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Query: UNIPROT:P06889 (Mol)
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Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2 slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2 gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.
Mol Gen Genet 1993 Dec
PMID:High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes. 826 27

Erwinia carotovora subsp. carotovora strain Ecc71 produces an array of extracellular enzymes including pectate lyase (Pel), polygalacturonase, cellulase, and protease. In strain Ecc71, these enzymes are coregulated by aepA, which encodes an activator of extracellular protein production (H. Murata, J. L. McEvoy, A. Chatterjee, A. Collmer, and A. K. Chatterjee, Mol. Plant-Microbe Interact, 4:239-246, 1991). The nucleotide sequence of a 2.7-kb aepA+ DNA segment revealed an open reading frame (ORF) of 1,395 bp which matches with the size of the aepA transcript determined by Northern blot analysis. aepA is predicted to encode a protein of 465 amino acid residues with a molecular mass of approximately 51 kDa and a pI of 6.52. The occurrence of a putative signal sequence and several hydrophobic domains suggest membrane localization of AepA. An aepA-lacZ operon fusion was constitutively expressed in E. coli (DH5 alpha) but inducible by pectate and celery extract in E. c. subsp. carotovora (AC5006). These findings suggest that aepA expression may be negatively regulated in E. c. subsp. carotovora. By assaying for the transcript of pel-1, which species a major secreted Pel species in strain Ecc71, and by following the expression of a pel1-lacZ operon fusion we determined that AepA activates pel-1 transcription. The characteristics of aepA including the lack of homology with other prokaryotic regulatory genes indicate that aepA encodes a novel regulatory protein required for extracellular protein production. Whereas homologs of Ecc71 aepA occur in E. c. subsp. carotovora and E. c. subsp. atroseptica strains, activation of exoenzyme production is markedly stimulated by aepA in E. c. subsp. carotovora than in E. c. subsp. atroseptica.
Mol Plant Microbe Interact
PMID:Characterization of a novel regulatory gene aepA that controls extracellular enzyme production in the phytopathogenic bacterium Erwinia carotovora subsp. carotovora. 832 48

Ethylene promotes the abscission of leaves and the ripening of fruits in pepper plants, and in both events an increase in cellulase activity is observed. However, two enzyme isoforms (pI 7.2 and 8.5, respectively) are differentially involved in the two physiological phenomena. The pI 8.5 form has been purified from ripe fruits. It is a glycoprotein with an apparent molecular mass of 54 kDa. Two short peptides were sequenced and a very high homology to a tomato cellulase was observed. Polyclonal antibodies, raised against the purified enzyme, have allowed us to demonstrate that the observed ethylene-induced increase in cellulase activity is paralleled by de novo synthesis of protein. Three cDNAs (CX1, CX2 and CX3), encoding different cellulases, were obtained and characterized and their expression investigated. Accumulation of all three mRNAs is induced by ethylene treatment, though to different levels. CX1 is mainly expressed in ripe fruits while CX2 is especially found in abscission zones. CX3 accumulates at very low levels in activated abscission zones. Comparisons with other known cellulases demonstrate clear heterogeneity within the higher plant cellulases. Differences in ethylene inducibility and molecular structure suggest different physiological roles for cellulase in pepper plants.
Plant Mol Biol 1995 Nov
PMID:Differential ethylene-inducible expression of cellulase in pepper plants. 854

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pelA, pelB, pelC, pelD and pelE genes. Recently, a new set of pectate lyases was identified in E. chrysanthemi mutants deleted of those pel genes. We cloned the pelL gene, encoding one of these secondary pectate lyases of E. chrysanthemi 3937, from a genomic bank of a strain deleted of the five major pel genes. The nucleotide sequence of the region containing the pelL gene was determined. The pelL reading frame is 1275 bases long, corresponding to a protein of 425 amino acids including a typical amino-terminal signal sequence of 25 amino acids. Comparison of the amino acid sequences of PelL and the exo-pectate lyase PelX of E. chrysanthemi EC16 revealed a low homology, limited to 220 residues of the central part of the proteins. No homology was detected with other bacterial pectinolytic enzymes. Regulation of pelL transcription was analysed using gene fusion. As shown for the other pel genes, the transcription of pelL is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, temperature, iron starvation, osmolarity, anaerobiosis, nitrogen starvation and catabolite repression. Regulation of pelL expression appeared to be independent of the KdgR repressor, which controls all the steps of pectin catabolism. In contrast, the pecS gene, which is involved in regulation of the synthesis of the major pectate lyases and of cellulase, also appeared to be involved in pelL expression. The PelL protein is able to macerate plant tissue. This enzyme has a basic isoelectric point, presents an endo-cleaving activity on polygalacturonate or partially methylated pectin, with a basic pH optimum and an absolute requirement for Ca2+. The pelL mutant displayed a reduced virulence on potato tubers and Saintpaulia ionantha plants, demonstrating the important role of this enzyme in soft-rot disease.
Mol Microbiol 1995 Jun
PMID:Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937. 857 52

The Streptomyces reticuli cellulase (Cell, Avicelase) hydrolyzes crystalline cellulose (Avicel) efficiently to cellobiose. The synthesis of the enzyme is induced by Avicel and repressed by glucose. DNA-binding proteins were purified from induced S. reticuli mycelia by affinity chromatography using the upstream region of the cell gene linked to Sepharose. The enriched protein(s) provoked a gel electrophoresis mobility shift of the upstream region, irrespective of the presence or absence of a 14-bp palindromic sequence, and enhanced the transcription of the cell gene by the S. reticuli RNA polymerase in vitro. The binding site (GTGACTGAGCGCCG) for the protein(s) was located in the vicinity of a DNA bend upstream of the transcriptional start site. Results of physiological studies, deletion and gel-shift analyses lead to the conclusion that a 14-bp palindrome (TGGGAGCGCTCCCA)--situated between the transcriptional start site and the structure gene--is the operator for a repressor protein. The data presented suggest that the two identified cis-acting elements, in cooperation with an activator and a repressor, mediate regulation of cell transcription.
Mol Gen Genet 1996 May 23
PMID:The synthesis of the Streptomyces reticuli cellulase (avicelase) is regulated by both activation and repression mechanisms. 866 29

The cre1 genes of the filamentous fungi Trichoderma reesei and T. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor gene creA of Aspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C2H2 type. The cre1 promoters contain several sequence elements that are identical to the previously identified binding sites for A. nidulans CREA. Steady-state mRNA levels for cre1 of the T. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest that cre1 expression may be autoregulated. The T. reesei strain Rut-C30, a hyper-producer of cellulolytic enzymes, was found to express a truncated form of the cre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-length cre1 gene into this strain caused glucose repression of cbh1 expression, demonstrating that cre1 regulates cellulase expression.
Mol Gen Genet 1996 Jun 24
PMID:The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form. 870 49

Bean leaf abscission (organ separation) correlates with the de novo accumulation of a pI9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5'-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between -1 and -200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5' to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar -210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the -210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.
Plant Mol Biol 1996 Jun
PMID:The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase. 879 Feb 92

Erwinia carotovora subsp. carotovora wild-type strain Ecc71 does not elicit the hypersensitive reaction (HR) in tobacco leaves. By mini-Tn5-Km and chemical mutagenesis we have isolated RsmA- mutants of Ecc71 that produce high basal levels of pectate lyases, polygalacturonase, cellulase, and protease; they also are hypervirulent. The RsmA- mutants, but not their parent strains, elicit an HR-like response in tobacco leaves. This reaction is characterized by the rapid appearance of water soaking followed by tissue collapse and necrosis. The affected areas remain limited to the region infiltrated with bacterial cells, and the symptoms closely resemble a typical HR, e.g., the reactions caused by Pseudomonas syringae pv. pisi. Moreover, low concentrations of cells of the mini-Tn5-Km insertion RsmA- mutant, AC5070, infiltrated into tobacco leaf tissue prevent elicitation of the rapid necrosis by AC5070 or by P. syringae pv. pisi. Elicitation of the HR-like response by the mutants is not affected by the deficiency of N-(3-oxohexanoyl)-L-homoserine lactone, the cell density (quorum) sensing signal. Cloning and sequence analysis have disclosed that E. carotovora subsp. carotovora strain Ecc71 possesses a homolog of E. chrysanthemi hrpN known to encode an elicitor of the HR; the corresponding Ecc71 gene is designated hrpNEcc. Northern (RNA) blot data show that the level of hrpNEcc mRNA is considerably higher in the RsmA- mutants than in the RsmA+ strains. Moreover, a low copy plasmid carrying the rsmA+ allele severely reduces the level of the hrpNEcc transcripts in the RsmA- mutants. These constructs, like the RsmA+ E. carotovora subsp. carotovora strains, do not elicit the HR-like response. These data taken along with the effects of rsmA on exoenzyme production and pathogenicity (A. Chatterjee et al., 1995, Appl. Environ. Microbiol. 61:1959-1967) demonstrate that this global regulator gene plays a critical role in plant interaction of E. carotovora subsp. carotovora.
Mol Plant Microbe Interact 1996 Sep
PMID:The RsmA- mutants of Erwinia carotovora subsp. carotovora strain Ecc71 overexpress hrpNEcc and elicit a hypersensitive reaction-like response in tobacco leaves. 881 71

Clostridium thermocellum produces a highly active cellulase system that consists of a high-M(r) multienzyme complex termed cellulosome. Hydrolytic components of the cellulosome are organized around a large, noncatalytic glycoprotein termed CipA that acts both as a scaffolding component and a cellulose-binding factor. Catalytic subunits of the cellulosome bear conserved, noncatalytic subdomains, termed dockerin domains, which bind to receptor domains of CipA, termed cohesin domains. CipA includes nine cohesin domains, a cellulose-binding domain, and a specialized dockerin domain. Proteins of the cell envelope carrying cohesin domains that specifically bind the dockerin domain of CipA have been identified. These proteins may mediate anchoring of the cellulosomes to the cell surface. Cellulase complexes similar to the cellulosome of C. thermocellum are produced by several cellulolytic clostridia. High-M(r) multienzyme complexes have also been identified in anaerobic rumen fungi. The architecture of the fungal complexes also seems to rely on the interaction of conserved, noncatalytic docking domains with a scaffolding component. However, the sequence of the fungal docking domains bears no resemblance to the clostridial dockerin domains, suggesting that the fungal and clostridial complexes arose independently.
Crit Rev Biochem Mol Biol 1996 Jun
PMID:The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation. 881 76

Phanerochaete chrysosporium completely degrades lignocellulose. The most recalcitrant component, lignin, is oxidized by the radical products of lignin and manganese peroxidases, whereas cellulose and hemicellulose are hydrolysed. Both peroxidases and cellulases exist as complex families at both the DNA and protein levels. The lignin peroxidases may function principally when mycelium-bound and, therefore, undetectable in culture supernatants. Moreover, methods for the study of P. chrysosporium must be applicable to solid substrate as well as liquid-culture conditions. For these reasons, detailed studies of gene expression, made possible by the reverse transcriptase-polymerase chain reaction method, are essential. Such studies reveal that gene families are subject to differential expression. The cellulase system has some differences from that of Trichoderma reesei; the distinction made between the activities of exocellobiohydrolases and endoglucanases needs to be re-appraised in both species. Current studies also seek to reconstruct the systems of degradation of lignocellulose and its individual components by heterologous expression of individual proteins in recombinant systems, and their use in mechanistic studies singly and in combinations.
Mol Microbiol 1996 Mar
PMID:Lignocellulose degradation by Phanerochaete chrysosporium: gene families and gene expression for a complex process. 883 Feb 73


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