Generation of a novel transposon promoter probe to study gene regulation in Porphyromonas gingivalis.

The transposon Tn4351, originally isolated from Bacteroides fragilis, has previously been used generate many isogenic mutants in the oral pathogen Porphyromonas gingivalis. Although such transposon mutants can determine the function of putative virulence genes they can not be used to study the regulation of these genes. To correct this deficiency Tn4351 was manipulated to include a reporter gene to generate a type I promoter probe with which to study the regulation of putative virulence genes in P. gingivalis under different environmental conditions. The manipulation of Tn4351 was achieved by using PCR to amplify IS4351R, one of the two directly repeated insertion elements that flank Tn4351. The PCR primers were designed to introduce a unique restriction enzyme site at the 5' end of IS4351R into which the reporter gene, a type III chloramphenicol acetyltransferase (catIII), could be cloned. Recreation of the entire transposon was achieved by cloning a 3.8 kb AvaI fragment from Tn4351 into a unique AvaI restriction site within IS4351R::catIII creating the transposon promoter probe Tn4351::catIIl. Tn4351::catIII was cloned into the Bacteroides suicide vector pJRD215 and transferred from E. coli into P. gingivalis by an optimised filter mating procedure, which resulted in the generation of transposon inserted mutants. Southern blot analysis of these mutants confirmed that transposition of Tn4351::catIII into the P. gingivalis genome occurred randomly. Screening of the transconjugants on media supplemented with chloramphenicol identified a number of mutants containing a transcriptional fusion with the catIII gene. A quantitative assay was also used to demonstrate CAT activity in these mutants. It is hoped that Tn4351::catIII will prove to be a useful genetic tool for studying the regulation of putative virulence determinants in P. gingivalis.