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.

Heterologous production, assembly, and secretion of a minicellulosome by Clostridium acetobutylicum ATCC 824.

The gene man5K encoding the mannanase Man5K from Clostridium cellulolyticum was cloned alone or as an operon with the gene cipC1 encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic Clostridium acetobutylicum. The expression of the heterologous gene(s) was under the control of a weakened thiolase promoter Pthl. The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter.

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

In recent work, 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.

Towards designer cellulosomes in Clostridia: mannanase enrichment of the cellulosomes produced by Clostridium cellulolyticum.

The man5K gene of Clostridium cellulolyticum was cloned and overexpressed in Escherichia coli. This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases. Mature Man5K displays 62% identity with ManA from Clostridium cellulovorans.

Cellulolysis is severely affected in Clostridium cellulolyticum strain cipCMut1.

Progress towards understanding the molecular basis of cellulolysis by Clostridium cellulolyticm was obtained through the study of the first cellulolysis defective mutant strain, namely cipCMut1. In this mutant, a 2 659 bp insertion element, disrupts the cipC gene at the sequence encoding the seventh cohesin of the scaffoldin CipC. cipC is the first gene in a large 'cel' gene cluster, encoding several enzymatic subunits of the cellulosomes, including the processive cellulase Cel48F, which is the major component.

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.

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.

X-Ray crystal structure of the multidomain endoglucanase Cel9G from Clostridium cellulolyticum complexed with natural and synthetic cello-oligosaccharides.

Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the beta-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.

Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum.

A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes.

Degradation of cellulose substrates by cellulosome chimeras. Substrate targeting versus proximity of enzyme components.

A library of 75 different chimeric cellulosomes was constructed as an extension of our previously described approach for the production of model functional complexes (Fierobe, H.-P., Mechaly, A.

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.

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.

Cel9M, a new family 9 cellulase of the Clostridium cellulolyticum cellulosome.

A new cellulosomal protein from Clostridium cellulolyticum Cel9M was characterized. The protein contains a catalytic domain belonging to family 9 and a dockerin domain. Cel9M is active on carboxymethyl cellulose, and the hydrolysis of this substrate is accompanied by a decrease in viscosity.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.