Abstract
Recent advances in fluorescent protein technology provide a wide variety of biological imaging applications; however current tools for bio-imaging in the Gram-positive bacterium Staphylococcus aureus has necessitated further developments for fluorescence intensity and for a multicolor palette of fluorescent proteins. To enhance the expression of multicolor fluorescent proteins in clinical S. aureus strains, we developed new fluorescent protein expression vectors, containing the blaZ/sodp promoter consisting of the β-lactamase gene (blaZ) promoter and the ribosome binding site (RBS) of superoxide dismutase gene (sod). We found S. aureus-adapted GFP (GFPsa) driven by the blaZ/sodp promoter was highly expressed in the S. aureus laboratory strain RN4220, but not in the clinical strains, MW2 and N315, harboring the endogenous blaI gene, a repressor of the blaZ gene promoter. We therefore constructed a constitutively induced blaZ/sodp promoter (blaZ/sodp(Con)) by introducing substitution mutations into the BlaI binding motif, and this modification allowed enhanced expression of the multicolor GFP variants (GFPsa, EGFP, mEmerald, Citrine, Cerulean, and BFP) as well as codon-optimized reef coral fluorescent proteins (mCherry and AmCyan) in the S. aureus clinical strains. These new fluorescent probes provide new tools to enhance expression of multicolor fluorescent proteins and facilitate clear visualization of clinical S. aureus strains.
Highlights
Fluorescent proteins are widely used as biological markers that enable visualization of subcellular protein localization, gene expressions, protein-protein interactions, and in vivo monitoring of bacterial infection[1,2,3]
For bio-imaging numerous green fluorescent protein (GFP) and reef coral fluorescent proteins (RCFP) variants are commercially available from many distributors; these FPs were not optimized for less common bacterial strains
S. aureus strain RN4220 colonies containing pS1GFP in which GFPmut3b was expressed under the control of the β-lactamase gene promoter exhibited faint fluorescence on trypticase soy broth (TSB) agar plates (Fig. 1b)
Summary
Fluorescent proteins are widely used as biological markers that enable visualization of subcellular protein localization, gene expressions, protein-protein interactions, and in vivo monitoring of bacterial infection[1,2,3]. This study developed fluorescent protein expression vectors with the PblaZ promoter to highly express GFP and RCFP variants in clinically isolated S. aureus. To improve the expression of an exogenous fluorescent protein in S. aureus clinical strains, we used the blaZ/sodp(Con) promoter and codon-optimized fluorescent protein genes, and showed that the fluorescence intensity in the series of GFP and RCFP variants were significantly enhanced in the clinical strains. These new tools efficiently expressing fluorescent proteins in clinical S. aureus strains are valuable for understanding the pathogenic mechanism of S. aureus
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