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Journal of Bacteriology, June 2008, p. 4301-4312, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.01308-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cooperative Binding of Phosphorylated DevR to Upstream Sites Is Necessary and Sufficient for Activation of the Rv3134c-devRS Operon in Mycobacterium tuberculosis: Implication in the Induction of DevR Target Genes ^,

Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India

Received 13 August 2007/ Accepted 8 March 2008

The DevR-DevS two-component system of Mycobacterium tuberculosis mediates bacterial adaptation to hypoxia, a condition believed to be associated with the initiation and maintenance of dormant bacilli during latent tuberculosis. The activity of the Rv3134c-devRS operon was studied in M. tuberculosis using several transcriptional fusions comprised of promoter regions and the gfp reporter gene under inducing and aerobic conditions. Aerobic transcription was DevR independent, while hypoxic induction was completely DevR dependent. The hypoxia transcriptional start point, T_H, was mapped at –40 bp upstream of Rv3134c. In contrast, the divergently transcribed Rv3135 gene was not induced under hypoxic conditions. DNase I footprinting and mutational analyses demonstrated that induction required the interaction of DevRP with binding sites centered at bp –42.5 and –63.5 relative to T_H. Binding to the distal site (D) was necessary to recruit another molecule of DevRP to the proximal site (P), and interaction with both sequences was essential for promoter activation. These sites did not bind to either unphosphorylated or phosphorylation-defective DevR protein, which was consistent with an essential role for DevRP in activation. Phosphorylated DevR also bound to three copies of the motif at the hspX promoter. The Rv3134c and hspX promoters have a similar architecture, wherein the proximal DevRP binding site overlaps with the promoter –35 element. A model for the likely mode of action of DevR at these promoters is discussed.

Published ahead of print on 21 March 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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