Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component.
Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose. The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies. However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J. H. D. Wu, W. H. Orme-Johnson, and A. L. Demain, Biochemistry 27:1703-1709, 1988). To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein. The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues. It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein. A putative ribosome binding site was identified at the 5' end of the gene. A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein. The predicted molecular weight of the secreted protein was 80,670. The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes. A palindromic structure was found downstream from the open reading frame. The celS gene is unique among the known cel genes of C. thermocellum. However, it is highly homologous to the partial open reading frame found in C. cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.
Wang
W K
WK
Department of Chemical Engineering, University of Rochester, New York 14627-0166.
Kruus
K
K
Wu
J H
JH
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002480:Cellulase / Q000235:genetics*; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D003062:Codon; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D003062:Codon; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; C071223:endoglucanase Ss; D002480:Cellulase