To gain insight in the molecular architecture of Actinomyces viscosus fimbriae, recombinant DNA clones expressing genes for fimbrial proteins were sought. One plasmid (pAV1402) was identified in a cosmid gene library prepared in Escherichia coli from A. viscosus T14V DNA expressed an antigen that reacted with polyclonal antibody against type 2 fimbriae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that pAV1402 expressed an immunoreactive 59 kDa protein (p59) that comigrated with a component of partially dissociated type 2 fimbriae. Fimbriae labeled by radioiodination showed virtually the same pattern as fimbriae stained with several type 2 specific monoclonal antibodies after SDS-PAGE. These monoclonal antibodies also reacted with p59, which indicates that the structural gene for p59 (fimA) encodes a major repeating subunit of type 2 fimbriae. Mapping and shotgun subcloning experiments with BamHI showed that fimA was contained within the 9.7-kb HindIII B fragment of pAV1402. This fragment was subcloned into pUC13. Expression of p59 was strongly orientation dependent, which indicates that the putative Actinomyces promoter from fimA has little or no activity in E. coli. Further mapping revealed that fimA expression from pAV1402 was depended upon the nearby P2 promoter of tet in the vector. Subsequent subcloning of the 9.7 kb HindIII fragment resulted in the identification of a 2.5 kb SmaI fragment that expressed p59. To raise expression, the SmaI fragment was cloned into the expression vector pKK223-3 to yield clone AV3463. p59 can be purified to homogeneity from IPTG-induced cultures of AV3463 by a series of steps including sonification, ultracentrifugation, DEAE-chromatography, gel filtration, and affinity chromatography.
