Objective: To identify the differential genes in Porphyromonas gingivalis (P.gingivalis) highly toxic strain W83 and minimally toxic strain ATCC 33277.
Methods: Using suppression subtractive hybridization (SSH) to compare P.gingivalis highly toxic strain W83 (tester) and minimally toxic strain ATCC 33277 (driver). The chromosomal DNAs were purified from P.gingivalis W83 and P.gingivalis ATCC 33277, and digested by restriction enzyme RsaI. The tester DNA samples were separated and ligated with adaptor 1 and adaptor 2R. Two subtractive hybridization and PCR profile were performed. Tester-specific DNAs also were selectively amplified. The mixture of subtracted DNA fragments were ligated with pMD-18T vector and transformed to competent cells E.coli JM109. The differential subtraction library was established. The positive clones were identified by PCR and then sequenced, and searched homologically.
Results: Subtractive library which had high subtractive efficiency was successfully set up and 36 positive clones were screened by SSH. The fragments from 88 bp to 372 bp were enriched in P.gingivalis highly toxic strain W83 sequences which were absent from P.gingivalis ATCC 33277. Through dot blot analysis confirmed that all these fragments were present in P.gingivalis W83 but absent from ATCC 33277. The GenBank homology search indicated that among them, several genes were associated with two paralogous regions of the chromosome; Some genes are associated with evasion of P.gingivalis W83; Another gene was related to antibiotic resistance and the products of some genes were virulence and acquisition of peptides.
Conclusions: Comparative whole-genome analysis of highly toxic and minimally toxic strains of P.gingivalis has identified the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277. These genes may provide an important clue for studying the mechanism of occurrence and development of periodontal disease.