ArticleApplied and environmental microbiology2012
Development of a double-crossover markerless gene deletion system in Bifidobacterium longum: functional analysis of the α-galactosidase gene for raffinose assimilation.
Article in Applied and environmental microbiology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 43 papers.
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Who cites it
43 citing papers in PubMed, 70 citations in OpenAlex.
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- Bifidobacterium response to lactulose ingestion in the gut relies on a solute-binding protein-dependent ABC transporter.Communications biology · 2021Article
- Broad Purpose Vector for Site-Directed Insertional Mutagenesis inFrontiers in microbiology · 2021Article
- A Resource for Cloning and Expression Vectors Designed for Bifidobacteria: Overview of Available Tools and Biotechnological Applications.Methods in molecular biology (Clifton, N.J.) · 2021Review
- Methionine utilization by bifidobacteria: possible existence of a reverse transsulfuration pathway.Bioscience of microbiota, food and health · 2021Article
- The importance of genetic research on the dominant species of human intestinal indigenous microbiota.Bioscience of microbiota, food and health · 2021Review
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Functional analysis of Bifidobacterium 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 in Bifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-ΔrepA, 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 vector 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 β-galactosidase gene as a target. Markerless gene deletion was tested using the aga 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 of aga. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based aga-complemented strain. These functional analyses revealed that aga is the only gene encoding a functional α-galactosidase enzyme in B. longum 105-A.
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