Tough nuts to crack: site-directed mutagenesis of bifidobacteria remains a challenge.

Most members of the genus Bifidobacterium are commensals of the human gastrointestinal tract and some strains were shown to exert beneficial effects on their host. Based on these effects and due to their status as GRAS (generally recognized as safe) microorganisms, specific strains of bifidobacteria are marketed as probiotics. Despite their important role in food and dairy industries, the mechanisms responsible for the probiotic effects of bifidobacteria are mostly unknown. Over the last decade, the genomes of a large number of bifidobacteria have been sequenced and analyzed. This has yielded a number of genes and their products that are speculated to contribute to the probiotic effects of bifidobacteria. The gold standard to demonstrate a role for specific genes is the analysis of mutants. At present, only a small number of mutants of bifidobacteria have been generated by targeted mutagenesis. This is owed to the genetic inaccessibility of most strains and a lack of appropriate molecular tools. Successful generation of mutants of bifidobacteria was achieved by various methods including classical suicide vector strategies, increase of transformation efficiencies by methylation of plasmids and the use of temperature-sensitive vectors. In this commentary, we will describe the methods successfully used for mutagenesis of bifidobacteria and discuss their advantages and limitations.