Guangming Xiong

Identification and Characterization of a Novel Translational Repressor of the Steroid-inducible 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase Gene in Comamonas testosteroni

Comamonas testosteroni 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-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, 3α-HSD/CR mediates the carbonyl reduction of non-steroidal aldehydes and ketones. Because the gene of 3α-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 3α-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 3α-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.

Testosterone-inducible Regulator Is a Kinase That Drives Steroid Sensing and Metabolism in Comamonas testosteroni

The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3α-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3α-HSD/CR which was paralleled by an enhanced catabolism of testosterone. We also found that TeiR responds to a variety of different steroids other than testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity.

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%.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: biological significance, three-dimensional structure and gene regulation

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) catalyses the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralisation of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. The enzyme was found to be functional towards a variety of steroid substrates, including the steroid antibiotic fusidic acid. The enzyme also catalyses the carbonyl reduction of non-steroidal aldehydes and ketones such as a novel insecticide. It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. The 3alpha-HSD/CR gene (hsdA) is 774 base pairs long and the deduced amino acid sequence comprises 258 residues with a calculated molecular mass of 26.4 kDa. A homology search revealed 3alpha-HSD/CR as a new member of the short-chain dehydrogenase/reductase (SDR) superfamily. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein indicating a dimeric nature of 3alpha-HSD/CR. The protein was crystallised and the structure solved by X-ray analysis. The crystal structure reveals one homodimer per asymmetric unit, thereby verifying its dimeric nature. Dimerisation takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far, this type of intermolecular contact has exclusively been observed in homotetrameric SDRs, but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSD/CR by the presence of a predominantly alpha-helical subdomain, which is missing in all other SDRs of known structure. The promoter domain was localised within the 93 bp region upstream of hsdA and the transcriptional start site was identified at 28 bp upstream of the translation start site. Interestingly, hsdA expression was found to be under negative control by two repressor proteins, the genes of which were found in opposite direction downstream or overlapping with hsdA. Based on our results, we propose that induction of hsdA expression in C. testosteroni by steroids actually appears to be a de-repression by preventing the binding of repressor proteins to regulatory regions.

Characterization of the steroid degrading bacterium S19-1 from the Baltic Sea at Kiel, Germany

Steroid contamination of sea water is an ever growing problem and impacts population dynamics of all kinds of sea animals. We have long experience with the soil bacterium Comamonas testosteroni ATCC11996 which is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons, and which might be used in the bioremediation of contaminated places. In C. testosteroni about 20 enzymes which are involved in steroid degradation could be induced by 0.5 mM testosterone. In this work, we isolated a marine bacterial strain, S19-1, from the Baltic Sea at Kiel (Germany) which degrades steroids and is able to use steroids as carbon source. S19-1 was characterized as being Gram negative, and 16S rRNA analysis showed that it belongs to the genus Buttiauxella of the Enterobacteriaceae family. Strain S19-1 could be best grown in SIN medium supplemented with 0.6-4.1% NaCl at 20°C. As a carbon source, testosterone, estradiol or cholesterol in minimal medium could be used by S19-1. Moreover, S19-1 could grow up in medium with 50 μg/ml erythromycin. Plasmids pK18 and other plasmids were successfully transformed into S19-1 by chemical transformation (CaCl(2)) and shown to replicate in the cells. Currently, work is ongoing to find the steroid inducible genes.

Identification and Characterization of the LysR-Type Transcriptional Regulator HsdR for Steroid-Inducible Expression of the 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase Gene in Comamonas testosteroni

ABSTRACT 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni is a key enzyme in steroid degradation in soil and water. 3α-HSD/CR gene (hsdA) expression can be induced by steroids like testosterone and progesterone. Previously, we have shown that the induction of hsdA expression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking of hsdA transcription and translation, respectively (G. Xiong and E. Maser, J. Biol. Chem. 276:9961-9970, 2001; G. Xiong, H. J. Martin, and E. Maser, J. Biol. Chem. 278:47400–47407, 2003). In the present study, a new LysR-type transcriptional factor, HsdR, for 3α-HSD/CR expression in C. testosteroni has been identified. The hsdR gene is located 2.58 kb downstream from hsdA on the C. testosteroni ATCC 11996 chromosome with an orientation opposite that of hsdA. The hsdR gene was cloned and recombinant HsdR protein was produced, as was anti-HsdR polyclonal antibodies. While heterologous transformation systems revealed that HsdR activates the expression of the hsdA gene, electrophoresis mobility shift assays showed that HsdR specifically binds to the hsdA promoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore, in vitro binding assays indicated that HsdR can come into contact with RNA polymerase. As expected, an hsdR knockout mutant expressed low levels of 3α-HSD/CR compared to that of wild-type C. testosteroni after testosterone induction. In conclusion, HsdR is a positive transcription factor for the hsdA gene and promotes the induction of 3α-HSD/CR expression in C. testosteroni.

Steroid degradation and two steroid-inducible enzymes in the marine bacterium H5.

Natural and synthetic steroid hormones excreted into the environment are potentially threatening the population dynamics of all kinds of animals and public health. We have previously isolated a steroid degrading bacterial strain (H5) from the Baltic Sea, at Kiel, Germany. 16S-rRNA analysis showed that bacterial strain H5 belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria. Bacterial strain H5 can degrade steroids such as testosterone and estrogens, which was shown in this study by determining the (3)H labeled steroid retaining in the bacterial H5 culture medium at incubation times of 5 h and 20 h. Since 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) is a key enzyme in adaptive steroid degradation in Comamonas testosteroni (C. testosteroni), in previous investigations, a meta-genomic system with the 3α-HSD/CR gene as a positive control was established. By this meta-genomic system, two estradiol inducible genes coding 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase, respectively, which are involved in steroid degradation, were found in marine strain H5. In the present work, the 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase genes were subcloned into plasmids pET38-12 and pET24-17, respectively. Overexpression in Escherichia coli (E. coli) strain BL21(DE3)pLysS cells resulted in corresponding proteins with an N-terminal His-tag sequence. After induction with isopropyl-β-D-thiogalactoside, 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were purified in one step using nickel-chelate chromatography. After protein determination, 3-ketosteroid-delta-1-dehydrogenase (0.48 mg/ml) and carboxylesterase (1.28 mg/ml) were used to prepare antibodies to determine steroid binding specificity in future research. In summary, we have shown that the marine strain H5 could metabolize steroids; have isolated two estradiol inducible genes from strain H5 chromosomal DNA, and purified the corresponding proteins for further research. The exact characterization and systematic classification of the marine steroid degrading bacterial strain H5 is envisaged. The strain might be used for the bioremediation of steroid contaminations in seawater.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase as a tool for isolation and characterization of a new marine steroid degrading bacterial strain

Steroid contamination of sea water is an ever growing problem and impacts population dynamics of all kinds of sea animals. We have long experience with the soil bacterium Comamonas testosteroni which is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons, and which might be used in the bioremediation of contaminated soil. For our studies, we use 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-HSD/CR) as an indicator system, since it is a key enzyme in adaptive steroid degradation in C. testosteroni. In this work, we have isolated a new bacterium (strain H5) from the Baltic Sea at Kiel, Germany, which is able to degrade steroids like testosterone, estradiol, and cholesterol. Strain H5 was characterized as being a gram-negative bacterium that could be best grown in SIN medium supplemented with 1.6-4.1% NaCl. More than 80% of cholesterol was digested when strain H5 was grown in SIN medium containing 0.05 mM cholesterol. ELISA revealed that this salt water strain expresses a 3 alpha-HSD/CR orthologous enzyme. Interestingly, 3 alpha-HSD/CR expression increased after induction with testosterone and estradiol. According to our results, H5 is a new bacterial strain which might be used for the bioremediation of steroid contamination in sea water. Isolation of the 3 alpha-HSD/CR orthologous gene, as well as studies on its regulation are currently in progress. In addition, the exact characterization and systematic classification of this marine steroid degrading bacterial strain is envisaged.

Cis- and trans-regulatory elements of 3α-hydroxysteroid dehydrogenase/carbonyl reductase as biosensor system for steroid determination in the environment

3Alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni is a key enzyme in the degradation of steroids in the environment. The encoding gene, hsdA, is expressed only at very low levels in the absence of steroids, but undergoes a several fold induction in the presence of steroid substrates. In previous investigations, we have elucidated the mechanism of hsdA regulation that involves several activators and repressors. In the present study, the hsdA gene was replaced by the green fluorescent protein (GFP) gene which was inserted downstream from the hsdA regulatory region. By homologous integration into the chromosomal DNA, the C. testosteroni mutant strain CT-GFP5-1 was generated and used as fluorescence based biosensor system for steroid determination.With this cell-based system we could determine testosterone in a range between 57 and 450 pg/ml, estradiol between 1.6 and 12.8 pg/mland cholesterol between 19.3 and 15.4 pg/ml.. With the resulting cell-free system we could determine testosterone in a range between 28 and 219 pg/ml, estradiol between 0.029 and 0.430 pg/mg and cholesterol between 9.7 and 77.2 pg/ml [DOSAGE ERROR CORRECTED].The recovery ratio of the extraction was around 95% and the maximum fluorescence signals were obtained as early as after 30 min. Limitations of the established steroid biosensor system were quenching at higher steroid concentrations and the relatively high background of fluorescence, which are currently being improved in our lab. Combined, by exploiting the regulatory region of the gene hsdA that codes for the enzyme 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase we have constructed a mutant C. testosteroni strain that can be used as a sensitive biosensor system for steroid determination in the environment.

Identification of microRNAs as a potential novel regulatory mechanism in HSD11B1 expression

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1, gene HSD11B1) converts glucocorticoid receptor-inert cortisone to receptor-active cortisol. Multiple evidence supports a causal role for 11β-HSD1 in the current obesity epidemic. In obese, HSD11B1 expression is increased in adipose tissue but typically decreased in liver, and the underlying tissue-specific mechanisms are largely unknown. In this context, we investigated a potential role of microRNAs (miRNAs). We used several miRNA target prediction tools to identify possible candidates and a publicly available miRNA expression atlas to further select candidates expressed in hepatocytes. Using a dual luciferase reporter assay, we identified three potential miRNAs, hsa-miR-340, -561 and -579, as potential negative regulators of HSD11B1 expression. Disruption of the corresponding microRNA response elements abolished repression of luciferase activity for hsa-miR-561 and -579, but not for hsa-miR-340. Furthermore, levels of firefly luciferase mRNA were not changed by miR-561 and -579, indicating a mechanism based on translational repression rather than mRNA degradation. Finally, we were able to detect both, miR-561 and -579, in human total liver RNA by reverse-transcription-polymerase chain reaction (RT-PCR). According to the presented results, miR-561 and -579 are likely to be involved in the tissue-specific regulation of HSD11B1 expression. Moreover, literature findings and a pathway enrichment analysis support a potential role of these two miRNAs in glucocorticoid metabolism and signalling and associated diseases.