Site-Directed Mutagenesis of Bifidobacterium Strains

At present, only a limited number of Bifidobacterium species are amenable to genetic manipulation using mutagenesis. This lack of genetic accessibility among the majority of bifidobacterial strains represents a significant roadblock for the study of gene function and expression in these potential probiotics. Genetic tools for generating mutants are difficult to develop for bifidobacteria, as they require workarounds for obstacles such as low transformation efficiencies, and the presence of differing and sometimes multiple restriction modification systems, in different strains. Site-directed mutagenesis is a frequently applied molecular strategy for the generation of targeted mutations, resulting in gene deletion or disruption, or alteration of their expression, thereby revealing information regarding their function. This strategy has been employed as a molecular tool in some Bifidobacterium strains and is typically achieved using a nonreplicating vector, harboring a DNA fragment corresponding to an internal part of the gene to be mutated. This vector is introduced into a bifidobacterial cell of the strain in question by electroporation. Through homologous recombination, this vector is integrated into the genomic DNA of said cell, disrupting the coding region of the targeted gene, thus preventing the expression of a functional protein product. Such mutant versions of Bifidobacterium strains may then be assessed for alterations in their phenotype or gene expression.