Porphryromonas (Bacteroides) gingivalis has been implicated as a major pathogen in adult periodontitis. The current proposal seeks to develop a system for genetic analysis of P. gingivalis. The primary goals of this study are: 1)(a) Development of transpositional mutagenesis system for P. gingivalis as a tool for genetic analysis. The Bacteroides fragilis transposon, TN4351 will be introduced into P. gingivalis by conjugation employing Escherichia coli R751::Tn4351 as the donor. Initial work will focus on maximizing the conjugal transfer of Tn4351 into P. gingivalis recipients. (b) Confirmation and characterization of transpositional events in P. gingivalis. P. gingivalis transconjugants carrying Tn4351 will be confirmed using pVOH1, a 10.2 kb plasmid containing the entire Tn4351. We will determine the nature of, as well as the number of insertions of Tn4351 by into the P. gingivalis chromosome. We will definitively confirm random insertion of Tn4351 by PCR amplification and sequencing of the P. gingivalis DNA adjacent to Tn4351 insertions. 2) Isolation and characterization of hemin utilization mutants generated by transpositional mutagenesis. Defining the mechanisms of hemin utilization in P. gingivalis is a major focus of our laboratory. After confirming the nature of the insertion of Tn4351 into the chromosome, we will screen for the inactivation of specific gene(s) required for hemin utilization. Potential hemin utilization mutants will be isolated by screening transconjugants for loss of pigmentation and/or inability to bind congo red, an aromatic sulfonated diazo dye. Development of this system will ultimately provide a valuable tool for the analysis of many virulence factors produced by P. gingivalis.
