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Query: UNIPROT:P06889 (Mol)
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Erwinia chrysanthemi mutants (designated as pecS) displaying derepressed pectate lyase and cellulase synthesis were isolated. In addition, the pecS mutation is responsible for production of an extracellular insoluble blue pigment whose synthesis is cryptic in the wild-type 3937 strain. Transduction analysis indicates that the phenotype is due to a single mutation located near the xyl marker on the strain 3937 chromosome. This mutation was complemented by an R-prime plasmid carrying the xyl and argG genes of E. chrysanthemi, suggesting that the pecS product acts in trans to modulate pectinase, cellulase and blue pigment production. Insertion mutagenesis of the cloned region and recombination of the corresponding mutations in the bacterial chromosome by marker exchange revealed the existence of two divergently transcribed genes, pecS and pecM, that are both involved in the pectate lyase and cellulase regulation. The nucleotide sequences of pecS and pecM were determined. The pecS gene encodes a 166 amino acid polypeptide that shows similarity to the MprA regulatory protein of Escherichia coli whereas the pecM gene encodes a 297 amino acid polypeptide that was shown to be an integral membrane protein. The possible functions of the PecS and PecM proteins derived from the mutant phenotype and sequence analysis are discussed in terms of signal transduction and transcription regulation.
Mol Microbiol 1994 Mar
PMID:pecS: a locus controlling pectinase, cellulase and blue pigment production in Erwinia chrysanthemi. 802 82

Most fungal cellulases are found in multiple forms varying in size and substrate specificity. Aspergillus aculeatus is known to produce nine cellulolytic enzymes including an endoglucanase (FI CM-cellulase, M(r) = 24,002) as the major component. Single crystals of FI CM-cellulase from Aspergillus aculeatus have been prepared by sitting-drop vapour diffusion using ammonium sulphate as a precipitant. The cellulase crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 52.79(2) A, b = 106.40(4) A and c = 33.15(1) A. The crystals contain one enzyme molecule per asymmetric unit. They diffract to at least 2.0 A resolution and are very stable against X-ray irradiation.
J Mol Biol 1994 Aug 12
PMID:Crystallization and preliminary X-ray diffraction studies of an endoglucanase from Aspergillus aculeatus. 805 68

Northern analysis showed that accumulation of Agaricus bisporus cel1 mRNA was regulated by two independent mechanisms: (i) induction by cellulose; and (ii) repression by glucose and other sugars. Isolated A. bisporus nuclei were transcriptionally active. Nuclei isolated from cellulose-grown mycelium synthesized six times more cel1 mRNA than nuclei from glucose-grown mycelium. The start point of transcription (tsp) was identified by primer extension and S1 nuclease analysis. Putative glucose-, and cAMP-responsive elements as well as regions with homology to promoter regions of other fungal cellulase genes were detected both upstream and downstream from the tsp of the cel1 gene.
Mol Microbiol 1994 Apr
PMID:Regulation of transcription of the cel1 gene in Agaricus bisporus. 805 39

The out gene cluster of Erwinia spp. encodes the proteins of the general secretory pathway (GSP) apparatus that is required for pectinase and cellulase secretion. We have used fusions between Erwinia carotovora subsp. carotovora (Ecc) out genes and the topology probe blaM to assess the ability of Out protein regions to export BlaM across the cytoplasmic membrane in Escherichia coli and Ecc. For the outO gene product (an NMePhe peptidase), seven transmembrane regions have been identified and one more is predicted. The region of OutO with the highest level of hydrophilicity is likely to exist as a large cytoplasmic loop, located between two hydrophobic domains, and is positioned towards the N-terminus of the protein. When BlaM was fused on the C-terminal side of the last hydrophobic stretch of OutO, the resulting hybrid protein transferred the BlaM moiety to the periplasm whilst retaining OutO activity. Removal of a portion of this hydrophobic stretch resulted in the loss of OutO activity, suggesting that there are tight constraints on the topological integrity of OutO for maintaining catalytic function. When outG, -H, -I, -J, -K and -N were fused to blaM, the resulting phenotype suggested that the majority of each protein was targeted to the periplasm. Our results indicate that these six Out proteins, when produced by E. coli or Ecc, each adopt, at least temporarily, a type II bitopic conformation in the cytoplasmic membrane. For OutG, -H, -I and -J this probably represents the membrane topology prior to processing by OutO in Ecc. When produced in vivo from a T7 gene 10 promoter construct, the outG product was processed in Ecc whereas the outO mutant RJP249 failed to process pre-OutG. BlaM fusions positioned on the C-terminal side of the hydrophobic stretches of pre-OutG, -H, -I, and -J were processed by wild-type Ecc but not RJP249 or E. coli DH1. Thus the periplasmic domains of these proteins play no role in the peptidase cleavage reaction. An OutG-BlaM fusion construct was used to demonstrate NMePhe peptidase activity in other bacterial strains including E. carotovora subsp. carotovora (ATCC39048), E. carotovora subsp. atroseptica (SCRI1043) and Erwinia chrysanthemi (3937).
Mol Microbiol 1994 May
PMID:beta-Lactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus. 806 62

Differential screening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7 degrees C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.
Plant Mol Biol 1993 Feb
PMID:Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage. 809 63

Secretion to the cell exterior of cellulase EGZ and of at least six pectinases enables the Gram-negative Erwinia chrysanthemi to cause severe plant disease. The C-terminal cellulose-binding domain (CBD) of EGZ was found to contain a disulphide bond which forms, in the periplasm, between residues Cys-325 and Cys-382. Dithiothreitol (DTT)-treatment of native EGZ showed that the disulphide bond was dispensable, both for catalysis and cellulose binding. Adding DTT to E. chrysanthemi cultures led to immediate arrest of secretion of EGZ which accumulated in the periplasm where the CBD was eventually proteolysed. Site-directed mutagenesis that affected Cys residues involved in disulphide bond formation resulted in molecules that were catalytically active and able to bind to cellulose but were no longer secreted. Instead they accumulated in the periplasm. Interestingly, the region around EGZ Cys-325 is conserved in two pectinases secreted by the same pathway as EGZ. We conclude that the conserved Cys, and possibly adjacent residues, bear essential information for EGZ to be secreted and that periplasmic disulphide bond formation is an obligatory step which provides a pre-folded functional form of EGZ with secretion competence.
Mol Microbiol 1994 Feb
PMID:Periplasmic disulphide bond formation is essential for cellulase secretion by the plant pathogen Erwinia chrysanthemi. 815 78

Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) contains a discrete N-terminal cellulose-binding domain (CBDCenA). Related CBDs occur in at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDCenA. The adsorption of CBDCenA to crystalline cellulose was compared with that of two Trp mutants (W14A and W68A). The affinities of the mutant CBDs for cellulose were reduced by approximately 50- and 30-fold, respectively, relative to the wild type. Physical measurements indicated that the mutant CBDs fold normally. Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface.
Mol Microbiol 1994 Feb
PMID:The cellulose-binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding. 819 46

A cDNA library was produced using mRNA extracted from ethylene-treated leaflet abscission zones of common elder (Sambucus nigra). Screening of the library with the insert from pBAC10, which encodes an abscission beta-1,4-glucanase (cellulase) from bean (Phaseolus vulgaris), resulted in the isolation of a near-full-length cDNA which was designated JET 1. Northern analysis, using JET 1 as a probe, detected a transcript of 1.9 kb that accumulated prior to the first visible signs of cell separation. Accumulation of the JET 1 transcript is promoted by ethylene and primarily restricted to the tissue comprising the abscission zone. Sequence analysis of JET 1 indicates it is 1768 bp in length and shares significant homology at the amino acid level with beta-1,4-glucanases from the leaf abscission zone of P. vulgaris (67%) and ripening avocado fruit (48%). The predicted peptide sequence of the S. nigra enzyme contains two potential glycosylation sites. Genomic Southern analysis of S. nigra DNA reveals that JET 1 may belong to a multi-gene family.
Plant Mol Biol 1994 Mar
PMID:Characterization and accumulation pattern of an mRNA encoding an abscission-related beta-1,4-glucanase from leaflets of Sambucus nigra. 820 32

The structural gene for the major cellulase of Erwinia carotovora subspecies carotovora (Ecc) was isolated and expressed in Escherichia coli. Sequencing of the gene (celV) revealed a typical signal sequence and two functional domains in the enzyme; a catalytic domain linked by a short proline/threonine-rich linker to a cellulose-binding domain (CBD). The deduced amino acid sequence of the catalytic domain showed homology with cellulases of Family A, including enzymes from Bacillus spp. and Erwinia chrysanthemi CelZ, whereas the CBD showed homology with cellulases from several diverse families, supporting a "mix-and-match" hypothesis for evolution of this domain. Analysis of the substrate specificity of CelV showed it to be an endoglucanase with some exoglucanase activity. The pH optimum is about 7.0 and the temperature optimum about 42 degrees C. CelV is secreted by Ecc and by the taxonomically related Erwinia carotovora subspecies atroseptica (Eca) but not by E. coli. Overproduction of the enzyme from multicopy plasmids in Ecc appears to overload the secretory mechanism.
Mol Gen Genet 1993 Nov
PMID:Molecular analysis of the major cellulase (CelV) of Erwinia carotovora: evidence for an evolutionary "mix-and-match" of enzyme domains. 824 88

The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37-63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.
Mol Gen Genet 1993 Dec
PMID:High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. 826 26


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