HPI毒力岛缺失的EAggEC17-2突变菌株的构建及其功能研究

目的 构建HPI毒力岛缺失的EAggEC17-2突变株，初步研究EAggEC菌株携带的耶尔森菌HPI毒力岛合成铁载体Ybt的功能。方法以EAggEC17-2为出发菌株，irp8基因部分序列作为同源重组的一侧序列，irp5基因序列作为同源重组的另一侧序列，中间插入有氯霉素（Can）抗性基因（cat基因）标记。通过接合转移和同源重组，构建了缺失约24kb的HPI毒力岛功能核心区区域EAggEC17-2的仝岛缺失株EA85。应用流式细胞技术（FACS）检测指示菌株WA-CS irp1：：KN（pC3G3．3N）荧光强度的变化情况，对EA85缺失株和出发菌株进行了合成Ybt的功能比较研究。结果成功构建了EAggEC17-2HPI全岛缺失株EA85。EAggEC17-2菌株具有表达Ybt的功能，而缺失株EA85丧失了合成Ybt的能力。结论EAggEC17-2HPI毒力岛的缺失，使Ybt的合成彻底阻断。EAggEC17-2具有的合成Ybt的功能是由其染色体携带的HPI毒力岛所决定的。

Analysis of HPI function by constructing EAggEC17-2 mutant deleted HPI full island

Objective To analyze the biosynthesis of siderophore yersiniabactin(Ybt) of high pathogenicity island(HPI) in EAggEC strain by constructing EAggEC17 2 mutant deleted HPI full island. Methods irp 8 and irp 5 genes by constructed by inserting a selectable substitution with chloramphenicol resistance gene( cat gene) were used as two homologous sequences. By homologous recombination and conjunction mobilization, we screened EA85 mutant which deleted about 24 kb HPI core part from irp 8 to irp 5 gene. Flow cytometry measurement was used to detect Ybt biosynthesis of EA85 mutant and EAggEC17 2 strain by the changing fluorescent signal of a reporter strain WA CS irp 1::KN(pCJG3.3N). Results We constructed EA85 strain successfully, a HPI deleted mutant of EAggEC17 2 strain. EAggEC17 2 strain was able to biosynthesize Ybt while EA85 mutant lost the ability. Conclusion Deletion of EAggEC17 2 HPI resulted in the disability of biosynthesizing Ybt. It suggested that Ybt biosynthesis of EAggEC17 2 was depended on the HPI located on its chromosome.