FIG. 1.

Schematic diagram of the P. multocida nrfE mutagenesis construct and confirmation of the nrfE mutant by PCR. (A) A single 1.8-kb fragment containing nrfE was amplified by PCR by using primers 1914 and 1915 (indicated by the arrows labeled 1914 and 1915). This fragment was digested with BamHI to obtain two 900-bp fragments that were then ligated to either end of tet(M) to produce the mutagenesis cassette used for allelic exchange. (B) Schematic diagram of the genome organization around nrfE after insertion of the tet(M) cassette. The labeled arrows indicate primers used for PCR. (C) The genotype of AL362 was investigated by PCR. Genomic DNA from AL362 (lanes 3, 5, and 7) was compared to genomic DNA from wild-type strain X-73 (lanes 2, 4, and 6). Lanes 2 and 3, amplification with 1914 and 1915; lanes 4 and 5, amplification with 2277 and 2278; lanes 6 and 7, amplification with tet(M) primer 683 together with genomic primer 2278. Lane 1 contained λ DNA digested with HindIII.