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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.


Cellular location of Xylan PUL enzymes containing a predicted N-terminal lipidation site or uncleaved transmembrane anchor.(a) Fluorescent microscopic images of B. ovatus cells cultured on WAX and incubated with polyclonal antibodies raised against recombinant BACOVA_03419 (GH3), BACOVA_03421 and BACOVA_03425 (GH43s), BACOVA_03431 (SGBP), BACOVA_03432 (GH30), BACOVA_03433 (GH98) and BACOVA_04390 (GH10). (b) Western blots of the B. ovatus cells shown in panel a, either untreated with Proteinase K (-) or incubated with 2 mg ml−1 Proteinase K for 16 h (+). The blots were probed with antibodies against the B. ovatus proteins indicated. The full blots are shown in Supplementary Fig. 8.
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f3: Cellular location of Xylan PUL enzymes containing a predicted N-terminal lipidation site or uncleaved transmembrane anchor.(a) Fluorescent microscopic images of B. ovatus cells cultured on WAX and incubated with polyclonal antibodies raised against recombinant BACOVA_03419 (GH3), BACOVA_03421 and BACOVA_03425 (GH43s), BACOVA_03431 (SGBP), BACOVA_03432 (GH30), BACOVA_03433 (GH98) and BACOVA_04390 (GH10). (b) Western blots of the B. ovatus cells shown in panel a, either untreated with Proteinase K (-) or incubated with 2 mg ml−1 Proteinase K for 16 h (+). The blots were probed with antibodies against the B. ovatus proteins indicated. The full blots are shown in Supplementary Fig. 8.

Mentions: Bioinformatic analysis, in conjunction with cell localization studies and whole-cell assays, was used to identify the enzymes encoded by PUL-XylS and PUL-XylL that are displayed on the bacterial surface. The presence of a predicted Type II lipoprotein signal sequence or uncleaved transmembrane (TM) anchor in seven GH family proteins indicated that they were membrane attached. The cell localization of these seven proteins was evaluated experimentally by (i) fluorescence microscopy of whole cells treated with labelled antibodies directed against the appropriate proteins and (ii) western blotting after removal of surface proteins with Proteinase K. The data indicated that BACOVA_03419 (GH3), BACOVA_03421 (GH43), BACOVA_03431 (inactive GH10, see later results), BACOVA_03432 (GH30), BACOVA_03433 (GH98) and BACOVA_04390 (GH10) were presented on the surface of B. ovatus (Fig. 3). The only lipoprotein facing the periplasm was the GH43 enzyme BACOVA_03425. These conclusions were further supported by whole-cell assays of the B. ovatus mutant lacking PUL-XylS (ΔPUL-XylS) incubated with BGX. The product profile of ΔPUL-XylS was extremely similar to that of recombinant BACOVA_03432 GH30 against the polysaccharide (Supplementary Fig. 1a). Thus, BGX degradation is mediated predominantly by BACOVA_03432 in the ΔPUL-XylS mutant, confirming the extracellular location of the GH30 enzyme. Whole-cell assays also showed that the PUL-XylL encoded GH43 arabinofuranosidase, BACOVA_03421 (see results below), was extracellular, as arabinose was released from WAX by the ΔPUL-XylS mutant (Supplementary Fig. 1b). It is surprising that BACOVA_03419, a GH3 β-xylosidase, is located on the surface as its obvious role is to contribute to the degradation of the xylooligosaccharides generated in the periplasm (Supplementary Fig. 2a). BACOVA_03419, however, displays only low activity against linear β1,4-xylooligosaccharides, but, unlike the cytoplasmic xylosidase, BACOVA_04386 (see results below), will cleave linkages in which the leaving group xylose is decorated (Supplementary Table 1 and Supplementary Fig. 2b,c). While the roles of BACOVA_03419 and BACOVA_03421 in xylan degradation are unclear, we speculate that the enzymes contribute to the surface ‘trimming' of complex xylans.


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)

Cellular location of Xylan PUL enzymes containing a predicted N-terminal lipidation site or uncleaved transmembrane anchor.(a) Fluorescent microscopic images of B. ovatus cells cultured on WAX and incubated with polyclonal antibodies raised against recombinant BACOVA_03419 (GH3), BACOVA_03421 and BACOVA_03425 (GH43s), BACOVA_03431 (SGBP), BACOVA_03432 (GH30), BACOVA_03433 (GH98) and BACOVA_04390 (GH10). (b) Western blots of the B. ovatus cells shown in panel a, either untreated with Proteinase K (-) or incubated with 2 mg ml−1 Proteinase K for 16 h (+). The blots were probed with antibodies against the B. ovatus proteins indicated. The full blots are shown in Supplementary Fig. 8.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4491172&req=5

f3: Cellular location of Xylan PUL enzymes containing a predicted N-terminal lipidation site or uncleaved transmembrane anchor.(a) Fluorescent microscopic images of B. ovatus cells cultured on WAX and incubated with polyclonal antibodies raised against recombinant BACOVA_03419 (GH3), BACOVA_03421 and BACOVA_03425 (GH43s), BACOVA_03431 (SGBP), BACOVA_03432 (GH30), BACOVA_03433 (GH98) and BACOVA_04390 (GH10). (b) Western blots of the B. ovatus cells shown in panel a, either untreated with Proteinase K (-) or incubated with 2 mg ml−1 Proteinase K for 16 h (+). The blots were probed with antibodies against the B. ovatus proteins indicated. The full blots are shown in Supplementary Fig. 8.
Mentions: Bioinformatic analysis, in conjunction with cell localization studies and whole-cell assays, was used to identify the enzymes encoded by PUL-XylS and PUL-XylL that are displayed on the bacterial surface. The presence of a predicted Type II lipoprotein signal sequence or uncleaved transmembrane (TM) anchor in seven GH family proteins indicated that they were membrane attached. The cell localization of these seven proteins was evaluated experimentally by (i) fluorescence microscopy of whole cells treated with labelled antibodies directed against the appropriate proteins and (ii) western blotting after removal of surface proteins with Proteinase K. The data indicated that BACOVA_03419 (GH3), BACOVA_03421 (GH43), BACOVA_03431 (inactive GH10, see later results), BACOVA_03432 (GH30), BACOVA_03433 (GH98) and BACOVA_04390 (GH10) were presented on the surface of B. ovatus (Fig. 3). The only lipoprotein facing the periplasm was the GH43 enzyme BACOVA_03425. These conclusions were further supported by whole-cell assays of the B. ovatus mutant lacking PUL-XylS (ΔPUL-XylS) incubated with BGX. The product profile of ΔPUL-XylS was extremely similar to that of recombinant BACOVA_03432 GH30 against the polysaccharide (Supplementary Fig. 1a). Thus, BGX degradation is mediated predominantly by BACOVA_03432 in the ΔPUL-XylS mutant, confirming the extracellular location of the GH30 enzyme. Whole-cell assays also showed that the PUL-XylL encoded GH43 arabinofuranosidase, BACOVA_03421 (see results below), was extracellular, as arabinose was released from WAX by the ΔPUL-XylS mutant (Supplementary Fig. 1b). It is surprising that BACOVA_03419, a GH3 β-xylosidase, is located on the surface as its obvious role is to contribute to the degradation of the xylooligosaccharides generated in the periplasm (Supplementary Fig. 2a). BACOVA_03419, however, displays only low activity against linear β1,4-xylooligosaccharides, but, unlike the cytoplasmic xylosidase, BACOVA_04386 (see results below), will cleave linkages in which the leaving group xylose is decorated (Supplementary Table 1 and Supplementary Fig. 2b,c). While the roles of BACOVA_03419 and BACOVA_03421 in xylan degradation are unclear, we speculate that the enzymes contribute to the surface ‘trimming' of complex xylans.

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.