A "terminal" case of glycan catabolism: Structural and enzymatic characterization of the sialidases of Clostridium perfringens.

Sialic acids are commonly found on the terminal ends of biologically important carbohydrates, including intestinal mucin O-linked glycans. Pathogens such as Clostridium perfringens, the causative agent of necrotic enteritis in poultry and humans, have the ability to degrade host mucins and colonize the mucus layer, which involves removal of the terminal sialic acid by carbohydrate-active enzymes (CAZymes). Here, we present the structural and biochemical characterization of the GH33 catalytic domains of the three sialidases of C. perfringens and probe their substrate specificity. The catalytically active domains, which we refer to as NanHGH33, NanJGH33, and NanIGH33, displayed differential activity on various naturally occurring forms of sialic acid. We report the X-ray crystal structures of these domains in complex with relevant sialic acid variants revealing the molecular basis of how each catalytic domain accommodates different sialic acids. NanHGH33 displays a distinct preference for α-2,3-linked sialic acid, but can process α-2,6-linked sialic acid. NanJGH33 and NanIGH33 both exhibit the ability to process α-2,3- and α-2,6-linked sialic acid without any significant apparent preference. All three enzymes were sensitive to generic and commercially available sialidase inhibitors, which impeded sialidase activity in cultures as well as the growth of C. perfringens on sialylated glycans. The knowledge gained in these studies can be applied to in vivo models for C. perfringens growth and metabolism of mucin O-glycans, with a view toward future mitigation of bacterial colonization and infection of intestinal tissues.

Metabolism of a hybrid algal galactan by members of the human gut microbiome.

Native porphyran is a hybrid of porphryan and agarose. As a common element of edible seaweed, this algal galactan is a frequent component of the human diet. Bacterial members of the human gut microbiota have acquired polysaccharide utilization loci (PULs) that enable the metabolism of porphyran or agarose. However, the molecular mechanisms that underlie the deconstruction and use of native porphyran remains incompletely defined. Here, we have studied two human gut bacteria, porphyranolytic Bacteroides plebeius and agarolytic Bacteroides uniformis, that target native porphyran. This reveals an exo-based cycle of porphyran depolymerization that incorporates a keystone sulfatase. In both PULs this cycle also works together with a PUL-encoded agarose depolymerizing machinery to synergistically reduce native porphyran to monosaccharides. This provides a framework for understanding the deconstruction of a hybrid algal galactan, and insight into the competitive and/or syntrophic relationship of gut microbiota members that target rare nutrients.

Methylation-GC-MS/FID-Based Glycosidic Linkage Analysis of Unfractionated Polysaccharides in Red Seaweeds.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more responses in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique's potential to differentiate various red seaweed species.

Characterization of Unfractionated Polysaccharides in Brown Seaweed by Methylation-GC-MS-Based Linkage Analysis.

This study introduces a novel approach to analyze glycosidic linkages in unfractionated polysaccharides from alcohol-insoluble residues (AIRs) of five brown seaweed species. GC-MS analysis of partially methylated alditol acetates (PMAAs) enables monitoring and comparison of structural variations across different species, harvest years, and tissues with and without blanching treatments. The method detects a wide array of fucose linkages, highlighting the structural diversity in glycosidic linkages and sulfation position in fucose-containing sulfated polysaccharides. Additionally, this technique enhances cellulose quantitation, overcoming the limitations of traditional monosaccharide composition analysis that typically underestimates cellulose abundance due to incomplete hydrolysis of crystalline cellulose. The introduction of a weak methanolysis-sodium borodeuteride reduction pretreatment allows for the detection and quantitation of uronic acid linkages in alginates.

Glycogen-Degrading Activities of Catalytic Domains of α-Amylase and α-Amylase-Pullulanase Enzymes Conserved in Gardnerella spp. from the Vaginal Microbiome.

Gardnerella spp. are associated with bacterial vaginosis in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria, including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina, and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake, a process that depends on the combined action of glycogen-degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose, and maltotetraose from glycogen was demonstrated. Carbohydrate-active enzymes (CAZymes) were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single-domain α-amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an α-amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules, and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the α-amylase-pullulanase and the single-domain α-amylase were each produced in Escherichia coli. The α-amylase domain from the α-amylase-pullulanase released maltose, maltotriose, and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella α-amylase-pullulanase is capable of hydrolyzing α-1,4 and α-1,6 glycosidic bonds. Similarly, the single-domain α-amylase protein also produced maltose, maltotriose, and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as "public goods" that can digest glycogen into maltose, maltotriose, and maltotetraose that can be used by the vaginal microbiota. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community, but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.

Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.

Corrigendum: Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

This corrects the article DOI: 10.1038/nature21725.

The pangenome of the wheat pathogen Pyrenophora tritici-repentis reveals novel transposons associated with necrotrophic effectors ToxA and ToxB.

BACKGROUND: In fungal plant pathogens, genome rearrangements followed by selection pressure for adaptive traits have facilitated the co-evolutionary arms race between hosts and their pathogens. Pyrenophora tritici-repentis (Ptr) has emerged recently as a foliar pathogen of wheat worldwide and its populations consist of isolates that vary in their ability to produce combinations of different necrotrophic effectors. These effectors play vital roles in disease development. Here, we sequenced the genomes of a global collection (40 isolates) of Ptr to gain insights into its gene content and genome rearrangements. RESULTS: A comparative genome analysis revealed an open pangenome, with an abundance of accessory genes (~ 57%) reflecting Ptr's adaptability. A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around effector coding genes, were detailed using long-read assemblies (PacBio RS II) generated in this work in addition to previously assembled genomes. We also discovered the involvement of large mobile elements associated with Ptr's effectors: ToxA, the gene encoding for the necrosis effector, was found as a single copy within a 143-kb 'Starship' transposon (dubbed 'Horizon') with a clearly defined target site and target site duplications. 'Horizon' was located on different chromosomes in different isolates, indicating mobility, and the previously described ToxhAT transposon (responsible for horizontal transfer of ToxA) was nested within this newly identified Starship. Additionally, ToxB, the gene encoding the chlorosis effector, was clustered as three copies on a 294-kb element, which is likely a different putative 'Starship' (dubbed 'Icarus') in a ToxB-producing isolate. ToxB and its putative transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and 'Icarus' were both present in a different non-coding isolate. This suggests that ToxB may have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption of ToxB vertical inheritance. Finally, the genome architecture of Ptr was defined as 'one-compartment' based on calculated gene distances and evolutionary rates. CONCLUSIONS: These findings together reflect on the highly plastic nature of the Ptr genome which has likely helped to drive its worldwide adaptation and has illuminated the involvement of giant transposons in facilitating the evolution of virulence in Ptr.

Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform.

Fluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a "Computational Identification of Non-disruptive Conjugation sites" (CINC) approach, an in silico pipeline utilizing molecular dynamics simulations for the rapid design and construction of novel protein-fluorophore conjugate-type biosensors. Here, we report an improved in silico scoring algorithm for use in CINC and its use in the construction of an oligogalacturonide-detecting biosensor set. Using both 4,5-unsaturated and saturated oligogalacturonides, we demonstrate that signal transmission from the ligand-binding pocket of the starting protein scaffold to the CINC-selected reporter positions is effective for multiple different ligands. The utility of an oligogalacturonide-detecting biosensor is shown in Carbohydrate Active Enzyme (CAZyme) activity assays, where the biosensor is used to follow product release upon polygalacturonic acid (PGA) depolymerization in real time. The oligogalacturonide-detecting biosensor set represents a novel enabling tool integral to our rapidly expanding platform for biosensor-based carbohydrate detection, and moving forward, the CINC pipeline will continue to enable the rational design of biomolecular tools to detect additional chemically distinct oligosaccharides and other solutes.

Characterization of an α-Glucosidase Enzyme Conserved in Gardnerella spp. Isolated from the Human Vaginal Microbiome.

Gardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, in which a lactobacillus-dominated community is replaced with mixed bacteria, including Gardnerella species. Co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, including glycogen, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up and further processed by bacteria requires the combined activities of several enzymes collectively known as amylases, which belong to glycoside hydrolase family 13 (GH13) within the CAZy classification system. GH13 is a large and diverse family of proteins, making prediction of their activities challenging. SACCHARIS annotation of the GH13 family in Gardnerella resulted in identification of protein domains belonging to eight subfamilies. Phylogenetic analysis of predicted amylase sequences from 26 genomes demonstrated that a putative α-glucosidase-encoding sequence, CG400_06090, was conserved in all Gardnerella spp. The predicted α-glucosidase enzyme was expressed, purified, and functionally characterized. The enzyme was active on a variety of maltooligosaccharides with maximum activity at pH 7. Km, kcat, and kcat/Km values for the substrate 4-nitrophenyl α-d-glucopyranoside were 8.3 µM, 0.96 min-1, and 0.11 µM-1 min-1, respectively. Glucose was released from maltose, maltotriose, maltotetraose, and maltopentaose, but no products were detected when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce an α-glucosidase enzyme that may contribute to the multistep process of glycogen metabolism by releasing glucose from maltooligosaccharides. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major carbon source available to vaginal bacteria. The significance of our research is characterizing the activity of an enzyme conserved in Gardnerella species that likely contributes to the ability of these bacteria to utilize glycogen.

The glycoconjugate-degrading enzymes of Clostridium perfringens: Tailored catalysts for breaching the intestinal mucus barrier.

The gastrointestinal (GI) tract of humans and animals is lined with mucus that serves as a barrier between the gut microbiota and the epithelial layer of the intestine. As the proteins present in mucus are typically heavily glycosylated, such as the mucins, several enteric commensal and pathogenic bacterial species are well-adapted to this rich carbon source and their genomes are replete with carbohydrate-active enzymes targeted toward dismantling the glycans and proteins present in mucus. One such species is Clostridium perfringens, a Gram-positive opportunistic pathogen indigenous to the gut of humans and animals. The genome of C. perfringens encodes numerous carbohydrate-active enzymes that are predicted or known to target glycosidic linkages within or on the termini of mucus glycans. Through this enzymatic activity, the degradation of the mucosal layer by C. perfringens has been implicated in a number of GI diseases, the most severe of which is necrotic enteritis. In this review, we describe the wide array of extracellular glycoside hydrolases, and their accessory modules, that is possessed by C. perfringens, and examine the unique multimodularity of these proteins in the context of degrading the glycoconjugates in mucus as a potential component of disease.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

Application of culturomics to characterize diverse anaerobic bacteria from the gastrointestinal tract of broiler chickens in relation to environmental reservoirs.

Characterization of the microbiota of chickens is of current interest. The goals of the current study were to apply anaerobic isolation methods to comprehensively isolate and identify bacteria from the gastrointestinal tract of chickens and their environment. Bacterial communities within the drinking water were dominated by Escherichia, whereas communities in litter were more representative of the cecum. The crop and small intestine (jejunum and ileum) were dominated by Lactobacillus and Enterococcus spp., and the cecum was dominated by Proteus spp. The collection of bacteria isolated was dominated by Enterococcus spp., Escherichia/Shigella spp., Lactobacillus spp., and Proteus spp.; however, many rare taxa were observed. These included members of the Clostridiales and Clostridium spp., which were commonly isolated from the ileum and cecum. Bacteria isolated by enrichment and direct plating differed. The selective de Man-Rogosa-Sharpe agar was commonly associated with the isolation of Lactobacillus spp. and yielded the lowest diversity of all methods utilized. Increased diversity and frequency of Clostridium spp. was observed in enrichments of blood and mucus or by plating on Columbia agar supplemented with 10% blood and gentamicin. The bacteria isolated from this study provide source material for genomic and functional studies in chicken hosts.

A surrogate structural platform informed by ancestral reconstruction and resurrection of a putative carbohydrate binding module hybrid illuminates the neofunctionalization of a pectate lyase.

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.

KdgF, the missing link in the microbial metabolism of uronate sugars from pectin and alginate.

Uronates are charged sugars that form the basis of two abundant sources of biomass-pectin and alginate-found in the cell walls of terrestrial plants and marine algae, respectively. These polysaccharides represent an important source of carbon to those organisms with the machinery to degrade them. The microbial pathways of pectin and alginate metabolism are well studied and essentially parallel; in both cases, unsaturated monouronates are produced and processed into the key metabolite 2-keto-3-deoxygluconate (KDG). The enzymes required to catalyze each step have been identified within pectinolytic and alginolytic microbes; yet the function of a small ORF, kdgF, which cooccurs with the genes for these enzymes, is unknown. Here we show that KdgF catalyzes the conversion of pectin- and alginate-derived 4,5-unsaturated monouronates to linear ketonized forms, a step in uronate metabolism that was previously thought to occur spontaneously. Using enzyme assays, NMR, mutagenesis, and deletion of kdgF, we show that KdgF proteins from both pectinolytic and alginolytic bacteria catalyze the ketonization of unsaturated monouronates and contribute to efficient production of KDG. We also report the X-ray crystal structures of two KdgF proteins and propose a mechanism for catalysis. The discovery of the function of KdgF fills a 50-y-old gap in the knowledge of uronate metabolism. Our findings have implications not only for the understanding of an important metabolic pathway, but also the role of pectinolysis in plant-pathogen virulence and the growing interest in the use of pectin and alginate as feedstocks for biofuel production.

Discovery and characterization of family 39 glycoside hydrolases from rumen anaerobic fungi with polyspecific activity on rare arabinosyl substrates.

Enzyme activities that improve digestion of recalcitrant plant cell wall polysaccharides may offer solutions for sustainable industries. To this end, anaerobic fungi in the rumen have been identified as a promising source of novel carbohydrate active enzymes (CAZymes) that modify plant cell wall polysaccharides and other complex glycans. Many CAZymes share insufficient sequence identity to characterized proteins from other microbial ecosystems to infer their function; thus presenting challenges to their identification. In this study, four rumen fungal genes (nf2152, nf2215, nf2523, and pr2455) were identified that encode family 39 glycoside hydrolases (GH39s), and have conserved structural features with GH51s. Two recombinant proteins, NF2152 and NF2523, were characterized using a variety of biochemical and structural techniques, and were determined to have distinct catalytic activities. NF2152 releases a single product, ß1,2-arabinobiose (Ara2) from sugar beet arabinan (SBA), and ß1,2-Ara2 and α-1,2-galactoarabinose (Gal-Ara) from rye arabinoxylan (RAX). NF2523 exclusively releases α-1,2-Gal-Ara from RAX, which represents the first description of a galacto-(α-1,2)-arabinosidase. Both ß-1,2-Ara2 and α-1,2-Gal-Ara are disaccharides not previously described within SBA and RAX. In this regard, the enzymes studied here may represent valuable new biocatalytic tools for investigating the structures of rare arabinosyl-containing glycans, and potentially for facilitating their modification in industrial applications.

Polysaccharide Utilization Loci: Fueling Microbial Communities.

The complex carbohydrates of terrestrial and marine biomass represent a rich nutrient source for free-living and mutualistic microbes alike. The enzymatic saccharification of these diverse substrates is of critical importance for fueling a variety of complex microbial communities, including marine, soil, ruminant, and monogastric microbiota. Consequently, highly specific carbohydrate-active enzymes, recognition proteins, and transporters are enriched in the genomes of certain species and are of critical importance in competitive environments. In Bacteroidetes bacteria, these systems are organized as polysaccharide utilization loci (PULs), which are strictly regulated, colocalized gene clusters that encode enzyme and protein ensembles required for the saccharification of complex carbohydrates. This review provides historical perspectives and summarizes key findings in the study of these systems, highlighting a critical shift from sequence-based PUL discovery to systems-based analyses combining reverse genetics, biochemistry, enzymology, and structural biology to precisely illuminate the molecular mechanisms underpinning PUL function. The ecological implications of dynamic PUL deployment by key species in the human gastrointestinal tract are explored, as well as the wider distribution of these systems in other gut, terrestrial, and marine environments.

The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot.

BACKGROUND: The nasopharyngeal (NP) microbiota plays an important role in bovine health, comprising a rich and diverse microbial community. The nasopharynx is also the niche for potentially pathogenic agents which are associated with bovine respiratory disease (BRD), a serious and costly illness in feedlot cattle. We used 14 beef heifers from a closed and disease-free herd to assess the dynamics of the NP microbiota of cattle that are transported to a feedlot. Cattle were sampled prior to transport to the feedlot (day 0) and at days 2, 7, and 14. RESULTS: The structure of the NP microbiota changed significantly over the course of the study, with the largest shift occurring between day 0 (prior to transport) and day 2 (P < 0.001). Phylogenetic diversity and richness increased following feedlot placement (day 2; P < 0.05). The genera Pasteurella, Bacillus, and Proteus were enriched at day 0, Streptococcus and Acinetobacter at day 2, Bifidobacterium at day 7, and Mycoplasma at day 14. The functional potential of the NP microbiota was assessed using PICRUSt, revealing that replication and repair, as well as translation pathways, were more relatively abundant in day 14 samples. These differences were driven mostly by Mycoplasma. Although eight cattle were culture-positive for the BRD-associated bacterium Pasteurella multocida at one or more sampling times, none were culture-positive for Mannheimia haemolytica or Histophilus somni. CONCLUSIONS: This study investigated the effect that feedlot placement has on the NP microbiota of beef cattle over a 14-d period. Within two days of transport to the feedlot, the NP microbiota changed significantly, increasing in both phylogenetic diversity and richness. These results demonstrate that there is an abrupt shift in the NP microbiota of cattle after transportation to a feedlot. This may have importance for understanding why cattle are most susceptible to BRD after feedlot placement.

Contributions of a unique ß-clamp to substrate recognition illuminates the molecular basis of exolysis in ferulic acid esterases.

Lignocellulosic biomass is a promising renewable resource; however, deconstruction of this material is still the rate-limiting step. Major obstacles in the biocatalytic turnover of lignocellulose are ester-linked decorations that prevent access to primary structural polysaccharides. Enzymes targeting these esters represent promising biotools for increasing bioconversion efficiency. Ruminant livestock are unique in their ability to degrade lignocellulose through the action of their gut microbiome. The anaerobic fungi (phylum Neocallimastigomycota) are key members of this ecosystem that express a large repertoire of carbohydrate-active enzymes (CAZymes) with little sequence identity with characterized CAZymes [Lombard, Golaconda, Drula, Coutinho and Henrissat (2014) Nucleic Acids Res. 42: , D490-D495]. We have identified a carbohydrate esterase family 1 (CE1) ferulic acid esterase (FAE) belonging to Anaeromyces mucronatus(AmCE1/Fae1a), and determined its X-ray structure in both the presence [1.55 Å (1 Å=0.1 nm)] and absence (1.60 Å) of ferulic acid. AmCE1 adopts an α/ß-hydrolase fold that is structurally conserved with bacterial FAEs, and possesses a unique loop, termed the ß-clamp, that encloses the ligand. Isothermal titration calorimetry reveals that substrate binding is driven by enthalpic contributions, which overcomes a large entropic penalty. A comparative analysis of AmCE1 with related enzymes has uncovered the apparent structural basis for differential FAE activities targeting cross-linking ferulic acid conjugates compared with terminal decorations. Based on comparisons to structurally characterized FAEs, we propose that the ß-clamp may define the structural basis of exolytic activities in FAEs. This provides a structure-based tool for predicting exolysis and endolysis in CE1. These insights hold promise for rationally identifying enzymes tailored for bioconversion of biomass with variations in cell wall composition.

Functional Analyses of Resurrected and Contemporary Enzymes Illuminate an Evolutionary Path for the Emergence of Exolysis in Polysaccharide Lyase Family 2.

Family 2 polysaccharide lyases (PL2s) preferentially catalyze the ß-elimination of homogalacturonan using transition metals as catalytic cofactors. PL2 is divided into two subfamilies that have been generally associated with secretion, Mg(2+) dependence, and endolysis (subfamily 1) and with intracellular localization, Mn(2+) dependence, and exolysis (subfamily 2). When present within a genome, PL2 genes are typically found as tandem copies, which suggests that they provide complementary activities at different stages along a catabolic cascade. This relationship most likely evolved by gene duplication and functional divergence (i.e. neofunctionalization). Although the molecular basis of subfamily 1 endolytic activity is understood, the adaptations within the active site of subfamily 2 enzymes that contribute to exolysis have not been determined. In order to investigate this relationship, we have conducted a comparative enzymatic analysis of enzymes dispersed within the PL2 phylogenetic tree and elucidated the structure of VvPL2 from Vibrio vulnificus YJ016, which represents a transitional member between subfamiles 1 and 2. In addition, we have used ancestral sequence reconstruction to functionally investigate the segregated evolutionary history of PL2 progenitor enzymes and illuminate the molecular evolution of exolysis. This study highlights that ancestral sequence reconstruction in combination with the comparative analysis of contemporary and resurrected enzymes holds promise for elucidating the origins and activities of other carbohydrate active enzyme families and the biological significance of cryptic metabolic pathways, such as pectinolysis within the zoonotic marine pathogen V. vulnificus.