Figure 3. Genetic strategy for conditional disruption of MSMEG_6382.

A. The recombination plasmid pPKC72 contained a cloned copy of MSMEG_6382 interrupted by a non-polar kanamycin resistance cassette (MSMEG_6382::aphA3), a gentamycin resistance marker (Gm^r), a temperature-sensitive replication origin for M. smegmatis (oriMs (ts)), a replication origin for E. coli (oriEc) and a counterselectable marker encoding sucrose sensitivity (sacB). The construct was introduced into M. smegmatis at the permissive temperature (30°C). Integration of the plasmid by a single crossover at the position indicated was detected by growing the cells at the non-permissive temperature (42°C) in the presence of kanamycin. B. Genetic map of the single crossover, showing key restriction sites and fragments. C. Culturing the single crossover strain containing a rescue plasmid encoding MSMEG_6382 gave rise to a disrupted copy of MSMEG_6382 in the chromosome, producing the conditional knockout (6382CKO). Since the disruption of MSMEG_6382 coincided with the loss of the sacB gene, the conditional knockout strain could be selected on sucrose plates. Double lines indicate chromosomal DNA, single lines indicate plasmid DNA. Restriction fragments hybridising to the MSMEG_6382-specific probe are shown in kilobases (kb).