scholarly journals Molecular Analysis of Cellulosome Organization in Ruminococcus Albus and Fibrobacter Intestinalis for Optimization of Fiber Digestibility in Ruminants

2004 ◽  
Author(s):  
Mark Morrison ◽  
Joshuah Miron ◽  
Edward A. Bayer ◽  
Raphael Lamed
Keyword(s):  
Plant Cell Wall ◽  
Cellulose Degradation ◽  
Structural Features ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Cellulolytic Bacteria ◽  
Modular Architecture ◽  
Host Animal ◽  
Ruminococcus Albus ◽  
Fiber Degradation

Improving plant cell wall (fiber) degradation remains one of the highest priority research goals for all ruminant enterprises dependent on forages, hay, silage, or other fibrous byproducts as energy sources, because it governs the provision of energy-yielding nutrients to the host animal. Although the predominant species of microbes responsible for ruminal fiber degradation are culturable, the enzymology and genetics underpinning the process are poorly defined. In that context, there were two broad objectives for this proposal. The first objective was to identify the key cellulosomal components in Ruminococcus albus and to characterize their structural features as well as regulation of their expression, in response to polysaccharides and (or) P AA/PPA. The second objective was to evaluate the similarities in the structure and architecture of cellulosomal components between R. albus and other ruminal and non-ruminal cellulolytic bacteria. The cooperation among the investigators resulted in the identification of two glycoside hydrolases rate-limiting to cellulose degradation by Ruminococcus albus (Cel48A and CeI9B) and our demonstration that these enzymes possess a novel modular architecture specific to this bacterium (Devillard et al. 2004). We have now shown that the novel X-domains in Cel48A and Cel9B represent a new type of carbohydrate binding module, and the enzymes are not part of a ceiluiosome-like complex (CBM37, Xu et al. 2004). Both Cel48A and Cel9B are conditionally expressed in response to P AA/PPA, explaining why cellulose degradation in this bacterium is affected by the availability of these compounds, but additional studies have shown for the first time that neither PAA nor PPA influence xylan degradation by R. albus (Reveneau et al. 2003). Additionally, the R. albus genome sequencing project, led by the PI. Morrison, has supported our identification of many dockerin containing proteins. However, the identification of gene(s) encoding a scaffoldin has been more elusive, and recombinant proteins encoding candidate cohesin modules are now being used in Israel to verify the existence of dockerin-cohesin interactions and cellulosome production by R. albus. The Israeli partners have also conducted virtually all of the studies specific to the second Objective of the proposal. Comparative blotting studies have been conducted using specific antibodies prepare against purified recombinant cohesins and X-domains, derived from cellulosomal scaffoldins of R. flavefaciens 17, a Clostridium thermocellum mutant-preabsorbed antibody preparation, or against CbpC (fimbrial protein) of R. albus 8. The data also suggest that additional cellulolytic bacteria including Fibrobacter succinogenes S85, F. intestinalis DR7 and Butyrivibrio fibrisolvens Dl may also employ cellulosomal modules similar to those of R. flavefaciens 17. Collectively, our work during the grant period has shown that R. albus and other ruminal bacteria employ several novel mechanisms for their adhesion to plant surfaces, and produce both cellulosomal and non-cellulosomal forms of glycoside hydrolases underpinning plant fiber degradation. These improvements in our mechanistic understanding of bacterial adhesion and enzyme regulation now offers the potential to: i) optimize ruminal and hindgut conditions by dietary additives to maximize fiber degradation (e.g. by the addition of select enzymes or PAA/PPA); ii) identify plant-borne influences on adhesion and fiber-degradation, which might be overcome (or improved) by conventional breeding or transgenic plant technologies and; iii) engineer or select microbes with improved adhesion capabilities, cellulosome assembly and fiber degradation. The potential benefits associated with this research proposal are likely to be realized in the medium term (5-10 years).

scholarly journals Ruminococcus albus 8 Mutants Defective in Cellulose Degradation Are Deficient in Two Processive Endocellulases, Cel48A and Cel9B, Both of Which Possess a Novel Modular Architecture

2004 ◽  
Vol 186 (1) ◽  
pp. 136-145 ◽  
Author(s):  
Estelle Devillard ◽  
Dara B. Goodheart ◽  
Sanjay K. R. Karnati ◽  
Edward A. Bayer ◽  
Raphael Lamed ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Sequence Data ◽  
Cellulose Degradation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Glycoside Hydrolases ◽  
Cellulolytic Bacterium ◽  
Cellulolytic Bacteria ◽  
Modular Architecture ◽  
Ruminococcus Albus ◽  
Mutant Strains

ABSTRACT The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B2 (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.

scholarly journals Cell Surface Enzyme Attachment Is Mediated by Family 37 Carbohydrate-Binding Modules, Unique to Ruminococcus albus

2008 ◽  
Vol 190 (24) ◽  
pp. 8220-8222 ◽  
Author(s):  
Anat Ezer ◽  
Erez Matalon ◽  
Sadanari Jindou ◽  
Ilya Borovok ◽  
Nof Atamna ◽  
...  
Keyword(s):  
Cell Surface ◽  
Bacterial Cell ◽  
Cellulose Degradation ◽  
Glycoside Hydrolases ◽  
Rumen Bacterium ◽  
Carbohydrate Binding ◽  
Ruminococcus Albus ◽  
System A ◽  
C Terminus ◽  
Carbohydrate Binding Modules

ABSTRACT The rumen bacterium Ruminococcus albus binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding.

scholarly journals The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

2003 ◽  
Vol 371 (3) ◽  
pp. 1027-1043 ◽  
Author(s):  
Deborah HOGG ◽  
Gavin PELL ◽  
Paul DUPREE ◽  
Florence GOUBET ◽  
Susana M. MARTÍN-ORÚE ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Molecular Architecture ◽  
Catalytic Domains ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

scholarly journals Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from theRuminococcus flavefacienscellulosome

2015 ◽  
Vol 71 (1) ◽  
pp. 45-48 ◽  
Author(s):  
Immacolata Venditto ◽  
Arun Goyal ◽  
Andrew Thompson ◽  
Luis M. A. Ferreira ◽  
Carlos M. G. A. Fontes ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Cellulolytic Bacterium ◽  
Modular Architecture ◽  
Carbohydrate Binding Modules ◽  
Fundamental Biological Process

Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connectedvialinker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium,Ruminococcus flavefaciensstrain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM-Rf6A and CBM-Rf6B) and a C-terminal dockerin. The novel CBM-Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Å resolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using theCCP4 program suite andSHELXC/D/E.

scholarly journals Domain Analysis of a Modular α-l-Arabinofuranosidase with a Unique Carbohydrate Binding Strategy from the Fiber-Degrading Bacterium Fibrobacter succinogenes S85

2010 ◽  
Vol 192 (20) ◽  
pp. 5424-5436 ◽  
Author(s):  
Shosuke Yoshida ◽  
Charles W. Hespen ◽  
Robert L. Beverly ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Plant Cell Wall ◽  
Catalytic Efficiency ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Biofuel Production ◽  
Carbohydrate Binding ◽  
Fibrobacter Succinogenes ◽  
Carbohydrate Binding Module ◽  
Degrading Bacterium ◽  
Catalytic Module

ABSTRACT Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique β-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher k cat for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the Km for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.

scholarly journals Phylogenetic Distribution of Potential Cellulases in Bacteria

2012 ◽  
Vol 79 (5) ◽  
pp. 1545-1554 ◽  
Author(s):  
Renaud Berlemont ◽  
Adam C. Martiny
Keyword(s):  
Plant Cell Wall ◽  
Cellulose Degradation ◽  
Microbial Community Composition ◽  
Glycoside Hydrolases ◽  
Phylogenetic Distribution ◽  
Soil Ecosystem ◽  
Bacterial Genomes ◽  
Genes Encoding ◽  
Plant Cell Wall Degradation ◽  
Mechanistic Basis

ABSTRACTMany microorganisms contain cellulases that are important for plant cell wall degradation and overall soil ecosystem functioning. At present, we have extensive biochemical knowledge of cellulases but little is known about the phylogenetic distribution of these enzymes. To address this, we analyzed the distribution of 21,985 genes encoding proteins related to cellulose utilization in 5,123 sequenced bacterial genomes. First, we identified the distribution of glycoside hydrolases involved in cellulose utilization and synthesis at different taxonomic levels, from the phylum to the strain. Cellulose degradation/utilization capabilities appeared in nearly all major groups and resulted in strains displaying various enzyme gene combinations. Potential cellulose degraders, having both cellulases and β-glucosidases, constituted 24% of all genomes whereas potential opportunistic strains, having β-glucosidases only, accounted for 56%. Finally, 20% of the bacteria have no relevant enzymes and do not rely on cellulose utilization. The latter group was primarily connected to specific bacterial lifestyles like autotrophy and parasitism. Cellulose degraders, as well as opportunists, have multiple enzymes with similar functions. However, the potential degraders systematically harbor about twice more β-glucosidases than their potential opportunistic relatives. Although scattered, the distribution of functional types, in bacterial lineages, is not random but mostly follows a Brownian motion evolution model. Degraders form clusters of relatives at the species level, whereas opportunists are clustered at the genus level. This information can form a mechanistic basis for the linking of changes in microbial community composition to soil ecosystem processes.

scholarly journals Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases

2020 ◽  
Author(s):  
Japheth E. Gado ◽  
Brent E. Harrison ◽  
Mats Sandgren ◽  
Jerry Ståhlberg ◽  
Gregg T. Beckham ◽  
...  
Keyword(s):  
Machine Learning ◽  
Active Site ◽  
Catalytic Domain ◽  
Cellulose Degradation ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Functional Variation ◽  
Functional Relationships ◽  
Experimental Findings ◽  
And Function

AbstractFamily 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These important enzymes are often bimodular, comprised of a catalytic domain attached to a carbohydrate binding module (CBM) via a flexible linker, and exhibit a long active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG). Despite the critical biological and industrial importance of GH7 enzymes, there remain gaps in our understanding of how GH7 sequence and structure relate to function. Here, we employed machine learning to gain insights into relationships between sequence, structure, and function across the GH7 family. Machine-learning models, using the number of residues in the active-site loops as features, were able discriminate GH7 CBHs and EGs with up to 99% accuracy. The lengths of the A4, B2, B3, and B4 loops were strongly correlated with functional subtype across the GH7 family. Position-specific classification rules were derived such that specific amino acids at 42 different sequence positions predicted the functional subtype with accuracies greater than 87%. A random forest model trained on residues at 19 positions in the catalytic domain predicted the presence of a CBM with 89.5% accuracy. We propose these positions play vital roles in the functional variation of GH7 cellulases. Taken together, our results complement numerous experimental findings and present functional relationships that can be applied when prospecting GH7 cellulases from nature, for sequence annotation, and to understand or manipulate function.

scholarly journals Insights into Plant Cell Wall Degradation from the Genome Sequence of the Soil Bacterium Cellvibrio japonicus

2008 ◽  
Vol 190 (15) ◽  
pp. 5455-5463 ◽  
Author(s):  
Robert T. DeBoy ◽  
Emmanuel F. Mongodin ◽  
Derrick E. Fouts ◽  
Louise E. Tailford ◽  
Hoda Khouri ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Hydrolytic Enzymes ◽  
Soil Bacterium ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus ◽  
And Storage

ABSTRACT The plant cell wall, which consists of a highly complex array of interconnecting polysaccharides, is the most abundant source of organic carbon in the biosphere. Microorganisms that degrade the plant cell wall synthesize an extensive portfolio of hydrolytic enzymes that display highly complex molecular architectures. To unravel the intricate repertoire of plant cell wall-degrading enzymes synthesized by the saprophytic soil bacterium Cellvibrio japonicus, we sequenced and analyzed its genome, which predicts that the bacterium contains the complete repertoire of enzymes required to degrade plant cell wall and storage polysaccharides. Approximately one-third of these putative proteins (57) are predicted to contain carbohydrate binding modules derived from 13 of the 49 known families. Sequence analysis reveals approximately 130 predicted glycoside hydrolases that target the major structural and storage plant polysaccharides. In common with that of the colonic prokaryote Bacteroides thetaiotaomicron, the genome of C. japonicus is predicted to encode a large number of GH43 enzymes, suggesting that the extensive arabinose decorations appended to pectins and xylans may represent a major nutrient source, not just for intestinal bacteria but also for microorganisms that occupy terrestrial ecosystems. The results presented here predict that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases. Most importantly, the genome of C. japonicus is remarkably similar to that of the gram-negative marine bacterium, Saccharophagus degradans 2-40T. Approximately 50% of the predicted C. japonicus plant-degradative apparatus appears to be shared with S. degradans, consistent with the utilization of plant-derived complex carbohydrates as a major substrate by both organisms.

scholarly journals High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
K. M. Singh ◽  
Bhaskar Reddy ◽  
Dishita Patel ◽  
A. K. Patel ◽  
Nidhi Parmar ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Metagenomic Sequencing ◽  
Environmental Aspects ◽  
Rumen Microbiome ◽  
Polysaccharide Lyases ◽  
Effective System ◽  
Plant Cell Wall Degradation ◽  
Buffalo Rumen

The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs). We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs), carbohydrate binding module (CBM: 23 contigs), glycosyl transferase (GT: 373 contigs), carbohydrate esterases (CE: 259 contigs), and polysaccharide lyases (PE: 16 contigs). The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry.

scholarly journals Functional Analyses of Multiple Lichenin-Degrading Enzymes from the Rumen Bacterium Ruminococcus albus 8

2011 ◽  
Vol 77 (21) ◽  
pp. 7541-7550 ◽  
Author(s):  
Michael Iakiviak ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Plant Cell Wall ◽  
Bioinformatic Analysis ◽  
Major Product ◽  
Glycoside Hydrolases ◽  
Rumen Bacterium ◽  
Cell Wall Polysaccharides ◽  
Ruminococcus Albus ◽  
Content Type ◽  
Wide Range ◽  
Functional Analyses

ABSTRACTRuminococcus albus8 is a fibrolytic ruminal bacterium capable of utilization of various plant cell wall polysaccharides. A bioinformatic analysis of a partial genome sequence ofR. albusrevealed several putative enzymes likely to hydrolyze glucans, including lichenin, a mixed-linkage polysaccharide of glucose linked together in β-1,3 and β-1,4 glycosidic bonds. In the present study, we demonstrate the capacity of four glycoside hydrolases (GHs), derived fromR. albus, to hydrolyze lichenin. Two of the genes encoded GH family 5 enzymes (Ra0453 and Ra2830), one gene encoded a GH family 16 enzyme (Ra0505), and the last gene encoded a GH family 3 enzyme (Ra1595). Each gene was expressed inEscherichia coli, and the recombinant protein was purified to near homogeneity. Upon screening on a wide range of substrates, Ra0453, Ra2830, and Ra0505 displayed different hydrolytic properties, as they released unique product profiles. The Ra1595 protein, predicted to function as a β-glucosidase, preferred cleavage of a nonreducing end glucose when linked by a β-1,3 glycosidic bond to the next glucose residue. The major product of Ra0505 hydrolysis of lichenin was predicted to be a glucotriose that was degraded only by Ra0453 to glucose and cellobiose. Most importantly, the four enzymes functioned synergistically to hydrolyze lichenin to glucose, cellobiose, and cellotriose. This lichenin-degrading enzyme mix should be of utility as an additive to feeds administered to monogastric animals, especially those high in fiber.

Sign in / Sign up

Export Citation Format

Share Document