Cloning and DNA sequencing of the genes encoding Clostridium josui scaffolding protein CipA and cellulase CelD and identification of their gene products as major components of the cellulosome - PubMed (original) (raw)

Cloning and DNA sequencing of the genes encoding Clostridium josui scaffolding protein CipA and cellulase CelD and identification of their gene products as major components of the cellulosome

M Kakiuchi et al. J Bacteriol. 1998 Aug.

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. josui CipA is similar to those of the scaffolding proteins reported so far, such as Clostridium thermocellum CipA, Clostridium cellulovorans CbpA, and Clostridium cellulolyticum CipC, 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 the C. josui cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome.

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Figures

FIG. 1

Schematic diagrams of the scaffolding proteins responsible for cellulosome assembly. The amino acid sequences of C. cellulolyticum CipC, C. cellulovorans CbpA, and C. thermocellum CipA and CipB were derived from their gene sequences. The complete nucleic acid sequences of C. cellulolyticum cipC and C. thermocellum cipB have not been reported.

FIG. 2

Restriction maps of pMK-1 and pMK-2 (A) and the gene cluster of cipA, celD, celB, and celE (B). Thin lines correspond to vector plasmid DNA. Arrows indicate DNA fragments used as probes for Southern and Northern hybridizations.

FIG. 3

Nucleotide and deduced amino acid sequences of cipA and celD. The putative promoter and Shine-Dalgarno (SD) sequences are underlined. The amino acid stretches marked with a broken line were identified in the cellulosomal proteins by automated gas-phase sequencing. A palindrome is indicated by arrows facing each other. The duplicated sequences in the dockerin domain are boxed. ▵, boundary between a CBD and a hydrophilic domain; ▴, boundary between a hydrophilic domain and a cohesin domain or between two contiguous cohesin domains.

FIG. 4

Alignment of dockerin domains of CelB and CelD of C. josui (Cj); CelA, CelC, CelD, CelE, CelF, CelG, CelH, and CelJ of C. cellulolyticum (Cc); and CelS of C. thermocellum (Ct). Sequences of C. cellulolyticum proteins are from reference . Asterisks indicate amino acid residues involved in calcium-binding. The NST motifs in C. thermocellum CelS are double underlined. Residues suspected of serving as selectivity determinants are indicated by pound signs (#). Amino acids which are conserved or have similar chemical properties (I, L, M, V, K, R, S, and T) in at least 7 of 10 C. josui and C. cellulolyticum sequences are printed in white on black. Numbers refer to amino acid residues at the start of the respective lines; all sequences are numbered from Met-1 of the peptide. Complete amino acid sequences of CelE, CelH, and CelJ are not known (19).

FIG. 5

SDS-PAGE analysis of the cellulase complex from culture fluid of C. josui. Lane 1, Coomassie brilliant blue staining; lane 2, activity staining with carboxymethylcellulose as a substrate; lane M, protein molecular mass standards. Sizes are shown on the left.

FIG. 6

Dendrogram of 16s rDNA sequences of cluster III Clostridium species and C. josui constructed by the GENETYX program. The accession numbers for the rDNA sequences are as follows: C. papyrosolvens, X71852; C. cellulolyticum, X71847; C. cellobioparum, X71856; C. termitidis, X71854; C. aldrichii, X71846; C. thermocellum, L09173; C. stercorarium, L09174; C. thermolacticum, X72870. Bar, 1% sequence divergence.

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Cloning and DNA sequencing of the genes encoding Clostridium josui scaffolding protein CipA and cellulase CelD and identification of their gene products as major components of the cellulosome - PubMed (original) (raw)