UPEC双组分信号系统KguS/KguR调控基因缺失株的构建及其生长特性研究
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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。
[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.
[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.
引文
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[2]Mobley H L,Green D M,Trifillis A L,et al.Pyelonephritogenic Escherichia coli and killing of cultured human renal proximal tubular epithelial cells:role of hemolysin in some strains[J].Infection and Immunity,1990,58(5):1281-1289.
[3]Johnson J R,Stell A L.Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise[J].Journal of Infectious Diseases,2000,181(1):261-272.
[4]Cai W,Wannemuehler Y,Dell'Anna G,et al.A novel two-component signaling system facilitates uropathogenic Escherichia coli's ability to exploit abundant host metabolites[J].PLoS Pathogens,2013,9(6):e1003428.
[5]Rédei G P.Encyclopedia of Genetics,Genomics,Proteomics and Informatics[M].3rd ed.Dordrecht:Springer,2008:1837.
[6]Datsenko K A,Wanner B L.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J].Proc Natl Acad Sci USA,2000,97(12):6640-6645.
[7]Tretter L,Adamvizi V.Alpha-ketoglutarate dehydrogenase:a target and generator of oxidative stress[J].Philosophical Transactions of the Royal Society B Biological Sciences,2005,360(1464):2335-2345.
[8]Buck D,Spencer M E,Guest J R.Primary structure of the succinyl-CoA synthetase of Escherichia coli[J].Biochemistry,1985,24(22):6245-6252.
[9]Yu B J,Sung B H,Ju Y L,et al.sucA B and sucC D are mutually essential genes in Escherichia coli[J].FEMS Microbiology Letters,2006,254(2):245-250.
[10]Martin M,Ferrier B,Baverel G.Transport and utilization of alpha-ketoglutarate by the rat kidney in vivo[J].Pflügers Archiv European Journal of Physiology,1989,413(3):217-224.
[11]Pritchard J B.Intracellular alpha-ketoglutarate controls the efficacy of renal organic anion transport[J].The Journal of Pharmacology and Experimental Therapeutics,1995,274(3):1278-1284.