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.
Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London SW7 2AZ, UK.
Marjan J
MJ
Smeulders
Jacquie
J
Keer
Kathryn M
KM
Gray
Huw D
HD
Williams
D004251:DNA Transposable Elements; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics; D016254:Mutagenesis, Insertional; D020102:Mycobacterium smegmatis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D026422:S-Nitrosoglutathione / Q000494:pharmacology*
D004251:DNA Transposable Elements; D026422:S-Nitrosoglutathione; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases