Determinants outside the DevR C-terminal domain are essential for cooperativity and robust activation of dormancy genes in Mycobacterium tuberculosis

Abstract

Background: DevR (also called as DosR) is a two-domain response regulator of the NarL subfamily that controls dormancy adaptation of Mycobacterium tuberculosis (M. tb). In response to inducing signals such as hypoxia and ascorbic acid, the N-terminal receiver domain of DevR (DevR_N) is phosphorylated at Asp54. This results in DevR binding to DNA via its C-terminal domain (DevR_C) and subsequent induction of the DevR regulon. The mechanism of phosphorylation-mediated activation is not known. The present study was designed to understand the role of the N- and C-terminal domains of DevR in DevR regulon genes activation. Methodology/Principal Findings: Towards deciphering the activation mechanism of DevR, we compared the DNA binding properties of DevR_C and DevR and correlated the findings with their ability to activate gene expression. We show that isolated DevR_C can interact with DNA, but only with the high affinity site of a representative target promoter. Therefore, one role of DevR_N is to mask the intrinsic DNA binding function of DevR_C. However, unlike phosphorylated DevR, isolated DevR_C does not interact with the adjacent low affinity binding site suggesting that a second role of DevR_N is in cooperative binding to the secondary site. Transcriptional analysis shows that consistent with unmasking of its DNA binding property, DevR_C supports the aerobic induction, albeit feebly, of DevR regulon genes but is unable to sustain gene activation during hypoxia. Conclusions/Significance: DevR is a unique response regulator that employs a dual activation mechanism including relief of inhibition and cooperative interaction with binding sites. Importantly, both these functions reside outside the C-terminal domain. DevR_N is also essential for stabilizing DevR and sustaining autoregulation under hypoxia. Hence, both domains of DevR are required for robust transcription activation.