The xylan degradation apparatus expressed by PUL-XylS and PUL-XylL are optimized to maximise the intracellular breakdown and uptake of hemicellulose, similar to previously characterised glycan utilisation systems of Bacteroides (Martens et al., 2009). The B. thetaiotamicron yeast mannan utilisation system adopts a selfish mechanism, where all breakdown products generated by the surface mannanases are transported into the periplasm, resulting in an absence of breakdown products in the growth supernatant (Cuskin et al., 2015). Here, when grown on BX, WX and CX, B. ovatus generates oligosaccharides in the growth supernatant during different phases of growth
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(Figure 3.10). The TLC data show products that match the XOS standards and an additional oligosaccharide which does not migrate with xylooligosaccharoides used here (Figure 3.10). The unknown oligosaccharide remains in the media at stationary phase indicating it is not used by B. ovatus for growth. In plant cell walls the xylan backbone in the hemicellulose fraction is built upon an oligosaccharide of 4-β-D-Xylp-(1→4)-β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-α-D-GalpA-(1→4)-D-Xylp at the reducing end of the chain (Pena et al., 2007). During synthesis of xylan in plant cell walls this oligosaccharide acts as a primer from which the backbone is built (Pena et al., 2007). As the
unknown oligosaccharide is present in the BX, WX and CX culture supernatants, it may be the primer oligosaccharide as this is a common feature of xylans. Utilisation of the xylan primer by Bi.
adolescentis was not tested here it is was found to be not possible to purify in suitable quantities for growths. It is unlikely that Bi. adolescentis would be capable of utilising a relatively complex
oligosaccharide such as this with four unique linkages requiring specific enzymes for breakdown of a sparse substrate. Bi. adolescentis was able to use oligosaccharides generated from a digest of BX and WX with recombinant BACOVA_04390 (Figure 3.11c&d), confirming Bi. adolescentis is capable of utilising xylooligosaccharides derived from these polysaccharides (Figure 3.11a). Bi. adolescentis, however, was unable to use CX digested with the GH98 xylanase BACOVA_03433 as a growth substrate, suggesting that the resultant oligosaccharides were too complex to be utilised by the Bifidobacterium (Figure 3.11a). BACOVA_03433 generates GAXOSs that are extensively decorated, and these side chains may block binding by the ESBP, or perhaps the oligosaccharides are simply too large to fit into the ABC transporter channel used for oligosaccharide import. Bioinformatic and biochemical studies of B. adolescentis xylanases show activity on only a relativity narrow range of xylans and/or xylooligosaccharides (van den Broek et al., 2005). Bi. adolescentis encodes two GH43, a GH120 β-xylosidase and a GH8 xylanase, which show activity on xylooligosaccharides and are vital to utilisation (Lagaert et al., 2007). The presence of GH43 α-L-arabinofuranosidases suggests Bi. adolescentis is capable of utilising AXOS in addition to linear XOS (Lagaert et al., 2010).
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In contrast with the selfish hypothesis proposed for mannan utilisation (Cuskin et al., 2015), B. ovatus is able to support the growth of Bi. adolescentis on BX and WX (Figure 3.13a,b). Despite differences in WX and BX complexity and the presence of arabinose substitutions in WX, similar levels of Bi. adolescentis were observed in the co-cultures on the two xylans. During growth Bacteroides spp generate metabolites which include short chain fatty acids (SCFAs) as waste products of anaerobic ATP generation (Salt et al., 1985). SCFAs are energy rich molecules that are used as an energy source by colonocytes in the gut (Donohoe et al., 2011).
Interestingly, B. ovatus viable counts are much higher in the CX co-culture than either of the BX or WX co-cultures (Figure 3.14d). As B. ovatus generates much larger oligosaccharides at the cell surface when cultured on CX, than those produced from WX and BX, each CX oligosaccharide transported contains higher potential energy yield than BX or WX oligosaccharides. Due to the nature of the SusCD transport system each oligosaccharide, regardless of size is imported at the cost of an ATP molecule, giving higher energy to cost ratio for the longer oligosaccharides, which in turn allows greater growth, hence the greater cell numbers observed in the CX co-culture. In this regard it may also be relevant that all the surface xylanases have evolved to cleave the xylan backbone infrequently generating large oligosaccharides.
CX, as a highly complex substrate, requires a suite of enzymes for full breakdown and utilisation by B. ovatus, which are not associated with typical xylan degradation systems (Rogowski et al., 2015). The inability of Bi. adolescentis to grow on CX oligosaccharides, and in co-culture with B. ovatus on CX, may simply reflect the lack of the enzymes or transport systems required to degrade the complex xylan. This view is supported by the observation that a mutant of B. ovatus, lacking the surface GH98 xylanase was unable to grow on CX in monoculture, but could utilise the
polysaccharide in co-culture with the wild type bacterium (Figure 3.17a). These data demonstrate oligosaccharides are released during B. ovatus growth on CX, but these molecules are not utilized by Bi. adolescentis. The inability to observe the CX oligosaccharides may be because they are too large
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to migrate from the origin on TLC (Figure 10c). It is possible that CX and other highly complex substrates are only used as a last resort during times of nutritional crisis, requiring a high transcriptional/translational investment from the cell to produce the required apparatus for utilisation. For Bifidobacteria the initial investment to use such substrates may be too high when host glycans, such as mucins, are readily available (Egan et al., 2014). B. thetaiotaomicron glycan utilisation preferences can be organised into a hierarchy showing PULs directed against host glycans and simpler glycans are preferentially upregulated over more complex gylcans (Rogers et al., 2013), which may hold true for B. ovatus as a relative of B. thetaiotaomicron.
3.4 Conclusion
B. ovatus expresses the full repertoire of enzymes required to fully degrade simple (BX, WX) or complex (CX) xylans in the human gut lumen. PUL-XylS orchestrates the degradation of simpler xylans expressing two GH10 xylanases displaying different levels of activity reflecting their
localisation to the cell surface, BACOVA_04390, or periplasm, BACOVA_04387, to minimise loss of breakdown products to the competitors in the gut. Despite this adaptation oligosaccharides are released that are used by other members of the gut microbiota. In this chapter, Bifidobacteria were shown to use the BX- and WX-derived oligosaccharides generated by B. ovatus. In turn the
Bifidobacteria promote gut health by helping to alleviate symptoms of inflammatory bowel disease (IBD) and modulating inflammation of the gut during immune responses (Kajander et al., 2008; Ishikawa et al., 2011). Although variable in size, it would appear that B. ovatus releases
xylooligosaccharides into the culture supernatant irrespective of the complexity of the xylan. B. ovatus appears to aid Bi. adolescentis when co-cultured on xylans, generating accessible substrate from inaccessible xylan polysaccharide, despite lack of an apparent benefit of promoting Bi.
adolescentis growth to the bacterium itself. This apparent altruism may be rewarded in the gut from currently unknown pathways due to the complexity of the interactions within the HGM. Data presented here demonstrates potential for enrichment of different members of the HGM by
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including different fractions of glycan in the human diet. Complex GAX, like CX, promote B. ovatus growth in the gut, which in turn produce propionate, a molecule implicated in lipogenesis reduction in the host (Hosseini et al., 2011). Utilisation of simple xylans by B. ovatus will promote growth of Bifidobacteria to increase butyrate, a molecule known to maintain the health of intestinal epithelium (Wachtershauser and Stein, 2000). Indeed such oligosaccharides have been shown to selectively promote Bifidobacteria, suggesting simple xylans and xylooligosaccharides can be used as
bifidogenic prebiotics, due to a relatively low dose required to have a positive effect (Finegold et al., 2014). Products of complex xylans are, however only used by a small subset of gut microbes due to the rarity of enzymes displaying relevant activities. Thus, the xylan-B. ovatus axis could be
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