Single-Copy Gene Editing of a Cell Wall-Anchored Pilin in Actinomyces oris

The Gram-positive bacterium Actinomyces oris expresses a unique cell wall-anchored fimbria comprised of the fimbrial shaft FimA and the tip fimbrillin CafA, whose gene is not genetically linked to the fimA locus, unlike many other fimbrial gene loci in Gram-positive bacteria. Mutational analyses of individual fimbrillins, FimA and CafA, in A. oris often rely on multi-copy plasmids that may alter the stoichiometry of fimbrillins in vivo, hence fimbrial assembly. Here, we provide a robust method for single-copy gene expression and mutagenesis in A. oris, using CafA as an experimental model. This method can be applied for single-copy gene editing in various bacterial systems.