RelZ-Mediated Stress Response in Mycobacterium smegmatis : pGpp Synthesis and Its Regulation

Abstract

Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate [collectively termed (p)ppGpp] to reprogram their cellular processes under stress. In mycobacteria, these alarmones govern a multitude of cellular phenotypes, such as cell division, biofilm formation, antibiotic tolerance, and long-term survival. Mycobacterium smegmatis possesses the bifunctional RelMsm as a (p)ppGpp synthetase and hydrolase. In addition, it contains a short alarmone synthetase MS_RHII-RSD (renamed RelZ), which contains an RNase H domain in tandem with the (p)ppGpp synthetase domain. The physiological functions of RelMsm have been well documented, but there is no clear picture about the cellular functions of RelZ in M. smegmatis. RelZ has been implicated in R-loop induced stress response due to its unique domain architecture. In this study, we elucidate the differential substrate utilization pattern of RelZ compared to that of RelMsm. We unveil the ability of RelZ to use GMP as a substrate to synthesize pGpp, thereby expanding the repertoire of second messengers known in mycobacteria. We have demonstrated that the pGpp synthesis activity of RelZ is negatively regulated by RNA and pppGpp. Furthermore, we investigated its role in biofilm formation and antibiotic tolerance. Our findings highlight the complex role played by the RelZ in cellular physiology of M. smegmatis and sheds light upon its functions distinct from those of RelMsm. IMPORTANCE Bacteria utilize nucleotide messengers to survive the hostile environmental conditions and the onslaught of attacks within the host. The second messengers guanosine tetraphosphate and pentaphosphate [(p)ppGpp] have a profound impact on the long-term survival, biofilm formation, antibiotic tolerance, virulence, and pathogenesis of bacteria. Therefore, understanding the stress response mechanism regulated by (p)ppGpp is essential for discovering inhibitors of stress response and potential drug targets. Mycobacterium smegmatis contains two (p)ppGpp synthetases: RelMsm and RelZ. Our study unravels the novel regulatory mechanisms of RelZ activity and its role in mediating antibiotic tolerance. We further reveal its ability to synthesize novel second messenger pGpp, which may have regulatory roles in mycobacteria.

The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p)ppGpp

“…Another understudied aspect of (p)ppGpp regulation is whether ppGpp and pppGpp, while commonly referred to and characterized as a single species, target the same or different cellular pathways 21 . In addition, there is evidence for potential existence of a third alarmone, guanosine-5′-monophosphate-3′-diphosphate (pGpp), since several small alarmone synthetases can synthesize pGpp in vitro 22 , 23 . However, the clear demonstration of pGpp in bacterial cells has been challenging.…”

Pleiotropic Effects of Bacterial Small Alarmone Synthetases: Underscoring the Dual-Domain Small Alarmone Synthetases in Mycobacterium smegmatis

“…Any R-loops formed are removed by the RNase HII and (p)ppGpp helps to destabilize the stalled RNA polymerase via an unknown mechanism to rescue the cells from replication stress ( Krishnan et al, 2016 ). In addition, RelZ mediates antibiotic tolerance in M. smegmatis but does not impact biofilm formation significantly ( Petchiappan et al, 2020 ).…”

“…The synthetic subdomain of RelZ is a tetramer in solution like the other solved structures of RelP and RelQ ( Steinchen et al, 2015 , 2018 , 2020 ; Steinchen and Bange, 2016 ). Petchiappan et al (2020) showed that RelZ prefers guanosine monophosphate (GMP) as a substrate and synthesizes pGpp. To understand the difference between pGpp and ppGp, the reaction mixture was treated with NaOH that hydrolyzes only pGpp.…”

“…From thin layer chromatography, it was shown that Rel hydrolyzes pGpp to GMP and pyrophosphate as evidenced by the comigration of the radiolabeled product with the purified pyrophosphate whereas RelZ showed weak hydrolysis. We found that ssRNA inhibits RelZ-mediated pGpp synthesis, but R-loop did not show any effect ( Petchiappan et al, 2020 ). The pGpp synthesis activity of the RelZ is inhibited by pppGpp whereas ppGpp and pGpp did not have significant effect.…”

“…Therefore, we infer that the cellular pppGpp levels determine the RelZ-mediated synthesis, whereas ssRNA and pppGpp carefully regulate it. The altered cell surface properties of Δ relZ strain indicated that RelZ plays a role in cell wall metabolism ( Petchiappan et al, 2020 ).…”