FIG. 2.

Application of pGF-EM in construction of gene replacement. A 5′ portion of ftsH was amplified with primers P1 and P2 and cloned into pGE-EM, yielding pGF-EM-FtsH1. ΔftsH indicates the truncated ftsH portion (open section in diagram). An additional genomic fragment (gray section in diagram) downstream of ftsH was amplified with primers P3 and P4 and cloned in pGF-EM-FtsH1, yielding pGF-EM-FtsH2, which was introduced into L. monocytogenes and used for construction of allelic exchange products. Primer positions and orientations are as indicated. Further details are provided in the text. Crosses indicate crossover events. Black arrows in the plasmid diagrams indicate gfp, cross-hatched arrows indicate the erythromycin resistance gene erm, and open arrows indicate the chloramphenicol resistance gene cat; arrows point in the direction of transcription.