Isolation and characterization of nocardia-like variants of <i>Mycobacterium smegmatis</i>

Hawley, Robert J.; Imaeda, T; Mann, Nora

doi:10.1139/m76-219

Cited by 6 publications

(8 citation statements)

References 32 publications

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“…5). A precedent exists in M. smegmatis for lysogeny causing a shift from rough to smooth colony morphology (11,14,15,17,21,34), and it has been proposed that M. smegmatis is a polylysogen (11,14,33). The correlation of these phage-like structures with inactivation of lsr2 suggests that an absence of Lsr2 may induce lysogens.…”

Section: Discussionmentioning

confidence: 99%

Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis

et al. 2008

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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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Section: Discussionmentioning

confidence: 99%

Inactivation of lsr2 Results in a Hypermotile Phenotype in Mycobacterium smegmatis

et al. 2008

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“…On the other hand, foreign DNA molecules, including mycobacterial and mycobacteriophage DNA as well as synthetic polynucleotides, were reportedly taken up by mycobacteria (15,33,36,39,42,(45)(46)(47). Consequently, the major difficulty encountered in transformation experiments with mycobacteria may not lie in the initial stages of the process.…”

Section: Transformationmentioning

confidence: 99%

“…(This work was presented at the 77th Annual Table 1. In addition to the nutritional and antibiotic markers presented, strains 607+, 607-1, and 607-2 form rough, cream-yelow colonies and are capable of growing at 43°C and sensitive to UV irradiation at a dose of 7.2 x 102 ergs/mm2 (15). Recipient strains (OR4 and ORuv) are characterized by their smooth, orange-red-pigmented colonies, inability to grow at 43°C, and resistance to UV irradiation at a dose of 7.2 x 102 ergs/mm2 (15).…”

Section: Transformationmentioning

confidence: 99%

“…Mycobacteria were cultivated in Penassay-glycerol (PG) (15) (for strains OR4, ORuv, and OR4-1) or PG-Tween (15) (for strains 607+, 607-1, and 607-2) broth. Agar plates (PGA) contained 1.5% (wt/vol) agar (Difco Laboratories, Detroit, Mich.) (15). For the selection of streptomycin-resistant organisms, 500 ,ug of dihydrostreptomycin sulfate (Sigma Chemical Co., St. Louis, Mo.)…”

Section: Transformationmentioning

confidence: 99%

“…Recipient strains in the early stationary phase were harvested from PG, washed once in TF buffer, and suspended in TF buffer at a concentration of about 109 CFU/ml. Suspensions were irradiated with 7.2 x 102 ergs of UV light per nmm2 (15). A 0.9-ml sample of each suspension was kept in the dark, treated with 0.1 ml of 0.5 M CaCl2 at 00C for 15 min, and mixed with 0.2 ml (100,ug/ml) of sheared, sterile DNA of the donor strain, 607-1, for 30 min at 37°C.…”

Section: Or4-1mentioning

confidence: 99%

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Physiological factors involved in the transformation of Mycobacterium smegmatis

Norgard¹,

Imaeda²

1978

J Bacteriol

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Transfer of streptomycin resistance and changes from methionine and leucine auxotrophy to prototrophy were achieved in Mycobacterium smegmatis by transformation. Recipient cells were more resistant to mitomycin C and methyl methanesulfonate treatments than were wild-type cells. A high level of calcium ions was essential for transformation, especially during DNA adsorption, whereas the presence of magnesium ions and the exposure of recipient cells to mild doses of UV light enhanced recombination frequencies. Transformants were not isolated when recipient cell-DNA mixtures were first treated with deoxyribonuclease. Recipient cells at various stages of growth showed similar transformabilities. Transformation was successful only when recipient cells were incubated on rich agar medium after mixture with DNA. Exposure of recipient cells to Pronase before treatment with donor DNA did not affect transformation, suggesting the absence of a protein competence factor. Throughout the present experiments, cotransformation frequencies were very low and unselected-marker segregation patterns were independent, indicating that the methionine, leucine, and streptomycin markers are not closely linked in M. smegmatis. Despite the medical importance of mycobacteria, knowledge of genetic recombination in this genus remains limited. Experimental procedures in transduction (12, 13, 20-22, 41) and conjugation (30, 32, 40, 44) have recently been established for various species of mycobacteria, but results of transformation experiments have been inconclusive (4, 14, 43). Transformation in mycobacteria was first reported by Katunuma and Nakasato (23), who claimed that streptomycin resistance was transferred to Mycobacterium avium through DNA extracted from streptomycin-resistant strains. Tsukamura et al. (48) reported the transfer of isoniazid and streptomycin resistance to M. avium after exposure for 5 days to high concentrations of DNA extracted from resistant strains. Gelbart and Juhasz (12) and Juhasz et al. (S. E. Juhasz, S. M. Gelbart, and L. DeSalle, Bacteriol. Proc., p. 35, 1971) described xylose utilization transferred to Mycobacterium phlei through transformation. In contrast to these observations, Bloch et al. (3) failed to transfer streptomycin and isoniazid resistance to M. phlei, M. smegmatis, M. bovis, and M. tuberculosis, using purified DNA from respective resistant strains. They were also unsuccessful in transferring the ability to synthesize pigments to M. smegmnatis from M. phlei or the virulence of M. tuberculosis H37Rv to the avirulent M. tuberculosis H37Ra through transformation. Similarly, Bradley (4) was unable to transfer isoniazid, streptomycin, or cycloserine resistance or mycobacteriophage susceptibility to M. smegmatis, M. kansasii, M. bovis, M. tuberculosis, and M. intracellulare through

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