Table_1_Identification and Application of a Panel of Constitutive Promoters for Gene Overexpression in Staphylococcus aureus.xlsx

Staphylococcus aureus is a leading pathogen that is currently the most common cause of infection in hospitalized patients. An in-depth genetic analysis of S. aureus virulence genes contributing to pathogenesis is needed to develop novel antimicrobial therapies. However, tools for genetic manipulation in S. aureus are limited, particularly those for gene expression. Here, 38 highly expressed genes were identified in S. aureus USA300_FPR3757 via RNA-seq. Promoter regions from 30 of these genes were successfully cloned, of which 20 promoters exhibited a wide range of activity. By utilizing these active promoters, 20 S. aureus-Escherichia coli shuttle vectors were constructed and evaluated by expressing an egfp reporter gene. Expression of the egfp gene under the control of different promoters was confirmed and quantified by Western blotting and qPCR, which suggested that the activity of these promoters varied from 18 to 650% of the activity of PsarA, a widely used promoter for gene expression. In addition, our constructed vectors were verified to be highly compatible with gene expression in different S. aureus strains. Furthermore, these vectors were evaluated and used to overexpress two endogenous proteins in S. aureus, namely, catalase and the transcriptional repressor of purine biosynthesis (PurR). Meanwhile, the physiological functions and phenotypes of overexpressed PurR and catalase in S. aureus were validated. Altogether, this evidence indicates that our constructed vectors provide a wide range of promoter activity on gene expression in S. aureus. This set of vectors carrying different constitutive promoters developed here will provide a powerful tool for the direct analysis of target gene function in staphylococcal cells.

金黄色葡萄球菌（Staphylococcus aureus）是目前住院患者最常见感染的首要病原菌。开发新型抗菌疗法亟需针对参与致病过程的金黄色葡萄球菌毒力基因开展深入遗传学分析。然而，当前用于金黄色葡萄球菌的遗传操作工具十分有限，尤以基因表达相关工具为甚。本研究通过RNA测序（RNA-seq）在金黄色葡萄球菌USA300_FPR3757菌株中鉴定出38个高表达基因，成功克隆其中30个基因的启动子区域，其中20个启动子展现出跨度宽泛的转录活性。本研究利用这些活性启动子构建了20株金黄色葡萄球菌-大肠杆菌（Escherichia coli）穿梭载体，并通过表达增强型绿色荧光蛋白（egfp）报告基因对其进行功能评估。通过蛋白质免疫印迹（Western blotting）与实时定量PCR（qPCR）验证并定量了不同启动子调控下egfp基因的表达水平，结果显示这些启动子的活性范围为通用表达启动子PsarA活性的18%至650%。此外，经验证，本研究构建的载体可在多种金黄色葡萄球菌菌株中高效适配基因表达。进一步地，本研究对这些载体进行了评估，将其用于过表达金黄色葡萄球菌的两种内源蛋白：过氧化氢酶与嘌呤生物合成转录阻遏蛋白（PurR），同时验证了过表达PurR与过氧化氢酶后金黄色葡萄球菌的生理功能与表型。综上，上述实验结果表明，本研究构建的载体可在金黄色葡萄球菌中为基因表达提供跨度宽泛的启动子活性选择。本研究所构建的这套携带不同组成型启动子的载体，将为直接分析葡萄球菌细胞内靶基因的功能提供一款强有力的研究工具。