FIG. 2.

Disruption of the gene and rescue of adjacent sequences. (A) A 615-bp internal ccpA fragment (open box) was PCR amplified from the L. monocytogenes chromosome with the primers CcpA-05R and CcpA-06L, cloned into the integrational vector pCON1, and conjugated into L. monocytogenes. (B) Shift of the pCON1-CCPA-containing strain to the non-permissive temperature forced integration of the vector into the chromosome at the ccpA locus, resulting in the truncation of the gene after codon 279 (arrow). (C) Sequences from upstream of the integrated vector were rescued by extracting chromosomal DNA from the mutant, digesting it with XbaI, and cloning it directly in E. coli. bla, β-lactamase gene; cat, chloramphenicol acetyltransferase gene; oriT, mobilization signal from plasmid RP4; pE194 ts, replication origin derived from plasmid pE194ts; ColE1, replication origin derived from pUC18.