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.

Crystal structure of the cellulase Cel9M enlightens structure/function relationships of the variable catalytic modules in glycoside hydrolases.

Cellulases cleave the beta-1.4 glycosidic bond of cellulose. They have been characterized as endo or exo and processive or nonprocessive cellulases according to their action mode on the substrate. Different types of these cellulases may coexist in the same glycoside hydrolase family, which have been classified according to their sequence homology and catalytic mechanism. The bacterium C. celluloyticum produces a set of different cellulases who belong mostly to glycoside hydrolase families 5 and 9. As an adaptation of the organism to different macroscopic substrates organizations and to maximize its cooperative digestion, it is expected that cellulases of these families are active on the various macroscopic organizations of cellulose chains. The nonprocessive cellulase Cel9M is the shortest variant of family 9 cellulases (subgroup 9(C)) which contains only the catalytic module to interact with the substrate. The crystal structures of free native Cel9M and its complex with cellobiose have been solved to 1.8 and 2.0 A resolution, respectively. Other structurally known family 9 cellulases are the nonprocessive endo-cellulase Cel9D from C. thermocellum and the processive endo-cellulase Cel9A from T. fusca, from subgroups 9(B1) and 9(A), respectively, whose catalytic modules are fused to a second domain. These enzymes differ in their activity on substrates with specific macroscopic appearances. The comparison of the catalytic module of Cel9M with the two other known GH family 9 structures may give clues to explain its substrate profile and action mode.

Cloning, sequencing and overexpression of a Sinorhizobium meliloti M5N1CS carboxymethyl-cellulase gene.

The EndS encoding sequence was isolated from Sinorhizobium meliloti M5N1CS DNA. Comparisons between the deduced amino acid sequence of the mature EndS (337 amino acids, molecular mass 36,418 Da, isoelectric point 4.92) and those of published beta-glycanases showed that this enzyme belongs to family 5 of the glycoside hydrolases. The protein was overproduced in Escherichia coli using a T7 expression system. When the purified overexpressed EndS protein was tested on cellulose-type components, the best substrate was CM-cellulose.

Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes.

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.

Cohesin-dockerin interaction in cellulosome assembly: a single hydroxyl group of a dockerin domain distinguishes between nonrecognition and high affinity recognition.

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(a) approximately 10(9) 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.

Structure of a family IIIa scaffoldin CBD from the cellulosome of Clostridium cellulolyticum at 2.2 A resolution.

The crystal structure of the family IIIa cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit (CipC) of Clostridium cellulolyticum has been determined. The structure reveals a nine-stranded jelly-roll topology which exhibits distinctive structural elements consistent with family III CBDs that bind crystalline cellulose. These include a well conserved calcium-binding site, a putative cellulose-binding surface and a conserved shallow groove of unknown function. The CipC CBD structure is very similar to the previously elucidated family IIIa CBD from the CipA scaffoldin of C. thermocellum, with some minor differences. The CipC CBD structure was also compared with other previously described CBD structures from families IIIc and IV derived from the endoglucanases of Thermomonospora fusca and Cellulomonas fimi, respectively. The possible functional consequences of structural similarities and differences in the shallow groove and cellulose-binding faces among various CBD families and subfamilies are discussed.

Identification of odorant receptors from the Alpine marmot (Marmota marmota).

Alpine Marmots (Marmota marmota) are a good model to study intraspecific chemical communication among mammals. This species has been subjected to several behavioural and biochemical studies regarding both their scent-marking behaviour by cheek-rubbing, and the chemical composition of their glandular secretions. However, no molecular study has been undertaken until today on proteins from the olfactory epithelium possibly implicated in chemical perception. In this study, we identified, to our knowledge for the first time, some olfatory receptors from this wild rodent. Starting with olfactory epithelium of an Alpine Marmot, and by mean of reverse transcriptase polymerase chain reaction technique (RT-PCR), we isolated fourteen partial sequences that exhibited a high degree of homology (45-92%) with olfactory receptors from other vertebrates. Conserved identities and structural features clearly defined these Alpine Marmot sequences as members of the seven transmembrane domain olfactory receptors. All sequences were observed as belonging to known olfactory receptor families and were classified into ten subfamilies of the tetrapods OR class. Finally, Northern blot analysis revealed specific expression of these sequences in the Alpine Marmot olfactory epithelium tissue.

Crystal structure of a cohesin module from Clostridium cellulolyticum: implications for dockerin recognition.

In the assembly of the Clostridium cellulolyticum cellulosome, the multiple cohesin modules of the scaffolding protein CipC serve as receptors for cellulolytic enzymes which bear a dockerin module. The X-ray structure of a type I C. cellulolyticum cohesin module (Cc-cohesin) has been solved using molecular replacement, and refined at 2.0 A resolution. Despite a rather low sequence identity of 32 %, this module has a fold close to those of the two Clostridium thermocellum cohesin (Ct-cohesin) modules whose 3D structures have been determined previously. Cc-cohesin forms a dimer in the crystal, as do the two Ct-cohesins. We show here that the dimer exists in solution and that addition of dockerin-containing proteins dissociates the dimer. This suggests that the dimerization interface and the cohesin/dockerin interface may overlap. The nature of the overall surface and of the dimer interface of Cc-cohesin differ notably from those of the Ct-cohesin modules, being much less polar, and this may explain the species specificity observed in the cohesin/dockerin interaction of C. cellulolyticum and C. thermocellum. We have produced a topology model of a C. cellulolyticum dockerin and of a Cc-cohesin/dockerin complex using homology modeling and available biochemical data. Our model suggests that a special residue pair, already identified in dockerin sequences, is located at the center of the cohesin surface putatively interacting with the dockerin.

Solution structure of the module X2 1 of unknown function of the cellulosomal scaffolding protein CipC of Clostridium cellulolyticum.

Multidimensional, homo- and heteronuclear magnetic resonance spectroscopy combined with dynamical annealing has been used to determine the structure of a 94 residue module (X2 1) of the scaffolding protein CipC from the anaerobic bacterium Clostridium cellulolyticum. An experimental data set comprising 1647 nuclear Overhauser effect-derived restraints, 105 hydrogen bond restraints and 66 phi torsion angle restraints was used to calculate 20 converging final solutions. The calculated structures have an average rmsd about the mean structure of 0.55(+/-0.11) A for backbone atoms and 1.40(+/-0.11) A for all heavy atoms when fitted over the secondary structural elements. The X2 1 module has an immunoglobulin-like fold with two beta-sheets packed against each other. One sheet contains three strands, the second contains four strands. An additional strand is intercalated between the beta-sandwich, as well as two turns of a 3(.10) helix. X2 1 has a surprising conformational stability and may act as a conformational linker and solubility enhancer within the scaffolding protein. The fold of X2 1 is very similar to that of telokin, titin Ig domain, hemolin D2 domain, twitchin immunoglobulin domain and the first four domains of the IgSF portion of transmembrane cell adhesion molecule. As a consequence, the X2 1 module is the first prokaryotic member assigned to the I set of the immunoglobulin superfamily even though no sequence similarity with any member of this superfamily could be detected.

Cohesin-dockerin recognition in cellulosome assembly: experiment versus hypothesis.

The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article (Pagés et al., Proteins 1997;29:517-527) we predicted that four amino acid residues of the approximately 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. Proteins 2000;39:170-177.

CelE, a multidomain cellulase from Clostridium cellulolyticum: a key enzyme in the cellulosome?

CelE, one of the three major proteins of the cellulosome of Clostridium cellulolyticum, was characterized. The amino acid sequence of the protein deduced from celE DNA sequence led us to the supposition that CelE is a three-domain protein. Recombinant CelE and a truncated form deleted of the putative cellulose binding domain (CBD) were obtained. Deletion of the CBD induces a total loss of activity. Exhibiting rather low levels of activity on soluble, amorphous, and crystalline celluloses, CelE is more active on p-nitrophenyl-cellobiose than the other cellulases from this organism characterized to date. The main product of its action on Avicel is cellobiose (more than 90% of the soluble sugars released), and its attack on carboxymethyl cellulose is accompanied by a relatively small decrease in viscosity. All of these features suggest that CelE is a cellobiohydrolase which has retained a certain capacity for random attack mode. We measured saccharification of Avicel and bacterial microcrystalline cellulose by associations of CelE with four other cellulases from C. cellulolyticum and found that CelE acts synergistically with all tested enzymes. The positive influence of CelE activity on the activities of other cellulosomal enzymes may explain its relative abundance in the cellulosome.

Cellulosome from Clostridium cellulolyticum: molecular study of the Dockerin/Cohesin interaction.

Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent. In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function. This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction. Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin. These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other. Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment. Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.

Sequence analysis of scaffolding protein CipC and ORFXp, a new cohesin-containing protein in Clostridium cellulolyticum: comparison of various cohesin domains and subcellular localization of ORFXp.

The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial sequence was published earlier (S. Pagès, A. Bélaïch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Bélaïch, J. Bacteriol. 178:2279-2286, 1996; C. Reverbel-Leroy, A. Bélaïch, A. Bernadac, C. Gaudin, J. P. Bélaïch, and C. Tardif, Microbiology 142:1013-1023, 1996), was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein, called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka = 4.8 x 10(9) M-1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 x 10(8) M-1). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly.

Isolation of putative olfactory receptor sequences from pig nasal epithelium.

Binding to olfactory receptors is the first step in odorant and pheromonal recognition and discrimination. These receptors constitute one of the most important, although poorly known, families of neuronal receptors. In this study we used degenerated oligonucleotides and a RT-PCR approach to selectively amplify olfactory receptors in the nasal epithelium of the domestic pig Sus scrofa. Several combinations of oligonucleotide were tested and allowed the isolation of eleven different partial sequences belonging to the seven transmembrane olfactory receptor family. These receptors formed a separate family within the seven transmembrane receptor superfamily in pigs. Using the criteria of Ben Arie et al. [Ben-Arie N., Lancet D., Taylor C., Khen M., Walker N., Ledbetter DH., Carrozzo R., Patel K., Sheer D., Lehrah H. and North M., Hum. Mol. Genet., 3 (1994) 229-235], the 11 receptors described here can be classified into three known families and seven subfamilies (one known and six new).

Species-specificity of the cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: prediction of specificity determinants of the dockerin domain.

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.

CelG from Clostridium cellulolyticum: a multidomain endoglucanase acting efficiently on crystalline cellulose.

The gene coding for CelG, a family 9 cellulase from Clostridium cellulolyticum, was cloned and overexpressed in Escherichia coli. Four different forms of the protein were genetically engineered, purified, and studied: CelGL (the entire form of CelG), CelGcat1 (the catalytic domain of CelG alone), CelGcat2 (CelGcat1 plus 91 amino acids at the beginning of the cellulose binding domain [CBD]), and GST-CBD(CelG) (the CBD of CelG fused to glutathione S-transferase). The biochemical properties of CelG were compared with those of CelA, an endoglucanase from C. cellulolyticum which was previously studied. CelG, like CelA, was found to have an endo cutting mode of activity on carboxymethyl cellulose (CMC) but exhibited greater activity on crystalline substrates (bacterial microcrystalline cellulose and Avicel) than CelA. As observed with CelA, the presence of the nonhydrolytic miniscaffolding protein (miniCipC1) enhanced the activity of CelG on phosphoric acid swollen cellulose (PASC), but to a lesser extent. The absence of the CBD led to the complete inactivation of the enzyme. The abilities of CelG and GST-CBD(CelG) to bind various substrates were also studied. Although the entire enzyme is able to bind to crystalline cellulose at a limited number of sites, the chimeric protein GST-CBD(CelG) does not bind to either of the tested substrates (Avicel and PASC). The lack of independence between the two domains and the weak binding to cellulose suggest that this CBD-like domain may play a special role and be either directly or indirectly involved in the catalytic reaction.

The cellulolytic system of Clostridium cellulolyticum.

Recent findings on the cellulolytic system of the mesophilic Clostridium cellulolyticum are reviewed. Six cellulases and the scaffolding protein, which are, at the present time, the known components of the cellulosome have been cloned. The catalytic and structural properties of the cloned enzymes CelA, CelC, CelD and CelF are described. It was shown that the grafting of the cellulases onto the scaffolding protein was performed using the dockerin-cohesin attachment device and was strictly dependent on the integrity of both components of the complex. The amino-acid sequences of dockerin and cohesin domains of C. cellulolyticum were compared to that of C. cellulovorans and C. thermocellum. This sequence analysis shows that domains belonging to the thermophilic or the mesophilic bacteria can be placed into two well defined groups. The genetic organization of the gene cluster of C. cellulolyticum is discussed.

Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation.

The role of a miniscaffolding protein, miniCipC1, forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated. The parameters of the binding of miniCipC1, which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined. At saturating concentrations, about 8.2 micromol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, colloidal Avicel, and phosphoric acid-swollen cellulose bound 0.28, 0.38, and 0.55 micromol of miniCipC1 per g, respectively. The dissociation constants measured varied between 1.3 x 10(-7) and 1.5 x 10(-8) M. These results are discussed with regard to the properties of the various substrates. The synergistic action of miniCipC1 and two forms of endoglucanase CelA (with and without the dockerin domain [CelA2 and CelA3, respectively]) in cellulose degradation was also studied. Although only CelA2 interacted with miniCipC1 (K(d), 7 x 10(-9) M), nonhydrolytic miniCipC1 enhanced the activities of endoglucanases CelA2 and CelA3 with all of the insoluble substrates tested. This finding shows that miniCipC1 plays two roles: it increases the enzyme concentration on the cellulose surface and enhances the accessibility of the enzyme to the substrate by modifying the structure of the cellulose, leading to an increased available cellulose surface area. In addition, the data obtained with a hybrid protein, CelA3-CBD(CipC), which was more active towards all of the insoluble substrates tested confirm that the CBD of the scaffolding protein plays an essential role in cellulose degradation.

Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum.

The cellulolytic complex was isolated from Clostridium cellulolyticum grown on cellulose. Upon gel filtration, the complex was found to consist mainly of 600-kDa units, along with a 16-MDa aggregate. Its ability to degrade various substrates and its capacity to bind to the crystalline cellulose were measured. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and blotting analysis showed that all of the known cellulases of this organism are present in this complex. Three major components were observed: the first component, a noncatalytic, large (160-kDa) protein, was identified based on its ability to bind to the dockerin-containing cellulases as scaffolding protein CipC. The other two components, which had molecular masses of 94 and 80.6 kDa, were identified as CelE and CelF, respectively. The identified cellulases and some other components of the cellulosome were able to bind to a miniCipC1 construct. In addition to providing an extensive description of the system, the results of the present study confirm that the dockerin-cohesin domain interaction plays an essential role in the constitution of the cellulosome.

The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form.

The recombinant form of the cellulase CelF of Clostridium cellulolyticum, tagged by a C-terminal histine tail, was overproduced in Escherichia coli. The fusion protein was purified by affinity chromatography on a Ni-nitrilotriacetic acid column. The intact form of CelF (Mr, 79,000) was rapidly degraded at the C terminus, giving a shorter stable form, called truncated CelF (Mr, 71,000). Both the entire and the truncated purified forms degraded amorphous cellulose (kcat = 42 and 30 min(-1), respectively) and microcrystalline cellulose (kcat = 13 and 10 min(-1), respectively). The high ratio of soluble reducing ends to insoluble reducing ends released by truncated CelF from amorphous cellulose showed that CelF is a processive enzyme. Nevertheless, the diversity of the cellodextrins released by truncated CelF from phosphoric acid-swollen cellulose at the beginning of the reaction indicated that the enzyme might randomly hydrolyze beta-1,4 bonds. This hypothesis was supported by viscosimetric measurements and by the finding that CelF and the endoglucanase CelA are able to degrade some of the same cellulose sites. CelF was therefore called a processive endocellulase. The results of immunoblotting analysis showed that CelF was associated with the cellulosome of C. cellulolyticum. It was identified as one of the three major components of cellulosomes. The ability of the entire form of CelF to interact with CipC, the cellulosome integrating protein, or mini-CipC1, a recombinant truncated form of CipC, was monitored by interaction Western blotting (immunoblotting) and by binding assays using a BIAcore biosensor-based analytical system.