Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation

Functional analysis ofBifidobacterium genes is essential for understanding host-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 inBifidobacterium longum . Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-ΔrepA , which lacks the plasmid replication generepA , was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harborsrepA , was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected inB. longum 105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using theaga gene, which encodes α-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 ofaga . Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-basedaga -complemented strain. These functional analyses revealed thataga is the only gene encoding a functional α-galactosidase enzyme inB. longum 105-A.