Construction and analysis of a Streptococcus parasanguis recA mutant: homologous recombination is not required for adhesion in an in vitro tooth surface model.
PCR was used to amplify an internal region of the recA gene from Streptococcus 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 was determined. The deduced product of the S. parasanguis recA gene showed a high degree of amino acid identity with other prokaryotic RecA proteins. The cloned recA sequence was disrupted in vitro by insertional mutagenesis, and the mutated allele was then introduced into the S. parasanguis chromosome by homologous recombination. Results of Southern 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 with a mutation in recA. The S. parasanguis recA mutant showed no reduction in 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. parasanguis FW213 to SHA and expression of Fap1 are recA independent.
Froeliger
E H
EH
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.
Tomich
M
M
Fives-Taylor
P
P
D018829:Adhesins, Bacterial / Q000235:genetics; D001422:Bacterial Adhesion / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D017886:Durapatite; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D010641:Phenotype; D011938:Rec A Recombinases / Q000235:genetics*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012463:Saliva / Q000382:microbiology; D013298:Streptococcus sanguis / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D014070:Tooth / Q000382:microbiology*
D018829:Adhesins, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D017886:Durapatite; D011938:Rec A Recombinases