Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

Abstract

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 ⌬rel Msm and ⌬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 ⌬rel Msm and ⌬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. IMPORTANCEOur 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 ⌬rel Msm and ⌬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. N ucleotide second messengers are utilized across all domains of life (1, 2). To counter innumerable threats and to regulate physiological functions, bacteria exploit a repertoire of signaling nucleotides, (p)ppGpp, cyclic di-GMP (c-di-GMP), c-di-AMP, cGMP, and cAMP (1-3). Each second messenger signaling module controls the response to a specific set of cues and brings about changes in gene...

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria

“…Earlier, biofilm formation has been shown to be defective in a Rel mutant strain of M. smegmatis, and bacterial cells were found to be elongated (14). As expected, cells treated with the compounds used here were not able to form biofilm and were elongated, which is consistent with studies in the Rel mutant (14).…”

“…Earlier, biofilm formation has been shown to be defective in a Rel mutant strain of M. smegmatis, and bacterial cells were found to be elongated (14). As expected, cells treated with the compounds used here were not able to form biofilm and were elongated, which is consistent with studies in the Rel mutant (14). The long treatment regime, antibiotic tolerance, and the emergence of multiple drug resistance in M. tuberculosis are attributed to its stress response (15).…”

“…Previously, it has been reported that the mycobacterial cells devoid of the rel gene are morphologically different and elongated (4,14). We analyzed the average lengths of the M. smegmatis cells treated with AB compound and found them to be elongated (see Fig.…”

Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle

“…burgdorferi stringent response in the first 24–48 h of nymph feeding would coincide with observed borrelial cell blebs, transient round forms and formation of biofilm‐like networks and aggregates, and expression of borrelial adhesins for tick epithelial cell receptors together with adhesion‐mediated motility through the gut epithelial cells (Fig. ) (Pal et al ., ; Ferullo and Lovett, ; Traxler et al ., ; Dunham‐Ems et al ., ; Srivastava and de Silva, ; Zhang et al ., ; Meriläinen et al ., ; Gupta et al ., ). It is plausible that the stringent response mediates these processes since the stringent response in B .…”

Rel Is Required for Morphogenesis of Resting Cells in Mycobacterium smegmatis

“…The transcript level of ftsZ , which encodes the septum formation protein, has been shown to be significantly down-regulated in rel -disrupted strains of M. smegmatis (Gupta et al, 2016). Likewise, the expression of two other septum-formation components FtsW and PBPB was four–fivefolds less in a M. tuberculosis rel deletion strain compared to the wild-type strain under starvation (Dahl et al, 2003).…”