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 ofMycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His6-tagged proteins fromEscherichia coli. DevS201underwent 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 His395and Asp54as the sites of phosphorylation in DevS and DevR, respectively. Mutations in Asp8and Asp9residues, postulated to form the acidic Mg2+-binding pocket, and the invariant Lys104of DevR, abrogated phosphoryl transfer from DevS201to DevR. DevR–DevS was thus established as a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Expression of theRv3134c–devR–devSoperon was induced at the RNA level in hypoxic cultures ofM. tuberculosisH37Rv and was associated with an increase in the level of DevR protein. However, in adevRmutant 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 ofdevStranscripts was not associated with an increase in protein level in either wild-type or mutant strains, reflecting differential regulation of this locus during hypoxia.