Cloning and DNA Sequencing of the Genes EncodingClostridium josui Scaffolding Protein CipA and Cellulase CelD and Identification of Their Gene Products as Major Components of the Cellulosome

ABSTRACT The Clostridium josui cipA and celD genes, encoding a scaffolding-like protein (CipA) and a putative cellulase (CelD), respectively, have been cloned and sequenced. CipA, with an estimated molecular weight of 120,227, consists of an N-terminal signal peptide, a cellulose-binding domain of family III, and six successive cohesin domains. The molecular architecture of C. josuiCipA is similar to those of the scaffolding proteins reported so far, such as Clostridium thermocellum CipA, Clostridium cellulovorans CbpA, and Clostridium cellulolyticumCipC, but C. josui CipA is considerably smaller than the other scaffolding proteins. CelD consists of an N-terminal signal peptide, a family 48 catalytic domain of glycosyl hydrolase, and a dockerin domain. N-terminal amino acid sequence analysis of theC. josui cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome.

[1]  C. Tardif,et al.  Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum , 1997, Applied and environmental microbiology.

[2]  A. Demain,et al.  Two components of an extracellular protein aggregate of Clostridium thermocellum together degrade crystalline cellulose , 1988 .

[3]  J. Wu,et al.  Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component , 1993, Journal of bacteriology.

[4]  R. Doi,et al.  The Clostridium cellulovorans cellulosome. , 1994, Critical reviews in microbiology.

[5]  E. Bayer,et al.  Expression, purification and subunit‐binding properties of cohesins 2 and 3 of the Clostridium thermocellum cellulosome , 1995, FEBS letters.

[6]  A. Demain,et al.  Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL‐protein reveals an unusual degree of internal homology , 1993, Molecular microbiology.

[7]  Arnold L. Demain,et al.  Saccharification of Complex Cellulosic Substrates by the Cellulase System from Clostridium thermocellum , 1982, Applied and environmental microbiology.

[8]  E. Bayer,et al.  Species‐specificity of the cohesin‐dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: Prediction of specificity determinants of the dockerin domain , 1997, Proteins.

[9]  D. Kilburn,et al.  Non–Hydrolytic Disruption of Cellulose Fibres by the Binding Domain of a Bacterial Cellulase , 1991, Bio/Technology.

[10]  O. Shoseyov,et al.  Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[11]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[12]  A. Demain,et al.  The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[13]  G. Heijne.  A new method for predicting signal sequence cleavage sites. , 1986 .

[14]  P. Dhurjati,et al.  Properties conferred on Clostridium thermocellum endoglucanase CelC by grafting the duplicated segment of endoglucanase CelD. , 1993, Protein engineering.

[15]  S. Karita,et al.  Nucleotide sequences of the celB gene encoding endo-1,4-β-β-glucanase-2, ORF1 and ORF2 forming a putative cellulase gene cluster of Clostridium josui , 1993 .

[16]  P. Béguin,et al.  The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation. , 1996, Critical reviews in biochemistry and molecular biology.

[17]  M. Fletcher.  Bacterial adhesion : molecular and ecological diversity , 1996 .

[18]  H. Heus,et al.  Phylogeny of the conserved 3' terminal structure of the RNA of small ribosomal subunits. , 1984, Nucleic acids research.

[19]  S. Shimizu,et al.  Clostridium josui sp. nov., a Cellulolytic, Moderate Thermophilic Species from Thai Compost , 1988 .

[20]  K. Sakka,et al.  Structure of cellulases and their applications. , 1997, Biotechnology & genetic engineering reviews.

[21]  E. Bayer,et al.  The cellulosome--a treasure-trove for biotechnology. , 1994, Trends in biotechnology.

[22]  M E Watson,et al.  Compilation of published signal sequences. , 1984, Nucleic acids research.

[23]  G P Hazlewood,et al.  Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS. , 1992, FEMS microbiology letters.

[24]  G. Hazlewood,et al.  Characterization of the subunits in an apparently homogeneous subpopulation of Clostridium thermocellum cellulosomes. , 1995, Enzyme and microbial technology.

[25]  J. Giallo,et al.  Clostridium cellulolyticum sp. nov., a cellulolytic, mesophilic species from decayed grass , 1984 .

[26]  D Court,et al.  Regulatory sequences involved in the promotion and termination of RNA transcription. , 1979, Annual review of genetics.

[27]  C. Tardif,et al.  Interaction between the endoglucanase CelA and the scaffolding protein CipC of the Clostridium cellulolyticum cellulosome , 1996, Journal of bacteriology.

[28]  C. Tardif,et al.  Molecular study and overexpression of the Clostridium cellulolyticum celF cellulase gene in Escherichia coli. , 1996, Microbiology.

[29]  Raphael Lamed,et al.  Dissociation of the cellulosome of Clostridium thermocellum under nondenaturing conditions , 1996 .

[30]  J. Aubert,et al.  Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA , 1994, Journal of bacteriology.

[31]  C. Tardif,et al.  The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form , 1997, Journal of bacteriology.

[32]  A Bairoch,et al.  Updating the sequence-based classification of glycosyl hydrolases. , 1996, The Biochemical journal.

[33]  P. Lawson,et al.  The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. , 1994, International journal of systematic bacteriology.

[34]  C. Tardif,et al.  The cellulolytic system of Clostridium cellulolyticum. , 1997, Journal of biotechnology.