Tough nuts to crack

Abstract

M ost members of the genusBifidobacterium 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.

Bifidobacteria Exhibit LuxS-Dependent Autoinducer 2 Activity and Biofilm Formation

“…The resulting plasmid pMgap_LuxS NCC was introduced into B. longum NCC2705 by electroporation as described previously [17]. Transformants were selected on MRSc agar containing 100 µg/mL spectinomycin.…”

Complete Genome Sequence of Bifidobacterium longum 105-A, a Strain with High Transformation Efficiency

“…Bifidobacteria are naturally found in the human large intestine and are used as probiotic bacteria due to their beneficial effects on human health ( 1 ). Bifidobacteria are known for their difficulties with gene manipulation, especially in the generation of gene knockout mutants ( 2 – 4 ). This is mainly caused by the low transformation efficiencies demonstrated by Bifidobacterium strains, in part due to their restriction-modification (R-M) systems ( 2 – 6 ).…”

Expression of Fluorescent Proteins in Bifidobacteria for Analysis of Host-Microbe Interactions

“…Transformation into B. bifidum S17, B. longum subsp. longum E18, and B. breve S27 was performed by electroporation as described previously (19).…”

“…While some progress has been made in improving transformation efficiencies, directed gene inactivation, and transposon mutagenesis for single strains (19,20), the vast majority of strains of interest remain difficult if not impossible to modify genetically. Moreover, the available vector systems for bifidobacteria have a rather limited host range (i.e., replicons function only in some species of the genus Bifidobacterium but not in others), and expression systems are poorly developed (2,21).…”