Functional analysis of bifidobacterial promoters in Bifidobacterium longum and Escherichia coli using the α-galactosidase gene as a reporter

Heterologous gene expression in bifidobacteria requires weak, strong, and inducible promoters depending on the objectives of different expression studies. Weak promoters in Escherichia coli can also be desirable for stable heterologous gene cloning. Here, we developed a reporter system using the Bifidobacterium longum α-galactosidase gene and investigated the activity and inducibility of seven bifidobacterial promoters in B. longum and their activities in E. coli. These studies revealed diverse promoter activities. Three promoters were highly active in B. longum, but only slightly active in E. coli. Among these, two phosphoketolase gene (xfp) promoters exhibited strong activity in B. longum cells grown on glucose. In contrast, the promoter activity of the fructose transporter operon (fruEKFG) was strongly induced by carbohydrates other than glucose, including fructose, xylose, and ribose. These promoters will allow strong or highly inducible expression in bifidobacteria and stable gene cloning in E. coli. In contrast to the functions of these promoters, the promoter of sucrose-utilization operon cscBA showed very high activity in E. coli but low activity in B. longum. Other three promoters were functional in both B. longum and E. coli. In particular, two sucrose phosphorylase gene (scrP) promoters showed inducible activity by sucrose and raffinose in B. longum, indicating their applicability for regulated expression studies. The diverse promoter functions revealed in this study will contribute to enabling the regulated expression of heterologous genes in bifidobacteria research.