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Title: Characterisation of the dextran utilisation system from the prominent gut bacterium Bacteroides thetaiotaomicron
Author: Norman, Joanna
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2012
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The gut microbiota play an important role in human health and nutrition. Despite this, the molecular mechanisms that underpin the ability of specific microorganisms to survive and flourish in the highly competitive environment of the large intestine are not well understood. The genome of Bacteroides thetaiotaomicron (Bt), a prominent member of our normal gut microbiota, contains a large number of genes that are predicted to encode products dedicated to polysaccharide sensing, acquisition and utilization. These genes are organised into co-regulated clusters, termed polysaccharide utilization loci (PULs). Microarray data from our collaborators revealed Bt activates a single PUL of 5 genes (Bt3086 to Bt3090) and an orphan gene (Bt458I) when grown on the a-I, 6-linked glucose-polymer, dextran. The dextran locus encodes two proteins predicted to be involved in polysaccharide binding and translocation (homologues of SusD and SusC, respectively), a hypothetical protein downstream of the susD homologue and glycoside hydrolases belonging to families GH66, GH3I and GH97. Here we have characterised all components of the dextran utilisation system to further our understanding of polysaccharide utilization by this prominent gut bacterium. Bt309I resides upstream of the locus and its putative topology is suggestive of a membrane bound one-component system (OCS), comprising of an N-terminal periplasmic domain, a transmembrane region and a C-terminal cytoplasmic domain. Here we reveal Bt309I to be the regulator of the dextran locus and demonstrate the first example of direct carbohydrate binding. to the periplasmic sensor domain (Bt309I pd) of this class of protein. In attempt to identify the activating ligand we characterised the putative glycoside hydrolases of the system which display endo-dextranase/transglycosidase (Bt3086), endo-dextranase (Bt3087) and a- glucosidase activity (Bt4581). We show that Bt3087 is likely surface located, whereas Bt3086 and Bt458I are likely periplasmic. Bt309Ipd binds to dextran degradation products of Bt3087 and Bt3086. Based on the respective activities of the GHs, we propose that the activating ligand is an a-I,6 linked oligosaccharide of 4 residues or more. The cytoplasmic domain of Bt309I binds directly to sequence specific regions of DNA upstream of the dextran PUL and the orphan gene; this binding interaction is mediated by residues residing on the third helix of the predicted helix-turn-helix domain. This is the first demonstration of direct DNA binding in a Bacteroides OCS. We also show that following signal perception the cytoplasmic domain remains tethered to the membrane and therefore postulate that the DNA is constitutively bound to Bt3091. Initial acquisition of extracellular polysaccharides occurs via proteins located on the cell surface, yet information on how these components contribute to polysaccharide utilization is sparse. Here we reveal Bt3089 - a SusD homolog - to be essential for growth on dextran oligosaccharides >2 glucoses and demonstrate direct glycan binding with a preference for dextran oligosaccharides with a degree of polymerization >6 glucoses. The susE- positioned gene product from the dextran locus, Bt3088, is essential for growth on dextran polysaccharide, but not oligosaccharides of <30 sugar residues; this targeted function is reflected in its high affinity for polymeric dextran. A model for dextran utilization in this prominent gut bacterium is proposed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available