表皮葡萄球菌双组分信号转导系统arlS基因生物学功能研究

Biological functions of arlS gene of two-component signal transduction system in Staphylococcus epidermidis

目的探讨表皮葡萄球菌arlS/R双组分信号转导系统(two-component signal transduction system,TCS)的组氨酸激酶arlS基因删除对细菌生物学的影响。方法构建含有红霉素抗性基因(ermB)的pBT2-△arlS质粒,随后将电转入表皮葡萄球菌1457的重组质粒在43℃条件下振摇培养,筛选表皮葡萄球菌1457菌株的arlS缺失突变株(SE1457-△arlS);采用微量板半定量方法检测arlS删除突变对细菌生物被膜形成的影响,通过检测A595值观察其生长曲线,检测过夜培养上清液对菌细胞裂解的活性,并用0.1%TritonX-100诱导细菌自溶的方法检测SE1457-△arlS突变株的自溶情况。结果成功构建pBT2-△arlS质粒,利用同源重组基因删除的方法表皮葡萄球菌1457 arlS基因被删除。△arlS突变株与野生株的生长曲线基本相似,提示arlS基因删除对细菌的增殖无明显影响,但SE1457-△arlS突变株形成生物被膜的能力明显低于野生株,降低90.96%;在0.1%Triton X-100诱导下SE1457-△arlS基因删除突变株的裂解率为97.83%,野生株为55.38%;而△arlS突变株过夜培养上清液对靶细菌裂解的活性则低于野生株,其裂解率为20.50%,野生株为56.12%。结论表皮葡萄球菌arlS/R双组分信号转导系统arlS基因可调控细菌生物被膜形成、菌细胞自溶以及胞外细菌裂解酶的活性。

Objective To investigate biological fimctions of arlS gene deletion which encodes a histidine kinase and is located in the arlSlR two-component signal transduction system of S. epidermidis. Methods A plasmid pBT2-AarlS was constructed with inserting erythromycin resistance gene （ermB）. Then S. epidermidis 1457 transformed with pBT2-AarLS by electropomtion was continuously subcultured at 43℃ until arlS deletion strain （SE1457-△arlS） was selected by homologous recombination. Effect of the arlS mutant on biofilm formation was examined by a semi-quantitative microtiter plate assay. Bacterial growth curve was determined by measuring the value of A595. The bacteriolytic activities of supematants from overnight culture were measured, and Triton X- 100-induced autolysis of SE1457-△arlS was performed. Results The plasmid pBT2-AarlS was constructed and arlS gene was deleted from the genome of S. epidermidis 1457 strain. The growth curve of wild type S. epidermidis strain and SE1457-△arlS were similar, which indicates that arlS mutant has no obvious effect on the prolifera- tion of S. epidermidis. However, as compared with wild type strain, SE1457-△arlS has lower capacity of biofilm formation. After induction by 0. l% Triton X-100, the lytic percentage of SE1457-△arlS and the wild strain was 97.83% and 55.38% respectively. However, bacteriolytic activity of the overnight culture supematant from SE1457-△arlS is lower than that of the wild strain. Conduslon arlS gene of the arlSlR two-component signal transduction system of S. epidermidis can regulate bacterial biofilm formation, autolysis and extracellular bacterio- lytic activity.