title: Catalytic site targeted mutagenesis of the alpha-gingivain gene of Porphyromonas gingivalis using Tn-4351 to generate isogenic mutants.
abstract: The extracellular proteinases of the anaerobe Porphyromonas gingivalis, are implicated in the destruction of host defence mechanisms in periodontitis. We have previously purified one of these enzymes, alpha-gingivain, and established that it belongs to the cysteine proteinase family of enzymes. In the present study, transposon Tn4351 was used to alter the open reading frame encoding a region that includes the catalytic site of alpha-gingivain by targeted mutagenesis. Escherichia coli HB101 which harbours R751 was used to introduce the transposon into P. gingivalis ATCC 33277 by conjugal transfer. E. coli was transformed using the altered plasmid with a Cla I site insertion of a sequence common to the catalytic site histidine or cysteine of many cysteine proteinases. 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 surrounding zones of hydrolysis and their normal catalytic activity towards L-BAPNA was destroyed.
authors.affiliation: Department of Microbiology, Eastman Dental Institute, University of London, UK.
authors.forename: S E
authors.initials: SE
authors.lastname: Gharbia
authors.forename: H N
authors.initials: HN
authors.lastname: Shah
authors.forename: S
authors.initials: S
authors.lastname: Sreedharan
authors.forename: K
authors.initials: K
authors.lastname: Brocklehurst