| The two-component regulatory systems (2CRs) are the most prevalent signal transduction systems in bacteria. Signal transduction by 2CRS comprised of a membrane-bound sensor kinase and a cytosolic response regulator, play a central role in the regulation of various cellular processes. The MtrAB two-component regulatory system is essential for Mycobacterium tuberculosis (M. tb) survival. With a goal towards of understanding the roles of the M. tb, MtrAB 2CRSs and regulatory interactions among them, we created six different MtrA mutant proteins containing mutations in the receiver domain. Site-directed mutagenesis was used to create the desired mutations and the resulting mutant proteins were expressed and purified from E. coli . The following mutant proteins were created: D13A, D56E, Y102C, D13AD56N, D13AD56E, and D13AY102C. We examined these mutant proteins with respect to their ability to be phosphorylated using the heterologus EnvZ kinase from E. coli. We found that with the exception of MtrA Y102C, proteins MtrAD13A, MtrAD56N, MtrA D56E, MtrAD13AD56N MtrAD13AD56E, and MtrA D13AY102C were phosphorylation defective as compared to the MtrA WT protein. The data generated in this project clearly showed that aspartic acid residue at positions 13 and 56 of the receiver domain were critical for MtrA phosphorylation and that affecting the intra-domain interactions by mutating the tyrosine residue at position 102 to cysteine did not affect the phosphorylation potential of MtrA. We further examined if the mutations in the MtrA protein that affect its phosphorylation also affect binding to one of its targets, namely, Ag85B promoter. This work confirmed and expanded MtrA interactions with its target, the Ag85B promoter. Our data indicated that the MtrA WT and the mutant proteins bound to the Ag85B promoter, but with differences in their binding patterns, with most of them binding less efficiently than the wild type. Using size exclusion Chromatography analysis, the MtrA WT, MtrAD56N and MtrAD13AD56N, MtrAD56E, and MtrAD13AD56E were found to be 100% monomer. However, MtrA D13A and MtrAD13AD56E preparations contained a mixture of monomer and dimer at the following ratios: 63.3%:36.7% for MtrAD13A; 57%:43% for MtrAD13AD56E. Our results revealed that the potential mutations in the receiver domain of MtrA had varying effects on the oligomeric forms of these proteins. Finally we examined the interaction between MtrA and MtrB. Pull-Down assay and Western blot analysis indicated distinct interactions between MtrA and MtrB of M. tb and the presence of a mutation at either "Asp13", "Asp56", or "Tyr102" of MtrA did not affect its interaction with MtrB, although we found differences with respect to the ratio of MtrA to MtrB in the various complexes.;These data will set a stage for understanding the roles of the essential MtrAB 2CRS in M. tb. This knowledge can potentially be used to develop a new generation of antibiotics targeted against M. tb infections. |
