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

The DevR response regulator of Mycobacterium tuberculosis 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 M. tuberculosis 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 Rv3134c-devR-devS operon using M. tuberculosis H37Ra and H37Rv devR 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 phoP(Ra) gene of LIX48 with the WT phoP(Rv) gene. PhoP(Rv) dampened the induced expression of the DevR regulon by >70-80%, implicating PhoP in the negative regulation of devR expression. Electrophoretic mobility shift assays confirmed phosphorylation-independent binding of PhoP(Rv) to the Rv3134c 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.