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

首次在一种重要的牙周病病原体Tannerella patienthia中发现了一种依赖于comF的天然能力。 在整个发病前将嗜热厌氧菌保持在生物膜中允许有效的DNA摄取和等位基因替换。 基于这一发现，一个简单的方法已经建立了有效的靶向诱变T.影响转化效率的关键条件之一是生物膜的新鲜度，建议转化前4 d为接种生物膜的继代时间。此外，一个自主的氯霉素抗性CAT盒，permF-CAT，已被构建并成功地应用于T. acetthia。使用permF-CAT，已经设计了共同表达载体的最终版本，其不仅在嗜热厌氧菌中而且在其他红色复合厌氧菌中应该是功能性的。

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.