FIG. 1.

(A) Construction of BS1539 (ΔspoVAF::Kan^r ΔlysA) and BS1541 (P_blaP lysA). pDML1539 and pDML1541 are pGEM-T-Easy and pCR-Script derivatives, respectively, constructed as described in Materials and Methods. P_lysA and P_blaP are B. subtilis and B. licheniformis promoters. The operator DNA sequences recognized by BlaI are indicated by shaded boxes. The Kan^r gene was used for kanamycin selection. An X indicates the recombination events leading to the chromosomal constructions of BS1539 and BS1541. In the latter strain, the B. licheniformis P_blaP promoter replaces the B. subtilis P_lysA promoter. (B) Southern Blot analyses of the BglII-digested chromosomal DNA from chromosomal BS168 (lanes 1, 4, and 7), BS1539 (lanes 2, 5, and 8), and BS1541 (lanes 3, 6, and 9) DNA. Panels I, II, and III contain the same patterns of DNA digests hybridized to lysA (I), P_blap (II), and Kan^r (III) probes. lysA, P_blaP, and Kan^r probes were generated by PCR with the following pairs of primers as amplimers: lysABamHI/lysAEnd, BlaIBam+/promblaPBglII, and kanaup/kanadown. pDML1539 or pDML1541 was the DNA template.