Enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum explored by two-dimensional analysis: identification of seven genes encoding new dockerin-containing proteins

Abstract

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.

FIG. 1.

Composition of the cellulolytic system of C. cellulolyticum and localization of the dockerin-containing proteins. (A and B) Proteins of the Fc fraction were separated on two-dimensional gels (12% polyacrylamide with IEF at pH 4 to 7 [A] or 8% polyacrylamide with IEF at pH 3.9 to 5.1 [B]) and silver stained. The gel images are oriented with the IEF dimension horizontal and the SDS-PAGE dimension vertical. (C and D) After blotting, proteins separated by 2D electrophoresis on 12% polyacrylamide with IEF at pH 4 to 7 (C) and proteins separated by 2D electrophoresis on 8% polyacrylamide with IEF at pH 3.9 to 5.1 (D) were incubated with biotinylated miniCipC. The positions of known proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, Cel5A, Cel5N, Cel9J, and Cel44O are indicated by arrows, as are the positions of some unknown proteins. Each unknown protein, indicated by bold type, is designated “P” followed by a number referring to the molecular mass of the protein. The N-terminal sequences of these proteins and their enzymatic activities as determined by zymogram assays are shown on Table 3. P32b, P41b, P42, P45, P66, P72, and P105 (in bold type with an asterisk) did not interact with biotinylated miniCipC and presumably lack dockerin.

FIG. 2.

Isolation and characterization of new genes encoding dockerin-containing proteins. Genes encoding Cel9P (P90) (A), Gal27A (B), and Xyn10A (P41a) (C) were amplified from the total genomic DNA of C. cellulolyticum by Trap-Dock PCR using convergent inosine-containing degenerate primers. A nonspecific fragment (length, 0.4 kb) was amplified with oligonucleotides p41dir and dock-In (D). Chromosomal DNA walking was performed to clone DNA flanking regions of each gene. S, signal sequence; GH, glycoside hydrolase; D, dockerin domain; X, domain with unknown function. For glycoside hydrolases and CBMs, the number indicates the family.