S-Nitrosoglutathione cytotoxicity to Mycobacterium smegmatis and its use to isolate stationary phase survival mutants.

We report that stationary phase Mycobacterium smegmatis is more sensitive than exponential phase cells to the nitric oxide donor S-Nitrosoglutathione (GSNO). This finding was used to select for both spontaneous and transposon mutants of M. smegmatis with increased resistance to GSNO in stationary phase. Some of these mutants were also defective in stationary phase survival, demonstrating a link between sensitivity to GSNO and stationary phase survival. Transduction of the disrupted region from seven selected mutants indicated that the transposon insertion was linked to the GSNO-resistance and stationary phase survival phenotypes. For five mutants, the disrupted genes were identified. Three were homologous to genes with possible roles in nutrient scavenging, including: (i) a putative amino acid efflux pump, (ii) a putative thioesterase and (iii) an enoyl-CoA-hydratase. One mutant was disrupted in the atpD gene, encoding the beta chain of F1 F0 ATP synthase. We independently isolated a stationary phase survival mutant disrupted in the atpA gene (encoding the alpha chain) of the F1 F0 ATP synthase of the same operon, suggesting an important role for efficient ATP synthesis in stationary phase survival.

Mutants of Mycobacterium smegmatis impaired in stationary-phase survival.

A bank of 600 insertional mutants of Mycobacterium smegmatis was screened for mutants defective in stationary-phase survival. Of 74 mutants picked by the initial screen, 21 had stationary-phase survival defects and 7 of these were studied in more detail. In general, mutants survived stationary phase significantly less well in rich medium than under carbon-starvation conditions. In all cases the loss of viability in stationary phase was not complete even after prolonged incubation. All mutants showed an initial decrease in viability, during the first 40 d in stationary phase, followed by an increase in viable counts that returned viability close to the levels of the wild-type. Southern hybridization experiments showed that recovery of viability was not a consequence of precise excision or movement of the transposon. Two of the survival mutants differed from the wild-type in their colony morphology, and recovery of their viability in stationary phase was coincident with the return of wild-type colony morphology. It is possible that second-site suppressor mutations accumulate that alleviate the effects of the original mutation. For five of the mutants the DNA flanking the site of transposition was amplified by ligation-mediated PCR and sequenced to identify the disrupted locus. In each case, homologous genes were identified in the Mycobacterium tuberculosis genome, three of which have clearly predicted functions in M. tuberculosis as a penicillin-binding protein, in biotin biosynthesis and as a polyketide synthase. This is the first identification of genes implicated in the stationary-phase survival of mycobacteria.