大肠杆菌CusS的生物信息学分析及对银离子胁迫的响应

Bioinformatics of CusS in Escherichia coli in response to silver ion stress

【目的】解析大肠杆菌(Escherichia coli)K-12菌株同型二聚体内膜传感器组氨酸激酶(sensor histidine kinase,CusS)蛋白在细菌应答金属银离子胁迫中的调控机制,为该菌的防治提供重要科学依据。【方法】利用ProtParam、ProtScale、Protein-Sol、TMHMM、SignalP、LocTree3、NetNGlyc-1.0、NetPhosBac-3.0、SOPMA、I-TASSERF、STRING和MEGA分别预测CusS的理化性质、亲水性、可溶性、跨膜域、信号肽、亚细胞定位、糖基化位点、磷酸化位点、二级结构、三级结构、蛋白互作的关系网络和蛋白在革兰阴性杆菌中的同源性。采用Red同源重组技术构建大肠杆菌ΔcusS,在不同培养基中连续监测ΔcusS的生长情况,观察该基因缺失后的细菌生长活性;通过最小抑菌浓度(minimal inhibitory concentration,MIC)试验评价该缺失株对金属铜、银离子和临床常见抗生素的敏感性变化;运用RT-qPCR检测cusS缺失后其下游基因cusCFBA和cusR转录水平。【结果】CusS蛋白由480个氨基酸组成,相对分子质量为53738.05,原子总数为7624,等电点为6.02,具有稳定性,是一种亲水性、不溶性蛋白;含有跨膜域;不存在信号肽,定位于细胞内膜中;存在2个糖基化位点、24个丝氨酸磷酸化位点、14个苏氨酸磷酸化位点和3个酪氨酸磷酸化位点;二级结构中α-螺旋占比55.42%,β-折叠占比11.67%,β-转角占比3.75%,无规则卷曲占比29.17%;cusS在埃希菌属和志贺菌属中的保守性高;菌落PCR和一代测序验证ΔcusS构建成功;连续检测生长曲线表明cusS缺失并不影响细菌的生长代谢,但CusS蛋白为大肠杆菌抵御金属银胁迫的关键基因。【结论】cusS作为一个关键基因,它的缺失并不影响大肠杆菌的生长活性,但会显著降低细菌抵御银离子胁迫的应答能力。缺失cusS将使下游基因cusCFBA和cusR的mRNA表达水平显著下降。对CusS蛋白进行生物信息学分析及表型初探,为深入了解CusS在大肠杆菌应答银离子胁迫的调控机制奠定了基础。

[Objective]To decipher the regulatory mechanism of a sensor histidine kinase(CusS)in Escherichia coli K-12 in response to silver ion stress and provide scientific evidence for the prevention and treatment of this bacterium.[Methods]ProtParam,ProtScale,Protein-Sol,TMHMM,SignalP,LocTree3,NetNGlyc-1.0,NetPhosBac-3.0,SOPMA,I-TASSERF,STRING,and MEGA were employed to predict the physicochemical properties,hydrophilicity,solubility,transmembrane domain,signal peptides,subcellular localization,glycosylation sites,phosphorylation sites,secondary structure,tertiary structure,protein-protein interaction network of CusS,and the homology of CusS in Gram-negative bacilli,respectively.After that,ΔcusS was constructed by the Red homologous recombination system,and the growth ofΔcusS in different media was monitored.In addition,we evaluated the sensitivity ofΔcusS to silver and copper ions and common antibiotics based on the minimum inhibitory concentration(MIC).RT-qPCR was employed to determine the transcription levels of cusCFBA and cusR after cusS deletion.[Results]CusS was composed of 480 amino acid residues,with the relative molecular weight of 53738.05,the atom number of 7624,and the isoelectric point of 6.02.It was a hydrophilic and insoluble protein containing transmembrane domain,and no signal peptide,located in the intracellular membrane.CusS had 2 glycosylation sites,24 serine phosphorylation sites,14 threonine phosphorylation sites,and 3 tyrosine phosphorylation sites.In the secondary structure,α-helixes,β-sheets,β-turns,and random coils accounted for 55.42%,11.67%,3.75%,and 29.17%,respectively.The gene cusS was highly conserved in Escherichia and Shigella.The colony PCR and first-generation sequencing confirmed the successful construction ofΔcusS.The deletion of cusS had no influence on the growth or metabolism of the strain.However,cusS was the key gene for E.coli in response to the silver ion stress.[Conclusion]The deletion of cusS did not affect the growth but attenuated the protective response of E.coli to silver ion stress.Furthermore,the deletion of cusS significantly down-regulated the mRNA levels of the downstream genes cusCFBA and cusR.The bioinformatics analysis and phenotype characterization of CusS lays a foundation for unveiling the regulatory mechanism of CusS in E.coli in response to silver ion stress.