Transcriptomic analyses of xylan degradation by Prevotella bryantii and insights into energy acquisition by xylanolytic bacteroidetes

Abstract

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 B(1)4 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.

FIGURE 1.

Carbohydrate esterase and glycoside hydrolase genes up-regulated on soluble WAX compared with XA. Each gene that was overexpressed at least 4-fold was used as a query in a BLASTp search of the nonredundant (nr) database at GenBank^TM. Genes were assigned to CAZy (43) families if they exhibited significant similarity (E-value <1 × 10⁻⁵) to biochemically characterized proteins already catalogued in a CAZy family. The GenBank^TM accession numbers for these genes are listed under “Experimental Procedures.”

FIGURE 2.

RNA-Seq coverage for the major xylanolytic gene cluster in P. bryantii B₁4. P. bryantii B₁4 was grown with either WAX or a XA as the sole carbohydrate source, and the transcriptomes were analyzed by RNA-Seq as described under “Experimental Procedures.” A detailed view of the nucleotide coverage for the major xylanolytic gene cluster in P. bryantii B₁4 during growth on WAX (red) or XA (green) is shown for two biological replicates (WAX1, WAX2; XA1, XA2). The portion of the gene cluster including ORF1907–1912 has been studied previously by Flint and co-workers (7). Asterisks denote genes for which biochemical activities have been demonstrated for their cognate gene products: xynB and xynA (7) and xynR (5).

FIGURE 3.

P. bryantii B₁4 ORF0150 encodes an enzyme with endoxylanase activity. A, purification of recombinant PbXyn5A. ORF0150 was cloned into an expression vector and expressed heterologously as a hexahistidine fusion protein in E. coli. The protein (PbXyn5A) was purified using cobalt affinity chromatography, and the eluate was analyzed by 12% SDS-PAGE, followed by Coomassie Brilliant Blue G-250 staining. B, depolymerization of soluble wheat arabinoxylan. PbXyn5A was assessed for its capacity to depolymerize soluble WAX by incubating the protein on an agar plate infused with WAX followed by staining and destaining with Congo red and 1 m NaCl, respectively. C, hydrolysis of xylohexaose. PbXyn5A-catalyzed hydrolysis of xylohexaose was assessed by incubating the enzyme with the substrate, removing aliquots at the indicated time points, and then resolving the products by thin layer chromatography followed by staining with methanolic orcinol. D, thin layer chromatography of products released from WAX by PbXyn5A. PbXyn5A (0.50 μm) was incubated with WAX (1% w/v), and the products were resolved by thin layer chromatography followed by staining with methanolic orcinol. Xylo-oligosaccharide standards X₁–X₅ and arabinose (A1) were spotted on the plate in lanes 2 and 1, respectively, to serve as markers for the identification of hydrolysis products. In lane 4, PbXyn5A was incubated with WAX at 37 °C for 15 h, and 2.5 μl of the reaction mixture were resolved on the TLC plate. E, reducing sugars released from WAX by PbXyn5A. Wild-type Xyn5A was incubated with WAX (1% w/v), and the amounts of reducing sugars released were determined by the para-hydroxybenzoic acid hydrazide assay. The reducing sugar concentrations were calculated from the absorbance at 410 nm by comparison to a standard curve generated with known concentrations of glucose. E, the values are reported as the means ± S.D. from three independent experiments.

FIGURE 4.

PbXyn5A functions synergistically with β-xylosidase and α-l-arabinofuranosidase enzymes. A and B, hydrolysis of WAX was assessed by incubating the respective enzymes with the substrate and then resolving the products by HPAEC followed by detection with a pulsed amperometric detector. The products of hydrolysis were identified by comparison of peaks with retention times of purified substrates. Abbreviations are as follows: FT, flow-through; A₁, arabinose; X₁–X₆, xylose through xylohexaose. The concentration of xylose and arabinose in each of the reactions was estimated by comparison with a calibration curve constructed with known concentrations of each sugar. The same chromatograms are depicted in A and B; however, the scale is adjusted in B to reveal changes in the oligosaccharide patterns between different reactions. These experiments were performed three times, and single representative curves are shown. The concentrations of xylose and arabinose are reported as means ± S.D.

FIGURE 5.

B. eggerthii ORF1299 and B. intestinalis ORF4213 encode endoxylanases. A, purification of recombinant BeXyn5A and BiXyn5A. B. eggerthii ORF1299 and B. intestinalis ORF4123 were cloned into expression vectors and expressed heterologously as hexahistidine fusion proteins in E. coli. The proteins were purified using cobalt affinity chromatography and gel filtration, and the elution fractions were pooled and analyzed by 12% SDS-PAGE, followed by Coomassie Brilliant Blue G-250 staining. B, thin layer chromatography of products released from WAX by BeXyn5A and BiXyn5A. BeXyn5A or BiXyn5A (0.50 μm each) was incubated with WAX (1% w/v) for 15 h at 37 °C, and the products were resolved by thin layer chromatography followed by staining with methanolic orcinol. Xylo-oligosaccharide standards X₁–X₅ and arabinose (A1) were spotted on the plate in lanes 2 and 1, respectively, to serve as markers for the identification of hydrolysis products. C, reducing sugars released from sWAX by BeXyn5A and BiXyn5A. BeXyn5A or BiXyn5A (0.50 μm each) was incubated with WAX (1% w/v) for 15 h at 37 °C, and the reducing sugars were detected by using the para-hydroxybenzoic acid hydrazide assay. The reducing sugar concentrations were calculated from the absorbance at 410 nm by comparison with a standard curve generated with known concentrations of glucose. C, the values are reported as the means ± S.D. from three independent experiments.

FIGURE 6.

BeXyn5A and BiXyn5A release long xylo-oligosaccharides from WAX. A–D, hydrolysis of wheat arabinoxylan was assessed by incubating the enzymes with WAX (1% w/v) and then resolving the products by HPAEC followed by detection with a pulsed amperometric detector. The products of hydrolysis were identified by comparison of peaks with retention times of purified substrates. Abbreviations are as follows: FT, flow-through; X₂, xylobiose, X₃, xylotriose; X₄, xylotetraose; X₅, xylopentaose; X₆, xylohexaose,. The concentration of xylose and arabinose in each of the reactions was estimated by comparison with a calibration curve constructed with known concentrations of each sugar. The same chromatograms are depicted in A and B as well as in C and D; however, the scale is adjusted in B and D to reveal changes in the oligosaccharide patterns between different reactions. These experiments were performed three times, and single representative curves are shown. The concentrations of xylose and arabinose are reported as means ± S.D.

FIGURE 7.

Core 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 GenBank^TM 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 GenBank^TM database.