The gut microbiota play a significant role in human health and nutrition, although the mechanisms these organisms use to survive in this densely populated environment are not well understood. Bacteroides thetaiotaomicron is a dominant member of the gut bacterial community whose genome sequence reveals large expansions in protein families involved in the sensing, acquisition and utilisation of complex carbohydrates, pointing to the ability to access a wide range of glycans as playing a significant role in becoming a successful resident of the human gut. Here we have characterised components of the B. thetaiotaomicron polysaccharide utilisation apparatus at molecular level, focusing mainly on fructan sensing, binding and degradation systems. Microarray data from our collaborators revealed that growth of B. thetaiotaomicron on inulin (β-2,1-linked fructan) specifically upregulated a locus of nine genes BT1757-BT1765 and an orphan gene BT3082, encoding a glycoside hydrolase from family 32 (GH32). The locus contains three other GH32s, two predicted polysaccharide binding outer membrane proteins (SusC and SusD homologues), a fructokinase, and an inner membrane monosaccharide transporter. Together these components form a polysaccharide utilisation locus (PUL). The nearest regulatory gene to the PUL is BT1754, a hybrid two component system. Here we show that the periplasmic sensor domain of BT1754 (BT1754peri) binds specifically to fructose, with a Kd of ~2 μM and a stoichiometry of 1:1, but not fructooligosaccharides or other monosaccharides. The crystal structure of BT1754peri revealed a two domain periplasmic binding protein (PBP)-fold with the ligand fructose sandwiched between the two domains. BT1754 is the first periplasmic sensor histidine kinase domain to display a non-PAS fold. The structure in combination with biophysical and site-directed mutagenesis studies also shows how the protein displays such specificity in ligand recognition and provides insights into the mechanism of signal transduction across the inner membrane. The four glycoside hydrolase family 32 members regulated by BT1754 were also biochemically and structurally characterised in this thesis. Three of four GH32 enzymes BT1759, BT1765 and BT3082, digest both β-2,1-linked (inulin) and β-2,6-linked (levan) fructans, indicating that levan is also utilised by the same locus. BT1759 and BT3082 are exo-acting enzymes releasing fructose from both long-chain and short-chain inulins and levans, while BT1765 is also exo-acting and produces fructose, but preferred short-chain sugars. BT1760 is unusual for GH32 as it is specific for levan and has an endo-like activity, releasing a range of different size oligosaccharides from the polysaccharide. The crystal structures of wild type BT3082 and a nucleophile mutant in complex with substrate (kestose) were solved to 2.2 Å and revealed a typical GH32 β-propeller fold as the catalytic domain. Like all other GH32s solved to date, the enzyme has a C-terminal β-sheet domain of unknown function that was shown to be necessary for correct folding of the enzyme. BT3082 also has a unique N-terminal β-sheet domain that was shown to be essential for enzyme activity but not correct folding. Extensive site-directed mutagenesis was carried out to provide insight into the relative importance of different residues in substrate binding and catalysis in BT3082. The crystal structure of wild type BT1760 was solved to 2.6 Å, revealing a surprisingly similar structure to that of the exo-acting enzymes. A rationale for the endo-like activity of this enzyme and the role each of the four GH32s in fructan utilisation by B. thetaiotaomicron is discussed. The outer membrane SusD homologue BT1762 from the fructan locus was shown to bind preferentially to long chain levans, with no recognition of inulin. The crystal structure of BT1762 was solved to 1.9 Å and was shown to share the same novel α-helical fold as SusD. Site-directed mutagenesis of a number of residues in the same region as the SusD binding site showed that while the location of the ligand binding sites are conserved between these two proteins, the identity of the residues involved in polysaccharide recognition are not the same. A model is proposed for levan recognition in BT1762 and also its role in polysaccharide utilisation. B. thetaiotaomicron also has at least 12 ECF sigma/anti-sigma factor gene pairs likely involved in polysaccharide utilisation. Here we show that these systems form a trans-envelope signalling apparatus with their cognate SusC-transducer homologue, but that there is no cross talk between different systems. The significance of this finding in relation to survival of this important gut bacterium is discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:500969 |
| Date | January 2009 |
| Creators | Zheng, Hongjun |
| Publisher | University of Newcastle Upon Tyne |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10443/275 |
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