Handling Several Sugars at a Time: a Case Study of Xyloglucan Utilization by Ruminiclostridium cellulolyticum.

Xyloglucan utilization by Ruminiclostridium cellulolyticum was formerly shown to imply the uptake of large xylogluco-oligosaccharides, followed by cytosolic depolymerization into glucose, galactose, xylose, and cellobiose. This raises the question of how the anaerobic bacterium manages the simultaneous presence of multiple sugars. Using genetic and biochemical approaches targeting the corresponding metabolic pathways, we observed that, surprisingly, all sugars are catabolized, collectively, but glucose consumption is prioritized. Most selected enzymes display unusual features, especially the GTP-dependent hexokinase of glycolysis, which appeared reversible and crucial for xyloglucan utilization. In contrast, mutant strains lacking either galactokinase, cellobiose-phosphorylase, or xylulokinase still catabolize xyloglucan but display variably altered growth. Furthermore, the xylogluco-oligosaccharide depolymerization process appeared connected to the downstream pathways through an intricate network of competitive and noncompetitive inhibitions. Altogether, our data indicate that xyloglucan utilization by R. cellulolyticum relies on an energy-saving central carbon metabolism deviating from current bacterial models, which efficiently prevents carbon overflow. IMPORTANCE The study of the decomposition of recalcitrant plant biomass is of great interest as the limiting step of terrestrial carbon cycle and to produce plant-derived valuable chemicals and energy. While extracellular cellulose degradation and catabolism have been studied in detail, few publications describe the complete metabolism of hemicelluloses and, to date, the published models are limited to the extracellular degradation and sequential entry of simple sugars. Here, we describe how the model anaerobic bacterium Ruminiclostridium cellulolyticum deals with the synchronous intracellular release of glucose, galactose, xylose, and cellobiose upon cytosolic depolymerization of imported xyloglucan oligosaccharides. The described novel metabolic strategy involves the simultaneous activity of different metabolic pathways coupled to a network of inhibitions controlling the carbon flux and is distinct from the ubiquitously observed sequential uptake and metabolism of carbohydrates known as the diauxic shift. Our results highlight the diversity of cellular responses related to a complex environment.

Engineering of a new Escherichia coli strain efficiently metabolizing cellobiose with promising perspectives for plant biomass-based application design.

The necessity to decrease our fossil energy dependence requests bioprocesses based on biomass degradation. Cellobiose is the main product released by cellulases when acting on the major plant cell wall polysaccharide constituent, the cellulose. Escherichia coli, one of the most common model organisms for the academy and the industry, is unable to metabolize this disaccharide. In this context, the remodeling of E. coli to catabolize cellobiose should thus constitute an important progress for the design of such applications. Here, we developed a robust E. coli strain able to metabolize cellobiose by integration of a small set of modifications in its genome. Contrary to previous studies that use adaptative evolution to achieve some growth on this sugar by reactivating E. coli cryptic operons coding for cellobiose metabolism, we identified easily insertable modifications impacting the cellobiose import (expression of a gene coding a truncated variant of the maltoporin LamB, modification of the expression of lacY encoding the lactose permease) and its intracellular degradation (genomic insertion of a gene encoding either a cytosolic ß-glucosidase or a cellobiose phosphorylase). Taken together, our results provide an easily transferable set of mutations that confers to E. coli an efficient growth phenotype on cellobiose (doubling time of 2.2 â€‹h in aerobiosis) without any prior adaptation.

A Novel Two-Component System, XygS/XygR, Positively Regulates Xyloglucan Degradation, Import, and Catabolism in Ruminiclostridium cellulolyticum.

Cellulolytic microorganisms play a key role in the global carbon cycle by decomposing structurally diverse plant biopolymers from dead plant matter. These microorganisms, in particular anaerobes such as Ruminiclostridium cellulolyticum that are capable of degrading and catabolizing several different polysaccharides, require a fine-tuned regulation of the biosynthesis of their polysaccharide-degrading enzymes. In this study, we present a bacterial regulatory system involved in the regulation of genes enabling the metabolism of the ubiquitous plant polysaccharide xyloglucan. The characterization of R. cellulolyticum knockout mutants suggests that the response regulator XygR and its cognate histidine kinase XygS are essential for growth on xyloglucan. Using in vitro and in vivo analyses, we show that XygR binds to the intergenic region and activates the expression of two polycistronic transcriptional units encoding an ABC transporter dedicated to the uptake of xyloglucan oligosaccharides and the two-component system itself together with three intracellular glycoside hydrolases responsible for the sequential intracellular degradation of the imported oligosaccharides into mono- and disaccharides. Interestingly, XygR also upregulates the expression of a distant gene coding for the most active extracellular cellulosomal xyloglucanase of R. cellulolyticum by binding to the upstream intergenic region.IMPORTANCERuminiclostridium cellulolyticum is a Gram-positive, mesophilic, anaerobic, cellulolytic, and hemicellulolytic bacterium. The last property qualifies this species as a model species for the study of hemicellulose degradation, import of degradation products, and overall regulation of these phenomena. In this study, we focus on the regulation of xyloglucan dextrin import and intracellular degradation and show that the two components of the two-component regulation system XygSR are essential for growth on xyloglucan and that the response regulator XygR regulates the transcription of genes involved in the extracellular degradation of the polysaccharide, the import of degradation products, and their intracellular degradation.

Catalytic subunit exchanges in the cellulosomes produced by Ruminiclostridium cellulolyticum suggest unexpected dynamics and adaptability of their enzymatic composition.

Cellulosomes are complex nanomachines produced by cellulolytic anaerobic bacteria such as Ruminiclostridium cellulolyticum (formerly known as Clostridium cellulolyticum). Cellulosomes are composed of a scaffoldin protein displaying several cohesin modules on which enzymatic components can bind to through their dockerin module. Although cellulosomes have been studied for decades, very little is known about the dynamics of complex assembly. We have investigated the ability of some dockerin-bearing enzymes to chase the catalytic subunits already bound onto a miniscaffoldin displaying a single cohesin. The stability of the preassembled enzyme-scaffoldin complex appears to depend on the nature of the dockerin, and we have identified a key position in the dockerin sequence that is involved in the stability of the complex with the cohesin. Depending on the residue occupying this position, the dockerin can establish with the cohesin partner either a nearly irreversible or a reversible interaction, independently of the catalytic domain associated with the dockerin. Site-directed mutagenesis of this residue can convert a dockerin able to form a highly stable complex with the miniscaffoldin into a reversible complex forming one and vice versa. We also show that refunctionalization can occur with natural purified cellulosomes. Altogether, our results shed light on the dynamics of cellulosomes, especially their capacity to be remodeled even after their assembly is 'achieved', suggesting an unforeseen adaptability of their enzymatic composition over time.

The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action.

BACKGROUND: The α-l-arabinofuranosidases (α-l-ABFs) are exoenzymes involved in the hydrolysis of α-l-arabinosyl linkages in plant cell wall polysaccharides. They play a crucial role in the degradation of arabinoxylan and arabinan and they are used in many biotechnological applications. Analysis of the genome of R. cellulolyticum showed that putative cellulosomal α-l-ABFs are exclusively encoded by the xyl-doc gene cluster, a large 32-kb gene cluster. Indeed, among the 14 Xyl-Doc enzymes encoded by this gene cluster, 6 are predicted to be α-l-ABFs belonging to the CAZyme families GH43 and GH62. RESULTS: The biochemical characterization of these six Xyl-Doc enzymes revealed that four of them are α-l-ABFs. GH4316-1229 (RcAbf43A) which belongs to the subfamily 16 of the GH43, encoded by the gene at locus Ccel_1229, has a low specific activity on natural substrates and can cleave off arabinose decorations located at arabinoxylan chain extremities. GH4310-1233 (RcAbf43Ad2,3), the product of the gene at locus Ccel_1233, belonging to subfamily 10 of the GH43, can convert the double arabinose decorations present on arabinoxylan into single O2- or O3-linked decorations with high velocity (k cat = 16.6 ± 0.6 s-1). This enzyme acts in synergy with GH62-1234 (RcAbf62Am2,3), the product of the gene at locus Ccel_1234, a GH62 α-l-ABF which hydrolyzes α-(1 → 3) or α-(1 → 2)-arabinosyl linkages present on polysaccharides and arabinoxylooligosaccharides monodecorated. Finally, a bifunctional enzyme, GH62-CE6-1240 (RcAbf62Bm2,3Axe6), encoded by the gene at locus Ccel_1240, which contains a GH62-α-l-ABF module and a carbohydrate esterase (CE6) module, catalyzes deacylation of plant cell wall polymers and cleavage of arabinosyl mono-substitutions. These enzymes are also active on arabinan, a component of the type I rhamnogalacturonan, showing their involvement in pectin degradation. CONCLUSION: Arabinofuranosyl decorations on arabinoxylan and pectin strongly inhibit the action of xylan-degrading enzymes and pectinases. α-l-ABFs encoded by the xyl-doc gene cluster of R. cellulolyticum can remove all the decorations present in the backbone of arabinoxylan and arabinan, act synergistically, and, thus, play a crucial role in the degradation of plant cell wall polysaccharides.

Restoration of cellulase activity in the inactive cellulosomal protein Cel9V from Ruminiclostridium cellulolyticum.

Ruminiclostridium cellulolyticum produces extracellular cellulosomes which contain interalia numerous family-9 glycoside hydrolases, including the inactive Cel9V. The latter shares the same organization and 79% sequence identity with the active cellulase Cel9E. Nevertheless, two aromatic residues and a four-residue stretch putatively critical for the activity are missing in Cel9V. Introduction of one Trytophan and the four-residue stretch restored some weak activity in Cel9V, whereas the replacement of its catalytic domain by that of Cel9E generated a fully active cellulase. Altogether our data indicate that a series of mutations in the catalytic domain of Cel9V lead to an essentially inactive cellulase.

Cel5I, a SLH-Containing Glycoside Hydrolase: Characterization and Investigation on Its Role in Ruminiclostridium cellulolyticum.

Ruminiclostridium cellulolyticum (Clostridium cellulolyticum) is a mesophilic cellulolytic anaerobic bacterium that produces a multi-enzymatic system composed of cellulosomes and non-cellulosomal enzymes to degrade plant cell wall polysaccharides. We characterized one of the non-cellulosomal enzymes, Cel5I, composed of a Family-5 Glycoside Hydrolase catalytic module (GH5), a tandem of Family-17 and -28 Carbohydrate Binding Modules (CBM), and three S-layer homologous (SLH) modules, where the latter are expected to anchor the protein on the cell surface. Cel5I is the only putative endoglucanase targeting the cell surface as well as the only putative protein in R. cellulolyticum containing CBM17 and/or CBM28 modules. We characterized different recombinant structural variants from Cel5I. We showed that Cel5I has an affinity for insoluble cellulosic substrates through its CBMs, that it is the most active endoglucanase on crystalline cellulose of R. cellulolyticum characterized to date and mostly localized in the cell envelope of R. cellulolyticum. Its role in vivo was analyzed using a R. cellulolyticum cel5I mutant strain. Absence of Cel5I in the cell envelope did not lead to a significant variation of the phenotype compared to the wild type strain. Neither in terms of cell binding to cellulose, nor for its growth on crystalline cellulose, thus indicating that the protein has a rather subtle role in tested conditions. Cel5I might be more important in a natural environment, at low concentration of degradable glucose polymers, where its role might be to generate higher concentration of short cellodextrins close to the cell surface, facilitating their uptake or for signalization purpose.

Combining free and aggregated cellulolytic systems in the cellulosome-producing bacterium Ruminiclostridium cellulolyticum.

BACKGROUND: Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans (formerly known as Clostridium cellulolyticum and Clostridium phytofermentans, respectively) are anaerobic bacteria that developed different strategies to depolymerize the cellulose and the related plant cell wall polysaccharides. Thus, R. cellulolyticum produces large extracellular multi-enzyme complexes termed cellulosomes, while L. phytofermentans secretes in the environment some cellulose-degrading enzymes as free enzymes. In the present study, the major cellulase from L. phytofermentans was introduced as a free enzyme or as a cellulosomal component in R. cellulolyticum to improve its cellulolytic capacities. RESULTS: The gene at locus Cphy_3367 encoding the major cellulase Cel9A from L. phytofermentans and an engineered gene coding for a modified enzyme harboring a R. cellulolyticum C-terminal dockerin were cloned in an expression vector. After electrotransformation of R. cellulolyticum, both forms of Cel9A were found to be secreted by the corresponding recombinant strains. On minimal medium containing microcrystalline cellulose as the sole source of carbon, the strain secreting the free Cel9A started to grow sooner and consumed cellulose faster than the strain producing the cellulosomal form of Cel9A, or the control strain carrying an empty expression vector. All strains reached the same final cell density but the strain producing the cellulosomal form of Cel9A was unable to completely consume the available cellulose even after an extended cultivation time, conversely to the two other strains. Analyses of their cellulosomes showed that the engineered form of Cel9A bearing a dockerin was successfully incorporated in the complexes, but its integration induced an important release of regular cellulosomal components such as the major cellulase Cel48F, which severely impaired the activity of the complexes on cellulose. In contrast, the cellulosomes synthesized by the control and the free Cel9A-secreting strains displayed similar composition and activity. Finally, the most cellulolytic strain secreting free Cel9A, was also characterized by an early production of lactate, acetate and ethanol as compared to the control strain. CONCLUSIONS: Our study shows that the cellulolytic capacity of R. cellulolyticum can be augmented by supplementing the cellulosomes with a free cellulase originating from L. phytofermentans, whereas integration of the heterologous enzyme in the cellulosomes is rather unfavorable.

Characterization of all family-9 glycoside hydrolases synthesized by the cellulosome-producing bacterium Clostridium cellulolyticum.

The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.

Are cellulosome scaffolding protein CipC and CBM3-containing protein HycP, involved in adherence of Clostridium cellulolyticum to cellulose?

Clostridium cellulolyticum, a mesophilic anaerobic bacterium, produces highly active enzymatic complexes called cellulosomes. This strain was already shown to bind to cellulose, however the molecular mechanism(s) involved is not known. In this context we focused on the gene named hycP, encoding a 250-kDa protein of unknown function, containing a Family-3 Carbohydrate Binding Module (CBM3) along with 23 hyaline repeat modules (HYR modules). In the microbial kingdom the gene hycP is only found in C. cellulolyticum and the very close strain recently sequenced Clostridium sp BNL1100. Its presence in C. cellulolyticum guided us to analyze its function and its putative role in adhesion of the cells to cellulose. The CBM3 of HycP was shown to bind to crystalline cellulose and was assigned to the CBM3b subfamily. No hydrolytic activity on cellulose was found with a mini-protein displaying representative domains of HycP. A C. cellulolyticum inactivated hycP mutant strain was constructed, and we found that HycP is neither involved in binding of the cells to cellulose nor that the protein has an obvious role in cell growth on cellulose. We also characterized the role of the cellulosome scaffolding protein CipC in adhesion of C. cellulolyticum to cellulose, since cellulosome scaffolding protein has been proposed to mediate binding of other cellulolytic bacteria to cellulose. A second mutant was constructed, where cipC was inactivated. We unexpectedly found that CipC is only partly involved in binding of C. cellulolyticum to cellulose. Other mechanisms for cellulose adhesion may therefore exist in C. cellulolyticum. In addition, no cellulosomal protuberances were observed at the cellular surface of C. cellulolyticum, what is in contrast to reports from several other cellulosomes producing strains. These findings may suggest that C. cellulolyticum has no dedicated molecular mechanism to aggregate the cellulosomes at the cellular surface.

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.

Regulation of cel genes of C. cellulolyticum: identification of GlyR2, a transcriptional regulator regulating cel5D gene expression.

Transcription and expression regulation of some individual cel genes (cel5A, cel5I, cel5D and cel44O) of Clostridium cellulolyticum were investigated. Unlike the cip-cel operon, these genes are transcribed as monocistronic units of transcription, except cel5D. The location of the transcription initiation sites was determined using RT-PCR and the mRNA 5'-end extremities were detected using primer extension experiments. Similarly to the cip-cel operon, cel5A and cel5I expressions are regulated by a carbon catabolite repression mechanism, whereas cel44O and cel5D expressions do not seem to be submitted to this regulation. The role of the putative transcriptional regulator GlyR2 in the regulation of cel5D expression was investigated. The recombinant protein GlyR2 was produced and was shown to bind in vitro to the cel5D and glyR2 promoter regions, suggesting that besides regulating its own expression, GlyR2 may regulate cel5D expression. To test this hypothesis in vivo, an insertional glyR2 mutant was generated and the effect of this disruption on cel5D expression was evaluated. Levels of cel5D mRNAs in the mutant were 16 fold lower than that of the wild-type strain suggesting that GlyR2 acts as an activator of cel5D expression.

Scaffoldin modules serving as "cargo" domains to promote the secretion of heterologous cellulosomal cellulases by Clostridium acetobutylicum.

The secretion of large heterologous cellulases by Clostridium acetobutylicum was formerly shown to be deleterious. To circumvent this issue, various scaffoldins' modules were grafted at their N termini. Family 3a cellulose binding module combined with an X2 module(s) was found to trigger the secretion of Clostridium cellulolyticum cellulases by the solventogenic bacterium.

The issue of secretion in heterologous expression of Clostridium cellulolyticum cellulase-encoding genes in Clostridium acetobutylicum ATCC 824.

The genes encoding the cellulases Cel5A, Cel8C, Cel9E, Cel48F, Cel9G, and Cel9M from Clostridium cellulolyticum were cloned in the C. acetobutylicum expression vector pSOS952 under the control of a Gram-positive constitutive promoter. The DNA encoding the native leader peptide of the heterologous cellulases was maintained. The transformation of the solventogenic bacterium with the corresponding vectors generated clones in the cases of Cel5A, Cel8C, and Cel9M. Analyses of the recombinant strains indicated that the three cellulases are secreted in an active form to the medium. A large fraction of the secreted cellulases, however, lost the C-terminal dockerin module. In contrast, with the plasmids pSOS952-cel9E, pSOS952-cel48F, and pSOS952-cel9G no colonies were obtained, suggesting that the expression of these genes has an inhibitory effect on growth. The deletion of the DNA encoding the leader peptide of Cel48F in pSOS952-cel48F, however, generated strains of C. acetobutylicum in which mature Cel48F accumulates in the cytoplasm. Thus, the growth inhibition observed when the wild-type cel48F gene is expressed seems related to the secretion of the cellulase. The weakening of the promoter, the coexpression of miniscaffoldin-encoding genes, or the replacement of the native signal sequence of Cel48F by that of secreted heterologous or endogenous proteins failed to generate strains secreting Cel48F. Taken together, our data suggest that a specific chaperone(s) involved in the secretion of the key family 48 cellulase, and probably Cel9G and Cel9E, is missing or insufficiently synthesized in C. acetobutylicum.

Synergy, structure and conformational flexibility of hybrid cellulosomes displaying various inter-cohesins linkers.

Cellulosomes are large extracellular multi-enzyme complexes that exhibit elevated activity on plant cell-wall polysaccharides. In the present study, the relationships between the conformational flexibility and efficacy of cellulosomes, and the inter-modules linkers of their scaffold protein were investigated. For this purpose, the length of the intrinsically disordered Ser/Thr-rich 50-residue linker connecting a Clostridium thermocellum and a Clostridium cellulolyticum cohesin in a hybrid scaffoldin (Scaf4) was changed by sequences ranging from 4 to 128 residues. The composition was also modified and new linkers composed of series of N, S or repeats of the EPPV motif were generated. Two model cellulases (Cel48F and Cel9G) appended with appropriate dockerins were subsequently bound to the engineered scaffoldins. All the resulting minicomplexes displayed the same activity on crystalline cellulose as the complex based on the initial Scaf4, and were found to be 2-fold more active than Cel48F and Cel9G bound to separate cohesins. Small-angle X-ray scattering assays of the engineered scaffoldins confirmed, however, that the size and the conformational flexibility of some of the new inter-cohesins linkers differed significantly from that of the initial 50 residue linker displayed by the parental Scaf4. Our data suggest that the synergy induced by proximity does not require a specific inter-cohesins sequence or distance. The present study reveals that complexation onto the hybrid scaffoldins modifies the type of soluble sugars released from crystalline cellulose by the selected cellulases, compared to the free enzyme system.

Random mutagenesis of Clostridium cellulolyticum by using a Tn1545 derivative.

Further understanding of the plant cell wall degradation system of Clostridium cellulolyticum and the possibility of metabolic engineering in this species highlight the need for a means of random mutagenesis. Here, we report the construction of a Tn1545-derived delivery tool which allows monocopy random insertion within the genome.

Modulation of cellulosome composition in Clostridium cellulolyticum: adaptation to the polysaccharide environment revealed by proteomic and carbohydrate-active enzyme analyses.

Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate-active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin-containing proteins bear catalytic modules from numerous carbohydrate-active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip-cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32-kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate-binding module 6 or carbohydrate-binding module 22 xylan-binding modules along dockerins. This newly identified xyl-doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip-cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.

The cellulosomes from Clostridium cellulolyticum: identification of new components and synergies between complexes.

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.

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.