A Polysaccharide Utilization Locus from an Uncultured Bacteroidetes Phylotype Suggests Ecological Adaptation and Substrate Versatility

Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against β-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward β-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.     

Molecular details of a starch utilization pathway in the human gut symbiont Eubacterium rectale

Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute‐binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, whereas the membrane‐associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute‐binding proteins display a range of glycan‐binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6‐branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch‐degrading Clostridium cluster XIVa organisms in the human gut.     

Genomic Potential for Polysaccharide Deconstruction in Bacteria

Glycoside hydrolases are important enzymes that support bacterial growth by enabling the degradation of polysaccharides (e.g., starch, cellulose, xylan, and chitin) in the environment. Presently, little is known about the overall phylogenetic distribution of the genomic potential to degrade these polysaccharides in bacteria. However, knowing the phylogenetic breadth of these traits may help us predict the overall polysaccharide processing in environmental microbial communities. In order to address this, we identified and analyzed the distribution of 392,166 enzyme genes derived from 53 glycoside hydrolase families in 8,133 sequenced bacterial genomes. Enzymes for oligosaccharides and starch/glycogen were observed in most taxonomic groups, whereas glycoside hydrolases for structural polymers (i.e., cellulose, xylan, and chitin) were observed in clusters of relatives at taxonomic levels ranging from species to genus as determined by consenTRAIT. The potential for starch and glycogen processing, as well as oligosaccharide processing, was observed in 85% of the strains, whereas 65% possessed enzymes to degrade some structural polysaccharides (i.e., cellulose, xylan, or chitin). Potential degraders targeting one, two, and three structural polysaccharides accounted for 22.6, 32.9, and 9.3% of genomes analyzed, respectively. Finally, potential degraders targeting multiple structural polysaccharides displayed increased potential for oligosaccharide deconstruction. This study provides a framework for linking the potential for polymer deconstruction with phylogeny in complex microbial assemblages.     

SusE facilitates starch uptake independent of starch binding in B. thetaiotaomicron

The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model system for nutrient acquisition by gut Bacteroidetes, a dominant phylum of gut bacteria. The Sus includes SusCDEFG, which assemble on the cell surface to capture, degrade and import starch. While SusD is an essential starch‐binding protein, the precise role(s) of the partially homologous starch‐binding proteins SusE and SusF has remained elusive. We previously reported that a non‐binding version of SusD (SusD*) supports growth on starch when other members of the multi‐protein complex are present. Here we demonstrate that SusE supports SusD* growth on maltooligosaccharides, and determine the domains of SusE essential for this function. Furthermore, we demonstrate that SusE does not need to bind starch to support growth in the presence of SusD*, suggesting that the assembly of SusCDE is most important for maltooligosaccharide uptake in this context. However, starch binding by proteins SusDEF directs the uptake of maltooligosaccharides of specific lengths, suggesting that these proteins equip the cell to scavenge a range of starch fragments. These data demonstrate that the assembly of core Sus proteins SusCDE is secondary to their glycan binding roles, but glycan binding by Sus proteins may fine tune the selection of glycans from the environment.     

Unusual β1-4-galactosidase activity of an α1-6-mannosidase from Xanthomonas manihotis in the processing of branched hybrid and complex glycans

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell–pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected β1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel β1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal β1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.     

1H, 15N and 13C backbone and side-chain resonance assignments of a family 32 carbohydrate-binding module from the Clostridium perfringens NagH

The Gram-positive anaerobe Clostridium perfringens is an opportunistic bacterial pathogen that secretes a battery of enzymes involved in glycan degradation. These glycoside hydrolases are thought to be involved in turnover of mucosal layer glycans, and in the spread of major toxins commonly associated with the development of gastrointestinal diseases and gas gangrene in humans. These enzymes employ multi-modularity and carbohydrate-binding function to degrade extracellular eukaryotic host sugars. Here, we report the full (1)H, (15)N and (13)C chemical shift resonance assignments of the first family 32 carbohydrate-binding module from NagH, a secreted family 84 glycoside hydrolase.     

The genome and surface proteome of Capnocytophaga canimorsus reveal a key role of glycan foraging systems in host glycoproteins deglycosylation

Capnocytophaga canimorsus are commensal Gram-negative bacteria from dog's mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the Flavobacteria-Bacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus-like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection.     

Several Genes Encoding Enzymes with the Same Activity Are Necessary for Aerobic Fungal Degradation of Cellulose in Nature

The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.     

Characterization of four outer membrane proteins involved in binding starch to the cell surface of Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. Moreover, SusG alone did not have detectable starch binding activity. Previous studies have shown that starch binding is essential for starch utilization. In this paper, we report that four other outer membrane proteins, SusC through SusF, are responsible for starch binding. Results of (14)C-starch binding assays show that SusC and SusD both contribute a significant amount of starch binding. SusE also appears to contribute substantially to starch binding. Using affinity chromatography, we show in vitro that these Sus proteins interact to bind starch. Moreover, protease accessibility of either SusC or SusD greatly increased when one was expressed without the other. This finding supports the hypothesis that SusC and SusD interact in the outer membrane. Evidence from additional protease accessibility studies suggests that SusC, SusE, and SusF are exposed on the cell surface. Our results demonstrate that SusC and SusD act as the major starch binding proteins on the cell surface, with SusE enhancing binding. SusF's role in starch utilization has yet to be determined, although the fact that starch protected it from proteolytic attack suggests that it does bind starch.     

Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.     

Analysis of the bovine rumen microbiome reveals a diversity of Sus-like polysaccharide utilization loci from the bacterial phylum Bacteroidetes

Several unique Sus-like polysaccharide utilization loci (PULs) were identified from bacteria resident in bovine rumen microbiomes through functional screening of a fosmid library. The loci were phylogenetically assigned to the genus Prevotella within the phylum Bacteroidetes. These findings were augmented by a bioinformatic re-evaluation of ruminal Prevotella genomes, revealing additional loci not previously reported in the literature. Analysis of Bacteroidales-affiliated genomes reconstructed from a bovine rumen metagenome in a previous study further expanded the diversity of Sus-like PULs resident in this microbiome. Our findings suggest that Sus-like systems represent an important mechanism for degradation of a range of plant-derived glycans in ruminants.     

The GH26 β-mannanase RsMan26H from a symbiotic protist of the termite Reticulitermes speratus is an endo-processive mannobiohydrolase: Heterologous expression and characterization

Symbiotic protists in the gut of termites are prominent natural resources for enzymes involved in lignocellulose degradation. Here we report expression, purification, and biochemical characterization of a glycoside hydrolase family 26 mannanase RsMan26H from the symbiotic protist of the lower termite, Reticulitermes speratus. Biochemical analysis of RsMan26H demonstrates that this enzyme is an endo-processive mannobiohydrolase producing mannobiose from oligo- and polysaccharides, followed by a minor accumulation of oligosaccharides larger than mannobiose. To our knowledge, this is the first report describing the unique mannobiohydrolase enzyme from the eukaryotic origin.     

Development and Validation of a Microarray for the Investigation of the CAZymes Encoded by the Human Gut Microbiome

Distal gut bacteria play a pivotal role in the digestion of dietary polysaccharides by producing a large number of carbohydrate-active enzymes (CAZymes) that the host otherwise does not produce. We report here the design of a custom microarray that we used to spot non-redundant DNA probes for more than 6,500 genes encoding glycoside hydrolases and lyases selected from 174 reference genomes from distal gut bacteria. The custom microarray was tested and validated by the hybridization of bacterial DNA extracted from the stool samples of lean, obese and anorexic individuals. Our results suggest that a microarray-based study can detect genes from low-abundance bacteria better than metagenomic-based studies. A striking example was the finding that a gene encoding a GH6-family cellulase was present in all subjects examined, whereas metagenomic studies have consistently failed to detect this gene in both human and animal gut microbiomes. In addition, an examination of eight stool samples allowed the identification of a corresponding CAZome core containing 46 families of glycoside hydrolases and polysaccharide lyases, which suggests the functional stability of the gut microbiota despite large taxonomical variations between individuals.     

Physiological characterization of SusG, an outer membrane protein essential for starch utilization by Bacteroides thetaiotaomicron

Results from previous studies had suggested that Bacteroides thetaiotaomicron utilizes starch by binding the polysaccharide to the bacterial surface and subsequently degrading the polymer by using cell-associated enzymes. Most of the starch-degrading activity was localized to the periplasm, but a portion appeared to be membrane associated. This raised the possibility that some breakdown might occur in the outer membrane prior to exposure of the polysaccharide to the periplasmic polysaccharide-degrading enzymes. In this study, we show that SusG, an outer membrane protein which has been shown genetically to be essential for starch utilization, has enzymatic activity. Results of protease accessibility experiments support the hypothesis that SusG is exposed on the cell surface. Results of [(14)C]starch binding assays, however, show that SusG plays a negligible role in binding of starch to the cell surface. Consistent with this, SusG has a relatively high K(m) for starch and by itself is not sufficient to allow cells to grow on starch or to bind starch. Hence, the main role of SusG is to hydrolyze starch, but the binding of starch to the cell surface is evidently mediated by other proteins presumably interacting with SusG.     

Physiological roles of plant glycoside hydrolases

The functions of plant glycoside hydrolases and transglycosidases have been studied using different biochemical and molecular genetic approaches. These enzymes are involved in the metabolism of various carbohydrates containing compounds present in the plant tissues. The structural and functional diversity of the carbohydrates implies a vast spectrum of enzymes involved in their metabolism. Complete genome sequence of Arabidopsis and rice has allowed the classification of glycoside hydrolases in different families based on amino acid sequence data. The genomes of these plants contain 29 families of glycoside hydrolases. This review summarizes the current research on plant glycoside hydrolases concerning their principal functional roles, which were attributed to different families. The majority of these plant glycoside hydrolases are involved in cell wall polysaccharide metabolism. Other functions include their participation in the biosynthesis and remodulation of glycans, mobilization of energy, defence, symbiosis, signalling, secondary plant metabolism and metabolism of glycolipids.