Publications

Autoproteolysis has been discovered to play key roles in various biological processes, but functional autoproteolysis has been rarely reported for transmembrane signaling in prokaryotes. In this study, an autoproteolytic effect was discovered in the conserved periplasmic domain of anti-σ factor RsgIs from Clostridium thermocellum, which was found to transmit extracellular polysaccharide-sensing signals into cells for regulation of the cellulosome system, a polysaccharide-degrading multienzyme complex. Crystal and NMR structures of periplasmic domains from three RsgIs demonstrated that they are different from all known proteins that undergo autoproteolysis. The RsgI-based autocleavage site was located at a conserved Asn-Pro motif between the β1 and β2 strands in the periplasmic domain. This cleavage was demonstrated to be essential for subsequent regulated intramembrane proteolysis to activate the cognate SigI, in a manner similar to that of autoproteolysis-dependent activation of eukaryotic adhesion G protein-coupled receptors. These results indicate the presence of a unique prevalent type of autoproteolytic phenomenon in bacteria for signal transduction.

Cellulosomes are multi-enzymatic nanomachines that have been fine-tuned through evolution to efficiently deconstruct plant biomass. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between the various enzyme-borne dockerin modules and the multiple copies of the cohesin modules located on the scaffoldin subunit. Recently, designer cellulosome technology was established to provide insights into the architectural role of catalytic (enzymatic) and structural (scaffoldin) cellulosomal constituents for the efficient degradation of plant cell wall polysaccharides. Owing to advances in genomics and proteomics, highly structured cellulosome complexes have recently been unraveled, and the information gained has inspired the development of designer-cellulosome technology to new levels of complex organization. These higher-order designer cellulosomes have in turn fostered our capacity to enhance the catalytic potential of artificial cellulolytic complexes. In this chapter, methods to produce and employ such intricate cellulosomal complexes are reported.

The arsenal of genes that microbes express reflect the way in which they sense their environment. We have previously reported that the rumen microbiome composition and its coding capacity are different in animals having distinct feed efficiency states, even when fed an identical diet. Here, we reveal that many microbial populations belonging to the bacteria and archaea domains show divergent proteome production in function of the feed efficiency state. Thus, proteomic data serve as a strong indicator of host feed efficiency state phenotype, overpowering predictions based on genomic and taxonomic information. We highlight protein production of specific phylogenies associated with each of the feed efficiency states. We also find remarkable plasticity of the proteome both in the individual population and at the community level, driven by niche partitioning and competition. These mechanisms result in protein production patterns that exhibit functional redundancy and checkerboard distribution that are tightly linked to the host feed efficiency phenotype. By linking microbial protein production and the ecological mechanisms that act within the microbiome feed efficiency states, our present work reveals a layer of complexity that bears immense importance to the current global challenges of food security and sustainability.

Designer cellulosomes are self-assembled chimeric enzyme complexes that can be used to improve lignocellulosic biomass degradation. They are composed of a synthetic multimodular backbone protein, termed the scaffoldin, and a range of different chimeric docking enzymes that degrade polysaccharides. Over the years, several functional designer cellulosomes have been constructed. Since many parameters influence the efficiency of these multi-enzyme complexes, there is a need to optimise designer cellulosome architecture by testing combinatorial arrangements of docking enzyme and scaffoldin variants. However, the modular cloning procedures are tedious and cumbersome.
VersaTile is a combinatorial DNA assembly method, allowing the rapid construction and thus comparison of a range of modular proteins. Here, we present the extension of the VersaTile platform to facilitate the construction of designer cellulosomes. We have constructed a tile repository, composed of dockerins, cohesins, linkers, tags and enzymatically active modules. The developed toolbox allows us to efficiently create and optimise designer cellulosomes at an unprecedented speed. As a proof of concept, a trivalent designer cellulosome able to degrade the specific hemicellulose substrate, galactomannan, was constructed and optimised. The main factors influencing cellulosome efficiency were found to be the selected dockerins and linkers and the docking enzyme ratio on the scaffoldin. The optimised designer cellulosome was able to hydrolyse the galactomannan polysaccharide and release mannose and galactose monomers.
We have eliminated one of the main technical hurdles in the designer cellulosome field and anticipate the VersaTile platform to be a starting point in the development of more elaborate multi-enzyme complexes.

A novel member of the family 3 carbohydrate-binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3-0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3-0271 was crystallized and its three-dimensional structure was solved and refined to a resolution of 1.8 Å. In order to learn more about the role of this type of CBM3, a comparative study with its orthologue from Clostridium clariflavum (encoded by the Clocl_1192 gene) was performed, and the three-dimensional structure of CcCBM3-1192 was determined to 1.6 Å resolution. Carbohydrate binding by CcCBM3-1192 was found to be similar to that by CtCBM3-0271; both exhibited binding to xylan rather than to cellulose. Comparative structural analysis of the two CBM3s provided a clear functional correlation of structure and binding, in which the two CBM3s lack the required number of binding residues in their cellulose-binding strips and thus lack cellulose-binding capabilities. This is an enigma, as CtCBM3-0271 was reported to be a highly expressed protein when the bacterium was grown on cellulose. An additional unexpected finding was that CcCBM3-1192 does not contain the calcium ion that was considered to play a structural stabilizing role in the CBM3 family. Despite the lack of calcium, the five residues that form the calcium-binding site are conserved. The absence of calcium results in conformational changes in two loops of the CcCBM3-1192 structure. In this context, superposition of the non-calcium-binding CcCBM3-1192 with CtCBM3-0271 and other calcium-binding CBM3s reveals a much broader two-loop region in the former compared with CtCBM3-0271.

Many important proteins undergo pH-dependent conformational changes resulting in "on-off" switches for protein function, which are essential for regulation of life processes and have wide application potential. Here, we report a pair of cellulosomal assembly modules, comprising a cohesin and a dockerin from Clostridium acetobutylicum, which interact together following a unique pH-dependent switch between two functional sites rather than on-off states. The two cohesin-binding sites on the dockerin are switched from one to the other at pH 4.8 and 7.5 with a 180 degrees rotation of the bound dockerin. Combined analysis by nuclear magnetic resonance spectroscopy, crystal structure determination, mutagenesis, and isothermal titration calorimetry elucidates the chemical and structural mechanism of the pH-dependent switching of the binding sites. The pH-dependent dual-binding-site switch not only represents an elegant example of biological regulation but also provides a new approach for developing pH-dependent protein devices and biomaterials beyond an on-off switch for biotechnological applications.

The multi-enzyme cellulosome complex can mediate the valorization of lignocellulosic biomass into soluble sugars that can serve in the production of biofuels and valuable products. A potent bacterial chassis for the production of active cellulosomes displayed on the cell surface is the bacteriumLactobacillus plantarum, a lactic acid bacterium used in many applications. Here, we developed a methodological pipeline to produce improved designer cellulosomes, using a cell-consortium approach, whereby the different components self-assemble on the surface ofL. plantarum.The pipeline served as a vehicle to select and optimize the secretion efficiency of potent designer cellulosome enzyme components, to screen for the most efficient enzymatic combinations and to assess attempts to grow the engineered bacterial cells on wheat straw as a sole carbon source. Using this strategy, we were able to improve the secretion efficiency of the selected enzymes and to secrete a fully functional high-molecular-weight scaffoldin component. The adaptive laboratory process served to increase significantly the enzymatic activity of the most efficient cell consortium. Internal plasmid re-arrangement towards a higher enzymatic performance attested for the suitability of the approach, which suggests that this strategy represents an efficient way for microbes to adapt to changing conditions.

Clostridium saccharoperbutylacetonicum is a mesophilic, anaerobic, butanol-producing bacterium, originally isolated from soil. It was recently reported that C. saccharoperbutylacetonicum possesses multiple cellulosomal elements and would potentially form the smallest cellulosome known in nature. Its genome contains only eight dockerin-bearing enzymes, and its unique scaffoldin bears two cohesins (Cohs), three X2 modules, and two carbohydrate-binding modules (CBMs). In this study, all of the cellulosome-related modules were cloned, expressed, and purified. The recombinant cohesins, dockerins, and CBMs were tested for binding activity using enzyme-linked immunosorbent assay (ELISA)-based techniques. All the enzymes were tested for their comparative enzymatic activity on seven different cellulosic and hemicellulosic substrates, thus revealing four cellulases, a xylanase, a mannanase, a xyloglucanase, and a lichenase. All dockerin-containing enzymes interacted similarly with the second cohesin (Coh2) module, whereas Coh1 was more restricted in its interaction pattern. In addition, the polysaccharide-binding properties of the CBMs within the scaffoldin were examined by two complementary assays, affinity electrophoresis and affinity pulldown. The scaffoldin of C. saccharoperbutylacetonicum exhibited high affinity for cellulosic and hemicellulosic substrates, specifically to microcrystalline cellulose and xyloglucan. Evidence that supports substrate-dependent in vivo secretion of cellulosomes is presented. The results of our analyses contribute to a better understanding of simple cellulosome systems by identifying the key players in this minimalistic system and the binding pattern of its cohesin-dockerin interaction. The knowledge gained by our study will assist further exploration of similar minimalistic cellulosomes and will contribute to the significance of specific sets of defined cellulosomal enzymes in the degradation of cellulosic biomass.IMPORTANCE Cellulosome-producing bacteria are considered among the most important bacteria in both mesophilic and thermophilic environments, owing to their capacity to deconstruct recalcitrant plant-derived polysaccharides (and notably cellulose) into soluble saccharides for subsequent processing. In many ecosystems, the cellulosome-producing bacteria are particularly effective "first responders." The massive amounts of sugars produced are potentially amenable in industrial settings to further fermentation by appropriate microbes to biofuels, notably ethanol and butanol. Among the solvent-producing bacteria, Clostridium saccharoperbutylacetonicum has the smallest cellulosome system known thus far. The importance of investigating the building blocks of such a small, multifunctional nanomachine is crucial to understanding the fundamental activities of this efficient enzymatic complex.

Can molecular dynamics simulations predict the mechanical behavior of protein complexes? Can simulations decipher the role of protein domains of unknown function in large macromolecular complexes? Here, we employ a wide-sampling computational approach to demonstrate that molecular dynamics simulations, when carefully performed and combined with single-molecule atomic force spectroscopy experiments, can predict and explain the behavior of highly mechanostable protein complexes. As a test case, we studied a previously unreported homologue from Ruminococcus flavefaciens called X-module-Dockerin (XDoc) bound to its partner Cohesin (Coh). By performing dozens of short simulation replicas near the rupture event, and analyzing dynamic network fluctuations, we were able to generate large simulation statistics and directly compare them with experiments to uncover the mechanisms involved in mechanical stabilization. Our single-molecule force spectroscopy experiments show that the XDoc-Coh homologue complex withstands forces up to 1 nN at loading rates of 10(5) pN/s. Our simulation results reveal that this remarkable mechanical stability is achieved by a protein architecture that directs molecular deformation along paths that run perpendicular to the pulling axis. The X-module was found to play a crucial role in shielding the adjacent protein complex from mechanical rupture. These mechanisms of protein mechanical stabilization have potential applications in biotechnology for the development of systems exhibiting shear enhanced adhesion or tunable mechanics.

Rapid decomposition of plant biomass in soda lakes is associated with microbial activity of anaerobic cellulose-degrading communities. The alkaliphilic bacterium, Clostridium alkalicellulosi, is the single known isolate from a soda lake that demonstrates cellulolytic activity. This microorganism secretes cellulolytic enzymes that degrade cellulose under anaerobic and alkaliphilic conditions. A previous study indicated that the protein fraction of cellulose-grown cultures showed similarities in composition and size to known components of the archetypical cellulosome Clostridium thermocellum. Bioinformatic analysis of the C. alkalicellulosi draft genome sequence revealed 44 cohesins, organized into 22 different scaffoldins, and 142 dockerin-containing proteins. The modular organization of the scaffoldins shared similarities to those of C. thermocellum and Acetivibrio cellulolyticus, whereas some exhibited unconventional arrangements containing peptidases and oxidative enzymes. The binding interactions among cohesins and dockerins assessed by ELISA, revealed a complex network of cellulosome assemblies and suggested both cell-associated and cell-free systems. Based on these interactions, C. alkalicellulosi cellulosomal systems have the genetic potential to create elaborate complexes, which could integrate up to 105 enzymatic subunits. The alkalistable C. alkalicellulosi cellulosomal systems and their enzymes would be amenable to biotechnological processes, such as treatment of lignocellulosic biomass following prior alkaline pretreatment.

Background(Pseudo)Bacteroides cellulosolvens is a cellulolytic bacterium that produces the most extensive and intricate cellulosomal system known in nature. Recently, the elaborate architecture of the B. cellulosolvens cellulosomal system was revealed from analysis of its genome sequence, and the first evidence regarding the interactions between its structural and enzymatic components were detected in vitro. Yet, the understanding of the cellulolytic potential of the bacterium in carbohydrate deconstruction is inextricably linked to its high-molecular-weight protein complexes, which are secreted from the bacterium.ResultsThe current proteome-wide work reveals patterns of protein expression of the various cellulosomal components, and explores the signature of differential expression upon growth of the bacterium on two major carbon sourcescellobiose and microcrystalline cellulose. Mass spectrometry analysis of the bacterial secretome revealed the expression of 24 scaffoldin structural units and 166 dockerin-bearing components (mainly enzymes), in addition to free enzymatic subunits. The dockerin-bearing components comprise cell-free and cell-bound cellulosomes for more efficient carbohydrate degradation. Various glycoside hydrolase (GH) family members were represented among 102 carbohydrate-degrading enzymes, including the omnipresent, most abundant GH48 exoglucanase. Specific cellulosomal components were found in different molecular-weight fractions associated with cell growth on different carbon sources. Overall, microcrystalline cellulose-derived cellulosomes showed markedly higher expression levels of the structural and enzymatic components, and exhibited the highest degradation activity on five different cellulosic and/or hemicellulosic carbohydrates. The cellulosomal activity of B. cellulosolvens showed high degradation rates that are very promising in biotechnological terms and were compatible with the activity levels exhibited by Clostridium thermocellum purified cellulosomes.ConclusionsThe current research demonstrates the involvement of key cellulosomal factors that participate in the mechanism of carbohydrate degradation by B. cellulosolvens. The powerful ability of the bacterium to exhibit different degradation strategies on various carbon sources was revealed. The novel reservoir of cellulolytic components of the cellulosomal degradation machineries may serve as a pool for designing new cellulolytic cocktails for biotechnological purposes.

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum. We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin-cohesin interactions persist. Using allatom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as "beads on a string," we propose that cohesincohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing cellulosomes for industrial applications.

Bacteria can adjust their genetic programs via alternative σ factors to face new environmental pressures. Here, we analyzed a unique set of paralogous alternative σ factors, termed σ
^Is, which fine-tune the regulation of one of the most intricate cellulolytic systems in nature, the bacterial cellulosome, that is involved in degradation of environmental polysaccharides. We combined bioinformatics with experiments to decipher the regulatory networks of five σ
^Is in Clostridium thermocellum, the epitome of cellulolytic microorganisms, and one σ
^I in Pseudobacteroides cellulosolvens which produces the cellulosomal system with the greatest known complexity. Despite high homology between different σ
^Is, our data suggest limited cross-talk among them. Remarkably, the major cross-talk occurs within the main cellulosomal genes which harbor the same σ
^I-dependent promoter elements, suggesting a promoter-based mechanism to guarantee the expression of relevant genes. Our findings provide insights into the mechanisms used by σ
^Is to differentiate among their corresponding regulons, representing a comprehensive overview of the regulation of the cellulosome to date. Finally, we show the advantage of using a heterologous host system for analysis of multiple σ
^Is, since information generated by their analysis in their natural host can be misinterpreted owing to a cascade of interactions among the different σ
^Is.

Cellulose deconstruction is achieved in nature through two main enzymatic paradigms, i.e., free enzymes and enzymatic complexes (called cellulosomes). Gaining insights into the mechanism of action and synergy among the different cellulases is of high interest, notably in the field of renewable energy, and specifically, for the conversion of cellulosic biomass to soluble sugars, en route to biofuels. In this context, designer cellulosomes are artificially assembled, chimaeric protein complexes that are used as a tool to comparatively study cellulose degradation by different enzymatic paradigms, and could also serve to improve cellulose deconstruction. Various molecular biology techniques are employed in order to design and engineer the various components of designer cellulosomes. In this chapter, we describe the cloning processes through which the appropriate modules are selected and assembled at the molecular level.

Cell wall degradation by cellulases is extensively explored owing to its potential contribution to biofuel production. The cellulosome is an extracellular multienzyme complex that can degrade the plant cell wall very efficiently, and cellulosomal enzymes are therefore of great interest. The cellulosomal cellulases are defined as enzymes that contain a dockerin module, which can interact with a cohesin module contained in multiple copies in a noncatalytic protein, termed scaffoldin. The assembly of the cellulosomal cellulases into the cellulosomal complex occurs via specific protein–protein interactions. Cellulosome systems have been described initially only in several anaerobic cellulolytic bacteria. However, owing to ongoing genome sequencing and metagenomic projects, the discovery of novel cellulosome-producing bacteria and the description of their cellulosomal genes have dramatically increased in the recent years. In this chapter, methods for discovery of novel cellulosomal cellulases from a DNA sequence by bioinformatics and biochemical tools are described. Their biochemical characterization is also described, including both the enzymatic activity of the putative cellulases and their assembly into mature designer cellulosomes.

Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.

Heterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the "adaptor scaffoldin" strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia of Lactobacillus plantarum, a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all produced in vivo by the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw.

Transformation of cellulose into monosaccharides can be achieved by hydrolysis of the cellulose chains, carried out by a special group of enzymes known as cellulases. The enzymatic mechanism of cellulases is well described, but the role of non-enzymatic components of the cellulose-degradation machinery is still poorly understood, and difficult to measure using experiments alone. In this study, we use a comprehensive set of atomistic molecular dynamics simulations to probe the molecular details of binding of the family-3a carbohydrate-binding module (CBM3a) and the bacterial expansin protein (EXLX1) to a range of cellulose substrates. Our results suggest that CBM3a behaves in a similar way on both crystalline and amorphous cellulose, whereas binding of the dual-domain expansin protein depends on the substrate crystallinity, and we relate our computed binding modes to the experimentally measured features of CBM and expansin action on cellulose.

Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. Clostridium clariflavum is an anaerobic, thermophilic bacterium that produces the largest assembled cellulosome complex in nature to date, comprising three types of scaffoldins: a primary scaffoldin, ScaA; an adaptor scaffoldin, ScaB; and a cell surface anchoring scaffoldin, ScaC. This complex can contain 160 polysaccharide-degrading enzymes. In previous studies, we proposed potential types of cellulosome assemblies in C. clariflavum and demonstrated that these complexes are released into the extracellular medium. In the present study, we explored the disposition of the highly structured, four-tiered cell-anchored cellulosome complex of this bacterium. Four separate, integral cellulosome components were subjected to immunolabeling: ScaA, ScaB, ScaC, and the cellulosome's most prominent enzyme, GH48. Imaging of the cells by correlating scanning electron microscopy and three-dimensional (3D) super-resolution fluorescence microscopy revealed that some of the protuberance-like structures on the cell surface represent cellulosomes and that the components are highly colocalized and organized by a defined hierarchy on the cell surface. The display of the cellulosome on the cell surface was found to differ between cells grown on soluble or insoluble substrates. Cell growth on microcrystalline cellulose and wheat straw exhibited dramatic enhancement in the amount of cellulosomes displayed on the bacterial cell surface.IMPORTANCE Conversion of plant biomass into soluble sugars is of high interest for production of fermentable industrial materials, such as biofuels. Biofuels are a very attractive alternative to fossil fuels, both for recycling of agricultural wastes and as a source of sustainable energy. Cellulosomes are among the most efficient enzymatic degraders of biomass known to date, due to the incorporation of a multiplicity of enzymes into a potent, multifunctional nanomachine. The intimate association with the bacterial cell surface is inherent in its efficient action on lignocellulosic substrates, although this property has not been properly addressed experimentally. The dramatic increase in cellulosome performance on recalcitrant feedstocks is critical for the design of cost-effective processes for efficient biomass degradation.

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.

Background: (Pseudo) Bacteroides cellulosolvens is an anaerobic, mesophilic, cellulolytic, cellulosome-producing clostridial bacterium capable of utilizing cellulose and cellobiose as carbon sources. Recently, we sequenced the B. cellulosolvens genome, and subsequent comprehensive bioinformatic analysis, herein reported, revealed an unprecedented number of cellulosome-related components, including 78 cohesin modules scattered among 31 scaffoldins and more than 200 dockerin-bearing ORFs. In terms of numbers, the B. cellulosolvens cellulosome system represents the most intricate, compositionally diverse cellulosome system yet known in nature. Results: The organization of the B. cellulosolvens cellulosome is unique compared to previously described cellulosome systems. In contrast to all other known cellulosomes, the cohesin types are reversed for all scaffoldins i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. Many of the type II dockerin-bearing ORFs include X60 modules, which are known to stabilize type II cohesin-dockerin interactions. In the present work, we focused on revealing the architectural arrangement of cellulosome structure in this bacterium by examining numerous interactions between the various cohesin and dockerin modules. In total, we cloned and expressed 43 representative cohesins and 27 dockerins. The results revealed various possible architectures of cell-anchored and cell-free cellulosomes, which serve to assemble distinctive cellulosome types via three distinct cohesin-dockerin specificities: type I, type II, and a novel-type designated R (distinct from type III interactions, predominant in ruminococcal cellulosomes). Conclusions: The results of this study provide novel insight into the architecture and function of the most intricate and extensive cellulosomal system known today, thereby extending significantly our overall knowledge base of cellulosome systems and their components. The robust cellulosome system of B. cellulosolvens, with its unique binding specificities and reversal of cohesin–dockerin types, has served to amend our view of the cellulosome paradigm. Revealing new cellulosomal interactions and arrangements is critical for designing high-efficiency artificial cellulosomes for conversion of plant-derived cellulosic biomass towards improved production of biofuels.

The robust plant cell wall polysaccharide-degrading properties of anaerobic bacteria are harnessed within elegant, marcomolecular assemblages called cellulosomes, in which proteins of complementary activities amass on scaffold protein networks. Research efforts have focused and continue to focus on providing detailed mechanistic insights into cellulosomal complex assembly, topology, and function. The accumulated information is expanding our fundamental understanding of the lignocellulosic biomass decomposition process and enhancing the potential of engineered cellulosomal systems for biotechnological purposes. Ongoing biochemical studies continue to reveal unexpected functional diversity within traditional cellulase families. Genomic, proteomic, and functional analyses have uncovered unanticipated cellulosomal proteins that augment the function of the native and designer cellulosomes. In addition, complementary structural and computational methods are continuing to provide much needed insights on the influence of cellulosomal interdomain linker regions on cellulosomal assembly and activity.

Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzymeborne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens' cellulosome is assembled from a repertoire of 223 Doccontaining proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative sigma(I) factors has been demonstrated in Clostridium (Ruminiclostridium) thermocellum. In the present report, we provide experimental evidence demonstrating that the C. thermocellum cipA gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative sigma(I) factors and by the vegetative sigma(A) factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of C. thermocellum cipA are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved sigma I and sigma(A)-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, Clostridium straminisolvens, Clostridium clariflavum, Acetivibrio cellulolyticus, and Clostridium sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of Clostridium cellulovorans matches the predicted sigma(I)-dependent promoter identified in the present work rather than the previously proposed sigma(A) promoter. With the exception of C. cellulovorans, both sigma(I) and sigma(A) promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5=untranslated regions (5'-TRs).Furthermore, these 5'-Rs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components.IMPORTANCE Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of C. thermocellum and related cellulosome-producing bacteria en route to the consolidated bioprocessing of biomass.

Cellulosomes are multienzyme complexes that are produced by anaerobic cellulolytic bacteria for the degradation of lignocellulosic biomass. They comprise a complex of scaffoldin, which is the structural subunit, and various enzymatic subunits. The intersubunit interactions in these multienzyme complexes are mediated by cohesin and dockerin modules. Cellulosome-producing bacteria have been isolated from a large variety of environments, which reflects their prevalence and the importance of this microbial enzymatic strategy. In a given species, cellulosomes exhibit intrinsic heterogeneity, and between species there is a broad diversity in the composition and configuration of cellulosomes. With the development of modern technologies, such as genomics and proteomics, the full protein content of cellulosomes and their expression levels can now be assessed and the regulatory mechanisms identified. Owing to their highly efficient organization and hydrolytic activity, cellulosomes hold immense potential for application in the degradation of biomass and are the focus of much effort to engineer an ideal microorganism for the conversion of lignocellulose to valuable products, such as biofuels.

Ruminococcus champanellensis is a keystone species in the human gut that produces an intricate cellulosome system of various architectures. A variety of cellulosomal enzymes have been identified, which exhibit a range of hydrolytic activities on lignocellulosic substrates. We describe herein a unique R. champanellensis scaffoldin, ScaK, which is expressed during growth on cellobiose and comprises a cohesin module and a family 25 glycoside hydrolase (GH25). The GH25 is non-autolytic and exhibits lysozyme-mediated lytic activity against several bacterial species. Despite the narrow acidic pH curve, the enzyme is active along a temperature range from 2 to 85°C and is stable at very high temperatures for extended incubation periods. The ScaK cohesin was shown to bind selectively to the dockerin of a monovalent scaffoldin (ScaG), thus enabling formation of a cell-free cellulosome, whereby ScaG interacts with a divalent scaffodin (ScaA) that bears the enzymes either directly or through additional monovalent scaffoldins (ScaC and ScaD). The ScaK cohesin also interacts with the dockerin of a protein comprising multiple Fn3 domains that can potentially promote adhesion to carbohydrates and the bacterial cell surface. A cell-free cellulosomal GH25 lysozyme may provide a bacterial strategy to both hydrolyze lignocellulose and repel eventual food competitors and/or cheaters.

Cohesins and dockerins are complementary interacting protein modules that form stable and highly specific receptor–ligand complexes. They play a crucial role in the assembly of cellulose-degrading multi-enzyme complexes called cellulosomes and have potential applicability in several technology areas, including biomass conversion processes. Here, we describe several exceptional properties of cohesin–dockerin complexes, including their tenacious biochemical affinity, remarkably high mechanostability and a dual-binding mode of recognition that is contrary to the conventional lock-and-key model of receptor-ligand interactions. We focus on structural aspects of the dual mode of cohesin–dockerin binding, highlighting recent single-molecule analysis techniques for its explicit characterization.

Background: The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable beta-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. Results: The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Conclusions: Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.

Designer cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes. The largest designer cellulosome reported to date is a chimeric scaffoldin that contains 6 cohesins. This scaffoldin represented a technical limit of sorts, since adding another cohesin proved problematic, owing to resultant low expression levels, instability (cleavage) of the scaffoldin polypeptide, and limited numbers of available cohesin-dockerin specificities-the hallmark of designer cellulosomes. Nevertheless, increasing the number of enzymes integrated into designer cellulosomes is critical, in order to further enhance degradation of plant cell wall material. Adaptor scaffoldins comprise an intermediate type of scaffoldin that can both incorporate various enzymes and attach to an additional scaffoldin. Using this strategy, we constructed an efficient form of adaptor scaffoldin that possesses three type I cohesins for enzyme integration, a single type II dockerin for interaction with an additional scaffoldin, and a carbohydrate-binding module for targeting to the cellulosic substrate. In parallel, we designed a hexavalent scaffoldin capable of connecting to the adaptor scaffoldin by the incorporation of an appropriate type II cohesin. The resultant extended designer cellulosome comprised 8 recombinant enzymes-4 xylanases and 4 cellulases-thereby representing a potent enzymatic cocktail for solubilization of natural lignocellulosic substrates. The contribution of the adaptor scaffoldin clearly demonstrated that proximity between the two scaffoldins and their composite set of enzymes is crucial for optimized degradation. After 72 h of incubation, the performance of the extended designer cellulosome was determined to be approximately 70% compared to that of native cellulosomes.IMPORTANCE: Plant cell wall residues represent a major source of renewable biomass for the production of biofuels such as ethanol via breakdown to soluble sugars. The natural microbial degradation process, however, is inefficient for achieving cost-effective processes in the conversion of plant-derived biomass to biofuels, either from dedicated crops or human-generated cellulosic wastes. The accumulation of the latter is considered a major environmental pollutant. The development of designer cellulosome nanodevices for enhanced plant cell wall degradation thus has major impacts in the fields of environmental pollution, bioenergy production, and biotechnology in general. The findings reported in this article comprise a true breakthrough in our capacity to produce extended designer cellulosomes via synthetic biology means, thus enabling the assembly of higher-order complexes that can supersede the number of enzymes included in a single multienzyme complex.

Ruminococcus champanellensis is considered a keystone species in the human gut that degrades microcrystalline cellulose efficiently and contains the genetic elements necessary for cellulosome production. The basic elements of its cellulosome architecture, mainly cohesin and dockerin modules from scaffoldins and enzyme-borne dockerins, have been characterized recently. In this study, we cloned, expressed and characterized all of the glycoside hydrolases that contain a dockerin module. Among the 25 enzymes, 10 cellulases, 4 xylanases, 3 mannanases, 2 xyloglucanases, 2 arabinofuranosidases, 2 arabinanases and one β-glucanase were assessed for their comparative enzymatic activity on their respective substrates. The dockerin specificities of the enzymes were examined by ELISA, and 80 positives out of 525 possible interactions were detected. Our analysis reveals a fine-tuned system for cohesin-dockerin specificity and the importance of diversity among the cohesin-dockerin sequences. Our results imply that cohesin-dockerin pairs are not necessarily assembled at random among the same specificity types, as generally believed for other cellulosome-producing bacteria, but reveal a more organized cellulosome architecture. Moreover, our results highlight the importance of the cellulosome paradigm for cellulose and hemicellulose degradation by R.champanellensis in the human gut.

A cellulolytic fiber-degrading bacterium, Ruminococcus champanellensis, was isolated from human faecal samples, and its genome was recently sequenced. Bioinformatic analysis of the R.champanellensis genome revealed numerous cohesin and dockerin modules, the basic elements of the cellulosome, and manual sequencing of partially sequenced genomic segments revealed two large tandem scaffoldin-coding genes that form part of a gene cluster. Representative R.champanellensis dockerins were tested against putative cohesins, and the results revealed three different cohesin-dockerin binding profiles which implied two major types of cellulosome architectures: (i) an intricate cell-bound system and (ii) a simplistic cell-free system composed of a single cohesin-containing scaffoldin. The cell-bound system can adopt various enzymatic architectures, ranging from a single enzyme to a large enzymatic complex comprising up to 11 enzymes. The variety of cellulosomal components together with adaptor proteins may infer a very tight regulation of its components. The cellulosome system of the human gut bacterium R.champanellensis closely resembles that of the bovine rumen bacterium Ruminococcus flavefaciens. The two species contain orthologous gene clusters comprising fundamental components of cellulosome architecture. Since R.champanellensis is the only human colonic bacterium known to degrade crystalline cellulose, it may thus represent a keystone species in the human gut.

Dimeric avidins are a newly discovered subgroup of the avidin family that bind biotin with high affinity. Their dimeric configuration is a quaternary substructure of the classical tetrameric avidins which lacks the requirement of the critical Trp that defines the tetramer and dictates the tenacious interaction with biotin. Hoefavidin, derived from the bacterium Hoeflea phototrophica DFL-43(T), is the third characterized member of the dimeric avidin subfamily. Like the other members of this group, hoefavidin is a thermostable protein that contains a disulfide bridge between Cys57 and Cys88, thereby connecting and stabilizing the L3,4 and L5,6 loops. This represents a distinctive characteristic of dimeric avidins that compensates for the lack of Trp and enables their dimeric configuration. The X-ray structure of the intact hoefavidin revealed unique crystal packing generated by an octameric cylindrical structure wherein the C-termini segments of each monomer is introduced into the entrance of the biotin-binding site of an adjacent non-canonical monomer. This anomaly in the protein structure served as a lead toward the design of specific binding peptides. We screened for specific hoefavidin binding peptides derived from the C-terminal region and two peptides were obtained that bind a truncated form of hoefavidin (lacking the last 10 amino acids) with dissociation constants of 10(-5) M. The crystal structure of short hoefavidin complexed with a C-terminal derived peptide revealed the mode of binding. These peptides may form the basis of novel and reversible binders for dimeric avidins.

Background: Cellulosomal cohesin-dockerin types are reversed in Bacteroides cellulosolvens. Results: Combined crystallographic and computational approaches of a lone cohesin yielded a structural model of the cohesin-dockerin complex that was verified experimentally. Conclusion: The dockerin dual-binding mode is not exclusive to enzyme integration into cellulosomes; it also characterizes cell-surface attachment. Significance: This combined approach provides a platform for generating testable hypotheses of the high affinity cohesin-dockerin interaction. Cohesin-dockerin interactions orchestrate the assembly of one of nature's most elaborate multienzyme complexes, the cellulosome. Cellulosomes are produced exclusively by anaerobic microbes and mediate highly efficient hydrolysis of plant structural polysaccharides, such as cellulose and hemicellulose. In the canonical model of cellulosome assembly, type I dockerin modules of the enzymes bind to reiterated type I cohesin modules of a primary scaffoldin. Each type I dockerin contains two highly conserved cohesin-binding sites, which confer quaternary flexibility to the multienzyme complex. The scaffoldin also bears a type II dockerin that anchors the entire complex to the cell surface by binding type II cohesins of anchoring scaffoldins. In Bacteroides cellulosolvens, however, the organization of the cohesin-dockerin types is reversed, whereby type II cohesin-dockerin pairs integrate the enzymes into the primary scaffoldin, and type I modules mediate cellulosome attachment to an anchoring scaffoldin. Here, we report the crystal structure of a type I cohesin from B. cellulosolvens anchoring scaffoldin ScaB to 1.84-angstrom resolution. The structure resembles other type I cohesins, and the putative dockerin-binding site, centered at -strands 3, 5, and 6, is likely to be conserved in other B. cellulosolvens type I cohesins. Combined computational modeling, mutagenesis, and affinity-based binding studies revealed similar hydrogen-bonding networks between putative Ser/Asp recognition residues in the dockerin at positions 11/12 and 45/46, suggesting that a dual-binding mode is not exclusive to the integration of enzymes into primary cellulosomes but can also characterize polycellulosome assembly and cell-surface attachment. This general approach may provide valuable structural information of the cohesin-dockerin interface, in lieu of a definitive crystal structure.

Degradation of cellulose is of major interest in the quest for alternative sources of renewable energy, for its positive effects on environment and ecology, and for use in advanced biotechnological applications. Due to its microcrystalline organization, celluose is extremely difficult to degrade, although numerous microbes have evolved that produce the appropriate enzymes. The most efficient known natural cellulolytic system is produced by anaerobic bacteria, such as C. thermocellum, that possess a multi-enzymatic complex termed the cellulosome. Our laboratory has devised and developed the designer cellulosome concept, which consists of chimaeric scaffoldins for controlled incorporation of recombinant polysaccharide-degrading enzymes. Recently, we reported the creation of a combinatorial library of four cellulosomal modules comprising a basic chimaeric scaffoldin, i.e., a CBM and 3 divergent cohesin modules. Here, we employed selected members of this library to determine whether the position of defined cellulolytic enzymes is important for optimized degradation of a microcrystalline cellulosic substrate. For this purpose, 10 chimaeric scaffoldins were used for incorporation of three recombinant Thermobifida fusca enzymes: the processive endoglucanase Cel9A, endoglucanase Cel5A and exoglucanase Cel48A. In addition, we examined whether the characteristic properties of the T. fusca enzymes as designer cellulosome components are unique to this bacterium by replacing them with parallel enzymes from Clostridium thermocellum. The results support the contention that for a given set of cellulosomal enzymes, their relative position within a scaffoldin can be critical for optimal degradation of microcrystaline cellulosic substrates.

Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (K_a ∼10¹² M). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes.

Cellulosomes are large multicomponent cellulose-degrading assemblies found on the surfaces of cellulolytic microorganisms. Often containing hundreds of components, the self-assembly of cellulosomes is mediated by the ultra-high-affinity cohesin-dockerin interaction, which allows them to adopt the complex architectures necessary for degrading recalcitrant cellulose. Better understanding of how the cellulosome assembles and functions and what kinds of structures it adopts will further effort to develop industrial applications of cellulosome components, including their use in bioenergy production. Ruminococcus flavefaciens is a well-studied anaerobic cellulolytic bacteria found in the intestinal tracts of ruminants and other herbivores. Key to cellulosomal self-assembly in this bacterium is the dockerin ScaADoc, found on the non-catalytic structural subunit scaffoldin ScaA, which is responsible for assembling arrays of cellulose-degrading enzymes. This work expands on previous efforts by conducting a series of binding studies on ScaADoc constructs that contain mutations in their cohesin recognition interface, in order to identify which residues play important roles in binding. Molecular dynamics simulations were employed to gain insight into the structural basis for our findings. A specific residue pair in the first helix of ScaADoc, as well as a glutamate near the C-terminus, was identified to be essential for cohesin binding. By advancing our understanding of the cohesin binding of ScaADoc, this study serves as a foundation for future work to more fully understand the structural basis of cellulosome assembly in R. flavefaciens. 

Cellulosomes are massive cell-bound multienzyme complexes tethered by macromolecular scaffolds that coordinate the efforts of many anaerobic bacteria to hydrolyze plant cell-wall polysaccharides, which are a major untapped source of carbon and energy. Integration of cellulosomal components occurs via highly ordered protein protein interactions between cohesin modules, located in the scaffold, and dockerin modules, found in the enzymes and other cellulosomal proteins. The proposed cellulosomal architecture for Ruminococcus flavefaciens strain FD-1 consists of a major scaffoldin (ScaB) that acts as the backbone to which other components attach. It has nine cohesins and a dockerin with a fused X-module that binds to the cohesin on ScaE, which in turn is covalently attached to the cell wall. The ScaA dockerin binds to ScaB cohesins allowing more carbohydrate-active modules to be assembled. ScaC acts as an adaptor that binds to both ScaA and selected ScaB cohesins, thereby increasing the repertoire of dockerin-bearing proteins that integrate into the complex. In previous studies, a screen for novel cohesin-dockerin complexes was performed which led to the identification of a total of 58 probable cohesin-dockerin pairs. Four were selected for subsequent structural and biochemical characterization based on the quality of their expression and the diversity in their specificities. One of these is C12D22, which comprises the cohesin from the adaptor ScaC protein bound to the dockerin of a CBM-containing protein. This complex has been purified and crystallized, and data were collected to resolutions of 2.5 angstrom (hexagonal, P6(5)), 2.16 angstrom (orthorhombic, P2(1)2(1)2(1)) and 2.4 angstrom (orthorhombic, P2(1)2(1)2) from three different crystalline forms.

Background: A complex community of microorganisms is responsible for efficient plant cell wall digestion by many herbivores, notably the ruminants. Understanding the different fibrolytic mechanisms utilized by these bacteria has been of great interest in agricultural and technological fields, reinforced more recently by current efforts to convert cellulosic biomass to biofuels. Methodology/Principal Findings: Here, we have used a bioinformatics-based approach to explore the cellulosome-related components of six genomes from two of the primary fiber-degrading bacteria in the rumen: Ruminococcus flavefaciens (strains FD-1, 007c and 17) and Ruminococcus albus (strains 7, 8 and SY3). The genomes of two of these strains are reported for the first time herein. The data reveal that the three R. flavefaciens strains encode for an elaborate reservoir of cohesin- and dockerin-containing proteins, whereas the three R. albus strains are cohesin-deficient and encode mainly dockerins and a unique family of cell-anchoring carbohydrate-binding modules (family 37). Conclusions/ Significance: Our comparative genome-wide analysis pinpoints rare and novel strain-specific protein architectures and provides an exhaustive profile of their numerous lignocellulose-degrading enzymes. This work provides blueprints of the divergent cellulolytic systems in these two prominent fibrolytic rumen bacterial species, each of which reflects a distinct mechanistic model for efficient degradation of cellulosic biomass.

Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass-deconstruction enzymes or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e. multienzyme complexes) and noncellulosomal (i.e. 'free' enzyme systems). Plant-biomass-deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (noncellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective.

Ruminococcus flavefaciens is a cellulolytic bacterium found in the rumen of herbivores and produces one of the most elaborate and variable cellulosome systems. The structure of an R. flavefaciens protein (RfCohG, ZP06142108), representing a freestanding (non-cellulosomal) type III cohesin module, has been determined. A selenomethionine derivative with a C-terminal histidine tag was crystallized and diffraction data were measured to 2.44 Å resolution. Its structure was determined by single-wavelength anomalous dispersion, revealing eight molecules in the asymmetric unit. RfCohG exhibits the most complex among all known cohesin structures, possessing four -helical elements and a topographical protuberance on the putative dockerin-binding surface.

Dockerin modules of the cellulosomal enzyme subunits play an important role in the assembly of the cellulosome by binding tenaciously to cohesin modules of the scaffoldin subunit. A previously reported NMR-derived solution structure of the type-I dockerin module from Cel48S of Clostridium thermocellum, which utilized two-dimensional homonuclear ¹H-¹H NOESY and three-dimensional ¹⁵N-edited NOESY distance restraints, displayed substantial conformational differences from subsequent structures of dockerin modules in complex with their cognate cohesin modules, raising the question whether the source of the observed differences resulted from cohesin-induced structural rearrangements. Here, we determined the solution structure of the Cel48S type-I dockerin based on ¹⁵N- and ¹³C-edited NOESY-derived distance restraints. The structure adopted a fold similar to X-ray crystal structures of dockerin modules in complex with their cohesin partners. A unique cis-peptide bond between Leu-65 and Pro-66 in the Cel48S type-I dockerin module was also identified in the present structure. Our structural analysis of the Cel48S type-I dockerin module indicates that it does not undergo appreciable cohesin-induced structural alterations but rather assumes an inherent calcium-dependent cohesin-primed conformation.

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand-receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand-receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600-750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass.

Although bacteria adhere to many different types of surfaces present in their habitat, this review focuses on bacterial adhesion to animal cells and tissues as a first step in the ability of pathogens to colonize and subsequently cause tissue damage. Accordingly, basic principles that govern the interaction of bacterial adhesins to their cognate receptors on animal cells are presented, such as fimbriae as adhesin structures. Significantly, we discuss the types of receptor-adhesin relationship, the phenomenon of multiple adhesins each specific for distinct receptors produced by pathogenic clones, the identity of glycoconjugates as receptors for lectins that serve as adhesins, and the interaction of bacterial adhesins with the extracellular matrix on animal tissues. Finally, a specific section is devoted to recent developments in preventing or treating infections by blocking bacterial adhesion to animal cells. In this context, a review of the different approaches of antiadhesion therapy is discussed, including the use of receptor and adhesin analogs, dietary constituents, sub-lethal concentrations of antibiotics, and adhesin-based vaccines. A discussion and summary of these topics focus on the in vivo data, including human trials, whereby plant extracts are used as a source of antiadhesion agents to prevent or treat urinary tract infections caused by Escherichia coli and infectious gastritis or peptic ulcer diseases induced by Helicobacter pylori and to maintain oral health.

Cellulosomes are multi-enzyme complexes produced by anaerobic bacteria for the efficient deconstruction of plant cell wall polysaccharides. The assembly of enzymatic subunits onto a central non-catalytic scaffoldin subunit is mediated by a highly specific interaction between the enzyme-bearing dockerin modules and the resident cohesin modules of the scaffoldin, which affords their catalytic activities to work synergistically. The scaffoldin also imparts substrate-binding and bacterial-anchoring properties, the latter of which involves a second cohesin-dockerin interaction. Recent structure-function studies reveal an ever-growing array of unique and increasingly complex cohesin-dockerin complexes and cellulosomal enzymes with novel activities. A 'build' approach involving multimodular cellulosomal segments has provided a structural model of an organized yet conformationally dynamic supramolecular assembly with the potential to form higher order structures.

The cellulosome of the cellulolytic bacterium Clostridium thermocellum has a structural multi-modular protein called CipA (cellulosome-integrating protein A) that includes nine enzyme-binding cohesin modules and a family 3 cellulose-binding module (CBM3a). In the CipA protein, the CBM3a module is located between the second and third cohesin modules and is connected to them via proline/threonine-rich linkers. The structure of CBM3a with portions of the C- and N-terminal flanking linker regions, CBM3a-L, has been determined to a resolution of 1.98 Å. The structure is a β-sandwich with a structural Ca²⁺ ion. The structure is consistent with the previously determined CipA CBM structure; however, the structured linker regions provide a deeper insight into the overall cellulosome structure and assembly.

The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (K_d = 20.83 nM) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.

Clostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.

The cellulosome is a large extracellular multi-enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme-integrating ScaA scaffoldin into the cohesin-bearing ScaB adaptor scaffoldin. In the absence of a high-resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N-terminal Trp and a C-terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NAIR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca2+-bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well-defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module.

Cellulosomes are modular, super-molecular enzyme systems secreted by anaerobic bacteria to degrade recalcitrant plant cell wall polysaccharides to simple sugars. The components of this molecular machine include a manifold of enzymatic units and carbohydrate-binding modules, as well as the cohesin and dockerin modules responsible for the system's unique self-assembly and connectivity Known to be one of the highest affinity protein protein interactions discovered to date, the cohesin dockerin interaction is also the key to engineering the cellulosome. By mixing and matching the spare parts of the cellulosome and connecting them via cohesins and dockerins, biotechnologists have begun pursuing the concept of 'designer cellulosomes', which one day may be an important contributor to production of sustainable biomass-derived fuels. By incorporating molecules from other systems into the cellulosome paradigm, nanotechnologists have begun to harness the potential of this molecular construction kit to create diverse, self-assembling nanostructures for a broad variety of biotechnological applications.

The interaction between the cohesin and dockerin modules serves to attach cellulolytic enzymes (carrying dockerins) to non-catalytic scaffoldin units (carrying multiple cohesins) in cellulosome, a multienzyme plant cell-wall degrading complex. This interaction is species-specific, for example, the enzyme-borne dockerin from Clostridium thermocellum bacteria binds to scaffoldin cohesins from the same bacteria but not to cohesins from Clostridium cellulolyticum and vice versa. We studied the role of interface residues, contributing either to affinity or specificity, by mutating these residues on the cohesin counterpart from C. thermocellum. The high affinity of the cognate interactions makes it difficult to evaluate the effect of these mutations by common methods used for measuring protein-protein interactions, especially when subtle discrimination between the mutants is needed. We described in this article an approach based on indirect enzyme-linked immunosorbent assay (ELISA) that is able to detect differences in binding between the various cohesin mutants, whereas surface plasmon resonance and standard ELISA failed to distinguish between high-affinity interactions. To be able to calculate changes in energy of binding (ΔΔG) and dissociation constants (K _d) of mutants relative to wild type, a pre-equilibrium step was added to the standard indirect ELISA procedure. Thus, the cohesin-dockerin interaction under investigation occurs in solution rather than between soluble and immobilized proteins. Unbound dockerins are then detected through their interaction with immobilized cohesins. Because our method allows us to assess the effect of mutations on particularly tenacious protein-protein interactions much more accurately than do other prevalent methods used to measure binding affinity, we therefore suggest this approach as a method of choice for comparing relative binding in high-affinity interactions.

In Ruminococcus flavefaciens, a predominant fibre-degrading bacterium found in ruminants, cellulosomal proteins are anchored to the bacterial cell wall through a relatively small ScaE scaffoldin which includes a single type III cohesin. The cotton-binding protein CttA consists of two cellulose-binding modules and a C-terminal modular pair (XDoc) comprising an X-module and a contiguous dockerin, which exhibits high affinity towards the ScaE cohesin. Seleno-l-methionine-labelled derivatives of the ScaE cohesin module and the XDoc from CttA have been expressed, copurified and cocrystallized. The crystals belonged to the tetragonal space group P4₃2₁2, with unit-cell parameters a = b = 78.7, c = 203.4 Å, and the unit cell contains a single cohesin-XDoc complex in the asymmetric unit. The diffraction data were phased to 2.0 Å resolution using the anomalous signal of the Se atoms.

The crystal structure of the family 3b carbohydrate-binding module (CBM3b) of the cellulosomal multimodular hydrolytic enzyme cellobiohydrolase 9A (Cbh9A) from Clostridium thermocellum has been determined. Cbh9A CBM3b crystallized in space group P41 with four molecules in the asymmetric unit and diffracted to a resolution of 2.20 Å using synchrotron radiation. The structure was determined by molecular replacement using C. thermocellum Cel9V CBM3b′ (PDB entry 2wnx) as a model. The C. thermocellum Cbh9A CBM3b molecule forms a nine-stranded antiparallel β-sandwich similar to other family 3 carbo-hydrate-binding modules (CBMs). It has a short planar array of two aromatic residues that are assumed to bind cellulose, yet it lacks the ability to bind cellulose. The molecule contains a shallow groove of unknown function that characterizes other family 3 CBMs with high sequence homology. In addition, it contains a calcium-binding site formed by a group of amino-acid residues that are highly conserved in similar structures. After determination of the three-dimensional structure of Cbh9A CBM3b, the site-specific N126W mutant was produced with the intention of enhancing the cellulose-binding ability of the CBM. Cbh9A CBM3b^N126W crystallized in space group P41212, with one molecule in the asymmetric unit. The crystals diffracted to 1.04 Å resolution using synchrotron radiation. The structure of Cbh9A CBM3b ^N126W revealed incorporation of the mutated Trp126 into the putative cellulose-binding strip of residues. Cellulose-binding experiments demonstrated the ability of Cbh9A CBM3b^N126W to bind cellulose owing to the mutation. This is the first report of the engineered conversion of a non-cellulose-binding CBM3 to a binding CBM3 by site-directed mutagenesis. The three-dimensional structure of Cbh9A CBM3b^N126W provided a structural correlation with cellulose-binding ability, revealing a longer planar array of definitive cellulose-binding residues.

Clostridium thermocellum wild-type strain YS is an anaerobic, thermophilic, cellulolytic bacterium capable of directly converting cellulosic substrates into ethanol. Strain YS and a derived cellulose adhesion-defective mutant strain, AD2, played pivotal roles in describing the original cellulosome concept. We present their draft genome sequences.

Background: Microbial degradation of plant cell walls and its conversion to sugars and other byproducts is a key step in the carbon cycle on Earth. In order to process heterogeneous plant-derived biomass, specialized anaerobic bacteria use an elaborate multi-enzyme cellulosome complex to synergistically deconstruct cellulosic substrates. The cellulosome was first discovered in the cellulolytic thermophile, Clostridium thermocellum, and much of our knowledge of this intriguing type of protein composite is based on the cellulosome of this environmentally and biotechnologically important bacterium. The recently sequenced genome of the cellulolytic mesophile, Acetivibrio cellulolyticus, allows detailed comparison of the cellulosomes of these two select cellulosome-producing bacteria.Results: Comprehensive analysis of the A. cellulolyticus draft genome sequence revealed a very sophisticated cellulosome system. Compared to C. thermocellum, the cellulosomal architecture of A. cellulolyticus is much more extensive, whereby the genome encodes for twice the number of cohesin- and dockerin-containing proteins. The A. cellulolyticus genome has thus evolved an inflated number of 143 dockerin-containing genes, coding for multimodular proteins with distinctive catalytic and carbohydrate-binding modules that play critical roles in biomass degradation. Additionally, 41 putative cohesin modules distributed in 16 different scaffoldin proteins were identified in the genome, representing a broader diversity and modularity than those of Clostridium thermocellum. Although many of the A. cellulolyticus scaffoldins appear in unconventional modular combinations, elements of the basic structural scaffoldins are maintained in both species. In addition, both species exhibit similarly elaborate cell-anchoring and cellulosome-related gene- regulatory elements.Conclusions: This work portrays a particularly intricate, cell-surface cellulosome system in A. cellulolyticus and provides a blueprint for examining the specific roles of the various cellulosomal components in the degradation of complex carbohydrate substrates of the plant cell wall by the bacterium.

β-Xylosidases are hemicellulases that hydrolyze short xylo-oligosaccharides into xylose units, thus complementing endoxylanase degradation of the hemicellulose component of lignocellulosic substrates. Here, we describe the cloning, characterization, and kinetic analysis of a glycoside hydrolase family 43 β-xylosidase (Xyl43A) from the aerobic cellulolytic bacterium, Thermobifida fusca. Temperature and pH optima of 55-60 °C and 5.5-6, respectively, were determined. The apparent K _m value was 0.55 mM, using p-nitrophenyl xylopyranoside as substrate, and the catalytic constant (k _cat) was 6.72 s ^-1. T. fusca Xyl43A contains a catalytic module at the N terminus and an ancillary module (termed herein as Module-A) of undefined function at the C terminus. We expressed the two recombinant modules independently in Escherichia coli and examined their remaining catalytic activity and binding properties. The separation of the two Xyl43A modules caused the complete loss of enzymatic activity, whereas potent binding to xylan was fully maintained in the catalytic module and partially in the ancillary Module-A. Nondenaturing gel electrophoresis revealed a specific noncovalent coupling of the two modules, thereby restoring enzymatic activity to 66.7% (relative to the wild-type enzyme). Module-A contributes a phenylalanine residue that functions as an essential part of the active site, and the two juxtaposed modules function as a single functional entity.

Background: Microorganisms employ a multiplicity of enzymes to efficiently degrade the composite structure of plant cell wall cellulosic polysaccharides. These remarkable enzyme systems include glycoside hydrolases (cellulases, hemicellulases), polysaccharide lyases, and the carbohydrate esterases. To accomplish this challenging task, several strategies are commonly observed either separately or in combination. These include free enzyme systems, multifunctional enzymes, and multi-enzyme self-assembled designer cellulosome complexes. Results: In order to compare these different paradigms, we employed a synthetic biology approach to convert two different cellulases from the free enzymatic system of the well-studied bacterium, Thermobifida fusca, into bifunctional enzymes with different modular architectures. We then examined their performance compared to those of the combined parental free-enzyme and equivalent designer-cellulosome systems. The results showed that the cellulolytic activity displayed by the different architectures of the bifunctional enzymes was somewhat inferior to that of the wild-type free enzyme system. Conclusions: The activity exhibited by the designer cellulosome system was equal or superior to that of the free system, presumably reflecting the combined proximity of the enzymes and high flexibility of the designer cellulosome components, thus enabling efficient enzymatic activity of the catalytic modules.

Viruses are the most abundant biological entities on the planet and play an important role in balancing microbes within an ecosystem and facilitating horizontal gene transfer. Although bacteriophages are abundant in rumen environments, little is known about the types of viruses present or their interaction with the rumen microbiome. We undertook random pyrosequencing of virus-enriched metagenomes (viromes) isolated from bovine rumen fluid and analysed the resulting data using comparative metagenomics. A high level of diversity was observed with up to 28000 different viral genotypes obtained from each environment. The majority (~78%) of sequences did not match any previously described virus. Prophages outnumbered lytic phages approximately 2:1 with the most abundant bacteriophage and prophage types being associated with members of the dominant rumen phyla (Firmicutes and Proteobacteria). Metabolic profiling based on SEED subsystems revealed an enrichment of sequences with putative functional roles in DNA and protein metabolism, but a surprisingly low proportion of sequences assigned to carbohydrate and amino acid metabolism. We expanded our analysis to include previously described metagenomic data and 14 reference genomes. Clustered regularly interspaced short palindromic repeats (CRISPR) were detected in most of the microbial genomes, suggesting previous interactions between viral and microbial communities.

Background: The bovine rumen maintains a diverse microbial community that serves to break down indigestible plant substrates. However, those bacteria specifically adapted to degrade cellulose, the major structural component of plant biomass, represent a fraction of the rumen microbiome. Previously, we proposed scaC as a candidate for phylotyping Ruminococcus flavefaciens, one of three major cellulolytic bacterial species isolated from the rumen. In the present report we examine the dynamics and diversity of scaC-types both within and between cattle temporally, following a dietary switch from corn-silage to grass-legume hay. These results were placed in the context of the overall bacterial population dynamics measured using the 16S rRNA. Principal Findings: As many as 117 scaC-types were estimated, although just nineteen were detected in each of three rumens tested, and these collectively accounted for the majority of all types present. Variation in scaC populations was observed between cattle, between planktonic and fiber-associated fractions and temporally over the six-week survey, and appeared related to scaC phylogeny. However, by the sixth week no significant separation of scaC populations was seen between animals, suggesting enrichment of a constrained set of scaC-types. Comparing the amino-acid translation of each scaC-type revealed sequence variation within part of the predicted dockerin module but strong conservation in the N-terminus, where the cohesin module is located. Conclusions: The R. flavefaciens species comprises a multiplicity of scaC-types in-vivo. Enrichment of particular scaC-types temporally, following a dietary switch, and between fractions along with the phylogenetic congruence suggests that functional differences exist between types. Observed differences in dockerin modules suggest at least part of the functional heterogeneity may be conferred by scaC. The polymorphic nature of scaC enables the relative distribution of R. flavefaciens strains to be examined and represents a gene-centric approach to investigating the intraspecific adaptation of an important specialist population.

The potent cellulose-binding modules of cellulosomal scaffoldin subunits belong to the greater family of carbohydrate-binding modules (CBMs). They have generally been classified as belonging to family 3a on the basis of sequence similarity. They form nine-stranded Β-sandwich structures with jelly-roll topology. The members of this family possess on their surface a planar array of aromatic amino-acid residues (known as the linear strip) that form stacking interactions with the glucose rings of cellulose chains and have a conserved Ca²⁺-binding site. Intriguingly, the CBM3 from scaffoldin A (ScaA) of Bacteroides cellulosolvens exhibits alterations in sequence that make it more similar to the CBMs of free cellulolytic enzymes, which are classified into CBM family 3b. X-ray structural analysis was undertaken in order to examine the structural consequences of the sequence changes and the consequent family affiliation. The CBM3 crystallized in space group I4122 with one molecule in the asymmetric unit, yielding diffraction to a resolution of 1.83 Å using X-ray synchrotron radiation. Compared with the known structures of other scaffoldin-borne CBMs, a sequence insertion and deletion appear to compensate for each other as both contained an aromatic residue that is capable of contributing to cellulose binding; hence, even though there are alterations in the composition and localization of the aromatic residues in the linear strip its binding ability was not compromised. Interestingly, no Ca²⁺ ions were detected in the conserved calcium-binding site, although the module was properly folded; this suggests that the structural role of Ca²⁺ is less important than originally supposed. These observations indicate that despite their conserved function the scaffoldin-borne CBMs are more diverse in their sequences and structures than previously assumed.

Understanding the molecular-level mechanisms that enzymes employ to deconstruct plant cell walls is a fundamental scientific challenge with significant ramifications for renewable fuel production from biomass. In nature, bacteria and fungi use enzyme cocktails that include processive and non-processive cellulases and hemicellulases to convert cellulose and hemicellulose to soluble sugars. Catalyzed by an accelerated biofuels R&D portfolio, there is now a wealth of new structural and experimental insights related to cellulases and the structure of plant cell walls. From this background, computational approaches commonly used in other fields are now poised to offer insights complementary to experiments designed to probe mechanisms of plant cell wall deconstruction. Here we outline the current status of computational approaches for a collection of critical problems in cellulose deconstruction. We discuss path sampling methods to measure rates of elementary steps of enzyme action, coarse-grained modeling for understanding macromolecular, cellulosomal complexes, methods to screen for enzyme improvements, and studies of cellulose at the molecular level. Overall, simulation is a complementary tool to understand carbohydrate-active enzymes and plant cell walls, which will enable industrial processes for the production of advanced, renewable fuels.

Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca²⁺-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K_A) of 7.7 × 10⁸ M^-1, calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum β-glucosidase, purified using this approach, was tested and found to be similar to that of a β-glucosidase preparation (without the ΔDoc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca²⁺-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification.

The thermostability of endoglucanase (E.C. 3.2.1.4) Cel8A, a major component of the cellulosome complex from Clostridium thermocellum, was significantly enhanced using a directed evolution strategy. To ensure that thermostability would not compromise enzyme activity, a two-step screening strategy was employed that involved consecutive activity and thermostability assays. We have combined three of the mutations from the thermostability screen to obtain a Cel8A variant with a significant increase in thermal resistance without substantial alteration of kinetic parameters. One of the three mutations (S329G) provided the highest contribution to enzyme stability. This single mutation served to increase the T_m by 7.0°C and the half-life of activity by eight fold at 85°C. Site-saturation mutagenesis at position 329 revealed that only the glycine residue could confer thermostability. The structural changes responsible for the properties of the mutant enzymes are discussed.

Genome analysis of the Gram-positive cellulolytic bacterium Clostridium thermocellum revealed the presence of multiple negative regulators of alternative σ factors. Nine of the deduced proteins share a strong similarity in their N-terminal sequences to the Bacillus subtilis membrane-associated anti-σ^I factor RsgI and have an unusual domain organization. In six RsgI-like proteins, the C-terminal sequences contain predicted carbohydrate-binding modules. Three of these modules were overexpressed and shown to bind specifically to cellulose and/or pectin. Bioinformatic analysis of >1200 bacterial genomes revealed that the C. thermocellum RsgI-like proteins are unique to this species and are not present in other cellulolytic clostridial species (e.g. Clostridium cellulolyticum and Clostridium papyrosolvens). Eight of the nine genes encoding putative C. thermocellum RsgI-like anti-σ factors form predicted bicistronic operons, in which the first gene encodes a putative alternative σ factor, similar to B. subtilis σ^I, but lacking in one of its domains. These observations suggest a novel carbohydrate-sensing mechanism in C. thermocellum, whereby the presence of polysaccharide biomass components is detected extracellularly and the signal is transmitted intracellularly, resulting in the disruption of the interaction between RsgI-like proteins and σ^I-like factors, the latter of which serve to activate appropriate genes encoding proteins involved in cellulose utilization.

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and similar to 1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.

The anaerobic, thermophilic cellulolytic bacterium Clostridium thermocellum is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from C. thermocellum. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and vice versa. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome. C. thermocellum is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.

Cellulosomes are large, multienzyme, plant cell wall-degrading protein complexes found affixed to the surface of a variety of anaerobic microbes. The core of the cellulosome is a noncatalytic scaffoldin protein, which contains several type-I cohesin modules that bind type-I dockerin-containing enzymatic subunits, a cellulose-binding module, an X module, and a type-II dockerin that interacts with type-II cohesin-containing cell surface proteins. The unique arrangement of the enzymatic subunits in the cellulosome complex, made possible by the scaffoldin subunit, promotes enhanced substrate degradation relative to the enzymes free in solution. Despite representative high-resolution structures of all of the individual modules of the cellulosome, this mechanism of enzymatic synergy remains poorly understood. Consequently, a model of the entire cellulosome and a detailed picture of intermodular contacts will provide more detailed insight into cellulosome activity. Toward this goal, we have solved the structure of a multimodular heterodimeric complex from Clostridium thermocellum composed of the type-II cohesin module of the cell surface protein SdbA bound to a trimodular C-terminal fragment of the scaffoldin subunit CipA to a resolution of 1.95 Å. The linker that connects the ninth type-I cohesin module and the X module has elevated temperature factors, reflecting an inherent flexibility within this region. Interestingly, a novel dimer interface was observed between CipA and a second, symmetry-related CipA molecule within the crystal structure, mediated by contacts between a type-I cohesin and an X module of a symmetry mate, resulting in two intertwined scaffoldins. Sedimentation velocity experiments confirmed that dimerization also occurs in solution. These observations support the intriguing possibility that individual cellulosomes can associate with one another via inter-scaffoldin interactions, which may play a role in the mechanism of action of the complex.

The cellulosome is an intriguing multienzyme complex found in cellulolytic bacteria that plays a key role in the degradation of plant cell-wall polysaccharides. In Ruminococcus flavefaciens, a predominant fiber-degrading bacterium found in ruminants, the cellulosome is anchored to the bacterial cell wall through a relatively short ScaE scaffoldin. Determination of the crystal structure of the lone type-III ScaE cohesin from R. flavefaciens (Rf-CohE) was initiated as a part of a structural effort to define cellulosome assembly. The structure was determined at 1.95 Å resolution by single-wavelength anomalous diffraction. This is the first detailed description of a crystal structure for a type-III cohesin, and its features were compared with those of the known type-I and type-II cohesin structures. The Rf-CohE module folds into a ninestranded β-sandwich with jellyroll topology, typically observed for cohesins, and includes two β-flaps in the midst of β-strands 4 and 8, similar to the type-II cohesin structures. However, the presence in Rf-CohE of an additional 13-residue a-helix located between β-strands 8 and 9 represents a dramatic divergence from other known cohesin structures. The prominent α-helix is enveloped by an extensive N-terminal loop, not observed in any other known cohesin, which embraces the helix presumably enhancing its stability. A planar surface at the upper portion of the front face of the molecule, bordered by β-flap 8, exhibits plausible dimensions and exposed amino acid residues to accommodate the dockerin-binding site.

Cellulosome complexes comprise an intercalated set of multimodular dockerin-containing enzymatic subunits connected to cohesin-containing nonenzymatic subunits called scaffoldins. The adjoining modules in each cellulosomal subunit are interconnected by a variety of linker segments of different lengths and composition. The exact role of the cellulosomal linkers has yet to be described, although it is assumed that they contribute to the architecture and action of the cellulosome by providing the protein subunits with flexibility and by providing spacers between the enzymatic modules that could enhance interactions with the cellulose substrate. Here we present four crystal structures of Acetivibrio cellulolyticus cellulosomal type II cohesins with linker extensions. Two of the structures represent two different crystal forms (trigonal and orthorhombic) of the same N-terminal cohesin module (CohB1) together with its full (6-residue) native C-terminal linker, derived from scaffoldin B. The other two structures belong to the adjacent (second) cohesin module (CohB2), each of which was crystallized with the same 6-residue linker segment, but now positioned at the N-terminus and with either a truncated (5-residue) or a full-length (45-residue) C-terminal linker, respectively. Comparison between the two CohB1 structures revealed significant differences in the conformation of their equivalent C-terminal linker segment. In one crystal form a helical conformation was observed, as opposed to an extended conformation in the other. The CohB2 structures also displayed diverse conformations in their linker segments. In these structures, different linker conformations were observed in the individual molecules within the asymmetric unit of each structure. This conformational diversity implies that the linkers may adopt alternative conformations in their natural environment, consistent with varying environmental conditions. The findings suggest that linkers can play an important role in the assembly, dynamics and function of the cellulosomal components.

Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification, A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure, A complementary Doc module was then fused to a model protein target: xylanase T-6 from Ceobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module.

The high-affinity cohesin-dockerin interaction was originally discovered as modular components, which mediate the assembly of the various subunits of the multienzyme cellulosome complex that characterizes some cellulolytic bacteria. Until recently, the presence of cohesins and dockerins within a bacterial proteome was considered a definitive signature of a cellulosome-producing bacterium. Widespread genome sequencing has since revealed a wealth of putative cohesin- and dockerin-containing proteins in Bacteria, Archaea, and in primitive eukaryotes. The newly identified modules appear to serve diverse functions that are clearly distinct from the classical cellulosome archetype, and the vast majority of parent proteins are not predicted glycoside hydrolases. In most cases, only a few such genes have been identified in a given microorganism, which encode proteins containing but a single cohesin and/or dockerin. In some cases, one or the other module appears to be missing from a given species, and in other cases both modules occur within the same protein. This review provides a bioinformatics-based survey of the current status of cohesin- and dockerin-like sequences in species from the Bacteria, Archaea, and Eukarya. Surprisingly, many identified modules and their parent proteins are clearly unrelated to cellulosomes. The cellulosome paradigm may thus be the exception rather than the rule for bacterial, archaeal, and eukaryotic employment of cohesin and dockerin modules.

The genome of the opportunistic pathogen Clostridium perfringens encodes a large number of secreted glycoside hydrolases. Their predicted activities indicate that they are involved in the breakdown of complex carbohydrates and other glycans found in the mucosal layer of the human gastrointestinal tract, within the extracellular matrix, and on the surface of host cells. One such group of these enzymes is the family 84 glycoside hydrolases, which has predicted hyaluronidase activity and comprises five members [C. perfringens glycoside hydrolase family 84 (CpGH84) A-E]. The first identified member, CpGH84A, corresponds to the μ-toxin whose modular architecture includes an N-terminal catalytic domain, four family 32 carbohydrate-binding modules, three FIVAR modules of unknown function, and a C-terminal putative calcium-binding module. Here, we report the solution NMR structure of the C-terminal modular pair from the μ-toxin. The three-helix bundle FIVAR module displays structural homology to a heparin-binding module within the N-terminal of the a C protein from group B Streptoccocus. The C-terminal module has a typical calcium-binding dockerin fold comprising two anti-parallel helices that form a planar face with EF-hand calcium-binding loops at opposite ends of the module. The size of the helical face of the μ-toxin dockerin module is approximately equal to the planar region recently identified on the surface of a cohesin-like X82 module of CpGH84C. Size-exclusion chromatography and heteronuclear NMR-based chemical shift mapping studies indicate that the helical face of the dockerin module recognizes the CpGH84C X82 module. These studies represent the structural characterization of a noncellulolytic dockerin module and its interaction with a cohesin-like X82 module. Dockerin/X82-mediated enzyme complexes may have important implications in the pathogenic properties of C. perfringens.

The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the μ-toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction (K _a = 1.44 × 10¹¹M^-1) between the X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrate-active enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The μ-toxin dockerin module in this complex is positioned ≈180° relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.

The cellulosome is a multiprotein complex, produced primarily by anaerobic microorganisms, which functions to degrade lignocellulosic materials. An important topic of current debate is whether cellulosomal systems display greater ability to deconstruct complex biomass materials (e.g. plant cell walls) than nonaggregated enzymes, and in so doing would be appropriate for improved, commercial bioconversion processes. To sufficiently understand the complex macromolecular processes between plant cell wall polymers, cellulolytic microbes, and their secreted enzymes, a highly concerted research approach is required. Adaptation of existing biophysical techniques and development of new science tools must be applied to this system. This review focuses on strategies likely to permit improved understanding of the bacterial cellulosome using biophysical approaches, with emphasis on advanced imaging and computational techniques.

The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules. We hypothesize that many of these features are adaptations that facilitate depolymerization of complex polysaccharides in the marine environment. This is the first sequenced genome of a marine bacterium that can degrade plant cell walls, an important component of the carbon cycle that is not well-characterized in the marine environment.

The cellulosome is an intricate multienzyme complex, designed for efficient degradation of plant cell wall polysaccharides, notably cellulose. The supramolecular cellulosome architecture in different bacteria is the consequence of the types and specificities of the interacting cohesin and dockerin modules, borne by the different cellulosomal subunits. In this study, we describe a microarray system for determining cohesin-dockerin specificity, which allows global comparison among the interactions between various members of these two complementary families of interacting protein modules. Matching recombinant fusion proteins were prepared that contained one of the interacting modules: cohesins were joined to an appropriate cellulose-binding module (CBM) and the dockerins were fused to a thermostable xylanase that served to enhance expression and proper folding. The CBM-fused cohesins were immobilized on cellulose-coated glass slides, to which xylanase-fused dockerin samples were applied. Knowledge of the specificity characteristics of native and mutated members of the cohesin and dockerin families provides insight into the architecture of the parent cellulosome and allows selection of suitable cohesin-dockein pairs for biotechnological and nanotechnological application. Using this approach, extensive cross-species interaction among type-II cohesins and dockerins is shown for the first time. Selective intraspecies binding of an archaeal dockerin to two complementary cohesins is also demonstrated.

The genomes of myonecrotic strains of Clostridium perfringens encode a large number of secreted glycoside hydrolases. The activities of these enzymes are consistent with degradation of the mucosal layer of the human gastrointestinal tract, glycosaminoglycans and other cellular glycans found throughout the body. In many cases this is thought to aid in the propagation of the major toxins produced by C. perfringens. One such example is the family 84 glycoside hydrolases, which contains five C. perfringens members (CpGH84A-E), each displaying a unique modular architecture. The smallest and most extensively studied member, CpGH84C, comprises an N-terminal catalytic domain with β-N-acetylglucosaminidase activity, a family 32 carbohydrate-binding module, a family 82 X-module (X82) of unknown function, and a fibronectin type-III-like module. Here we present the structure of the X82 module from CpGH84C, determined by both NMR spectroscopy and X-ray crystallography. CpGH84C X82 adopts a jell-roll fold comprising two β-sheets formed by nine β-strands. CpGH84C X82 displays distant amino acid sequence identity yet close structural similarity to the cohesin modules of cellulolytic anaerobic bacteria. Cohesin modules are responsible for the assembly of numerous hydrolytic enzymes in a cellulose-degrading multi-enzyme complex, termed the cellulosome, through a high-affinity interaction with the calcium-binding dockerin module. A planar surface is located on the face of the CpGH84 X82 structure that corresponds to the dockerin-binding region of cellulolytic cohesin modules and has the approximate dimensions to accommodate a dockerin module. The presence of cohesin-like X82 modules in glycoside hydrolases of C. perfringens is an indication that the formation of novel X82-dockerin mediated multi-enzyme complexes, with potential roles in pathogenesis, is possible.

The second type II cohesin module of the cellulosomal scaffoldin polypeptide ScaB from Acetivibrio cellulolyticus (CohB2) was cloned into two constructs: one containing a short (five-residue) C-terminal linker (CohB2_S) and the second incorporating the full native 45-residue linker (CohB2_L). Both constructs encode proteins that also include the full native six-residue N-terminal linker. The CohB2_S and CohB2_L proteins were expressed, purified and crystallized in the orthorhombic crystal system, but with different unit cells and symmetries: space group P2₁2₁2₁ with unit-cell parameters a = 90.36, b = 68.65, c = 111.29 Å for CohB2_S and space group P2₁2₁2 with unit-cell parameters a = 68.76, b = 159.22, c = 44.21 Å for CohB2_L. The crystals diffracted to 2.0 and 2.9 Å resolution, respectively. The asymmetric unit of CohB2_S contains three cohesin molecules, while that of CohB2_L contains two molecules.

Family 3 carbohydrate-binding modules (CBM3s) are associated with the scaffoldin subunit of the multi-enzyme cellulosome complex and with the family 9 glycoside hydrolases, which are multimodular enzymes that act on plant cell-wall polysaccharides, notably cellulose. Here, the crystallization of CBM3b from cellobiohydrolase 9A is reported. The crystals are tetragonal and belong to space group P41 or P43. X-ray diffraction data for CBM3b have been collected to 2.68 Å resolution on beamline ID14-4 at the ESRF.

Ruminococcus flavefaciens produces a cellulosomal enzyme complex, based on the structural proteins ScaA, -B, and -C, that was recently shown to attach to the bacterial cell surface via the wall-anchored protein ScaE. ScaA, -B, -C, and -E are all cohesin-bearing proteins encoded by linked genes in the sca cluster. The product of an unknown open reading frame within the sea cluster, herein designated CttA, is similar in sequence at its C terminus to the corresponding region of ScaB, which contains an X module together with a dockerin sequence. The ScaB-XDoc dyad was shown previously to interact tenaciously with the cohesin of ScaE. Likewise, avid binding was confirmed between purified recombinant fragments of the CttA-XDoc dyad and the ScaE cohesin. In addition, the N-terminal regions of CttA were shown to bind to cellulose, thus suggesting that CttA is a cell wall-anchored, cellulose-binding protein. Proteomic analysis showed that the native CttA protein (similar to 130 kDa) corresponds to one of the three most abundant polypeptides binding tightly to insoluble cellulose in cellulose-grown R. flavefaciens 17 cultures. Interestingly, this protein was also detected among cellulose-bound proteins in the related strain R. flavefaciens 007C but not in a mutant derivative, 007S, that was previously shown to have lost the ability to grow on dewaxed cotton fibers. In R. flavefaciens, the presence of CttA on the cell surface is likely to provide an important mechanism for substrate binding, perhaps compensating for the absence of an identified cellulose-binding module in the major cellulosomal scaffolding proteins of this species.

Lignocellulose is the most abundant plant cell wall component of the biosphere and the most voluminous waste produced by our society. Fortunately, it is not toxic or directly harmful, but our major waste disposal facilities - the landfills - are rapidly filling up with few realistic alternatives. Because cellulose is pure glucose, its conversion to fine products or fuels has remained a romantic and popular notion; however, the heterogeneous and recalcitrant nature of cellulosic waste presents a major obstacle for conventional conversion processes. One paradigm for the conversion of biomass to products in nature relies on a multienzyme complex, the cellulosome. Microbes that produce cellulosomes convert lignocelluose to microbial cell mass and products (e.g. ethanol) simultaneously. The combination of designer cellulosomes with novel production concepts could in the future provide the breakthroughs necessary for economical conversion of cellulosic biomass to biofuels.

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.

The hydrolysis of biotinyl p-nitrophenyl ester (BNP) by a series of avidin derivatives was examined. Surprisingly, a hyperthermostable avidin-related protein (AVR4) was shown to display extraordinary yet puzzling hydrolytic activity. In order to evaluate the molecular determinants that contribute to the reaction, the crystal structure of AVR4 was compared with those of avidin, streptavidin and key mutants of the two proteins in complex with biotinyl p-nitroanilide (BNA), the inert amide analogue of BNP. The structures revealed that a critical lysine residue contributes to the hydrolysis of BNP by avidin but has only a minor contribution to the AVR4-mediated reaction. Indeed, the respective rates of hydrolysis among the different avidins reflect several molecular parameters, including binding-site architecture, the availability of the ligand to solvent and the conformation of the ligand and consequent susceptibility to efficient nucleophilic attack. In avidin, the interaction of BNP with Lys111 and disorder of the L3,4 loop (and consequent solvent availability) together comprise the major driving force behind the hydrolysis, whereas in AVR4 the status of the ligand (the pseudo-substrate) is a major distinguishing feature. In the latter protein, a unique conformation of the L3,4 loop restrains the pseudo-substrate, thereby exposing the carbonyl carbon atom to nucleophilic attack. In addition, due to its conformation, the pseudo-substrate in the AVR4 complex cannot interact with the conserved lysine analogue (Lys109); instead, this function is superseded by polar interactions with Arg112. The results demonstrate that, in highly similar proteins, different residues can perform the same function and that subtle differences in the active-site architecture of such proteins can result in alternative modes of reaction.

During the course of our studies on the structure-function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in 'designer cellulosomes'. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.

Sequence extension of the scaffoldin gene cluster from Ruminococcus flavefaciens revealed a new gene (scaE) that encodes a protein with an N-terminal cohesin domain and a C terminus with a typical gram-positive anchoring signal for sortase-mediated attachment to the bacterial cell wall. The recombinant cohesin of ScaE was recovered after expression in Escherichia coli and was shown to bind to the C-terminal domain of the cellulosomal structural protein ScaB, as well as to three unknown polypeptides derived from native cellulose-bound Ruminococcus flavefaciens protein extracts. The ScaB C terminus includes a cryptic dockerin domain that is unusual in its sequence, and considerably larger than conventional dockerins. The ScaB dockerin binds to ScaE, suggesting that this interaction occurs through a novel cohesin-dockerin pairing. The novel ScaB dockerin was expressed as a xylanase fusion protein, which was shown to bind tenaciously and selectively to a recombinant form of the ScaE cohesin. Thus, ScaE appears to play a role in anchoring the cellulosomal complex to the bacterial cell envelope via its interaction with ScaB. This sortase-mediated mechanism for covalent cell-wall anchoring of the cellulosome in R. flavefaciens differs from those reported thus far for any other cellulosome system.

Non-DNA microarrays, such as protein, peptide and small molecule microarrays, can potentially revolutionize the high-throughput screening tools currently used in basic and pharmaceutical research. However, fundamental obstacles remain that limit their rapid and widespread implementation as an alternative bioanalytical approach. These include the prerequisite for numerous proteins in active and purified form, ineffectual immobilization strategies and inadequate means for quality control of the considerable numbers of multiple reagents. This study describes a simple yet efficient strategy for the production of non-DNA microarrays, based on the tenacious affinity of a carbohydrate-binding module (CBM) for its three-dimensional substrate, i.e., cellulose. Various microarray formats are described, e.g., conventional and single-chain antibody microarrays and peptide microarrays for serodiagnosis of human immunodeficiency virus patients. CBM-based microarray technology overcomes many of the previous obstacles that have hindered fabrication of non-DNA microarrays and provides a technically simple but effective alternative to conventional microarray technology.

The expression of scaffoldin-anchoring genes and one of the major processive endoglucanases (CeIS) from the cellulosome of Clostridium thermocellum has been shown to be dependent on the growth rate. For the present work, we studied the gene regulation of selected cellulosomal endoglucanases and a major xylanase in order to examine the previously observed substrate-linked alterations in cellulosome composition. For this purpose, the transcript levels of genes encoding endoglucanases CelB, CelG, and CelD and the family 10 xylanase XynC were determined in batch cultures, grown on either cellobiose or cellulose, and in carbon-limited continuous cultures at different dilution rates. Under all conditions tested, the transcript levels of celB and celG were at least 10-fold higher than that of celD. Like the major processive endoglucanase CelS, the transcript levels of these endoglucanase genes were also dependent on the growth rate. Thus, at a rate of 0.04 h^-1, the levels of celB, celG, and celD were threefold higher than those obtained in cultures grown at maximal rates (0.35 h^-1) on cellobiose. In contrast, no clear correlation was observed between the transcript level of xynC and the growth rate - the levels remained relatively high, fluctuating between 30 and 50 transcripts per cell. The results suggest that the regulation of C. thermocellum endoglucanases is similar to that of the processive endoglucanase celS but differs from that of a major cellulosomal xylanase in that expression of the latter enzyme is independent of the growth rate.

The high affinity cohesin-dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The connection between affinity and species specificity was studied by exploring the recognition properties of two structurally related cohesin species of divergent specificity. The cohesins were examined by progressive rounds of swapping, in which corresponding homologous stretches were interchanged. The specificity of binding of the resultant chimeric cohesins was determined by enzyme-linked affinity assay and complementary protein microarray. In succeeding rounds, swapped segments were systematically contracted, according to the binding behavior of previously generated chimeras. In the fourth and final round we discerned three residues, reputedly involved in interspecies binding specificity. By replacing only these three residues, we were able to convert the specificity of the resultant mutated cohesin, which bound preferentially to the rival dockerin with ∼20% capacity of the wild-type interaction. These residues represent but 3 of the 16 contact residues that participate in the cohesin-dockerin interaction. This approach allowed us to differentiate, in a structure-independent fashion, between residues critical for interspecies recognition and binding residues per se.

The cohesive cellulosome complex is sustained by the high-affinity cohesin-dockerin interaction. In previous work [J. Biol. Chem. 276 (2001) 9883], we demonstrated that a single Thr-to-Leu replacement in the Clostridium thermocellum dockerin component differentiates between non-recognition and high-affinity recognition by the interspecies rival cohesin from C. cellulolyticum. In this report, we show that a single Asp-to-Asn substitution on the cohesin counterpart also disrupts normal recognition of the dockerin. The Asp34 carboxyl group of the cohesin appears to play a central role in the resultant hydrogen-bonding network as an acceptor of two crucial hydrogen bonds from Ser45 of the dockerin domain. The results underscore the fragile nature of the intermolecular contact interactions that maintain this very high-affinity protein-protein interaction.

We recently showed that some of the enzymes underpinning cellulose solubilization by Ruminococcus albus 8 lack the conventional type of dockerin module characteristic of cellulosomal proteins and instead, bear an "X" domain of unknown function at their C-termini. We have now subcloned and expressed six X domains and showed that five of them bind to xylan, chitin, microcrystalline and phosphoric-acid swollen cellulose, as well as more heterogenous substrates such as alfalfa cell walls, banana stem and wheat straw. The X domain that did not bind to these substrates was derived from a family-5 glycoside hydrolase (Cel5G), which possesses two X domains in tandem. Whereas the internal X domain failed to bind to the substrates, the recombinant dyad exhibited markedly enhanced binding relative to that observed for the C-terminal X domain alone. The evidence supports a distinctive carbohydrate-binding role of broad specificity for this type of domain, and we propose a novel family (designated family 37) of carbohydrate-binding modules that appear to be peculiar to R. albus.

A large gene downstream of the primary Bacteroides cellulosolvens cellulosomal scaffoldin (cipBc, now renamed scaA) was sequenced. The gene, termed scaB, contained an N-terminal leader peptide followed by 10 type I cohesins, an "X" domain of unknown structure and function, and a C-terminal S-layer homology (SLH) surface-anchoring module. In addition, a previously identified gene in a different part of the genome, encoding for a dockerin-borne family 48 cellulosomal glycoside hydrolase (Cel48), was sequenced completely, and a putative cellulosome-related family 9 glycosyl hydrolase was detected. Recombinant fusion proteins, comprising dockerins derived from either the ScaA scaffoldin or Cel48, were overexpressed. Their interaction with ScaA and ScaB cohesins was examined by immunoassay. The results indicated that the ScaB type I cohesin of the new anchoring protein binds selectively to the ScaA dockerin, whereas the Cel48 dockerin binds specifically to the type II ScaA cohesin 5. Thus, by virtue of the 11 type II ScaA cohesins and the 10 type I ScaB cohesins, the relatively simple two-component cellulosome-integrating complex would potentially incorporate 110 enzyme molecules onto the cell surface via the ScaB SLH module. Compared to previously described cellulosome systems, the apparent roles of the B. cellulosolvens cohesins are reversed, in that the type II cohesins are located on the enzyme-binding primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldin. The results underscore the extensive diversity in the supramolecular architecture of cellulosome systems in nature.

Clostridium thermocellum produces an extracellular multienzyme complex, termed the cellulosome, that allows efficient solubilization of crystalline cellulose. The complex is organized around a large noncatalytic protein subunit, termed CipA or scaffoldin, and is found either free in the supernatant or cell bound. The binding of the complex to the cell is mediated by three cell surface anchoring proteins, OlpB, Orf2p, and SdbA, that interact with the CipA scaffoldin. The transcriptional level of the olpB, orf2, sdbA, and cipA genes was determined quantitatively by RNase protection assays in batch and continuous cultures, under carbon and nitrogen limitation. The mRNA level of olpB, orf2, and cipA varied with growth rate, reaching 40 to 60 transcripts per cell under carbon limitation at a low growth rate of 0.04 h^-1 and 2 to 10 transcripts per cell at a growth rate of 0.35 h^-1 in batch culture. The mRNA level of sdbA was about three transcripts per cell and was not influenced by growth rate. Primer extension analysis revealed two major transcriptional start sites, at -81 and -50 bp, upstream of the translational start site of the cipA gene. The potential promoters exhibited homology to the known sigma factors σ^A and σ^L (σ ⁵⁴) of Bacillus subtilis. Transcription from the σ ^L-like promoter was found under all growth conditions, whereas transcription from the σ^A-like promoter was significant only under carbon limitation. The overall expression level obtained in the primer extension analysis was in good agreement with the results of the RNase-protection assays.

The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W→K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.

Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1 → 2 interface was mutated to lysine and asparagine 54 in the 1 → 4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K_d ∼10^-15 M of the wild-type tetramer to K_d ∼10^-7 M, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of intersubunit interactions on the binding of ligands.

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcusflavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in R. flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an ∼ 110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.

The assembly of enzyme components into the cellulosome complex is dictated by the cohesin-dockerin interaction. In a recent article (Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000) Proteins 39, 170-177), we provided experimental evidence that four previously predicted dockerin residues play a decisive role in the specificity of this high affinity interaction, although additional residues were also implicated. In the present communication, we examine further the contributing factors for the recognition of a dockerin by a cohesin domain between the respective cellulosomal systems of Clostridium thermocellum and Clostridium cellulolyticum. In this context, the four confirmed residues were analyzed for their individual effect on selectivity. In addition, other dockerin residues were discerned that could conceivably contribute to the interaction, and the suspected residues were similarly modified by site-directed mutagenesis. The results indicate that mutation of a single residue from threonine to leucine at a given position of the C. thermocellum dockerin differentiates between its nonrecognition and high affinity recognition (K_α ∼ 10⁹ M^-1) by a cohesin from C. cellulolyticum. This suggests that the presence or absence of a single decisive hydroxyl group is critical to the observed biorecognition. This study further implicates additional residues as secondary determinants in the specificity of interaction, because interconversion of selected residues reduced intraspecies self-recognition by at least three orders of magnitude. Nevertheless, as the latter mutageneses served to reduce but not annul the cohesin-dockerin interaction within this species, it follows that other subtle alterations play a comparatively minor role in the recognition between these two modules.

Bacterial streptavidin and chicken avidin are homotetrameric proteins that share an exceptionally high affinity towards the vitamin biotin. The biotin-binding sites in both proteins contain a crucial tryptophan residue contributed from an adjacent subunit. This particular tryptophan (W110 in avidin and W120 in streptavidin) plays an important role in both biotin binding and in the quaternary stabilities of the proteins. An intriguing naturally occurring alteration of tryptophan to lysine was previously described in the C-terminal domain of sea-urchin fibropellins, which share a relatively high sequence similarity with avidin and streptavidin. Avidin (Avm-W110K) and streptavidin (Savm-W120K) mutations show substantially reduced affinities towards biotin as well as the dissociation of their tetrameric structure into stable avidin and streptavidin dimers. Savm-W120K was crystallized at 293 K using the hanging-drop vapour-diffusion method. The crystals diffract to 1.7 resolution using synchrotron radiation and belong to the monoclinic space group P2₁, with unit-cell parameters a = 50.43, b = 100.41, c = 52.51 Å, β = 112.12°. The asymmetric unit contains four molecules of Savm-W120K, with a corresponding V_M of 2.3 ų Da^-1 and a solvent content of 46%.

The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex, In a previous article (Pages et al., Proteins 1997;29:517-527) we predicted that four amino acid residues of the similar to 70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohesin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction.

A recombinant non-glycosylated and acidic form of avidin was designed and expressed in soluble form in baculovirus-infected insect cells. The mutations were based on the same principles that guided the design of the chemically and enzymatically modified avidin derivative, known as NeutraLite Avidin. In this novel recombinant avidin derivative, five out of the eight arginine residues were replaced with neutral amino acids, and two of the lysine residues were replaced by glutamic acid. In addition, the carbohydrate-bearing asparagine-17 residue was altered to an isoleucine, according to the known sequences of avidin-related genes. The resultant mutant protein, termed recombinant NeutraLite Avidin, exhibited superior properties compared to those of avidin, streptavidin and the conventional NeutraLite Avidin, prepared by chemo-enzymatic means. In this context, the recombinant mutant is a single molecular species, which possesses strong biotin-binding characteristics. Due to its acidic pI, it is relatively free from non-specific binding to DNA and cells. The recombinant NeutraLite Avidin retains seven lysines per subunit, which are available for further conjugation and derivatization. Copyright (C) 2000 Federation of European Biochemical Societies.

The distribution of cellulosomal cohesin domains among the sequences currently compiled in various sequence databases was investigated. Two cohesin domains were detected in two consecutive open reading frames (ORFs) of the recently sequenced genome of the archaeon Archaeoglobus fulgidus. Otherwise, no cohesin-like sequence could be detected in organisms other than those of the Eubacteria. One of the A. fulgidus cohesin-containing ORFs also harbored a dockerin domain, but the additional modular portions of both genes are undefined, both with respect to sequence homology and function. It is currently unclear what function(s) the putative cohesin and dockerin- containing proteins play in the life cycle of this organism. In particular, since A. fulgidus contains no known glycosyl hydrolase gene, the presence of a cellulosome can be excluded. The results suggest that cohesin and dockerin signature sequences cannot be used alone for the definitive identification of cellulosomes in genomes.

We describe a method for the isolation of recombinant single-chain antibodies in a biologically active form. The single-chain antibodies are fused to a cellulose binding domain as a single-chain protein that accumulates as insoluble inclusion bodies upon expression in Escherichia coli. The inclusion bodies are then solubilized and denatured by an appropriate chaotropic solvent, then reversibly immobilized onto a cellulose matrix via specific interaction of the matrix with the cellulose binding domain (CBD) moiety. The efficient immobilization that minimizes the contact between folding protein molecules, thus preventing their aggregation, is facilitated by the robustness of the Clostridium thermocellum CBD we use. This CBD is unique in retaining its specific cellulose binding capability when solubilized in up to 6 M urea, while the proteins fused to it are fully denatured. Refolding of the fusion proteins is induced by reducing with time the concentration of the denaturing solvent while in contact with the cellulose matrix. The refolded single-chain antibodies in their native state are then recovered by releasing them from the cellulose matrix in high yield of 60% or better, which is threefold or higher than the yield obtained by using published refolding protocols to recover the same scFvs. The described method should have general applicability for the production of many protein-CBD fusions in which the fusion partner is insoluble upon expression.

The cellulosome is an extracellular supramolecular machine that can efficiently degrade crystalline cellulosic substrates and associated plant cell wall polysaccharides. The cellulosome arrangement can also promote adhesion to the insoluble substrate, thus providing individual microbial cells with a direct competitive advantage in the utilization of the soluble hydrolysis products.

The cellulosome concept is one of the major paradigms by which microorganisms bring about the efficient enzymatic degradation of crystalline cellulosic substrates. The principal structural element of a cellulosome is a scaffoldin subunit which integrates the other enzymatic subunits into the cellulosome complex. Ln recent years, the three-dimensional structures of various functional modules, which characterize the typical cellulosome, and in some cases free cellulases as well, have been determined. Currently, phylogenetic analysis of cellulosome-related sequences is contributing novel evidence concerning the intra- and interspecies relationship of the functional modules that comprise a typical cellulosome.

Avidin, a positively charged egg-white glycoprotein, is a widely used tool in biotechnological applications because of its ability to bind biotin strongly, The high pI of avidin (similar to 10.5), however, is a hindrance in certain applications due to non-specific (charge-related) binding. Here we report a construction of a series of avidin charge mutants with pIs ranging from 9.4 to 4.7. Rational design of the avidin mutants was based on known crystallographic data together with comparative sequence alignment of avidin, streptavidin and a set of avidin-related genes which occur in the chicken genome. All charge mutants retained the ability to bind biotin tightly according to optical biosensor interaction analysis. In most cases, their thermal stability characteristics were indistinguishable from those of the wild-type avidin, Our results demonstrate that the charge properties of avidin can be modified without disturbing the crucial biotin-binding activity. (C) 1998 Federation of European Biochemical Societies.

The cross-species specificity of the cohesin-dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated. Cohesin-containing segments from the cellulosomes of two different species, Clostridium thermocellum and Clostridium cellulolyticum, were allowed to interact with cellulosomal (dockerin-containing) enzymes from each species. In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium-dependent manner, but the same cohesin failed to recognize enzymes from the other species. Thus, in the case of these two bacteria, the cohesin- dockerin interaction seems to be species-specific. Based on intra- and cross- species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino-acid residues critical to recognition of the cohesins. The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium-binding motif of two duplicated sequences, characteristic of the dockerin domain. According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface.

Recombinant cohesin-2, a unique type of protein-recognition domain from the cellulosome of Clostridium thermocellum, has been crystallized by the hanging-drop vapor-diffusion method. The crystals are monoclinic, space group C2 with unit-cell dimensions a = 79.91, b = 47.86, c = 51.13 Å, β = 126.77. There is most likely to be one molecule per asymmetric unit, corresponding to a packing density of 2.16 ų Da^-1. The crystals diffract to beyond 2.3 Å on a conventional laboratory rotating anode source.

Chemically modified forms of egg-white avidin and bacterial streptavidin (termed nitro-avidin and nitro-streptavidin, respectively), in which the binding-site tyrosine was nitrated, were used for several biotechnological applications. The fundamental difference between nitro-avidin and the native protein is that interaction of the modified protein with biotin can be reversed under relatively mild conditions. Consequently, nitro-avidin affinity columns or immobilizing matrices can be reused. Three examples are given to demonstrate the possible uses of such columns: (a) biotinylated protein A was attached to a nitro-avidin affinity column, and immunoglobulin was purified directly from whole rabbit serum; (b) biotinylated transferrin was attached to a nitro-streptavidin column, and anti-transferrin was isolated directly from rabbit anti-serum; and (c) biotinylated beta-glucosidase was immobilized onto a nitro-avidin column and used as an enzyme reactor. In each example, the immobilized biotinylated probe could be released selectively from the column and recovered following its utilization. Reusable nitro-avidin thus provides an easy and attractive reversible form of avidin and thereby serves to expand the versatility of avidin-biotin technology. (C) 1996 Academic Press, Inc.

The cellulosome of the cellulolytic bacterium, Clostridium thermocellum, is a multi-enzyme complex in which the enzymatic (cellulolytic) subunits are attached to a unique nonhydrolytic subunit called scaffoldin. The attachment is mediated by two mutually interacting domains: namely, multiple cohesin domains on the scaffoldin subunit and a dockerin domain on each of the enzymatic subunits. Knowledge of the three-dimensional structure of each of the interacting components would be critical to a better understanding of the cohesin-dockerin interaction at the molecular level. In this report, we describe the purification of one of the nine cohesin domains of the scaffoldin subunit from C. thermocellum. A DNA segment containing the cohesin 2 sequence was fused to a hexa-histidine tag, and the resultant construct was expressed in Escherichia coli. The expressed peptide was efficiently isolated by metal-chelate affinity chromatography. The purified recombinant form of the cohesin was crystallized pending determination of its structure.

Avidin, a positively charged egg-white protein, aggregates extensively when mixed at ambient temperatures with anionic detergents, such as sodium dodecyl sulfate (SDS). The resultant aggregates fail to penetrate the stacking gel during polyacrylamide gel electrophoresis (PAGE). To prevent the formation of such aggregates, avidin was acetylated and the pI was thus reduced. Acetylated avidin was found to behave in a manner similar to that of streptavidin; under nondenaturing conditions (i.e., incubation of samples at room temperature), both proteins normally migrated mainly as tetramers with a tendency to form oligomers of the tetramer. When samples were boiled, both proteins migrated mainly as the monomer. The comparative stability properties of avidin and streptavidin were also examined using SDS-PAGE by heating samples and determining the extent of dissociation of tetramers to monomers as a function of temperature. A distinctive transition temperature could be defined for individual samples. Using this assay, it was determined that, in the absence of biotin, the quaternary structure of streptavidin is more stable than that of avidin. Biotin appears to stabilize structures of both avidin and streptavidin to a similar degree. Acetylation of avidin thus provides a simple means to analyze the quaternary structure of the molecule using SDS-PAGE.

A simple procedure for the preparation of deglycosylated avidin is described. Commercially obtained avidin was treated with a mixed microbial culture. The cells were capable of growing on the oligosaccharide residues, but generally ignored the polypeptide portion of the egg white glycoprotein. The resultant deglycosylated avidin retained its biotin-binding characteristics. The major bacterial strain (strain BECH080), responsible for the deglycosylation, was isolated. On the basis of elementary biochemical tests, fatty acid, and phenotypic analyses, the isolate was identified as a strain of Flavobacterium meningosepticum. The primary enzymatic activity that caused the removal of the oligosaccharide residues of avidin appeared to be similar to endoglycosidase F.

A heat-stable enzyme was isolated from the cellulase complex of a thermophilic strain of the micromycete Thielavia terrestris. The purified enzyme exhibited both endoglucanase and xylanase activities and had a mol mass of 69,000 Daltons and an isoelectric point of 6.4. When the cells were grown at 48°C, the initial activity of the purified enzyme using carboxymethylcellulose as a substrate was 150 nkat/mg and the Michaelis constant was 6.6 g/L. The heat stability of the enzyme was high, losing only 20% of the initial activity after a 6-h incubation at 65 °C. When cultures were grown on microcrystalline cellulose and xylose was added after 48 h of growth, endoglucanase and xylanase activities were more than doubled. Similar increases in these activities were observed by growing the cultures on straw.

The controversy regarding the identity of a major cellulosomal component type from two different strains of Clostridium thermocellum has been resolved. The principal cellobiohydrolase, subunit S8, from the cellulosome of strain YS has been demonstrated to be synonymous with cellulase component S_s (CelS) from the cellulosome of ATCC strain 27405. This component is not related to any other cellulosomal subunit or cloned endoglucanase in this organism.

Keywords: Biochemistry & Molecular Biology

The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 A, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel β-barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis.

Streptavidin is a biotin‐binding analogue of egg‐white avidin which is secreted by the bacterium Streptomyces avidinii. We have recently reported that streptavidin contains an Arg‐Tyr‐Asp‐Ser (RYDS) sequence which exhibits structural homology to the Arg‐Gly‐Asp‐Ser (RGDS) cell adhesion domain of fibronectin and other matrix‐associated glycoproteins. Competition studies with RGD peptides indicated that streptavidin binds to cells via this site and that the binding is independent of biotin recognition. Since the RGD‐containing peptide has been shown to play a key role in integrin‐mediated cell adhesion, we assumed that streptavidin may utilize the RYDS site to bind to immune cells and thereby abrogate their adhesion‐dependent functions. We now report that streptavidin modulates several atrix‐dependent interactions of immune cells. In this context, immobilized streptavidin was found to support activated human CD4⁺ T cell adhesion in an RGD‐specific, α₅β₁ dependent manner. In addition, soluble streptavidin (the commercially available or biotin‐blocked forms) inhibited T cell adhesion to fibronectin and interfered with its co‐stimulatory effect on tumor necrosis factor‐a secretion by co‐cultures of CD4+ T cells and macrophages. These results suggest that streptavidin is a novel example of a bacterial protein which utilizes RGD mimicry to interfere with integrin‐mediated immune responses.

The interaction of streptavidin with various cell systems was studied using fluorescent derivatives of the protein. The native unprocessed form of streptavidin bound to cells at low levels and in a nonspecific manner. In contrast, both the truncated "core" streptavidin (the commercially available form) and the biotin-blocked unprocessed protein bound to cells in enhanced levels and in a specific, saturable manner. This suggests that the binding of biotin or cleavage of the terminal portion(s) of the native protein molecule causes conformational changes which lead to the exposure of sites which presumably interact with cell surface receptors. Peptide inhibition studies demonstrated that the majority of binding to cells appears to be dependent on RGD-like specificity, suggesting that the GRYDS sequence of the streptavidin molecule may exhibit such specificity. Indirect immunofluorescence assays revealed that the protein is associated mainly with the cell surface. Moreover, streptavidin was demonstrated to compete with specific monoclonal antibodies to the RGD-binding site on the GpIIbIIIa integrin of activated platelets, thus suggesting that streptavidin may facilitate binding to ubiquitous cell-surface adhesion receptors via RGD mimicry.

The multiple cellulase‐containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O‐linked mainly to a 230‐kDa subunit. The oligosaccharide chains were liberated by alkaline‐borohydride treatment and fractionated as oligosaccharide alditols via gel‐permeation chromatography and HPLC. The fractions were investigated by one‐ and two‐dimensional (correlation, homonuclear Hartmann‐Hahn, rotating‐frame nuclear Overhauser enhancement) 500‐MHz ¹H‐NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast‐atom‐bombardment mass spectrometry. The following carbohydrate structures could be established: (Formula Presented.) (Formula Presented.) (Formula Presented.) (Formula Presented.) (Formula Presented.) and (Formula Presented.) . The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115–122], having 3, 5 and 6 as common elements. The furanose form of a terminal α‐d‐galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome‐like entity of B. cellulosolvens.

The extensive use of antibody-containing affinity columns in the purification of biologically active compounds (e.g., genetically engineered proteins) is severely hampered by the leaching of antibody (or portions thereof) from the immunoaffinity resin during elution of the target antigen. One of the major problems in this context is the combined use of reducing (i.e., thiols) and chaotropic (e.g., detergents and denaturants) agents in the elution step, which causes the disassociation of heavy and/or light chains from the immobilized antibody, thereby contaminating the resultant product. In order to overcome this problem, we have cross-linked the four antibody chains at their sites of disulfide interlinkage, thus producing a single antibody chain. To accomplish this, interchain disulfide bonds were reduced, and the resultant thiol groups were cross-linked by using Afunctional SH-specific reagents (particularly bismaleimides). Cross-linking of up to 95% of the available SH groups produced was achieved with concomitant retention of antigen-binding activity. The cross-linked antibody was immobilized onto CNBr-activated Sepharose, and the resultant column was found to be substantially more stable to harsh elution conditions than similar columns which contain the un-cross-linked antibody.

The cell‐free forms of the multiple cellulase‐containing protein complex (cellulosome), isolated from the celluloytic bacterium Clostridium thermocellum strains YS, ATCC 27405 and LQRI, have a total carbohydrate content of 5–7% (by mass), consisting of O‐linked oligosaccharide chains. The carbohydrate chains were liberated by alkaline‐borohydride treatment and fractionated as oligosaccharide alditols via gel‐permeation chromatography and HPLC. The fractions were investigated by 500‐MHz ¹H‐NMR spectroscopy in combination with monosaccharide and methylation analysis and with fast‐atom‐bombardment mass spectrometry (FAB‐MS). In addition to the previously described major oligosaccharide, (Formula Presented.) [Gerwig, G. J., de Waard, P., Kamerling, J. P., Vliegenthart, J. F. G., Morgenstern, E., Lamed, R. & Bayer, E. A. (1989) J. Biol. Chem. 264, 1027 – 10351, the following partial structures of this compound could be established: (Formula Presented.) Cell‐free and cell‐associated forms of the cellulosome of C. thermocellum, as determined for strain YS, have the same oligosaccharide pattern. Based on the oligosaccharide structures, a biosynthetic pathway is suggested.

In the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum, efficient solubilization of the insoluble cellulose substrate is accomplished largely through the action of a cellulose-binding multienzyme complex, the cellulosome. A major cellobiohydrolase activity from the cellulosome has been traced to its M(r) 75,000 S8 subunit, and an active fragment of this subunit was prepared by a novel procedure involving limited proteolytic cleavage. The truncated M(r) 68,000 fragment, termed S8-tr, was purified by gel filtration and high-performance ion-exchange chromatography. The purified protein adsorbed weakly to amorphous cellulose, and its enzymatic action yielded cellobiose as the major end product from both amorphous and crystalline cellulose preparations. The high ratio of exo- to endo-β-glucanase activities was supported by viscosimetric measurements. The use of model substrates showed that the smallest cellodextrin to be degraded was cellotetraose, but cellopentaose was degraded at a much greater rate. Cellobiose dramatically inhibited the cellulolytic activities. In the absence of calcium or other bivalent metal ions, both the truncated cellobiohydrolase activity of S8-tr and the true cellulase activity of the parent cellulosome were relatively unstable at temperatures above 50°C. Cysteine further enhanced the stabilizing effect of calcium. This is the first report of a defined cellobiohydrolase in C. thermocellum. Its association with the cellulosome and the correspondence of several of their major distinctive properties suggest that this cellobiohydrolase plays a key role in the solubilization of cellulose by the intact cellulosomal complex.

A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20-28% of the initial fluorescence with (K_SV+V)≤0.55 M^-1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka=5 M^-1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weber et al., 1989).

Keywords: Chemistry, Analytical

This chapter describes a method for the isolation of the non-glycosylated tetramer. This method is based on the removal of glycosylated forms using appropriate lectin columns. Belovo avidin is dissolved in 192 ml distilled water and the solution is centrifuged. The clarified solution is applied to a Con A column. Tris buffer is used to wash the column, after which the nonglycosylated avidin peak appears. Elution of the adsorbed glycosylated forms is accomplished with 5% α-methyl-D-mannopyranoside in the same buffer. The nonspecific adsorption of egg-white avidin is usually attributed to two inherent characteristics of the molecule—namely, the high pI and the presence of sugar residues. The nonglycosylated avidin has the same biotin-binding properties as the fully glycosylated form, which indicates that the sugars are not important for the biological activity.

This chapter discusses the procedures for the immobilization of a biotinylated antibody specific for transferrin. A solution (1 ml) containing 1.7 mg biotinylated antitransferrin antibody is applied to a 1 ml avidin column during immobilization of biotinylated antibody on avidin-containing column. Using this procedure, quantitative binding of the biotinylated antibody can be achieved. The acetic acid wash removes about 15% of the bound antibody but the remaining fraction is stable. The column is washed with five column volumes of acetic acid and the PBS washing procedure is repeated. A 1-ml affinity column is washed successively with PBS, acetic acid, water, and PBS again. The plasma is diluted to 5 ml with phosphate buffered saline (PBS) and applied to the column. Following passage of the effluent fraction, the column is washed with 25 ml of PBS. The purified transferrin contained even lower levels of contaminating antibody than that of the commercially available (Sigma) protein.

This chapter discusses the principles and advantages of the avidin–biotin system. The major distinguishing feature of the avidin–biotin system is the extraordinary affinity that characterizes the complex formed between the vitamin biotin and the egg-white protein avidin. The rationale in using the avidin–biotin system is based on the premise that if a compound biologically active with biotin is modified through its valeric acid side chain, the biological and physicochemical properties of the biotin-modified molecule are be changed significantly. If a reporter group of some sort is attached to the avidin molecule, the conjugate can be used for many different purposes. The avidin-conjugated probe can be added as a single chemically conjugated entity, or avidin can be applied in native form together with a biotinylated probe. The exceptionally high affinity and stability of the avidin–biotin complex ensures the desired conjunction of binder and probe. Biotin can readily be attached to most binders and probes; following biotinylation, the biological activity and physical characteristics are commonly retained.

This chapter discusses the procedures by which researchers have incorporated biotin moieties into proteins. Several laboratories have reported the use of an avidin-based assay for detecting the extent of biotinylation. An effective method for detecting the average number of biotin groups per protein molecule would be to first remove biotin from the protein and then to determine the amount of biotin in the sample. The percentage of biotinylated peanut agglutinin (PNA) molecules that are accessible for binding avidin in a given sample is assessed by quantitative affinity chromatography on an avidin column. An easy and effective way to determine nonspecific interactions of the biotinylated lectin is to use dot blots. A variety of target materials can be employed for this purpose. Because PNA recognizes the exposed penultimate galactose residues in desialylated sialoglycoconjugates, asialofetuin is an excellent positive control for this assay. Altering the relative concentration or ratio of reagent and/or antibody alters the extent or efficiency of biotinylation.

The avidin–biotin system was initially introduced with the intention of applying the technique in a unified fashion for analysis by various disciplines. At the cellular level, the distribution of the target can be analyzed using a fluorescent form of avidin combined with flow cytometry. At the ultrastructural level of resolution, the localization of the molecule of interest can be accomplished under the electron microscope using avidin–gold conjugates. Avidin–Sepharose can be employed for the isolation of the target molecule and avidin–enzyme or radioactive avidin can be used with blotting techniques for information at the molecular level of resolution. The epitome of such application would be to have a group of well-characterized biotinylated binders and a series of well-characterized avidin-conjugated probes for routine work in different areas. However, the interests and technological expertise of a given laboratory are often limited to a particular area as very few laboratories have devoted their research to composite studies involving avidin–biotin technology.

The use of the avidin––biotin system as DNA probes in nucleic acid research is very broad, owing to its potential for replacement of radioactive DNA probes. Nonetheless, the number of new methods for introducing biotin into DNA is quite limited. A biotinyl derivative of dUTP is incorporated into a suitable DNA probe via nick translation. Following hybridization of the biotinylated probe with the target DNA, an avidin-associated probe is used for localization and isolation. Many investigations have been published in which different enzymatic modes of incorporation of a biotinylated nucleotide have been described. In addition, various chemical methods are accumulating in the literature for incorporating the biotin moiety into DNA. In future, additional methods for labeling DNA will be developed. It is expected that the system will also become applicable for in vivo DNA diagnosis.