Chronic periodontitis is an inflammatory, plaque-associated disease of the supporting tissues of the teeth which results in destruction of the tooth’s supporting tissues. It is believed to be initiated by changes in the species composition of subgingival plaque, and subsequent alteration of the host immune response. Treponema denticola, together with Porphyromonas gingivalis and Tannerella forsythia form the ‘Red Complex’ bacteria, which have strong association with severe periodontal disease.
T. denticola is a proteolytic obligate anaerobe that is one of only a few motile bacteria in the oral cavity. The molecular details of virulence are poorly characterized in this bacterium due to the limitations in its genetic manipulation. Transformation of the type strain ATCC 35405 is impeded by: limited selectable markers; an inability to accept available shuttle plasmids; a unique DNA restriction-modification system and a very low transformation efficiency. A number of T. denticola isogenic mutants have been reported in recent years, however the transformation protocols are not routinely reproducible and there remains a need for the development of a reliable transformation system for T. denticola.
In this study, we systematically investigated a number of variables including washing conditions and storage of competent cells, number of transformable cells, amount and methylation status of DNA. Competent cells were electroporated with plasmid pCF382 and after 10-14 days of incubation, erythromycin-resistant colonies were enumerated and screened by PCR. Results show that ATCC 33520 was routinely transformed but no transformants were generated for ATCC 35405. Furthermore, the transformation efficiency of ATCC 33520 was increased by approximately 10-fold when the competent cells were prepared under strictly anaerobic conditions. This study confirms the difficulty in transformation of ATCC 35405 compared to ATCC 33520 and shows that preparation of competent cells under strictly anaerobic conditions is the most important factor in improving the transformation efficiency of T. denticola.