Transposon Mutagenesis in Bifidobacterium breve: Construction and Characterization of a Tn5 Transposon Mutant Library for Bifidobacterium breve UCC2003

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.

Bifidobacterium breve UCC2003 metabolises the human milk oligosaccharides lacto-N-tetraose and lacto-N-neo-tetraose through overlapping, yet distinct pathways

“…Growth of the insertion mutants was not impaired on lactose, where all strains reached final OD 600nm levels comparable to that reached by UCC2003, except for B. breve UCC2003-lacZ2 and B. breve UCC2003-lacS, as the interrupted genes in these mutants are known to be crucial for lactose metabolism4249 (Fig. 4a).…”

“…The nahS and nahA insertion mutants mentioned above were used for this purpose, in addition to insertion mutants in the genes Bbr_1551 ( lacS ), Bbr_1552 ( lacZ6 ), constructed in a previous study42, as well as a Tn5 transposon mutant in Bbr_0010 ( lacZ2 ), from a separate study49. These mutant strains were analysed for their ability to grow in mMRS supplemented with LNnT with a lactose (and ribose, in the case of B. breve UCC2003-lacZ2 and B. breve UCC2003-lacS) control, as compared to B. breve UCC2003.…”

A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS 3 Family Insertion Sequence, IS Blo11

“…Transposon mutagenesis is currently available only for a small number of intestinal bacteria, including bifidobacteria (1,2,7,(24)(25)(26), mainly because of their low transformation efficiency. Hence, high efficiency is essential for transposon mutagenesis of these species.…”

“…Hence, genetic factors that mediate bifidobacterial intestinal colonization have not been sufficiently identified. To address this issue, a transposon mutagenesis system for two strains of Bifidobacterium breve (1,15) was recently developed using a commercial EZ-Tn5 Transposome kit (Epicentre Biotechnologies). Although the EZ-Tn5 system is potentially applicable to other Bifidobacterium species, including our host strain, B. longum 105-A, alternative transposon mutagenesis systems are still required to broaden researchers' choices.…”

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

“…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).…”