FIG. 2.

Construction and confirmation of the vimF-defective mutant. (A) Construction of mutant. Plasmid pFLL87 contains the vimF gene interrupted by an ermF-ermAM cassette (the vimF gene with flanking sequences was amplified by PCR; the ermF-ermAM cassette was purified from pVA2198). The circular recombinant plasmid pFLL87 was integrally introduced into P. gingivalis W83 by electroporation. A reciprocal recombination event between areas of homology on the chromosome and regions flanking the ermAM-ermF cassette on pFLL87 replaced the intact vimF with an inactivated copy. (B) PCR analysis of allelic exchange mutants of P. gingivalis carrying the vimF gene inactivated with the ermF-ermAM cassette. Oligonucleotide primers specific for the vimF gene or erythromycin cassette were used to amplify that gene or erythromycin from total chromosomal DNA from P. gingivalis. Lane 1, P. gingivalis FLL95.1 (vimF::ermF-ermAM; vimF primers); lane 2, P. gingivalis FLL95.2 (vimF::ermF-ermAM; vimF primers); lane 3, P. gingivalis W83 (wild type); lane 4, P. gingivalis FLL95.1 (vimF::ermF-ermAM; erythromycin primers); lane 5, P. gingivalis FLL95.2 (vimF::ermF-ermAM; erythromycin primers); lane 6, pVA2198 plasmid (plasmid carrying the erythromycin cassette; erythromycin primers) as control. (C) Transcriptional profile of vimF-defective mutant. Total RNA isolated from P. gingivalis W83 and the vimF-defective mutant isolated from stationary phase (OD₆₀₀ of 1.4 to 1.5) was subjected to RT-PCR using primers for vimF and pro-kgp. No transcription or vimF was detected in P. gingivalis FLL95.