Abundance and Diversity of Dockerin-Containing Proteins in the Fiber-Degrading Rumen Bacterium,Ruminococcus flavefaciens FD-1

Background

The cellulosome is a multi-enzyme machine, which plays a key role in the breakdown of plant cell walls in many anaerobic cellulose-degrading microorganisms. Ruminococcus flavefaciens FD-1, a major fiber-degrading bacterium present in the gut of herbivores, has the most intricate cellulosomal organization thus far described. Cellulosome complexes are assembled through high-affinity cohesin-dockerin interactions. More than two-hundred dockerin-containing proteins have been identified in the R. flavefaciens genome, yet the reason for the expansion of these crucial cellulosomal components is yet unknown.

Methodology/Principal Findings

We have explored the full spectrum of 222 dockerin-containing proteins potentially involved in the assembly of cellulosome-like complexes of R. flavefaciens. Bioinformatic analysis of the various dockerin modules showed distinctive conservation patterns within their two Ca²⁺-binding repeats and their flanking regions. Thus, we established the conceptual framework for six major groups of dockerin types, according to their unique sequence features. Within this framework, the modular architecture of the parent proteins, some of which are multi-functional proteins, was evaluated together with their gene expression levels. Specific dockerin types were found to be associated with selected groups of functional components, such as carbohydrate-binding modules, numerous peptidases, and/or carbohydrate-active enzymes. In addition, members of other dockerin groups were linked to structural proteins, e.g., cohesin-containing proteins, belonging to the scaffoldins.

Conclusions/Significance

This report profiles the abundance and sequence diversity of the R. flavefaciens FD-1 dockerins, and provides the molecular basis for future understanding of the potential for a wide array of cohesin-dockerin specificities. Conserved differences between dockerins may be reflected in their stability, function or expression within the context of the parent protein, in response to their role in the rumen environment.     

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.     

Characterization of four endophytic fungi as potential consolidated bioprocessing hosts for conversion of lignocellulose into advanced biofuels

Applied Microbiology and Biotechnology - Recently, several endophytic fungi have been demonstrated to produce volatile organic compounds (VOCs) with properties similar to fossil fuels, called...     

The crystal structure of the family 6 carbohydrate binding module from Cellvibrio mixtus endoglucanase 5a in complex with oligosaccharides reveals two distinct binding sites with different ligand specificities

Glycoside hydrolases that release fixed carbon from the plant cell wall are of considerable biological and industrial importance. These hydrolases contain non-catalytic carbohydrate binding modules (CBMs) that, by bringing the appended catalytic domain into intimate association with its insoluble substrate, greatly potentiate catalysis. Family 6 CBMs (CBM6) are highly unusual because they contain two distinct clefts (cleft A and cleft B) that potentially can function as binding sites. Henshaw et al. (Henshaw, J., Bolam, D. N., Pires, V. M. R., Czjzek, M., Henrissat, B., Ferreira, L. M. A., Fontes, C. M. G. A., and Gilbert, H. J. (2003) J. Biol. Chem. 279, 21552-21559) show that CmCBM6 contains two binding sites that display both similarities and differences in their ligand specificity. Here we report the crystal structure of CmCBM6 in complex with a variety of ligands that reveals the structural basis for the ligand specificity displayed by this protein. In cleft A the two faces of the terminal sugars of beta-linked oligosaccharides stack against Trp-92 and Tyr-33, whereas the rest of the binding cleft is blocked by Glu-20 and Thr-23, residues that are not present in CBM6 proteins that bind to the internal regions of polysaccharides in cleft A. Cleft B is solvent-exposed and, therefore, able to bind ligands because the loop, which occludes this region in other CBM6 proteins, is much shorter and flexible (lacks a conserved proline) in CmCBM6. Subsites 2 and 3 of cleft B accommodate cellobiose (Glc-beta-1,4-Glc), subsite 4 will bind only to a beta-1,3-linked glucose, whereas subsite 1 can interact with either a beta-1,3- or beta-1,4-linked glucose. These different specificities of the subsites explain how cleft B can accommodate beta-1,4-beta-1,3- or beta-1,3-beta-1,4-linked gluco-configured ligands.     

Identification of the catalytic residues in family 52 glycoside hydrolase,a beta-xylosidase from Geobacillus stearothermophilus T-6

beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Br?nsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.     

A novel, noncatalytic carbohydrate-binding module displays specificity for galactose-containing polysaccharides through calcium-mediated oligomerization

The enzymic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The catalytic modules of enzymes that catalyze this process are generally appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs potentiate the rate of catalysis by bringing their cognate enzymes into intimate contact with the target substrate. A powerful plant cell wall-degrading system is the Clostridium thermocellum multienzyme complex, termed the "cellulosome." Here, we identify a novel CBM (CtCBM62) within the large C. thermocellum cellulosomal protein Cthe_2193 (defined as CtXyl5A), which establishes a new CBM family. Phylogenetic analysis of CBM62 members indicates that a circular permutation occurred within the family. CtCBM62 binds to d-galactose and l-arabinopyranose in either anomeric configuration. The crystal structures of CtCBM62, in complex with oligosaccharides containing α- and β-galactose residues, show that the ligand-binding site in the β-sandwich protein is located in the loops that connect the two β-sheets. Specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located in plant cell walls. CtCBM62 displays tighter affinity for multivalent ligands compared with molecules containing single galactose residues, which is associated with precipitation of these complex carbohydrates. These avidity effects, which confer the targeting of polysaccharides, are mediated by calcium-dependent oligomerization of the CBM.     

A Metagenomic Investigation of the Duodenal Microbiota Reveals Links with Obesity

Background

Few studies have tested the small intestine microbiota in humans, where most nutrient digestion and absorption occur. Here, our objective was to examine the duodenal microbiota between obese and normal volunteers using metagenomic techniques.

Methodology/Principal Findings

We tested duodenal samples from five obese and five normal volunteers using 16S rDNA V6 pyrosequencing and Illumina MiSeq deep sequencing. The predominant phyla of the duodenal microbiota were Firmicutes and Actinobacteria, whereas Bacteroidetes were absent. Obese individuals had a significant increase in anaerobic genera (p < 0.001) and a higher abundance of genes encoding Acyl-CoA dehydrogenase (p = 0.0018) compared to the control group. Obese individuals also had a reduced abundance of genes encoding sucrose phosphorylase (p = 0.015) and 1,4-alpha-glucan branching enzyme (p = 0.05). Normal weight people had significantly increased FabK (p = 0.027), and the glycerophospholipid metabolism pathway revealed the presence of phospholipase A1 only in the control group (p = 0.05).

Conclusions/Significance

The duodenal microbiota of obese individuals exhibit alterations in the fatty acid and sucrose breakdown pathways, probably induced by diet imbalance.     

Evolution of symbiotic bacteria in the distal human intestine

The adult human intestine contains trillions of bacteria, representing hundreds of species and thousands of subspecies. Little is known about the selective pressures that have shaped and are shaping this community's component species, which are dominated by members of the Bacteroidetes and Firmicutes divisions. To examine how the intestinal environment affects microbial genome evolution, we have sequenced the genomes of two members of the normal distal human gut microbiota, Bacteroides vulgatus and Bacteroides distasonis, and by comparison with the few other sequenced gut and non-gut Bacteroidetes, analyzed their niche and habitat adaptations. The results show that lateral gene transfer, mobile elements, and gene amplification have played important roles in affecting the ability of gut-dwelling Bacteroidetes to vary their cell surface, sense their environment, and harvest nutrient resources present in the distal intestine. Our findings show that these processes have been a driving force in the adaptation of Bacteroidetes to the distal gut environment, and emphasize the importance of considering the evolution of humans from an additional perspective, namely the evolution of our microbiomes.