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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, Ce148F, Ce18C, Ce19G, Ce19E, Man5K, Ce19M, and Ce15A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Ce15N, Ce19J, and Ce1440 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.

Two chimeric enzymes, FLX and FLXLC, are constructed and successfully overproduced in Aspergillus niger. The results have shown that the fusion of naturally free cell wall hydrolases and an A. niger-derived carbohydrate-binding module (CBM) onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates.

The gene encoding Man5K from Clostridium cellulolyticum was cloned and expressed to examine the capacity of Clostridium acetobutylicum to produce and secrete a heterologous complex mimicking a cellulosome. The results show that C. acetobutylicum is a suitable host for the production, assembly and secretion of heterologous minicellulosomes.

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. Two forms of the protein were purified from E. coli; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module. Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less. The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme. Man5K was successfully overproduced in C. cellulolyticum by using expression vectors. The trans-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components. Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20%. This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.

The three-dimensional crystal structure of the cohesin-dockerin heterodimer that sheds light on the critical amino acids that contribute to high affinity protein-protein interaction is reviewed. It is concluded that the regulation of cellulosome-related genes, budding genetic tools, and emerging genomics of cellulosome-producing bacteria promise new insight into the assembly and consequences of the multienzyme complex.

Clostridium cellulolyticum secretes large multienzymatic complexes with plant cell wail-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[vector]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.

Research has been conducted on the cellulosome protein from Clostridium cellulolyticum Ce19M. The characterization of this protein has been carried out and the results indicate that it contains a catalytic domain which belongs to family 9 and a dockerin domain.

Results demonstrate that CelE is one of the three major cellulosome proteins and is a cellobiohydrolase. Data show that CelE acts synergistically with other bacterial cellulosomal enzymes indicating that it is relatively abundant in the cellulosome.

The HndA subunit of NADP-inhibiting hydrogenase of Desulfovibrio fructuosovorans overproduced in Escherichia coli was purified and characterized. The purified subunit was found to have 1.7 moles of acid-labile sulphide per mole and 1.7 moles of non-heme iron. Analysis of the electron paramagnetic resonance of the reduced form of HndA revealed the presence of a single binuclear cluster that displays a spectrum of rhombic symmetry.

Cloning and overexpression were conducted in Escherichia coli of the gene coding for CelG, a family 9 cellulase from Clostridium cellulolyticum. Purification of the proteins to homogeneity was done to examine their biochemical properties. The lack of independence between the domains and the weak binding to cellulose indicate that this cellulose binding domain may play a special part and be either directly or indirectly involved in the catalytic reaction.

The cellulosomal endogluconase, CelA, was utilized to determine the role of the cellulose-binding domain (CBD) of the CipC scaffolding protein of Clostridium cellulolyticum on cellulose degradation. Analysis of the truncated form of CipC indicated the role of the CipC CBD in anchoring CelA to the cellulose surface and modifying the structure of the cellulose. Modification of cellulose structure in the presence of CelA also involves the exposure of new sites that can be hydrolyzed by the enzyme.

The catalytic binding properties of the purified cellusome of Clostridium cellulolyticum were analyzed to determine the molecular nature of the mesophilic bacterium's native cellulolytic complex. The native cellulolytic system of Clostridium cellulolyticum is composed of a 16-MDa aggregate and 600-kDa cellulosome units that do not have similar compositions. The relative proportions of the various cellulosome units are also dependent on culture conditions such as substrate type and starvation times.

The T7 expression system was utilized for overproduction and characterization of recombinant CelF of Clostridium cellulolyticum from Escherichia coli. Analysis of the truncated and intact forms of the purified 79 kilodalton fusion protein characterized CelF as one of the three major components of the Clostridium cellulolyticum cellulosome. Furthermore, CelF was more active on swollen Avicel than on microcrystalline Avicel and substituted soluble carboxymethyl-cellulose.

The 5' end of the cipC gene coding for the N-terminal portion of CipC, the scaffolding protein of Clostridium cellulolyticum ATCC 35319, was cloned and sequenced. It was found to encode a 586-amino-acid-peptide including a cellulose-binding domain, a hydrophilic domain and two hydrophobic domains. The N terminus of CipC and CbpA of C. cellulovorans have the same structure. Only the endoglucanase CelA was found to interact with cohesin domain 1 of CipC. There seems to be a high affinity between these two proteins.

The cloning, sequencing and expression of the celF gene of Clostridium cellulolyticum corresponding to the open reading frame-1 in the cel gene cluster are discussed. Expression of celF in Escherichia coli under standard conditions caused the recombinant protein to aggregate and form inclusion bodies. A mature form of CelF was generated after optimizing the induction conditions. CelF hydrolyzed cellulose but was poorly active on substituted cellulose.

The hnd operon (the genomic DNA fragment from Desulfovibrio fructosovorans) encodes nicotinamide adenine dinucleotide phosphate-reducing hydrogenase in D.fructosovorans. The hndA, hndB, hndC and hndD are the four genes found in the fragment and were present on the same transcription unit. Immunoblotting studies on cell extracts from D. fructosovorans wild type and one hndC deletion mutant (SM3) shows that 52-kDa protein is recognized only in wild-type strains.

The operon structure of the genes (hyn) coding for the periplasmic (NiFe) hydrogenase in Desulfovibrio fructosovorans was investigated. Northern blot analysis showed that the hyn operon transcribes three different mRNAs corresponding to hynA, hynB and hynD, encoding three subunits of NiFe dehydrogenase. Sequence analysis showed that the promoter region of the hyn operon bears strong resemblance to the Escherichia coli sigma-70-type of promoter.

A chimeric enzyme associating feruloyl esterase A from Aspergillus Niger and dockerin from Clostridium thermocellum is produced. The conclusion states that translational fusion to glucoamylase improved the secretion efficiency of a himeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin.

Research has been conducted on insertion sequence elements found in Clostridium cellulolyticum cipC gene. The description of these elements is presented.

Cellulolytic complexes produced by Clostridium cellulolyticum were investigated using site-directed mutagenesis, surface plasmon resonance, calcium overlay and circular dichroism. Results showed that both cohesin and dockerin were functionally independent domains. Using primary sequence analysis, each conserved segment of the dockerin domain was also shown to bind calcium. It was also proven that two segments of the dockerin were required for an interaction with the cohesin to occur.

The complete sequence of the gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum was determined. The corresponding protein, CipC, was found to be comprised of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains, a second hydrophilic domain, and a final cohesin domain which is only 57 to 60% similar to the seven other cohesin domains.

The crystal structure of the family lIla 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. [ABSTRACT FROM AUTHOR]