新发现鸭疫里默氏杆菌RA0C_0556蛋白参与自然转化的作用机制

鸭疫里默氏杆菌(RA)是一种致感染鸭、鹅等禽类严重发病和死亡的革兰氏阴性病原菌。项目组首次报道了RA具有自然转化功能，并确认其自然转化系统组成有别于已知细菌。我们通过构建和筛选RA突变体文库，发现一个1344 bp的ORF编码基因(注释为RA0C_0556)被转座子插入失活后，RA的自然转化能力随即丧失，提示RA0C_0556基因编码蛋白是RA具备自然转化能力的关键蛋白。为阐明其作用机制，拟采用基因敲除/回补、蛋白定位、蛋白-DNA互作、随机点突变、异源回补等方法，确定RA0C_0556蛋白在RA菌体的定位，分析该蛋白优先结合双链DNA还是单链DNA、结合DNA关键区域和关键氨基酸位点，探索其参与外源DNA转运的具体过程，最终确定这种新型蛋白在RA自然转化中所发挥的作用机制及其同源物的进化分布。项目完成后能够发现RA自然转化通路，为阐明革兰氏阴性菌自然转化发生的新机制提供明确的科学依据。

Riemerella anatipestifer(RA), a gram-negative pathogen, mainly infects ducks, geese and cause high morbidity and mortality. In our previous study, it has been shown that RA was natural competent. However, the components of natural transformation system of RA are different from that of the classical natural transformation system. Through screening the transposon insertion mutant library of RA ATCC11845, it was found that the RA0C_0556 mutant was not able to uptake DNA any more. The RA0C_0556 (1344 bp) was annotated as a putative protein in the genome, suggesting that the gene RA0C_0556 encode a novel protein involved in natural transformation. To identify the working mechanism of RA0C_0556, a series of methods including gene knockout/complementation, protein location, protein-DNA interaction, random point mutation, heterologous complement will be employed in this study. The aims of this study are to determine the location of the RA0C_0556 in RA ATCC11845, to explore the location of the protein, the protein bind dsDNA or ssDNA and the key DNA binding domains and the key amino acid site, to reveal the process mediated by this protein in natural transformation of RA ATCC11845. Finally, the working mechanism of RA0C_0556 and its phylogenetic distribution will be determined. This study will not only lay a key foundation for revealing the natural transformation pathway of R. anatipestifer, but also enrich the natural transformation mechanism of Gram-negative bacteria.