How Does the Alarmone ppGpp Change Bacterial Cell Metabolism? From Genome-wide Approaches to Structure to Physiology.

Wang et al. (2020) show that binding of the second messenger ppGpp to inosine-guanosine kinase (Gsk) in E. coli modulates the levels of the key metabolite phosphoribosyl pyrophosphate (pRpp), decreasing purine synthesis to favor amino acid synthesis during stress adaptation.

Tracking the homeostasis of second messenger cyclic-di-GMP in bacteria.

Cyclic-di-GMP (c-di-GMP) is an important second messenger in bacteria which regulates the bacterial transition from motile to sessile phase and also plays a major role in processes such as cell division, exopolysaccharide synthesis, and biofilm formation. Due to its crucial role in dictating the bacterial phenotype, the synthesis and hydrolysis of c-di-GMP is tightly regulated via multiple mechanisms. Perturbing the c-di-GMP homeostasis affects bacterial growth and survival, so it is necessary to understand the underlying mechanisms related to c-di-GMP metabolism. Most techniques used for estimating the c-di-GMP concentration lack single-cell resolution and do not provide information about any heterogeneous distribution of c-di-GMP inside cells. In this review, we briefly discuss how the activity of c-di-GMP metabolising enzymes, particularly bifunctional proteins, is modulated to maintain c-di-GMP homeostasis. We further highlight how fluorescence-based methods aid in understanding the spatiotemporal regulation of c-di-GMP signalling. Finally, we discuss the blind spots in our understanding of second messenger signalling and outline how they can be addressed in the future.

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

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 RelMsmIMPORTANCE 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.

Pup recycling regulates the proteasome.

The Pup proteasome system (PPS) in bacteria is equivalent to the eukaryotic ubiquitin proteasome system (UPS) that allows controlled protein degradation. Unlike the UPS, however, the PPS machinery and regulation is still poorly understood. In this issue of The FEBS Journal, Gur and colleagues combine experimental and modelling analyses to show how the PPS maintains steady-state levels of protein pupylation and consequently tightly controlled protein degradation.

Antibiotic Resistance: Current Perspectives.

Antibiotic resistance is one of the most serious challenges that the world is currently facing. The number of people succumbing to drug-resistant infections is increasing every day, but the rate of drug discovery has failed to match the requisite demands. Most of the currently known antibiotics target the three essential pathways of central dogma. However, bacteria have evolved multiple mechanisms to survive these antibiotics. Consequently, there is an urgent necessity to target auxiliary pathways for the discovery of new drugs. Metabolism-related and stress-associated pathways are ideal in this regard. The stringent response pathway regulated by the signaling nucleotides (p)ppGpp is an attractive target as inhibition of the pathway would in turn decrease the persistence and long-term survival of pathogenic bacteria. In this perspective, we focus on the recent design of small molecule analogues of (p)ppGpp that have yielded promising results in terms of growth and biofilm inhibition. Additionally, we discuss how targeting small RNAs and riboswitches, as well as antimicrobial peptides, would help combat drug-resistant infections in the near future.

R-loop induced stress response by second (p)ppGpp synthetase in Mycobacterium smegmatis: functional and domain interdependence.

Persistent R-loops lead to replicative stress due to RNA polymerase stalling and DNA damage. RNase H enzymes facilitate the organisms to survive in the hostile condition by removing these R-loops. MS_RHII-RSD was previously identified to be the second (p)ppGpp synthetase in Mycobacterium smegmatis. The unique presence of an additional RNase HII domain raises an important question regarding the significance of this bifunctional protein. In this report, we demonstrate its ability to hydrolyze R-loops in Escherichia coli exposed to UV stress. MS_RHII-RSD gene expression was upregulated under UV stress, and this gene deleted strain showed increased R-loop accumulation as compared to the wild type. The domains in isolation are known to be inactive, and the full length protein is required for its function. Domain interdependence studies using active site mutants reveal the necessity of a hexamer form with high alpha helical content. In previous studies, bacterial RNase type HI has been mainly implicated in R-loop hydrolysis, but in this study, the RNase HII domain containing protein showed the activity. The prospective of this differential RNase HII activity is discussed. This is the first report to implicate a (p)ppGpp synthetase protein in R-loop-induced stress response.

Quorum sensing and biofilm formation in mycobacteria: role of c-di-GMP and methods to study this second messenger.

Bacteria have evolved to survive the ever-changing environment using intriguing mechanisms of quorum sensing (QS). Very often, QS facilitates formation of biofilm to help bacteria to persist longer and the formation of such biofilms is regulated by c-di-GMP. It is a well-known second messenger also found in mycobacteria. Several methods have been developed to study c-di-GMP signaling pathways in a variety of bacteria. In this review, we have attempted to highlight a connection between c-di-GMP and biofilm formation and QS in mycobacteria and several methods that have helped in better understanding of c-di-GMP signaling.