Singh, N.; Basra, V.; Sharma, C.

The stringent response in bacteria is an important defense mechanism in which hyperphosphorylated forms of guanosine, also known as molecular alarmones, are synthesized by RelA/SpoT Homolog (RSH) proteins. Rel protein in Mycobacterium tuberculosis (RelMtb) also regulates expression of persistence or virulence associated genes. Loss of RelMtb leads to higher expression of few of the virulence and cell wall remodeling factors in addition to upregulating many secreted antigens and proteins. The Enhanced Intracellular Survival (MtbEis) protein is one of the upregulated virulence factors. Based on this information, the precise role of MtbEis, a GNAT family acetyltransferase, in the stringent response in M. tuberculosis was explored. To begin with, MtbEis has been confirmed to enhance the guanosine pentaphosphate (pppGpp) synthesis activity of RelMtb by acetylating RelMtb at K513. Next, the MtbEis gene was knocked out in Mtb. The deletion of MtbEis resulted in a compromised survival accompanied by elevated levels of rRNAs under starvation conditions. Furthermore, the reduced expression of RelMtb and subsequent decrease in pppGpp synthesis was also observed in Mtb{Delta}Eis cells. Complementation of Mtb{Delta}Eis with full length MtbEis restored the expression of RelMtb, rRNAs, pppGpp levels and survival of M. tuberculosis. However, MtbEis lacking acetyltransferase domain ({Delta}AT) failed to restore this, confirming the role of MtbEis mediated acetylation in regulating stringent response. In sum, our findings not only report the unexplored role of MtbEis in the starvation survival but also the acetylation of RelMtb as a novel mechanistic aspect of stringent response in M. tuberculosis. In addition, this being the first post translational modification (PTM) report on any of the bacterial Rel proteins opens up the field for the discovery of new PTMs of Rel proteins.