β-Mannans are plant cell wall polysaccharides that are commonly found in human diets. However, a mechanistic understanding into the key populations that degrade this glycan is absent, especially for the dominant Firmicutes phylum. Here, we show that the prominent butyrate-producing Firmicute Roseburia intestinalis expresses two loci conferring metabolism of β-mannans. We combine multi-“omic” analyses and detailed biochemical studies to comprehensively characterize loci-encoded proteins that are involved in β-mannan capturing, importation, de-branching and degradation into monosaccharides. In mixed cultures, R. intestinalis shares the available β-mannan with Bacteroides ovatus, demonstrating that the apparatus allows coexistence in a competitive environment. In murine experiments, β-mannan selectively promotes beneficial gut bacteria, exemplified by increased R. intestinalis, and reduction of mucus-degraders. Our findings highlight that R. intestinalis is a primary degrader of this dietary fiber and that this metabolic capacity could be exploited to selectively promote key members of the healthy microbiota using β-mannan-based therapeutic interventions.

The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from Bifidobacterium longum subspecies infantis. Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2′-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of Bifidobacterium to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans.

Metabolism of dietary glycans is pivotal in shaping the human gut microbiota. However, the mechanisms that promote competition for glycans among gut commensals remain unclear. Roseburia intestinalis, an abundant butyrate-producing Firmicute, is a key degrader of the major dietary fibre xylan. Despite the association of this taxon to a healthy microbiota, insight is lacking into its glycan utilization machinery. Here, we investigate the apparatus that confers R. intestinalis growth on different xylans. R. intestinalis displays a large cell-attached modular xylanase that promotes multivalent and dynamic association to xylan via four xylan-binding modules. This xylanase operates in concert with an ATP-binding cassette transporter to mediate breakdown and selective internalization of xylan fragments. The transport protein of R. intestinalis prefers oligomers of 4-5 xylosyl units, whereas the counterpart from a model xylan-degrading Bacteroides commensal targets larger ligands. Although R. intestinalis and the Bacteroides competitor co-grew in a mixed culture on xylan, R. intestinalis dominated on the preferred transport substrate xylotetraose. These findings highlight the differentiation of capture and transport preferences as a possible strategy to facilitate co-growth on abundant dietary fibres and may offer a unique route to manipulate the microbiota based on glycan transport preferences in therapeutic interventions to boost distinct taxa.

Escherichia coli strains with inactivated rluD genes were previously found to lack the conserved pseudouridines in helix 69 of 23S ribosomal RNA and to grow slowly. A suppressor mutant was isolated with a near normal growth rate that had changed the conserved Glu-172 codon to a Lys codon in prfB, encoding translation termination factor RF2. When nonsense suppression in strains with all combinations of prfB ؉ /prfBE172K and rluD ؉ /rluD::cat was analyzed, misreading of all three stop codons as sense codons was found to be increased by rluD inactivation: Nonsense suppression was increased 2-fold at UAG codons, 9-fold at UAA, and 14-fold at UGA. The increased read-through at UGA corresponds to reading UGA as a sense codon in 30% of the cases. In contrast, the accuracy of reading sense codons appeared unaffected by loss of rluD. When the inactivated rluD gene was combined with the altered prfB, wild-type levels of termination were restored at UAA codons and termination was more efficient than wild type at UGA. These results strongly suggest that at least one of the helix 69 pseudouridines has a function in translation termination. To our knowledge, this is the first described function for a ribosomal RNA pseudouridine modification.nonsense suppression ͉ release factor 2 ͉ rluD ͉ rRNA modification P seudouridine is found in many species of stable RNA. Its formation is catalyzed by several pseudouridine synthases and takes place after transcription (1). No function has so far been identified for any of the pseudouridines in ribosomal RNA (2). In Escherichia coli 7 enzymes catalyze the formation of the 11 pseudouridines in rRNA (3). The rluD gene product catalyzes the formation of the pseudouridine in helix 69 (h69) at positions 1911 , 1915 , and 1917. In the structure of the 70S ribosome, h69 contacts both A and P site tRNA and also contributes to the universally conserved bridge B2a between the two ribosomal subunits (5). Changes in h69 can result in defects in subunit association (6, 7) and in an altered translational accuracy (8). Recently, Ali et al. (9) were able to prepare ribosomes in which h69 was deleted. These retained the ability to perform all separate ribosome functions, including proper translational accuracy and ability to translate a natural mRNA. Only peptide chain termination catalyzed by RF1 and ribosome recycling catalyzed by RRF were affected. It was therefore concluded that the main function of h69 was in translation initiation, translation termination, or ribosome recycling (9).The presence of pseudouridines in h69 is a highly conserved feature of cytoplasmic ribosomes (4) even if the modification mechanism in eukaryotes is very different from the mechanism used by bacteria (10, 11). The question of whether all h69 pseudouridines are equally important was addressed by Hirabayashi et al. (7), who isolated strains where the ribosomal RNA did not have U1915 but retained normal cellular growth rate. They were unable to isolate variants with an altered U1917 and did not address the essentiality of U...

SummaryGlycan utilization plays a key role in modulating the composition of the gut microbiota, but molecular insight into oligosaccharide uptake by this microbial community is lacking. Arabinoxylo-oligosaccharides (AXOS) are abundant in the diet, and are selectively fermented by probiotic bifidobacteria in the colon. Here we show how selectivity for AXOS uptake is established by the probiotic strain Bifidobacterium animalis subsp. lactis Bl-04. The binding protein BlAXBP, which is associated with an ATP-binding cassette (ABC) transporter that mediates the uptake of AXOS, displays an exceptionally broad specificity for arabinosyl-decorated and undecorated xylooligosaccharides, with preference for tri-and tetrasaccharides. Crystal structures of BlAXBP in complex with four different ligands revealed the basis for this versatility. Uniquely, the protein was able to recognize oligosaccharides in two opposite orientations, which facilitates the optimization of interactions with the various ligands. Broad substrate specificity was further enhanced by a spacious binding pocket accommodating decorations at different mainchain positions and conformational flexibility of a lid-like loop. Phylogenetic and genetic analyses show that BlAXBP is highly conserved within Bifidobacterium, but is lacking in other gut microbiota members. These data indicate niche adaptation within Bifidobacterium and highlight the metabolic syntrophy (cross-feeding) among the gut microbiota.