FIG. 6.

Verification of the deletion mutants. Examples are shown of the methods used to verify the deletion of the regions. Positions of the molecular size markers are shown in kilobases for each gel. (A) Deletion of region 2 from strain ROU. Chromosomal DNA from the parental strain (lanes 1 and 3) and the region 2 deletion mutant (lanes 2 and 4) was digested with ClaI. Lanes 1 and 2 were probed with a PCR product corresponding to the gene fhaC; lanes 3 and 4 were probed with the cassette “omega” used to replace the region. Due to its size (about 25 kb) region 2 encompassed several ClaI fragments; similar results were obtained after probing with PCR products corresponding to several of the other ORFs. (B) Deletion of region 3 from strain Z5463. Chromosomal DNA from the parental strain (lanes 1 and 3) and the region 2 deletion mutant (lanes 2 and 4) was digested with SgfI. Lanes 1 and 2 are the pulsed-field gel electrophoresis analysis of the deletion. Note the disappearance of a band at about 194 kb in the mutant and the new band appearing with a size of about 146 kb; this corresponds to the deletion of about 50 kb (region 3). Lanes 3 and 4 were probed with a PCR product corresponding to a portion of the phage transposase gene. (C) Deletion of region 8 from strains MC58 and ROU. Lane 1, MC58 parental strain; lane 2, MC58 region 8 deletion; lane 3, ROU; lane 4, ROU region 8 deletion. Chromosomal DNA was digested with ClaI, and Southern blots were probed with a PCR product corresponding to the entire region 8 from strain Z2491.