Construction of a reporter system for bifidobacteria using chloramphenicol acetyltransferase and its application for evaluation of promoters and terminators.

Abstract

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 (cat) from Staphylococcus aureus. 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^2-) 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 Bifidobacterium longum 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 B. longum, Bifidobacterium breve, and Bifidobacterium adolescentis. 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 B. longum 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.