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Figures
- FIG. 1.
Southern blot analysis of M. smegmatis. (A-1) Southern blot of DNA from the first-crossover strain, M. smegmatis FP201 (Table 1). DNA was cleaved with SmaI, and the probe was rmlD. (A-2) Origins of the fragments. (B-1) Southern blot of DNA from the second-crossover strain, M. smegmatis YM202 (Table 1). DNA was cleaved with SmaI, and the probe was rmlD. (B-2) Origins of the fragments. In the original blot, a fragment at 10 kb that originated from the rescue plasmid was faintly visible. Values to the sides of the blots are molecular sizes (in kilobases).
- FIG. 2.
Growth curves of M. smegmatis strains at 30 and 42°C. The growth of the control strain, M. smegmatis mc2155, containing plasmid pYM201 at 30°C (▴) and at 42°C (▵), and the growth of the rmlD knockout strain, M. smegmatis YM202, containing plasmid pYM201 at 30°C (•) and at 42°C (○), are illustrated.
Tables
- TABLE 1.
Key bacterial strains and plasmids
Strain or plasmid Relevant characteristic(s) Reference or source Strains M. smegmatis mc2 155 Harbors all plasmids used herein 22 M. smegmatis FP201 M. smegmatis mc2 155 with pFP201 integrated into the rmlD locus (Fig. 1) This study M. smegmatis YM202 M. smegmatis FP201 which has undergone a second-crossover event in the presence of pYM201 (Fig. 1) This study Plasmids pPR27 TS mycobacterial origin of replication; carries sacB gene, gen, E. coli origin of replication 21 pFP201 pPR27 derivative carrying rmlD::kan and the xylE gene This study pCG76 Escherichia coli/Mycobacterium shuttle vector carrying a TS mycobacterial origin of replication and streptomycin resistance cassette (str) 11 pYM201 TS rescue plasmid pCG76 carrying the M. tuberculosis rmlD gene under the control of the rmlD natural promoter, str This study
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- Article
- ABSTRACT
- Constructing the rmlD replacement plasmid (pFP201) and obtaining the first homologous recombination event.
- Construction of an rmlD rescue plasmid.
- Second-crossover attempts and events.
- M. smegmatis YM202 will not grow at 42°C.
- The wbbL gene is transcribed and translated in YM202.
- Application of results to M. tuberculosis.
- ACKNOWLEDGMENTS
- FOOTNOTES
- REFERENCES
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