Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima, 734-8551, Japan.
Sci Rep. 2017 Jun 6;7(1):2865. doi: 10.1038/s41598-017-02930-7.
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 (GFP) 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 (GFP, 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.
荧光蛋白技术的最新进展为生物成像应用提供了多种多样的选择；然而，目前革兰氏阳性菌金黄色葡萄球菌的生物成像工具需要进一步开发，以提高荧光强度和实现多色荧光蛋白调色板。为了增强临床金黄色葡萄球菌菌株中多色荧光蛋白的表达，我们开发了新的荧光蛋白表达载体，其中包含 blaZ/sodp 启动子，该启动子由β-内酰胺酶基因 (blaZ) 启动子和超氧化物歧化酶基因 (sod) 的核糖体结合位点 (RBS) 组成。我们发现，由 blaZ/sodp 启动子驱动的金黄色葡萄球菌适应型 GFP（GFP）在金黄色葡萄球菌实验室菌株 RN4220 中高度表达，但在含有内源性 blaI 基因的临床菌株 MW2 和 N315 中不表达，该基因是 blaZ 基因启动子的抑制剂。因此，我们通过引入 BlaI 结合基序中的取代突变构建了一个组成型诱导的 blaZ/sodp 启动子（blaZ/sodp(Con)），该修饰允许多色 GFP 变体（GFP、EGFP、mEmerald、Citrine、Cerulean 和 BFP）以及优化密码子的珊瑚礁荧光蛋白（mCherry 和 AmCyan）在金黄色葡萄球菌临床菌株中的表达增强。这些新的荧光探针为增强多色荧光蛋白的表达提供了新的工具，并有助于清晰地可视化临床金黄色葡萄球菌菌株。
