Interplay of PhoP and DevR response regulators defines expression of the dormancy regulon in virulent Mycobacterium tuberculosis

Abstract

The DevR response regulator of <i>Mycobacterium tuberculosis</i> is an established regulator of the dormancy response in mycobacteria and can also be activated during aerobic growth conditions in avirulent strains, suggesting a complex regulatory system. Previously, we reported culture medium-specific aerobic induction of the DevR regulon genes in avirulent <i>M. tuberculosis</i> H37Ra that was absent in the virulent H37Rv strain. To understand the underlying basis of this differential response, we have investigated aerobic expression of the <i>Rv3134c-devR-devS</i> operon using <i>M. tuberculosis</i> H37Ra and H37Rv <i>devR</i> overexpression strains, designated as LIX48 and LIX50, respectively. Overexpression of DevR led to the up-regulation of a large number of DevR regulon genes in aerobic cultures of LIX48, but not in LIX50. To ascertain the involvement of PhoP response regulator, also known to co-regulate a subset of DevR regulon genes, we complemented the naturally occurring mutant <i>phoP_Ra</i> gene of LIX48 with the WT <i>phoP_Rv</i> gene. PhoP_Rv dampened the induced expression of the DevR regulon by >70-80%, implicating PhoP in the negative regulation of <i>devR</i> expression. Electrophoretic mobility shift assays confirmed phosphorylation-independent binding of PhoP_Rv to the <i>Rv3134c</i> promoter and further revealed that DevR and PhoP_Rv proteins exhibit differential DNA binding properties to the target DNA. Through co-incubations with DNA, ELISA, and protein complementation assays, we demonstrate that DevR forms a heterodimer with PhoP_Rv but not with the mutant PhoP_Ra protein. The study puts forward a new possible mechanism for coordinated expression of the dormancy regulon, having implications in growth adaptations critical for development of latency.