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.
Blouzard
Jean-Charles
JC
Laboratoire de Bioénergétique et Ingénierie des Protéines, IBSM, Centre National de la Recherche Scientifique and Université de Provence, Marseille, France.
Bourgeois
Caroline
C
de Philip
Pascale
P
Valette
Odile
O
Bélaïch
Anne
A
Tardif
Chantal
C
Bélaïch
Jean-Pierre
JP
Pagès
Sandrine
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000145:classification* / Q000235:genetics* / Q000378:metabolism; D044602:Cellulases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D016386:Chromosome Walking; D046973:Ruminiclostridium cellulolyticum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015180:Electrophoresis, Gel, Two-Dimensional; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000145:classification* / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C473132:cellulosome, Clostridium; D002482:Cellulose; D044602:Cellulases; D006026:Glycoside Hydrolases; D002480:Cellulase