Development of an efficient transformation and gene expression system for a fastidi-ous oral anaerobe

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A comF-dependent natural competence has been discovered for the first time in an important periodontal pathogen, Tannerella forsythia. Keeping T.forsythia in a biofilm throughout the pr-ocedure allowed efficient DNA uptake and allelic replacement. Based on this finding, a simple method has been established for efficient targeted mutagenesis in T.forsythia. One of the critical conditions affect-ing the efficiency of transformation is the freshness of biofilm, and the recommended subculture duration of input biofilm is 4 days just before transformation. Furthermore, an autonomous chloramphenicol-resista-nt CAT cassette, permF-CAT, has been constructed and successfully applied to T.forsythia. Using permF-CAT, a final version of the common expression vector has been designed which should be functional not only in T.forsythia but also in the other Red Complex anaerobes.