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.     

Characterization of the CipA scaffolding protein and in vivo production of a minicellulosome in Clostridium acetobutylicum

The cipA gene encoding the Clostridium acetobutylicum scaffolding protein CipA was cloned and expressed in Escherichia coli. CipA contains an N-terminal signal peptide, a family 3a cellulose-binding domain (CBD), five type I cohesin domains, and six hydrophilic domains. The uniqueness of CipA lies in the enchainment of cohesin domains that are all separated by a hydrophilic domain. Affinity-purified CipA was used in equilibrium-binding experiments to characterize the interaction of CipA with crystalline cellulose. A K(d) of 0.038 micro M and a [C](max) of 0.43 micro mol of CipA bound per g of Avicel were determined. A mini-CipA polypeptide consisting of a CBD3a and two cohesin domains was overexpressed in C. acetobutylicum, yielding the in vivo formation of a minicellulosome. This is to our knowledge the first demonstration of the in vivo assembly of a recombinant minicellulosome.     

Cohesin-dockerin interactions within and between Clostridium josui and Clostridium thermocellum: binding selectivity between cognate dockerin and cohesin domains and species specificity

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.     

Characterization and subcellular localization of the Clostridium thermocellum scaffoldin dockerin binding protein SdbA.

This article reports the characterization of the Clostridium thermocellum SdbA protein thought to anchor the cellulosome to the bacterial cell surface. The NH2-terminal region of SdbA consists of a cohesin domain which specifically binds the dockerin domain of the cellulosomal scaffolding protein CipA. The COOH-terminal region consists of a triplicated segment, termed SLH repeats, which is present in the sequence of many bacterial cell surface polypeptides. The binding parameters of the interaction between the dockerin domain of CipA and the cohesin domain of SdbA were studied by using, as a probe, the chimeric polypeptide CelC-DSCipA, which carries the dockerin domain of CipA fused to endoglucanase CelC. In the presence of Ca2+, CelC-DSCipA bound to SdbA with an affinity constant of 1.26 x 10(7) M(-1). Binding of CelC-DSCipA to SdbA as a function of Ca2+ concentration was sigmoidal, corresponding to a Hill coefficient of 2 and an affinity constant for Ca2+ of 4 x 10(6) M(-2). This suggested the presence of two cooperatively bound Ca2+ ions in the cohesin-dockerin complex. Immunoblotting of C. thermocellum subcellular fractions and electron microscopy of immunocytochemically labeled cells indicated that SdbA is located on the cell surface and is a component of the cellulosome. Together, the data confirm that SdbA could mediate anchoring of the cellulosome to the surface of C. thermocellum cells by interacting with the dockerin domain of CipA.     

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.     

Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose,as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome

The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.     

Interactions of the CelS binding ligand with various receptor domains of the Clostridium thermocellum cellulosomal scaffolding protein, CipA.

The Clostridium thermocellum cellulosomal scaffolding protein, CipA, acts as an anchor on the cellulose surface for the various catalytic subunits of the cellulosome, a large extracellular cellulase complex. CipA contains nine repeated domains that serve as receptors for the cellulosomal catalytic subunits, each of which carries a conserved, duplicated ligand sequence (DS). Four representative CipA receptor domains with sequence dissimilarity were cloned and expressed in Escherichia coli. The interaction of these cloned receptor domains with the duplicated ligand sequence of CelS (expressed as a thioredoxin fusion protein, TRX-DSCelS), was studied by nondenaturing polyacrylamide gel electrophoresis. TRX-DSCelS formed a stable complex with each of the four receptor domains, indicating that CelS, the most abundant cellulosomal catalytic subunit, binds nonselectively to all of the CipA receptors. Conversely, the duplicated sequence of CipA (in the form of TRX-DSCipA), which is homologous to that of CelS, did not bind to any of the receptors under the experimental conditions.     

Transposon Tn10: genetic organization, regulation, and insertion specificity

Transposon Tn10 is a composite element in which two individual insertion sequence (IS)-like sequences cooperate to mediate transposition of the intervening material. The two flanking IS10 elements are not identical; IS10-right is responsible for functions required to promote transposition, and IS10-left is defective in transposition functions. We suggest that the two IS10 elements were originally identical in sequence and have subsequently diverged. IS10-right is compactly organized with structural gene(s), promoters, and sites important for transposition and (presumably) its regulation all closely linked and, in some cases, overlapping. IS10 has a single major coding region that almost certainly encodes an essential transposition function. A pair of opposing promoters flank the start of this coding region. One of these promoters is responsible for expression in vivo of transposon-encoded transposition functions. We propose that the second promoter is involved in modulation of Tn10 transposition. Genetic analysis suggests that transposon-encoded function(s) may be preferentially cis-acting. Insertion of Tn10 into particular preferred target sites is due primarily to the occurrence of a particular six-base pair target DNA sequence. The properties of this sequence suggest that symmetrically disposed subunits of a single protein may be responsible for both recognition and cleavage of target DNA during insertion.     

Production of heterologous and chimeric scaffoldins by Clostridium acetobutylicum ATCC 824

Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.     

An Acetobacter xylinum insertion sequence element associated with inactivation of cellulose production.

An insertion sequence (IS) element, IS1031, caused insertions associated with spontaneous cellulose deficient (Cel-) mutants of Acetobacter xylinum ATCC 23769. The element was discovered during hybridization analysis of DNAs from Cel- mutants of A. xylinum ATCC 23769 with pAXC145, an indigenous plasmid from a Cel- mutant of A. xylinum NRCC 17005. An IS element, IS1031B, apparently identical to IS1031, was identified on pAXC145. IS1031 is about 950 bp. DNA sequencing showed that the two elements had identical termini with inverted repeats of 24 bp containing two mismatches and that they generated 3-bp target sequence duplications. The A. xylinum ATCC 23769 wild type carries seven copies of IS1031. Southern hybridization showed that 8 of 17 independently isolated spontaneous Cel- mutants of ATCC 23769 contained insertions of an element homologous to IS1031. Most insertions were in unique sites, indicating low insertion specificity. Significantly, two insertions were 0.5 kb upstream of a recently identified cellulose synthase gene. Attempts to isolate spontaneous cellulose-producing revertants of these two Cel- insertion mutants by selection in static cultures were unsuccessful. Instead, pseudorevertants that made waxlike films in the liquid-air interface were obtained. The two pseudorevertants carried new insertions of an IS1031-like element in nonidentical sites of the genome without excision of the previous insertions. Taken together, these results suggest that indigenous IS elements contribute to genetic instability in A. xylinum. The elements might also be useful as genetic tools in this organism and related species.     

Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction

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 Geobacillus 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.     

Isolation of a new insertion sequence, IS13655, and its application to Corynebacterium glutamicum genome mutagenesis

A new functional Corynebacterium glutamicum insertion sequence (IS) element, IS13655, was isolated using a suicide vector. The IS element was 1,293 bp in size and contained 26-bp imperfect inverted repeats (IRs) and 3-bp target site duplication as direct repeats (DRs). IS13655 harbored two ORFs with high similarity to the transposase of IS1206, an IS3 family element. IS13655 revealed relatively high transposition efficiency, with low target site selectivity along the Corynebacterium glutamicum R genome, making it a potentially useful genetic engineering tool.     

A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA.

The cellulosome-integrating protein CipA, which serves as a scaffolding protein for the cellulolytic complex produced by Clostridium thermocellum, comprises a COOH-terminal duplicated segment termed the dockerin domain. This paper reports the cloning and sequencing of a gene, termed sdbA (for scaffoldin dockerin binding), encoding a protein which specifically binds the dockerin domain of CipA. The sequenced fragment comprises an open reading frame of 1,893 nucleotides encoding a 631-amino-acid polypeptide, termed SdbA, with a calculated molecular mass of 68,577 kDa. SAA comprises an NH2-terminal leader peptide followed by three distinct regions. The NH2-terminal region is similar to the NH2-terminal repeats of C. thermocellum OlpB and ORF2p. The central region is rich in lysine and harbors a motif present in Streptococcus M proteins. The COOH-terminal region consists of a triplicated sequence present in several bacterial cell surface proteins. The NH2-terminal region of SdbA and a fusion protein carrying the first NH2-terminal repeat of OlpB were shown to bind the dockerin domain of CipA. Thus, a new type of cohesin domain, which is present in one, two, and four copies in SdbA, ORF2p, and OlpB, respectively, can be defined. Since OlpB and most likely SdbA and ORF2p are located in the cell envelope, the three proteins probably participate in anchoring CipA (and the cellulosome) to the cell surface.     

IS186 Insertion at a Hot Spot in the lon Promoter as a Basis for Lon Protease Deficiency of Escherichia coli B: Identification of a Consensus Target Sequence for IS186 Transposition

The radiation sensitivity of Escherichia coli B was first described more than 50 years ago, and the genetic locus responsible for the trait was subsequently identified as lon (encoding Lon protease). We now show that both E. coli B and the first reported E. coli K-12 lon mutant, AB1899, carry IS186 insertions in opposite orientations at a single site in the lon promoter region and that this site represents a natural hot spot for transposition of the insertion sequence (IS) element. Our analysis of deposited sequence data for a number of other IS186 insertion sites permitted the deductions that (i) the consensus target site sequence for IS186 transposition is 5'-(G)(> or =4)(N)(3-6)(C)(> or =4)-3', (ii) the associated host sequence duplication varies within the range of 6 to 12 bp and encompasses the N(3-6) sequence, and (iii) in a majority of instances, at least one end of the duplication is at the G-N (or N-C) junction. IS186-related sequences were absent in closely related bacterium Salmonella enterica serovar Typhimurium, indicating that this IS element is a recent acquisition in the evolutionary history of E. coli.     

Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome.

The ORF3 gene of Clostridium thermocellum encodes a polypeptide (ORF3p) which contains a receptor domain for the docking sequence borne by the catalytic subunits of the cellulosome and a triplicated domain related to some bacterial cell surface proteins. It was thus surmised that ORF3p is a surface protein. In this study, this hypothesis was confirmed. Subcellular fractionation, Western blotting (immunoblotting), and electron microscopy of immunocytochemically labeled cells indicated that ORF3p produced by C. thermocellum was located in the outer surface layer of the bacterium. This layer appeared to consist of a soft matrix shedding off particulate fragments. Nonsedimenting ORF3p derived from sonicated cells was associated with high-molecular-mass fractions (> 20 MDa), probably corresponding to fragments of the outer cell layer. The same high-molecular-mass fractions also contained the cellulosomal marker CipA. Contrary to CipA, however, ORF3p was not associated with 2- to 4-MDa fractions corresponding to individual cellulosomes, and a significant fraction of ORF3p failed to bind to cellulose. It is proposed that ORF3 and ORF3p be renamed olpA and OlpA, respectively (for outer layer protein).     

Analysis of cohesin-dockerin interactions using mutant dockerin proteins

Clostridial cellulosomes are cellulolytic complexes that are formed by highly specific interactions between one of the repeated cohesin modules present in the scaffolding protein and a dockerin module of the catalytic components. Although Clostridium thermocellum Xyn11A dockerin has a typical C. thermocellum dockerin sequence, in which two amino acid residues are species specifically conserved within the two segments of the dockerin modules, it can recognize Clostridium josui cohesin modules in a non-species-specific manner. The importance of these two conserved amino acids, which are part of the recognition site of the cohesin and dockerin interaction, was investigated by introducing mutations into the first and/or the second segments of the Xyn11A dockerin. Mutations in the first segment did not affect the interactions between dockerin and C. thermocellum and C. josui cohesins. However, mutations in the second segment prevented binding to cohesin proteins. A second round of mutations within the first segment re-established the affinity for both the C. thermocellum and the C. josui cohesins. However, this was not observed for a 'conventional' dockerin, Xyn10C. These results suggest that the combination of the first and second dockerin segments is important for the target recognition.     

Interaction between a type-II dockerin domain and a type-II cohesin domain from Clostridium thermocellum cellulosome

The interaction between the type-II dockerin domain of the scaffoldin protein CipA and the type-II cohesin domain of the outer layer protein SdbA is the fundamental mechanism for anchoring the cellulosome to the cell surface of Clostridium thermocellum. We constructed and purified a dockerin polypeptide and a cohesin polypeptide, and determined affinity constants of the interaction between them by the surface plasmon resonance method. The dissociation constant (K(D)) value was 1.8 x 10(-9) M, which is a little larger than that for the combination of a type-I dockerin and a type-I cohesin.     

Unusual binding properties of the dockerin module of Clostridium thermocellum endoglucanase CelJ (Cel9D-Cel44A)

Cellulosomes are cellulolytic complexes produced by anaerobic bacteria, and are composed of a scaffolding protein and several catalytic components. The complexes are formed by highly specific interactions of one of the reiterated cohesin modules of the scaffolding protein with a dockerin module of the catalytic components. The affinities of a dockerin module of Clostridium thermocellum CelJ (Cel9D-Cel44A) for several cohesin modules from C. thermocellum and Clostridium josui scaffolding proteins were quantitatively measured by surface plasmon resonance analysis. The recombinant CelJ dockerin-containing protein interacted with three recombinant C. josui cohesin proteins as well as recombinant C. thermocellum cohesin proteins beyond the so-called 'species specificity' of the dockerin and cohesin interactions. However, this protein did not recognize a second cohesin module from the C. josui scaffolding protein, suggesting that the catalytic components are not necessarily arranged randomly on a scaffolding protein in native cellulosomes.     

Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface.

The nucleotide sequence was determined for a 9.4-kb region of Clostridium thermocellum DNA extending from the 3' end of the gene (now termed cipA), encoding the S1/SL component of the cellulosome. Three open reading frames (ORFs) belonging to two operons were detected. They encoded polypeptides of 1,664, 688, and 447 residues, termed ORF1p, ORF2p, and ORF3p, respectively. The COOH-terminal regions of the three polypeptides were highly similar and contained three reiterated segments of 60 to 70 residues each. Similar segments have been found at the NH2 terminus of the S-layer proteins of Bacillus brevis and Acetogenium kivui, suggesting that ORF1p, ORF2p, and ORF3p might also be located on the cell surface. Otherwise, the sequence of ORF1p and ORF2p gave little clue concerning their potential function. However, the NH2-terminal region of ORF3p was similar to the reiterated domains previously identified in CipA as receptors involved in binding the duplicated segment of 22 amino acids present in catalytic subunits of the cellulosome. Indeed, it was found previously that ORF3p binds 125I-labeled endoglucanase CelD containing the duplicated segment (T. Fujino, P. Béguin, and J.-P. Aubert, FEMS Microbiol. Lett. 94:165-170, 1992). These findings suggest that ORF3p might serve as an anchoring factor for the cellulosome on the cell surface by binding the duplicated segment that is present at the COOH end of CipA.