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

Abstract

The genetic and biochemical aspects of the essential Mycobacterium tuberculosis MtrAB twocomponent 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.

Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression

“…We recently reported that the MtrB fragment containing the C-terminal 334 residues (amino acid 234 to the end, designated as MtrBsol) is soluble and competent for exhibiting autophosphorylation and transphosphorylation activities (14). We found that the MtrBsol-GFP protein localized to midcell sites in M. smegmatis (Msmeg-Ptet::mtrBsol-gfp) but also showed an overall diffuse fluorescence throughout the cell ( Fig.…”

“…MVM877-MVM878 primers were used to amplify the mtrBsol coding regions of mtrB, mtrB H305D , and mtrB H305Y , and these were cloned into the pMALc4E vector to express the corresponding maltose-binding protein (MBP) fusions (see pDS21, pSVM16, and pSVM17 in supplemental Table S1). MtrA Y102C was created by site-specific mutagenesis using Y102C-F and Y102C-R primers essentially as described for MtrA D13A (14). The coding regions of mtrA D13A and mtrA Y102C were cloned into pJFR19 and pMALc4E (14).…”

“…MtrA Y102C was created by site-specific mutagenesis using Y102C-F and Y102C-R primers essentially as described for MtrA D13A (14). The coding regions of mtrA D13A and mtrA Y102C were cloned into pJFR19 and pMALc4E (14).…”

“…Purification of FtsZ, MtrB, and MtrA Proteins-His-FtsZ TB , MBP fusion derivatives of MtrA WT , MtrA D13A , MtrA Y102C , soluble MtrB, MtrB H305D , and MtrB H305Y , and His-MtrA D13A were purified following the protocols as previously described (14,17). Preliminary experiments indicated that MBPMtrA D13A was not active, whereas His-MtrA D13A was active; hence, His-MtrA D13A was used to evaluate the promoter DNA binding experiments.…”

“…Phosphorylation Assay-Phosphorylation of WT and mutant MtrB proteins (5 M) with [␥-32 P]ATP was performed essentially as described (14).…”