Abstract
3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17β-HSD. Previous reports showed that three tetR genes are located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first tetR gene encoded a potential repressor of 3,17β-HSD by sensing environmental signals. However, whether the other proposed tetR genes act as repressors of 3,17β-HSD are still unknown. In the present study, we cloned the second tetR gene and analyzed the regulatory mechanism of the protein on 3,17β-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second tetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17β-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17β-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second tetR gene knocked-out mutant expresses good biological genetic stability, and the expression of 3,17β-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction. The second tetR gene acts as a negative regulator in 3,17β-HSD expression, and the mutant has potential application in bioremediation of steroids contaminated environment.
Highlights
Comamonas testosteroni is a gram-negative bacterium with various steroid compounds or polycyclic aromatic hydrocarbons as its carbon source [12, 15]
The results showed that the second Tetracycline repressor (TetR) gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17βHSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17β-HSD gene
The mutant generated in this study can be used to treat environmental pollution caused by steroid hormones
Summary
Comamonas testosteroni is a gram-negative bacterium with various steroid compounds or polycyclic aromatic hydrocarbons as its carbon source [12, 15]. 3,17β-HSD is a member of short-chain dehydrogenase/reductase (SDR) superfamily, which can catalyze the conversion of hydroxyl groups of steroids into oxygen groups [16, 3]. TetR works as a homodimer and binds to two α-helix-β-turn-α-helix motifs (HTH) of two conserved DNA operators, blocking the expression of the target genes [10, 5, 14]. The regulatory network involved by the TetR family members can be simple or complex [14]. TetR inhibits membrane-associated protein (tetA) transcription by binding to the target operator upstream of the tetA gene, while the expression of the TetR family member is regulated by another regulator through a series of regulatory cascades [14]. The TetR family member can trigger a cell response to react to environmental signals [14]
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