IDENTIFICATION OF A GENE INVOLVED IN ASSEMBLY OF ACTINOMYCES-NAESLUNDII T14V TYPE-2 FIMBRIAE

The nucleotide sequence of the Actinomyces naeslundii T14V type 2 fimb rial structural subunit gene, fimA, and the 3' flanking DNA region was determined. The fimA gene encoded a 535-amino-acid precursor subunit protein (FimA) which included both N-terminal leader and C-terminal ce ll wall sorting sequences. A second gene, designated orf365, that enco ded a 365-amino-acid protein which contained a putative transmembrane segment was identified immediately 3' to fimA. Mutants in which either fimA or orf365 was replaced with a kanamycin resistance gene did not participate in type 2 fimbriae-mediated coaggregation with Streptococc us oralis 34. Type 2 fimbrial antigen was not detected in cell extract s of the fimA mutant by Western blotting with anti-A. naeslundii type 2 fimbrial antibody, but the subunit protein was detected in extracts of the orf365 mutant. The subunit protein detected in this mutant also was immunostained by an antibody raised against a synthetic peptide r epresenting the C-terminal 20 amino acid residues of the predicted Fim A. The antipeptide antibody reacted with FimA isolated from the recomb inant Escherichia coli clone containing fimA but did not react with pu rified type 2 fimbriae in extracts of the wild-type strain, These resu lts indicate that synthesis of type 2 fimbriae in A. naeslundii T14V m ay involve posttranslational cleavage of both the N-terminal and C-ter minal peptides of the precursor subunit and also the expression of orf 365.