NUCLEOTIDE-SEQUENCE OF THE PORPHYROMONAS-GINGIVALIS W83 RECA HOMOLOG AND CONSTRUCTION OF A RECA-DEFICIENT MUTANT

Degenerate oligonucleotide primers were used in PCR to amplify a regio n of the recA homolog-from Porphyromonas gingivalis W83., The resultin g PCR fragment was used as a probe to identify a recombinant lambda DA SH phage (L10) carrying the P. gingivalis recA homolog. The recA homol og was localized to a 2.1-kb BamHI fragment. The nucleotide sequence o f this 2.1-kb fragment was determined, and a 1.02-kb open reading fram e (341 amino acids) was detected. The predicted amino acid sequence wa s strikingly similar (90% identical residues) to the RecA protein from Bacteroides fragilis, No SOS box, characteristic of LexA-regulated pr omoters, was found in the 5' upstream region of the P. gingivalis recA homolog, In both methyl methanesulfonate and UV survival experiments the recA homolog from P. gingivalis complemented the recA mutation of Escherichia coli HB101. The cloned P. gingivalis recA gene was inserti onally inactivated with the ermF-ermAM antibiotic resistance cassette to create a recA-deficient mutant (FLL33) by allelic exchange. The rec A-deficient mutant was significantly more sensitive to UV irradiation than the wild-type strain, W83. W83 and FLL33 showed the same level of virulence in in vivo experiments using a mouse model. These results s uggest that the recA gene in P. gingivalis W83 plays the expected role of repairing DNA damage caused by UV irradiation. However, inactivati on of this gene did not alter the virulence of P. gingivalis in the mo use model.