Regulation of testosterone degradation in Comamonas testosteroni

Recently, we have identified a gene encoding a LuxR-type factor, TeiR (Testosterone-inducible Regulator), which positively regulates steroid degradation in Comamonas testosteroni. Herein, we demonstrate that TeiR interacts in vivo with steroid catabolic gene promoters. The presence of testosterone induces a significant TeiR protein increase at the early logarithmic phase of growth. Interestingly, it is not until the early stationary phase where the activation of a steroid-inducible gene promoter is observed, indicating that testosterone might not be the true inductor of the steroid degradation pathway. In addition, beta-galactosidase expression driven by a testosterone-inducible promoter is prematurely activated in cells cultured in medium supplemented with ethyl acetate extracts obtained from the early stationary phase cell-free supernatants of C. testosteroni grown in presence of testosterone. Complementation experiments of C. testosteroni wild type performed with teiR deletion constructs indicate that extra-copies of deleted-TeiR exert a dominant negative effect on the wild-type TeiR protein. While, when C. testosteroni teiR mutants were used to carry out complementation assays only the full length gene can overcome the teiR mutant phenotype. Altogether these findings indicate that TeiR regulates steroid catabolic genes interacting with their promoters and suggest that this interaction requires the presence of a testosterone-derived metabolite to induce the system.     

Genetics of naphthalene and phenanthrene degradation by Comamonas testosteroni

Naphthalene and phenanthrene have long been used as model compounds to investigate the ability of bacteria to degrade polycyclic aromatic hydrocarbons. The catabolic pathways have been determined, several of the enzymes have been purified to homogeneity, and genes have been cloned and sequenced. However, the majority of this work has been performed with fast growing Pseudomonas strains related to the archetypal naphthalene-degrading P. putida strains G7 and NCIB 9816-4. Recently Comamonas testosteroni strains able to degrade naphthalene and phenanthrene have been isolated and shown to possess genes for polycyclic aromatic hydrocarbon degradation that are different from the canonical genes found in Pseudomonas species. For instance, C. testosteroni GZ39 has genes for naphthalene and phenanthrene degradation which are not only different from those found in Pseudomonas species but are also arranged in a different configuration. C. testosteroni GZ42, on the other hand, has genes for naphthalene and phenanthrene degradation which are arranged almost the same as those found in Pseudomonas species but show significant divergence in their sequences. Received 10 August 1997/ Accepted in revised form 15 August 1997     

Conjugative plasmids and the degradation of arylsulfonates in Comamonas testosteroni.

Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate. The appropriate genes are expressed in at least five regulatory units, some of which are also found in C. testosteroni PSB-4 (F. Junker, R. Kiewitz, and A. M. Cook, J. Bacteriol. 179:919-927, 1997). C. testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T. C. testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp). The type strain contained no plasmids. Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates. Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071. Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071. In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome. We presume the PSB genes to be located in a composite transposon. The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.     

Steroid degradation gene cluster of Comamonas testosteroni consisting of 18 putative genes from meta-cleavage enzyme gene tesB to regulator gene tesR

Steroid degradation genes of Comamonas testosteroni TA441 are encoded in at least two gene clusters: one containing the meta-cleavage enzyme gene tesB and ORF1, 2, 3; and another consisting of ORF18, 17, tesI, H, A2, and tesA1, D, E, F, G (tesA2 to ORF18 and tesA1 to tesG are encoded in opposite directions). Analysis of transposon mutants with low steroid degradation revealed 13 ORFs and a gene (ORF4, 5, 21, 22, 23, 25, 26, 27, 28, 30, 31, 32, 33, and tesR) involved in steroid degradation in the downstream region of ORF3. TesR, which is almost identical to that of TeiR, a positive regulator of Delta1-dehydrogenase (corresponds to TesH in TA441) and 3alpha-dehydrogenase (currently not identified in TA441), in C. testosteroni ATCC11996 (Pruneda-Paz, 2004), was shown to be necessary for induction of the steroid degradation gene clusters identified in TA441, tesB to tesR, tesA1 to tesG, and tesA2 to ORF18. At least some of the ORFs from ORF3 to ORF33 were suggested to be involved in 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid degradation.     

Genome sequence of Comamonas testosteroni ATCC 11996, a representative strain involved in steroid degradation

Comamonas testosteroni strains belong to the family of Comamonadaceae and are known for their ability to utilize steroid compounds as carbon source. Here, we present the draft genome sequence of strain ATCC 11996, with a G+C content of 61.48%.     

Mechanism for phenol tolerance in phenol-degrading Comamonas testosteroni strain

Comamonas testosteroni P15 and its mutant strain E23 can tolerate and utilize phenol as the sole source of carbon and energy at up to 15 mM and 20 mM, respectively. Compared to the wild type P15, mutant E23 showed higher values of K _s and K _i but a lower μ_max value, and had lower phenol hydroxylase and catechol 2,3-dioxygenase activities. Without phenol exposure, mutant E23 demonstrated a two-fold greater amount of cardiolipin than the wild type P15. Upon exposure to phenol, an increase in cardiolipin at the expense of phosphatidylethanolamine was observed in the wild type P15. However, there was no significant difference in major phospholipid contents between mutant E23 cells grown in the presence or absence of phenol. It was noted that the ratio of trans/cis fatty acids of phosphatidylethanolamine and cardiolipin in mutant E23 was 65–70% higher than that in the wild type P15. In the absence of phenol, the degree of saturation of cardiolipin in mutant E23 was 33% higher than that in wild type P15. In contrast to earlier findings, an increase in C16:1 9trans with a simultaneous decrease in C18:1 11cis instead of C16:1 9cis was observed in specific classes of phospholipids. Received: 30 July 1998 / Received last revision: 16 November 1998 / Accepted: 12 December 1998     

Gene encoding the hydrolase for the product of the meta-cleavage reaction in testosterone degradation by Comamonas testosteroni

In a previous study we isolated the meta-cleavage enzyme gene, tesB, that encodes an enzyme that carries out a meta-cleavage reaction in the breakdown of testosterone by Comamonas testeroni TA441 (M. Horinouchi et al., Microbiology 147:3367-3375, 2001). Here we report the isolation of a gene, tesD, that encodes a hydrolase which acts on the product of the meta-cleavage reaction. We isolated tesD by using a Tn5 mutant of TA441 that showed limited growth on testosterone. TesD exhibited ca. 40% identity in amino acid sequence with BphDs, known hydrolases of biphenyl degradation in Pseudomonas spp. The TesD-disrupted mutant showed limited growth on testosterone, and the culture shows an intense yellow color. High-pressure liquid chromatography analysis of the culture of TesD-disrupted mutant incubated with testosterone detected five major intermediate compounds, one of which, showing yellow color under neutral conditions, was considered to be the product of the meta-cleavage reaction. The methylation product was analyzed and identified as methyl-4,5-9,10-diseco-3-methoxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oate, indicating that the substrate of TesD in testosterone degradation is 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid. 4,5-9,10-Diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid was transformed by Escherichia coli-expressed TesD. Downstream of tesD, we identified tesE, F, and G, which encode for enzymes that degrade one of the products of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid converted by TesD.     

Folding mechanism of ketosteroid isomerase from Comamonas testosteroni

Ketosteroid isomerase (KSI) from Comamonas testosteroni is a homodimeric enzyme with 125 amino acids in each monomer catalyzing the allylic isomerization reaction at rates comparable to the diffusion limit. Kinetic analysis of KSI refolding has been carried out to understand its folding mechanism. The refolding process as monitored by fluorescence change revealed that the process consists of three steps with a unimolecular fast, a bimolecular intermediate, and most likely unimolecular slow phases. The fast refolding step might involve the formation of structured monomers with hydrophobic surfaces that seem to have a high binding capacity for the amphipathic dye 8-anilino-1-naphthalenesulfonate. During the refolding process, KSI also generated a state that can bind equilenin, a reaction intermediate analogue, at a very early stage. These observations suggest that the KSI folding might be driven by the formation of the apolar active-site cavity while exposing hydrophobic surfaces. Since the monomeric folding intermediate may contain more than 83% of the native secondary structures as revealed previously, it is nativelike taking on most of the properties of the native protein. Urea-dependence analysis of refolding revealed the existence of folding intermediates for both the intermediate and slow steps. These steps were accelerated by cyclophilin A, a prolyl isomerase, suggesting the involvement of a cis-trans isomerization as a rate-limiting step. Taken together, we suggest that KSI folds into a monomeric intermediate, which has nativelike secondary structure, an apolar active site, and exposed hydrophobic surface, followed by dimerization and prolyl isomerizations to complete the folding.     

Permanent draft genome sequence of Comamonas testosteroni KF-1

Comamonas testosteroni KF-1 is a model organism for the elucidation of the novel biochemical degradation pathways for xenobiotic 4-sulfophenylcarboxylates (SPC) formed during biodegradation of synthetic 4-sulfophenylalkane surfactants (linear alkylbenzenesulfonates, LAS) by bacterial communities. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,026,527 bp long chromosome (one sequencing gap) exhibits an average G+C content of 61.79% and is predicted to encode 5,492 protein-coding genes and 114 RNA genes.     

Characterization of Comamonas thiooxidans sp. nov., and Comparison of Thiosulfate Oxidation with Comamonas testosteroni and Comamonas composti

Comamonas thiooxidans (strain S23^T) capable of oxidizing thiosulfate under a mixotrophic growth condition was isolated from a sulfur spring. DNA–DNA homology study showed 55% similarity with Comamonas testosteroni KCTC2990^T and 52% with Comamonas composti LMG24008^T, the nearest phylogenetic relative (16S rRNA sequence similarity <97%). Comparative genomic fingerprinting by using ERIC and Rep-PCR further delineated species identity of the strain S23^T for which Comamonas thiooxidans sp. nov. is proposed. In addition, thiosulfate oxidation potential of the strain S23^T was compared with Comamonas testosteroni and Comamonas composti.     

Membrane topology of aspartate:alanine antiporter AspT from Comamonas testosteroni

We cloned the aspT gene encoding the L-aspartate:L-alanine antiporter AspTCt in Comamonas testosteroni genomic DNA. Analysis of the nucleotide sequence revealed that C. testosteroni has an asp operon containing aspT upstream of the l-aspartate 4-decarboxylase gene, and that the gene order of the asp operon of C. testosteroni is the inverse of that of Tetragenococcus halophilus. We used proteoliposomes to confirm the transport processes of AspTCt. To elucidate the two-dimensional structure of AspTCt, we analysed its membrane topology by means of alkaline phosphatase (PhoA) and beta-lactamase (BlaM) fusion methods. The fusion analyses revealed that AspTCt has seven transmembrane segments (TMs), a large cytoplasmic loop containing approximately 200 amino acid residues between TM4 and TM5, a cytoplasmic N-terminus, and a periplasmic C-terminus. These results suggest that the orientation of the N-terminus of AspTCt differs from that of tetragenococcal AspT, even though these two AspT orthologues catalyse the same transport reactions.     

A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Delta(1)-dehydrogenase and 3-ketosteroid-Delta(4)(5alpha)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9alpha-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.     

Comamonas testosteroni bacteremia in a patient with perforated acute appendicitis. Short communication

Comamonas testosteroni is an uncommon isolate in the clinical laboratory as a human pathogen. C. testosteroni most commonly emerges in abdominal pathologies especially in perforated appendicitis. In Turkey we report first time a case of bacteremia due to this organism, in a 22-year-old man with perforated acute appendicitis. The organism was shown to be susceptible to routine antibiotics so it was easily eliminated even after having caused a bacteremia.     

Identification of a novel Comamonas testosteroni gene encoding a steroid-inducible extradiol dioxygenase

Insulin-like growth factor-1 (IGF-1) both promotes survival and activates protein synthesis in neurons. In the present paper, we investigate the effect of IGF-1 treatment on cap-dependent translation in primary cultured neuronal cells. IGF-1 treatment increased the phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein 1 (4E-BP1), exclusively at Thr-36 and Thr-45 residues, and eIF-4G phosphorylation at Ser-1108. In contrast, a significant eIF-4E dephosphorylation was found. In parallel, increased eIF-4E/4G assembly and protein synthesis activation in response to IGF-1 treatment were observed. The phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, but not the mitogen-activated protein kinase (MAPK)-activating kinase (MEK) inhibitor PD98059, reversed the IGF-1-induced effects observed on eIF-4E/4G assembly and phosphorylation status of 4E-BP1, eIF-4E, and eIF-4G. Therefore, our findings show that the IGF-1-induced regulation of cap-dependent translation is largely dependent on the PI-3K and mTOR pathway in neuronal cells.     

Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni

Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis.     

睾酮丛毛单胞菌JA1菌株羟基化烟酸产生6-羟基烟酸的发酵产酶条件研究

睾酮丛毛单胞菌Comamonas testosteroni JA1是一株具有氰基吡啶羟基化酶活性的菌株,研究表明它还具有很高的烟酸羟基化酶的活性,是未报道过的具有烟酸羟基化酶活性的新菌种。该菌最适生长和产酶的碳源为1%葡萄糖、氮源为1%蛋白胨、诱导物为1%烟酸,此外,它的发酵条件优越,在温度25~37℃、初始pH6.5~7.0、装液量(100mL锥形瓶)10mL~40mL范围内,产酶能力均保持很高水平,很具有工业化生产应用的前景。