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Functional analysis of Bifidobacterium genes is essential for understanding bost-Bifidobacterium interactions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover in Bifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-&Dgr;repA, which lacks the plasmid replication gene repA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors repA, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vec-tor from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected in B. longum 105-A using the chromosomal full-length &bgr;-galactosidase gene as a target. Markerless gene deletion was tested using the aga gene, which en-codes &agr;-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion of aga. Carbohydrate assimilation analysis and &agr;-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based aga-comple-mented strain. These functional analyses revealed that aga is the only gene encoding a functional &agr;-galactosidase enzyme in B. longum 105-A. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]