The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel Msm . The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.The ability of Mycobacterium tuberculosis to persist in the human host is a major challenge for both vaccine-and drugbased strategies for controlling the spread of tuberculosis (TB) (17). Persistent M. tuberculosis cells are capable of initiating active growth in the host, a condition known as reactivation TB. Although it is somewhat controversial, it is generally believed that the site of viable M. tuberculosis in the host is inside caseous, necrotic, granulomatous lesions in the lungs. It has been reported that M. tuberculosis cells persisting inside granulomas lack the acid-fast staining characteristic of bacteria recovered from sputa and lesions of patents with active disease, indicating that changes occur in the bacterial cell wall during metabolic adaptation to a state of dormancy in the host (18,22). Various models have been developed to create in vitro growth conditions that mimic the presumed state of M. tuberculosis inside granulomas, including oxygen limitation (32, 33) and nutrient starvation (1,20). For example, when M. tuberculosis cultures are suspended in distilled water, they appear to lose their acid-fast staining ability, but the cells can remain viable for over 2 years in this extreme nutrient-deprived environment (22). This implies that in vitro observations of mycobacterial physiology may provide important insights into understanding how pathogenic mycobacteria survive in hosts. In addition, several mutants of M. tuberculosis that are impaired in de novo biosynthesis of various amino acids and vitamins (14) are attenuated in mouse models of tuberculosis infection, suggesting that the organism resides in a nutrient-poor environment. This supports the significance of in vitro nutrient starvation models for understanding in vivo persistence.Recently, the stringent response of M. tuberculosis has been shown to play an important role in the in vitro and in vivo survival of this bacterium (9, 29). Escherichia coli has two homologous genes that are responsible for initiating the stringent response, namely, relA and spoT. Collectively, RelA and SpoT can sense nutrient deprivation and respond by synthesizing guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which can alter the promoter specificity of RNA polymeras...

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...

Adaptation to environmental stress is an important survival characteristic of any bacterial species. As a soil-dwelling saprophyte, Mycobacterium smegmatis is exposed to factors such as UV light and rounds of freezing and thawing that occur in temperate climates. Numerous studies in Escherichia coli have linked histone-like proteins to stress resistance and adaptation. We hypothesized that the 'histone-like' protein Hlp might likewise be involved in the stress response of M. smegmatis. The hlp gene was inactivated and the M. smegmatis Dhlp strain was found to be more susceptible to UV light and to the stress created by repeated cycles of freezing and thawing. In addition, loss of Hlp altered the colony morphology and allowed the organism to grow dispersed in the absence of a detergent, suggesting changes in the cell wall composition. As cell wall changes could affect permeability to certain antibiotics, the susceptibility of M. smegmatis Dhlp to kanamycin, rifamipicin, ethambutol and isoniazid (INH) was tested. M. smegmatis Dhlp was more susceptible to INH, but loss of Hlp did not affect susceptibility to the other antibiotics tested. This suggests that the increased sensitivity of M. smegmatis Dhlp to INH was unlikely to be the result of alterations in cell permeability.