金葡菌转座子测序文库的构建及促蛋白酶的活性基因鉴定

Generating a collection of insertion mutations in the Staphylococcus aureus genome for transposon sequencing and Identification of Genes contributing to the production of extracellular proteases

背景金黄色葡萄球菌能产生多种蛋白酶引起侵袭性感染,转座子测序技术是发掘基因功能的有力工具,从基因组层面分析促进金葡菌蛋白酶活性基因对其预防及治疗具有重要作用。目的构建能用于转座子测序的突变文库,筛选及鉴定促进蛋白水解活性基因。方法采用基因合成及无缝克隆技术构建带有mariner转座酶的质粒pSATn,并使用脱水四环素红霉素诱导转座并富集形成突变文库。通过牛奶平板对金葡菌蛋白酶水解活性相关基因进行筛选并使用半随机PCR对转座子插入位点进行鉴定。结果成功构建转座子末端均带有MmeⅠ酶切位点金葡菌转座子突变文库,共筛选出26株蛋白酶缺陷克隆菌株,涉及22个基因。其中,新基因pde2转座子插入株显示蛋白水解活性减弱,生物被膜形成能力显著增强,大蜡螟幼虫致死能力显著降低的特征。结论本研究构建的转座子突变文库为后续转座子测序技术的开展奠定基础,为了解金葡菌蛋白水解活性调控的分子基础提供了新的见解,新筛选的基因pde2有望成为控制金黄色葡萄球菌感染的新靶标。

Background Staphylococcus aureus establish an invasive infection by produce a variety of proteases.Transposon sequencing provides a powerful tool to explore gene functions.Genomic analysis of genes that increase proteolytic active play a key role in prevention and treatment of infections.Objective To construct mutation library for transposon sequencing and identify proteolytic active genes.Methods The plasmid pSATn containing mariner transposase was constructed by gene synthesis and Seamless Cloning,transposon mutagenesis was induced by erythromycin and dehydrating tetracycline enrichment for mutational libraries.The genes involved in the hydrolytic activity were screened by milk plate and the transposon insertion sites were identified by semi-random PCR.Results A library of transposon mutation mutations with MmeI restriction site at the ends of transposons was successfully constructed,and a total of 26 protease-deficient clonal strains involving 22 genes were screened.Among them,the new gene pde2 transposon insertion showed that the proteolytic activity was weakened,biofilm formation was significantly enhanced,and the lethal ability of C.mellonella was significantly reduced.Conclusion The transposon mutation library constructed in this study lay the groundwork for such future transposon sequencing technology,provides new insight into the molecular basis of the regulation of staphylococcus aureus hydrolytic activity,and the newly characterization of pde2 is expected to be a novel target for controlling Staphylococcus aureus infection.