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Transcriptomic Analyses of Xylan Degradation by Prevotella bryantii and Insights into Energy Acquisition by Xylanolytic Bacteroidetes*

  • Dylan Dodd
    Affiliations
    Department of Microbiology, University of Illinois, Urbana, Illinois 61801

    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801
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  • Young-Hwan Moon
    Affiliations
    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801
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  • Kankshita Swaminathan
    Affiliations
    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801

    Departments of Crop Sciences, University of Illinois, Urbana, Illinois 61801
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  • Roderick I. Mackie
    Affiliations
    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801

    Departments of Animal Sciences, University of Illinois, Urbana, Illinois 61801
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  • Isaac K.O. Cann
    Correspondence
    To whom correspondence should be addressed: 1105 Institute for Genomic Biology, 1206 West Gregory Dr., University of Illinois at Urbana-Champaign, Urbana, IL 61801. Tel.: 217-333-2090; Fax: 217-333-8286
    Affiliations
    Department of Microbiology, University of Illinois, Urbana, Illinois 61801

    Energy Biosciences Institute, University of Illinois, Urbana, Illinois 61801

    Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801

    Departments of Animal Sciences, University of Illinois, Urbana, Illinois 61801
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  • Author Footnotes
    * This work was supported by the Energy Biosciences Institute. D. D. was partially supported by National Institutes of Health Fellowship 1F30DK084726 from NIDDK.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S8 and Tables S1 and S2.
Open AccessPublished:July 09, 2010DOI:https://doi.org/10.1074/jbc.M110.141788
      Enzymatic depolymerization of lignocellulose by microbes in the bovine rumen and the human colon is critical to gut health and function within the host. Prevotella bryantii B14 is a rumen bacterium that efficiently degrades soluble xylan. To identify the genes harnessed by this bacterium to degrade xylan, the transcriptomes of P. bryantii cultured on either wheat arabinoxylan or a mixture of its monosaccharide components were compared by DNA microarray and RNA sequencing approaches. The most highly induced genes formed a cluster that contained putative outer membrane proteins analogous to the starch utilization system identified in the prominent human gut symbiont Bacteroides thetaiotaomicron. The arrangement of genes in the cluster was highly conserved in other xylanolytic Bacteroidetes, suggesting that the mechanism employed by xylan utilizers in this phylum is conserved. A number of genes encoding proteins with unassigned function were also induced on wheat arabinoxylan. Among these proteins, a hypothetical protein with low similarity to glycoside hydrolases was shown to possess endoxylanase activity and subsequently assigned to glycoside hydrolase family 5. The enzyme was designated PbXyn5A. Two of the most similar proteins to PbXyn5A were hypothetical proteins from human colonic Bacteroides spp., and when expressed each protein exhibited endoxylanase activity. By using site-directed mutagenesis, we identified two amino acid residues that likely serve as the catalytic acid/base and nucleophile as in other GH5 proteins. This study therefore provides insights into capture of energy by xylanolytic Bacteroidetes and the application of their enzymes as a resource in the biofuel industry.

      Introduction

      The hydrolysis and fermentation of plant cell wall polysaccharides are important metabolic processes that occur within the gut ecosystem of ruminants as well as humans. Xylan is the most abundant plant polysaccharide after cellulose, and microbes within the bovine rumen have evolved to efficiently degrade this hemicellulosic substrate. The depolymerization of xylan requires the coordinated action of a number of enzymes, including endoxylanases, β-xylosidases, α-l-arabinofuranosidases, α-glucuronidases, acetylxylan esterases, and ferulic acid esterases (
      • Dodd D.
      • Cann I.O.
      ). Prevotella spp. are the most numerically dominant xylanolytic bacteria in the rumen (
      • Stevenson D.M.
      • Weimer P.J.
      ,
      • Edwards J.E.
      • McEwan N.J.
      • Travis A.J.
      • Wallace R.J.
      ); therefore, the mechanism by which they degrade and utilize xylan has been an important topic of investigation (
      • Dodd D.
      • Kiyonari S.
      • Mackie R.I.
      • Cann I.K.
      ,
      • Miyazaki K.
      • Miyamoto H.
      • Mercer D.K.
      • Hirase T.
      • Martin J.C.
      • Kojima Y.
      • Flint H.J.
      ,
      • Miyazaki K.
      • Martin J.C.
      • Marinsek-Logar R.
      • Flint H.J.
      ,
      • Gasparic A.
      • Martin J.
      • Daniel A.S.
      • Flint H.J.
      ,
      • Gasparic A.
      • Marinsek-Logar R.
      • Martin J.
      • Wallace R.J.
      • Nekrep F.V.
      • Flint H.J.
      ,
      • Dodd D.
      • Kocherginskaya S.A.
      • Spies M.A.
      • Beery K.E.
      • Abbas C.A.
      • Mackie R.I.
      • Cann I.K.
      ). Prevotella bryantii B14 is frequently isolated from the rumen microbiome and can grow with xylan as the sole carbohydrate source (
      • Miyazaki K.
      • Martin J.C.
      • Marinsek-Logar R.
      • Flint H.J.
      ). However, to date only six genes with roles in xylan hydrolysis have been cloned and characterized from P. bryantii B14, including two glycoside hydrolase (GH)
      The abbreviations used are: GH
      glycoside hydrolase
      WAX
      wheat arabinoxylan
      HPAEC
      high performance anion exchange chromatography
      XA
      xylose and arabinose.
      family 10 endoxylanases (
      • Gasparic A.
      • Martin J.
      • Daniel A.S.
      • Flint H.J.
      ,
      • Gasparic A.
      • Marinsek-Logar R.
      • Martin J.
      • Wallace R.J.
      • Nekrep F.V.
      • Flint H.J.
      ,
      • Flint H.J.
      • Whitehead T.R.
      • Martin J.C.
      • Gasparic A.
      ), three GH family 3 β-xylosidases (
      • Dodd D.
      • Kiyonari S.
      • Mackie R.I.
      • Cann I.K.
      ), and one GH family 43 β-xylosidase enzyme (
      • Gasparic A.
      • Martin J.
      • Daniel A.S.
      • Flint H.J.
      ,
      • Gasparic A.
      • Marinsek-Logar R.
      • Martin J.
      • Wallace R.J.
      • Nekrep F.V.
      • Flint H.J.
      ). Given the capacity of this organism to grow efficiently with xylan substrates and considering the complexity in chemical linkages within natural xylans, it is likely that P. bryantii B14 uses additional as yet unidentified enzymes to degrade xylan.
      The genome of P. bryantii B14 has recently been partially sequenced (
      • Morrison M.
      • Daugherty S.C.
      • Nelson W.C.
      • Davidsen T.
      • Nelson K.E.
      ), and this bacterium harbors at least 109 genes predicted to encode either glycoside hydrolases or carbohydrate esterases. In this study, a transcriptional approach was employed to identify the genes harnessed by P. bryantii B14 to degrade xylan. Because it has been reported that endoxylanase activity of P. bryantii B14 is induced by medium to large sized xylo-oligosaccharides and not by monosaccharides (
      • Miyazaki K.
      • Hirase T.
      • Kojima Y.
      • Flint H.J.
      ), the transcriptional profile of P. bryantii B14 cultured either with soluble wheat arabinoxylan (WAX) or with a mixture of xylose and arabinose (XA) was investigated. The studies allowed us to assemble the enzymes that likely constitute the xylan-degrading machinery of P. bryantii B14. In addition, we have assigned biochemical function to two hypothetical proteins that were up-regulated in cells metabolizing wheat arabinoxylan compared with a mixture of its monosaccharide components. Genes encoding similar polypeptides, which invariably exhibited endoxylanase activity, were identified in several members of the Bacteroidetes, suggesting that this group of enzymes is critical to the capture of energy from xylan in this phylum. More importantly, our analyses have led to the discovery of a gene cluster, composed of an invariant core of six genes flanked by either biochemically characterized or putative glycoside hydrolases and carbohydrate esterases in P. bryantii B14. The genes within the cluster and their collective response during wheat arabinoxylan utilization suggest that the cluster is critical to xylan utilization in this bacterium. Furthermore, the conservation of the gene cluster in other xylanolytic Prevotella and Bacteroides spp. derived from the bovine rumen and the human colonic microbiomes suggests a conserved mechanism for xylan utilization by xylanolytic Bacteroidetes.

      DISCUSSION

      Xylans are an abundant group of plant polysaccharides that are hydrolyzed and fermented by commensal microbes within the rumen (
      • Hespell R.B.
      • Wolf R.
      • Bothast R.J.
      ,
      • Dehority B.A.
      ,
      • Dehority B.A.
      ) and the human gut (
      • Salyers A.A.
      • Balascio J.R.
      • Palmer J.K.
      ,
      • Chassard C.
      • Goumy V.
      • Leclerc M.
      • Del'homme C.
      • Bernalier-Donadille A.
      ,
      • Salyers A.A.
      • Gherardini F.
      • O'Brien M.
      ,
      • Chassard C.
      • Bernalier-Donadille A.
      ,
      • Chassard C.
      • Delmas E.
      • Lawson P.A.
      • Bernalier-Donadille A.
      ,
      • Mirande C.
      • Kadlecikova E.
      • Matulova M.
      • Capek P.
      • Bernalier-Donadille A.
      • Forano E.
      • Bera-Maillet C.
      ,
      • Weaver J.
      • Whitehead T.R.
      • Cotta M.A.
      • Valentine P.C.
      • Salyers A.A.
      ). Despite the abundance of xylanolytic bacteria in these microbiomes, relatively little is known about the mechanisms underlying the degradation and utilization of xylan by these bacteria.
      In this study, a whole genome transcriptomic approach was employed to evaluate the repertoire of genes that the rumen hemicellulolytic bacterium P. bryantii B14 employs to degrade xylan. The regulation of xylanase activity has been studied previously in P. bryantii B14, and it was found that xylanase activity is not induced by glucuronic acid, arabinose, xylose, or small xylo-oligosaccharides (X2–X5) (
      • Miyazaki K.
      • Hirase T.
      • Kojima Y.
      • Flint H.J.
      ). Rather, the major inducers of xylanase activity were found to be medium to large sized xylo-oligosaccharides. Our data confirmed the results from this previous study and reveal that the two previously studied endoxylanase genes (xyn10A and xyn10C) are highly induced during growth on soluble WAX compared with the component monosaccharides, XA. Although these genes were among the top 10 most highly induced genes, a large number of additional genes were also induced under these conditions. This observation underscores the complexity in the transcriptional response of P. bryantii B14 during growth on this arabinoxylan polysaccharide.
      The major xylanolytic gene cluster in P. bryantii B14 identified in this study contains a genomic DNA fragment that was previously cloned by Gasparic et al. (
      • Gasparic A.
      • Martin J.
      • Daniel A.S.
      • Flint H.J.
      ). The genes previously identified include open reading frames 1907 (xynR), 1908 (xynB), 1909 (xyn10A), 1910 (xynD), 1911 (xynE), and 1912 (xynF), although our data suggest that two genes (xynR and xynF) on either end of the DNA fragment reported by Gasparic et al. (
      • Gasparic A.
      • Martin J.
      • Daniel A.S.
      • Flint H.J.
      ) were truncated in their study. A translation start site (ATG) that occurs 1602 bp upstream of the start site predicted for this gene by Gasparic et al. (
      • Gasparic A.
      • Martin J.
      • Daniel A.S.
      • Flint H.J.
      ) was identified for xynR (ORF1907), and the assignment of this alternative start site was corroborated by the RNA-Seq coverage, which is continuous throughout the entire length of ORF1907 (Fig. 2). This result suggests that the xylan catabolism regulator (XynR) in P. bryantii B14 is 534 amino acids longer than originally reported. Analysis of the domain architecture for this protein revealed three functional domains, including a putative N-terminal periplasmic sensing domain, followed by a histidine kinase domain and a response regulator domain (supplemental Fig. S6). Thus, this hybrid two-component system regulator contains all of the regulatory elements found in “classical” two-component regulatory systems, but rather than encoding the sensor and effector domains in separate genes as in the classical system, xynR sencodes all of these functions within a single polypeptide. The domain organization for XynR is analogous to that for a hybrid two-component system regulator (BT3172) characterized from the related bacterium, Bacteroides thetaiotaomicron VPI-5482 (
      • Sonnenburg E.D.
      • Sonnenburg J.L.
      • Manchester J.K.
      • Hansen E.E.
      • Chiang H.C.
      • Gordon J.I.
      ); however, the sequence similarity across the entire polypeptide is relatively low (23%, 1405 amino acids aligned). The gene xynR was shown to be important for coordinating the transcription of a xylan utilization gene cluster in response to growth on xylan in P. bryantii B14 (
      • Miyazaki K.
      • Miyamoto H.
      • Mercer D.K.
      • Hirase T.
      • Martin J.C.
      • Kojima Y.
      • Flint H.J.
      ); thus, this protein may be the primary regulator responsible for the transcriptional response identified in this study. This hypothesis can be tested by constructing a P. bryantii B14 strain carrying a deletion in this gene and examining its growth on xylan. However, the lack of a genetic system for manipulating P. bryantii B14 precludes this analysis at the current time. The gene xynR is conserved in the human colonic xylan utilizing bacterium Bacteroides ovatus, which is amenable to genetic manipulation. Thus this bacterium can be used as a model to investigate the role of the hybrid two-component system in the catabolism of xylan.
      The xylan utilization gene cluster previously characterized by Gasparic et al. (
      • Gasparic A.
      • Martin J.
      • Daniel A.S.
      • Flint H.J.
      ) was found to be part of a larger xylanolytic gene cluster that includes the previously characterized endoxylanase gene, xyn10C (
      • Flint H.J.
      • Whitehead T.R.
      • Martin J.C.
      • Gasparic A.
      ). The gene for Xyn10C (ORF1893) occurs in a group of six genes that the RNA-Seq data suggest are co-transcribed within a single polycistronic mRNA molecule. This operon includes six of the seven most highly induced genes during growth on WAX relative to XA, which indicates that it is likely to be of critical importance to xylan utilization by P. bryantii B14. Contained within this operon are two tandem repeats of genes predicted to encode outer membrane proteins that share homology to the starch utilization system (Sus) components, SusC and SusD, followed by a hypothetical gene that is then followed by xyn10C. This arrangement of genes is similar in certain respects to the starch utilization system identified by Salyers and co-workers (
      • Shipman J.A.
      • Berleman J.E.
      • Salyers A.A.
      ) in B. thetaiotaomicron. SusC is predicted to encode an outer membrane porin that transports oligosaccharides into the periplasm in a TonB-dependent fashion. SusD harbors a signal peptidase II cleavage site that may facilitate the tethering of this protein onto the outer leaflet of the outer membrane where it may play a role in oligosaccharide binding (
      • Koropatkin N.M.
      • Martens E.C.
      • Gordon J.I.
      • Smith T.J.
      ). The hypothetical protein (ORF1894) and Xyn10C both possess putative signal peptidase II cleavage sites, which suggests that these two proteins are also tethered on the outer surface of the cell. The function of the hypothetical protein (ORF1894) has yet to be determined; however, its location within this highly induced xylan utilization operon suggests that it is involved in the degradation and utilization of xylan. The gene product has been made and purified, and when tested it exhibited a zone of Congo red exclusion following incubation on a WAX-infused agar plate (data not shown). Analysis of the activity of this protein is an ongoing focus in our laboratory. These observations suggest that this cluster of six proteins may be critical for the binding, depolymerization, and transport of extracellular xylan fragments into the periplasmic space, although further functional studies must be performed to verify this hypothesis.
      The arrangement of this operon is conserved among a number of bacteria for which genome sequences are available, including Prevotella ruminicola, Prevotella copri, Prevotella buccae, Prevotella bergensis, B. intestinalis, Bacteroides cellulosilyticus, Bacteroides sp. 2_2_4, B. ovatus, B. eggerthii, Bacteroides plebeius, and an additional Bacteroidetes member, Spirosoma linguale that is commonly isolated from soil or freshwater (Fig. 7 and supplemental Fig. S7). These organisms are members of the phylum Bacteroidetes and, with the exception of S. linguale, they were isolated from the bovine rumen or the human alimentary tract (supplemental Fig. S7). This is the first evidence of a xylan utilization cluster that is strictly conserved across xylan degrading members of the rumen Prevotella and the human-associated Bacteroides spp. The conservation of this cluster is highly suggestive that xylanolytic members from these two bacterial genera employ a conserved mechanism to degrade and utilize xylan. Apart from the high level of conservation in the orientation of genes within this operon, there are no other strictly conserved arrangements of genes in the regions nearby on the chromosome (Fig. 2). It has been proposed that the Sus system, initially identified by Salyers and co-workers (
      • Shipman J.A.
      • Berleman J.E.
      • Salyers A.A.
      ,
      • D'Elia J.N.
      • Salyers A.A.
      ), represents a paradigm for oligo- and polysaccharide utilization by Bacteroidetes (
      • Martens E.C.
      • Koropatkin N.M.
      • Smith T.J.
      • Gordon J.I.
      ), and the core xylan utilization cluster identified in this study provides further support for this prediction.
      Figure thumbnail gr7
      FIGURE 7Core xylan utilization system is conserved among certain species within the phylum Bacteroidetes. The P. bryantii B14 endoxylanase, Xyn10C, was used as the query sequence in a BLASTp search of the GenBankTM database. The genomic context is shown for each of the top BLASTp hits. Only the region that contains genes with predicted roles associated with xylan deconstruction are shown. Genes are color-coded based on their predicted roles as indicated in the legend. ORF numbers are indicated within each of the genes as derived from the genome project for each organism in the GenBankTM database.
      The majority of the genes that were induced by P. bryantii B14 during growth on WAX compared with XA code for proteins that are currently designated hypothetical proteins. This observation underscores the fact that there are large gaps in our current understanding of how polysaccharides are metabolized by P. bryantii B14 and other gut-associated bacteria. Whole genome transcriptional profiling represents a useful approach to gain insight into the potential roles of genes with unassigned functions. In this study, a subset of genes with low homology to glycoside hydrolases has been shown to belong to GH family 5 based on biochemical data. These genes were only found within certain members of the Bacteroidetes phylum, which are resident within the alimentary tract of humans or ruminants. Most of these GH5 genes occur near the conserved xylan utilization cluster in the genome (Fig. 7; P. copri, ORF6092; P. buccae, ORF0844, Bacteroides sp. 2_2_4, ORF3750; B. intestinalis, ORF4213; B. cellulosilyticus, ORF3415; B. eggerthii, ORF1299), which suggests that they may be directly involved in the degradation of xylan. BeXyn5A and BiXyn5A contain putative signal peptidase II cleavage sites, raising the possibility that the two proteins are anchored on the outside of the cell and perhaps function coordinately with the core xylan utilization cluster.
      All of the GH5 proteins identified in this study possess N- or C-terminal stretches of amino acids that did not align with known domains in either the Pfam or the NCBI conserved domains database (Table 3). Whether or not these regions represent functional domains is currently unclear. The two Bacteroides proteins (BeXyn5A and BiXyn5A) share 78% identity at the amino acid level and also share similar domain organizations with both proteins possessing a C-terminal bacterial Ig2-like domain. The function of this C-terminal extension is currently unknown; however, the Ig2-like domain is predicted to be involved in cell surface adhesion (
      • Kelly G.
      • Prasannan S.
      • Daniell S.
      • Fleming K.
      • Frankel G.
      • Dougan G.
      • Connerton I.
      • Matthews S.
      ). Many xylan-degrading enzymes are associated with carbohydrate-binding modules within a single polypeptide, and it is possible that the Ig2-like domain serves a carbohydrate binding function. Further studies in our laboratory will focus on delineating the functional role of this domain in BeXyn5A and BiXyn5A.
      Despite the fact that the three enzymes characterized in this study are clearly related to each other at the amino acid sequence level, they exhibited differences in the products released from WAX. The observation that BeXyn5A (ORF1299, Table 3) synergizes with Ara43A, whereas BiXyn5A (ORF4213, Table 3) and PbXyn5A (ORF0150, Table 3) do not exhibit synergy with Ara43A, clearly indicates that there is a fundamental difference in the enzymatic activities among these enzymes, most likely originating from the GH5 active site domain.
      Two additional genes (P. bryantii B14 ORF0336 and B. intestinalis ORF1125, Table 3), encoding similar GH5 modules as those found in the enzymes described above, were expressed. Both proteins exhibited endoxylanase activity, and the gene products were named PbXyn5B and BiXyn5B (supplemental Fig. S8). In addition to the GH5 module, BiXyn5B also contains a GH43 domain and is therefore different from BiXyn5A.
      Recently, a large number of genome sequences have been made available for human colonic bacteria through the human microbiome project (
      • Turnbaugh P.J.
      • Ley R.E.
      • Hamady M.
      • Fraser-Liggett C.M.
      • Knight R.
      • Gordon J.I.
      ). These genome sequences provide a wealth of information on the genome contents of commensal microbes within the human gut microbiome; however, proper annotation of these genes is critical to interpreting the genomic data in terms of the metabolic repertoire of the microbial community. In this study, transcriptional profiling of a rumen bacterium led to the assignment of function to a group of hypothetical proteins within the rumen Prevotella spp. and human colonic Bacteroides spp. Furthermore, this study has provided insights that suggest a conserved mechanism for xylan utilization among members of the phylum Bacteroidetes.

      Acknowledgments

      We thank Shosuke Yoshida, Yejun Han, Michael Iakiviak, and Xiaoyun Su of the Energy Biosciences Institute for valuable scientific discussions and Hiroshi Miyagi for technical assistance. We also thank members of the North American Consortium for Genomics of Fibrolytic Ruminal Bacteria for access to the partial genome sequence of P. bryantii B14, Alvaro Hernandez and Chris Wright of the W. M. Keck Center for Comparative and Functional Genomics for assistance with Illumina sequencing, and Mark Band from the same center for assistance with microarray analyses.

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