Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal S_L‐protein reveals an unusual degree of internal homology

Abstract

It is known that two proteins of the cellulosomal complex of Clostridium thermocellum (SL and SS) together degrade crystalline cellulose. SL is a glycoprotein of 210,000 Da which enhances the binding to cellulose and the activity of SS, an endoglucanase of 83,000 Da. We have previously reported the cloning of a DNA fragment encoding the N-terminal end of the SL protein using antibodies raised against the native protein. A chromosomal walking approach using an EcoRI and a Bam HI-Sau3A gene library allowed us to isolate the C-terminal end of the gene. Sequencing of both fragments revealed the existence of a leader peptide as has been found in cellulases of the same organism. This leader sequence is followed by a stretch of 14 amino acids that is identical to the N-terminal amino acid sequence of the native secreted protein. The open reading frame (ORF) of this gene encodes a protein of 196,800 Da and is followed by a hairpin loop that could be involved in transcription termination. Within the open reading frame (ORF), we found nine internal repeated elements (IREs) of about 500 nucleotides each. Seven of these sequences displayed 98-100% homology and were located adjacent to each other within the structural gene without intervening regions. The remaining two, located on the N-terminal end of the gene, showed a significantly lower homology. Bearing in mind the inherent instability of reiterated regions, we confirmed the authenticity of our clones by Southern blot analysis using chromosomal C. thermocellum DNA and ruled out the possibility of rearrangements during the cloning and sequencing process. The sequenced gene is designated cipA and the encoded SL protein CipA.

Crystallization and preliminary X-ray analysis of a cohesin domain of the cellulosome from Clostridium thermocellum

“…Its cellulase activity is carried out predominantly by a multi-functional, multi-enzyme complex, termed the cellulosome (Lamed, Setter, Kenig & Bayer, 1983;Lamed & Bayer, 1988). In order to form this very large complex, the bacterium produces a special multipledomain polypeptide, called scaffoldin, which organizes the catalytic (enzymatic) subunits into the complex (Bayer, Setter & Lamed, 1985;Bayer, Morag & Lamed, 1994;Wu, OrmeJohnson & Demain, 1988;Gerngross, Romaniec, Kobayashi, Huskisson & Demain, 1993). The 210kDa scaffoldin subunit comprises a single cellulose-binding domain and nine distinct but closely related copies of a strong protein-recognizing domain, the cohesin domain.…”

Evidence for a dual binding mode of dockerin modules to cohesins

“…The grafting of the catalytic entities, primarily glycoside hydrolases but also carbohydrate esterases and polysaccharide lyases, onto the macromolecular scaffold CipA, contributes to enzyme-substrate targeting and enhances the synergistic interactions between the hydrolases. CipA is a noncatalytic protein composed of nine modules known as type I cohesins (13) which display high affinity for the type I dockerins present in the cellulosomally destined plant cell wall degrading enzymes (14). CipA also contains a type II dockerin at its C terminus, which maintains the cellulosome on the bacterial cell surface by its binding to the type II cohesin modules located in proteins anchored to the bacterial proteoglycan layer (15).…”

Lignocellulosic Biomass Utilization Toward Biorefinery Using Meshophilic Clostridial Species

“…Cellulosomal genes among clostridial genomes were identified and classified as cohesincontaining scaffolding proteins and dockerin-containing proteins. So far, the scaffolding proteins for constructing cellulosomes were found in C. acetobutylicum [42], C. cellulolyticum [43], C. cellulovorans [44], C. josui [45], and C. thermocellum [46]. Among a total of 57 cellulosomal genes of the C. cellulovorans genome, 53 dockerincontaining proteins and four cohesin-containing scaffolding proteins were found, respectively [40].…”