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The paper “Cell wall damage reveals spatial flexibility in peptidoglycan synthesis and a nonredundant role for RodA in mycobacteria” by Melzer et al. (E. S. Melzer, T. Kado, A. Garcia-Heredia, K. R. Gupta, et al., J Bacteriol 204:e00540-21, 2022, https://doi.org/10.1128/JB.00540-21) presents several new observations about the localization and function of cell wall enzymes in Mycobacterium smegmatis and their responses to stress. This work illustrates some important aspects of cell wall physiology in mycobacteria and also points to a new model for how peptidoglycan synthesis may be organized in pole-growing bacteria.

Mycobacterium tuberculosis is a human pathogen that can thrive inside the host immune cells for several years and cause tuberculosis. This is due to the propensity of M. tuberculosis to synthesize a sturdy cell wall, shift metabolism and growth, secrete virulence factors to manipulate host immunity, and exhibit stringent response. These attributes help M. tuberculosis to manage the host response, and successfully establish and maintain an infection even under nutrient-deprived stress conditions for years. In this review, we will discuss the importance of mycobacterial stringent response under different stress conditions. The stringent response is mediated through small signaling molecules called alarmones “(pp)pGpp”. The synthesis and degradation of these alarmones in mycobacteria are mediated by Rel protein, which is both (p)ppGpp synthetase and hydrolase. Rel is important for all central dogma processes—DNA replication, transcription, and translation—in addition to regulating virulence, drug resistance, and biofilm formation. Rel also plays an important role in the latent infection of M. tuberculosis. Here, we have discussed the literature on alarmones and Rel proteins in mycobacteria and highlight that (p)ppGpp-analogs and Rel inhibitors could be designed and used as antimycobacterial compounds against M. tuberculosis and non-tuberculous mycobacterial infections.

The bacterial second messengers (p)ppGpp and bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p)ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (Δ rel Msm ) or c-di-GMP synthesis (Δ dcpA ) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Δ rel Msm and Δ dcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis . Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis .

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis , both (p)ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel Msm and DcpA, encoded by rel Msm and dcpA genes, respectively. We have previously shown that the Δ rel Msm and Δ dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571–2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14 ). In this work, we have explored the phenotypes that are affected by both (p)ppGpp and c-di-GMP in mycobacteria. We have shown that both (p)ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Δ rel Msm and Δ dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p)ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis , thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p)ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p)ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Δ rel Msm and Δ dcpA strains are defective in biofilm formation. In this work, the overproduction of (p)ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p)ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p)ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.

Background Many studies on M. tuberculosis have emerged from using M. smegmatis MC2155 (Msm), since they share significant similarities and yet Msm is non-pathogenic and faster growing. Although several individual molecules have been studied from Msm, many questions remain open about its metabolism as a whole and its capability to be versatile. Adaptability and versatility are emergent properties of a system, warranting a molecular systems perspective to understand them. Results We identify feasible metabolic pathways in Msm in reference condition with transcriptome, phenotypic microarray, along with functional annotation of the genome. Together with transcriptome data, specific genes from a set of alternatives have been mapped onto different pathways. About 257 metabolic pathways can be considered to be feasible in Msm. Next, we probe cellular metabolism with an array of alternative carbon and nitrogen sources and identify those that are utilized and favour growth as well as those that do not support growth. In all, about 135 points in the entire metabolic map are probed. Analyzing growth patterns under these conditions, lead us to hypothesize different pathways that can become active in various conditions and possible alternate routes that may be induced, thus explaining the observed physiological adaptations. Conclusions The study provides the first detailed analysis of feasible pathways towards adaptability. We obtain mechanistic insights that explain observed phenotypic behaviour by studying gene-expression profiles and pathways inferred from the genome sequence. Comparison of transcriptome and phenome analysis of Msm and Mtb provides a rationale for understanding commonalities in metabolic adaptability. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0276-5) contains supplementary material, which is available to authorized users.