Determinants Outside the DevR C-Terminal Domain Are Essential for Cooperativity and Robust Activation of Dormancy Genes in Mycobacterium tuberculosis

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.