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Query: EC:3.2.1.15 (pectinase)
 2,440 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.
Plant Mol Biol 1994 Jul
PMID:Structure and expression of an inhibitor of fungal polygalacturonases from tomato. 806 15

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

The Erwinia chrysanthemi kdgR gene encodes a repressor that negatively regulates the expression of genes involved in pectinolysis and in pectinase secretion. The cloned kdgR gene was overexpressed in Escherichia coli by using a phage T7 system. Overproduced repressor was purified to homogeneity by two chromatographic steps. Gel retardation and DNase I protection experiments demonstrated the specific binding of the KdgR protein to the operators of pectinase genes (pelA, pelB, pelC, pelE), to the operator of genes involved in pectin catabolism (kdgT, ogl, kduI-kdgF) and to that of the outT gene involved in pectinase secretion. These interactions involved one (pelA, pelB, kduI-kdgF, outT) or several operator sites (pelC, pelE, ogl, kdgT) that generally overlap the promoter. Despite the presence of potential KdgR binding sites (KdgR-box) in the regulatory regions of four genes involved in pectin catabolism (kdgC, kduD, pem, kdgA) and in a pectinase secretion gene outC, no DNA-repressor complex could be observed by in vitro experiments. By using a missing contact experiment on the coding strand of ogl and pelE regulatory regions, a new KdgR-binding consensus was proposed. This new consensus, constituted by two half motifs (AATGAAAACT)N(NTCGATTTCTA), is well conserved in the operators which interact in vitro with the KdgR repressor. In contrast, this repressor-recognized motif is degenerated in the other operators that cannot interact in vitro with the repressor. These results suggest the existence of different regulation mechanisms mediated by the KdgR protein for the two classes of operators.
J Mol Biol 1994 Feb 18
PMID:Specific interactions of Erwinia chrysanthemi KdgR repressor with different operators of genes involved in pectinolysis. 810 32

Tomatoes (Lycopersicon esculentum Mill cv. Ailsa Craig) were transformed with a gene construct having 244 bp of the 5' end of a polygalacturonase (PG) cDNA, coding for a 71 amino acid N-terminal extension to the mature protein, fused to 1320 bp of a pectin-esterase (PE) cDNA encoding the full sequence of the mature PE protein. This chimaeric gene was inserted in a sense orientation between a CaMV 35S promoter and terminator for constitutive expression. In transformed tomato plants expression of the endogenous PG and PE genes in the fruit was inhibited; there was little or no observable PG and PE mRNA and a substantial reduction in the level of PG and PE enzyme activity. The transgene was expressed in the leaves of the transformed plants as demonstrated by the accumulation of mRNA, but no protein product could be identified. However, no transgene mRNA or protein were observed in the transgenic fruit. The paper represents the first report of the down-regulation of two non-homologous endogenous genes using a single gene construct. A sense gene construct was responsible for these effects. These findings are discussed in relation to possible mechanisms of action of co-suppression.
Plant Mol Biol 1993 Oct
PMID:Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct. 821 42

A putative defective transposable element has been identified in tobacco. This element has been found and characterised in two separate parts of the tobacco genome, specifically within the 3rd intron of the pollen-specific polygalacturonase gene (Npg1) and upstream of the endochitinase gene (Chn50). The element is ca. 0.4 kb in length and is bounded by conserved inverted repeats and putative target site duplications. It appears to fall into the category of non-autonomous transposable elements.
Plant Mol Biol 1993 Oct
PMID:Identification of a defective transposable element in tobacco. 821 73

Five isozymes (four acidic and one basic) of polygalacturonase were separated by chromatofocusing from the culture filtrate of Botrytis cinerea grown on apple pectin. The isozymes, designated as Polygalacturonase I to V, have isoelectric points of 9.7, 4.9, 4.6, 3.7, and 2.7, respectively, with Polygalacturonase III exhibiting the highest specific activity. Polygalacturonase I appeared to function as an endo-polygalacturonase while the other four isozymes act as exo-polygalacturonases. The pH optima of the isozymes range from pH 4.5 to 5.5 with Polygalacturonase V being less sensitive to higher pH compared with the rest of the isozymes.
Biochem Mol Biol Int 1993 Aug
PMID:Polygalacturonase isozymes from Botrytis cinerea grown on apple pectin. 822 Feb 35

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.
Plant Mol Biol 1993 Dec
PMID:Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen. 829 91

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


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