FIG. 1.

Construction and genotypic characterization of M. smegmatis cyd mutants. (A) Restriction map of the cyd locus of M. smegmatis showing the insertion sites of the aph cassette in the cyd knockout mutants. The 2.4-kb KpnI fragment from pBK1, used as the probe for Southern blot analysis (see panel B), is denoted by a dashed, double-headed arrow. Restriction sites: Bg, BglII; H, HindIII; K, KpnI; P, PstI; S, SphI. The sequence extending from the PstI site upstream of cydA to a SalI site downstream of cydC has been deposited in GenBank under accession no. AF196488. A hypothetical open reading frame that showed high homology to Rv1624c from M. tuberculosis was found upstream of cydA, confirming that the genetic organization at this locus is conserved between M. smegmatis and M. tuberculosis. (B) Southern blot analysis of cyd mutants. Genomic DNA from the various strains was digested with the indicated enzyme(s) and probed with the 2.4-kb KpnI fragment from pBK1. In all cases, the sizes of the hybridizing fragments derived from the mutant strains were consistent with site-specific, allelic exchange.