FIG. 1.

Construction of markerless yycH, yycI, yycJ, and yycHI deletion strains. Deletion strains JH25021 (ΔyycH), JH25022 (ΔyycI), JH25029 (ΔyycJ), and JH2531 (ΔyycHI) were constructed by adopting the Bacillus anthracis method of Janes and Stibitz to Bacillus subtilis (19). This method involves single-crossover integration of a suicide plasmid featuring chromosomal regions flanking the gene to be deleted (∼500 bp each site) and an I-SceI site. The Cm^r pJM103 integrative plasmid was modified by introducing an I-SceI site (M. Perego, unpublished). Following single-crossover integration (shown here for yycH) and selection for Cm^r, the I-SceI gene was expressed from a second plasmid pBKJ223 conferring Tet^r. This introduced a DNA double-stranded break, which was repaired by homologous crossover recombination resulting in Cm^s strains. Roughly 50% of all Cm^s transformants contained the correct deletion, whereas the other 50% are wild type. Deletion strains were identified by colony PCR. The pBKJ223 plasmid was lost by repeated growth without antibiotic selection in liquid media.