CATALYTIC SITE TARGETED MUTAGENESIS OF THE ALPHA-GINGIVAIN GENE OF PORPHYROMONAS-GINGIVALIS USING TN-4351 TO GENERATE ISOGENIC MUTANTS

The extracellular proteinases of the anaerobe Porphyromonas gingivalis , are implicated in the destruction of host defence mechanisms in peri odontitis. We have previously purified one of these enzymes, alpha-gin givain, and established that it belongs to the cysteine proteinase fam ily of enzymes. In the present study, transposon Tn4351 was used to al ter the open reading frame encoding a region that includes the catalyt ic site of alpha-gingivain by targeted mutagenesis. Escherichia coli H B101 which harbours R751 was used to introduce the transposon into P. gingivalis ATCC 33277 by conjugal transfer. E, coli was transformed us ing the altered plasmid with a Cla I site insertion of a sequence comm on to the catalytic site histidine or cysteine of many cysteine protei nases. The frequency of the transconjugation was 4.5 x 10(5) while the recipient viable counts comprised 60% of the original P. gingivalis. The result of this targeted mutagenesis was inactivation of gingivains such that some colonies on skimmed-milk agar plates showed no clear s urrounding zones of hydrolysis and their normal catalytic activity tow ards L-BAPNA was destroyed.