Identification and Characterization of a Novel Translational Repressor of the Steroid-inducible 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase Gene in Comamonas testosteroni

Abstract

Comamonas testosteroni 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) is a key enzyme in the degradation of steroid compounds in soil and may therefore play a significant role in the bioremediation of hormonally active compounds in the environment. The enzyme is also involved in the degradation of the steroid antibiotic fusidic acid. In addition, 3α-HSD/CR mediates the carbonyl reduction of non-steroidal aldehydes and ketones. Because the gene of 3α-HSD/CR (hsdA) is inducible by steroids, we were interested in the mode of its molecular regulation. Recently, we could identify the first molecular determinant in procaryotic steroid signaling, i.e. a repressor protein (RepA), which acts as a negative regulator by binding to upstream operator sequences of hsda, thereby blocking hsdA transcription. In this work, we identified and cloned a second novel regulator gene that we named RepB. The gene locates 932 bp downstream from hsdA on the C. testosteroni chromosome with an orientation opposite to that of hsdA. The open reading frame of RepB consists of 237 bp and translates into a protein of 78 amino acids that was found to act as a repressor that regulates hsdA expression on the translational level. Northern blot analysis, UV-cross linking, gel-shift assays, and competition experiments proved that RepB binds to a 16-nucleotide sequence downstream of AUG at the 5' end of the 3α-HSD/CR mRNA, thereby blocking hsdA translation. Testosterone, on the other hand, was shown to specifically bind to RepB, thereby yielding the release of RepB from the 3α-HSD/CR mRNA such that hsdA translation could proceed. Data bank searches with the RepB primary structure yielded a 46.2% identity to the regulator of nucleoside diphosphate kinase, a formerly unknown protein from Escherichia coli that can restore a growth defect in alginate production in Pseudomonas aeruginosa. In conclusion, the induction of hsdA by steroids in fact is a derepression where steroidal inducers bind to two repressor proteins, RepA and RepB, thereby preventing blocking of hsdA transcription and translation, respectively.