The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.

Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.

Comamonas testosteroni as the cause of mortality in embryonated chicken eggs of breeding broiler hens in Costa Rica.

Mortality of chicken embryos and first-week chickens was reported in a commercial incubator company in Costa Rica. Six 1-day-old Cobb chickens and twenty-four embryonated chicken eggs were examined in the Laboratory of Avian Pathology and the Laboratory of Bacteriology of the National University of Costa Rica. Twelve dead-in-shell embryos showed maceration and were immersed in a putrid, turbid, slightly thick brown liquid. Additionally, the other 12 embryonated eggs had milky yellow-orange content. The livers of those embryos had congestion, haemorrhages and multifocal cream foci of necrosis. Granulocytic infiltration was observed in the bursa of Fabricius, myocardium, liver, lung and kidney. Livers and egg yolks from six embryonated chickens and all 1-day-old chickens were aseptically collected and cultured. In addition, tissues from six better conserved embryos and all 1-day-old chickens were fixed in buffered formalin and embedded in paraffin. Biochemical and molecular tests identified Comamonas testosteroni as the cause of the early, middle and late embryo mortality. As all the eggshells from the sampled embryonated eggs were dirty with soiled a fecal matter, contamination after manipulating the eggs was considered the source of infection. C. testosteroni is an environmental microorganism that has rarely been reported to cause human disease. To our knowledge, this is the first report of C. testosteroni causing mortality in a hatchery. Cleaning and disinfection using ozone were implemented in the hatchery to eliminate the embryo mortality associated with C. testosteroni.

Chicken manure-derived biochar enhanced the potential of Comamonas testosteroni ZG2 to remediate Cd contaminated soil.

The combined remediation of Cd-contaminated soil using biochar and microorganisms has a good application value. In this study, the effect of chicken manure-derived biochar on CdCO3 precipitation induced by Comamonas testosteroni ZG2 was investigated. The results showed that biochar could be used as the carrier of strain ZG2, enhance the resistance of strain ZG2 to Cd, and reduce the toxicity of Cd to bacterial cells. Cd adsorbed by biochar could be induced by strain ZG2 to form CdCO3 precipitation. Strain ZG2 could also induce CdCO3 precipitation when biochar was added during precipitation formation and fermentation broth formation. The CdCO3 precipitation could enter the pores of the biochar and attach to the surface of the biochar. The single and combined effects of strain ZG2 and biochar could realize the remediation of Cd-contaminated soil to a certain extent. The overall effect was in the order of strain ZG2 with biochar > biochar > strain ZG2. The combination of strain ZG2 and biochar reduced soil available Cd by 48.2%, the aboveground biomass of pakchoi increased by 72.1%, and the aboveground Cd content decreased by 73.3%. At the same time, it promoted the growth and development of the root system and improved the microbial community structure of the rhizosphere soil. The results indicated that chicken manure-derived biochar could enhance the stability of CdCO3 precipitation induced by strain ZG2, and strain ZG2 combined with biochar could achieve a more stable remediation effect on Cd-contaminated soil.

Identification of "missing links" in C- and D-ring cleavage of steroids by Comamonas testosteroni TA441.

Comamonas testosteroni TA441 is capable of aerobically degrading steroids through the aromatization and cleavage of the A- and B-rings, followed by D- and C-ring cleavage via ß-oxidation. While most of the degradation steps have been previously characterized, a few intermediate compounds remained unidentified. In this study, we proposed that the cleavage of the D-ring at C13-17 required the ScdY hydratase, followed by C-ring cleavage via the ScdL1L2 transferase. The anticipated reaction was expected to yield 6-methyl-3,7-dioxo-decane-1,10-dioic acid-coenzyme A (CoA) ester. To confirm this hypothesis, we constructed a plasmid enabling the induction of targeted genes in TA441 mutant strains. Induction experiments of ScdL1L2 revealed that the major product was 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester. Similarly, induction experiments of ScdY demonstrated that the substrate of ScdY was a geminal diol, 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester. These findings suggest that ScdY catalyzes the addition of a water molecule at C14 of 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester, leading to D-ring cleavage at C13-17. Subsequently, the C9 ketone of the D-ring cleavage product is converted to a hydroxyl group, followed by C-ring cleavage, resulting in the production of 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester.IMPORTANCEStudies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain substrates for steroid drugs. In recent years, the role of steroid-degrading bacteria in relation to human health has gained significant attention, as emerging evidence suggests that the intestinal microflora plays a crucial role in human health. Furthermore, cholic acid, a major component of bile acid secreted in the intestines, is closely associated with the gut microbiota. While Comamonas testosteroni TA441 is recognized as the leading bacterial model for aerobic steroid degradation, the involvement of aerobic steroid degradation in the intestinal microflora remains largely unexplored. Nonetheless, the presence of C. testosteroni in the cecum suggests the potential influence of aerobic steroid degradation on gut microbiota. To establish essential information about the role of these bacteria, here, we identified the missing compounds and propose more details of C-, and D-ring cleavage, which have remained unclear until now.

Comprehensive summary of steroid metabolism in Comamonas testosteroni TA441: entire degradation process of basic four rings and removal of C12 hydroxyl group.

Comamonas testosteroni is one of the representative aerobic steroid-degrading bacteria. We previously revealed the mechanism of steroidal A,B,C,D-ring degradation by C. testosteroni TA441. The corresponding genes are located in two clusters at both ends of a mega-cluster of steroid degradation genes. ORF7 and ORF6 are the only two genes in these clusters, whose function has not been determined. Here, we characterized ORF7 as encoding the dehydrase responsible for converting the C12ß hydroxyl group to the C10(12) double bond on the C-ring (SteC), and ORF6 as encoding the hydrogenase responsible for converting the C10(12) double bond to a single bond (SteD). SteA and SteB, encoded just upstream of SteC and SteD, are in charge of oxidizing the C12α hydroxyl group to a ketone group and of reducing the latter to the C12ß hydroxyl group, respectively. Therefore, the C12α hydroxyl group in steroids is removed with SteABCD via the C12 ketone and C12ß hydroxyl groups. Given the functional characterization of ORF6 and ORF7, we disclose the entire pathway of steroidal A,B,C,D-ring breakdown by C. testosteroni TA441.IMPORTANCEStudies on bacterial steroid degradation were initiated more than 50 years ago, primarily to obtain materials for steroid drugs. Now, their implications for the environment and humans, especially in relation to the infection and the brain-gut-microbiota axis, are attracting increasing attention. Comamonas testosteroni TA441 is the leading model of bacterial aerobic steroid degradation with the ability to break down cholic acid, the main component of bile acids. Bile acids are known for their variety of physiological activities according to their substituent group(s). In this study, we identified and functionally characterized the genes for the removal of C12 hydroxyl groups and provided a comprehensive summary of the entire A,B,C,D-ring degradation pathway by C. testosteroni TA441 as the representable bacterial aerobic degradation process of the steroid core structure.

Engineering Comamonas testosteroni for the production of 2-pyrone-4,6-dicarboxylic acid as a promising building block.

BACKGROUND: Plastics are an indispensable part of our daily life. However, mismanagement at their end-of-life results in severe environmental consequences. The microbial conversion of these polymers into new value-added products offers a promising alternative. In this study, we engineered the soil-bacterium Comamonas testosteroni KF-1, a natural degrader of terephthalic acid, for the conversion of the latter to the high-value product 2-pyrone-4,6-dicarboxylic acid. RESULTS: In order to convert terephthalic acid to 2-pyrone-4,6-dicarboxylic acid, we deleted the native PDC hydrolase and observed only a limited amount of product formation. To test whether this was the result of an inhibition of terephthalic acid uptake by the carbon source for growth (i.e. glycolic acid), the consumption of both carbon sources was monitored in the wild-type strain. Both carbon sources were consumed at the same time, indicating that catabolite repression was not the case. Next, we investigated if the activity of pathway enzymes remained the same in the wild-type and mutant strain. Here again, no statistical differences could be observed. Finally, we hypothesized that the presence of a pmdK variant in the degradation operon could be responsible for the observed phenotype and created a double deletion mutant strain. This newly created strain accumulated PDC to a larger extent and again consumed both carbon sources. The double deletion strain was then used in a bioreactor experiment, leading to the accumulation of 6.5 g/L of product in 24 h with an overall productivity of 0.27 g/L/h. CONCLUSIONS: This study shows the production of the chemical building block 2-pyrone-4,6-dicarboxylic acid from terephthalic acid through an engineered C. testosteroni KF-1 strain. It was observed that both a deletion of the native PDC hydrolase as well as a pmdK variant is needed to achieve high conversion yields. A product titer of 6.5 g/L in 24 h with an overall productivity of 0.27 g/L/h was achieved.

Comparative metabolomic analysis reveals Ni(II) stress response mechanism of Comamonas testosteroni ZG2.

The focus on the toxicity of nickel (Ni(II)) in animal and human cells has increased recently. Ni(II) contamination hazards to animals and humans can be reduced by bioremediation methods. However, one of the limitation of bioremediation bacteria in soil remediation is that they cannot survive in moderate and heavy contamination Ni(II)-contaminated environments. Therefore, the Ni(II) response mechanism of Comamonas testosteroni ZG2 which has soil remediation ability in high-concentration Ni(II) environment must be elucidated. The results demonstrated that the ZG2 strain can survive at 350 mg/L concentration of Ni(II), but the growth of ZG2 was completely inhibited under the concentration of 400 mg/L Ni(II) with significant alterations in the membrane morphology, adhesion behavior, and functional groups and serious membrane damage. Furthermore, the metabolic analysis showed that Ni(II) may affect the adhesion behavior and biofilm formation of the ZG2 strain by affecting the abundance of metabolites in amino acid biosynthesis, aminoacyl-tRNA biosynthesis, ABC transporter, and cofactor biosynthesis pathways, and inhibiting its growth. This study provides new evidence clarifying the response mechanism of Ni(II) stress in the ZG2 strain, thus playing a significant role in designing the strategies of bioremediation.

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni.

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDOKF1 from Comamonas testosteroni KF1 and found that it had an apparent kcat/Km for phthalate of 0.58 ± 0.09 µM-1s-1, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α3 trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDOKF1 larger than that of other characterized ROs. Complexes of PDOKF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.

Functional and structural characterization of AntR, an Sb(III) responsive transcriptional repressor.

The ant operon of the antimony-mining bacterium Comamonas testosterone JL40 confers resistance to Sb(III). The operon is transcriptionally regulated by the product of the first gene in the operon, antR. AntR is a member of ArsR/SmtB family of metal/metalloid-responsive repressors resistance. We purified and characterized C. testosterone AntR and demonstrated that it responds to metalloids in the order Sb(III) = methylarsenite (MAs(III) >> As(III)). The protein was crystallized, and the structure was solved at 2.1 Å resolution. The homodimeric structure of AntR adopts a classical ArsR/SmtB topology architecture. The protein has five cysteine residues, of which Cys103a from one monomer and Cys113b from the other monomer, are proposed to form one Sb(III) binding site, and Cys113a and Cys103b forming a second binding site. This is the first report of the structure and binding properties of a transcriptional repressor with high selectivity for environmental antimony.

Assessment of Comamonas testosteroni strain PT9 as a rapid phthalic acid degrader for industrial wastewaters.

In this study, characterization of industry-borne Comamonas testosteroni strain PT9 isolate was performed by determining degradation ability on phthalic acid (PA). High-performance liquid chromatography analyses showed that strain PT9 completely degraded 102.94 mg/L of PA within 6 h. Viability polymerase chain reaction (vPCR) was performed with propidium monoazide treatment. vPCR showed that the PA has positively stimulated the cell growth during degradation. To consider the fate of PA, the proposed catalytic genes (ophA2, iphA2, tphA2, tphA3, pmdA, and pmdB) for the degradation pathways of PA isomers for C. testosteroni were screened in strain PT9. All genes except iphA2 were detected in strain PT9, and expression levels of related genes were analyzed by Real-Time PCR (qPCR).

Molecular Research of Lipid Peroxidation and Antioxidant Enzyme Activity of Comamonas testosteroni Bacterial Cells under the Hexachlorobenzene Impact.

The species of Comamonas testosteroni is the most common human pathogen of the genus, which can be associated with acute appendicitis, infections of the bloodstream, the peritoneal cavity, cerebrospinal fluid, inflammatory bowel disease, and in general, bacteremia. According to the literature, Comamonas testosteroni has destructive activity to a wide range of toxic chemical compounds, including chlorobenzenes. The specified strains were isolated from the soil of the organochlorine waste landfill, where hexachlorobenzene (HCB) was predominant. These strains were expected to be capable of degrading HCB. Microbiological (bacterial enrichment and cultivating, bacterial biomass obtaining), molecular biology, biochemical (enzymatic activities, malondialdehyde measuring, peroxidation lipid products measuring), and statistical methods were carried out in this research. The reaction of both strains (UCM B-400 and UCM B-401) to the hexachlorobenzene presence differed in the content of diene and triene conjugates and malondialdehyde, as well as different catalase and peroxidase activity levels. In terms of primary peroxidation products, diene conjugates were lower, except conditions with 20 mg/L HCB, where these were higher up to two times, than the pure control. Malondialdehyde in strain B-400 cells decreased up to five times, in B-401, but increased up to two times, compared to the pure control. Schiff bases in strain B-400 cells were 2-3 times lower than the pure control. However, in B-401 cells Schiff bases under higher HCB dose were in the same level with the pure control. Catalase activity was 1.5 times higher in all experimental variants, compared to the pure control (in the strain B-401 cells), but in the B-400 strain, cells were 2 times lower, compared to the pure control. The response of the two strains to hexachlorobenzene was similar only in peroxidase activity terms, which was slightly higher compared to the pure control. The physiological response of Comamonas testosteroni strains to hexachlorobenzene has a typical strain reaction. The physiological response level of these strains to hexachlorobenzene confirms its tolerance, and indirectly, the ability to destroy the specified toxic compound.

Identification of the Coenzyme A (CoA) Ester Intermediates and Genes Involved in the Cleavage and Degradation of the Steroidal C-Ring by Comamonas testosteroni TA441.

Comamonas testosteroni TA441 degrades steroids aerobically via aromatization of the A-ring accompanied by B-ring cleavage, followed by D- and C-ring cleavage. We previously revealed major enzymes and intermediate compounds in A,B-ring cleavage, the ß-oxidation cycle of the cleaved B-ring, and partial C,D-ring cleavage. Here, we elucidate the C-ring cleavage and the ß-oxidation cycle that follows. ScdL1L2, a 3-ketoacid coenzyme A (CoA) transferase which belongs to the SugarP_isomerase superfamily, was thought to cleave the C-ring of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid-CoA ester, the key intermediate compound in the degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (3aα-H-4α [3'-propionic acid]-7aß-methylhexahydro-1,5-indanedione; HIP)-CoA ester in our previous study; however, the present study suggested that ScdL1L2 is the isomerase of the derivative with a hydroxyl group at C-14 which cleaves the C-ring. The subsequent ring-cleaved product was indicated to be converted to 4-methyl-5-oxo-octane-1,8-dioic acid-CoA ester mainly by ORF33-encoded CoA-transferase (named ScdJ), followed by dehydrogenation by ORF21- and 22-encoded acyl-CoA dehydrogenase (named ScdM1M2). Then, a water molecule is added by ScdN for further degradation by ß-oxidation. ScdN is proposed to catalyze the last reaction in C,D-ring degradation by the enzymes encoded in the steroid degradation gene cluster tesB to tesR. IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment, as well as in humans, is attracting attention. The overall degradation process of the four steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aimed to uncover the whole steroid degradation process in C. testosteroni, which is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption.

High expression of ring-hydroxylating dioxygenase genes ensure efficient degradation of p-toluate, phthalate, and terephthalate by Comamonas testosteroni strain 3a2.

Para-toluic acid, a major pollutant in industrial wastewater, is hazardous to human health. It has been demonstrated that Gram-negative bacteria are among the most effective degraders of para-toluic acid. In this study, the ability of Comamonas testosteroni strain 3a2, isolated from a petrochemical industry wastewater, to degrade para-toluic acid was investigated. The effect of different carbon (glucose and ethylene glycol) and nitrogen sources (urea, yeast extract, peptone, NaNO3, NH4NO3) on the biodegradation of para-toluic acid by the isolate 3a2 was evaluated. Furthermore, ring hydroxylating dioxygenase genes were amplified by PCR and their expression was evaluated during the biodegradation of para-toluic acid. The results indicated that strain 3a2 was able to degrade up to 1000 mg/L of para-toluic acid after 14 h. The highest degradation yield was recorded in the presence of yeast extract as nitrogen source. However, the formation of terephthalic acid and phthalic acid was noted during para-toluic acid degradation by the isolate 3a2. Toluate 1,2-dioxygenase, terephthalate 1,2 dioxygenase, and phthalate 4,5 dioxygenase genes were detected in the genomic DNA of 3a2. The induction of ring hydroxylating dioxygenase genes was proportional to the concentration of each hydrocarbon. This study showed that the isolate 3a2 can produce terephthalate and phthalate during the para-toluic acid biodegradation, which were also degraded after 24 h.

Cloning and identification of a new repressor of 3,17ß-Hydroxysteroid dehydrogenase of Comamonas testosteroni.

BACKGROUND: 3,17ß-hydroxysteroid dehydrogenase (3,17ß-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17ß-HSD. Previous reports showed that three tetR genes are located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first tetR gene encoded a potential repressor of 3,17ß-HSD by sensing environmental signals. However, whether the other proposed tetR genes act as repressors of 3,17ß-HSD are still unknown. METHODS AND RESULTS: In the present study, we cloned the second tetR gene and analyzed the regulatory mechanism of the protein on 3,17ß-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second tetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17ß-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17ß-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second tetR gene knocked-out mutant expresses good biological genetic stability, and the expression of 3,17ß-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction. CONCLUSIONS: The second tetR gene acts as a negative regulator in 3,17ß-HSD expression, and the mutant has potential application in bioremediation of steroids contaminated environment.

How bioaugmentation with Comamonas testosteroni accelerates pyridine mono-oxygenation and mineralization.

Pyridine is a common heterocycle found in industrial wastewaters. Its biodegradation begins with a mono-oxygenation reaction, and bioaugmentation with bacteria able to carry out this mono-oxygenation is one strategy to improve pyridine removal and mineralization. Although bioaugmentation has been used to enhance the biodegradation of recalcitrant organic compounds, the specific role played by the bioaugmented bacteria usually has not been addressed. We acclimated activated-sludge biomass for pyridine biodegradation and then isolated a strain -- Comamonas testosteroni -- based on its ability to biodegrade and grow on pyridine alone. Pyridine was removed faster by C. testosteroni, compared to pyridine-acclimated biomass, but pyridine mineralization was slower. Pyridine biodegradation and mineralization rates were accelerated when C. testosteroni was bioaugmented into the acclimated biomass, which increased the amount of C. testosteroni, but otherwise had minimal effects on the microbial community. The key role of C. testosteroni was to accelerate the first step of pyridine biodegradation, mono-oxygenation to 2-hydroxylpyridine (2HP), and the acclimated biomass was better able to complete downstream reactions leading to mineralization. Thus, bioaugmentation increased the rates of pyridine mono-oxygenation and subsequent mineralization through the synergistic roles of C. testosteroni and the main community in the acclimated biomass.

The ligand-binding domain of a chemoreceptor from Comamonas testosteroni has a previously unknown homotrimeric structure.

Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell interior. Here, we report the structure of the periplasmic ligand-binding domain (LBD) of the transmembrane chemoreceptor MCP2201, which governs chemotaxis to citrate and other organic compounds in Comamonas testosteroni. The apo-form LBD crystal revealed a typical four-helix bundle homodimer, similar to previously well-studied chemoreceptors such as Tar and Tsr of Escherichia coli. However, the citrate-bound LBD revealed a four-helix bundle homotrimer that had not been observed in bacterial chemoreceptor LBDs. This homotrimer was further confirmed with size-exclusion chromatography, analytical ultracentrifugation and cross-linking experiments. The physiological importance of the homotrimer for chemotaxis was demonstrated with site-directed mutations of key amino acid residues in C. testosteroni mutants.

Steroid Degradation in Comamonas testosteroni TA441: Identification of the Entire ß-Oxidation Cycle of the Cleaved B Ring.

Comamonas testosteroni TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by ß-oxidation. In this study, we revealed that 7ß,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3S)-3-hydroxylacyl CoA-dehydrogenase, encoded by scdE (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by scdF (ORF23). With these results, the whole cycle of ß-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1C2, followed by hydration by ScdD. 7ß,9α-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-CoA ester then is dehydrogenated by ScdE to be converted to 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid-CoA ester and acetyl-CoA by ScdF. ScdF is an ortholog of FadA6 in Mycobacterium tuberculosis H37Rv, which was reported as a 3-ketoacyl-CoA transferase involved in C ring cleavage. We also obtained results suggesting that ScdF is also involved in C ring cleavage, but further investigation is required for confirmation. ORF25 and ORF26, located between scdF and scdE, encode enzymes belonging to the amidase superfamily. Disrupting either ORF25 or ORF26 did not affect steroid degradation. Among the bacteria having gene clusters similar to those of tesB to tesR, some have both ORF25- and ORF26-like proteins or only an ORF26-like protein, but others do not have either ORF25- or ORF26-like proteins. ORF25 and ORF26 are not crucial for steroid degradation, yet they might provide clues to elucidate the evolution of bacterial steroid degradation clusters.IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the four basic steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in Comamonas testosteroni TA441 as a model of steroid-degrading bacteria. C. testosteroni is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway, because the involvement of degradation-related genes can be determined by gene disruption. Here, we elucidated the entire ß-oxidation cycle of the cleaved B ring. This cycle is essential for the following C and D ring cleavage.

A microfluidic gradient mixer-flow chamber as a new tool to study biofilm development under defined solute gradients.

Understanding the dynamics of biofilm development in response to chemical cues and signals is required toward the development of controllable biofilm-mediated bioprocesses. In this study, we report a new biofilm growth system that integrates a microfluidic gradient mixer with a biofilm growth chamber. The biofilm growth system allows biofilms to grow under defined solute gradients and enables nondestructive monitoring of the biofilm development dynamics in response to the defined gradients. The solute gradients generated in the system were simulated and then validated experimentally. We then demonstrated the applicability of the biofilm growth system in studying biofilm development under defined solute gradients. Specifically, we examined biofilm development of Shewanella oneidensis and Comamonas testosteroni under a defined calcium and nitrate gradient, respectively. Using two C. testosteroni strains (WDL7 and I2), we further demonstrated the applicability of our biofilm growth system to study the development of coculture biofilms under a defined solute gradient. Our results show that the biofilm growth system we have developed here can be a promising tool to reveal the dynamics of biofilm development in response to chemical cues and signals as well as the interorganism interactions in coculture biofilms.

Chemotaxis Towards Aromatic Compounds: Insights from Comamonas testosteroni.

Chemotaxis is an important physiological adaptation that allows many motile bacteria to orientate themselves for better niche adaptation. Chemotaxis is best understood in Escherichia coli. Other representative bacteria, such as Rhodobacter sphaeroides, Pseudomonas species, Helicobacter pylori, and Bacillus subtilis, also have been deeply studied and systemically summarized. These bacteria belong to α-, γ-, ε-Proteobacteria, or Firmicutes. However, ß-Proteobacteria, of which many members have been identified as holding chemotactic pathways, lack a summary of chemotaxis. Comamonas testosteroni, belonging to ß-Proteobacteria, grows with and chemotactically responds to a range of aromatic compounds. This paper summarizes the latest research on chemotaxis towards aromatic compounds, mainly from investigations of C. testosteroni and other Comamonas species.

Steroid Degradation in Comamonas testosteroni TA441: Identification of Metabolites and the Genes Involved in the Reactions Necessary before D-Ring Cleavage.

Bacterial steroid degradation has been studied mainly with Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni as representative steroid degradation bacteria for more than 50 years. The primary purpose was to obtain materials for steroid drugs, but recent studies showed that many genera of bacteria (Mycobacterium, Rhodococcus, Pseudomonas, etc.) degrade steroids and that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment. The role of bacterial steroid degradation in the environment is, however, yet to be revealed. To uncover the whole steroid degradation process in a representative steroid-degrading bacterium, C. testosteroni, to provide basic information for further studies on the role of bacterial steroid degradation, we elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage in C. testosteroni TA441. In bacterial oxidative steroid degradation, A- and B-rings of steroids are cleaved to produce 2-hydroxyhexa-2,4-dienoic acid and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. The latter compound was revealed to be degraded to the coenzyme A (CoA) ester of 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid, which is converted to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid by ORF31-encoded hydroxylacyl dehydrogenase (ScdG), followed by conversion to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid by ORF4-encoded acyl-CoA dehydrogenase (ScdK). Then, a water molecule is added by the ORF5-encoded enoyl-CoA hydratase (ScdY), which leads to the cleavage of the D-ring. The conversion by ScdG is presumed to be a reversible reaction. The elucidated pathway in C. testosteroni TA441 is different from the corresponding pathways in Mycobacterium tuberculosis H37Rv.IMPORTANCE Studies on representative steroid degradation bacteria Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni were initiated more than 50 years ago primarily to obtain materials for steroid drugs. A recent study showed that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment, but the role of bacterial steroid degradation in the environment is yet to be revealed. This study aimed to uncover the whole steroid degradation process in C. testosteroni TA441, in which major enzymes for steroidal A- and B-ring cleavage were elucidated, to provide basic information for further studies on bacterial steroid degradation. C. testosteroni is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption. We elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage, which appeared to differ from those present in Mycobacterium tuberculosis H37Rv.