Celgene Research Articles

Immunogold labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant vigna angularis

The catalytic subunit of cellulose synthase is shown to be associated with the putative cellulose-synthesizing complex (rosette terminal complex [TC]) in vascular plants. The catalytic subunit domain of cotton cellulose synthase was cloned using a primer based on a rice expressed sequence tag (D41261) from which a specific primer was constructed to run a polymerase chain reaction that used a cDNA library from 24 days postanthesis cotton fibers as a template. The catalytic region of cotton cellulose synthase was expressed in Escherichia coli, and polyclonal antisera were produced. Colloidal gold coupled to goat anti-rabbit secondary antibodies provided a tag for visualization of the catalytic region of cellulose synthase during transmission electron microscopy. With a freeze-fracture replica labeling technique, the antibodies specifically localized to rosette TCs in the plasma membrane on the P-fracture face. Antibodies did not specifically label any structures on the E-fracture face. Significantly, a greater number of immune probes labeled the rosette TCs (i.e., gold particles were 20 nm or closer to the edge of the rosette TC) than did preimmune probes. These experiments confirm the long-held hypothesis that cellulose synthase is a component of the rosette TC in vascular plants, proving that the enzyme complex resides within the structure first described by freeze fracture in 1980. In addition, this study provides independent proof that the CelA gene is in fact one of the genes for cellulose synthase in vascular plants.

Structural and putative regulatory genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii.

Six genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii were identified using Tn5 mutagenesis. Four of them displayed homology to the previously cloned and sequenced Agrobacterium tumefaciens cellulose genes celA, celB, celC and celE. These genes are organized similarly in R. leguminosarum bv. trifolii. In addition, there were strong indications that two tandemly located genes, celR1 and celR2, probably organized as one operon, are involved in the regulation of cellulose synthesis. The deduced amino acid sequences of these genes displayed a high degree of similarity to the Caulobacter crescentus DivK and PleD proteins that belong to the family of two-component response regulators. This is to our knowledge the first report of genes involved in the regulation of cellulose synthesis. Results from attachment assays and electron microscopic studies indicated that cellulose synthesis in R. leguminosarum bv. trifolii is induced upon close contact with plant roots during the attachment process.

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12.

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6-7 and its highest measured initial activity at 100 degrees C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 degrees C.

Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile ‘Anaerocellum thermophilum’ with separate glycosyl hydrolase family 9 and 48 catalytic domains

A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium 'Anaerocellum thermophilum'. Full-length CelA and a truncated enzyme species designated CelA' were purified to homogeneity from culture supernatants. CelA has an apparent molecular mass of 230 kDa. The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and beta-glucan. Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 degrees C (CM-cellulase) and 85 degrees C (Avicelase). Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation. The CelA-encoding gene was isolated from genomic DNA of 'A. thermophilum' by PCR and the nucleotide sequence was determined. The celA gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the N-terminus of CelA purified from 'A. thermophilum'. Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family III cellulose-binding domain linked by Pro-Thr-Ser-rich regions. The enzyme is most closely related to CelA of Caldicellulosiruptor saccharolyticus (sequence identities of 96 and 97% were found for the N- and C-terminal catalytic domains, respectively). Endoglucanase CelZ of Clostridium stercorarium shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain. Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound to microcrystalline cellulose. Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.

Effect of carbon source on the expression of celA 1, a cellulase-encoding gene from Streptomyces halstedii JM8

The production of the cellulase Cel1 from Streptomyces halstedii JM8 was studied in cells grown in the presence of glucose, cellobiose or microcrystalline cellulose (Avicel). Among these, glucose repressed expression while cellobiose and Avicel exerted a clear induction. The gene celA 1 was cloned in several heterologous Streptomyces hosts and its expression analyzed. S. parvulus transformed with the plasmid pJM11, a pIJ702 derivative, was the best producer. A region which includes the sequence ATTGGGACCGCTTCC located between positions −161 and −147 upstream from the translation initiation codon [Fernández-Abalos et al. (1992) J. Bacteriol. 174, 6368–6376] was deleted and its effect was studied in the presence of different carbon sources. Although the observed effect depends on the host used, this region seems to be involved in activation of the expression of this gene.

Identification of 5' flanking sequences that affect human pancreatic cholesterol esterase gene expression

Pancreatic cholesterol esterase (CEL) is shown to play a significant role in cholesterol metabolism. As the hydrolytic property of CEL is important for transport of lipid esters, the extent of its expression is an important factor in the metabolism of lipids. Therefore, to identify the elements that modulate the transcription of its mRNA, we obtained several cosmid clones carrying the CEL gene. From one of these cosmid clones a 6.5-kb SmaI fragment that hybridizes to the 5' untranslated region of CEL cDNA was subcloned. Primer extension and S1 protection assays revealed that the 5' untranslated region is relatively short (only 20 nucleotides long). An analysis of the 5' flanking sequence revealed typical TATA and CCAAT boxes that impart tissue specificity. Further, consensus sequences of several cis elements described earlier could also be detected in this region. To identify the promoter sequences, various deletion constructs of the 5' region were made using polymerase chain reaction. These constructs were subcloned into a bacterial plasmid vector carrying chloramphenicol acetyltransferase (CAT) as the reporter gene and transfected into HepG-2 cells. CAT activity in the cell homogenate of the transfected cells was measured 48 h after transfection. Results showed that the promoter activity of human pancreatic CEL mRNA is in a large segment of 5' flanking sequences spanning the -10 and -930 nucleotides of its gene.

An endoglucanase from the anaerobic fungus Orpinomyces joyonii: characterization of the gene and its product.

An endoglucanase gene (celA) was isolated from a genomic library of the ruminal fungus Orpinomyces joyonii. DNA sequence analysis of celA revealed an intronless gene encoding a typical signal sequence, an N-terminal catalytic domain, two repeated regions linked by a short Ser/Thr-rich linker and a domain of unknown function. The deduced amino acid sequence of the catalytic domain showed homology with the family 5 cellulases. While the catalytic domain of CelA was not homologous to the catalytic domain of the endoglucanase gene (EG3) from the ruminal bacterium Fibrobacter succinogenes, the repeated regions of CelA were very similar to the noncatalytic domain of EG3. This suggests that evolutionary shuffling of endoglucanase domains might occur among bacteria and fungi within the anaerobic ecosystem of the rumen. The celA gene was expressed in Escherichia coli, and the periplasmic endoglucanase was used for the characterization studies of the enzyme. CelA exhibited both endoglucanase and xylanase activities. Its pH optimum was 4 and the temperature optimum was 40 degrees C. Deletion analysis showed that the repeated sequences and C-terminal domain of CelA were not required for enzyme activity.

Pathways and genes involved in cellulose biosynthesis.

For many years, progress in the field of cellulose biosynthesis was hampered by inability to characterize enzymes or genes involved in the process. However, two breakthroughs led the study of this process into a new era. The first was the discovery in 1987 of cyclic diguanylic acid as a unique activator of a bacterial cellulose synthase, a finding that opened the way for enzyme purification and characterization (1). The second breakthrough came in 1990 with the first cloning of genes involved in the process (2,3). In this review, we have attempted to provide an overview of this complex process for those new to the field. In addition, we have made efforts to review critically genes and proteins, and motifs within those proteins, that could be potential future targets for genetic engineering to improve or alter cellulose production in plants and bacteria. We have also tried to point out some of the many remaining unanswered questions in cellulose synthesis and discuss them from different angles, hoping that this review will initiate constructive debates and stimulate new directions for research. We will focus on the following issues: (i) a discussion of various models for the pathway of cellulose synthesis in bacteria and plants; (ii) bacterial operons involved in cellulose synthesis; (iii) celA genes isolated recently from cotton and rice; (iv) an analysis of motifs for binding of the substrate UDP-glucose (UDP-Glc); and (v) mechanisms involved in the crystallization and deposition of cellulose microfibrils in the plant cell wall. Since determination of cellulose structure and mode of biosynthesis are not new subjects of study, a large number of reviews on the subject has been published (1,4–15). For those new to the field, we highly encourage the reader to read at least some of these other reviews to comprehend the complexity and controversy that has been characteristic of this field since each review has a different emphasis and viewpoint.KeywordsCotton FiberCellulose MicrofibrilCellulose SynthesisCellulose BiosynthesisDirect PolymerizationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Tagging a Vibrio cholerae El Tor candidate vaccine strain by disruption of its hemagglutinin/protease gene using a novel reporter enzyme: Clostridium thermocellum endoglucanase A

The celA gene encoding Clostridium thermocellum endoglucanase A was expressed in Vibrio cholerae on its own promoter and used to tag a candidate El Tor biotype cholera vaccine strain. Colonies of the tagged strain could be unequivocally distinguished by overlaying them with CM-cellulose indicator agar and Congo Red staining. Expression of celA did not affect growth of V. cholerae in vitro and in vivo. The celA gene was inserted in the chromosomal hap locus encoding V. cholerae hemagglutinin/protease, a putative “detachase”, to create a hap − mutant that could be identified and scored by its halo of cellulolytic activity. The inactivation of hap had a positive effect on colonization in the infant mice model. The above results indicate that celA is a suitable marker gene for V. cholerae and hap is an appropriate locus for insertion of foreign DNA in vaccine development. Inactivation of hap, by increasing the duration of adherence, might decrease excretion of the resulting vaccine vector strain and thus increase its immunogenicity.

Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase.

In spite of much effort, no one has succeeded in isolating and characterizing the enzyme(s) responsible for synthesis of cellulose, the major cell wall polymer of plants. We have characterized two cotton (Gossypium hirsutum) cDNA clones and identified one rice (Oryza sativa) cDNA that are homologs of the bacterial celA genes that encode the catalytic subunit of cellulose synthase. Three regions in the deduced amino acid sequences of the plant celA gene products are conserved with respect to the proteins encoded by bacterial celA genes. Within these conserved regions, there are four highly conserved subdomains previously suggested to be critical for catalysis and/or binding of the substrate UDP-glucose (UDP-Glc). An overexpressed DNA segment of the cotton celA1 gene encodes a polypeptide fragment that spans these domains and binds UDP-Glc, while a similar fragment having one of these domains deleted does not. The plant celA genes show little homology at the N- and C-terminal regions and also contain two internal insertions of sequence, one conserved and one hypervariable, that are not found in the bacterial gene sequences. Cotton celA1 and celA2 genes are expressed at high levels during active secondary wall cellulose synthesis in developing cotton fibers. Genomic Southern blot analyses in cotton demonstrate that celA forms a small gene family.

Mutants of Trichoderma reesei are defective in cellulose induction, but not basal expression of cellulase-encoding genes

Four mutants of Trichoderma reesei defective in cellulose utilization were characterized at the molecular level. Genomic analysis of the cellulase-encoding genes (cel) and transcript induction using two well-established inducers of the cel system the insoluble polymer, cellulose and the soluble inducer, sophorose, - revealed that these mutants are defective in the transcription of cel genes. The results also indicate that the cel genes are coordinately expressed and most probably are regulated by the same mechanism. Using a heterologous gene construct, in which the hygromycin-B-resistance-encoding gene was placed under the control of the promoter of the major cel gene, cbhl, we showed that the mutants synthesize basic levels of cellulase, but are defective in the cel induction.

Analysis of a Thermotoga maritima DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes.

Recombinant Escherichia coli clones displaying thermostable beta-glucanase activity were isolated from two different gene libraries of the hyperthermophilic bacterium Thermotoga maritima MSB8 (DSM 3109), and the nucleotide sequence of a 1,4-beta-glucanase gene designated celA was determined. Amino-terminal sequencing of cellulase I previously detected in T. maritima cells indicated that the celA gene encodes this beta-glucanase, which is now designated CelA. CelA, which has a calculated molecular mass of 29,732 Da, was purified from a recombinant E. coli strain to apparent homogeneity as judged by SDS-PAGE with a 44% yield. The enzyme was most active against soluble substrates such as mixed-linkage beta-glucan and CM-cellulose. CelA displayed remarkable thermostability, which was enhanced in the presence of high concentrations of salt. Downstream of the celA gene we found a second open reading frame, celB, whose nucleotide sequence was 58% identical to celA. Experimental proof that celB also encodes a beta-glucanase was obtained by separation from celA and expression in E. coli under the control of an efficient host promoter. According to the deduced amino acid sequences, CelB, in contrast to CelA, contains a signal peptide at the amino terminus. CelB and CelA had similar substrate specificities and temperature optima, but differed in their pH optima. Also, the addition of salt had a less stabilizing effect on CelB than on CelA. Nine 30 bp direct repeats, each itself representing a sequence with imperfect dyad symmetry, were detected upstream of the celA-celB cellulase gene cluster.

Cloning and expression of a Bacillus sp. 79-23 cellulase gene

A gene for encoding cellulase was cloned from Bacillus sp. 79-23 into Escherichia coli and the nucleotide sequence was determined. The cellulase gene, designated as celS, was composed of 1,497 base pairs and the nucleotide sequence of the celS gene was highly homologous to those of other B. subtilis cellulase genes. The enzyme encoded by celS was highly active on carboxymethylcellulose but also exhibited activity towards avicel and p-nitrophenyl-β-spd-cellobiopyranoside. When its native promoter was replaced with a strong B. subtilis promoter, the extracellular cellulase was produced up to 8.5 units per ml in B. subtilis DB104.

Two overlapping SOS-boxes in ColE operons are responsible for the viability of cells harboring the Col plasmid.

In this study, oligonucleotide-directed site-specific mutagenesis was used to change the consensus sequences of the LexA binding motifs in either one of the two SOS-boxes of the ColE7 operon. The results indicated that both mutants produced larger amounts of colicin than cells harboring the wild-type ColE7 plasmid. This finding would imply that two biologically functional SOS boxes exist in the ColE7 operon. In the non-induced state, no lysis of cells harboring wild-type plasmids occurred at 37 degrees C, whereas, cells harboring recombinant plasmids containing either one of the mutated SOS boxes underwent lysis within 100 min under the same conditions. This result indicated that adaptation of two SOS boxes of the ColE operon would obviously tightly control the expression of ColE operons. In such a way that it may prevent excessive expression of the lysis (cel) gene, thus safeguard the host cells from being lysed in ordinary living conditions.

Dietary Free and Esterified Cholesterol Absorption in Cholesterol Esterase (Bile Salt-stimulated Lipase) Gene-targeted Mice

The involvement of pancreatic cholesterol esterase (bile salt-stimulated lipase) in cholesterol absorption through the intestine has been controversial. We have addressed this issue by using homologous recombination in embryonic stem cells to produce mice lacking a functional cholesterol esterase gene. Cholesterol esterase knockout mice and their wild type counterparts were fed a bolus dose of [3H]cholesterol and a trace amount of [beta-14C]sitosterol by gavage. The ratio of the two radiolabels excreted in the feces over a 24-h period was found to be similar in the control and cholesterol esterase-null mice. Similar results were observed when the radiolabeled sterols were supplied in an emulsion with phospholipid and triolein or in lipid vesicles with phosphatidylcholine. Cholesterol absorption results were similar between the control and cholesterol esterase-null mice regardless of whether the animals were fed a low fat diet or a high fat/high cholesterol diet. The rate of [3H]cholesterol appearance in the serum of the gene-targeted mice paralleled that observed in control animals. In contrast to these results, when experiments were performed with [3H]cholesteryl oleate instead of [3H]cholesterol, a higher amount of the 3H radiolabel was found excreted in feces and dramatically less of the radiolabel was detected in the serum of the cholesterol esterase-null mice in comparison with that detected in control animals. Serum cholesterol levels were not significantly different between control and cholesterol esterase-null mice fed either control or an atherogenic diet. These results indicate that cholesterol esterase is responsible for mediating intestinal absorption of cholesteryl esters but does not play a primary role in free cholesterol absorption.

An endo-beta-1,4-glucanase gene (celA) from the rumen anaerobe Ruminococcus albus 8: cloning, sequencing, and transcriptional analysis.

A genomic library of Ruminococcus albus 8 DNA was constructed in Escherichia coli using bacteriophage lambda ZapII. This library was screened for cellulase components and several Ostazin brilliant red/carboxymethyl cellulose positive clones were isolated. All of these clones contained a common 3.4-kb insert, which was recovered as a plasmid by helper phage excision. The carboxymethyl cellulase coding region was localized to a 1.4-kb region of DNA by nested deletions, and a clone containing the entire celA gene was sequenced. Analysis of the sequence revealed a 1231-bp open reading frame, coding for a protein of 411 amino acids with a predicted molecular weight of 45 747. This protein, designated CelA, showed extensive homology with family 5 endoglucanases by both primary amino acid sequence alignment and hydrophobic cluster analysis. Cell-free extracts of E. coli containing the celA clone demonstrated activity against carboxymethyl cellulose and acid swollen cellulose but not against any of the p-nitrophenol glycosides tested, indicating an endo-beta-1,4-glucanase type of activity. In vitro transcription-translation experiments showed that three proteins of 48,000, 44,000, and 23,000 molecular weight were produced by clones containing the celA gene. Northern analysis of RNA extracted from R. albus 8 grown on cellulose indicated a celA transcript of approximately 2700 bases, whereas when R. albus 8 was grown on cellobiose, celA transcripts of approximately 3000 and 600 bases were detected. Primer extension analysis of these RNAs revealed different transcription initiation sites for the celA gene when cells were grown with cellulose or cellobiose as the carbon source. These two sites differed by 370 bases in distance. A model, based on transcription and sequence data, is proposed for celA regulation.

A novel family 9 endoglucanase gene (celD), whose product cleaves substrates mainly to glucose, and its adjacent upstream homolog (celE) from Fibrobacter succinogenes S85.

Two adjacent, highly homologous endoglucanase genes, celD and celE from Fibrobacter succinogenes S85, which were separated by an AT-rich 223-nucleotide intergenic region were characterized. The celD gene codes for endoglucanase D (EGD), a protein of 668 residues with a molecular mass of 71.7 kDa, while the celE gene encodes endoglucanase E, a protein of 467 amino acids with a molecular mass of 50.7 kDa. Both gene products belong to family 9 of glycosyl hydrolases. EGD displays an array of serine-rich periodic sequences (SRPS) near its C terminus which separate the catalytic domain from a basic terminal domain (BTD) rich in positively charged amino acids. Endoglucanase E has a BTD which is homologous to that of EGD, but it lacks the SRPS and 151 residues present at the N terminus of EGD. The SRPS structures may function as flexible linkers which facilitate interactions between the BTDs and acidic membrane proteins from F. succinogenes S85. The recombinant EGD showed pH and temperature optima of 5.5 and 35 degrees C, respectively. The enzyme cleaved barley-beta-glucan, carboxymethyl cellulose, and acid-swollen cellulose with specific activities of 19.1, 11.5 and 1.7 micromol x min-1 x mg of protein-1, respectively. There was a rapid drop in viscosity during hydrolyses of carboxymethyl cellulose, which is characteristic of an endoglucanase. Glucose was the main hydrolysis product of acid-swollen cellulose. Monospecific polyclonal antibodies against EGD detected the expression of a 68-kDa cellulose-inducible protein corresponding in size to the recombinant EGD in the culture fluid of F. succinogenes S85 and several larger proteins. The celE gene appeared to have little activity when expressed from the beta-galactosidase promoter in pBluescript in Escherichia coli; however, reverse transcriptase PCR analysis with internal primers for the gene revealed that a cellulose-inducible message was made in F. succinogenes, thereby documenting expression of the gene.

Characterization of a Bifunctional Cellulase and Its Structural Gene: THE cel GENE OF BACILLUS SP. D04 HAS EXO- AND ENDOGLUCANASE ACTIVITY

Bacillus sp. D04 secreted a bifunctional cellulase that had a molecular weight of 35,000. This cellulase degraded Cm-cellulose, cellotetraose, cellopentaose, p-nitrophenyl-beta-D-cellobioside, and avicel PH101. Based on the high performance liquid chromatography analysis of the degradation products, this cellulase randomly cleaved internal beta-1, 4-glycosidic bonds in cellotetraose and cellopentaose as an endoglucanase. It also hydrolyzed the aglycosidic bond in p-nitrophenyl-beta-D-cellobioside and cleaved avicel to cellobiose as an exoglucanase. Cellobiose competitively inhibited the p-nitrophenyl-beta-D-cellobioside degrading activity but not Cm-cellulose degrading activity. Ten mM p-chloromercuribenzoate inhibited p-nitrophenyl-beta-D-cellobioside degrading activity completely, but Cm-cellulose degrading activity incompletely. Cm-cellulose increased p-nitrophenyl-beta-D-cellobioside degrading activity, and vice versa, whereas methylumbelliferyl-beta-D-cellobiose strongly inhibited p-nitrophenyl-beta-D-cellobioside degrading activity. The cellulase gene (cel gene), 1461 base pairs, of Bacillus sp. D04 was cloned. The nucleotide sequence of the cel gene was highly homologous to those of Bacillus subtilis DLG and B. subtilis BSE616. The cel gene was overexpressed in Escherichia coli, and its product was purified. The substrate specificity and substrate competition pattern of the purified recombinant cellulase were the same as those of the purified cellulase from Bacillus sp. D04. These results suggest that a single polypeptide cellulase had both endo- and exoglucanase activities and each activity exists in a separate site.

Transcriptional induction and expression of the endoglucanase celA gene from a ruminal Clostridium sp. ("C. longisporum").

Northern (RNA) blot analysis of RNA from Clostridium sp. revealed induction of transcription of the celA gene when barley beta-glucan was used as carbon source, while no celA mRNA was detected after growth on cellobiose. Western blots (immunoblots), prepared by using a rabbit antiserum raised against CelA protein purified from Escherichia coli, revealed the extracellular location of CelA in Clostridium sp. Despite the absence of detectable celA mRNA, significant quantities of CelA were detected in the culture supernatant during growth on cellobiose. This finding indicated a low constitutive expression of celA. A 6.7-fold increase in the total beta-glucanase specific activity in the extracellular fraction was observed during growth on beta-glucan. The transcriptional start site of celA was mapped by extension and was found to be the same in Clostridium sp. and in E. coli expressing the cloned celA gene. A consensus E. coli -10 promoter region (AATAAT), but not a -35 promoter region, could be identified. Two direct repeats (TATTGAATTTAT) separated by 15 nucleotides flank the region where the consensus -35 promoter regions would have been. The size of the celA mRNA transcript corresponded with the size of the open reading frame. A potential stem-loop structure was found 18 nucleotides downstream of the 3' stop codon, which could be responsible for termination of transcription.

Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp

The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.