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At least 19 recordsLinked to original sources

PubMed2004

Identification of a novel steroid inducible gene associated with the beta hsd locus of Comamonas testosteroni.

Comamonas testosteroni is a soil bacterium, which can use a variety of steroids as carbon and energy source. Even if it can be estimated that the complete degradation of the steroid nucleus requires more than 20 enzymatic reactions, the complete molecular characterization of the genes encoding these steroid degradative enzymes as well as the genetic organization of them remain to be elucidated. We have previously reported the cloning and nucleotide sequence of two steroid-inducible genes, beta hsd and stdC encoding 3 beta-17 beta-hydroxysteroid dehydrogenase and a hypothetical protein respectively, located in both ends of a 3.2kb HindIII fragment. Herein, we report the cloning and characterization of another steroid-inducible gene, called sip48 (steroid inducible protein), located between these two genes. The analysis of Sip48 amino acid sequence predicts a protein of 438 amino acids with a molecular mass of 48.5 kDa. This protein bears high homology with conserved hypothetical proteins of unknown function described in Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Burkholderia fungorum, Shewanella oneidensis, Pseudomonas fluorescens and Thauera aromatica. The predicted protein shows a typical structure of a leader peptide at its N-terminus. A 48.5 kDa protein encoded by the recombinant plasmid was detected by SDS-PAGE analysis of in vitro [35S]-methionine labeled polypeptides. Analysis of gene expression indicates that Sip48 is tightly controlled at the transcriptional level by several steroid compounds. In addition, transcriptional analysis of sip48 and beta hsd in a sip48 mutant strain, indicates that both genes are transcribed as a polycistronic mRNA. lacZ transcriptional fusions integrated into the chromosome of C. testosteroni demonstrate that a steroid-inducible promoter located upstream of sip48 regulates the expression of both genes.

3-Hydroxysteroid Dehydrogenases↗

PubMed2003

Identification and characterization of a novel translational repressor of the steroid-inducible 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni.

Comamonas testosteroni 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-HSD/CR) is a key enzyme in the degradation of steroid compounds in soil and may therefore play a significant role in the bioremediation of hormonally active compounds in the environment. The enzyme is also involved in the degradation of the steroid antibiotic fusidic acid. In addition, 3 alpha-HSD/CR mediates the carbonyl reduction of non-steroidal aldehydes and ketones. Because the gene of 3 alpha-HSD/CR (hsdA) is inducible by steroids, we were interested in the mode of its molecular regulation. Recently, we could identify the first molecular determinant in procaryotic steroid signaling, i.e. a repressor protein (RepA), which acts as a negative regulator by binding to upstream operator sequences of hsdA, thereby blocking hsdA transcription. In this work, we identified and cloned a second novel regulator gene that we named repB. The gene locates 932 bp downstream from hsdA on the C. testosteroni chromosome with an orientation opposite to that of hsdA. The open reading frame of repB consists of 237 bp and translates into a protein of 78 amino acids that was found to act as a repressor that regulates hsdA expression on the translational level. Northern blot analysis, UV-cross linking, gel-shift assays, and competition experiments proved that RepB binds to a 16-nucleotide sequence downstream of AUG at the 5' end of the 3 alpha-HSD/CR mRNA, thereby blocking hsdA translation. Testosterone, on the other hand, was shown to specifically bind to RepB, thereby yielding the release of RepB from the 3 alpha-HSD/CR mRNA such that hsdA translation could proceed. Data bank searches with the RepB primary structure yielded a 46.2% identity to the regulator of nucleoside diphosphate kinase, a formerly unknown protein from Escherichia coli that can restore a growth defect in alginate production in Pseudomonas aeruginosa. In conclusion, the induction of hsdA by steroids in fact is a derepression where steroidal inducers bind to two repressor proteins, RepA and RepB, thereby preventing blocking of hsdA transcription and translation, respectively.

Alcohol Oxidoreductases↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed2001

Regulation of the steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni.

The Comamonas testosteroni 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene (hsdA) codes for an adaptive enzyme in the degradation of steroid compounds. However, no information was available on the molecular regulation of steroid-inducible genes nor on the mechanism of steroid signaling in procaryotes. We, therefore, investigated the cis- and trans-acting elements of hsdA expression to infer the mechanism of its molecular regulation by steroids. The gene was localized on a 5.257-kilobase EcoRI fragment of C. testosteroni chromosomal DNA. The promoter was characterized, and the transcriptional start site was identified. Two palindromic operator domains were found upstream of hsdA. A new gene coding for a trans-acting negative regulator (repressor A, RepA) of hsdA expression was characterized. The specific interaction between RepA, testosterone, and the operator domain is demonstrated. From our results we conclude that hsdA is under negative transcriptional control by an adjacent gene product (RepA). Accordingly, induction of hsdA by steroids in fact is a derepression, where steroidal inducers bind to the repressor, thereby preventing its binding to the hsdA operator.

Alcohol Oxidoreductases↗

PubMed2005

Identification of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, 4-hydroxy-2-oxohexanoic acid, and 2-hydroxyhexa-2,4-dienoic acid and related enzymes involved in testosterone degradation in Comamonas testosteroni TA441.

Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.

Bacterial Proteins↗

PubMed2001

Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441.

Comamonas testosteroni metabolizes testosterone as the sole carbon source via a meta-cleavage reaction. A meta-cleavage enzyme gene, tesB, was cloned from C. testosteroni TA441. The deduced N-terminal amino acid sequence of tesB matched that of the purified meta-cleavage enzyme which is induced in TA441 during growth on testosterone as the sole carbon source. The tesB-disrupted mutant did not show growth on testosterone, suggesting that tesB is necessary for TA441 to grow on testosterone. Downstream from tesB, three putative ORFs which encode products also necessary for growth of TA441 on testosterone were identified. The usual substrate of TesB is probably 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione. Although this compound was not identified in the gene disrupted mutants, accumulation of upstream metabolites of testosterone degradation, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione, was shown by TLC analysis.

Bacterial Proteins↗

PubMed1999

Genetic organization and characteristics of the 3-(3-hydroxyphenyl)propionic acid degradation pathway of Comamonas testosteroni TA441.

Comamonas testosteroni TA441 degrades 3-(3-hydroxyphenyl)propionate (3HPP) via the meta pathway. A gene cluster required for degradation of 3HPP was cloned from strain TA441 and sequenced. The genes encoding six catabolic enzymes, a flavin-type hydroxylase (mhpA), extradiol dioxygenase (mhpB), 2-keto-4-pentenoate hydratase (mhpD), acetaldehyde dehydrogenase (acylating) (mhpF), 4-hydroxy-2-ketovalerate aldolase (mhpE) and the meta cleavage compound hydrolase (mhpC), were found in this cluster, encoded in this order. mhpD and mhpF were separated by two genes, orf4 and orf5, which were not necessary for growth on 3HPP. The gene mhpR, encoding a putative transcriptional activator of the IcIR family, was located adjacent to mhpA in the opposite orientation. Disruption of the mhpB or mhpR genes affected growth on 3HPP or trans-3-hydroxycinnamate. The mhpB and mhpC gene products showed high specificity for 3-(2,3-dihydroxyphenyl)propionate (DHPP) and the meta cleavage compound produced from DHPP, respectively.

Aldehyde Oxidoreductases↗

PubMed2000

Arrangement and regulation of the genes for meta-pathway enzymes required for degradation of phenol in Comamonas testosteroni TA441.

Comamonas testosteroni TA441 degrades phenol by a meta-cleavage pathway after the occurrence of a spontaneous mutation that derepresses the aphKLMNOPQB operon encoding phenol hydroxylase and catechol 2,3-dioxygenase, the enzymes for the initial two steps of the degradation pathway. A gene cluster, aphCEFGHJI, encoding the meta-pathway enzymes for degradation of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates was found downstream of the aphK operon. The upstream operon and the downstream gene cluster were found to be separated by two open reading frames of unknown function and an oppositely oriented aphT gene, which is similar to regulatory genes for ortho-cleavage of catechol or chlorinated catechols. A promoter assay using an aphC::lacZ transcriptional fusion plasmid revealed that the aphC promoter activity is induced by both phenol and HMS. The phenol-dependent induction was mediated by AphR and the HMS-dependent induction was mediated by AphT. The aphC promoter in strain TA441 was not silenced, unlike the cases of the aphK and aphR promoters, and was highly induced by HMS.

Biodegradation, Environmental↗

PubMed2002

An AraC/XylS family member at a high level in a hierarchy of regulators for phenol-metabolizing enzymes in Comamonas testosteroni R5.

Comamonas testosteroni strain R5 expresses a higher level of phenol-oxygenating activity than any other bacterial strain so far characterized. The expression of the operon encoding multicomponent phenol hydroxylase (mPH), which is responsible for the phenol-oxygenating activity, is controlled by two transcriptional regulators, PhcS and PhcR, in strain R5. In this study, we identified a third transcriptional regulator for the mPH operon (PhcT) that belongs to the AraC/XylS family. While the disruption of phcT in strain R5 significantly reduced the expression of the mPH operon, it did not eliminate the expression. However, the disruption of phcT in strain R5 increased the expression of phcR. The phenol-oxygenating activity was abolished by the disruption of phcR, indicating that PhcT alone was not sufficient to activate the expression of the mPH operon. The disruption of phcS has been shown in our previous study to confer the ability of strain R5 to express the mPH operon in the absence of the genuine substrate for mPH. PhcT was not involved in the gratuitous expression. Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.

Bacterial Proteins↗

PubMed1994

Regulation of chloro- and methylphenol degradation in Comamonas testosteroni JH5.

Comamonas testosteroni JH5 was isolated from a mixed bacterial culture enriched on different chloro- and methylphenols. The strain completely mineralized a mixture consisting of 4-chlorophenol (4-CP) and 4-methylphenol (4-MP). During degradation of the mixture, 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and 4-chlorocatechol were detected as short-lived intermediates. Mineralization of 4-CP and that of 4-MP occurred successively and were accompanied by diauxic growth, whereas 4-CP and 2-methylphenol were mineralized simultaneously. It was ascertained that neither a reversible enzyme inhibition nor potential toxic intermediates caused the observed diauxie. Some facts support the hypothesis that the successive degradation of 4-CP and 4-MP is regulated on the level of transcription. C. testosteroni JH5 contained a meta-cleaving enzyme when pregrown on 4-CP and the isomeric monomethylphenols. Inactivation of this enzyme in the presence of 3-chlorocatechol was observed.

Biodegradation, Environmental↗

PubMed1997

Degradation of 4-Chlorophenol via the meta Cleavage Pathway by Comamonas testosteroni JH5.

Comamonas testosteroni JH5 used 4-chlorophenol (4-CP) as its sole source of energy and carbon up to a concentration of 1.8 mM, accompanied by the stoichiometric release of chloride. The degradation of 4-CP mixed with the isomeric 2-CP by resting cells led to the accumulation of 3-chlorocatechol (3-CC), which inactivated the catechol 2,3-dioxygenase. As a result, further 4-CP breakdown was inhibited and 4-CC accumulated as a metabolite. In the crude extract of 4-CP-grown cells, catechol 1,2-dioxygenase and muconate cycloisomerase activities were not detected, whereas the activities of catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-oxopent-4-enoate hydratase were detected. These enzymes of the meta cleavage pathway showed activity with 4-CC and with 5-chloro-2-hydroxymuconic semialdehyde. The activities of the dioxygenase and semialdehyde dehydrogenase were constitutive. Two key metabolites of the meta cleavage pathway, the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde) and 5-chloro-2-hydroxymuconic acid, were detected. Thus, our previous postulation that C. testosteroni JH5 uses the meta cleavage pathway for the complete mineralization of 4-CP was confirmed.

Journal Article↗

PubMed1997

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

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

Alcohol Dehydrogenase↗

PubMed2006

Metabolism of isovanillate, vanillate, and veratrate by Comamonas testosteroni strain BR6020.

In Comamonas testosteroni strain BR6020, metabolism of isovanillate (iVan; 3-hydroxy-4-methoxybenzoate), vanillate (Van; 4-hydroxy-3-methoxybenzoate), and veratrate (Ver; 3,4-dimethoxybenzoate) proceeds via protocatechuate (Pca; 3,4-dihydroxybenzoate). A 13.4-kb locus coding for the catabolic enzymes that channel the three substrates to Pca was cloned. O demethylation is mediated by the phthalate family oxygenases IvaA (converts iVan to Pca and Ver to Van) and VanA (converts Van to Pca and Ver to iVan). Reducing equivalents from NAD(P)H are transferred to the oxygenases by the class IA oxidoreductase IvaB. Studies using whole cells, cell extracts, and reverse transcriptase PCR showed that degradative activity and expression of vanA, ivaA, and ivaB are inducible. In succinate- and Pca-grown cells, there is negligible degradative activity towards Van, Ver, and iVan and little to no expression of vanA, ivaA, and ivaB. Growth on Van or Ver results in production of oxygenases with activity towards Van, Ver, and iVan and expression of vanA, ivaA, and ivaB. With iVan-grown cultures, ivaA and ivaB are expressed, and in assays with whole cells, production of the iVan oxygenase is observed, but there is little activity towards Van or Ver. In cell extracts, though, Ver metabolism is observed, which suggests that the system mediating iVan uptake in whole cells does not mediate Ver uptake.

Comamonas testosteroni↗

PubMed2006

Characterization of MobR, the 3-hydroxybenzoate-responsive transcriptional regulator for the 3-hydroxybenzoate hydroxylase gene of Comamonas testosteroni KH122-3s.

Comamonas testosteroni KH122-3s is an aerobic soil bacterium that utilizes 3-hydroxybenzoate as a sole carbon and energy source. In this strain, 3-hydroxybenzoate hydroxylase (MobA) acts on the initial step of the degradation to produce 3,4-dihydroxybenzoate, which is subsequently subjected to the meta-cleavage pathway leading to tricarboxylic acid cycle intermediates. Gene walking analysis of the upstream region of mobA revealed an open reading frame (mobR) that encodes a transcriptional regulator of the MarR family. Here, we report that MobR negatively regulates the expression of mobA, and that the repression is relieved by binding of 3-hydroxybenzoate, the substrate for MobA. A primer extension experiment was performed to determine the transcription start site for mobA and identified it at 83 bp upstream of the mobA start codon, accompanied by a typical sigma70-type promoter. The mobR gene was expressed in Escherichia coli cells and the recombinant product was purified to homogeneity. Gel mobility-shift assays and DNase I footprinting analyses indicated that MobR binds as a homodimer to an imperfect inverted repeat within the mobA-mobR intergenic region, with an apparent dissociation constant of 11.5(+/- 0.5) nM. The operator site is located between the start codon and the promoter region for mobA, suggesting that MobR functions as a transcriptional repressor for mobA expression. The results of effector-binding assays indicated that MobR, but not its isomers 4-hydroxybenzoate and salicylate, is released from the operator site by the addition of 3-hydroxybenzoate. This dissociation process is highly cooperative, with a Hill coefficient of approximately 2. In addition, CD spectroscopic studies demonstrated that MobR adopts two conformational states corresponding to the effector-bound and unbound forms. These results suggest that the MobR dimer possesses at least two effector-binding sites, and that the effector binding to MobR induces an allosteric conformational change required for dissociation of the protein-DNA complex.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1989

Degradation of p-toluenesulphonic acid via sidechain oxidation, desulphonation and meta ring cleavage in Pseudomonas (Comamonas) testosteroni T-2.

Pseudomonas (Comamonas) testosteroni T-2 completely converted p-toluenesulphonic acid (TS) or p-sulphobenzoic acid (PSB) to cell material, CO2 and sulphate, with growth yields of about 5 g protein (mol C)-1. PSB and sulphite were excreted as transient intermediates during growth in TS-salts medium. All reactions of a catabolic pathway involving sidechain oxidation and cleavage of the sulphonate moiety as sulphite were measurable in the soluble portion of cell extracts. Degradation of TS and PSB was inducible and apparently involved at least two regulons. TS was converted to p-sulphobenzyl alcohol in a reaction requiring NAD(P)H and 1 mol O2 (mol TS)-1. This alcohol was in an equilibrium (in the presence of NAD+) with p-sulphobenzaldehyde, which was converted to PSB in an NAD(P)+-dependent reaction. PSB was desulphonated to protocatechuic acid in a reaction requiring NAD(P)H and 1 mol O2 (mol PSB)-1. Experiments with 18 O2 confirmed involvement of a dioxygenase, because both atoms of this molecular oxygen were recovered in protocatechuate. Protocatechuate was converted to 2-hydroxy-4-carboxymuconate semialdehyde by a 4.5-dioxygenase.

Benzenesulfonates↗

PubMed1995

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

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

Base Sequence↗

PubMed2003

Regulation of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase in Comamonas testosteroni: function and relationship of two operators.

Comamonas testosteroni 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) is a key enzyme in the degradation of steroid compounds in soil, and may therefore play a significant role in the bioremediation of hormonally active substances in the environment. We previously reported the isolation of the 3alpha-HSD/CR gene (hsdA) from C. testosteroni and two repressor genes, repA and repB, for hsdA transcriptional and translational regulation, respectively. In this work, we found that the expression of 3alpha-HSD/CR is closely connected to the distance between two 10 bp operator elements (OP1 and OP2) to which the RepA protein binds and therefore blocks the transcription of hsdA. The two 10 bp palindromic operator sequences are located upstream of hsdA (at 0.935 kb and at 2.568 kb on an EcoRI fragment) and are separated by 1.644 kb. In order to elucidate how the distance between OP1 and OP2 influences the repression of hsdA expression, we used E. coli cells transformed with plasmids carrying a set of deletions between OP1 and OP2. Our theory that a 'loop-structure' between the two operators is formed, which strongly influences hsdA transcriptional regulation, was proved by the increasing amounts of 3alpha-HSD/CR expression when the distance between the operators was too small to form the 'loop' structure. Moreover, additional -3, -5 and -7 nt deletions in each construct, known to result in DNA rotations and leading to altered orientations between the two operator sequences, reveal strong influences on hsdA expression the shorter the distance between the operators was. Our results demonstrate that a cis-regulating 'stem-loop' operator system is an important determinant in the regulation of the 3alpha-HSD/CR gene in Comamonas testosteroni.

3-Hydroxysteroid Dehydrogenases↗

PubMed2001

Endotoxic properties of lipid A from Comamonas testosteroni.

The lipid A from Comamonas testosteroni has been isolated and its complete chemical structure determined [Iida, T., Haishima, Y., Tanaka, A., Nishijima, K., Saito, S. & Tanamoto, K. (1996). Eur J Biochem 237, 468-475]. In this work, the relationship between its chemical structure and biological activity was studied. The lipid A was highly homogeneous chemically and was characterized by the relatively short chain length (C(10)) of the 3-hydroxy fatty acid components directly bound to the glucosamine disaccharide backbone by either amide or ester linkages. The lipid A exhibited endotoxic activity in all of the assay systems tested (mitogenicity in mouse spleen cells; induction of tumour necrosis factor alpha release from both mouse peritoneal macrophages and mouse macrophage-like cell line J774-1, as well as from the human monocytic cell line THP-1; induction of nitric oxide release from J774-1 cells; Limulus gelation activity and lethal toxicity in galactosamine-sensitized mice) to the same extent as did 'Salmonella minnesota' lipid A or Escherichia coli LPS used as controls. The strong endotoxic activity of the C. testosteroni lipid A indicates that the composition of 3-hydroxydecanoic acid is not responsible for the low endotoxicity of the lipid A observed in members of the genus Rhodopseudomonas, as has previously been suggested. Furthermore, both the lack of a second acylation of the 3-hydroxy fatty acid attached at the 3' position, and the substitution of the hydroxyl group of the 3-hydroxy fatty acid attached at position 2, do not affect the manifestation of endotoxic activity or species specificity.

Animals↗