PT Unknown
AU Gong, W
TI Characterization of the LysR-type Transcriptional Regulator HsdR Gene and Its Adjacent Short-chain Dehydrogenase/Reductase SDRx Gene in Comamonas testosteroni ATCC 11996
PY 2011
PU Christian-Albrechts-Universität zu Kiel
WP https://macau.uni-kiel.de/receive/diss_mods_00007303
LA en
DE Comamonas testosteroni; HsdR; SDRx; transcriptional regulation
AB 3a-hydroxysteroid dehydrogenase/carbonyl reductase (3a-HSD/CR) from Comamonas testosteroni (C. testosteroni) is a key enzyme in the degradation of steroid compounds in soil and water. Interestingly, 3a-HSD/CR gene (hsdA) expression can be induced by steroids like testosterone and progesterone. Thus, the regulatory mechanism of 3a-HSD/CR induction has attracted considerable attention of our group. Previously, it has been shown that induction of hsdA expression by steroids is a derepression where steroid inducers bind to two repressors, RepA and RepB, thereby preventing blocking of hsdA transcription and translation, respectively. 

In the present study, a new LysR-type transcriptional factor HsdR for 3a-HSD/CR expression in C. testosteroni was identified. The hsdR gene locates 2.58 kb downstream of the hsdA gene on the C. testosteroni ATCC 11996 chromosome with an orientation opposite to hsdA. The hsdR gene was cloned and the recombinant HsdR protein was overproduced, and an anti-HsdR polyclonal antibody was subsequently prepared. While heterologous transformation systems revealed that HsdR activates the expression of the hsdA gene, electrophoretic mobility shift assays (EMSA) showed that HsdR specifically binds to the hsdA promoter region. Furthermore, the activity of HsdR is dependent on the decreased repression by RepA. in vitro binding assays clearly indicated that HsdR can contact with RNA polymerase. Interestingly, an hsdR disrupted mutant expressed low levels of 3a-HSD/CR compared to wild type C. testosteroni after testosterone induction. In addition, HsdR itself cannot be induced by testosterone. As a member of LysR-type regulators, HsdR may also repress it own expression. Here, electrophoretic mobility shift assays indicated that HsdR specifically binds to its own promoter. As expected, mutated HsdR expression in an hsdR-gfp fusion mutant and an hsdR gene disrupted mutant of C. testosteroni increased compared to that in the wild type strain, largely because autorepression of HsdR in these mutants is prevented. This result revealed that HsdR negatively regulates its own expression. Phylogenetic analyses indicated that HsdR is related to the contact-regulated gene A (CrgA) from Neisseria meningitidis, which exists as an octamer. To detect the active form of HsdR, three truncated proteins, HsdRΔN (residues 1-86 deleted), HsdRΔC (residues 221-303 deleted), and HsdRΔNC (residues 1-86 and 221-303 deleted), were constructed and purified. These deleted domains are important for the positive control of HsdR on 3a-HSD/CR expression. Western blotting indicated that HsdR may also exist as an octamer, where the central domain is crucial for the multimerization of HsdR. Unexpectedly, gel filtration chromatography showed that there are two dominant oligomers (octamer and hexamer) present for HsdR and its truncated proteins. Taken together, HsdR is a positive transcription factor for 3a-HSD/CR expression in C. testosteroni, and it may also negatively regulate its own expression. 

In addition, a novel gene SDRx, which is divergently transcribed from the hsdR gene, was found to be a member of the short-chain dehydrogenase/reductase (SDR) superfamily. The open reading frame of this SDRx consists of 768 bp and translates into a protein of 255 amino acids. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 160-164 in the SDRx sequence) essential for catalytic activity of SDR proteins. Phylogenetic analyses indicated that the novel SDRx gene from C. testosteroni, which is active in steroid metabolism, is related to 7α-hydroxysteroid dehydrogenase (7α-HSD). Degradation of the steroids testosterone and estradiol decreased in the SDRx knock-out mutant strain. Furthermore, growth on the steroids cholic acid, estradiol and testosterone was impaired in the SDRx knock-out strain. Combined, the novel SDRx in C. testosteroni was identified as 7α-HSD that is involved in steroid degradation.
PI Kiel
ER