Construction and use of integration plasmids to generate site-specific mutations in the Actinomyces viscosus T14V...

Stable transformants of Actinomyces viscosus T14V carrying heterologous DNA were obtained with the aid of integration plasmids. These plasmids contained a kanamycin resistance (Kmr) gene flanked by A. viscosus T14V genomic DNA, including parts of the type 1 structural fimbrial subunit gene (fimP) on one or both sides of the antibiotic marker. Significantly more Kmr transformants were obtained with a plasmid carrying longer segments of homologous strain T14V DNA. Integration of this plasmid into the A. viscosus T14V genome affected the expression and function of type 1 fimbriae in the transformants. In the transformant strain designated A. viscosus MY50D, the inactivated fimP replaced the wild-type fimP via allelic replacement. A. viscosus MY51S and MY52S each contained a copy of the plasmid integrated into the genome by a Campbell-like insertion mechanism. A. viscosus MY50D and MY51S lacked type 1 fimbriae and did not bind to proline-rich proteins (the fimbrial receptors) immobilized on nitrocellulose. In contrast, strain MY52S synthesized the structural subunit protein, as detected by immunostaining with anti-A. viscosus T14V type 1 fimbria antibodies. However, the high-molecular-weight proteins observed in sodium dodecyl sulfate-polyacrylamide gels of fimbriae from the cell wall of the wild-type strain T14V were absent in cell wall preparations of this strain. Moreover, A. viscosus MY52S failed to bind, in vitro, to proline-rich proteins. Thus, these results demonstrate that insertion of heterologous DNA at specific sites of the Actinomyces genome can be facilitated with integratable plasmids and that the transformants and mutants generated will aid in the delineation of the roles and contributions of specific genes to the structure and function of any macromolecule produced by these organisms.