Isolation and identification of a repressor TetR for 3,17β-HSD expressional regulation in Comamonas testosteroni

•Comamonas testosteroni harbours several genes for steroid degrading enzymes.•The genes code for hydroxysteroid dehydrogenases and are inducible by steroids.•We have identified a new regulatory gene (tetR) for 3,17β-HSD expression.•TetR works in concert with other cis- and trans-acting regulators.

Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a key enzyme in steroid degradation. Understanding the mechanism of 3,17β-HSD gene (βhsd) induction may help us to elucidate its complete molecular regulation. Sequencing the C. testosteroni ATCC11996 genome lead us to identify the tetR (522 bp) downstream of βhsd. Two repeat sequences (RS; 13 bp), that are separated to each other by 1661 bp, were found upstream of βhsd. A bioinformatic analysis revealed that TetR family proteins act as transcriptional repressors which are sensitive to environmental signals. Since, C. testosteroni responds to environmental steroid induction and upregulates steroid catabolic genes, we hypothesized that TetR might act in C. testosteroni as repressor for βhsd expression. The tetR was cloned into different plasmids, including an EGFP reporter system, for functional characterization and/or overexpression. The data indicate that, indeed, TetR acts as a repressor for 3,17β-HSD expression. Testosterone in turn, which is known to induce βhsd expression, could not resolve TetR repression. To further substantiate TetR as repressor for βhsd expression, a tetR gene knock-out mutant of C. testosteroni was generated. TetR gene knock-out mutants showed the same basal low level of βhsd expression as the C. testosteroni wild type cells. Interestingly, testosterone induction leads to a strong increase in βhsd expression, especially in the tetR gene knock-out mutants. The result with the knock-out mutant, in principle, supports our hypothesis that TetR is a repressor for βhsd expression, but the exact role of testosterone in this context remains unknown. Finally, it turned out that TetR is obviously also involved in the regulation of the hsdA gene.