Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis

Abstract

Mycobacterial species are characterized by the presence of lipid-rich, hydrophobic cell envelopes. These cell envelopes contribute to properties such as roughness of colonies, aggregation of cells in liquid culture without detergent, and biofilm formation. We describe here a mutant strain of Mycobacterium smegmatis, called DL1215, which demonstrates marked deviations from the above-mentioned phenotypes. DL1215 arose spontaneously from a strain deficient for the stringent response (M. smegmatis ⌬rel Msm strain) and is not a reversion to a wild-type phenotype. The nature of the spontaneous mutation was a single base-pair deletion in the lsr2 gene, leading to the formation of a truncated protein product. The DL1215 strain was complicated by having both inactivated rel Msm and lsr2 genes, and so a single lsr2 mutant was created to analyze the gene's function. The lsr2 gene was inactivated in the wild-type M. smegmatis mc 2 155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and ⌬rel Msm strains, some of which include a greatly enhanced ability to slide over agar surfaces (referred to here as "hypermotility"), greater resistance to phage infection and to the antibiotic kanamycin, and an inability to form biofilms. Complementation of the DL2008 mutant with a plasmid containing lsr2 (pLSR2) reverts the strain to the mc 2 155 phenotype. Although these phenotypic differences allude to changes in cell surface lipids, no difference is observed in glycopeptidolipids, polar lipids, apolar lipids, or mycolic acids of the cell wall.Mycobacterium smegmatis is a fast-growing, saprophytic mycobacterial species. Although considered nonpathogenic, M. smegmatis provides a popular model for studying virulence mechanisms of slow-growing, pathogenic relatives such as Mycobacterium tuberculosis (9,16,37,41) and Mycobacterium leprae (35,42). An important aspect of mycobacterial pathogenesis is the ability of the pathogen to establish latent infections in hosts lasting for several years. Persistent M. tuberculosis bacilli in the host manifest drastic changes in gene expression that set the cells apart from actively growing tubercle bacilli (36,40). One bacterial regulatory network that coordinates nutrient deprivation with adaptive metabolism is the stringent response. In mycobacteria this global regulatory system is controlled by a single gene called rel, and deletion of this gene in M. tuberculosis results in a severe defect in both long-term in vitro and in vivo survival (10, 30). We recently reported that the rel gene of M. smegmatis (rel Msm ) is involved in the regulation of cellular and colony morphologies (9). As seen with M. tuberculosis, the stringent response is required for long-term survival of M. smegmatis in culture, since the rel Msm mutant readily dies over a month-long period while in stationary phase.Here we report the appearance of a mutant strain, called DL1215, that arose spontaneously from the parental M. smegmatis ⌬rel Msm strain. We...

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