Iida Haruka, Nishikawa Kiyoshi, Sato Takuma, Kawaguchi Misuzu, Miyazawa Ken, Hasegawa Yoshiaki
Department of Orthodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Japan.
Department of Microbiology, School of Dentistry, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan.
Pathogens. 2024 Apr 10;13(4):309. doi: 10.3390/pathogens13040309.
() utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing infection. However, the specific environmental signal received by FimS remains unknown. We constructed random mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of , and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the -knockout strain using the - conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of , including the elucidation of structure-function relationships of proteins of interest.
（）利用菌毛粘附素定殖于龈沟并逃避宿主免疫系统。所有与菌毛粘附素相关成分的生物合成均受FimS-FimR双组分系统的正向调控，这使得FimS传感蛋白成为预防感染的一个有吸引力的靶点。然而，FimS所接收的特定环境信号仍不清楚。我们构建了随机突变文库以鉴定FimS信号感知的关键氨基酸残基。使用优化的易错聚合酶链反应（PCR）在（此处原文缺失具体基因名称）的周质结构域编码序列中引入有限数量的随机突变，并通过反向PCR生成携带各种突变体的表达载体。使用（此处原文缺失具体转移系统名称）接合转移系统从（此处原文缺失具体基因名称）敲除菌株中获得了500多个转化体，而使用电穿孔仅获得了约100个转化体。分别有4个和6个转化体菌株表现出表达增加和减少。6个菌株在周质结构域中有单个氨基酸替换，表明这些是FimS信号感知的关键残基。这种新开发的策略应具有普遍适用性，并有助于（此处原文缺失具体基因名称）的分子遗传学研究，包括阐明感兴趣蛋白质的结构-功能关系。
