FIG. 1.

(A) Schematic representation of the construct used to disrupt the msl6 gene in M. tuberculosis. Hatched, checkered, and unshaded regions represent msl6 coding sequences, an internal segment replaced with the hyg gene (black box), and regions of the gene outside those used to make the disruption construct, respectively. Primer pair A-B was used to amplify the msl6 region chosen to generate the disruption construct. Primer pairs C-H1, D-H2, and E-F were used to test for allelic exchange by PCR analysis. P₁ and P₂, DNA segments used as probes in Southern blot analysis (P₁, msl6 segment deleted in making the construct; P₂, hyg gene). WT, wild type; M, mutant. (B) PCR analysis of internal and flanking regions of msl6 showing products consistent with allelic exchange. Lanes 1, 3, and 5, wild type; lanes 2, 4, and 6, mutant. Lane 1 and 2, 5′-flanking product with primers C and H1; lanes 3 and 4, 3′-flanking product with primers D and H2; lanes 5 and 6, internal deletion segment with primers E and F. (C) Southern blot analysis of M. tuberculosis H37Rv and msl6 mutants. Genomic DNA was digested with PstI. Left, DNA hybridized with an msl6 segment that was deleted in making the construct (probe P₁); right, DNA probed with the hyg gene (probe P₂). WT, wild type; M1 and M2, mutants.