Related Experiment Videos
Mutants of Mycobacterium smegmatis impaired in stationary-phase survival
Jacquie Keer1, Marjan J Smeulders1, Kathryn M Gray1
1Department of Biology, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, UK1.
Abstract:
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.
Insights
Researchers screened Mycobacterium smegmatis mutants for defects in stationary-phase survival. They identified key genes involved in mycobacterial survival, including those for penicillin-binding protein and biotin biosynthesis.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Mycobacteria possess complex survival mechanisms during stationary phase.
- Understanding stationary-phase survival is crucial for developing effective treatments against mycobacterial infections.
Purpose of the Study:
- To identify genes essential for stationary-phase survival in Mycobacterium smegmatis.
- To investigate the genetic basis of survival defects in mycobacteria.
Main Methods:
- Screening of 600 insertional mutants of Mycobacterium smegmatis.
- Detailed analysis of 7 mutants with survival defects.
- Ligation-mediated PCR and DNA sequencing to identify disrupted loci.
Main Results:
- 21 mutants exhibited stationary-phase survival defects.
- Viability recovery was observed in some mutants, linked to changes in colony morphology.
- Five mutants had their disrupted loci identified, revealing homologous genes in Mycobacterium tuberculosis.
Conclusions:
- This study is the first to identify specific genes involved in mycobacterial stationary-phase survival.
- Identified genes include those for penicillin-binding protein, biotin biosynthesis, and polyketide synthase.
- Findings provide new targets for understanding and combating mycobacterial persistence.