In vivo inactivation of the Streptococcus mutans recA gene mediated by PCR amplification and cloning of a recA DNA fragment.
The inactivation of the RecA protein in pathogenic oral streptococci would facilitate genetic analysis of potential virulence factors in these strains. Comparison of recA nucleotide (nt) sequences from a number of bacteria has suggested that two regions of highly conserved RecA amino acid (aa) sequence could be used as a basis for synthesizing degenerate oligodeoxyribonucleotide primers with which to amplify recA homologues from the streptococci. Accordingly, primer mixtures were used to amplify a 693-bp fragment of the Streptococcus mutans chromosome by PCR. The amplified fragment was cloned and its identity confirmed via hybridization to an Escherichia coli recA gene probe and by nt sequence determination. The recA homologue fragment from S. mutans GS-5 was 63% and 75% homologous to the deduced aa sequences of the E. coli and Bacillus subtilis RecA enzymes, respectively. The S. mutans recA fragment was mutagenized in vitro via insertional inactivation and returned to the chromosome using allelic exchange. The resulting strains of S. mutans were shown to be substantially more sensitive to UV irradiation than the wild-type strain. Further, the ability to incorporate linear markers into the chromosome was abolished in putative S. mutans recA strains, thus indicating the functional inactivation of RecA in these microorganisms.
Quivey
R G
RG
Department of Dental Research, University of Rochester School of Medicine and Dentistry, NY 14642.
Faustoferri
R C
RC
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013295:Streptococcus mutans / Q000235:genetics* / Q000528:radiation effects
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011938:Rec A Recombinases