D. Wade Abbott

Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts

Competition for nutrients contained in diverse types of plant cell wall-associated polysaccharides may explain the evolution of substrate-specific catabolic gene modules in common bacterial members of the human gut microbiota.

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.

Histone variant H2ABbd confers lower stability to the nucleosome.

The histone H2ABbd is a novel histone variant of H2A with a totally unknown function. We have investigated the behaviour of the H2ABbd nucleosomes. Nucleosomes were reconstituted with recombinant histone H2ABbd and changes in their conformations at different salt concentrations were studied by analytical centrifugation. The data are in agreement with H2ABbd being less tightly bound compared with conventional H2A in the nucleosome. In addition, stable cell lines expressing either green fluorescent protein (GFP)–H2A or GFP–H2ABbd were established and the mobility of both fusions was measured by fluorescence recovery after photobleaching. We show that GFP–H2ABbd exchanges much more rapidly than GFP–H2A within the nucleosome. The reported data are compatible with a lower stability of the variant H2ABbd nucleosome compared with the conventional H2A particle.

Structural Biology of Pectin Degradation by Enterobacteriaceae

SUMMARY Pectin is a structural polysaccharide that is integral for the stability of plant cell walls. During soft rot infection, secreted virulence factors from pectinolytic bacteria such as Erwinia spp. degrade pectin, resulting in characteristic plant cell necrosis and tissue maceration. Catabolism of pectin and its breakdown products by pectinolytic bacteria occurs within distinct cellular environments. This process initiates outside the cell, continues within the periplasmic space, and culminates in the cytoplasm. Although pectin utilization is well understood at the genetic and biochemical levels, an inclusive structural description of pectinases and pectin binding proteins by both extracellular and periplasmic enzymes has been lacking, especially following the recent characterization of several periplasmic components and protein-oligogalacturonide complexes. Here we provide a comprehensive analysis of the protein folds and mechanisms of pectate lyases, polygalacturonases, and carbohydrate esterases and the binding specificities of two periplasmic pectic binding proteins from Enterobacteriaceae. This review provides a structural understanding of the molecular determinants of pectin utilization and the mechanisms driving catabolite selectivity and flow through the pathway.

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.

H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis

Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals.

The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae

The genome of Streptococcus pneumoniae strains, as typified by the TIGR4 strain, contain several genes encoding proteins putatively involved in α‐glucan degradation, modification and synthesis. The extracellular components comprise an ATP binding cassette‐transporter with its solute binding protein, MalX, and the hydrolytic enzyme SpuA. We show that of the commonly occurring exogenous α‐glucans, S. pneumoniae TIGR4 is only able to grow on glycogen in a MalX‐ and SpuA‐dependent manner. SpuA is able to degrade glycogen into a ladder of α‐1,4‐glucooligosaccharides while the high‐affinity interaction (Ka ∼ 106 M−1) of MalX with maltooligosaccharides plays a key role in promoting the selective uptake of the glycogen degradation products that are produced by SpuA. The X‐ray crystallographic analyses of apo‐ and complexed MalX illuminate the proteins specificity for the degradation products of glycogen and its striking ability to recognize the helical structure of the ligand. Overall, the results of this work provide new structural and functional insight into streptococcal α‐glucan metabolism while supplying biochemical support for the hypothesis that the substrate of the S. pneumoniaeα‐glucan metabolizing machinery is glycogen, which in a human host is abundant in lung epithelial cells, a common target for invasive S. pneumoniae.

Quantitative Approaches to The Analysis of Carbohydrate-Binding Module Function

Carbohydrate-binding modules (CBMs) are important components of carbohydrate-active enzymes. Their primary functions are to assist in substrate turnover by targeting appended catalytic modules to substrate and concentrating appended catalytic modules on the surface of substrate. Presented here are four well-established methodologies for investigating and quantifying the CBM-polysaccharide binding relationship. These methods include: (1) the solid state depletion assay, (2) affinity gel electrophoresis, (3) UV difference and fluorescence spectroscopy, and (4) isothermal titration calorimetry. In addition, entropy-driven CBM-crystalline cellulose binding events and differential approaches to calculating stoichiometry with polyvalent polysaccharide ligands are also discussed.

Using structure to inform carbohydrate binding module function

Generally, non-catalytic carbohydrate binding module (CBM) specificity has been shown to parallel the catalytic activity of the carbohydrate active enzyme (CAZyme) module it is appended to. With the rapid expansion in metagenomic sequence space for the potential discovery of new CBMs in addition to the recent emergence of several new CBM families that display diverse binding profiles and novel functions, elucidating the function of these protein modules has become a much more challenging task. This review summarizes several approaches that have been reported for using primary structure to inform CBM specificity and streamlining their biophysical characterization. In addition we discuss general trends in binding site architecture and several newly identified functions for CBMs. Streams of investigation that will facilitate the development and refinement of sequence-based prediction tools are suggested.

Polysaccharide Utilization Loci: Fuelling 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.