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Glycan complexity dictates microbial resource allocation in the large intestine.

Rogowski A, Briggs JA, Mortimer JC, Tryfona T, Terrapon N, Lowe EC, Baslé A, Morland C, Day AM, Zheng H, Rogers TE, Thompson P, Hawkins AR, Yadav MP, Henrissat B, Martens EC, Dupree P, Gilbert HJ, Bolam DN - Nat Commun (2015)

Bottom Line: We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan.Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains.Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

ABSTRACT
The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

No MeSH data available.


The activity of the GH98 enzyme BACOVA_03433.(a) Different xylans at 1% (w/v) were incubated with 1 μM of the GH98 enzyme in PBS for 16 h at 37 °C. The reaction products were subjected to thin layer chromatography and compared with xylooligosaccharide standards with a d.p. ranging from 1 to 5 (X1-X5). (b) HPAEC-PAD trace showing the high molecular weight oligosaccharide products of CX digestion by the GH98 endo-xylanase, separated using a sodium acetate gradient of 0-500 mM. The reaction conditions used are the same as in panel a Control reactions of substrate and enzyme only are shown. (c) CX was incubated with 5 μM of the GH98 enzyme for 24 h in 50 mM sodium phosphate buffer, pH 7.0, at 37 °C. The reaction products, and CX that was not incubated with the GH98 enzyme, were then treated with sodium borohydride, to convert the reducing end monosaccharide unit into its alditol, followed by acid hydrolysis to convert the oligosaccharides (or in the case of control CX alone) into their monosaccharide constituents. The hydrolysed products were subjected to HPAEC and compared to the migration of standard monosaccharides as well as the alditol of xylose (xylitol) and galactose (galactitol). (d) The GH98 enzyme was incubated with CX (lane CX vs GH98), or CX that had been pre-treated with all the side chain cleaving enzymes encoded by PUL-XylL (lane CX vs MIX), or CX that had been pre-treated with all the side chain cleaving enzymes encoded by PUL-XylL, except the GH43 enzyme BACOVA_03417 that removes O3 linked arabinose from double substituted xylose (lane CX vs MIX no double abfase).
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f4: The activity of the GH98 enzyme BACOVA_03433.(a) Different xylans at 1% (w/v) were incubated with 1 μM of the GH98 enzyme in PBS for 16 h at 37 °C. The reaction products were subjected to thin layer chromatography and compared with xylooligosaccharide standards with a d.p. ranging from 1 to 5 (X1-X5). (b) HPAEC-PAD trace showing the high molecular weight oligosaccharide products of CX digestion by the GH98 endo-xylanase, separated using a sodium acetate gradient of 0-500 mM. The reaction conditions used are the same as in panel a Control reactions of substrate and enzyme only are shown. (c) CX was incubated with 5 μM of the GH98 enzyme for 24 h in 50 mM sodium phosphate buffer, pH 7.0, at 37 °C. The reaction products, and CX that was not incubated with the GH98 enzyme, were then treated with sodium borohydride, to convert the reducing end monosaccharide unit into its alditol, followed by acid hydrolysis to convert the oligosaccharides (or in the case of control CX alone) into their monosaccharide constituents. The hydrolysed products were subjected to HPAEC and compared to the migration of standard monosaccharides as well as the alditol of xylose (xylitol) and galactose (galactitol). (d) The GH98 enzyme was incubated with CX (lane CX vs GH98), or CX that had been pre-treated with all the side chain cleaving enzymes encoded by PUL-XylL (lane CX vs MIX), or CX that had been pre-treated with all the side chain cleaving enzymes encoded by PUL-XylL, except the GH43 enzyme BACOVA_03417 that removes O3 linked arabinose from double substituted xylose (lane CX vs MIX no double abfase).

Mentions: The surface GH98 enzyme BACOVA_03433 also generates very large oligosaccharides but exclusively from CX (Fig. 4a,b). As BACOVA_03433 is a member of GH98, a family that hitherto contained only endo-β-D-galactosidases29, it was formally possible that the enzyme targets rare β-D-Gal residues in the backbone of CX. Converting the reducing end aldehyde of the products generated by BACOVA_03433 to the alditol followed by acid hydrolysis revealed the presence of xylitol but not galactitol (Fig. 4c). These data show that BACOVA_03433 is an endo-xylanase that hydrolyses xylosidic bonds in a context found only in very complex xylans. It is possible that the enzyme targets an unusual linkage in the xylan backbone; however, this is unlikely as the enzyme was not active against β1,4-β1,3-mixed linked xylans and CX is reported to contain a backbone comprising exclusively β1,4-Xyl units13. To explore the specificity determinants of the GH98 enzyme further, CX was pre-treated with cocktails of side-chain cleaving hydrolases (arabinofuranosidases, xylosidases, glucuronidases and galactosidases) encoded by PUL-XylL, and the debranched forms of the xylan were then incubated with the GH98 xylanase. Only GH consortia lacking BACOVA_03417 generated a variant of CX that was hydrolysed by the GH98 enzyme (Fig. 4d). As described below BACOVA_03417 is an α-L-arabinofuranosidase that targets the O3 linked Araf on backbone Xyl residues decorated at both O2 and O3 with α-L-Araf. It would appear, therefore, that the double Araf decoration is a component of the specificity determinant for BACOVA_03433. Wheat arabinoxylan, however, also contains this double arabinose decoration and is not a substrate for the GH98 xylanase (Fig. 4a). Thus, the specificity of BACOVA_03433 is more complex than simply Xyl units linked O2 and O3 to arabinose. Interestingly, the CX backbone contains low levels of doubly substituted residues with Xylp as the O3 linked side chain (see Fig. 1 and results below). This structure is also a substrate for BACOVA_03417 (Supplementary Fig. 4a), suggesting Xylp at O3 (and Araf at O2) may be the critical specificity determinant of the GH98 enzyme. These Xylp decorations are absent in WAX (Supplementary Fig. 4a–c), providing a possible explanation for the lack of activity of the GH98 enzyme against this substrate.


Glycan complexity dictates microbial resource allocation in the large intestine.

Rogowski A, Briggs JA, Mortimer JC, Tryfona T, Terrapon N, Lowe EC, Baslé A, Morland C, Day AM, Zheng H, Rogers TE, Thompson P, Hawkins AR, Yadav MP, Henrissat B, Martens EC, Dupree P, Gilbert HJ, Bolam DN - Nat Commun (2015)

The activity of the GH98 enzyme BACOVA_03433.(a) Different xylans at 1% (w/v) were incubated with 1 μM of the GH98 enzyme in PBS for 16 h at 37 °C. The reaction products were subjected to thin layer chromatography and compared with xylooligosaccharide standards with a d.p. ranging from 1 to 5 (X1-X5). (b) HPAEC-PAD trace showing the high molecular weight oligosaccharide products of CX digestion by the GH98 endo-xylanase, separated using a sodium acetate gradient of 0-500 mM. The reaction conditions used are the same as in panel a Control reactions of substrate and enzyme only are shown. (c) CX was incubated with 5 μM of the GH98 enzyme for 24 h in 50 mM sodium phosphate buffer, pH 7.0, at 37 °C. The reaction products, and CX that was not incubated with the GH98 enzyme, were then treated with sodium borohydride, to convert the reducing end monosaccharide unit into its alditol, followed by acid hydrolysis to convert the oligosaccharides (or in the case of control CX alone) into their monosaccharide constituents. The hydrolysed products were subjected to HPAEC and compared to the migration of standard monosaccharides as well as the alditol of xylose (xylitol) and galactose (galactitol). (d) The GH98 enzyme was incubated with CX (lane CX vs GH98), or CX that had been pre-treated with all the side chain cleaving enzymes encoded by PUL-XylL (lane CX vs MIX), or CX that had been pre-treated with all the side chain cleaving enzymes encoded by PUL-XylL, except the GH43 enzyme BACOVA_03417 that removes O3 linked arabinose from double substituted xylose (lane CX vs MIX no double abfase).
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f4: The activity of the GH98 enzyme BACOVA_03433.(a) Different xylans at 1% (w/v) were incubated with 1 μM of the GH98 enzyme in PBS for 16 h at 37 °C. The reaction products were subjected to thin layer chromatography and compared with xylooligosaccharide standards with a d.p. ranging from 1 to 5 (X1-X5). (b) HPAEC-PAD trace showing the high molecular weight oligosaccharide products of CX digestion by the GH98 endo-xylanase, separated using a sodium acetate gradient of 0-500 mM. The reaction conditions used are the same as in panel a Control reactions of substrate and enzyme only are shown. (c) CX was incubated with 5 μM of the GH98 enzyme for 24 h in 50 mM sodium phosphate buffer, pH 7.0, at 37 °C. The reaction products, and CX that was not incubated with the GH98 enzyme, were then treated with sodium borohydride, to convert the reducing end monosaccharide unit into its alditol, followed by acid hydrolysis to convert the oligosaccharides (or in the case of control CX alone) into their monosaccharide constituents. The hydrolysed products were subjected to HPAEC and compared to the migration of standard monosaccharides as well as the alditol of xylose (xylitol) and galactose (galactitol). (d) The GH98 enzyme was incubated with CX (lane CX vs GH98), or CX that had been pre-treated with all the side chain cleaving enzymes encoded by PUL-XylL (lane CX vs MIX), or CX that had been pre-treated with all the side chain cleaving enzymes encoded by PUL-XylL, except the GH43 enzyme BACOVA_03417 that removes O3 linked arabinose from double substituted xylose (lane CX vs MIX no double abfase).
Mentions: The surface GH98 enzyme BACOVA_03433 also generates very large oligosaccharides but exclusively from CX (Fig. 4a,b). As BACOVA_03433 is a member of GH98, a family that hitherto contained only endo-β-D-galactosidases29, it was formally possible that the enzyme targets rare β-D-Gal residues in the backbone of CX. Converting the reducing end aldehyde of the products generated by BACOVA_03433 to the alditol followed by acid hydrolysis revealed the presence of xylitol but not galactitol (Fig. 4c). These data show that BACOVA_03433 is an endo-xylanase that hydrolyses xylosidic bonds in a context found only in very complex xylans. It is possible that the enzyme targets an unusual linkage in the xylan backbone; however, this is unlikely as the enzyme was not active against β1,4-β1,3-mixed linked xylans and CX is reported to contain a backbone comprising exclusively β1,4-Xyl units13. To explore the specificity determinants of the GH98 enzyme further, CX was pre-treated with cocktails of side-chain cleaving hydrolases (arabinofuranosidases, xylosidases, glucuronidases and galactosidases) encoded by PUL-XylL, and the debranched forms of the xylan were then incubated with the GH98 xylanase. Only GH consortia lacking BACOVA_03417 generated a variant of CX that was hydrolysed by the GH98 enzyme (Fig. 4d). As described below BACOVA_03417 is an α-L-arabinofuranosidase that targets the O3 linked Araf on backbone Xyl residues decorated at both O2 and O3 with α-L-Araf. It would appear, therefore, that the double Araf decoration is a component of the specificity determinant for BACOVA_03433. Wheat arabinoxylan, however, also contains this double arabinose decoration and is not a substrate for the GH98 xylanase (Fig. 4a). Thus, the specificity of BACOVA_03433 is more complex than simply Xyl units linked O2 and O3 to arabinose. Interestingly, the CX backbone contains low levels of doubly substituted residues with Xylp as the O3 linked side chain (see Fig. 1 and results below). This structure is also a substrate for BACOVA_03417 (Supplementary Fig. 4a), suggesting Xylp at O3 (and Araf at O2) may be the critical specificity determinant of the GH98 enzyme. These Xylp decorations are absent in WAX (Supplementary Fig. 4a–c), providing a possible explanation for the lack of activity of the GH98 enzyme against this substrate.

Bottom Line: We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan.Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains.Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

ABSTRACT
The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

No MeSH data available.