Bifidobacterial physiology and metabolism in the gut environment
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Date
2020-12
Authors
Kelly, Sandra M.
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Publisher
University College Cork
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Abstract
Bifidobacteria represent Gram-positive gut commensals of mammals, birds and insects. Certain bifidobacterial species are associated with various health benefits if present in adequate amounts in the human gastro-intestinal tract (GIT). Bifidobacteria are highly prevalent and abundant in the infant gut, though they decrease in abundance with increasing age of their human host. Bifidobacteria must overcome many challenges in order to survive in the human gut, such as competition by other gut microbes, exposure to bile salts/acids and a fluctuating pH, and nutrient starvation. One key factor to bifidobacterial survival in the gut environment is the ability of members of the genus Bifidobacterium to metabolise complex carbohydrates indigestible to the human host. In the infant gut such carbohydrates are supplied in breast milk, whilst in the adult diet complex carbohydrates are often derived from plant-based oligosaccharides ingested from the diet. In particular, members of the Bifidobacterium longum subsp. longum taxon are associated with plant-derived poly- and oligo-saccharide utilisation. The research described in this thesis studied the metabolism of certain plant-derived oligosaccharides by different strains in the B. longum subsp. longum taxon. Bile salts possess major anti-microbial activity and act by disrupting the integrity of the bacterial cell membrane. Concentrations of bile salts/acids are highest in the small intestine and starting from the duodenum a decreasing gradient exists along the GIT with lowest bile concentrations in the large intestine. Therefore, if bifidobacteria are ingested as an active ingredient of a functional food, they encounter and must be able to survive bile stress if they are to be effective as a probiotic. This thesis examined biofilm formation of Bifidobacterium breve UCC2003 in response to bile stress and assessed this ability in terms of survival and genetic requirements.
Chapter II of this thesis focused on an in silico annotated hydroxycinnamic acid esterase encoded within a genetic locus present in B. longum subsp. longum NCIMB8809 suspected to be involved in plant-derived poly/oligosaccharide utilisation. Both this locus and encoded esterase were found to be conserved amongst several strains of the B. longum subsp. longum taxon. Through a number of experiments the function of the esterase was proven and the enzyme was characterised. Therefore, it is likely this esterase cleaves offhydroxycinnamic acids commonly present as substituents on arabinose moieties present in certain plant-derived poly/oligosaccharides.
Chapter III investigates the enzymatic degradation of plant-derived poly/oligosaccharides by B. longum subsp. longum NCIMB 8809. This strain was found to grow on various plant-derived glycans including arabinoxylan, arabinogalactan and XOS. In this chapter, three glycosyl hydrolase (GH) 43 enzymes, from a presumed plant-oligosaccharide cluster described in Chapter II, were found to possess exo-α-L-arabinofuranosidase or α-endo-arabinanase activity. Furthermore, these enzymes were found to elicit activity against arabino-oligosaccharide (AOS) substrates. Therefore, a novel locus involved in AOS plant-oligosaccharide utilisation was identified in certain members of the B. longum subsp. longum taxon.
Chapter IV describes the investigation of a LacI-type regulator, designated here as AauR, present in the aau locus of B. longum subsp. longum CCUG 30698 and predicted to regulate transcription of the aau locus encoding various GH43 enzymes. The consensus AauR recognition motif previously predicted for this regulator in another B. longum subsp. longum representative was indeed also shown to be present in strain CCUG 30698. This study showed that AauR binds to its predicted operator sequences located at specific positions within the aau locus. Therefore, AauR is presumed to transcriptionally regulate this locus, likely mediated through an as yet unidentified effector.
Chapter V explores biofilm formation of B. breve UCC2003 in response to high concentrations of bile and bile salts. Through experimentation it was found that various factors are involved in biofilm formation including extracellular polysaccharide (EPS) production, LuxS and fatty acid biosynthesis. The formation of biofilm was found to be protective against high concentrations of bile. Extracellular DNA production, proteins and EPS were all found to impact on biofilm formation. Therefore, the formation of biofilms in bifidobacteria is presumed to constitute a multi-factorial process in response to high concentrations of bile.
This thesis represents novel information on the metabolism of plant-derived oligosaccharides, specifically HCA removal, AOS metabolism and AOS transcriptional regulation in B. longum subsp. longum taxon. The mechanism of biofilm formation in B.
breve UCC2003 was also investigated and this is the first report on the molecular players important for biofilm formation in bifidobacteria.
Future research is required to further elaborate on the enzymatic steps that are required for AOS and arabinan metabolism by various B. longum subsp. longum strains in order to explain strain-specific differences on the basis of encoded GH activities. Also, further detailed analysis of transcriptional regulation of the aau genetic locus is needed to identify the molecular effector of the AauR regulator, as well as the precise location of the relevant promoter sequences and the manner by which AauR controls transcription of its target genes. Finally, additional experimentation is required to further investigate the biological roles of bifidobacterial biofilm formation in the gut. It will in particular be important to learn how biofilm formation is triggered by certain bile components and to what extent biofilm formation is important for colonisation and survival under in vivo conditions. Ultimately, the generated knowledge on plant glycan metabolism and biofilm formation will contribute to our understanding of how a bacterium can take up residence and survive in a very crowded and sometimes hostile environment.
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Keywords
Bifidobacteria , Fibre , Plant glycan , Carbohydrate metabolism , Bile , Biofilm , Bifidobacterium , Bifidobacterium longum , Bifidobacterium breve , Bile resistance
Citation
Kelly, S. M. 2020. Bifidobacterial physiology and metabolism in the gut environment. PhD Thesis, University College Cork.