Article

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

Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, TX 75708, USA.
Plasmid (Impact Factor: 1.76). 02/2011; 65(3):210-8. DOI: 10.1016/j.plasmid.2011.01.002
Source: PubMed

ABSTRACT The genetic and biochemical aspects of the essential Mycobacteriumtuberculosis 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 [Mg²+, Ca²+ or both] promote MtrB kinase autophosphorylation activity, but only Mg²+ 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.tuberculosismtrA 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.

Download full-text

Full-text

Available from: Malini Rajagopalan, Dec 19, 2014
0 Followers
 · 
99 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The mechanisms responsible for activation of the MtrAB two-component regulatory signal transduction system, which includes sensor kinase MtrB and response regulator MtrA, are unknown. Here, we show that an MtrB-GFP fusion protein localized to the cell membrane, the septa, and the poles in Mycobacterium tuberculosis and Mycobacterium smegmatis. This localization was independent of MtrB phosphorylation status but dependent upon the assembly of FtsZ, the initiator of cell division. The M. smegmatis mtrB mutant was filamentous, defective for cell division, and contained lysozyme-sensitive cell walls. The mtrB phenotype was complemented by either production of MtrB protein competent for phosphorylation or overproduction of MtrA(Y102C) and MtrA(D13A) mutant proteins exhibiting altered phosphorylation potential, indicating that either MtrB phosphorylation or MtrB independent expression of MtrA regulon genes, including those involved in cell wall processing, are necessary for regulated cell division. In partial support of this observation, we found that the essential cell wall hydrolase ripA is an MtrA target and that the expression of bona fide MtrA targets ripA, fbpB, and dnaA were compromised in the mtrB mutant and partially rescued upon MtrA(Y102C) and MtrA(D13A) overproduction. MtrB septal assembly was compromised upon FtsZ depletion and exposure of cells to mitomycin C, a DNA damaging agent, which interferes with FtsZ ring assembly. Expression of MtrA targets was also compromised under the above conditions, indicating that MtrB septal localization and MtrA regulon expression are linked. We propose that MtrB septal association is a necessary feature of MtrB activation that promotes MtrA phosphorylation and MtrA regulon expression.
    Journal of Biological Chemistry 05/2012; 287(28):23887-99. DOI:10.1074/jbc.M112.346544 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The MtrAB histidine-aspartate signal transduction of mycobacteria includes the response regulator MtrA and sensor kinase MtrB. We recently showed that Mycobacterium smegmatis ΔmtrB is filamentous, defective for cell division, cell shape maintenance and shows compromised MtrA target gene expression. Interestingly, overproduction of phosphorylation competent M. tuberculosis MtrAY102C reverses the ΔmtrB mutant phenotype, although the genetic basis of phenotype reversal is unknown. Here we show that introduction of D56N mutation in MtrAY102C completely abolished its phosphorylation potential yet the double mutant protein retained a partial ability to reverse the mtrB mutant phenotype indicating that phosphorylation activity is not necessary for the function of MtrAY102C. The phosphorylation-defective MtrAD56N-Y102C protein bound its target promoters ripA and fbpB efficiently. Together, these results support a hypothesis that the gain-of-function phenotype of MtrAY102C is in part due to its ability to function as a constitutively active protein in the absence of phosphorylation.
    Tuberculosis (Edinburgh, Scotland) 12/2013; 93S:S28-S32. DOI:10.1016/S1472-9792(13)70007-6 · 3.50 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The septal association of Mycobacterium tuberculosis MtrB, the kinase partner of the MtrAB two-component signal transduction system, is necessary for the optimal expression of the MtrA-regulon targets, including ripA, fbpB and ftsI which are involved in cell division and cell-wall synthesis. Here, we show that MtrB, irrespective of its phosphorylation status, interacts with Wag31, whereas only phosphorylation-competent MtrB interacts with FtsI. We provide evidence that FtsI-depletion compromises the MtrB-septal assembly and MtrA-regulon expression; likewise, the absence of MtrB compromises FtsI localization and, possibly, FtsI activity. We conclude from these results that FtsI and MtrB are codependent for their activities and that FtsI functions as a positive modulator of MtrB activation and MtrA-regulon expression. In contrast to FtsI, Wag31-depletion does not affect MtrB-septal assembly and MtrA-regulon expression, whereas the loss of MtrB increased Wag31 localization and the levels of PknA/PknB serine-threonine protein kinase-mediated Wag31 phosphorylation (Wag31∼P). Interestingly, we found that FtsI decreased Wag31∼P and that MtrB interacted with PknA/B. Overall, our results indicate that MtrB interactions with FtsI, Wag31 and PknA/B are required for its optimal localization, MtrA-regulon expression and Wag31∼P. Our results emphasize a new role for MtrB in cell division and cell wall synthesis distinct from that regulating the MtrA phosphorylation activities.
    Journal of Bacteriology 09/2014; 196(23). DOI:10.1128/JB.01795-14 · 2.69 Impact Factor