Construction and analysis of a Streptococcus parasanguis recA mutant: Homologous recombination is not required for adhesion in an in vitro tooth surface model

PCR,vas used to amplify an internal region of the recA gene from Streptococ cus parasanguis FW213. The PCR fragment was used as a probe to recover the entire streptococcal recA gene from an S. parasanguis genomic library, and the sequence of the gene aas determined. The deduced product of the S. para sanguis recA gene showed a high degree of amino acid identity with other pr okaryotic RecA proteins. The cloned recA sequence was disrupted in vitro by insertional mutagenesis, and the mutated allele was then introduced into t he S. parasanguis chromosome by homologous recombination. Results of Southe rn hybridizations confirmed the replacement of the wild-type recA gene with the mutated allele. The recA mutant strain was considerably more sensitive to UV light than the parental strain, and this phenotype was consistent wi th a mutation in recA. The S. parasanguis recA mutant showed no reduction i n its ability to adhere in the in vitro tooth surface model, saliva-coated hydroxylapatite (SHA), or in its ability to express the fimbria-associated adhesin Fap1. These results demonstrate that in vitro attachment of S. para sanguis FW213 to SHA and expression of Fap1 are recA independent.