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UNLABELLED: Bacteroides species are successful colonizers of the human colon and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in polysaccharide utilization loci (PULs). While recent work has uncovered the PULs required for the use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-β-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron. Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under the control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron. Examining the genomes of other Bacteroides species, we found homologs of BT1871 in a subset and showed that representative strains of species with a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide. IMPORTANCE: The gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the microbiota in the colon since they remain undigested by the host. Here, we study how the model commensal organism, Bacteroides thetaiotaomicron utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource.

Glycoside hydrolase family 97 (GH97) comprises enzymes like anomer‐inverting α‐glucoside hydrolases (i.e., glucoamylase) and anomer‐retaining α‐galactosidases. In a soil bacterium, Flavobacterium johnsoniae, we previously identified a GH97 enzyme (FjGH97A) within the branched dextran utilization locus. It functions as an α‐glucoside hydrolase, targeting α‐(1→6)‐glucosidic linkages in dextran and isomaltooligosaccharides (i.e., glucodextranase). FjGH97A exhibits a preference for α‐(1→6)‐glucoside linkages over α‐(1→4)‐linkages, while Bacteroides thetaiotaomicron glucoamylase SusB (with 69% sequence identity), which is involved in the starch utilization system, exhibits the highest specificity for α‐(1→4)‐glucosidic linkages. Here, we examined the crystal structures of FjGH97A in complexes with glucose, panose, or isomaltotriose, and analyzed the substrate preferences of its mutants to identify the amino acid residues that determine the substrate specificity for α‐(1→4)‐ and α‐(1→6)‐glucosidic linkages. The overall structure of FjGH97A resembles other GH97 enzymes, with conserved catalytic residues similar to anomer‐inverting GH97 enzymes. A comparison of active sites between FjGH97A and SusB revealed differences in amino acid residues at subsites +1 and +2 (specifically Ala195 and Ile378 in FjGH97A). Among the three mutants (A195S, I378F, and A195S‐I378F), A195S and A195S‐I378F exhibited increased activity toward α‐(1→4)‐glucoside bonds compared to α‐(1→6)‐glucoside bonds. This suggests that Ala195, located on the Gly184‐Thr203 loop (named loop‐N) conserved within the GH97 subgroup, including FjGH97A and SusB, holds significance in determining linkage specificity. The conservation of alanine in the active site of the GH97 enzymes, within the same gene cluster as the putative dextranase, indicates its crucial role in determining the specificity for α‐(1→6)‐glucoside linkage. [ABSTRACT FROM AUTHOR]

Glycogen, an α-glucan polymer serving as an energy storage compound in microorganisms, is synthesized through distinct pathways (GlgC-GlgA or GlgE pathway). Both pathways involve multiple enzymes, with a shared glycogen branching enzyme (GBE). GBEs play a pivotal role in establishing α-1,6-linkages within the glycogen structure. GBEs are also used for starch modification. Understanding how these enzymes work is interesting for both glycogen synthesis in microorganisms, as well as novel applications for starch modification. This study focuses on a putative enzyme GH13_9 GBE (PoGBE13), present in a polysaccharide utilization locus (PUL) of Pontibacter sp. SGAir0037, and related to the GlgE glycogen synthesis pathway. While the PUL of Pontibacter sp. SGAir0037 contains glycogen-degrading enzymes, the branching enzyme (PoGBE13) was also found due to genetic closeness. Characterization revealed that PoGBE13 functions as a typical branching enzyme, exhibiting a relatively high branching over non-branching (hydrolysis and α-1,4-transferase activity) ratio on linear maltooctadecaose (3.0 ± 0.4). Besides the GH13_9 GBE, a GH57 (PoGH57) enzyme was selected for characterization from the same PUL due to its undefined function. The combined action of both GH13 and GH57 enzymes suggested 4-α-glucanotransferase activity for PoGH57. The characterization of these unique enzymes related to a GlgE glycogen synthesis pathway provides a more profound understanding of their interactions and synergistic roles in glycogen synthesis and are potential enzymes for use in starch modification processes. Due to the structural similarity between glycogen and starch, PoGBE13 can potentially be used for starch modification with different applications, for example, in functional food ingredients. [ABSTRACT FROM AUTHOR]

Polysaccharide Utilization Loci (PULs) are physically linked gene clusters conserved in the Gram-negative phylum of Bacteroidota and are valuable sources for Carbohydrate Active enZyme (CAZyme) discovery. This study focuses on BD-β-Gal, an enzyme encoded in a metagenomic PUL and member of the Glycoside Hydrolase family 154 (GH154). BD-β-Gal showed exo- β-galactosidase activity with regiopreference for hydrolyzing β- d -(1,6) glycosidic linkages. Notably, it exhibited a preference for d -glucopyranosyl (d -Glc p) over d -galactopyranosyl (d -Gal p) and d -fructofuranosyl (d -Fru f) at the reducing end of the investigated disaccharides. In addition, we determined the high resolution crystal structure of BD-β-Gal, thus providing the first structural characterization of a GH154 enzyme. Surprisingly, this revealed an (α/α) 6 topology, which has not been observed before for β-galactosidases. BD-β-Gal displayed low structural homology with characterized CAZymes, but conservation analysis suggested that the active site was located in a central cavity, with conserved E73, R252, and D253 as putative catalytic residues. Interestingly, BD-β-Gal has a tetrameric structure and a flexible loop from a neighboring protomer may contribute to its reaction specificity. Finally, we showed that the founding member of GH154, BT3677 from Bacteroides thetaiotaomicron , described as β-glucuronidase, displayed exo -β-galactosidase activity like BD-β-Gal but lacked a tetrameric structure. [Display omitted] • New β-galactosidase activity in Glycoside Hydrolase family 154 (GH154). • BD-β-Gal shows β-galactosidase activity with preference for β-(1,6)-linkages. • First crystal structure of GH154 with novel (α/α) 6 topology for β-galactosidase. • Novel active site topology for BD-β-Gal, different from canonical β-galactosidases. • BD-β-Gal shows transglycosylation activity on lactose. [ABSTRACT FROM AUTHOR]

Introduction: Research on the mechanism of marine polysaccharide utilization by Bacteroides thetaiotaomicron has drawn substantial attention in recent years. Derived from marine algae, the marine algae polysaccharides could serve as prebiotics to facilitate intestinal microecological balance and alleviate colonic diseases. Bacteroides thetaiotaomicron, considered the most efficient degrader of polysaccharides, relates to its capacity to degrade an extensive spectrum of complex polysaccharides. Polysaccharide utilization loci (PULs), a specialized organization of a collection of genes-encoded enzymes engaged in the breakdown and utilization of polysaccharides, make it possible for Bacteroides thetaiotaomicron to metabolize various polysaccharides. However, there is still a paucity of comprehensive studies on the procedure of polysaccharide degradation by Bacteroides thetaiotaomicron. Methods: In the current study, the degradation of four kinds of marine algae polysaccharides, including sodium alginate, fucoidan, laminarin, and Pyropia haitanensis polysaccharides, and the underlying mechanism by Bacteroides thetaiotaomicron G4 were investigated. Pure culture of Bacteroides thetaiotaomicron G4 in a substrate supplemented with these polysaccharides were performed. The change of OD600, total carbohydrate contents, and molecular weight during this fermentation were determined. Genomic sequencing and bioinformatic analysis were further performed to elucidate the mechanisms involved. Specifically, Gene Ontology (GO) annotation, Clusters of Orthologous Groups (COG) annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were utilized to identify potential target genes and pathways. Results: Underlying target genes and pathways were recognized by employing bioinformatic analysis. Several PULs were found that are anticipated to participate in the breakdown of these four polysaccharides. These findings may help to understand the interactions between these marine seaweed polysaccharides and gut microorganisms. Discussion: The elucidation of polysaccharide degradation mechanisms by Bacteroides thetaiotaomicron provides valuable insights into the utilization of marine polysaccharides as prebiotics and their potential impact on gut health. Further studies are warranted to explore the specific roles of individual PULs and their contributions to polysaccharide metabolism in the gut microbiota. [ABSTRACT FROM AUTHOR]

BACKGROUND: Alginate lyases are important tools for alginate biodegradation and oligosaccharide production, which have great potential in food and biofuel fields. The alginate polysaccharide utilization loci (PUL) typically encode a series of alginate lyases with a synergistic action pattern. Exploring valuable alginate lyases and revealing the synergistic effect of enzymes in the PUL is of great significance. RESULTS: An alginate PUL was discovered from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T, and a repertoire of alginate lyases within it was cloned, expressed and characterized. The four alginate lyases in PUL demonstrated similar optimal reaction conditions: maximum enzyme activity at 35–50 °C and pH 8.0–9.0. The results of action pattern indicated that they were two PL7 endolytic bifunctional enzymes (Aly7A and Aly7B), a PL6 exolytic bifunctional enzyme (Aly6A) and a PL17 exolytic M‐specific enzyme (Aly17A). Ultra‐performance liquid chromatography–mass spectrometry was employed to reveal the synergistic effect of the four enzymes. The end products of Aly7A were further degraded by Aly7B and eventually generated oligosaccharides, from disaccharide to heptasaccharide. The oligosaccharide products were completely degraded to monosaccharides by Aly6A, but it was unable to directly degrade alginate. Aly17A could also produce monosaccharides by cleaving the M‐blocks of oligosaccharide products, as well as the M‐blocks of polysaccharides. The combination of these enzymes resulted in the complete degradation of alginate to monosaccharides. CONCLUSION: A new alginate PUL was mined and four novel alginate lyases in the PUL were expressed and characterized. The four cooperative alginate lyases provide novel tools for alginate degradation and biological fermentation. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Humans consume alginate in the form of seaweed, food hydrocolloids, and encapsulations, making the digestion of this mannuronic acid (M) and guluronic acid (G) polymer of key interest for human health. To increase knowledge on alginate degradation in the gut, a gene catalog from human feces was mined for potential alginate lyases (ALs). The predicted ALs were present in nine species of the Bacteroidetes phylum, of which two required supplementation of an endo-acting AL, expected to mimic cross-feeding in the gut. However, only a new isolate grew on alginate. Wholegenome sequencing of this alginate-utilizing isolate suggested that it is a new Bacteroides ovatus strain harboring a polysaccharide utilization locus (PUL) containing three ALs of families: PL6, PL17, and PL38. The BoPL6 degraded polyG to oligosaccharides of DP 1-3, and BoPL17 released 4,5-unsaturated monouronate from polyM. BoPL38 degraded both alginates, polyM, polyG, and polyMG, in endo-mode; hence, it was assumed to deliver oligosaccharide substrates for BoPL6 and BoPL17, corresponding well with synergistic action on alginate. BoPL17 and BoPL38 crystal structures, determined at 1.61 and 2.11 Å, respectively, showed (a/a)6-barrel + anti-parallel ß-sheet and (a/a)7-barrel folds, distinctive for these PL families. BoPL17 had a more open active site than the two homologous structures. BoPL38 was very similar to the structure of an uncharacterized PL38, albeit with a different triad of residues possibly interacting with substrate in the presumed active site tunnel. Altogether, the study provides unique functional and structural insights into alginate-degrading lyases of a PUL in a human gut bacterium. [ABSTRACT FROM AUTHOR]

Special geographical location and abundant organic matter profiles in tidal flats have resulted in great microbial diversity, in which Bacteroidota strains are considered as one of the primary degraders of polysaccharides, playing a crucial role in the carbon cycle. In this study, we collected sediment or sand samples from 34 bare tidal flats in China and investigated the profile of culturable bacteria, selected three Bacteroidota for polyphasic taxonomic analysis and revealed their polysaccharide metabolic potential. Totally, we isolated 352 pure cultured bacteria and they mainly distributed in Bacteroidota, Pseudomonadota, Bacillota, and Actinomycetota. It is shown that the bare tidal flats contained a large number of potential novel species, mainly distributed in Flavobacteriales and Cytophagales within Bacteroidota. Three Bacteroidota strains, M17T, M82T, and M415T, isolated from mudflat were selected for polyphasic taxonomic analysis. The 16S rRNA gene sequence similarity between strain M17T and Mangrovivirga cuniculi KCTC 72349T was 99.28%, and less than 90.09% with other species; strain M82T shared the highest 16S rRNA gene sequence similarity of 97.85% with Pontibacter litorisediminis KCTC 52252T, and less than 97.43% with other species; strain M415T had higher 16S rRNA gene sequence similarities with type species of genera Eudoraea (92.62-93.68%), Zeaxanthinibacter (92.02-92.91%), and Muriicola (92.21-92.83%). Phylogenetic analysis based on 16S rRNA gene sequences and single-copy orthologous clusters showed that strains M17T and M82T represent novel species within the genus Mangrovivirga and Pontibacter, respectively, and strain M415T represents a novel species of a novel genus within the family Flavobacteriaceae. The potential in polysaccharide metabolism of all these three strains was analyzed by genomes. The analysis revealed that glycoside hydrolases and glycosyltransferases account for more than 70% of the total CAZymes. Additionally, the numbers of polysaccharide utilization loci (PULs) and annotated CAZymes in Cytophagales spp. M17T and M82T were found to be higher than those in Flavobacteriales sp. M415T. Highly specialized saccharolytic systems and the presence of numerous diversified CAZymes for obtaining energy through polysaccharide metabolism were speculated to help the three novel strains adapt to the utilization of both terrestrial and marine polysaccharides.

Acute lower gastrointestinal GVHD (aLGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation. Although the intestinal microbiota is associated with the incidence of aLGI-GVHD, how the intestinal microbiota impacts treatment responses in aLGI-GVHD has not been thoroughly studied. In a cohort of patients with aLGI-GVHD (n = 37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and a disrupted fecal microbiome characterized by reduced abundances of Bacteroides ovatus. In a murine GVHD model aggravated by carbapenem antibiotics, introducing B. ovatus reduced GVHD severity and improved survival. These beneficial effects of Bacteroides ovatus were linked to its ability to metabolize dietary polysaccharides into monosaccharides, which suppressed the mucus-degrading capabilities of colonic mucus degraders such as Bacteroides thetaiotaomicron and Akkermansia muciniphila , thus reducing GVHD-related mortality. Collectively, these findings reveal the importance of microbiota in aLGI-GVHD and therapeutic potential of B. ovatus. [Display omitted] • Clinical steroid-refractory GVHD is associated with reduced Bacteroides ovatus • Introduction of B. ovatus after meropenem reduces experimental GVHD-related mortality • The beneficial effects of B. ovatus are linked to its ability to produce xylose • B. ovatus suppresses the mucus-degrading capabilities of colonic mucus degraders Hayase et al. discover that Bacteroides ovatus reduces the severity of graft-versus-host disease (GVHD), a complication of hematopoietic cell transplantation, by suppressing mucus-degrading gut microbes. These beneficial effects of B. ovatus are linked to the metabolism of dietary polysaccharides into monosaccharides and highlight the therapeutic potential of B. ovatus. [ABSTRACT FROM AUTHOR]

Marine bacteria usually contain polysaccharide utilization loci (PUL) for metabolizing red algae polysaccharides. They are of great significance in the carbon cycle of the marine ecosystem, as well as in supporting marine heterotrophic bacterial growth. Here, we described the whole k-carrageenan (KC), i-carrageenan (IC), and partial l-carrageenan (LC) catabolic pathways in a marine Gram-negative bacterium, Flavobacterium algicola, which is involved carrageenan polysaccharide hydrolases, oligosaccharide sulfatases, oligosaccharide glycosidases, and the 3,6-anhydro-D-galactose (D-AHG) utilizationrelated enzymes harbored in the carrageenan-specific PUL. In the pathways, the KC and IC were hydrolyzed into 4-sugar-unit oligomers by specific glycoside hydrolases. Then, the multifunctional G4S sulfatases would remove their nonreducing ends' G4S sulfate groups, while the i-neocarratetrose (Ni4) product would further lose the nonreducing end of its DA2S group. Furthermore, the neocarrageenan oligosaccharides (NCOSs) with no G4S and DA2S groups in their nonreducing ends would completely be decomposed into D-Gal and D-AHG. Finally, the released D-AHG would enter the cytoplasmic four-step enzymatic process, and an L-rhamnose-H1 transporter (RhaT) was preliminarily verified for the function for transportation of D-AHG. Moreover, comparative analysis with the reported carrageenan metabolism pathways further implied the diversity of microbial systems for utilizing the red algae carrageenan. [ABSTRACT FROM AUTHOR]

Two Gram-stain-negative, aerobic, non-motile, and rod-shaped bacterial strains, designated SM1352T and A20T, were isolated from intertidal sediments collected from King George Island, Antarctic. They shared 99.8% 16S rRNA gene sequence similarity with each other and had the highest sequence similarity of 98.1% to type strain of Aureibaculum marinum but < 93.4% sequence similarity to those of other known bacterial species. The genomes of strains SM1352T and A20T consisted of 5,108,092 bp and 4,772,071 bp, respectively, with the G + C contents both being 32.0%. They respectively encoded 4360 (including 37 tRNAs and 6 rRNAs) and 4032 (including 36 tRNAs and 5 rRNAs) genes. In the phylogenetic trees based on 16S rRNA gene and single-copy orthologous clusters (OCs), both strains clustered with Aureibaculum marinum and together formed a separate branch within the family Flavobacteriaceae. The ANI and DDH values between the two strains and Aureibaculum marinum BH-SD17T were all below the thresholds for species delineation. The major cellular fatty acids (> 10%) of the two strains included iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH. Their polar lipids predominantly included phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified aminolipid, and two unidentified lipids. Genomic comparison revealed that both strains possessed much more glycoside hydrolases and sulfatase-rich polysaccharide utilization loci (PULs) than Aureibaculum marinum BH-SD17T. Based on the above polyphasic evidences, strains SM1352T and A20T represent two novel species within the genus Aureibaculum, for which the names Aureibaculum luteum sp. nov. and Aureibaculum flavum sp. nov. are proposed. The type strains are SM1352T (= CCTCC AB 2014243 T = JCM 30335 T) and A20T (= CCTCC AB 2020370 T = KCTC 82503 T), respectively. [ABSTRACT FROM AUTHOR]

Flavobacterium suncheonense is a member of the family Flavobacteriaceae in the phylum Bacteroidetes. Strain GH29-5(T) (DSM 17707(T)) was isolated from greenhouse soil in Suncheon, South Korea. F. suncheonense GH29-5(T) is part of the G enomic E ncyclopedia of B acteria and A rchaea project. The 2,880,663 bp long draft genome consists of 54 scaffolds with 2739 protein-coding genes and 82 RNA genes. The genome of strain GH29-5(T) has 117 genes encoding peptidases but a small number of genes encoding carbohydrate active enzymes (51 CAZymes). Metallo and serine peptidases were found most frequently. Among CAZymes, eight glycoside hydrolase families, nine glycosyl transferase families, two carbohydrate binding module families and four carbohydrate esterase families were identified. Suprisingly, polysaccharides utilization loci (PULs) were not found in strain GH29-5(T). Based on the coherent physiological and genomic characteristics we suggest that F. suncheonense GH29-5(T) feeds rather on proteins than saccharides and lipids.

Xyloglucan (XyG) is a ubiquitous plant cell wall hemicellulose that is targeted by a range of syntenic, microheterogeneous xyloglucan utilization loci (XyGUL) in Bacteroidetes species of the human gut microbiota (HGM), including Bacteroides ovatus and B. uniformis. Comprehensive biochemical and biophysical analyses have identified key differences in the protein complements of each locus that confer differential access to structurally diverse XyG side chain variants. A second, nonsyntenic XyGUL was previously identified in B. uniformis, although its function in XyG utilization compared to its syntenic counterpart was unclear. Here, complementary enzymatic product profiles and bacterial growth curves showcase the notable preference of BuXyGUL2 surface glycan-binding proteins (SGBPs) to bind full-length XyG, as well as a range of oligosaccharides produced by the glycoside hydrolase family 5 (GH5_4) endo-xyloglucanase from this locus. We use isothermal titration calorimetry (ITC) to characterize this binding capacity and pinpoint the specific contributions of each protein to nutrient capture. The high-resolution structure of BuXyGUL2 SGBP-B reveals remarkable putative binding site conservation with the canonical XyG-binding BoXyGUL SGBP-B, supporting similar roles for these proteins in glycan capture. Together, these data underpin the central role of complementary XyGUL function in B. uniformis and broaden our systems-based and mechanistic understanding of XyG utilization in the HGM. [ABSTRACT FROM AUTHOR]

Background: Honey bee gut microbiota transmitted via social interactions are beneficial to the host health. Although the microbial community is relatively stable, individual variations and high strain-level diversity have been detected across honey bees. Although the bee gut microbiota structure is influenced by environmental factors, the heritability of the gut members and the contribution of the host genetics remains elusive. Considering bees within a colony are not readily genetically identical due to the polyandry of the queen, we hypothesize that the microbiota structure can be shaped by host genetics. Results: We used shotgun metagenomics to simultaneously profile the microbiota and host genotypes of bees from hives of four different subspecies. Gut composition is more distant between genetically different bees at both phylotype- and "sequence-discrete population" levels. We then performed a successive passaging experiment within colonies of hybrid bees generated by artificial insemination, which revealed that the microbial composition dramatically shifts across batches of bees during the social transmission. Specifically, different strains from the phylotype of Snodgrassella alvi are preferentially selected by genetically varied hosts, and strains from different hosts show a remarkably biased distribution of single-nucleotide polymorphism in the Type IV pili loci. Genome-wide association analysis identified that the relative abundance of a cluster of Bifidobacterium strains is associated with the host glutamate receptor gene specifically expressed in the bee brain. Finally, mono-colonization of Bifidobacterium with a specific polysaccharide utilization locus impacts the alternative splicing of the gluR-B gene, which is associated with an increased GABA level in the brain. Conclusions: Our results indicated that host genetics influence the bee gut composition and suggest a gut-brain connection implicated in the gut bacterial strain preference. Honey bees have been used extensively as a model organism for social behaviors, genetics, and the gut microbiome. Further identification of host genetic function as a shaping force of microbial structure will advance our understanding of the host-microbe interactions. 45gpjtUuBYRAxeW78Mk2MG Video abstract [ABSTRACT FROM AUTHOR]

ABSTRACT A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL’s specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBPMLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage β1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBPMLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.

Symbiotic bacteria, including members of the Bacteroides genus, are known to digest dietary fibers in the gastrointestinal tract. The metabolism of complex carbohydrates is restricted to a specified subset of species and is likely orchestrated by polysaccharide utilization loci (PULs) in these microorganisms. β-Mannans are plant cell wall polysaccharides that are commonly found in human nutrients. Here, we report the structural basis of a PUL cluster, BdPUL12, which controls β-mannan-like glycan catabolism in Bacteroides dorei. Detailed biochemical characterization and targeted gene disruption studies demonstrated that a key glycoside hydrolase, BdP12GH26, performs the initial attack on galactomannan or glucomannan likely via an endo -acting mode, generating mannooligosaccharides and mannose. Importantly, coculture assays showed that the B. dorei promoted the proliferation of Lactobacillus helveticus and Bifidobacterium adolescentis , likely by sharing mannooligosaccharides and mannose with these gut probiotics. Our findings provide new insights into carbohydrate metabolism in gut-inhabiting bacteria and lay a foundation for novel probiotic development. • The gene cluster, BdPUL12, exclusively contributes to β-mannan-like substrates utilization in Bacteroides dorei. • The mannanase BdP12GH26 involved in BdPUL12 hydrolyzes galactomannan or glucomannan likely via an endo -acting mode, generating mannooligosaccharides and mannose. • B. dorei is capable of promoting proliferation of gut probiotics. [ABSTRACT FROM AUTHOR]

SAR92 is one of the few examples of a widely distributed, abundant oligotroph that can be cultivated to study pathways of carbon oxidation in ocean systems. Genomic evidence for SAR92 suggests that this gammaproteobacterium might be a primary consumer of polysaccharides in the epipelagic zone, its main habitat. Here, we investigated cell growth, polysaccharide utilization gene expression, and carbohydrate-active enzyme abundance of a culturable SAR92 strain, HTCC2207, grown with different polysaccharides. Xylan and laminarin, two polysaccharides mainly produced by phytoplankton, supported the growth of HTCC2207 better than other polysaccharides. HTCC2207 possessed polysaccharide utilization loci (PULs) consisting of TonB-dependent receptor (TBDR) and glycoside hydrolase (GH) family genes. GH genes such as GH17 and GH3 presented no substrate-specificity and were induced by different sugar substrates, while expressions of GH16, GH10 and GH30 were enhanced in the glucose-treatment but suppressed in the polysaccharide-treatment, indicating complex polysaccharide utilization by HTCC2207. Metabolic pathways for laminarin and xylan were re-constructed in HTCC2207 based on the PULs genes and other predicted carbohydrate-active enzymes. This study reveals features of the epipelagic niche of SAR92 and provide insight into the biogeochemical cycling of labile, high-molecular carbohydrate compounds in the surface ocean. [ABSTRACT FROM AUTHOR]

Flavobacterium rivuli Ali et al. 2009 emend. Dong et al. 2013 is one of about 100 species in the genus Flavobacterium (family Flavobacteriacae, phylum Bacteroidetes) with a validly published name, and has been isolated from the spring of a hard water rivulet in Northern Germany. Including all type strains of the genus Myroides and Flavobacterium into the 16S rRNA gene sequence phylogeny revealed a clustering of members of the genus Myroides as a monophyletic group within the genus Flavobacterium. Furthermore, F. rivuli WB 3.3-2(T) and its next relatives seem more closely related to the genus Myroides than to the type species of the genus Flavobacterium, F. aquatile. The 4,489,248 bp long genome with its 3,391 protein-coding and 65 RNA genes is part of the G enomic E ncyclopedia of B acteria and A rchaea project. The genome of F. rivuli has almost as many genes encoding carbohydrate active enzymes (151 CAZymes) as genes encoding peptidases (177). Peptidases comprised mostly metallo (M) and serine (S) peptidases. Among CAZymes, 30 glycoside hydrolase families, 10 glycosyl transferase families, 7 carbohydrate binding module families and 7 carbohydrate esterase families were identified. Furthermore, we found four polysaccharide utilization loci (PUL) and one large CAZy rich gene cluster that might enable strain WB 3.3-2(T) to decompose plant and algae derived polysaccharides. Based on these results we propose F. rivuli as an interesting candidate for further physiological studies and the role of Bacteroidetes in the decomposition of complex polymers in the environment.

Glycosaminoglycans (GAGs), as a class of biopolymers, play pivotal roles in various biological metabolisms such as cell signaling, tissue development, cell apoptosis, immune modulation, and growth factor activity. They are mainly present in the colon in free forms, which are essential for maintaining the host's health by regulating the colonization and proliferation of gut microbiota. Therefore, it is important to explain the specific members of the gut microbiota for GAGs' degradation and their enzymatic machinery in vivo. This review provides an outline of GAGs-utilizing entities in the Bacteroides , highlighting their polysaccharide utilization loci (PULs) and the enzymatic machinery involved in chondroitin sulfate (CS) and heparin (Hep)/heparan sulfate (HS). While there are some variations in GAGs' degradation among different genera, we analyze the reputed GAGs' utilization clusters in lactic acid bacteria (LAB), based on recent studies on GAGs' degradation. The enzymatic machinery involved in Hep/HS and CS metabolism within LAB is also discussed. Thus, to elucidate the precise mechanisms utilizing GAGs by diverse gut microbiota will augment our understanding of their effects on human health and contribute to potential therapeutic strategies for diseases. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA) is a negatively charged and linear polysaccharide existing in the tissues and body fluids of all vertebrates. Some pathogenic bacteria target hyaluronic acid for adhesion and/or infection to host cells. Vibrio alginolyticus is an opportunistic pathogen related to infections of humans and marine animals, and the hyaluronic acid-degrading potential of Vibrio spp. has been well-demonstrated. However, little is known about how Vibrio spp. utilize hyaluronic acid. In this study, a marine bacterium V. alginolyticus LWW-9 capable of degrading hyaluronic acid has been isolated. Genetic and bioinformatic analysis showed that V. alginolyticus LWW-9 harbors a gene cluster involved in the degradation, transport, and metabolism of hyaluronic acid. Two novel PL8 family hyaluronate lyases, VaHly8A and VaHly8B, are the key enzymes for the degradation of hyaluronic acid. VaHly8A and VaHly8B have distinct biochemical properties, reflecting the adaptation of the strain to the changing parameters of the aquatic habitats and hosts. Based on genomic and functional analysis, we propose a model for the complete degradation of hyaluronic acid by V. alginolyticus LWW-9. Overall, our study expands our knowledge of the HA utilization paradigm within the Proteobacteria, and the two novel hyaluronate lyases are excellent candidates for industrial applications.

Marine microorganisms encode a complex repertoire of carbohydrate-active enzymes (CAZymes) for the catabolism of algal cell wall polysaccharides. While the core enzyme cascade for degrading agar is conserved across agarolytic marine bacteria, gain of novel metabolic functions can lead to the evolutionary expansion of the gene repertoire. Here, we describe how two less-abundant GH96 a-agarases harbored in the agar-specific polysaccharide utilization locus (PUL) of Colwellia echini strain A3T facilitate the versatility of the agarolytic pathway. The cellular and molecular functions of the a-agarases examined by genomic, transcriptomic, and biochemical analyses revealed that a-agarases of C. echini A3T create a novel auxiliary pathway. a-Agarases convert even-numbered neoagarooligosaccharides to odd-numbered agaro- and neoagarooligosaccharides, providing an alternative route for the depolymerization process in the agarolytic pathway. Comparative genomic analysis of agarolytic bacteria implied that the agarolytic gene repertoire in marine bacteria has been diversified during evolution, while the essential core agarolytic gene set has been conserved. The expansion of the agarolytic gene repertoire and novel hydrolytic functions, including the elucidated molecular functionality of a-agarase, promote metabolic versatility by channeling agar metabolism through different routes. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA) is a negatively charged and linear polysaccharide existing in the tissues and body fluids of all vertebrates. Some pathogenic bacteria target hyaluronic acid for adhesion and/or infection to host cells. Vibrio alginolyticus is an opportunistic pathogen related to infections of humans and marine animals, and the hyaluronic acid-degrading potential of Vibrio spp. has been well-demonstrated. However, little is known about how Vibrio spp. utilize hyaluronic acid. In this study, a marine bacterium V. alginolyticus LWW-9 capable of degrading hyaluronic acid has been isolated. Genetic and bioinformatic analysis showed that V. alginolyticus LWW-9 harbors a gene cluster involved in the degradation, transport, and metabolism of hyaluronic acid. Two novel PL8 family hyaluronate lyases, VaHly8A and VaHly8B, are the key enzymes for the degradation of hyaluronic acid. VaHly8A and VaHly8B have distinct biochemical properties, reflecting the adaptation of the strain to the changing parameters of the aquatic habitats and hosts. Based on genomic and functional analysis, we propose a model for the complete degradation of hyaluronic acid by V. alginolyticus LWW-9. Overall, our study expands our knowledge of the HA utilization paradigm within the Proteobacteria , and the two novel hyaluronate lyases are excellent candidates for industrial applications. [ABSTRACT FROM AUTHOR]

Bacteroides species are successful colonizers of the human gut and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in Polysaccharide Utilization Loci (PULs). While recent work has uncovered the PULs required for use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-β-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron . Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron . Examining the genomes of other Bacteroides species, we found homologs of BT1871 in subset and show that representative strains of species containing a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide.

Abstract Background Acetylated 4-O-(methyl)glucuronoxylan (GX) is the main hemicellulose in deciduous hardwood, and comprises a β-(1→4)-linked xylopyranosyl (Xylp) backbone substituted by both acetyl groups and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Whereas enzymes that target singly acetylated Xylp or doubly 2,3-O-acetyl-Xylp have been well characterized, those targeting (2-O-MeGlcpA)3-O-acetyl-Xylp structures in glucuronoxylan have remained elusive. Results An unclassified carbohydrate esterase (FjoAcXE) was identified as a protein of unknown function from a polysaccharide utilization locus (PUL) otherwise comprising carbohydrate-active enzyme families known to target xylan. FjoAcXE was shown to efficiently release acetyl groups from internal (2-O-MeGlcpA)3-O-acetyl-Xylp structures, an activity that has been sought after but lacking in known carbohydrate esterases. FjoAcXE action boosted the activity of α-glucuronidases from families GH67 and GH115 by five and nine times, respectively. Moreover, FjoAcXE activity was not only restricted to GX, but also deacetylated (3-O-Araf)2-O-acetyl-Xylp of feruloylated xylooligomers, confirming the broad substrate range of this new carbohydrate esterase. Conclusion This study reports the discovery and characterization of the novel carbohydrate esterase, FjoAcXE. In addition to cleaving singly acetylated Xylp, and doubly 2,3-O-acetyl-Xylp, FjoAcXE efficiently cleaves internal 3-O-acetyl-Xylp linkages in (2-O-MeGlcpA)3-O-acetyl-Xylp residues along with densely substituted and branched xylooligomers; activities that until now were missing from the arsenal of enzymes required for xylan conversion.

Lignocellulose is one of the most abundant renewable carbon sources, representing an alternative to petroleum for the production of fuel and chemicals. Nonetheless, the lignocellulose saccharification process, to release sugars for downstream applications, is one of the most crucial factors economically challenging to its use. The synergism required among the various carbohydrate-active enzymes (CAZymes) for efficient lignocellulose breakdown is often not satisfactorily achieved with an enzyme mixture from a single strain. To overcome this challenge, enrichment strategies can be applied to develop microbial communities with an efficient CAZyme arsenal, incorporating complementary and synergistic properties, to improve lignocellulose deconstruction. We report a comprehensive and deep analysis of an enriched rumen anaerobic consortium (ERAC) established on sugarcane bagasse (SB). The lignocellulolytic abilities of the ERAC were confirmed by analyzing the depolymerization of bagasse by scanning electron microscopy, enzymatic assays, and mass spectrometry. Taxonomic analysis based on 16S rRNA sequencing elucidated the community enrichment process, which was marked by a higher abundance of Firmicutes and Synergistetes species. Shotgun metagenomic sequencing of the ERAC disclosed 41 metagenome-assembled genomes (MAGs) harboring cellulosomes and polysaccharide utilization loci (PULs), along with a high diversity of CAZymes. The amino acid sequences of the majority of the predicted CAZymes (60% of the total) shared less than 90% identity with the sequences found in public databases. Additionally, a clostridial MAG identified in this study produced proteins during consortium development with scaffoldin domains and CAZymes appended to dockerin modules, thus representing a novel cellulosome-producing microorganism. [ABSTRACT FROM AUTHOR]

The rumen of ruminants is a natural anaerobic fermentation system that efficiently degrades lignocellulosic biomass and mainly depends on synergistic interactions between multiple microbes and their secreted enzymes. Ruminal microbes have been employed as biomass waste converters and are receiving increasing attention because of their degradation performance. To explore the application of ruminal microbes and their secreted enzymes in biomass waste, a comprehensive understanding of these processes is required. Based on the degradation capacity and mechanism of ruminal microbes and their secreted lignocellulose enzymes, this review concentrates on elucidating the main enzymatic strategies that ruminal microbes use for lignocellulose degradation, focusing mainly on polysaccharide metabolism-related gene loci and cellulosomes. Hydrolysis, acidification, methanogenesis, interspecific H 2 transfer, and urea cycling in ruminal metabolism are also discussed. Finally, we review the research progress on the conversion of biomass waste into biofuels (bioethanol, biohydrogen, and biomethane) and value-added chemicals (organic acids) by ruminal microbes. This review aims to provide new ideas and methods for ruminal microbe and enzyme applications, biomass waste conversion, and global energy shortage alleviation. • Rumen microbes have genetic potential in degradation and exhibit diversity in enzymes. • Cellulosome and polysaccharide utilization loci explain enzyme degradation mechanism. • Rumen microbes together contribute to metabolism exchange of ecosystem functions. • Synergy of rumen microbes and enzymes effectively converts biomass to bioenergy. • A future biorefinery based on rumen microbes and digestive strategies is proposed. [ABSTRACT FROM AUTHOR]

Studying the physiology and genomics of cultured hydrolytic bacteria is a valuable approach to decipher the biogeochemical cycling of marine polysaccharides, major nutrients derived from phytoplankton and macroalgae. We herein describe the profound potential of Alteromonas sp. 76-1, isolated from alginate-enriched seawater at the Patagonian continental shelf, to degrade the algal polysaccharides alginate and ulvan. Phylogenetic analyses indicated that strain 76-1 might represent a novel species, distinguished from its closest relative (Alteromonas naphthalenivorans) by adaptations to their contrasting habitats (productive open ocean vs. coastal sediments). Ecological distinction of 76-1 was particularly manifested in the abundance of carbohydrate-active enzymes (CAZymes), consistent with its isolation from alginate-enriched seawater and elevated abundance of a related OTU in the original microcosm. Strain 76-1 encodes multiple alginate lyases from families PL6, PL7, PL17, and PL18 largely contained in two polysaccharide utilization loci (PUL), which may facilitate the utilization of different alginate structures in nature. Notably, ulvan degradation relates to a 126 Kb plasmid dedicated to polysaccharide utilization, encoding several PL24 and PL25 ulvan lyases and monomer-processing genes. This extensive and versatile CAZyme repertoire allowed substantial growth on polysaccharides, showing comparable doubling times with alginate (2 h) and ulvan (3 h) in relation to glucose (3 h). The finding of homologous ulvanolytic systems in distantly related Alteromonas spp. suggests CAZyme plasmids as effective vehicles for PUL transfer that mediate niche gain. Overall, the demonstrated CAZyme repertoire substantiates the role of Alteromonas in marine polysaccharide degradation and how PUL exchange influences the ecophysiology of this ubiquitous marine taxon.

The secretion of extracellular enzymes by soil microbes is rate-limiting in the recycling of biomass. Fungi and bacteria compete and collaborate for nutrients in the soil, with wide ranging ecological impacts. Within soil microbiota, the Bacteroidetes tend to be a dominant phylum, just like in human and animal intestines. The Bacteroidetes thrive because of their ability to secrete diverse arrays of carbohydrate-active enzymes (CAZymes) that target the highly varied glycans in the soil. Bacteroidetes use an energy-saving system of genomic organization, whereby most of their CAZymes are grouped into Polysaccharide Utilization Loci (PULs). These loci enable high level production of specific CAZymes only when their substrate glycans are abundant in the local environment. This gives the Bacteroidetes a clear advantage over other species in the competitive soil environment, further enhanced by the phylum-specific Type IX Secretion System (T9SS). The T9SS is highly effective at secreting CAZymes and/or tethering them to the cell surface, and is tightly coupled to the ability to rapidly glide over solid surfaces, a connection that promotes an active hunt for nutrition. Although the soil Bacteroidetes are less well studied than human gut symbionts, research is uncovering important biochemical and physiological phenomena. In this review, we summarize the state of the art on research into the CAZymes secreted by soil Bacteroidetes in the contexts of microbial soil ecology and the discovery of novel CAZymes for use in industrial biotechnology. We hope that this review will stimulate further investigations into the somewhat neglected enzymology of non-gut Bacteroidetes. [ABSTRACT FROM AUTHOR]

Studying the physiology and genomics of cultured hydrolytic bacteria is a valuable approach to decipher the biogeochemical cycling of marine polysaccharides, major nutrients derived from phytoplankton and macroalgae. We herein describe the profound potential of Alteromonas sp. 76-1, isolated from alginate-enriched seawater at the Patagonian continental shelf, to degrade the algal polysaccharides alginate and ulvan. Phylogenetic analyses indicated that strain 76-1 might represent a novel species, distinguished from its closest relative (Alteromonas naphthalenivorans) by adaptations to their contrasting habitats (productive open ocean vs. coastal sediments). Ecological distinction of 76-1 was particularly manifested in the abundance of carbohydrate-active enzymes (CAZymes), consistent with its isolation from alginate-enriched seawater and elevated abundance of a related OTU in the original microcosm. Strain 76-1 encodes multiple alginate lyases from families PL6, PL7, PL17, and PL18 largely contained in two polysaccharide utilization loci (PUL), which may facilitate the utilization of different alginate structures in nature. Notably, ulvan degradation relates to a 126 Kb plasmid dedicated to polysaccharide utilization, encoding several PL24 and PL25 ulvan lyases and monomer-processing genes. This extensive and versatile CAZyme repertoire allowed substantial growth on polysaccharides, showing comparable doubling times with alginate (2 h) and ulvan (3 h) in relation to glucose (3 h). The finding of homologous ulvanolytic systems in distantly related Alteromonas spp. suggests CAZyme plasmids as effective vehicles for PUL transfer that mediate niche gain. Overall, the demonstrated CAZyme repertoire substantiates the role of Alteromonas in marine polysaccharide degradation and how PUL exchange influences the ecophysiology of this ubiquitous marine taxon. [ABSTRACT FROM AUTHOR]

Members of the genera Proteiniphilum and Petrimonas were speculated to represent indicators reflecting process instability within anaerobic digestion (AD) microbiomes. Therefore, Petrimonas mucosa ING2-E5AT was isolated from a biogas reactor sample and sequenced on the PacBio RSII and Illumina MiSeq sequencers. Phylogenetic classification positioned the strain ING2-E5AT in close proximity to Fermentimonas and Proteiniphilum species (family Dysgonomonadaceae). ING2-E5AT encodes a number of genes for glycosyl-hydrolyses (GH) which are organized in Polysaccharide Utilization Loci (PUL) comprising tandem susCD-like genes for a TonB-dependent outer-membrane transporter and a cell surface glycan-binding protein. Different GHs encoded in PUL are involved in pectin degradation, reflecting a pronounced specialization of the ING2-E5AT PUL systems regarding the decomposition of this polysaccharide. Genes encoding enzymes participating in amino acids fermentation were also identified. Fragment recruitments with the ING2-E5AT genome as a template and publicly available metagenomes of AD microbiomes revealed that Petrimonas species are present in 146 out of 257 datasets supporting their importance in AD microbiomes. Metatranscriptome analyses of AD microbiomes uncovered active sugar and amino acid fermentation pathways for Petrimonas species. Likewise, screening of metaproteome datasets demonstrated expression of the Petrimonas PUL-specific component SusC providing further evidence that PUL play a central role for the lifestyle of Petrimonas species.

Proteiniphilum saccharofermentans str. M3/6T is a recently described species within the family Porphyromonadaceae (phylum Bacteroidetes), which was isolated from a mesophilic laboratory-scale biogas reactor. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding biomass degradation and fermentation pathways. The P. saccharofermentans str. M3/6T genome consists of a 4,414,963 bp chromosome featuring an average GC-content of 43.63%. Genome analyses revealed that the strain possesses 3396 protein-coding sequences. Among them are 158 genes assigned to the carbohydrate-active-enzyme families as defined by the CAZy database, including 116 genes encoding glycosyl hydrolases (GHs) involved in pectin, arabinogalactan, hemicellulose (arabinan, xylan, mannan, β-glucans), starch, fructan and chitin degradation. The strain also features several transporter genes, some of which are located in polysaccharide utilization loci (PUL). PUL gene products are involved in glycan binding, transport and utilization at the cell surface. In the genome of strain M3/6T, 64 PUL are present and most of them in association with genes encoding carbohydrate-active enzymes. Accordingly, the strain was predicted to metabolize several sugars yielding carbon dioxide, hydrogen, acetate, formate, propionate and isovalerate as end-products of the fermentation process. Moreover, P. saccharofermentans str. M3/6T encodes extracellular and intracellular proteases and transporters predicted to be involved in protein and oligopeptide degradation. Comparative analyses between P. saccharofermentans str. M3/6T and its closest described relative P. acetatigenes str. DSM 18083T indicate that both strains share a similar metabolism regarding decomposition of complex carbohydrates and fermentation of sugars.

Importance: Pathogenic Xanthomonas bacteria can affect a variety of economically relevant crops causing losses in productivity, limiting commercialization and requiring phytosanitary measures. These plant pathogens exhibit high level of host and tissue specificity through multiple molecular strategies including several secretion systems, effector proteins, and a broad repertoire of carbohydrate-active enzymes (CAZymes). Many of these CAZymes act on the plant cell wall and storage carbohydrates, such as cellulose and starch, releasing products used as nutrients and modulators of transcriptional responses to support host colonization by mechanisms yet poorly understood. Here, we reveal that structural and storage β-glucans from the plant cell function as spatial markers, providing distinct chemical stimuli that modulate the transition between higher and lower motility states in Xanthomonas citri , a key virulence trait for many bacterial pathogens., Competing Interests: The authors declare no conflict of interest.

Prevotella copri is an abundant member of the human gastrointestinal microbiome, whose relative abundance has curiously been associated with positive and negative impacts on diseases, such as Parkinson's disease and rheumatoid arthritis. Yet, the verdict is still out on the definitive role of P. copri in human health, and on the effect of different diets on its relative abundance in the gut microbiome. The puzzling discrepancies among P. copri studies have only recently been attributed to the diversity of its strains, which substantially differ in their encoded metabolic patterns from the commonly used reference strain. However, such strain differences cannot be resolved by common 16S rRNA amplicon profiling methods. Here, we scrutinize P. copri , its versatile metabolic potential, and the hypotheses behind the conflicting observations on its association with diet and human health. We also provide suggestions for designing studies and bioinformatics pipelines to better research P. copri .

ABSTRACT When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron, we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs) were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome. IMPORTANCE The microorganisms that reside in the human colon fulfill their energy requirements mainly from diet- and host-derived complex carbohydrates. Members of this ecosystem possess poorly understood strategies to prioritize and compete for these nutrients. Based on direct carbohydrate measurements and corresponding transcriptional analyses, our findings showed that individual bacterial species exhibit different preferences for the same set of glycans and that this prioritization is maintained in a competitive environment, which may promote stable coexistence. Such understanding of gut bacterial glycan utilization will be essential to eliciting predictable changes in the gut microbiota to improve health through the diet.

Members of phylum Bacteroidetes are distributed across diverse marine niches and Flavobacteria is often the predominant bacterial class decomposing algae-derived polysaccharides. Here, we report the complete genome of Gramella flava JLT2011 (Flavobacteria) isolated from surface water of the southeastern Pacific. A remarkable genomic feature is that the number of glycoside hydrolase (GH) genes in the genome of G. flava JLT2011 is more than 2-fold higher than that of other Gramella species. The functional profiles of the GHs suggest extensive variation in Gramella species. Growth experiments revealed that G. flava JLT2011 has the ability to utilize a wide range of polysaccharides for growth such as xylan and homogalacturonan in pectin. Nearly half of all GH genes were located on the multi-gene polysaccharide utilization loci (PUL) or PUL-like systems in G. flava JLT2011. This species was also found to harbor the two xylan PULs and a pectin PUL, respectively. Gene expression data indicated that more GHs and sugar-specific outer-membrane susC-susD systems were found in the presence of xylan than in the presence of pectin, suggesting a different strategy for heteropolymeric xylan and homoglacturonan utilization. Multi-omics data (transcriptomics, proteomics, and metabolomics) indicated that xylan PULs and pectin PUL are respectively involved in the catabolism of their corresponding polysaccharides. This work presents a comparison of polysaccharide decomposition within a genus and expands current knowledge on the diversity and function of PULs in marine Bacteroidetes, thereby deepening our understanding of their ecological role in polysaccharide remineralization in the marine system. [ABSTRACT FROM AUTHOR]

The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta)genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a β-glucosidase, and two α-l-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.

Flavobacterium suncheonense is a member of the family Flavobacteriaceae in the phylum Bacteroidetes. Strain GH29-5T (DSM 17707T) was isolated from greenhouse soil in Suncheon, South Korea. F. suncheonense GH29-5T is part of the Genomic Encyclopedia of Bacteria and Archaea project. The 2,880,663 bp long draft genome consists of 54 scaffolds with 2739 protein-coding genes and 82 RNA genes. The genome of strain GH29-5T has 117 genes encoding peptidases but a small number of genes encoding carbohydrate active enzymes (51 CAZymes). Metallo and serine peptidases were found most frequently. Among CAZymes, eight glycoside hydrolase families, nine glycosyl transferase families, two carbohydrate binding module families and four carbohydrate esterase families were identified. Suprisingly, polysaccharides utilization loci (PULs) were not found in strain GH29-5T. Based on the coherent physiological and genomic characteristics we suggest that F. suncheonense GH29-5T feeds rather on proteins than saccharides and lipids. [ABSTRACT FROM AUTHOR]

Flavobacterium suncheonense is a member of the family Flavobacteriaceae in the phylum Bacteroidetes. Strain GH29-5T (DSM 17707T) was isolated from greenhouse soil in Suncheon, South Korea. F. suncheonense GH29-5T is part of the Genomic Encyclopedia of Bacteria and Archaea project. The 2,880,663 bp long draft genome consists of 54 scaffolds with 2739 protein-coding genes and 82 RNA genes. The genome of strain GH29-5T has 117 genes encoding peptidases but a small number of genes encoding carbohydrate active enzymes (51 CAZymes). Metallo and serine peptidases were found most frequently. Among CAZymes, eight glycoside hydrolase families, nine glycosyl transferase families, two carbohydrate binding module families and four carbohydrate esterase families were identified. Suprisingly, polysaccharides utilization loci (PULs) were not found in strain GH29-5T. Based on the coherent physiological and genomic characteristics we suggest that F. suncheonense GH29-5T feeds rather on proteins than saccharides and lipids. [ABSTRACT FROM AUTHOR]

Complex carbohydrates are ubiquitous in all kingdoms of life. As major components of the plant cell wall they constitute both a rich renewable carbon source for biotechnological transformation into fuels, chemicals and materials, and also form an important energy source as part of a healthy human diet. In both contexts, there has been significant, sustained interest in understanding how microbes transform these substrates. Classical perspectives of microbial polysaccharide degradation are currently being augmented by recent advances in the discovery of lytic polysaccharide monooxygenases (LPMOs) and polysaccharide utilization loci (PULs). Fundamental discoveries in carbohydrate enzymology are both advancing biological understanding, as well as informing applications in industrial biomass conversion and modulation of the human gut microbiota to mediate health benefits. [ABSTRACT FROM AUTHOR]

Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron , via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality., Competing Interests: Declaration of interests R.R.J. has served as a consultant or advisory board member for Merck, Microbiome DX, Karius, MaaT Pharma, LISCure, Seres, Kaleido, and Prolacta and has received patent license fee or stock options from Seres and Kaleido. E.J.S. has served as a consultant or advisory board member for Adaptimmune, Axio, Navan, Fibroblasts and FibroBiologics, NY Blood Center, and Celaid Therapeutics and has received patent license fee from Takeda and Affimed. E.H., M.A.J., J.L.K., and R.R.J. are inventors on a patent application by The University of Texas MD Anderson Cancer Center supported by results of the current study entitled, “Methods and Compositions for Treating Cancer therapy-induced Neutropenic Fever and/or GVHD.”

Background Efficient industrial processes for converting plant lignocellulosic materials into biofuels are a key to global efforts to come up with alternative energy sources to fossil fuels. Novel cellulolytic enzymes have been discovered in microbial genomes and metagenomes of microbial communities. However, the identification of relevant genes without known homologs, and the elucidation of the lignocellulolytic pathways and protein complexes for different microorganisms remain challenging. Results We describe a new computational method for the targeted discovery of functional modules of plant biomass-degrading protein families, based on their co-occurrence patterns across genomes and metagenome datasets, and the strength of association of these modules with the genomes of known degraders. From approximately 6.4 million family annotations for 2,884 microbial genomes, and 332 taxonomic bins from 18 metagenomes, we identified 5 functional modules that are distinctive for plant biomass degraders, which we term «plant biomass degradation modules» (PDMs). These modules incorporate protein families involved in the degradation of cellulose, hemicelluloses, and pectins, structural components of the cellulosome, and additional families with potential functions in plant biomass degradation. The PDMs were linked to 81 gene clusters in genomes of known lignocellulose degraders, including previously described clusters of lignocellulolytic genes. On average, 70% of the families of each PDM were found to map to gene clusters in known degraders, which served as an additional confirmation of their functional relationships. The presence of a PDM in a genome or taxonomic metagenome bin furthermore allowed us to accurately predict the ability of any particular organism to degrade plant biomass. For 15 draft genomes of a cow rumen metagenome, we used cross-referencing to confirmed cellulolytic enzymes to validate that the PDMs identified plant biomass degraders within a complex microbial community. Conclusions Functional modules of protein families that are involved in different aspects of plant cell wall degradation can be inferred from co-occurrence patterns across (meta-)genomes with a probabilistic topic model. PDMs represent a new resource of protein families and candidate genes implicated in microbial plant biomass degradation. They can also be used to predict the plant biomass degradation ability for a genome or taxonomic bin. The method is also suitable for characterizing other microbial phenotypes. [ABSTRACT FROM AUTHOR]

Metagenomic and bioinformatic approaches were used to characterize' plant biomass conversion within the foregut microbiome of Australia's "model" marsupial, the Tammar wallaby (Macropus eugenhi). Like the termite hindgut and bovine rumen, key enzymes and modular structures characteristic of the "free enzyme" and "cellulosome" paradigms of cellulose solubilization remain either poorly represented or elusive to capture by shotgun sequencing methods. Instead, multigene polysaccharide utilization loci-like systems coupled with genes encoding 4-1,4-endoglucanases and -1,4- endoxylanases-which have not been previously encountered in metagenomic datasets-were identified, as were a diverse set of glycoside hydrolases targeting noncellulosic polysacchar- ides. Furthermore, both rrs gene and other phylogenetic analyses confirmed that unique clades of the Lachnospiraceae, Bacteroi- dales, and Gammaproteobacteria are predominant in the Tammar foregut microbiome. Nucleotide composition-based sequence binding facilitated the assemblage of more than two megabase pairs of genomic sequence for one of the novel Lachnospiraceae clades (WG-2). These analyses show that WG-2 possesses numerous glycoside hydrolases targeting noncellulosic polysaccharides. These collective data demonstrate that Australian macropods not only harbor unique bacterial lineages underpinning plant biomass con- version, but their repertoire of glycoside hydrolases is distinct from those of the microbiomes of higher termites and the bovine rumen. [ABSTRACT FROM AUTHOR]

Polysaccharide utilization loci (PULs) are co-regulated bacterial genes that sense nutrients and enable glycan digestion. Human gut microbiome members, notably Bacteroides , contain numerous PULs that enable glycan utilization and shape ecological dynamics. To investigate the role of PULs on fitness and inter-species interactions, we develop a CRISPR-based genome editing tool to study 23 PULs in Bacteroides uniformis (BU). BU PULs show distinct glycan-degrading functions and transcriptional coordination that enables the population to adapt upon loss of other PULs. Exploiting a BU mutant barcoding strategy, we demonstrate that in vitro fitness and BU colonization in the murine gut are enhanced by deletion of specific PULs and modulated by glycan availability. PULs mediate glycan-dependent interactions with butyrate producers that depend on the degradation mechanism and glycan utilization ability of the butyrate producer. Thus, PULs determine community dynamics and butyrate production and provide a selective advantage or disadvantage depending on the nutritional landscape. [Display omitted] • CRISPR genome-editing tool examines the impact of 23 PULs in B. uniformis • PULs in B. uniformis display transcriptional coordination • B. uniformis PULs provide a nutrient-dependent fitness advantage or disadvantage • B. uniformis PULs determine community dynamics and butyrate production Polysaccharide utilization loci (PULs) in the human gut microbiome determine microbial fitness and community-level functions. Feng et al. show that PULs in Bacteroides uniformis provide a fitness advantage or disadvantage that depends on nutrient availability. B. uniformis PULs display transcriptional coordination and play key roles in shaping butyrate production. [ABSTRACT FROM AUTHOR]