Sandrine Pagès

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. Proteins 29:517–527, 1997.

Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin.

In recent work (Fierobe, H.-P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaïch, A., Lamed, R., Shoham, Y., and Belaich, J.-P. (2002) J. Biol. Chem. 277, 49621–49630), we reported the self-assembly of a comprehensive set of defined “bifunctional” chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.

The cellulosomes from Clostridium cellulolyticum

Cellulosomes produced by Clostridium cellulolyticum grown on cellulose were purified and separated using anion‐exchange chromatography. SDS/PAGE analysis of six fractions showed variations in their cellulosomal protein composition. Hydrolytic activity on carboxymethyl cellulose, xylan, crystalline cellulose and hatched straw differed from one fraction to another. Fraction F1 showed a high level of activity on xylan, whereas fractions F5 and F6 were most active on crystalline cellulose and carboxymethyl cellulose, respectively. Several cellulosomal components specific to fractions F1, F5 and F6 were investigated using MS analysis. Several hemicellulases were identified, including three xylanases in F1, and several cellulases belonging to glycoside hydrolase families 9 and 5 and, a cystein protease inhibitor were identified in F5 and F6. Synergies were observed when two or three fractions were combined. A mixture containing fractions F1, F3 and F6 showed the most divergent cellulosomal composition, the most synergistic effects and the highest level of activity on straw (the most heterogeneous substrate tested). These findings show that on complex substrates such as straw, synergies occur between differently composed cellulosomes and the degradation efficiency of the cellulosomes is correlated with their enzyme diversity.

A Rhamnogalacturonan Lyase in the Clostridium cellulolyticum Cellulosome

Clostridium cellulolyticum secretes large multienzymatic complexes with plant cell wall-degrading activities named cellulosomes. Most of the genes encoding cellulosomal components are located in a large gene cluster: cipC-cel48F-cel8C-cel9G-cel9E-orfX-cel9H-cel9J-man5K-cel9M. Downstream of the cel9M gene, a new open reading frame was discovered and named rgl11Y. Amino acid sequence analysis indicates that this gene encodes a multidomain pectinase, Rgl11Y, containing an N-terminal signal sequence, a catalytic domain belonging to family 11 of the polysaccharide lyases, and a C-terminal dockerin domain. The present report describes the biochemical characterization of a recombinant form of Rgl11Y. Rgl11Y cleaves the alpha-L-Rhap-(1-->4)-alpha-D-GalpA glycosidic bond in the backbone of rhamnogalacturonan I (RGI) via a beta-elimination mechanism. Its specific activity on potato pectic galactan and rhamnogalacturonan was found to be 28 and 3.6 IU/mg, respectively, indicating that Rgl11Y requires galactan decoration of the RGI backbone. The optimal pH of Rgl11Y is 8.5 and calcium is required for its activity. Rgl11Y was shown to be incorporated in the C. cellulolyticum cellulosome through a typical cohesin-dockerin interaction. Rgl11Y from C. cellulolyticum is the first cellulosomal rhamnogalacturonase characterized.

Transcriptional Regulation of the Clostridium cellulolyticum cip-cel Operon: a Complex Mechanism Involving a Catabolite-Responsive Element

The cip-cel cluster of genes plays an important role in the catabolism of the substrate cellulose by Clostridium cellulolyticum. It encodes several key components of the cellulosomes, including the scaffolding protein CipC and the major cellulase Cel48F. All the genes of this cluster display linked transcription, focusing attention on the promoter upstream from the first gene, cipC. We analyzed the regulation of the cipC promoter using a transcriptional fusion approach. A single promoter is located between nucleotides -671 and -643 with respect to the ATG start codon, and the large mRNA leader sequence is processed at position -194. A catabolite-responsive element (CRE) 414 nucleotides downstream from the transcriptional start site has been shown to be involved in regulating this operon by a carbon catabolite repression mechanism. This CRE is thought to bind a CcpA-like regulator complexed with a P-Ser-Crh-like protein. Sequences surrounding the promoter sequence may also be involved in direct (sequence-dependent DNA curvature) or indirect (unknown regulator binding) regulation.

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.

Design and Production in Aspergillus niger of a Chimeric Protein Associating a Fungal Feruloyl Esterase and a Clostridial Dockerin Domain

ABSTRACT A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergillus niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was located upstream of the FAEA (Doc-FAEA). Northern blot analysis showed similar transcript levels for the two constructs, indicating a posttranscriptional bottleneck for Doc-FAEA production. The sequence encoding the first 514 amino acids from A. niger glucoamylase and a dibasic proteolytic processing site (kex-2) were fused upstream of the Doc-FAEA sequence. By using this fusion strategy, the esterase activity found in the extracellular medium was 20-fold-higher than that of the wild-type reference strain, and the production yield was estimated to be about 100 mg of chimeric protein/liter. Intracellular and extracellular production was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dockerin-cohesin interaction assays, and Western blotting. Labeled cohesins detected an intact extracellular Doc-FAEA of about 43 kDa and a cleaved-off dockerin domain of about 8 kDa. In addition, an intracellular 120-kDa protein was recognized by using labeled cohesins and antibodies raised against FAEA. This protein corresponded to the unprocessed Doc-FAEA form fused to glucoamylase. In conclusion, these results indicated that translational fusion to glucoamylase improved the secretion efficiency of a chimeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin.

Enzyme Diversity of the Cellulolytic System Produced by Clostridium cellulolyticum Explored by Two-Dimensional Analysis: Identification of Seven Genes Encoding New Dockerin-Containing Proteins

The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.

Unraveling enzyme discrimination during cellulosome assembly independent of cohesin–dockerin affinity

Bacterial cellulosomes are generally believed to assemble at random, like those produced by Clostridium cellulolyticum. They are composed of one scaffolding protein bearing eight homologous type I cohesins that bind to any of the type I dockerins borne by the 62 cellulosomal subunits, thus generating highly heterogeneous complexes. In the present study, the heterogeneity and random assembly of the cellulosomes were evaluated with a simpler model: a miniscaffoldin containing three C. cellulolyticum cohesins and three cellulases of the same bacterium bearing the cognate dockerin (Cel5A, Cel48F, and Cel9G). Surprisingly, rather than the expected randomized integration of enzymes, the assembly of the minicellulosome generated only three distinct types of complex out of the 10 possible combinations, thus indicating preferential integration of enzymes upon binding to the scaffoldin. A hybrid scaffoldin that displays one cohesin from C. cellulolyticum and one from C. thermocellum, thus allowing sequential integration of enzymes, was exploited to further characterize this phenomenon. The initial binding of a given enzyme to the C. thermocellum cohesin was found to influence the type of enzyme that subsequently bound to the C. cellulolyticum cohesin. The preferential integration appears to be related to the length of the inter‐cohesin linker. The data indicate that the binding of a cellulosomal enzyme to a cohesin has a direct influence on the dockerin‐bearing proteins that will subsequently interact with adjacent cohesins. Thus, despite the general lack of specificity of the cohesin–dockerin interaction within a given species and type, bacterial cellulosomes are not necessarily assembled at random.

A Two-Component System (XydS/R) Controls the Expression of Genes Encoding CBM6-Containing Proteins in Response to Straw in Clostridium cellulolyticum

The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by Clostridium cellulolyticum differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called xyl-doc) seems to be induced by the presence of straw and not of cellulose. Genes encoding a putative two-component regulation system (XydS/R) were found upstream of xyl-doc. First evidence for the involvement of the response regulator, XydR, part of this two-component system, in the expression of xyl-doc genes was given by the analysis of the cellulosomes produced by a regulator overproducing strain when grown on cellulose. Nano-LC MS/MS analysis allowed the detection of the products of all xyl-doc genes and of the product of the gene at locus Ccel_1656 predicted to bear a carbohydrate binding domain targeting hemicellulose. RT-PCR experiments further demonstrated that the regulation occurs at the transcriptional level and that all xyl-doc genes are transcriptionally linked. mRNA quantification in a regulator knock-out strain and in its complemented derivative confirmed the involvement of the regulator in the expression of xyl-doc genes and of the gene at locus Ccel_1656 in response to straw. Electrophoretic mobility shift assays using the purified regulator further demonstrated that the regulator binds to DNA regions located upstream of the first gene of the xyl-doc gene cluster and upstream of the gene at locus Ccel_1656.