Powerful induction of divergent tgs1-Rv3131 genes in Mycobacterium tuberculosis is mediated by DevR interaction with a high-affinity site and an adjacent cryptic low-affinity site

Abstract

DevR activates the transcription of ∼48 genes in response to hypoxia and other stresses and triggers metabolic downshift and dormancy development in Mycobacterium tuberculosis. tgs1 and Rv3131 encode triacylglycerol synthase and a putative nitroreductase, respectively, and both are members of the DevR regulon. This study aimed to understand how a single putative DevR binding site identified previously could sustain powerful induction of divergent tgs1-Rv3131 genes. DNase I footprinting revealed that phosphorylated DevR in fact binds to two sites symmetrically located at −42.5 and −63.5 bp from transcription start points of both genes. DevR first bound to the high-affinity site, P, and cooperatively recruited another DevR molecule to the secondary low-affinity site, S, to activate tgs1-Rv3131 transcription by ~210- and ~110-fold, respectively. The presence of a single P site significantly reduced activation of tgs1 expression and abolished Rv3131 activity, reinforcing the requirement of two binding sites for robust expression in both directions. P site inversion abolished tgs1 but not Rv3131 transcription despite DevR occupancy at both sites. The lack of tgs1 expression is most likely due to disruption of its −35 promoter element rather than inversion of the binding site per se. We conclude that (i) an overlap of a DevR binding site and −35 sequence is indispensable for promoter activation, (ii) DevR interaction with two binding sites is obligatory for synergistic activation of tgs1-Rv3131 promoters, and (iii) DevR interaction with binding sites of different affinities offers scope for temporal and differential expression of target genes.