AUTHOR=Liu Qiang , Li Daiyu , Wang Ning , Guo Gang , Shi Yun , Zou Quanming , Zhang Xiaokai 

TITLE=Identification and Application of a Panel of Constitutive Promoters for Gene Overexpression in Staphylococcus aureus

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.818307

DOI=10.3389/fmicb.2022.818307

ISSN=1664-302X

ABSTRACT=<p><italic>Staphylococcus aureus</italic> is a leading pathogen that is currently the most common cause of infection in hospitalized patients. An in-depth genetic analysis of <italic>S. aureus</italic> virulence genes contributing to pathogenesis is needed to develop novel antimicrobial therapies. However, tools for genetic manipulation in <italic>S. aureus</italic> are limited, particularly those for gene expression. Here, 38 highly expressed genes were identified in <italic>S. aureus</italic> 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 <italic>S. aureus</italic>-<italic>Escherichia coli</italic> shuttle vectors were constructed and evaluated by expressing an <italic>egfp</italic> reporter gene. Expression of the <italic>egfp</italic> 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 P<italic><sub><italic>sarA</italic></sub></italic>, a widely used promoter for gene expression. In addition, our constructed vectors were verified to be highly compatible with gene expression in different <italic>S. aureus</italic> strains. Furthermore, these vectors were evaluated and used to overexpress two endogenous proteins in <italic>S. aureus</italic>, namely, catalase and the transcriptional repressor of purine biosynthesis (PurR). Meanwhile, the physiological functions and phenotypes of overexpressed PurR and catalase in <italic>S. aureus</italic> were validated. Altogether, this evidence indicates that our constructed vectors provide a wide range of promoter activity on gene expression in <italic>S. aureus</italic>. 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.</p>