SYNTHESIS AND FUNCTION OF ACTINOMYCES-NAESLUNDII T14V TYPE-1 FIMBRIAEREQUIRE THE EXPRESSION OF ADDITIONAL FIMBRIA-ASSOCIATED GENES

The nucleotide sequence of the chromosomal DNA flanking the Actinomyce s naeslundii (formerly A. viscosus) T14V type 1 fimbrial structural su bunit gene (fimP) was determined. Six open reading frames (ORFs), in t he order 5' ORF3, ORF2, ORF1, fimP, ORF4, ORF5, ORF6 3', were identifi ed. ORF1 encoded a protein of 408 amino acid residues (M-r = 39,270) a nd had significant sequence homology with the A. naeslundii T14V type 1 and A. naeslundii WVU45 type 2 fimbrial structural subunits. An in-f rame fusion of ORF1 to the malE gene of the expression vector, pMAL-c2 , yielded a protein that was immunostained with antibodies raised agai nst the maltose binding protein and A. naeslundii T14V whole bacteria. Digestion of the fusion protein with factor Xa released a protein (ap parent molecular mass of 34 kDa) that was immunostained only with the antibody directed against A. naeslundii T14V whole bacterial cells. In tegration plasmids carrying a kanamycin resistance gene (kan) that was used to substitute for ORF1 or for DNA fragments internal to the codi ng region of the other five ORFs were used to transform A. naeslundii T14V. Neither type 1 fimbriae nor the 65-kDa fimbrial structural subun it was detected in mutants obtained by allelic replacement of ORF1 or ORF2. Mutants obtained by allelic replacement of ORF3 or ORF4 expresse d only the 65-kDa fimbrial structural subunit. These mutants did not b ind, in vitro, to proline-rich proteins that serve as the receptors fo r Actinomyces type 1 fimbriae. In contrast, a mutant in which the inte gration plasmid DNA had been inserted at a site close to the carboxyl terminus of ORF6 expressed type 1 fimbriae and had adherence propertie s similar to those observed in the wild-type strain. These results dem onstrate the existence of additional genes near fimP that are likely t o be involved in the synthesis and function of cell surface fimbriae o f A. naeslundii T14V.