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
ABSTRACT3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) fromComamonas testosteroniis 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 ofhsdAexpression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking ofhsdAtranscription 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. testosteronihas been identified. ThehsdRgene is located 2.58 kb downstream fromhsdAon theC. testosteroniATCC 11996 chromosome with an orientation opposite that ofhsdA. ThehsdRgene 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 thehsdAgene, electrophoresis mobility shift assays showed that HsdR specifically binds to thehsdApromoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore,in vitrobinding assays indicated that HsdR can come into contact with RNA polymerase. As expected, anhsdRknockout mutant expressed low levels of 3α-HSD/CR compared to that of wild-typeC. testosteroniafter testosterone induction. In conclusion, HsdR is a positive transcription factor for thehsdAgene and promotes the induction of 3α-HSD/CR expression inC. testosteroni.