In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.

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.

Bacteria evolved over millennia in the presence of the vital micronutrient iron. Iron is involved in numerous processes within the cell and is essential for nearly all living organisms. The importance of iron to the survival of bacteria is obvious from the large variety of mechanisms by which iron may be acquired from the environment. Random mutagenesis and global gene expression profiling led to the identification of a number of genes, which are essential for Bifidobacterium breve UCC2003 survival under iron-restrictive conditions. These genes encode, among others, Fe-S cluster-associated proteins, a possible ferric iron reductase, a number of cell wall-associated proteins, and various DNA replication and repair proteins. In addition, our study identified several presumed iron uptake systems which were shown to be essential for B. breve UCC2003 growth under conditions of either ferric and/or ferrous iron chelation. Of these, two gene clusters encoding putative iron-uptake systems, bfeUO and sifABCDE, were further characterised, indicating that sifABCDE is involved in ferrous iron transport, while the bfeUO-encoded transport system imports both ferrous and ferric iron. Transcription studies showed that bfeUO and sifABCDE constitute two separate transcriptional units that are induced upon dipyridyl-mediated iron limitation. In the anaerobic gastrointestinal environment ferrous iron is presumed to be of most relevance, though a mutation in the sifABCDE cluster does not affect B. breve UCC2003's ability to colonise the gut of a murine model.

In the current study, we show that biofilm formation by various strains and species belonging to Bifidobacterium, a genus that includes gut commensals with reported health-promoting activities, is induced by high concentrations of bile (0.5% (w/v) or higher) and individual bile salts (20 mM or higher), rather than by acid or osmotic stress. The transcriptomic response of a bifidobacterial prototype Bifidobacterium breve UCC2003 to such high bile concentrations was investigated and a random transposon bank of B. breve UCC2003 was screened for mutants that affect biofilm formation in order to identify genes involved in this adaptive process. Eleven mutants affected in their ability to form a biofilm were identified, while biofilm formation capacity of an insertional mutation in luxS and an exopolysaccharide (EPS) negative B. breve UCC2003 was also studied. Reduced capacity to form biofilm also caused reduced viability when exposed to porcine bile. We propose that bifidobacterial biofilm formation is an adaptive response to high concentrations of bile in order to avoid bactericidal effects of high bile concentrations in the gastrointestinal environment. Biofilm formation appears to be a multi-factorial process involving EPS production, proteins and extracellular DNA release, representing a crucial strategy in response to bile stress in order to enhance fitness in the gut environment. Biofilms are microbial communities that are attached to a surface and are enclosed/structured by an extracellular matrix 1. Biofilms may also form when free floating cells clump together or adhere to particulate matter, this being referred to as microcolony formation 2. Biofilm formation is a complex process involving multiple steps, including initial attachment, accumulation, maturation and dispersal 3. Initial attachment is reversible and can be driven by electrostatic interactions; attachment to a surface can also be mediated by cell wall-associated proteins that bind to a substrate-covered surface or extracellular DNA (eDNA) release, where DNA released by cell lysis coats the surface and changes surface properties to allow attachment 4,5. The accumulation phase of a biofilm can be mediated by cell wall associated protein interactions or extracellular matrix (EM) secretion 5,6. The EM of biofilms may be composed of extracellular polysaccharide (EPS), DNA and/or proteins 7-9. Finally, following maturation, individual cells may disperse from the biofilm to resume planktonic growth 10. Biofilm formation is often triggered in response to environmental stresses, such as nutrient starvation, antibiotics, pH and bile, or induced through quorum sensing systems, such as the Agr or autoinducer-2 (AI-2) systems 11-14. Bifidobacteria are non-motile gut commensals, some of which are purported to exert health-promoting or probiotic properties (see review 15). Particular bifidobacterial strains are for this reason included in certain 'functional foods' so as to bestow these benefits to the host that ingests them 16. However, whether bifidobacteria ...