Oligomerization and negative autoregulation of the LysR-type transcriptional regulator HsdR from Comamonas testosteroni

Abstract

“3α-Hydroxysteroid dehydrogenase/carbonyl reductase regulator” (HsdR) from Comamonas testosteroni (C. testosteroni) was identified as a member of the LysR-type transcriptional regulator (LTTR) family. We have shown previously that HsdR activates the expression of the hsdA gene, encoding 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR), which is an important enzyme involved in the degradation of steroid compounds. Phylogenetic analysis indicated that HsdR is related to the contact-regulated gene A (CrgA) from Neisseria meningitidis, which exists as a homooctamer. Therefore, to further elucidate the regulatory mechanism of HsdR, we investigated the oligomeric state and autoregulation of this transcriptional factor in the present study. To identify the active domains of HsdR, three truncated forms, HsdRΔN (N-terminus deleted), HsdRΔC (C-terminus deleted), and HsdRΔNC (both N- and C-terminus deleted), were constructed and purified. 3α-HSD/CR expression was measured by ELISA to detect the function of HsdR. Functional and biochemical analyses of wild type HsdR and its truncated forms indicated that HsdR may exist as an oligomer where the central domain is crucial for its oligomerization. Gel filtration chromatography revealed that there are two dominant oligomer forms which may be octamers and hexamers. According to electrophoretic mobility shift assays, HsdR specifically binds to its own promoter, where it negatively regulates its own expression. Therefore, the expression of non-functional HsdR variants (an hsdR-gfp fusion mutant and a hsdR gene disrupted mutant) increased compared to the wild type strain, because autorepression of HsdR was prevented. As a consequence, 3α-HSD/CR expression in these hsdR mutant strains was impaired. Combined, in our study we provide evidence that the transcription factor HsdR is a component of the steroid degradation machinery in C. testosteroni, which is active as an oligomer and negatively regulates its own expression.