Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

Nucleotide sequence of the cellulase gene celD encoding endoglucanase D of Clostridium thermocellum.

The nucleotide sequence of the celD gene, encoding the previously crystallized endoglucanase D of Clostridium thermocellum, is reported. The enzyme shares a conserved, reiterated domain with the COOH-terminal end of endoglucanases A and B from the same organism. The overexpression in Escherichia coli of celD subcloned in pUC8 appears to result from a translational fusion of the NH2-terminal end of the endoglucanase with the NH2-terminal end of beta-galactosidase.     

Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum

The complete nucleotide sequence of the celH gene of Clostridium thermocellum was determined. The open reading frame extended over 2.7-kb DNA fragment and encoded a 900-amino acid (aa) protein (Mr 102,301) which hydrolyzes carboxymethylcellulose, p-nitrophenyl-beta-D-cellobioside, methylumbelliferyl- beta-D-cellobioside, barley beta-glucan, and larchwood xylan. The N terminus showed a typical signal peptide, and a cleavage site after Ser44 was predicted. Two Pro-Thr-Ser-rich regions divided the protein into three approximately equal domains. The central 328-aa region was similar to the N-terminal part, carrying the active site, of C. thermocellum endoglucanase E (EGE; 30.2%). The C-terminal region ended with two conserved 24-aa stretches showing close similarity with those previously described in EGA, EGB, EGD, EGE, EGX, and xylanase from C. thermocellum. Deletions of celH removing up to 327 codons from the 5' end and up to 245 codons from the 3' end of the coding sequence did not affect enzyme activity, confirming that the central domain was indeed responsible for catalytic activity. Production of truncated EGH in Escherichia coli was increased up to 120-fold by fusing fragments containing the 3' portion of the gene with the start of lacZ' present in pTZ19R.     

Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum.

The nucleotide sequence of the xynZ gene, encoding the extracellular xylanase Z of Clostridium thermocellum, was determined. The putative xynZ gene was 2,511 base pairs long and encoded a polypeptide of 837 amino acids. A region of 60 amino acids containing a duplicated segment of 24 amino acids was found between residues 429 and 488 of xylanase Z. This region was strongly similar to the conserved domain found at the carboxy-terminal ends of C. thermocellum endoglucanases A, B, and D. Deletions removing up to 508 codons from the 5' end of the gene did not affect the activity of the encoded polypeptide, showing that the active site was located in the C-terminal half of the protein and that the conserved region was not involved in catalysis. Expression of xylanase activity in Escherichia coli was increased up to 220-fold by fusing fragments containing the 3' end of the gene with the start of lacZ present in pUC19. An internal translational initiation site which was efficiently recognized in E. coli was tentatively identified 470 codons downstream from the actual start codon.     

Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG.

The nucleotide sequence of the celG gene of Clostridium thermocellum, encoding endoglucanase CelG, was determined. The open reading frame extended over 1,698 bp and encoded a 566-amino-acid polypeptide (molecular weight of 63,128) similar to the C. thermocellum endoglucanase CelB (51.5% identical residues). The N terminus displayed a typical signal peptide, followed by a catalytic domain. The C terminus, which was separated from the catalytic domain by a 25-amino-acid segment rich in Pro, Thr, and Ser, contained two conserved stretches of 22 amino acids closely similar to those previously described in other cellulases from the same organism. Expression of the gene in Escherichia coli was increased by fusing the fragment coding for the catalytic domain in frame with the start of the lacZ' gene present in the vector. A low- and a high-M(r) form of the protein were purified. The two forms displayed identical enzymatic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that both forms consist of a major polypeptide of M(r) 50,000 and two minor polypeptides of M(r)s 49,000 and 48,000, resulting from heterogeneous proteolytic cleavage at the C terminus. An antiserum raised against the forms purified from E. coli reacted with an immunoreactive polypeptide of M(r) 66,000, which was associated with the extracellular cellulolytic complex of C. thermocellum known as the cellulosome.     

Nucleotide sequence of the Clostridium thermocellum bgIB gene encoding thermostable beta-glucosidase B: homology to fungal beta-glucosidases

Summary The nucleotide sequence of the bglB gene, coding for the thermostable -glucosidase B of Clostridium thermocellum was determined. The coding region of 2265 bp was identified by comparison with the N-terminal amino acid sequence of -glucosidase B purified from Escherichia coli. The derived amino acid sequence corresponding to a polypeptide of M_r 84100 was confirmed by sequencing of the C-terminal peptide generated by cleavage with cyanogen bromide. The protein bears no resemblance to other bacterial -glucosidase sequences. However, extensive regions of homology were identified between the C. thermocellum enzyme and fungal -glucosidases. The N-terminal homologous region contains an amino acid sequence very similar to the active site of -glucosidase A₃ from Aspergillus wentii. The striking sequence similarities between C. thermocellum -glucosidase B and Kluyveromyces fragilis -glucosidase suggest the possibility of a genetic exchange between thermophilic anaerobic bacteria and yeasts.     

Nucleotide sequence of the alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum

The nucleotide sequence of the gene (apu) encoding the thermostable alpha-amylase-pullulanase of Clostridium thermohydrosulfuricum was determined. An open reading frame of 4425 bp was present. The deduced polypeptide (Mr 165,600), including a 31 amino acid putative signal sequence, comprised 1475 amino acids, with no cysteine residues. The structural gene was preceded by the consensus promoter sequence TTGACA TATAAT, a putative regulatory sequence and a putative ribosome-binding sequence AAAGGGGG. The codon usage resembled that of Bacillus genes. The deduced sequence of the mature apu product showed similarities to various amylolytic enzymes, especially the neopullulanase of Bacillus stearothermophilus, whereas the signal sequence showed similarity to those of the alpha-amylases of B. stearothermophilus and B. subtilis. Three regions thought to be highly conserved in the primary structure of alpha-amylases could also be distinguished in the apu product, two being partly 'duplicated' in this alpha-1,4/alpha-1,6-active enzyme.     

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

An endoglucanase gene, engB, from Clostridium cellulovorans, previously cloned into pUC19, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichia coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosome-binding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.     

Nucleotide sequence analysis of the endoglucanase-encoding gene, celCCD, of Clostridium cellulolyticum

The nucleotide sequence of the Clostridium cellulolyticum endo-beta-1,4- glucanase (EGCCD)-encoding gene, celCCD, and its flanking regions, was determined. The open reading frame encodes a protein (Mr 66,061) which consists of 584 amino acids (aa). The N terminus shows the features of the typical signal peptide, with a cleavage site after Gly24. The protein could be divided into N-terminal and C-terminal regions by an intermediate Pro + Thr-rich sequence. Deletion analysis suggests the C-terminal region is not necessary for EG activity. The predicted aa sequence of the mature protein was similar to those of the central catalytic and the following C-terminal regions of the C. thermocellum endoglucanase H (EGH; identity, 58.8%). The N-terminal region resembled that of the endoglucanase, EGCCA, from C. cellulolyticum (identity, 24.7%; 336 aa) and the endoglucanase, EGE, from C. thermocellum (identity, 31.4%; 373 aa). The C-terminal regions ended with two conserved 21-aa stretches which had close similarity to each other. The C-terminal sequence was also highly similar to the reiterated domain of several EG and a xylanase from C. thermocellum, and of an EG from C. cellulolyticum.     

Complete genome sequence of the cellulolytic thermophile Clostridium thermocellum DSM1313

Clostridium thermocellum DSM1313 is a thermophilic, anaerobic bacterium with some of the highest rates of cellulose hydrolysis reported. The complete genome sequence reveals a suite of carbohydrate-active enzymes and demonstrates a level of diversity at the species level distinguishing it from the type strain ATCC 27405.     

Cloning theClostridium thermocellum thermostable laminarinase gene inEscherichia coli: The properties of the enzyme thus produced

Summary We have cloned a 1.9-kb-long fragment ofClostridium thermocellum DNA which encodes laminarinase (EC 3.2.1.39). The enzyme hydrolyzes the -1,3-glucoside bonds in -1,3-and in mixed -1,3-1,4-polyglucans. The enzyme's optimum pH value is around 8.5, temperature optimum –70°C. PAGE-determined mol. weight –32 kDa.Abbreviations used CMC carboxymethyl cellulose - pNPC p-nitrophenyl D cellobioside - pNPLac p-nitrophenyl- D-lactoside - pNPG p-nitrophenyl D glucopyranoside - pNPGal p-nitrophenyl- D galactopyranoside - pNPXyl p-nitrophenyl- - D xylopyranoside - Ap ampicillin - SDS-PAGE SDS polyacrylamide gel electrophoresis