In the context of the gut, the HGM is flooded with a mixture of polysaccharides from plant cell walls, which are major components of the human diet (Backhed et al., 2005). Here, RGI consists of a rhamnogalaturonic acid backbone decorated with long chains of galactan, and
branched/unbranched arabinan (Mohnen, 2008). When B. thetaiotaomicron comes into contact with RGI it can liberate the arabinan and galactan side chains by the action of surface endo-acting
enzymes, BT_4668 (galactan), BT_0360 (arabinan) and BT_0367 (arabinan). The data presented above describe each component of the galactan utilisation system in isolation. These data in
conjunction with other publications can be used to create a model for the degradation and import of galactan by B. thetaiotaomicron (summarised in Figure 4.30). As with all PULs, the galactan PUL is constitutively expressed to survey the environment for potential polysaccharide nutrients (Martens et al., 2011). B. thetaiotaomicron encounters galactan at the external face of the outer membrane where it is bound by BT_4669 a lipoprotein SGBP. BT_4669 shows preference for the polysaccharide over oligosaccharides (Table 4.3), suggesting the presence of an extended binding site. Binding of galactan to the cell surface increases local concentrations of the glycan, enhances the proximity of the endo-galactanase (BT_4668) to its substrate leading to increased catalytic efficiency. Deletion of BT_4669 demonstrates the importance of the SGBP in galactan utilisation. Binding of galactan may open up the structure, uncoiling the helical conformation and allowing BT_4668 to cleave the glycan backbone. It should be emphasised, however, that the endo-galactanase (BT_4668) displays typical activity for GH53 enzymes and thus appears to be functional in the absence of accessory
carbohydrate binding proteins. Another possibility is BT_4669 fulfils an as yet unclear structural role in stabilising the surface apparatus involved in binding and deconstructing galactan, without which surface activity does not occur.
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Figure 4.30 B. thetaiotaomicron galactan utilisation. Diagram summarising the breakdown and
utilisation of galactan at the cell surface and in the periplasm of B. thetaiotaomicron.
The surface lipoprotein BT_4668, is an endo-β1,4-galactanase generating long
galactooligosaccharides from galactan polysaccharide (Figure 4.5b ). The oligosaccharide products from BT_4668 digestion of galactan at the cell surface are bound by BT_4670 (Figure 4.10, Table 4.3), the galactan PUL SusD-homologue and channelled through BT_4671, the galactan PUL SusC- homologue, into the periplasm. The small difference in affinity for Gal6 and galactan suggests the ligand binding site is able to accommodate around six galactosyl residues (Table 4.3). Galactan forms a helical structure with each turn approximately six residues long (Cid et al., 2010). Ligand specificity of BT_4670 may be based on recognition of the structure and chemical composition, similar to the starch utilisation system where SusD binds more tightly to cyclic than linear oligosaccharides of the same length (Koropatkin et al., 2008). This process is not efficient, leading to oligosaccharide release
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at the cell surface (Figure 4.25a). The inefficiency derives from an imbalance between the rate at which oligosaccharides are generated by BT_4668 (Table 4.2), and the rate at which the SusCD pair transport these saccharides. Saturation of the transport system does not appear to cause any detrimental effects on B. thetaiotaomicron growth on galactan in vitro, perhaps transporter expression is limited to allow for maximal growth without flooding the periplasm with undigested oligosaccharides which may cause unwanted interactions at high concentrations in the
compartment. The action of BT_4668 is vital to galactan utilisation as very large oligosaccharides cannot be transported into the periplasm by the SusCD-homologue pair. Once in the periplasm oligosaccharides are sequestered away from any potential competitors, here the oligosaccharides are degraded to their monosaccharide constituents by BT_4667 (Figure 4.11), an exo-acting β1,4- galactosidase. Interestingly BT_4667 is much more active against Gal2 than lactose (Table 4.4), indicating a preference for galactose over glucose at the +1 subsite; lactose comprises Gal-β1,4-Glc. This preference for homogenous galactooligosaccharide substrates indicates a specialisation consistent with its location in the galactan degrading apparatus of B. thetaiotaomicron; the enzyme encounters galactooligosaccharides rather than lactose in the periplasm. The intermediate products of galactan utilisation can then be bound by the ligand binding domain of the HTCS sensor, BT_4673 (Figure 4.12, Table 4.3). Binding affinity data of BT_4673 shows a slight preference for Gal4 over Gal5 (Table 4.3). Thus, mid-range oligosaccharides are the activating ligand for the galactan PUL. This specificity protects B. thetaiotaomicron from inappropriate PUL upregulation from ligands which may contain galactose but are not derived from the polysaccharide. The galactooligosaccharides are completely degraded to mono- or di- saccharides in the periplasm and transported into the
cytoplasm for entry into fermentation and energy generation pathways.
4.3.1.1 BACOVA_05493
BACOVA_05493, the glycoside hydrolase found in the B. ovatus galactan PUL, but not in the
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homology to GH2 family enzymes (Figure 4.13). BACOVA_05493 most likely belongs to a GH2 subfamily. BACOVA_05493 displays slight preference for the longer oligosaccharides tested and requires a minimum substrate length of 3 DP for activity (Figure 4.14a, Table 4.5). A preference for the longer galactooligosaccharides may indicate a divergence in the galactan utilisation systems of B. thetaiotaomicron and Bacteroides spp. possessing a BACOVA_05493 homologue. Those with a BACOVA_05493 homologue may import longer galactooligosaccharides than B. thetaiotaomicron requiring another β1,4-galactosidase to efficiently degrade these extended substrates in the
periplasm. Interestingly, when BACOVA_05493 is inactivated the Δbacova_05493 B. ovatus mutant is unable to utilise galactan (Figure 4.15b), despite possessing the same machinery encoded by the B. thetaiotaomicron galactan PUL. A possible explanation is that the HTCS, which activates the galactan PUL in B. ovatus, requires products generated by BACOVA_05493 to upregulate the PUL. An
alternative explanation is that the GH53 and/or GH2 present in the B. ovatus galactan PULs have divergent activities from their counterparts in the B. thetaiotaomicron galactan PUL, hence making the presence of BACOVA_05493 a requirement for utilisation. However this seems unlikely as the B. ovatus homologues to BT_4667 and BT_4668 are practically identical to the corresponding enzymes in the B. thetaiotaomicron galactan PUL based on sequence alignment (data not shown). It is possible that the GH2 and/or GH53 enzyme is poorly expressed in species containing the additional galactosidase, or the outer membrane importer is specific to long galactooligosaccharides.
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Figure 4.31 RGI galactooligosaccharide sidechain breakdown. Diagram summarising deconstruction
of galactan sidechains of RGI by BT_4151, BT_4156, BT_4181 and BT_4160 in the periplasm of B. thetaiotaomicron.