Cloning and Identification of a New Repressor of 3,17Β-Hydroxysteroid Dehydrogenase of Comamonas Testosteroni

3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme inthe metabolic pathway for steroid compounds catabolism in Comamonastestosteroni. Tetracycline repressor (TetR) family, repressors existing inmost microorganisms, may play key roles in regulating the expression of 3,17β-HSD. Previous reportsshowed that three tetR genes arelocated in the contig58 of C. testosteroni ATCC 11996 (GenBank:AHIL01000049.1), among which the first tetR gene encoded a potentialrepressor of 3,17β-HSD by sensing environmental signals. However, whether theother proposed tetR genes act asrepressors of 3,17β-HSD are still unknown. In the present study,we cloned the second tetR gene andanalyzed the regulatory mechanism of the protein on 3,17β-HSD usingelectrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-basedassay, and loss-of-function analysis. The results showed that the second tetRgene was 660-bp, encoding a 26 kD protein, which could regulate the expressionof 3,17β-HSD gene via binding to theconserved consensus sequences located 1100-bp upstream of the 3,17β-HSD gene. Furthermore, the mutantstrain of C. testosteroni with the second tetR gene knocked-out mutant expresses good biological geneticstability, and the expression of 3,17β-HSD in the mutant strain is slightlyhigher than that in the wild type under testosterone induction. The second tetR gene acts as a negative regulatorin 3,17β-HSD expression, and the mutant has potential application in bioremediation ofsteroids contaminated environment.