Characterization of the KstR2 regulator responsible of the lower cholesterol degradative pathway in Mycobacterium smegmatis.

The interaction of KstR2-dependent promoters of the divergon constituted by the MSMEG_6000-5999 and MSMEG_6001-6004 operons of Mycobacterium smegmatis which encode the genes involved in the lower cholesterol degradative pathway has been characterized. Footprint analyses have demonstrated experimentally for the first time that KstR2 specifically binds to an operator region of 29 nucleotides containing the palindromic sequence AAGCAAGNNCTTGCTT. This region overlaps with the -10 and -35 boxes of the putative P(6000) and P(6001) divergent promoters, suggesting that KstR2 represses their transcription by preventing the binding of the ribonucleic acid polymerase. A three-dimensional model of the KstR2 protein revealed a typical TetR-type regulator folding with two domains, a deoxyribonucleic acid (DNA)-binding N-terminal domain and a regulator-binding C-terminal domain composed by three and six helices respectively. KstR2 is an all alpha protein as confirmed by circular dichroism. We have determined that M. smegmatis is able to grow using sitolactone (HIL) as the only carbon source and that this compound induces the kstR2 regulon in vivo. HIL or its open form 5OH-HIP were unable to release in vitro the KstR2-DNA operator interaction, suggesting that 5OH-HIP-CoA or a further derivative would induce the lower cholesterol catabolic pathway.