Restoration of BilRI expression in the bilRI-deletion mutant

Because we did not succeed in restoring the bilRI gene to the markerless bilRI deletion mutant despite many attempts, we decided to restore BilRI expression using an A. actinomycetemcomitans/E. coli shuttle plasmid under a constitutively expressed leucotoxin promoter (ltxP). The bilRI gene was amplified from A. actinomycetemcomitans strain D7S by PCR using the 5′-ATACTCGAGTTTAGGAGTAACGATG-3′ forward primer and the 5′-TTTCTGCAGTTAT-TTGCTTTCAGTT-3′ reverse primer, which contained XhoI and PstI restriction sites, respectively. The bilRI gene was inserted into ltxP from the pVT1296 plasmid⁶⁵ by ligating the bilRI PCR product into XhoI- and PstI-digested pVT1296. The final ltxP-bilRI construct was moved to the pPK1-based ⁶⁶ pVT1503 plasmid⁶⁷ by cutting the pVT1-296-based construct with PstI, blunting the ends with Klenow, cutting ltxP-bilRI from the plasmid with KpnI, and ligating ltxP-bilRI to KpnI- and EcoRV-digested pVT1503 (KanR). The correct insert size was confirmed through KpnI-EcoRI double digestion of the final expression plasmid pVT1503-ltxP-bilRI. The bilRI-containing product of the KpnI-EcoRI-digested expression plasmid pVT1503-ltxP-bilRI was ligated into the KpnI-EcoRI-digested pUC19 plasmid (New England Biolabs, Ipswich, MA, USA) and sequenced (Eurofins Genomics). The expression plasmid pVT1503-ltxP-bilRI was then transformed into a markerless bilRI-deletion mutant through natural transformation as described above. This time, 300 ng of DNA was mixed with cells and supplemented with 1 mM CaCl₂ to improve the transformation efficiency.⁶⁸ After incubation, the transformants were screened on TSA plates supplemented with 30 μg/mL kanamycin to select a BilRI-overexpressing variant containing the pVT1503-based expression plasmid.