Bifidobacterium breve adaption to the gut environment

Certain strains and species of the genus Bifidobacterium are considered probiotic organisms, whose presence in the gastrointestinal tract (GIT) may elicit one or more health benefits to the host. A number of factors impact on the ability of bifidobacteria to survive transit through the upper parts of the GIT, and to colonize and persist in the colon (where they are believed to exert their beneficial effects). They must for example be able to resist various environmental stresses, including oxidative stress and stresses imposed by low pH, bile exposure and nutrient starvation (such as iron or carbohydrate limitation). This thesis will take a focused view on the ability of the gut commensal Bifidobacterium breve UCC2003 to combat many of the rigours it faces upon gastrointestinal transit as a prototypical representative of its genus. Chapter 2 of this thesis will investigate which genes are important for the survival of B. breve UCC2003 under iron-limiting conditions. Phenotypic screening of a Tn5- based random mutant library in B. breve UCC2003 and transcriptomic analysis of B. breve UCC2003 when exposed to iron-limiting conditions identified a number of genes, of diverse predicted cellular functions, that were implicated in the survival of the strain under iron restriction. Among the identified genes were two putative iron-uptake systems: (i) a presumed ferrous iron uptake system, designated here as bfeUO, and (ii) a predicted ferric iron/siderophore uptake system, designated sifABCDE. In silico analysis also illustrated that these two clusters are highly conserved across members of the genus Bifidobacterium and are invariably co-located. Murine colonization studies demonstrated that B. breve UCC2003-bfeU and B. breve UCC2003-sifA insertion mutants are able to colonize a healthy murine gut as efficiently as the wild type B. breve strain, indicating that while bfeUO and sifABCDE are important for in vitro growth under iron-limiting conditions, they are not crucial for gut survival or gut colonization of a healthy host. Chapter 3 describes the B. breve UCC2003 global genome response to long-term iron starvation, which appears to be associated with an increased ability to resist bile stress. Analysis of the response of B. breve UCC20003 to chronic iron starvation was found to be distinct from the response of B. breve UCC2003 following exposure to iron- limiting conditions as described Chapter 2. Chronic iron starvation caused in/decreased transcription of genes associated with carbon and nitrogen metabolism, genes predicted to be responsible for iron uptake, genes encoding putative DPS proteins (which are involved in iron storage/DNA protection) and Fe-S cluster-associated proteins, as well as a gene (bshB) encoding a bile salt hydrolase. Insertional mutagenesis and survival assays demonstrate that iron limitation imposed on B. breve UCC2003 evokes increased resistance to bile stress, being partly due to the iron-inducible transcription of the bshB gene. These findings therefore link bile salt hydrolase activity of B. breve UCC2003 to its ability to survive the adverse effects of bile exposure and suggests that this strain uses iron availability as a signal to adapt to the variable environment of the small intestine. Being able to rapidly adapt to changing and/or adverse conditions is necessary for bifidobacteria to be able to survive and persist in the gut environment. Chapter 4 describes a novel mechanism by which bifidobacteria controls its central carbon metabolic pathway, again employing B. breve UCC2003 as the prototypical representative of its genus. Adaption to the gut environment requires rapid, energy-consuming adjustments in gene transcription, and B. breve UCC2003 is believed to achieve this through the utilisation of two predicted LacI-type transcription factors (TFs), designated BifR1 and BifR2. BifR1 and BifR2 were shown to be involved in regulating the central, carbohydrate-associated metabolic pathway, and thus, carbon flux of B. breve UCC2003. BifR1 and BifR2, though being encoded by two distinctly different genes, were found to be functionally very similar, due to their common control of genes within the central metabolic pathways, such as tkt, tal, pyk, ldH, eno, pflA and the pflB. Along with regulating the transcription of their own genes and each other, these TFs also appear to transcriptionally control additional genes. This complex network of BifR1/BifR2-mediated gene regulation provides novel insights into the decision-making process governing cell metabolism and physiology in bifidobacteria. Chapter 5 describes the characterisation of four B. breve UCC2003-encoded LacI-type transcriptional regulators, namely MalR2, MalR3, MalR5 and MalR6. These transcription factors (TFs) have previously been proposed to be involved in the utilisation of maltose, maltodextrins and related polysaccharides, such as starch, amylopectin, amylose, glycogen and pullulan. However, MalR2, MalR3 and MalR5 were found to have a more diverse role and were shown to participate in transcriptional regulation of a number of other carbon sources such as ribose and cellobiose. Interestingly, our in vitro data indicate that these regulators often cross-regulate the same carbohydrate utilization genes, while also regulating each other. This hierarchical regulatory system controls the transcription of genes involved in carbohydrate uptake, storage, breakdown and central metabolism. These four LacItype regulators were shown to respond to differing carbohydrate effectors, such as turanose or galactose, thus indicating that each regulator is responsible for a different aspect of (α-glucoside-containing) carbohydrate metabolism. This complex network of gene regulation provides intriguing insights into the decision-making process of the cell with regards to carbohydrate utilisation, and into bifidobacterial metabolic adaption to and competitiveness in its environment