Two extracellular α-arabinofuranosidases are required for cereal-derived arabinoxylan metabolism by Bifidobacterium longum subsp. longum

Abstract

Members of the genus Bifidobacterium are commonly found in the human gut and are known to utilize complex carbohydrates that are indigestible by the human host. Members of the Bifidobacterium longum subsp. longum taxon can metabolize various plant-derived carbohydrates common to the human diet. To metabolize such polysaccharides, which include arabinoxylan, bifidobacteria need to encode appropriate carbohydrate-active enzymes in their genome. In the current study, we describe two GH43 family enzymes, denoted here as AxuA and AxuB, which are encoded by B. longum subsp. longum NCIMB 8809 and are shown to be required for cereal-derived arabinoxylan metabolism by this strain. Based on the observed hydrolytic activity of AxuA and AxuB, assessed by employing various synthetic and natural substrates, and based on in silico analyses, it is proposed that both AxuA and AxuB represent extracellular α-L-arabinofuranosidases with distinct substrate preferences. The variable presence of the axuA and axuB genes and other genes previously described to be involved in the metabolism of arabinose-containing glycans can in the majority cases explain the (in)ability of individual B. longum subsp. longum strains to grow on cereal-derived arabinoxylans and arabinan.

Keywords: Dietary fiber; bifidobacterial; gut microbiota; prebiotic; probiotic.

Figure 1.

Growth profiles after 24 hours of incubation (top panel) and corresponding gene map (bottom panel) of B. longum subsp. longum strains B. longum subsp. longum strains (names indicated below the top panel) were cultivated for 24 h in mMRS containing particular carbohydrates (indicated on the right-hand margin in the top panel) and growth is indicated in blue color coding based on OD readings as indicated. Bottom panel displays the presence/absence of genes involved in the metabolism of plant glycans in the corresponding genomes of the B. longum strains, where yellow indicates presence and black absence. The cutoff presence or absence of the gene/enzyme was set to E-value of 0.0001, with at least 50% identity across at least 50% of either protein sequence.

Figure 2.

Growth abilities of B. longum subsp. longum NCIMB 8809 and derivatives with genes from the abu gene clusterOd600nm after 24 hours anaerobic growth in mMRS + 0.5% (w/v) arabinan or arabinose. Asterisks represent a significant difference (p ≤ 0.001***, p ≤ 0.01**) and NS indicates no significant difference (p ≥ 0.05).

Figure 3.

Heatmap of differential expressed genes differential expressed genes (DEGs) in B. longum subsp. longum NCIMB 8809, with significant levels of change in their degree of log₂ fold expression, when comparing arabinose (left) or AXR (right) to a lactose control. Red: negative log₂ fold change (down-regulated), gray: zero log₂ fold change, blue positive log₂ fold change (up-regulated), and white no significant change under these conditions.

Figure 4.

Schematic display of the axu gene cluster and neighboring genes in B. longum subsp. longum NCIMB 8809. (panel A). Illustration of the domains present in AxuA (panel B) and AxuB (panel C), predicted by TMHMM, SignalP, and HMMER; T: transmembrane domain; S: signal peptide; CBD: carbohydrate binding domain; M: sortase motif.

Figure 5.

HPAEC-PAD analysis of hydrolysis products of AX incubated with AxuA_His/AxuB_His–AXW (panel A), AXR (panel B). From bottom to top: substrate only (gray), with AxuBHis (red), with AxuAHis (black), with both (blue) after 30 min. In box: amount (g/L) of release monomeric L-arabinose of 1 mM enzyme after 30 min of incubation.

Figure 6.

HPAEC-PAD analysis of products from enzymatic reactions of synthetic AX with and without incubation with AxuA_His/AxuB_His see SM6; from the bottom to top: substrate only (gray), with AxuBHis (red), with AxuAHis (black), with both (blue); utilizing A2XX (panel A), A3X (panel B) and A23XX (panel C); the structures of tested synthetic AX are shown in the top-right hand corner. Green Stars indicated arabinose residues and orange stars correspond to xylose units. (structures created with BioRender.com).

Figure 7.

Growth profile of B. longum subsp. longum NCIMB 8809 mutant and transformants with genes from the axu gene cluster OD_600 nm after 24 hours anaerobically grown in mMRS + 0.5% (w/v) arabinose, AXR, AXW, and lactose. Asterisks represent a significant difference (p ≤ 0.001***) and NS indicates no significant difference (p ≥ 0.05).

Figure 8.

Growth profile of B. longum subsp. longum MM0464 transformants with genes from the axu gene cluster OD_600 nm after 24 hours anaerobically grown in mMRS + 0.5% (w/v) arabinose, AXR, AXW, and lactose. Asterisks represent a significant difference (p ≤ 0.001***, p ≤ 0.01**, p ≤ 0.05*) and NS indicates no significant difference (p ≥ 0.05).

Figure 9.

Model of AX degradation of AxuA and AxuB and arabinose metabolism in B. longum susp. longum locus tags for genes in B. longum susp. longum NCIMB8809, see text for further details. Created with BioRender.com.