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.

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.

Exploration of potential driving mechanisms of Comamonas testosteroni in polycyclic aromatic hydrocarbons degradation and remodelled bacterial community during co-composting.

Polycyclic aromatic hydrocarbons (PAHs) are a cluster of highly hazardous organic pollutants that are widespread in ecosystems and threaten human health. Composting has been shown to be an effective strategy for PAHs degredation. Here, we used Comamonas testosteroni as an inoculant in composting and investigated the potential mechanisms of PAHs degradation by co-occurrence network and structural equation modelling analysis. The results showed that more than 60% of PAHs were removed and the bacterial community responded to the negative effects of PAHs by upgrading the network. Inoculation with C. testosteroni altered bacterial community succession, intensified bacterial response to PAHs, improved metabolic activity, and promoted the degradation of PAHs during co-composting. The increased in the positive cohesion index of the community suggested that agents increased the cooperative behaviour between bacteria and led to changes in keystones of the bacterial network. However, the topological values of C. testosteroni in the network were not elevated, which confirmed that C. testosteroni altered communities by affecting other bacterial growth rather than its own colonisation. This study strengthens our comprehension of the potential mechanisms for the degradation of PAHs in inoculated composting.

Effects of Comamonas testosteroni on dissipation of polycyclic aromatic hydrocarbons and the response of endogenous bacteria for soil bioremediation.

Bioremediation is a promising method of treating polycyclic aromatic hydrocarbons (PAHs) in contaminated soil; however, the understanding of the efficiency and the way of microbial inoculants work in complex soil environments is limited. Comamonas testosteroni (Ct) strains could efficiently degrade PAHs, especially naphthalene (Nap) and phenanthrene (Phe). This study aimed to explore the functional role of Ct in soil indigenous microorganisms and analyze the effect of Ct addition on PAHs concentration in PAH-contaminated soil. The results showed that inoculation with Ct degraded naphthalene (Nap), phenanthrene (Phe), and benzo [α] pyrene (BaP) significantly; the degradation rates were 63.38%, 81.18%, and 37.98% on day 25, respectively, suggesting that the low molecular weights of Nap and Phe were more easily degraded by microorganisms than those of BaP. We speculated that BaP and Phe might be converted into Nap for further degradation, which is the main reason for the low degradation rate of Nap detected after 10-25 days. Network analysis showed that inoculation with Ct significantly increased bacteria community abundance closely related to PAHs. Structural equation models confirmed that Steroidobacter, as functional bacteria, could affect the degradation of Nap and BaP. Inoculated Ct effectively enhanced the synergy among indigenous bacteria to degrade PAHs. This finding will help understand the function of inoculated Ct strains in PAH-contaminated soil at the laboratory level.

Analogous Metabolic Decoupling in Pseudomonas putida and Comamonas testosteroni Implies Energetic Bypass to Facilitate Gluconeogenic Growth.

Gluconeogenic carbon metabolism is not well understood, especially within the context of flux partitioning between energy generation and biomass production, despite the importance of gluconeogenic carbon substrates in natural and engineered carbon processing. Here, using multiple omics approaches, we elucidate the metabolic mechanisms that facilitate gluconeogenic fast-growth phenotypes in Pseudomonas putida and Comamonas testosteroni, two Proteobacteria species with distinct metabolic networks. In contrast to the genetic constraint of C. testosteroni, which lacks the enzymes required for both sugar uptake and a complete oxidative pentose phosphate (PP) pathway, sugar metabolism in P. putida is known to generate surplus NADPH by relying on the oxidative PP pathway within its characteristic cyclic connection between the Entner-Doudoroff (ED) and Embden-Meyerhoff-Parnas (EMP) pathways. Remarkably, similar to the genome-based metabolic decoupling in C. testosteroni, our 13C-fluxomics reveals an inactive oxidative PP pathway and disconnected EMP and ED pathways in P. putida during gluconeogenic feeding, thus requiring transhydrogenase reactions to supply NADPH for anabolism in both species by leveraging the high tricarboxylic acid cycle flux during gluconeogenic growth. Furthermore, metabolomics and proteomics analyses of both species during gluconeogenic feeding, relative to glycolytic feeding, demonstrate a 5-fold depletion in phosphorylated metabolites and the absence of or up to a 17-fold decrease in proteins of the PP and ED pathways. Such metabolic remodeling, which is reportedly lacking in Escherichia coli exhibiting a gluconeogenic slow-growth phenotype, may serve to minimize futile carbon cycling while favoring the gluconeogenic metabolic regime in relevant proteobacterial species. IMPORTANCE Glycolytic metabolism of sugars is extensively studied in the Proteobacteria, but gluconeogenic carbon sources (e.g., organic acids, amino acids, aromatics) that feed into the tricarboxylic acid (TCA) cycle are widely reported to produce a fast-growth phenotype, particularly in species with biotechnological relevance. Much remains unknown about the importance of glycolysis-associated pathways in the metabolism of gluconeogenic carbon substrates. Here, we demonstrate that two distinct proteobacterial species, through genetic constraints or metabolic regulation at specific metabolic nodes, bypass the oxidative PP pathway during gluconeogenic growth and avoid unnecessary carbon fluxes by depleting protein investment into connected glycolysis pathways. Both species can leverage instead the high TCA cycle flux during gluconeogenic feeding to meet NADPH demand. Importantly, lack of a complete oxidative pentose phosphate pathway is a widespread metabolic trait in Proteobacteria with a gluconeogenic carbon preference, thus highlighting the important relevance of our findings toward elucidating the metabolic architecture in these bacteria.

Imbalance in Carbon and Nitrogen Metabolism in Comamonas testosteroni R2 Is Caused by Negative Feedback and Rescued by L-arginine.

The collapse of Comamonas testosteroni R2 under chemostat conditions and the aerobic growth of strain R2 under batch conditions with phenol as the sole carbon source were investigated using physiological and transcriptomic techniques. Phenol-/catechol-degrading activities under chemostat conditions gradually decreased, suggesting that metabolites produced from strain R2 accumulated in the culture, which caused negative feedback. The competitive inhibition of phenol hydroxylase and catechol dioxygenase was observed in a crude extract of the supernatant collected from the collapsed culture. Transcriptomic analyses showed that genes related to nitrogen transport were up-regulated; the ammonium transporter amtB was up-regulated approximately 190-fold in the collapsed status, suggesting an increase in the concentration of ammonium in cells. The transcriptional levels of most of the genes related to gluconeogenesis, glycolysis, the pentose phosphate pathway, and the TCA and urea cycles decreased by ~0.7-fold in the stable status, whereas the activities of glutamate synthase and glutamine synthetase increased by ~2-fold. These results suggest that ammonium was assimilated into glutamate and glutamine via 2-oxoglutarate under the limited supply of carbon skeletons, whereas the synthesis of other amino acids and nucleotides was repressed by 0.6-fold. Furthermore, negative feedback appeared to cause an imbalance between carbon and nitrogen metabolism, resulting in collapse. The effects of amino acids on negative feedback were investigated. L-arginine allowed strain R2 to grow normally, even under growth-inhibiting conditions, suggesting that the imbalance was corrected by the stimulation of the urea cycle, resulting in the rescue of strain R2.

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.

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.

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.

Comamonas testosteroni antA encodes an antimonite-translocating P-type ATPase.

Antimony, like arsenic, is a toxic metalloid widely distributed in the environment. Microbial detoxification of antimony has recently been identified. Here we describe a novel bacterial P1B-type antimonite (Sb(III))-translocating ATPase from the antimony-mining bacterium Comamonas testosterone JL40 that confers resistance to Sb(III). In a comparative proteomics analysis of strain JL40, an operon (ant operon) was up-regulated by Sb(III). The ant operon includes three genes, antR, antC and antA. AntR belongs to the ArsR/SmtB family of metalloregulatory proteins that regulates expression of the ant operon. AntA belongs to the P1B family of the P-type cation-translocating ATPases. It has both similarities to and differences from other members of the P1B-1 subfamily and appears to be the first identified member of a distinct subfamily that we designate P1B-8. Expression AntA in E. coli AW3110 (Δars) conferred resistance to Sb(III) and reduced the intracellular concentration of Sb(III) but not As(III) or other metals. Everted membrane vesicles from cells expressing antA accumulated Sb(III) but not As(III), where uptake in everted vesicles reflects efflux from cells. AntC is a small protein with a potential Sb(III) binding site, and co-expression of AntC with AntA increased resistance to Sb(III). We propose that AntC functions as an Sb(III) chaperone to AntA, augmenting Sb(III) efflux. The identification of a novel Sb(III)-translocating ATPase enhances our understanding of the biogeochemical cycling of environmental antimony by bacteria.

Quantitative analysis of the surficial and adhesion properties of the Gram-negative bacterial species Comamonas testosteroni modulated by c-di-GMP.

Cyclic diguanylate monophosphate (c-di-GMP) is a ubiquitous intracellular secondary messenger which governs the transition from a bacterial cell's planktonic state to biofilm formation by stimulating the production of a variety of exopolysaccharide material by the bacterial cell. A range of genes involved in c-di-GMP signaling in the Gram-negative species Comamonas testosteroni have been identified previously, yet the physical-chemical properties of the produced extracellular polymeric substances (EPS) and the bacterial adhesion characteristics regulated by c-di-GMP are not well understood. Here, we modulated the in vivo c-di-GMP levels of Comamonas testosteroni WDL7 through diguanylate cyclase (YedQ) and phosphodiesterase (YhjH) gene editing. The strains and their adhesion properties were characterized by Fourier-transform infrared and two-dimensional correlation spectroscopy analysis (FTIR-2D CoS), contact angle and zeta potential measurements, atomic force microscopy (AFM) and extended-Derjaguin-Landau-Verwey-Overbeek (ExDLVO) analysis. Our results show that high c-di-GMP levels promoted the secretion of long-chain hydrophobic and electroneutral extracellular polysaccharides and proteins. The protein molecules on WDL7/pYedQ2 promoted the bacterial self-aggregation and adhesion onto negatively charged surfaces. In contrast, the reduction of intracellular c-di-GMP concentrations resulted in a nearly 80 % decrease in the adhesion of bacterial cells, although little change in the surface hydrophobicity or surface charge properties were observed for these cells relative to the wild type. These results indicate that the reduced adsorption of WDL7/YhjH that we observed may be caused by the flagellum-accelerated mobility at low c-di-GMP concentrations. Taken together, these results improve our mechanistic understanding of the effects of c-di-GMP in controlling bacterial physical-chemical properties and initial biofilm development.

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases.

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta-gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.

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.

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.

Identification of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid as a metabolite of steroid degradation in Comamonas testosteroni TA441 and the genes involved in the conversion.

Comamonas testosteroni TA441 degrades steroid compounds via aromatization of the A-ring to produce 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (a metabolite with C- and D-rings), which is presumed to be further degraded via ß-oxidation. In elucidating the complete steroid degradation process in C. testosteroni, we isolated 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid and several other metabolites containing only C-ring. For conversion of the CoA-ester of this compound, a two-subunit ß -ketoacyl-CoA-transferase encoded by ORF1 and ORF2 was shown to be indispensable. ORF1 and ORF2 are located just after tesB, the meta-cleavage enzyme gene in one of the two major steroid degradation gene clusters of strain TA441. Conversion by the CoA-transferase leads to cleavage of the remaining C-ring, and the product was suggested to be further degraded by ß-oxidation involving other genes in the cluster. ORF1 and ORF2 are considered orthologues of ipdAB gene in Mycobacterium tuberculosis H37Rv, which is recently reported as the CoA-transferase of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid (Crowe AM, Casabon I, Brown KL, Liu J, Lian J, Rogalski JC, Hurst TE, Snieckus V, Foster LJ, Eltis LD. 2017. MBio 8).

Identification of 4-methyl-5-oxo-octane-1,8-dioic acid and the derivatives as metabolites of steroidal C,D-ring degradation in Comamonas testosteroni TA441.

Comamonas testosteroni TA441 degrades steroids via 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, which is presumed to be further degraded by ß-oxidation. In the ß-oxidation process, Coenzyme A (CoA)-ester of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid is produced and converted by ß-ketoacyl-CoA-transferase encoded by ORF1 and ORF2 (scdL1L2) to cleave the remaining C-ring. In this study, we isolated and identified 4-methyl-5-oxo-octane-1,8-dioic acid and 4-methyl-5-oxo-3-octene-1,8-dioic acid from the culture of the ORF3 (scdN)-null mutant as metabolites of steroid degradation (ADD and cholic acid analogues; cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid). In addition of these compounds, UHPLC/MS analysis of the culture of the scdN-null mutant revealed significant accumulation of another compound, which was detected as a dominant peak of m/z 155 ([M-CO2]-) accompanied by a small peak of parental ion (m/z 199 [M-]). On the bases of experimental data, this compound was presumed to be 4-methyl-5-oxo-2-octene-1,8-dioic acid, whose CoA-ester was indicated to be converted by scdN-encoded CoA-hydratase into the CoA-ester of 3-hydroxy-4-methyl-5-oxooctan-1,7-carboxylic acid.

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.

Novel mechanisms of selenate and selenite reduction in the obligate aerobic bacterium Comamonas testosteroni S44.

Selenium oxyanion reduction is an effective detoxification or/and assimilation processes in organisms, but little is known the mechanisms in aerobic bacteria. Aerobic Comamonas testosteroni S44 reduces Se(VI)/Se(IV) to less-toxic elemental selenium nanoparticles (SeNPs). For Se(VI) reduction, sulfate and Se(VI) reduction displayed a competitive relationship. When essential sulfate reducing genes were respectively disrupted, Se(VI) was not reduced to red-colored SeNPs. Consequently, Se(VI) reduction was catalyzed by enzymes of the sulfate reducing pathway. For Se(IV) reduction, one of the potential periplasm molybdenum oxidoreductase named SerT was screened and further used to analyze Se(IV) reduction. Compared to the wild type and the complemented mutant strain, the ability of Se(IV) reduction was reduced 75% in the deletion mutant ΔserT. Moreover, the Se(IV) reduction rate was significantly enhanced when the gene serT was overexpressed in Escherichia coli W3110. In addition, Se(IV) was reduced to SeNPs by the purified SerT with the presence of NADPH as the electron donor in vitro, showing a Vmax of 61 nmol/min·mg and a Km of 180 µmol/L. A model of Se(VI)/Se(IV) reduction was generated in aerobic C. testosteroni S44. This work provides new insights into the molecular mechanisms of Se(VI)/Se(IV) reduction activities in aerobic bacteria.

Developing a Synthetic Biology Toolkit for Comamonas testosteroni, an Emerging Cellular Chassis for Bioremediation.

Synthetic biology is rapidly evolving into a new phase that emphasizes real-world applications such as environmental remediation. Recently, Comamonas testosteroni has become a promising chassis for bioremediation due to its natural pollutant-degrading capacity; however, its application is hindered by the lack of fundamental gene expression tools. Here, we present a synthetic biology toolkit that enables rapid creation of functional gene circuits in C. testosteroni. We first built a shuttle system that allows efficient circuit construction in E. coli and necessary phenotypic testing in C. testosteroni. Then, we tested a set of wildtype inducible promoters, and further used a hybrid strategy to create engineered promoters to expand expression strength and dynamics. Additionally, we tested the T7 RNA Polymerase-PT7 promoter system and reduced its leaky expression through promoter mutation for gene expression. By coupling random library construction with FACS screening, we further developed a synthetic T7 promoter library to confer a wider range of expression strength and dynamic characteristics. This study provides a set of valuable tools to engineer gene circuits in C. testosteroni, facilitating the establishment of the organism as a useful microbial chassis for bioremediation purposes.