ISOLATION AND CHARACTERIZATION OF TRANSPOSON-INDUCED MUTANTS OF PORPHYROMONAS-GINGIVALIS DEFICIENT IN FIMBRIATION

Fimbriae are considered to be an important virulence factor of Porphyr omonas gingivalis. In order to identify genes essential for fimbriatio n, other than fimA which encodes the major subunit protein of fimbriae , transposon mutagenesis and immunological screening techniques were u sed to isolate fimbria-deficient mutants. R751::Omega 4, a suicide ve ctor that carries Tn4351, was transferred from Escherichia coil to P. gingivalis by conjugation. Twenty-two independent fimbria-deficient mu tants were identified among the resulting transformants. Southern hybr idization analysis with pBlue 4351, a transposon-specific probe, and R 751 indicated that 45% of the mutants resulted from single transposon insertions and that the remaining 55% of the mutants resulted from coi ntegration of R751 sequences. Southern hybridization analysis with pUC Bg12.1, a probe for the fimA region, indicated that nine of the mutant s contained insertions within the 2.5 kb Sad DNA fragment of P. gingiv alis that contains fimA, ORF1 (which encodes a 15 kDa protein), and th e C-terminal portion of ORF5 (which encodes a 63 kDa protein). Polymer ase chain reaction (PCR) analysis and further Southern hybridization a nalysis indicated that the insertion site(s) for all nine of these mut ants was within the fimA gene. Southern hybridization analysis also in dicated that the remaining thirteen mutants contained insertions somew here outside the 10 kb fimA region. Analysis by pulsed field gel elect rophoresis (PFGE) revealed that insertions for most of the thirteen mu tants mapped to a 300 kb Notl fragment and are located at least approx imately 200 kb away from fimA. These results identify genetic loci oth er than fimA, that are required for fimbriation of P. gingivalis. Futu re cloning and characterization of these genetic loci should be straig htforward since they are now marked by antibiotic resistance genes car ried by the transposon. (C) 1998 Academic Press Limited.