Tn4351-generated non-haemolytic and/or non-pigmented mutants of Porphyromonas gingivalis

Escherichia coli-Porphyromonas gingivalis plasmid-shuttle vectors R751::* omega 4, pVOH1, and pVAL-1 were used for isolation of non-pigmented, defective protease, or non-haemolytic activity phenotypes in P. gingivalis. Transfer frequencies for R751::* omega 4, pVOH1, and pVAL-1 varied from 1.7 x 10(-9) to 5.3 x 10(-11) depending on the P. gingivalis 381 and ATCC 33277 strains. Two erythromycin-resistant transconjugants were screened from P. gingivalis 381 and eighteen were screened from ATCC 33277. Among isolated transconjugants, two from ATCC 33277 contained only one transposon insertion, while others included both Tn4351 and R751 sequences. The one transconjugant which contained a single insertion of Tn4351, designated NUM-T14, demonstrated defective pigmentation, and defective protease and haemolytic activities. The other transconjugant, designated NUM-T29, demonstrated defective haemolytic activity, but had black pigmentation and protease activity. Cell surface protein analysis by SDS-PAGE indicated that 40 and 18 kD proteins were lost or reduced and that 45 and 36 kD proteins were made to appear in both NUM-T14 and NUM-T29 transconjugants.