Kozakai Tomoya, Shimofusa Yoko, Nomura Izumi, Suzuki Tohru
United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan.
Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan.
Biosci Microbiota Food Health. 2021;40(2):115-122. doi: 10.12938/bmfh.2020-070. Epub 2021 Jan 19.
A reporter assay system is an essential tool for investigating gene expression mechanisms. In the case of bifidobacteria, several convenient and sensitive reporter systems have been developed. Here, we developed a new reporter system for bifidobacteria using the chloramphenicol acetyltransferase gene () from . This enzyme stoichiometrically produced free CoA-SH, which was analyzed quantitatively with Ellman's test using 2-nitrobenzoic acid (DTNB). The 2-nitro-5-thiobenzoate (TNB) produced showed a strong yellowish color with maximum absorbance at 412 nm. We also constructed a new pBCMAT plasmid series for CAT assays in bifidobacteria to evaluate promoters and terminators. Analyses using promoters from NCC2705 indicated that the CAT assay using these promoters is quantitative, has a wide measurement range, and is stable. In addition, this assay was useful for several bifidobacterial species, including , , and . Compared with evoglow-Bs2, a fluorescent protein used under anaerobic conditions, the CAT assay showed about 0.25% background activity. In analyses using this CAT assay, we identified 11 promoters and 12 terminators of NCC2705. The genes encoding ribosomal proteins, elongation factors, and transfer RNAs possessed strong promoters, and terminators that include strong stem-loops and poly-U tails structures tended to show high activities. Although the abovementioned promoters made stronger contributions to expression activities than the terminators, the maximum fold difference in the activities among the tested terminators was approximately 17-fold. Modification of the -10 box and 5-UTR in the promoters and the structure around the stem-loop in the terminators affected expression levels. These results suggest that the CAT assay is useful for various analyses of bifidobacterial gene expression.
报告基因检测系统是研究基因表达机制的重要工具。对于双歧杆菌而言，已经开发出了几种便捷且灵敏的报告系统。在此，我们利用来自[具体来源未提及]的氯霉素乙酰转移酶基因（）为双歧杆菌开发了一种新的报告系统。该酶按化学计量产生游离的辅酶A - SH，使用2 - 硝基苯甲酸（DTNB）通过埃尔曼测试对其进行定量分析。产生的2 - 硝基 - 5 - 硫代苯甲酸（TNB）呈现出强烈的淡黄色，在412 nm处有最大吸光度。我们还构建了一个新的pBCMAT质粒系列，用于双歧杆菌中的氯霉素乙酰转移酶（CAT）检测，以评估启动子和终止子。使用来自[具体菌株未提及]NCC2705的启动子进行的分析表明，使用这些启动子的CAT检测是定量的，具有较宽的测量范围且稳定。此外，该检测方法对几种双歧杆菌菌株，包括[具体菌株未提及]、[具体菌株未提及]和[具体菌株未提及]都很有用。与在厌氧条件下使用的荧光蛋白evoglow - Bs2相比，CAT检测显示出约0.25%的背景活性。在使用该CAT检测进行的分析中，我们鉴定出了[具体菌株未提及]NCC2705的11个启动子和12个终止子。编码核糖体蛋白、延伸因子和转运RNA的基因具有较强的启动子，而包含强茎环和多聚 - U尾结构的终止子往往表现出较高的活性。尽管上述启动子对表达活性的贡献比终止子更大，但测试的终止子之间活性的最大倍数差异约为17倍。启动子中 - 10框和5 - 非翻译区（5 - UTR）的修饰以及终止子中茎环周围的结构会影响表达水平。这些结果表明，CAT检测对于双歧杆菌基因表达的各种分析很有用。
