Mycobacterium tuberculosis mtrA merodiploid strains with point mutations in the signal-receiving domain of MtrA exhibit growth defects in nutrient broth

The genetic and biochemical aspects of the essential Mycobacterium tuberculosis MtrAB two-component regulatory signal transduction (2CRS) system have not been extensively investigated. We show by bacterial two-hybrid assay that the response regulator (RR) MtrA and the sensor kinase MtrB interact. We further demonstrate that divalent metal ions [Mg(2+), Ca(2+) or both] promote MtrB kinase autophosphorylation activity, but only Mg(2+) promotes phosphotransfer to MtrA. Replacement of the conserved aspartic acid residues at positions 13 and 56 with alanine (D13A), glutamine (D56E) or asparagine (D56N) prevented MtrA phosphorylation, indicating that these residues are important for phosphorylation. The MtrA_D56E and MtrA_D13A proteins bound to the promoter of fbpB, the gene encoding antigen 85B protein, efficiently in the absence of phosphorylation, whereas MtrA_D56N did not. We also show that M. tuberculosis mtrA merodiploids overproducing MtrA_D13A, unlike cells overproducing wild-type MtrA, grow poorly in nutrient broth and show reduced expression of fbpB. These latter findings are reminiscent of a phenotype associated with MtrA overproduction during intramacrophage growth. Our results suggest that MtrA_D13A behaves like a constitutively active response regulator and that further characterization of mtrA merodiploid strains will provide valuable clues to the MtrAB system.