Isolation and characterization of transposon-induced mutants ofPorphyromonas gingivalisdeficient in fimbriation

Abstract

Fimbriae are considered to be an important virulence factor ofPorphyromonas gingivalis. In order to identify genes essential for fimbriation, other thanfimAwhich encodes the major subunit protein of fimbriae, transposon mutagenesis and immunological screening techniques were used to isolate fimbria-deficient mutants. R751::ast;Ω4, a suicide vector that carries Tn4351, was transferred fromEscherichia colitoP. gingivalisby conjugation. Twenty-two independent fimbria-deficient mutants were identified among the resulting transformants. Southern hybridization analysis with pBlue 4351, a transposon-specific probe, and R751 indicated that 45% of the mutants resulted from single transposon insertions and that the remaining 55% of the mutants resulted from cointegration of R751 sequences. Southern hybridization analysis with pUCBg12.1, a probe for thefimAregion, indicated that nine of the mutants contained insertions within the 2.5 kbSacI DNA fragment ofP. gingivalisthat containsfimA, ORF1 (which encodes a 15 kDa protein), and the C-terminal portion of ORF5 (which encodes a 63 kDa protein). Polymerase chain reaction (PCR) analysis and further Southern hybridization analysis indicated that the insertion site(s) for all nine of these mutants was within thefimAgene. Southern hybridization analysis also indicated that the remaining thirteen mutants contained insertions somewhere outside the 10 kbfimAregion. Analysis by pulsed field gel electrophoresis (PFGE) revealed that insertions for most of the thirteen mutants mapped to a 300 kbNotI fragment and are located at least approximately 200 kb away fromfimA. These results identify genetic loci other thanfimA, that are required for fimbriation ofP. gingivalis. Future cloning and characterization of these genetic loci should be straightforward since they are now marked by antibiotic resistance genes carried by the transposon.