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, B. breve, and B. 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.

Recent Development of Probiotic Bifidobacteria for Treating Human Diseases

“…The naturally occurring promoter sequences contains promoter core motifs located at the -35 to -10 region from the start of the coding sequence. These sequences conventionally contain the TTGNNN and the TANNNT conserved sequences respectively ( Kozakai et al, 2021 ).…”

“…Various constitutive promoters for Bifidobacterium expression were developed by sifting through the gene sequences within the Bifidobacterium genome ( Sakanaka et al, 2014 ; Kozakai et al, 2021 ). The promoter core motif sequences were identified in silico using the hidden Markov model on the upstream sequences of the transcriptional start site of various coding genes in Bifidobacterium.…”

“…The promoter core motif sequences were identified in silico using the hidden Markov model on the upstream sequences of the transcriptional start site of various coding genes in Bifidobacterium. This approach was used to identify the various putative constitutive promoter sequences in B. longum NCC2705 ( Kozakai et al, 2020 ; Kozakai et al, 2021 ) and B. longum 105-A ( Sakanaka et al, 2014 ). Additionally, the space between the -35 and -10 sequences can range from 11 to 18 nucleotides in length, where a shorter space length was found to improve the transcription levels ( Kozakai et al, 2020 ).…”

“…In investigating the various terminators used in Bifidobacterium, the gene sequences were identified using WebGeSTer DB terminator database ( Kozakai et al, 2021 ). These include the canonical (L-shaped hairpin structure) and non-canonical (I-, U-, V-, and X-shaped hairpin structures) terminators used across different phyla of microorganisms ( Mitra et al, 2011 ).…”

A bacterial sulfoglycosidase highlights mucin O-glycan breakdown in the gut ecosystem

“…An In-Fusion HD Cloning kit (Takara Bio, Shiga, Japan) was used for ligation unless otherwise stated. First, the Sp R gene of an E. coli-Bifidobacterium shuttle vector pMSK187, a derivative of pKKT427 47 , was replaced with the Cm R gene that was placed under the control of the rpmB promoter 48 . The Cm R gene was synthesized at Thermo Fisher Scientific (Waltham, MA, US) (1260-1969 bp of pC194, GenBank accession: V01277.1), while the rpmB promoter was amplified by PCR from the B. longum genome.…”