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 P _sarA , 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.

Keywords: PurR; S. aureus; catalase; constitutive promoter; gene expression; highly expressed gene.

FIGURE 1

Identification of highly expressed genes from Staphylococcus aureus USA300_FPR3757. (A) Growth curve of S. aureus USA300_FPR3757 growing at 37°C in TSB medium. (B) Normalized expression level (FPKM) of genes in the LP, EP, and SP growth phases. Each point represents a gene, and the dashed line indicates the cutoff value of FPKM 1,500. (C) Venn diagram representing the number of shared highly expressed genes (FPK ≥ 1,500) in the LP, EP, and SP growth phases.

FIGURE 2

Evaluation of the activity of selected promoters by beta-galactosidase assay. (A) Selected active promoters were cloned into the promoter-probe vector pQLV003 carrying a lacZ reporter gene. The pQLV003 vector contains the Escherichia coli origin ColE1, pAMα1 replicon for propagation in Gram-positive bacteria, the selectable marker tetracycline (tet) in E. coli and chloramphenicol (cat) in S. aureus. (B) The vector pQLV003 carrying each selected promoter in front of the lacZ gene was transformed into S. aureus USA300. The resulting strains were grown on TSB X-gal plates at 37°C for 24 h. (C) The beta-galactosidase activity of the cell lysate of S. aureus USA300 transformed with each promoter_lacZ plasmid at lag phase (LP), exponential phase (EP), and stationary phase (SP). NG represents the negative control in which the egfp gene was driven by no promoter.

FIGURE 3

Construction of overexpression vectors by using the active promoters. (A) The backbone vector pBUS1_P_cap_HC was used for the development of the set of overexpression vectors. The elements in the backbone vector pBUS1_P_cap_HC include the P_cap promoter, E. coli origin ColE1, the terminator sequence (rrnB T1)5 and the selectable marker tetracycline tet (L). The elements required for rolling-circle replication are indicated: the replication initiator protein gene (repB), the double-strand origin (oriU), and the single-strand origin (oriL). (B) The chloramphenicol cassette (cat) was inserted into pBUS1_P_cap_HC at the BglII site to generate the plasmid pBUS1_P_cap_HC_cat. (C) The DNA fragment containing each individual selected promoter sequence and a ribosomal-binding site (RBS) was cloned into pBUS1_P_cap_HC_cat at the KpnI and NdeI sites, which replaced the original P_cap promoter sequence, to generate a set of expression vectors carrying different constitutive and active promoters.

FIGURE 4

Evaluation and quantification of the strength of constructed vectors by expressing an egfp reporter gene. (A) Fluorescence-based promoter activity assay. The egfp reporter gene was cloned into each vector under the control of different promoters. The resulting strain was transformed into S. aureus USA300, and the fluorescence intensity and OD₆₀₀ value of each strain were measured by a Bioreader at the LP, EP, and SP growth phases. The value of the ratio fluorescence intensity/OD₆₀₀ ratio was calculated. (B) Additionally, the transcriptional level of the egfp gene under different promoters was assessed by RT-qPCR. The expression level was normalized to the internal control sigA gene. Data shown are the mean ± SD of three experiments. (C) The production of recombinant eGFPs was checked by SDS-PAGE and (D) Western blotting using an anti-6 × His tag antibody. NG represents the negative control wherein the egfp gene was driven by no promoter.

FIGURE 5

The constructed vectors were evaluated in different S. aureus strains. The egfp reporter gene was cloned into each vector under the control of different promoters. The resulting plasmid was transformed into different S. aureus strains, and the fluorescence intensity and OD₆₀₀ value of each strain were measured by a Bioreader at the exponential phase. The fluorescence intensity/OD₆₀₀ ratio was calculated. NG represents the negative control in which the egfp gene was driven by no promoter.

FIGURE 6

Evaluation and application of the developed expression vectors on endogenous gene expression in S. aureus. (A) The expression level of the purR gene in three overexpression vectors was validated by RT-qPCR. (B) The production of the PurR protein was evaluated by SDS-PAGE and western blotting. (C) The expression levels of the fnbA and fnbB genes, the target genes repressed by the transcriptional repressor PurR, were assessed by RT-qPCR in three purR overexpression and WT strains. (D) The expression level of the catalase gene in three overexpression vectors was assessed by RT-qPCR. (E) The production of PurR protein was evaluated by SDS-PAGE and western blotting. (F) The catalase activity of cell lysates from each overexpression strain and WT was measured by H₂O₂ and ammonium molybdate-based assays. The undecomposed hydrogen peroxide reacts with ammonium molybdate to produce a yellowish color. More transparency in the reacted mixture represents less hydrogen peroxide left, indicating higher catalase activity. Data shown are the mean ± SD of three experiments. (****P ≤ 0.0001, ***P ≤ 0.005; *P ≤ 0.05 relative to the WT).