Analysis of mmpL4b and mbtH allelic exchange mutants. (a), top: map of genomic region for mmpL4b. F1 and F2 are locations for primers used to amplify region for the construction of the deletion alleles, while D1 and D2 are the locations for primers used for diagnostic PCR. The grey box indicates the probe fragment used for the Southern blot. (a), middle: ethidium bromide-stained gel of PCR reactions using D1 and D2 diagnostic primers for mmpL4b and DNA prepared from the parental strain and two mutants: Lane 1 (PM3044, mmpL4b+ parental strain), Lane 2 (PM3366, mmpL4b mutant), Lane 3 (PM3365, mmpL4b mutant). (a), bottom: Southern blot of genomic DNA digested with BglII and probed with the DNA fragment shown in the map in (a), top. Lane 1 (PM3044, mmpL4b+ parental strain), Lane 2 (PM3366, mmpL4b mutant), Lane 3 (PM3365, mmpL4b mutant). (b), top: map of genomic region for mbtH. F1 and F2 are locations for primers used to amplify region for the construction of the deletion alleles, while D1 and D2 are the locations for primers used for diagnostic PCR. (b), bottom: ethidium bromide stained gel of PCR reactions using D1 and D2 diagnostic primers for mbtH and DNA prepared from the parental strain and the mbtH mutant: Lane 1 (PM3044, mbtH+ parental strain), Lane 2 (PM3492, ΔmbtH mutant). M is the 1 kb DNA ladder marker lane.