3,4-dioxygenase. 2,3-Dihydroxybiphenyl 1,2-dioxygenase activity was also found to vary among the C. testosteroni isolates tested in this study. Eleven strains showed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity specific for 2,3-dihydroxybiphenyl, whereas isolate BW 169 could metabolize both 2,3-dihydroxybiphenyl and 4-methylcatechol, and isolate BW74 had the ability to metabolize all three substrates (2,3-dihydroxybiphenyl, 4-chlorocatechol, and 4-methylcatechol).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

Molecular analysis of isophthalate and terephthalate degradation by Comamonas testosteroni YZW-D.

Comamonas testosteroni YZW-D was isolated from Passaic River sediment for its ability to degrade isophthalate and terephthalate. Degradation of the two isomeric compounds proceeds via separately inducible catabolic pathways that converge at protocatechuate. Analysis of the catabolic pathways by which these two isomers are degraded demonstrated that a cis-dihydrodiol intermediate is involved in both pathways. The genes for the conversion of isophthalate and terephthalate to protocatechuate were cloned on a single fragment of genomic DNA from C. testosteroni YZW-D. The two operons were located by subcloning and mutant complementation experiments. The regions coding for the two degradative pathways were sequenced. Analysis of the nucleotide sequence for the isophthalate degradation operon located genes for a dioxygenase, a transport protein, a cis-dihydrodiol dehydrogenase, and a reductase. Analysis of the nucleotide sequence for the terephthalate degradation operon located genes for a regulatory protein, a transport protein, a dioxygenase large subunit, a dioxygenase small subunit, a cis-dihydrodiol dehydrogenase, and a reductase.

Base Sequence↗

Microbial metabolism of quinoline and related compounds. XVII. Degradation of 3-methylquinoline by Comamonas testosteroni 63.

A bacterial strain which utilizes 3-methylquinoline as sole source of carbon, nitrogen and energy was isolated from activated sludge. On the basis of its morphological and physiological characteristics, this isolate was classified as Comamonas testosteroni. Four metabolites of 3-methylquinoline degradation were isolated from the culture supernatant and identified as 3-methyl-2-oxo-1,2-dihydroquinoline, 6-hydroxy-3-methyl-2-oxo-1,2-dihydroquinoline, 5,6-dihydroxy-3-methyl-2-oxo-1,2-dihydroquinoline and 2,5,6-trihydroxy-3-methylpyridine. Based on these results, a degradation pathway for 3-methylquinoline is proposed.

Culture Media↗

Biphenyl-associated meta-cleavage dioxygenases from Comamonas testosteroni B-356.

In addition to 2,3-dihydroxybiphenyl 1,2-dioxygenase (B1,2O), biphenyl-grown cells of Comamonas testosteroni B-356 were shown to produce a catechol 2,3-dioxygenase (C2,3O). B1,2O showed strong sequence homology with B1,2Os found in other biphenyl catabolic pathways, while partial sequence analysis of the C2,3O of B-356 suggested a relationship with xylEII-encoded C2,3O. The coexistence of two meta-cleavage dioxygenases in this strain prompted a comparison between the catalytic properties of the two enzymes. C2,3O has a much broader substrate specificity than native or His-tagged B1,2O: both enzymes were inhibited by chlorocatechols, but B1,2O was more sensitive than C2,3O. The results are discussed in terms of the physiological implications of interaction between metabolites from the lower biphenyl-chlorobiphenyl pathway and enzymes of the upper pathway.

Amino Acid Sequence↗