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

Abstract

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.

Identification and Characterization of a Novel Major Facilitator Superfamily Efflux Pump, SA09310, Mediating Tetracycline Resistance in Staphylococcus aureus

“…Genomic isolation and plasmid preparation from E. coli or S. aureus were performed as previously described ( 36 ). PCRs were performed using OneTaq 2× master mix or Q5 high-fidelity 2× master mix from New England Biolabs (NEB) according to the manufacturer’s instructions.…”

“…The strain with repair of Δ sa09310 (Δ sa09310 _com) was generated by complementing the sa09310 gene at its original site on the chromosome in the Δ sa09310 mutant using the same method as gene knockout. Overexpression of sa09310 was achieved by using the replicative vector pQLV1025 with the strong constitutive promoter P 08825 from S. aureus ( 36 ).…”

Identification and Characterization of a Novel Major Facilitator Superfamily (MFS) Efflux Pump SA09310 Mediating Tetracycline Resistance inStaphylococcus aureus

“…The complementary strain of Δ sa09310 (Δ sa09310_ com) was generated by complementing the sa09310 gene at its original site on the chromosome in Δ sa09310 using the same method as gene knockout. Overexpression of sa09310 was achieved by using the replicative vector pQLV1025 with the strong constitutive promoter P 08825 from S. aureus (26).…”

“…Genomic isolation and plasmid preparation from E. coli or S. aureus were performed as previously described (26). PCRs were performed using OneTaq 2´ Master Mix or Q5 High-Fidelity 2´ Master Mix from NEB according to the manufacturer's instructions.…”

Toolbox of Characterized Genetic Parts for Staphylococcus aureus

“…We characterized 24 constitutive promoters in S. aureus, to control gene expression at the level of transcription. Several libraries of native gene promoter fragments have been characterized previously in S. aureus, 41,42 but these genetic parts have used large DNA fragments (>100 base pairs (bp)) that contain both a promoter and putative RBS. Thus, we aimed to characterize short promoters (<65 bp) to minimize the inclusion of unknown regulatory elements and for easier cloning of synthetic constructs.…”