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Multienzyme complexes having high activity against crystalline cellulose, known as a cellulosome, have been identified and characterized in cellulolytic clostridia such as Clostridium cellulolyticum (5), Clostridium cellulovorans (8), and Clostridium thermocellum (3, 4, 18) and in anaerobic cellulolytic fungi such as Neocallimastix patriciarum and Piromyces sp. (29). A common feature of the clostridial cellulosomes is that they consist of a large number of catalytic components arranged around noncatalytic scaffolding proteins. The scaffolding proteins have been identified and termed CipA (or scaffoldin) and CipB in C. thermocellum (2, 11, 22), CbpA in C. cellulovorans (27), and CipC in C. cellulolyticum (20). These proteins consist of multiple noncatalytic domains but do not contain any catalytic domains (Fig. 1); e.g., C. thermocellum CipA comprises a cellulose-binding domain (CBD) classified in family III, a docking domain, termed dockerin, and nine cohesin domains (11). Each cohesin domain is a subunit-binding domain which interacts with a dockerin domain of each catalytic component. Therefore, cohesin domains are responsible for integrating catalytic components into the cellulosome (15, 26, 34). C. cellulovorans CbpA is similar to CipA in that both of them contain a family III CBD and nine cohesin domains, although the former contains additional repeated domains of unknown function, termed hydrophilic domains, and does not contain a dockerin domain (27). The strong cellulolytic activity of the clostridial cellulosome systems may be ascribed to the function of the CBD of the scaffolding proteins and/or to the ordered structure of the cellulosomes, because the activity of the dissociated catalytic components was shown to be only 25 to 30% of the activity of the intact cellulosome from C. thermocellum (17).

View larger version (29K): [in this window] [in a new window]   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.

Clostridium josui is a moderately thermophilic cellulolytic bacterium (28). Previously, we cloned a cluster of cellulase genes (9), the celB gene encoding endoglucanase CelB (formerly EG-2), classified into family 8 of glycosyl hydrolases (12), and two incomplete open reading frames (ORFs), ORF1 and ORF2 upstream and downstream of celB, respectively (Fig. 2). The presence of the dockerin domain sequences in the amino acid sequences deduced from celB and ORF1 strongly suggests that C. josui also secretes the cellulosome into the culture medium, although the C. josui cellulosome has not yet been characterized. The organization of this gene cluster, ORF1-celB-ORF2, is similar to that of the cellulase gene cluster, celF-celC-celG, from C. cellulolyticum (5, 9) although the physiological characteristics of C. josui (28) isolated from compost in Thailand are significantly different from those of C. cellulolyticum (21) in several points, especially in optimum growth temperature, i.e., 45°C for the former but 32 to 35°C for the latter. The deduced amino acid sequences of the gene products of ORF1, celB, and ORF2 exhibit extremely high sequence identities89.9, 92.3, and 96.1%with those of CelF, CelC, and CelG, respectively, from C. cellulolyticum. In C. cellulolyticum, the cipC gene, encoding a scaffolding protein, was identified upstream of the celF gene and a part of this gene was cloned by an inverse PCR technique (20). These findings suggested that the gene encoding a scaffolding protein of C. josui exists in the region upstream of ORF1.

View larger version (25K): [in this window] [in a new window]   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.

In this study, we report cloning and DNA sequencing of the cipA gene encoding the scaffolding protein and the celD gene, formerly ORF1, of C. josui. We also describe the existence of the CelD and CipA proteins as major components of the C. josui cellulosome.

Cloning and DNA sequencing of the celD and cipA genes. Plasmid pUCJ2E is a pUC118 derivative that contains a 3.8-kb EcoRI fragment (9) carrying the celB gene along with the two incomplete ORFs of C. josui (Fig. 2). Southern hybridization analysis with the EcoRI-BamHI fragment of pUCJ2E as a probe indicated that NheI digestion of C. josui chromosomal DNA gave a 5.8-kb fragment which was associated with the probe (data not shown). Therefore, we cloned this fragment into the XbaI site of pBluescript II KS+ (Stratagene) upon screening by colony hybridization with the same probe. The recombinant plasmid obtained was named pMK-1 (Fig. 2). DNA sequencing of the genomic DNA insert of pMK-1 revealed that the ORF of cipA extended beyond the 5' end of this DNA fragment, although the full-length celD gene and a part of cipA encoding the C-terminal region of CipA were contained in this region. Southern hybridization was carried out again, with the 2.0-kb NheI-PstI fragment of pMK-1 as a probe, to determine the restriction sites upstream of the 5.8-kb fragment cloned in pMK-1. As a result, we expected that a 6.0-kb EcoRI fragment would contain the full-length cipA gene, and we isolated this DNA fragment (Fig. 2).

View larger version (88K): [in this window] [in a new window]   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.

Amino acid sequences of CipA and CelD. The N terminus of the deduced amino acid sequence of CipA contains a typical signal peptide characteristic of bacterial extracellular proteins (32). Comparison of the amino acid sequence of CipA with entries in the SWISS-PROT and PIR sequence databases revealed that the mature CipA has a molecular structure similar to the scaffolding proteins reported so far, C. cellulovorans CbpA (27), C. thermocellum CipA (11), and C. cellulolyticum CipC (20); i.e., it contains a CBD at its N terminus, followed by a hydrophilic domain and six type I cohesin domains (Fig. 1). In general, scaffolding proteins, which comprise a CBD and a number of cohesin domains, are thought to have mainly two functions: first, they bind a series of catalytic subunits together to form a complex through cohesin-dockerin interaction (15, 26, 30, 34); second, they cause the complex to adsorb onto cellulose and, furthermore, may disorder the crystalline structure of cellulose to produce an easily hydrolyzable substrate through the function of its CBD (7). In C. josui CipA, cohesin domains are located in amino acid 284 to 428, 429 to 576, 577 to 724, 725 to 872, 873 to 1020, and 1021 to 1062. Sequence identities between respective cohesin domains are 63 to 97%. In addition, CipA contains a single hydrophilic domain, which is conserved in C. cellulovorans CbpA (27) and C. cellulolyticum CipC (20) but not in C. thermocellum CipA. Despite the overall similarity of the scaffolding proteins, C. josui CipA is different from C. cellulovorans CbpA and C. thermocellum CipA in that the former is considerably smaller than the others; i.e., the numbers of amino acid residues constituting C. josui CipA, C. cellulovorans CbpA (27), and C. thermocellum CipA (11) are 1,162, 1,848, and 1,854, respectively. Although the full-length scaffolding protein gene, cipC, from C. cellulolyticum has not been cloned, DNA fragments encoding the N- and C-terminal regions of CipC were cloned and sequenced (20). The deduced amino acid sequence of the N-terminal region of CipC, comprising a succession of a signal peptide, a CBD, and two cohesin domains, is highly homologous with the corresponding region of C. josui CipA; the overall sequence identity between them is 82.5%. On the other hand, comparison of the C-terminal regions of CipA and CipC revealed an apparent difference (Fig. 1); i.e., the last (sixth) cohesin domain is connected to another (fifth) cohesin domain in C. josui CipA, although in CipC the last cohesin domain is connected to a hydrophilic domain. Furthermore, N-terminal amino acid sequence analysis of the C. cellulolyticum cellulosomal proteins showed that CipC is a protein of 160 kDa (10), which is larger than the predicted molecular mass (120 kDa) of C. josui CipA. These findings indicate that the molecular architecture of CipA is different from that of CipC. The difference can be explained by assuming that C. josui cipA arose from a common ancestral gene of C. josui cipA and C. cellulolyticum cipC through the deletion of the region encoding its C-terminal moiety, since the N-terminal moieties between these scaffolding protein genes are highly conserved.

View larger version (80K): [in this window] [in a new window]   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 19. 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).

Isolation of the cellulosome from culture of C. josui and identification of CipA and CelD as major components of it. C. josui was cultivated at 45°C for 7 days in 500 ml of GS medium (13) containing 0.5% ball-milled cellulose as a carbon source. The cells were removed by centrifugation at 10,000 × g for 10 min. The supernatant was concentrated 50-fold by ultrafiltration through an XM50 membrane (nominal molecular weight cutoff of 50,000) (Amicon). A portion (3 ml) of the concentrated culture supernatant was subjected to HiPrep Sephacryl S-300HR column (16/60) (Amersham Pharmacia Biotech) chromatography with 50 mM Tris-HCl buffer (pH 7.5) containing 12 mM CaCl₂ and 500 mM NaCl as an eluent. A fraction containing protein complexes with a high molecular mass (ca. 700 kDa) was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (16). All protein samples were prepared by heating at 100°C for 3 min in SDS gel loading buffer to denature the proteins. As shown in Fig. 5, a large number of proteins were visualized by Coomassie brilliant blue staining, and many of them showed endoglucanase activity in the gel containing carboxymethylcellulose as a substrate, by the activity staining assay (1). When the proteins in this fraction were incubated with insoluble cellulose, all of the proteins were bound to the cellulose (data not shown). These results suggest that an extracellular multienzyme complex, cellulosome, may be present in the supernatant of the C. josui culture. After separation of the cellulosomal proteins by SDS-PAGE, the proteins were blotted onto an Immobilon P transfer membrane (Millipore) and subjected to automated amino acid sequencing (model 476A protein sequencer, PE; Applied Biosystems). The N-terminal amino acid sequences of two proteins with molecular masses of 115 and 70 kDa were identified as ADTGVISVQF and AASPVNKVYQERFESMYNKI, respectively, which were found in the amino acid sequences deduced from the cipA and celD genes. These results suggest that CipA and CelD may be components of a C. josui cellulosome and confirm that C. josui CipA is quite smaller than C. cellulolyticum CipC.

View larger version (70K): [in this window] [in a new window]   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.

Taxonomic relatedness between C. josui and C. cellulolyticum. High-level resemblance in the organization of the cellulase gene clusters of C. josui and C. cellulolyticum allows us to expect close taxonomic relatedness between them despite the differences in optimum temperature for their growth, i.e., 45°C for C. josui and 32 to 35°C for C. cellulolyticum. Therefore, we determined the nucleotide sequence of the 16s ribosomal DNA (rDNA) (registered with accession no. AB011057) of C. josui by amplification of rDNA by PCR and direct sequencing of the PCR products. Since C. cellulolyticum was placed in cluster III of the genus Clostridium (6), which consists of cellulolytic species, the 16s rDNA sequences of C. josui and the species of cluster III were aligned and analyzed by the GENETYX computer program. As shown in Fig. 6, the dendrogram places C. josui in cluster III at a position close to C. papyrosolvens and C. cellulolyticum, suggesting that the cellulase gene clusters arise from a common ancestor with some evolutionary modifications.

View larger version (13K): [in this window] [in a new window]   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.

Nucleotide sequence accession number. The nucleotide sequence data reported herein appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB004845.