Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis

Abstract

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 (⌬rel Msm ) or c-di-GMP synthesis (⌬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 ⌬rel Msm and ⌬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. N ucleotide-based second messengers regulate various biological processes in all domains of life. Pentaphosphate guanosine (pppGpp) or tetraphosphate guanosine (ppGpp), collectively referred to as (p)ppGpp, and bis-(3=-5=)-cyclic dimeric GMP (cdi-GMP) are two such bacterial second messengers (1, 2). The alarmone (p)ppGpp is synthesized when bacteria are stressed or starved and regulates important processes, like stringent response, quorum sensing, virulence, and biofilm formation (2-5). In mycobacteria, (p)ppGpp is synthesized and broken down by the dual-function enzyme Rel, encoded by the rel gene (6). The Mycobacterium smegmatis rel gene knockout (⌬rel Msm ) strain is compromised for long-term survival during nutrient starvation and progressive hypoxia (7). Similarly, Mycobacterium tuberculosis lacking the Rel Mtb protein has reduced long-term survival in the lungs of mice and in a mouse hypoxic granuloma model and fails to form tubercle lesions in a guinea pig model of infection (8-11). Both the M. tuberculosis and M. smegmatis genomes encode a second (p)ppGpp synthetase called small alarmone synthetase (12, 13). However, its role in mycobacterial physiology has not been completely elucidated.The signaling nucleotide c-di-GMP is s...

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

“…7). Thus, differential expression of genes involved in cell-wall-related processes in ⌬rel Msm and ⌬dcpA strains might also explain our previous work where we have shown that the ⌬rel Msm and ⌬dcpA strains are defective in synthesis of cell wall glycopeptidolipids and polar lipid synthesis (33).…”

“…Hence, we report a novel role for c-di-GMP in mycobacterial growth. We have reported earlier that both ⌬rel Msm and ⌬dcpA strains of M. smegmatis are defective in biofilm formation (33). It is well known that high levels of (p)ppGpp or c-di-GMP are required for biofilm formation (1,4,5,43).…”

“…In our previous work, we have shown that both ⌬rel Msm and ⌬dcpA strains are relatively more antibiotic tolerant, show altered cell surface properties, and have reduced amounts of glycopeptidolipids (GPLs) and polar lipids in their cell walls compared to the wild-type (WT) M. smegmatis (33). We found that impairing (p)ppGpp and c-di-GMP signaling in M. smegmatis caused similar effects on "macroscopic" surface-related properties such as colony morphology, sliding motility, and biofilm formation, which gave us the hint that there could be more phenotypes that could be commonly regulated by these second messengers.…”

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c

“…The requirement for 1-3 g of mycobacterial cells to conduct analytical measurement of cyclic di-GMP in M. tuberculosis or M. smegmatis (Bharati et al, 2012;Hong et al, 2013) does not provide a robust system to correlate cyclic di-GMP levels with phenotypes. Thus, given the ambiguity of cyclic di-GMP production in M. smegmatis and the reported cyclic di-GMP phenotype(s) of this bacterium (Bharati et al, 2012;Gupta et al, 2015), future efforts will focus on defining the function and significance of DGC activity in M. leprae, using a model system that allows for genetic analyses of individual DGCs. Towards this goal, all of the known and predicted DGCs of M. leprae (Fig.…”

“…Protein interaction studies have also suggested that cyclic di-GMP production in M. tuberculosis is involved in regulation of rhamnose biosynthesis, a key sugar in the formation of the mycobacterial cell wall (Deng et al, 2014). Moreover, studies of the Mycobacterium smegmatis homologue of rv1354c demonstrated that the cyclic di-GMP was involved in colony morphology and long-term survival during nutrient starvation (Sharma et al, 2014;Bharati et al, 2012;Gupta et al, 2015).…”