CONSTRUCTION AND USE OF INTEGRATION PLASMIDS TO GENERATE SITE-SPECIFIC MUTATIONS IN THE ACTINOMYCES-VISCOSUS T14V CHROMOSOME

Stable transformants of Actinomyces viscosus T14V carrying heterologou s DNA were obtained with the aid of integration plasmids. These plasmi ds contained a kanamycin resistance (Km(r)) gene flanked by A, viscosu s T14V genomic DNA, including parts of the type 1 structural fimbrial submit gene (fimP) on one or both sides of the antibiotic marker. Sign ificantly more Km(r) transformants were obtained with a plasmid carryi ng longer segments of homologous strain T14V DNA. Integration of this plasmid into the A. viscosus T14V genome affected the expression and f unction of type 1 fimbriae in the transformants. In the transformant s train designated A. viscosus MY50D, the inactivated fimP replaced the wild-type fimP via allelic replacement. A. viscosus MY51S and MY52S ea ch contained a copy of the plasmid integrated into the genome by a Cam pbell-like insertion mechanism. A. viscosus MY50D and MY51S lacked typ e 1 fimbriae and did not bind to proline-rich proteins (the fimbrial r eceptors) immobilized on nitrocellulose. In contrast, strain MY52S syn thesized the structural subunit protein, as detected by immunostaining with anti-A. viscosus T14V type 1 fimbria antibodies. However, the hi gh-molecular-weight proteins observed in sodium dodecyl sulfate-polyac rylamide gels of fimbriae from the cell wail of the wild-type strain T 14V were absent in cell wall preparations of this strain. Moreover, A. viscosus MY52S failed to bind, in vitro, to proline-rich proteins. Th us, these results demonstrate that insertion of heterologous DNA at sp ecific sites of the Actinomyces genome can be facilitated with integra table plasmids and that the transformants and mutants generated will a id in the delineation of the roles and contributions of specific genes to the structure and function of any macromolecule produced by these organisms.