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contract DL-57/2016 WT108430/Z/15/Z WT218304/Z/19/Z 22-FY18-821 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. publishersversion published

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.

The lignocellulolytic ERAC displays a unique set of plant polysaccharide-degrading enzymes (with multimodular characteristics), cellulosomal complexes, and PULs. The MAGs described here represent an expansion of the genetic content of rumen bacterial genomes dedicated to plant polysaccharide degradation, therefore providing a valuable resource for the development of biocatalytic toolbox strategies to be applied to lignocellulose-based biorefineries., 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. IMPORTANCE The lignocellulolytic ERAC displays a unique set of plant polysaccharide-degrading enzymes (with multimodular characteristics), cellulosomal complexes, and PULs. The MAGs described here represent an expansion of the genetic content of rumen bacterial genomes dedicated to plant polysaccharide degradation, therefore providing a valuable resource for the development of biocatalytic toolbox strategies to be applied to lignocellulose-based biorefineries.

The genome and natural habitat of Chitinophaga pinensis suggest it has the ability to degrade a wide variety of carbohydrate-based biomass. Complementing our earlier investigations into the hydrolysis of some plant polysaccharides, we now show that C. pinensis can grow directly on spruce wood and on the fungal fruiting body. Growth was stronger on fungal material, although secreted enzyme activity was high in both cases, and all biomass-induced secretomes showed a predominance of β-glucanase activities. We therefore conducted a screen for growth on and hydrolysis of β-glucans isolated from different sources. Most noncrystalline β-glucans supported good growth, with variable efficiencies of polysaccharide deconstruction and oligosaccharide uptake, depending on the polysaccharide backbone linkage. In all cases, β-glucan was the only type of polysaccharide that was effectively hydrolyzed by secreted enzymes. This contrasts with the secretion of enzymes with a broad range of activities observed during growth on complex heteroglycans. Our findings imply a role for C. pinensis in the turnover of multiple types of biomass and suggest that the species may have two metabolic modes: a "scavenging mode," where multiple different types of glycan may be degraded, and a more "focused mode" of β-glucan metabolism. The significant accumulation of some types of β-gluco-oligosaccharides in growth media may be due to the lack of an appropriate transport mechanism, and we propose that this is due to the specificity of expressed polysaccharide utilization loci. We present a hypothetical model for β-glucan metabolism by C. pinensis that suggests the potential for nutrient sharing among the microbial litter community.IMPORTANCE It is well known that the forest litter layer is inhabited by a complex microbial community of bacteria and fungi. However, while the importance of fungi in the turnover of natural biomass is well established, the role of their bacterial counterparts is less extensively studied. We show that Chitinophaga pinensis, a prominent member of an important bacterial genus, is capable of using both plant and fungal biomass as a nutrient source but is particularly effective at deconstructing dead fungal material. The turnover of dead fungus is key in natural elemental cycles in the forest. We show that C. pinensis can perform extensive degradation of this material to support its own growth while also releasing sugars that may serve as nutrients for other microbial species. Our work adds detail to an increasingly complex picture of life among the environmental microbiota.

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. Electronic supplementary material The online version of this article (10.1186/s13068-018-1074-3) contains supplementary material, which is available to authorized users.

International audience; Glycoside phosphorylases (EC 2.4.x.x) carry out the reversible phosphorolysis of glucan polymers, producing the corresponding sugar 1-phosphate and a shortened glycan chain. β-1,3-Glucan phosphorylase activities have been reported in the photosynthetic euglenozoan Euglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology of E. gracilis, we identified a candidate phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 in Escherichia coli and characterized it in vitro as a β-1,3-glucan phosphorylase. BLASTP identified several hundred EgP1 orthologs, most of which were from Gram-negative bacteria and had 37-91% sequence identity to EgP1. We heterologously expressed a bacterial metagenomic sequence, Pro_7066 in E. coli and confirmed it as a β-1,3-glucan phosphorylase, albeit with kinetics parameters distinct from those of EgP1. EgP1, Pro_7066, and their orthologs are classified as a new glycoside hydrolase (GH) family, designated GH149. Comparisons between GH94, EgP1, and Pro_7066 sequences revealed conservation of key amino acids required for the phosphorylase activity, suggesting a phosphorylase mechanism that is conserved between GH94 and GH149. We found bacterial GH149 genes in gene clusters containing sugar transporter and several other GH family genes, suggesting that bacterial GH149 proteins have roles in the degradation of complex carbohydrates. The Bacteroidetes GH149 genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial β-1,3-glucan phosphorylase and uncovered a new family of phosphorylases that we name GH149.

Highlights • P. saccharofermentans str. M3/6T harbors several carbohydrate active enzymes. • The strain encodes 64 polysaccharide utilization loci (PULs). • Several PULs are associated with genes encoding carbohydrate active enzymes. • P. saccharofermentans M3/6T harbors two CRISPR-cas systems., 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.

International audience; Polysaccharide degradation by heterotrophic microbes is a key process within Earth’s carbon cycle. Here, we use environmental proteomics and metagenomics in combination with cultivation experiments and biochemical characterizations to investigate the molecular details of in situ polysaccharide degradation mechanisms during microalgal blooms. For this, we use laminarin as a model polysaccharide. Laminarin is a ubiquitous marine storage polymer of marine microalgae and is particularly abundant during phytoplankton blooms. In this study, we show that highly specialized bacterial strains of the Bacteroidetes phylum repeatedly reached high abundances during North Sea algal blooms and dominated laminarin turnover. These genomically streamlined bacteria of the genus Formosa have an expanded set of laminarin hydrolases and transporters that belonged to the most abundant proteins in the environmental samples. In vitro experiments with cultured isolates allowed us to determine the functions of in situ expressed key enzymes and to confirm their role in laminarin utilization. It is shown that laminarin consumption of Formosa spp. is paralleled by enhanced uptake of diatom-derived peptides. This study reveals that genome reduction, enzyme fusions, transporters, and enzyme expansion as well as a tight coupling of carbon and nitrogen metabolism provide the tools, which make Formosa spp. so competitive during microalgal blooms.

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 two-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.

Polysaccharides are the most abundant and the most complex organic molecules in the ocean. In contrast to land polysaccharides, many marine polysaccharides are highly sulfated; in particular, the cell walls of macroalgae harbor a high diversity of sulfated polysaccharides (carrageenans, agarans, fucoidans, ulvans, etc.). These sulfated polysaccharides, biosynthesized by macroalgal primary producers, represent an important food source for heterotrophic organisms. Their biodegradation requires a set of enzymes that can cleave the glycosidic linkages of the carbohydrate backbone (called glycoside hydrolases) and the sulfate ester groups (called polysaccharide sulfatases). This review first provides on overview of the current state of knowledge on the classification and mechanisms of sulfatases in general. Then, based on an exploration of marine genomic and metagenomics data that reveals the diversity of carbohydrate sulfatases, it focuses on strategies to predict these sulfatases. In particular, the modularity of sulfatases and their location in marine polysaccharide utilization loci (PUL) provide clues as to their potential substrates and can drive future functional assays. Finally, the review underscores the low number of currently biochemically characterized marine carbohydrate sulfatases (e.g., agarases, carrageenanases, and fucose sulfatases). Bottlenecks encountered in studies on sulfatases likely lie in the difficulties in purifying them and producing them in heterologous systems.

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 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 GH29-5T 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.

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 3.3-2T 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 3.3-2T 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.