Determination of subunit composition of Clostridium cellulovorans cellulosomes that degrade plant cell walls

Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of C. cellulovorans and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.     

Degradation of corn fiber by Clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein CbpA

Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.     

Three Surface Layer Homology Domains at the N Terminus of the Clostridium cellulovorans Major Cellulosomal Subunit EngE

The gene engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been isolated and sequenced. engE is comprised of an open reading frame (ORF) of 3,090 bp and encodes a protein of 1,030 amino acids with a molecular weight of 111,796. The amino acid sequence derived from engE revealed a structure consisting of catalytic and noncatalytic domains. The N-terminal-half region of EngE consisted of a signal peptide of 31 amino acid residues and three repeated surface layer homology (SLH) domains, which were highly conserved and homologous to an S-layer protein from the gram-negative bacterium Caulobacter crescentus. The C-terminal-half region, which is necessary for the enzymatic function of EngE and for binding of EngE to the scaffolding protein CbpA, consisted of a catalytic domain homologous to that of family 5 of the glycosyl hydrolases, a domain of unknown function, and a duplicated sequence (DS or dockerin) at its C terminus. engE is located downstream of an ORF, ORF1, that is homologous to the Bacillus subtilis phosphomethylpyrimidine kinase (pmk) gene. The unique presence of three SLH domains and a DS suggests that EngE is capable of binding both to CbpA to form a CbpA-EngE cellulosome complex and to the surface layer of C. cellulovorans.     

Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of Clostridium cellulovorans EngE

engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been cloned and sequenced (Y. Tamaru and R. H. Doi, J. Bacteriol. 181:3270-3276, 1999). The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins. Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA. Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface. This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in Escherichia coli and cell wall preparations from C. cellulovorans. When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall. However, rEngEs without SLH domains lost their ability to bind to cell walls. When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE. However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions. These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin.     

Determination of Subunit Composition of Clostridium cellulovorans Cellulosomes That Degrade Plant Cell Walls

Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of C. cellulovorans and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.     

Hydrophilic domains of scaffolding protein CbpA promote glycosyl hydrolase activity and localization of cellulosomes to the cell surface of Clostridium cellulovorans

CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.     

Analysis of selective, high protein-protein binding interaction of cohesin-dockerin complex using biosensing methods

Optical biosensors that use fluorescence are promising tools for the analysis of target materials such as protein, DNA and other biomaterial. To analyze the binding properties of a protein-protein interaction, we constructed fluorescent biomarkers based on the cohesin-dockerin interaction, which coordinates the assembly of cellulolytic enzymes and scaffolding proteins to produce a cell surface multiprotein complex known as the "cellulosome" in some anaerobic bacteria. Our 2D-PAGE results displayed diverse binding profiles to the dockerin containing cellulosomal proteins produced by Clostridium cellulovorans grown on different carbon sources, such as Avicel, xylan and AXP (Avicel:xylan:pectin (3:1:1)). Fluorescence intensity analysis indicated that EngE and EngH bound more efficiently to Coh6 than to Coh2 or Coh9 (2-fold to 6-fold and 1.5-fold to 5-fold, respectively), while others cellulosomal proteins displayed similar results. In addition, both an enzyme-linked interaction assay (ELIA) and surface plasmon resonance (SPR) analyses demonstrated that both EngE and EngH preferentially bound cohesin6 versus the other two cohesin molecules. This work demonstrated the analysis of the binding patterns between interacting proteins using fluorescent biomarkers. We also illustrated the potential of this sensitive approach to quantify specific target analytical materials via the example of the cohesin-dockerin interaction.     

Degradation of Corn Fiber by Clostridium cellulovorans Cellulases and Hemicellulases and Contribution of Scaffolding Protein CbpA

Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.     

Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans

Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.     

Characterization of a cellulose binding domain from Clostridium cellulovorans endoglucanase-xylanase D and its use as a fusion partner for soluble protein expression in Escherichia coli

Different chimeric proteins combining the non-catalytic C-terminal putative cellulose binding domain of Clostridium cellulovorans endoglucanase-xylanase D (EngD) with its proline-threonine rich region PT-linker, PTCBD(EngD), cellulose binding domain of C. cellulovorans cellulose binding protein A, CBD(CbpA), cohesin domains Cip7, Coh6 and CipC1 from different clostridial species and recombinant antibody binding protein LG were constructed, expressed, purified and analyzed. The solubilities of chimeric proteins containing highly soluble domains Cip7, CipC1 and LG were not affected by fusion with PTCBD(EngD). Insoluble domain Coh6 was solubilized when fused with PTCBD(EngD). In contrast, fusion with CBD(CbpA) resulted in only a slight increase in solubility of Coh6 and even decreased solubility of CipC1 greatly. PTCBD(EngD) and Cip7-PTCBD(EngD) were shown to bind regenerated commercial amorphous cellulose Cuprophan. The purity of Cip7-PTCBD(EngD) eluted from Cuprophan was comparable to that purified by conventional ion exchange chromatography. The results demonstrated that PTCBD(EngD) can serve as a bi-functional fusion tag for solubilization of fusion partners and as a domain for the immobilization, enrichment and purification of molecules or cells on regenerated amorphous cellulose.     

In vitro reconstitution of the complete Clostridium thermocellum cellulosome and synergistic activity on crystalline cellulose

Artificial cellulase complexes active on crystalline cellulose were reconstituted in vitro from a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of a C. thermocellum cipA mutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. The in vitro-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities for in vitro complex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases.     

Synergistic interaction of Clostridium cellulovorans cellulosomal cellulases and HbpA

Clostridium cellulovorans, an anaerobic bacterium, produces a small nonenzymatic protein called HbpA, which has a surface layer homology domain and a type I cohesin domain similar to those found in the cellulosomal scaffolding protein CbpA. In this study, we demonstrated that HbpA could bind to cell wall fragments from C. cellulovorans and insoluble polysaccharides and form a complex with cellulosomal cellulases endoglucanase B (EngB) and endoglucanase L (EngL). Synergistic degradative action of the cellulosomal cellulase and HbpA complexes was demonstrated on acid-swollen cellulose, Avicel, and corn fiber. We propose that HbpA functions to bind dockerin-containing cellulosomal enzymes to the cell surface and complements the activity of cellulosomes.     

Resonance assignments of cohesin and dockerin domains from Clostridium acetobutylicum ATCC824

Cohesin and dockerin domains are critical assembling components of cellulosome, a large extracellular multienzyme complex which is used by anaerobic cellulolytic bacteria to efficiently degrade lignocellulose. According to sequence homology, cohesins can be divided into three major groups, whereas cohesins from Clostridium acetobutylicum are beyond these groups and emanate from a branching point between the type I and type III cohesins. Cohesins and dockerins from C. acetobutylicum show low sequence homology to those from other cellulolytic bacteria, and their interactions are specific in corresponding species. Therefore the interactions between cohesins and dockerins from C. acetobutylicum are meaningful to the studies of both cellulosome assembling mechanism and the construction of designer cellulosome. Here we report the NMR resonance assignments of one cohesin from cellulosome scaffoldin cipA and one dockerin from a cellulosomal glycoside hydrolase (family 9) of C. acetobutylicum for further structural determination and functional studies.     

The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has specific interactions with endoglucanases.

We overexpressed one of the hydrophobic repeated domains (HBDs) (110 amino acid residues) of the cellulose-binding protein (CbpA) from Clostridium cellulovorans by making a hybrid protein with the Escherichia coli maltose-binding protein (MalE). The HBD was purified to homogeneity, and interactions between the HBD and endoglucanases were analyzed by a novel interaction Western blotting (immunoblotting) method. The HBD had specific interactions with endoglucanases (EngB and EngD) from C. cellulovorans. These results indicated that the HBD was an endoglucanase binding site of CbpA.     

Cellulose promotes extracellular assembly of Clostridium cellulovorans cellulosomes.

Cellulosome synthesis by Clostridium cellulovorans was investigated by growing the cells in media containing different carbon sources. Supernatant from cells grown with cellobiose contained no cellulosomes and only the free forms of cellulosomal major subunits CbpA, P100, and P70 and the minor subunits with enzymatic activity. Supernatant from cells grown on pebble-milled cellulose and Avicel contained cellulosomes capable of degrading crystalline cellulose. Supernatants from cells grown with cellobiose, pebble-milled cellulose, and Avicel contained about the same amount of carboxymethyl cellulase activity. Although the supernatant from the medium containing cellobiose did not initially contain active cellulosomes, the addition of crystalline cellulose to the cell-free supernatant fraction converted the free major forms to cellulosomes with the ability to degrade crystalline cellulose. The binding of P100 and P70 to crystalline cellulose was dependent on their attachment to the endoglucanase-binding domains of CbpA. These data strongly indicate that crystalline cellulose promotes cellulosome assembly.     

Novel Clostridium thermocellum Type I Cohesin-Dockerin Complexes Reveal a Single Binding Mode

Protein-protein interactions play a pivotal role in a large number of biological processes exemplified by the assembly of the cellulosome. Integration of cellulosomal components occurs through the binding of type I cohesin modules located in a non-catalytic molecular scaffold to type I dockerin modules located at the C terminus of cellulosomal enzymes. The majority of type I dockerins display internal symmetry reflected by the presence of two essentially identical cohesin-binding surfaces. Here we report the crystal structures of two novel Clostridium thermocellum type I cohesin-dockerin complexes (CohOlpC-Doc124A and CohOlpA-Doc918). The data revealed that the two dockerins, Doc918 and Doc124A, are unusual because they lack the structural symmetry required to support a dual binding mode. Thus, in both cases, cohesin recognition is dominated by residues located at positions 11, 12, and 19 of one of the dockerin binding surfaces. The alternative binding mode is not possible (Doc918) or highly limited (Doc124A) because residues that assume the critical interacting positions, when dockerins are reoriented by 180°, make steric clashes with the cohesin. In common with a third dockerin (Doc258) that also presents a single binding mode, Doc124A directs the appended cellulase, Cel124A, to the surface of C. thermocellum and not to cellulosomes because it binds preferentially to type I cohesins located at the cell envelope. Although there are a few exceptions, such as Doc918 described here, these data suggest that there is considerable selective pressure for the evolution of a dual binding mode in type I dockerins that direct enzymes into cellulosomes.     

Cohesin-dockerin microarray: Diverse specificities between two complementary families of interacting protein modules

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.     

解纤维梭菌纤维小体黏附域与锚定域相互作用的差异性

黏附域(cohesin)与锚定域(dockerin)的相互作用是纤维素酶复合体-纤维小体(cellulosome)组装的基础,该作用是自然界已知最强的相互作用力之一。为解析纤维小体的装配机制,本研究以解纤维梭菌(Ruminiclostridium cellulolyticum)纤维小体为研究对象,通过Pull-down和等温滴定量热(ITC)的方法,分析并比较不同簇的3个黏附域与7个锚定域之间的相互作用。结果表明,不同簇黏附域与锚定域的相互作用具有显著差异。其中,Coh1与Doc-0729的相互作用最强,K_a为10⁸ M^-1,Coh7与Doc-0729、0931作用力强,K_a为10⁷ M^-1,而Coh8与Doc-0931、1656、0752作用强,K_a也达到10⁷ M^-1。总之,Doc-0729、0931、1656与3个Coh的结合力均较高。本研究揭示了纤维小体黏附域与锚定域的组装具有偏爱性...     

Cell-surface binding domains from Clostridium cellulovorans can be used for surface display of cellulosomal scaffoldins in Lactococcus lactis

Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2^nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.     

A scaffoldin of the Bacteroides cellulosolvens cellulosome that contains 11 type II cohesins

A cellulosomal scaffoldin gene, termed cipBc, was identified and sequenced from the mesophilic cellulolytic anaerobe Bacteroides cellulosolvens. The gene encodes a 2,292-residue polypeptide (excluding the signal sequence) with a calculated molecular weight of 242,437. CipBc contains an N-terminal signal peptide, 11 type II cohesin domains, an internal family III cellulose-binding domain (CBD), and a C-terminal dockerin domain. Its CBD belongs to family IIIb, like that of CipV from Acetivibrio cellulolyticus but unlike the family IIIa CBDs of other clostridial scaffoldins. In contrast to all other scaffoldins thus far described, CipBc lacks a hydrophilic domain or domain X of unknown function. The singularity of CipBc, however, lies in its numerous type II cohesin domains, all of which are very similar in sequence. One of the latter cohesin domains was expressed, and the expressed protein interacted selectively with cellulosomal enzymes, one of which was identified as a family 48 glycosyl hydrolase on the basis of partial sequence alignment. By definition, the dockerins, carried by the cellulosomal enzymes of this species, would be considered to be type II. This is the first example of authentic type II cohesins that are confirmed components of a cellulosomal scaffoldin subunit rather than a cell surface anchoring component. The results attest to the emerging diversity of cellulosomes and their component sequences in nature.