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3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) fromComamonas 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 ofhsdA expression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking ofhsdA 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 inC. testosteroni has been identified. ThehsdR gene is located 2.58 kb downstream fromhsdA on theC. testosteroni ATCC 11996 chromosome with an orientation opposite that ofhsdA . ThehsdR 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 thehsdA gene, electrophoresis mobility shift assays showed that HsdR specifically binds to thehsdA 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, anhsdR knockout mutant expressed low levels of 3α-HSD/CR compared to that of wild-typeC. testosteroni after testosterone induction. In conclusion, HsdR is a positive transcription factor for thehsdA gene and promotes the induction of 3α-HSD/CR expression inC. testosteroni .

applied and environmental microbiology

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