Biofilms of Porphyromonas gingivalis Contain Extracellular DNA

Objective: Porphyromonas gingivalis is a Gram-negative anaerobe that colonizes dental biofilms and is an etiological agent in the development of periodontitis. Previously, we established that an efficient natural competence system, relying on the comF gene, was present in strain W83, and DNA can also be transferred by conjugation mediated by genes traA-Q. In this study we investigate the conditions regulating release of extracellular DNA (eDNA) as a substrate for natural transformation. Method: P. gingivalis cells grown planktonically or as biofilms were challenged for 24 hours with a PCR-generated DNA fragment containing tetQ, and DNA transformation frequencies calculated after recovery on selective media. Biofilms of wild-type strain W83, W83 comF mutant, W83 traA-Q mutant, strain 33277, or a mix of strains W83/33277 were grown for 24 hours or 7 days anaerobically, and the presence of live/dead bacteria and eDNA detected via fluorescent staining and epifluorescent microscopy.  The presence of eDNA in biofilms was confirmed by treatment with DNase I prior to staining. Relative proportions of eDNA produced by wild-type and mutant strains was determined by purification and quantification of DNA from the supernatant of 24 hour biofilms. Group comparisons for statistical significance were done using the unpaired T-test, using greater than three independent replicates for each group. Results: P. gingivalis grown as biofilms were transformed at significantly higher frequencies than planktonic cells. eDNA was detected in biofilms of strains W83 and 33277, and was sensitive to digestion with DNaseI. Biofilm eDNA was associated equally with live and dead bacterial cells; live cells dominated biofilms at both time points except in the mixed strain biofilm. eDNA was significantly less in the W83 traA-Q mutant biofilm. Conclusion: P. gingivalis biofilm conditions favor enhanced production and uptake of eDNA, and is a likely mechanism for the generation of genetic diversity within the species.