DevR-DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR

Abstract

Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS₂₀₁) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of Mycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His₆-tagged proteins from Escherichia coli. DevS₂₀₁ underwent autophosphorylation and participated in rapid phosphotransfer to DevR in a Mg²⁺-dependent manner. Chemical stability analysis and site-directed mutagenesis implicated the highly conserved residues His³⁹⁵ and Asp⁵⁴ as the sites of phosphorylation in DevS and DevR, respectively. Mutations in Asp⁸ and Asp⁹ residues, postulated to form the acidic Mg²⁺-binding pocket, and the invariant Lys104 of DevR, abrogated phosphoryl transfer from DevS₂₀₁ to DevR. DevR-DevS was thus established as a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Expression of the Rv3134c-devR-devS operon was induced at the RNA level in hypoxic cultures of M. tuberculosis H37Rv and was associated with an increase in the level of DevR protein. However, in a devR mutant strain expressing the N-terminal domain of DevR, induction was observed at the level of RNA expression but not at that of protein. DevS was translated independently of DevR and induction of devS transcripts was not associated with an increase in protein level in either wild-type or mutant strains, reflecting differential regulation of this locus during hypoxia.