Abstract
Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.
Highlights
Members of the genus Bifidobacterium are anaerobic Grampositive bacteria, constituting one of the members of Actinobacteria phylum that inhabit the human gastrointestinal tract (GIT) [1], [2]
When electrocompetent cells were prepared from cells that had been grown in modified de Man Rogosa and Sharpe (mMRS) containing lactose as the sole carbohydrate source higher amounts of transposon insertion mutants were obtained per electroporation reaction
Only a few targeted mutations have been reported in certain strains [22], [45], [48], [49] and no systems for random mutagenesis in bifidobacteria have yet been described
Summary
Members of the genus Bifidobacterium are anaerobic Grampositive bacteria, constituting one of the members of Actinobacteria phylum that inhabit the human gastrointestinal tract (GIT) [1], [2]. Lactis DN-173 010, or B. longum Bb536), being widely included in functional foods [10], [12]. Their status as ‘‘generally recognized as safe’’ (GRAS) organisms makes them potential candidates for the development of delivery systems to produce peptides or proteins of interest at intestinal level as well as for the use as live oral vaccines [13], [14], [15], [16]
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