单 位：
山东省农业科学院农产品研究所;山东省临沂市相公中心医院;齐鲁工业大学(山东省科学院)生物工程学院/山东省微生物工程重点实验室
关键词：
尿道致病性大肠杆菌;Kgu S/KguR;c5032—c5037基因;Red重组系统
摘 要：
[目的]构建尿道致病性大肠杆菌(uropathogenic Escherichia coli,UPEC) CFT073株的双组分信号系统Kgu S/KguR调控的c5032—c5037基因的缺失株,并研究基因缺失株在有氧和厌氧条件下的生长特性。[方法]运用Red重组系统将带有c5032—c5037基因同源臂的氯霉素抗性基因取代c5032—c5037基因,在温度敏感型质粒p CP20作用下消除氯霉素抗性基因,构建基因缺失株CFT073Δc5032—c5037,用PCR和基因测序验证基因敲除是否成功,测定有氧和厌氧条件下CFT073Δc5032—c5037在以α-酮戊二酸为唯一碳源的M9培养基中培养不同时间的菌液D600值。[结果]构建了CFT073Δc5032—c5037,PCR验证及基因测序结果均表明已成功敲除c5032—c5037基因;厌氧条件下CFT073Δc5032—c5037在以α-酮戊二酸为唯一碳源的M9培养基中生长比野生型菌株CFT073缓慢,且差异极显著(P<0.01),但在有氧条件下二者的生长无显著差异。[结论]成功构建了基因缺失株CFT073Δc5032—c5037,并试验证实Kgu S/KguR调控的c5032—c5037基因在厌氧条件下参与α-酮戊二酸的利用,为进一步研究Kgu S/KguR在UPEC中的代谢适应机制奠定了基础。
译 名：
Construction and growth characteristics analysis of the deletion mutant of the target gene regulated by two-component signaling system KguS/KguR of UPEC
作 者：
LI Guoqiang;ZHU Liping;ZHU Lizhen;CHEN Leilei;YAN Shigan;School of Bioengineering/Shandong Key Laboratory of Microbial Bioengineering,Qilu University of Technology(Shandong Academy of Sciences);Xianggong Central Hospital of Linyi City;Institute of Agricultural Food,Shandong Academy of Agricultural Sciences;
关键词：
uropathogenic Escherichia coli;;Kgu S/KguR;;c5032-c5037 genes;;Red recombination system
摘 要：
[Objectives]This study aims to delete c5032-c5037 genes regulated by two-component signaling system KguS/KguR of uropathogenic Escherichia coli(UPEC) strain CFT073,and study the growth characteristics of the mutant under aerobic or anaerobic conditions.[Methods]Red recombination system was used to replace c5032-c5037 genes with chloramphenicol resistance gene containing the homologous arms of c5032-c5037 genes,and then chloramphenicol resistance genes was eliminated by introducing a temperature sensitive plasmid p CP20.Finally c5032-c5037 genes deletion mutant CFT073Δc5032-c5037 was constructed.PCR and gene sequencing were used to confirm whether genes were successfully knocked out.The growth characteristics of the mutant CFT073Δc5032-c5037 were monitored in M9 minimal medium with α-ketoglutarate as the sole carbon source under aerobic or anaerobic conditions.[Results]The results of PCR and DNA sequencing of the mutant CFT073Δc5032-c5037 accorded with theoretical results.No significant growth differences were observed between the wild strain CFT073 and the mutant CFT073Δc5032-c5037 under aerobic conditions,while significant differences appeared between them under anaerobic conditions(P<0.01).[Conclusions]Genes deletion mutant CFT073Δc5032-c5037 was constructed successfully and the growth result revealed c5032-c5037 genes were closely correlated with the utilization of α-ketoglutarate under anaerobic conditions.This study could establish the foundation for further research in the function of KguS/KguR.
