The human gut microbiota (HGM) makes an important contribution to health and disease. It is a complex microbial community of trillions of microbes with a majority of its members represented within ...two phyla, the Bacteroidetes and Firmicutes, although it also contains species of Actinobacteria and Proteobacteria. Reflecting its importance, the HGM is sometimes referred to as an 'organ' as it performs functions analogous to systemic tissues within the human host. The major nutrients available to the HGM are host and dietary complex carbohydrates. To utilise these nutrient sources, the HGM has developed elaborate, variable and sophisticated systems for the sensing, capture and utilisation of these glycans. Understanding nutrient acquisition by the HGM can thus provide mechanistic insights into the dynamics of this ecosystem, and how it impacts human health. Dietary nutrient sources include a wide variety of simple and complex plant and animal-derived glycans most of which are not degraded by enzymes in the digestive tract of the host. Here we review how various adaptive mechanisms that operate across the major phyla of the HGM contribute to glycan utilisation, focusing on the most complex carbohydrates presented to this ecosystem.
Abstract
The Polysaccharide Utilization Loci (PUL) database was launched in 2015 to present PUL predictions in ∼70 Bacteroidetes species isolated from the human gastrointestinal tract, as well as ...PULs derived from the experimental data reported in the literature. In 2018 PULDB offers access to 820 genomes, sampled from various environments and covering a much wider taxonomical range. A Krona dynamic chart was set up to facilitate browsing through taxonomy. Literature surveys now allows the presentation of the most recent (i) PUL repertoires deduced from RNAseq large-scale experiments, (ii) PULs that have been subjected to in-depth biochemical analysis and (iii) new Carbohydrate-Active enzyme (CAZyme) families that contributed to the refinement of PUL predictions. To improve PUL visualization and genome browsing, the previous annotation of genes encoding CAZymes, regulators, integrases and SusCD has now been expanded to include functionally relevant protein families whose genes are significantly found in the vicinity of PULs: sulfatases, proteases, ROK repressors, epimerases and ATP-Binding Cassette and Major Facilitator Superfamily transporters. To cope with cases where susCD may be absent due to incomplete assemblies/split PULs, we present 'CAZyme cluster' predictions. Finally, a PUL alignment tool, operating on the tagged families instead of amino-acid sequences, was integrated to retrieve PULs similar to a query of interest. The updated PULDB website is accessible at www.cazy.org/PULDB_new/
•Carbohydrate-binding modules (CBMs) display diverse specificity and structure.•A refined classification of CBM types is presented.•Advances in understanding plant cell wall carbohydrate binding are ...discussed.•Metals play diverse roles in CBM function.•CBMs can drive substrate specificity.
Plant cell walls are complex configurations of polysaccharides that are recalcitrant to degradation. The enzymes deployed by microbes to degrade these materials comprise glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and polysaccharide oxidases. Non-catalytic carbohydrate-binding modules (CBMs) are found as discretely folded units within the multi-modular structures of these enzymes where they play critical roles in the recognition of plant cell wall components and potentiating the activity of the enzymes. Here we propose a refinement to the Types A, B, and C classification of CBMs whereby the Type A CBMs remain those that bind the surfaces of crystalline polysaccharides but the Type B CBMs are redefined as those that bind internally on glycan chains (endo-type), CBMs that bind to the termini of glycan chains are defined as Type C modules (exo-type). In this context, we discuss recent advances, primarily driven by structural studies, which reveal the molecular modes of CBM–sugar interactions and how this specifically underpins and influences the biological function of CBMs in cell wall recognition and degradation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Symbiotic bacteria inhabiting the human gut have evolved under intense pressure to utilize complex carbohydrates, primarily plant cell wall glycans in our diets. These polysaccharides are not ...digested by human enzymes, but are processed to absorbable short chain fatty acids by gut bacteria. The Bacteroidetes, one of two dominant bacterial phyla in the adult gut, possess broad glycan-degrading abilities. These species use a series of membrane protein complexes, termed Sus-like systems, for catabolism of many complex carbohydrates. However, the role of these systems in degrading the chemically diverse repertoire of plant cell wall glycans remains unknown. Here we show that two closely related human gut Bacteroides, B. thetaiotaomicron and B. ovatus, are capable of utilizing nearly all of the major plant and host glycans, including rhamnogalacturonan II, a highly complex polymer thought to be recalcitrant to microbial degradation. Transcriptional profiling and gene inactivation experiments revealed the identity and specificity of the polysaccharide utilization loci (PULs) that encode individual Sus-like systems that target various plant polysaccharides. Comparative genomic analysis indicated that B. ovatus possesses several unique PULs that enable degradation of hemicellulosic polysaccharides, a phenotype absent from B. thetaiotaomicron. In contrast, the B. thetaiotaomicron genome has been shaped by increased numbers of PULs involved in metabolism of host mucin O-glycans, a phenotype that is undetectable in B. ovatus. Binding studies of the purified sensor domains of PUL-associated hybrid two-component systems in conjunction with transcriptional analyses demonstrate that complex oligosaccharides provide the regulatory cues that induce PUL activation and that each PUL is highly specific for a defined cell wall polymer. These results provide a view of how these species have diverged into different carbohydrate niches by evolving genes that target unique suites of available polysaccharides, a theme that likely applies to disparate bacteria from the gut and other habitats.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A bacterial polysaccharide utilization locus (PUL) is a set of physically linked genes that orchestrate the breakdown of a specific glycan. PULs are prevalent in the Bacteroidetes phylum and are key ...to the digestion of complex carbohydrates, notably by the human gut microbiota. A given Bacteroidetes genome can encode dozens of different PULs whose boundaries and precise gene content are difficult to predict.
Here, we present a fully automated approach for PUL prediction using genomic context and domain annotation alone. By combining the detection of a pair of marker genes with operon prediction using intergenic distances, and queries to the carbohydrate-active enzymes database (www.cazy.org), our predictor achieved above 86% accuracy in two Bacteroides species with extensive experimental PUL characterization.
PUL predictions in 67 Bacteroidetes genomes from the human gut microbiota and two additional species, from the canine oral sphere and from the environment, are presented in our database accessible at www.cazy.org/PULDB/index.php.
The enzymic degradation of insoluble polysaccharides is one of the most important reactions on earth. Despite this, glycoside hydrolases attack such polysaccharides relatively inefficiently as their ...target glycosidic bonds are often inaccessible to the active site of the appropriate enzymes. In order to overcome these problems, many of the glycoside hydrolases that utilize insoluble substrates are modular, comprising catalytic modules appended to one or more non-catalytic CBMs (carbohydrate-binding modules). CBMs promote the association of the enzyme with the substrate. In view of the central role that CBMs play in the enzymic hydrolysis of plant structural and storage polysaccharides, the ligand specificity displayed by these protein modules and the mechanism by which they recognize their target carbohydrates have received considerable attention since their discovery almost 20 years ago. In the last few years, CBM research has harnessed structural, functional and bioinformatic approaches to elucidate the molecular determinants that drive CBM-carbohydrate recognition. The present review summarizes the impact structural biology has had on our understanding of the mechanisms by which CBMs bind to their target ligands.
Cellulosomes can be described as one of nature's most elaborate and highly efficient nanomachines. These cell bound multienzyme complexes orchestrate the deconstruction of cellulose and ...hemicellulose, two of the most abundant polymers on Earth, and thus play a major role in carbon turnover. Integration of cellulosomal components occurs via highly ordered protein:protein interactions between cohesins and dockerins, whose specificity allows the incorporation of cellulases and hemicellulases onto a molecular scaffold. Cellulosome assembly promotes the exploitation of enzyme synergism because of spatial proximity and enzyme-substrate targeting. Recent structural and functional studies have revealed how cohesin-dockerin interactions mediate both cellulosome assembly and cell-surface attachment, while retaining the spatial flexibility required to optimize the catalytic synergy within the enzyme complex. These emerging advances in our knowledge of cellulosome function are reviewed here.
Fresh strawberries (Fragaria x ananassa) are valued for their characteristic red color, juicy texture, distinct aroma, and sweet fruity flavor. In this study, genetic and environmentally induced ...variation is exploited to capture biochemically diverse strawberry fruit for metabolite profiling and consumer rating. Analyses identify fruit attributes influencing hedonics and sensory perception of strawberry fruit using a psychophysics approach. Sweetness intensity, flavor intensity, and texture liking are dependent on sugar concentrations, specific volatile compounds, and fruit firmness, respectively. Overall liking is most greatly influenced by sweetness and strawberry flavor intensity, which are undermined by environmental pressures that reduce sucrose and total volatile content. The volatile profiles among commercial strawberry varieties are complex and distinct, but a list of perceptually impactful compounds from the larger mixture is better defined. Particular esters, terpenes, and furans have the most significant fits to strawberry flavor intensity. In total, thirty-one volatile compounds are found to be significantly correlated to strawberry flavor intensity, only one of them negatively. Further analysis identifies individual volatile compounds that have an enhancing effect on perceived sweetness intensity of fruit independent of sugar content. These findings allow for consumer influence in the breeding of more desirable fruits and vegetables. Also, this approach garners insights into fruit metabolomics, flavor chemistry, and a paradigm for enhancing liking of natural or processed products.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans ...heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron, a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS, and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides, indicating that the model developed is of generic relevance to this important microbial community.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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