Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis
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RelMtb, a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, RelMtb has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel Mtb gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The RelMtb and its truncated derivatives were expressed from an arabinose inducible promoter (P BAD ), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188–430 residues) containing fragment are sufficient for RelMtb (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of RelMtb by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.
KeywordsMycobacterium tuberculosis Rel enzyme (p)ppGpp Stringent response RSD
We thank Prof. Siddhartha Roy for his constant support and encouragement. We are grateful to Dr. M. Cashel, National Institute of Health, Bethesda, Md., for the generous gift of E. coli strains CF1648 the CF1693. We thank T. Muruganandan, Pratap C. Koyal and Sib Prasad Sharma for their sincere technical assistance in this work. The work was supported by the research grant (MLP107) from the Council of Scientific and Industrial Research (CSIR), Government of India (GOI), New Delhi. SB and SDG are grateful for research fellowships from Indian Council of Medical Research, GOI, New Delhi and CSIR, respectively.
Conflict of interest
The authors declare that they have no conflict of interest.
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