MtrAB is one of the important two-component regulatory systems (2CRS) in mycobacteria and consists of MtrB sensor kinase and MtrA response regulator. Mycobacterium smegmatis mtrB mutant is filamentous and shows defects in cell division, cell shape and optimal expression of the MtrA-regulon. In an effort to identify M. tuberculosis sensor kinases that work with MtrA and/or bypass the need for MtrB, we attempted to overexpress other M. tuberculosis sensor kinases in M. smegmatis mtrB background and reverse the mtrB phenotype. Overexpression of trcS kinase, but not nine others tested, reversed the mtrB mutant phenotype including the expression of the MtrA-regulon. However, the overexpression of trcS kinase did not reverse the mutant phenotype of a mtrA mutant. Bacterial-two hybrid assays revealed that the TrcS kinase interacts with both MtrB kinase and the response regulator MtrA. Recombinant TrcS protein exhibits autophosphorylation and transphosphorylation of its cognate response regulator TrcR, and MtrA. Together, these results support a model involving cross-talk between the MtrAB and TrcRS two-component systems.