Multiple Transporters and Glycoside Hydrolases Are Involved in Arabinoxylan-Derived Oligosaccharide Utilization in Bifidobacterium pseudocatenulatum

Abstract

Arabinoxylan hydrolysates (AXH) are the hydrolyzed products of the major components of the dietary fiber arabinoxylan. AXH include diverse oligosaccharides varying in xylose polymerization and side residue modifications with arabinose at the O-2 and/or O-3 position of the xylose unit. Previous studies have reported that AXH exhibit prebiotic properties on gut bifidobacteria; moreover, several adult-associated bifidobacterial species (e.g., Bifidobacterium adolescentis and Bifidobacterium longum subsp. longum) are known to utilize AXH. In this study, we tried to elucidate the molecular mechanisms of AXH utilization by Bifidobacterium pseudocatenulatum, which is a common bifidobacterial species found in adult feces. We performed transcriptomic analysis of B. pseudocatenulatum YIT 4072^T, which identified three upregulated gene clusters during AXH utilization. The gene clusters encoded three sets of ATP-binding cassette (ABC) transporters and five enzymes belonging to glycoside hydrolase family 43 (GH43). By characterizing the recombinant proteins, we found that three solute-binding proteins of ABC transporters showed either broad or narrow specificity, two arabinofuranosidases hydrolyzed either single- or double-decorated arabinoxylooligosaccharides, and three xylosidases exhibited functionally identical activity. These data collectively suggest that the transporters and glycoside hydrolases, encoded in the three gene clusters, work together to utilize AXH of different sizes and with different side residue modifications. Thus, our study sheds light on the overall picture of how these proteins collaborate for the utilization of AXH in B. pseudocatenulatum and may explain the predominance of this symbiont species in the adult human gut.IMPORTANCE Bifidobacteria commonly reside in the human intestine and possess abundant genes involved in carbohydrate utilization. Arabinoxylan hydrolysates (AXH) are hydrolyzed products of arabinoxylan, one of the most abundant dietary fibers, and they include xylooligosaccharides and those decorated with arabinofuranosyl residues. The molecular mechanism by which B. pseudocatenulatum, a common bifidobacterial species found in adult feces, utilizes structurally and compositionally variable AXH has yet to be extensively investigated. In this study, we identified three gene clusters (encoding five GH43 enzymes and three solute-binding proteins of ABC transporters) that were upregulated in B. pseudocatenulatum YIT 4072^T during AXH utilization. By investigating their substrate specificities, we revealed how these proteins are involved in the uptake and degradation of AXH. These molecular insights may provide a better understanding of how resident bifidobacteria colonize the colon.

Keywords: ABC transporters; GH43; arabinoxylan; bifidobacteria; dietary fiber; glycoside hydrolase; oligosaccharides.

FIG 1

Gene clusters responsible for utilizing AXH. (A) Growth curves of B. pseudocatenulatum YIT 4072^T on AX-related carbohydrates. (B) (Top) Ratio of the RPKM value of B. pseudocatenulatum YIT 4072^T grown on the different carbon sources. The ratios of the RPKM values (AXH/lactose [Lac]) are aligned in the order of the gene locus. For genes with an RPKM value of zero under the lactose condition, the ratio was calculated with the number of reads set equal to 1. The vertical axis shows the part with values below 100. (Bottom) The gene organization of the three clusters which displayed high levels expression during growth on the AXH are shown. (C) The RPKM values of genes encoded by the three clusters are represented by a heat map. The numbers represent abbreviated locus tags (BBPC_RSXXXXX).

FIG 2

Affinities of the BpAXBP for the differently decorated AXOS or XOS with different polymerizations. (A to C) The affinity of each BpAXBP toward AXOS or XOS was analyzed by surface plasmon resonance, and their representative sensorgrams are shown. The sensorgrams of all oligosaccharides tested are shown in Fig. S3 in the supplemental material. (D) The affinity of BpAXBP for AXOS or XOS is represented by a heat map based on the calculated K_d value (in micromolar). Each box is colored according to the legend shown on the right. ND^(a), no SPR signals were detected; ND^(b), the K_d values were too high to be determined. The graphical representations of the glycans are based on reference . The stars represent xylose (orange) or arabinose (green).

FIG 3

Activity of the five GH43 enzymes toward AX-related oligosaccharides. (A) Concentrations of monosaccharides liberated from AXH by the recombinant GH43 enzymes. The concentration increase from the initial solution is represented. (B) TLC analysis of AXOS by the two recombinant BpAbf43. The substrates are indicated at the top. (C) Time course of hydrolysis of XOS4 by the three recombinant BpXyl43. The numbers indicate the reaction time of incubation. Lanes Std, a mixture of xylose, XOS2, XOS3, and XOS4. The stars represent xylose (orange) or arabinose (green).

FIG 4

Model of AXH utilization by B. pseudocatenulatum YIT 4072^T. (Top) Mechanism of AXH uptake; (bottom) model for the degradation of AXH. AXH derived from AX are taken up in the cells by ABC transporters. The lines surrounding the AXOS or XOS indicate that the K_d values were less than 10 μM, and their color corresponds to the color of the BpAXBP. In the bottom part, the arrows from each enzyme indicate the targeted linkages. The broken line from BpAbf43A indicates weak activity compared to that of BpAbf43B. The stars represent xylose (orange) or arabinose (green).