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The phytopathogenic enterobacterium Erwinia chrysanthemi secretes a number of enzymes involved in plant-tissue degradation, notably the five isoenzymes of pectate lyase. We have cloned a region involved in pectate lyase and cellulase secretion by complementation of non-secretory outJ mutants of E. chrysanthemi strain 3937 using the RP4::miniMu plasmid pULB110. The cloned region maps near the ade-22 marker on the E. chrysanthemi 3937 chromosome. An R-prime containing a chromosomal DNA insert of about 30kb was first obtained; subcloning into pBR325 permitted the isolation of a 4kb ClaI/SspI fragment able to complement outJ mutations in E. chrysanthemi. The isolation of phoA fusions in this fragment allowed us to determine the direction of transcription of the encoding region, which extends over about 2.5kb, and demonstrate that this region encodes exported protein(s). When the TnphoA insertions were transferred back into E. chrysanthemi chromosome, the recombined strains no longer secreted pectate lyases or cellulases. Identification of the products encoded by the ClaI/SspI fragment demonstrated that outJ encodes an 83 kD polypeptide which is processed to an 81 kD polypeptide by cleavage of a signal sequence. The cloned DNA fragment did not endow Escherichia coli with the ability to secrete pectate lyases.
Mol Microbiol 1989 Mar
PMID:Molecular cloning of the outJ gene involved in pectate lyase secretion by Erwinia chrysanthemi. 254 3

In this paper, we have used filter hybridization and nucleotide sequencing to analyse the relationship between the three genes of the pelADE cluster in the Erwinia chrysanthemi (Ech) strain B374. This cluster encodes for three of the five pectate lyase proteins that are involved in the maceration and soft-rotting of plant tissue, an important trait in Ech pathogenicity. Southern hybridization revealed homology between each of the three pel genes. A 3560 bp DNA fragment containing the pelE and pelD genes was sequenced. These two genes show extensive homology in the coding regions but only low homology in the 5' and 3' non-coding regions. However both genes exhibit sequences homologous to the Escherichia coli CAP-binding site consensus sequence upstream of the start codon and an inverted repeat sequence which may act as a rho-independent transcriptional terminator after the translational stop. The pel genes of Ech B374 were also compared with the already sequenced pel genes of EC16, another Ech strain.
Mol Microbiol 1989 Oct
PMID:Relationship between the pel genes of the pelADE cluster in Erwinia chrysanthemi strain B374. 261 52

Various mutations in the pectin catabolic pathway of Erwinia chrysanthemi were isolated by selection of Mu-lac insertions, resulting in expression of the lac genes inducible by pectin degradation products. This approach allowed us to isolate lacZ fusions with the genes pelC, pelD, ogl and pem, encoding pectate lyases PLc and PLd, oligogalacturonate lyase and pectin methylesterase, respectively. Moreover, we obtained mutations affecting the regulation of pectinolytic enzymes; a locus called pecl appeared to be involved in induction of pectate lyases and pectin methylesterase. A second locus, called pecL, may encode an activator protein acting on pectate lyase production. Both pecl and pecL expression are induced in the presence of pectic polymers. The expression of the pem gene was studied in more detail by analysis of the pem-lacZ fusions. The expression of pem appears to be controlled by the negative regulatory gene kdgR, which controls all the genes involved in pectin degradation (pem, pel, ogl, kduD, kdul, kdgK, kdgA). This study confirmed that 2-keto-3-deoxygluconate is a key intermediate for the induction of the pectin catabolic pathway. The three genes pem, pelD and pecl were localized in the same region, near the ade-377 marker on the genetic map of the E. chrysanthemi strain 3937. The pem gene was located more precisely on an 18kb DNA fragment containing the pelADE cluster. However, this 18kb DNA fragment did not complement the pecl mutation. The pecL mutations were located near the ile-2 marker on the genetic map of E. chrysanthemi strain 3937.
Mol Microbiol 1989 Nov
PMID:Isolation of Erwinia chrysanthemi mutants altered in pectinolytic enzyme production. 269 3

Pectate lyase is a saccharide-binding enzyme that lyitically depolymerizes polypectate in higher plant cell walls, thus causing soft-rot diseases in food crops. A pectate lyase from Erwinia chrysanthemi, EC16 (PLe), crystallizes in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimension of a = 39.0 A, b = 91.0 A and c = 103.4 A. The asymmetric unit consists of one molecule with a molecular mass of 38,118 daltons and the X-ray diffraction extends to a resolution of 1.8 A. The crystals reproducibly grow to large dimensions and are suitable for a high-resolution X-ray diffraction analysis.
J Mol Biol 1989 Jul 20
PMID:Preliminary crystallographic analysis of a plant pathogenic factor: pectate lyase. 276 65

The gene for a pectate lyase of E. chrysanthemi ENA49 cloned in a recombinant plasmid pPTL1 (a derivative of RSF1010) was transferred into E. carotovora. The pectate lyase determined by the cloned gene was secreted into the cultural medium from the cells of E. crysanthemi EC16. Partial secretion of the enzyme was registered for E. carotovora cells. The major part of EC1 E. chrysanthemi pectate lyase synthesized by E. carotovora cells is accumulated in periplasmic and cytoplasmic fractions. The obtained results suggest the different specificity or efficiency of pectate lyase secretion systems in the studied Erwinia strains.
Mol Gen Mikrobiol Virusol 1987 May
PMID:[Expression of the pectate lyase gene from Erwinia chrysanthemi ENA49 in cells of other Erwinia species]. 303 59

E.atroseptica 36A cells were transformed by the recombinant plasmids p27-1 and pEA364 (derivatives of the vector plasmid pUC19) containing pectate lyase genes of E.carotovora 17A and E.atroseptica 36A, respectively. The synthesis of pectate lyases determined by the cloned genes of bacteria of both subspecies, as well as the synthesis of the native enzymes, were induced by sodium poly pectate. Increase of the dose of pectate lyase genes did not result in alteration of pectate lyase secretion by E.atroseptica 36ApEA364 cells. At the same time, the efficiency of secretion of heterologous pectate lyases by E.atroseptica 36Ap27-1 cells was lower. The synthesis and secretion of the resident isoenzymes are as efficient as those of the parental cells. The results indicate a high specificity of the pectinase secretory system in Erwinia of different species and, moreover, subspecies.
Mol Gen Mikrobiol Virusol
PMID:[Expression of pectate lyase genes of Erwinia carotovora subsp. carotovora 17A and Erwinia carotovora subsp. atroseptica 36A in Erwinia carotovor substp. atroseptica 36A cells]. 773 92

The pectate lyase (Pel, EC 4.2.2.2) isoenzyme profile of Erwinia carotovora subsp. carotovora was characterized by isoelectric focusing, and the corresponding genes coding for four different exported Pels were cloned. The nucleotide sequence of the pelB gene encoding one of these isoenzymes was determined and was shown to contain 1,040-bp open reading frame coding for a 37,482-Da protein with a putative cleavable amino terminal signal peptide. Overexpression and selective labeling experiments with the pelB clone demonstrated the synthesis of a 35-kDa polypeptide, which is in accordance with the deduced size of the processed PelB. The predicted amino acid sequence of PelB was very similar to that of Pel-3 of another E.c. subsp. carotovora strain 71, but showed no similarity to other previously characterized pectinolytic enzymes. The pelB gene is located next to the previously characterized pehA gene encoding an endopolygalacturonase. The two genes are divergently transcribed from a common control region and are subject to similar global regulation by the central virulence regulator expI. Inactivation of pelB did not appear to reduce the virulence of the mutant strain, suggesting that pelB does not have a major role in pathogenicity. Unlike other Pels, PelB required partially methyl esterified pectin as substrate suggesting that PelB represents a novel isoform of pectate lyase.
Mol Plant Microbe Interact
PMID:Characterization of a novel pectate lyase from Erwinia carotovora subsp. carotovora. 775 91

Of the various exoproteins secreted by Erwinia carotovora subsp. carotovora strain 71, Pel1 is the major pectate lyase species with tissue macerating activity. Nucleotide sequencing of a 2.2-kb pel1+ DNA segment revealed a 1,122 base pair open reading frame which could encode pre-Pel1 of 374 amino acid residues. A signal peptide of 22 amino acid residues is present within the NH2-terminal region of pre-Pel1. Transcription of pel1 was initiated at the guanine residue 111 base pairs upstream of the start codon. Consensus sequences for the binding of KdgR, a negative regulatory factor known to control some of the E. chrysanthemi pectinases, flank the promoter of pel1. Although pel1 belongs to the pelBC family, it is more closely related to the pel genes of E. carotovora than to the pelBC genes of E. chrysanthemi.
Mol Plant Microbe Interact
PMID:Nucleotide sequence of a pectate lyase structural gene, pel1 of Erwinia carotovora subsp. carotovora strain 71 and structural relationship of pel1 with other pel genes of Erwinia species. 777 8

The production of pectolytic enzymes (pectate lyase [Pel] and polygalacturonase [Peh]), cellulase (Cel), and protease (Prt) is activated in the soft rot bacterium Erwinia carotovora subsp. carotovora by aepA (activator of extracellular protein production) and celery extract (Y. Liu, H. Murata, A. Chatterjee, and A. K. Chatterjee, Mol. Plant-Microbe Interact. 6:299-308, 1993). We recently isolated a new class of mutants of strain E. carotovora subsp. carotovora 71 which overproduces Pel, Peh, Cel, and Prt. From the overproducing strain AC5034, we identified an activator locus, designated aepH*, which stimulated Pel, Peh, Cel, and Prt production in E. carotovora subsp. carotovora 71 or its derivatives. The nucleotide sequence of the aepH* DNA segment revealed an open reading frame of 141 bp that could encode a small (5.45-kDa) highly basic (pI 11.7) protein of 47 amino acid residues. Analyses of deletions and MudI insertions indicated that the activator function required the 508-bp DNA segment which contains this open reading frame. The wild-type locus, aepH+, is localized within a DNA segment upstream of aepA. An AepH- strain constructed by exchanging aepH+ with aepH*::MudI was deficient in Pel, Peh, Cel, and Prt production; exoenzyme production was restored upon the introduction of a plasmid carrying aepH+ or aepH*. Plasmids carrying either aepH+ or aepH* activated the production of Pel-1, Peh-1, and Cel in Escherichia coli HB101 carrying the cognate genes. The aepH effect in E. coli was due to the activation of transcription, as indicated by assays of pel-1 and peh-1 mRNAs. The aepH+ and aepH* plasmids also stimulated Pel, Peh, Cel, and Prt production in other wild-type E. carotovora subsp. carotovora strains as well as in E. carotovora subsp. atroseptica. Although the stimulatory effect was generally more pronounced with aepH* than with aepH+, the extent of activation in the wild-type strains depended upon the bacterial strain and the growth medium. Southern blot hybridization revealed the presence of aepH homologs in E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, and provided physical evidence for linkage between aepA and aepH homologs in genomes of these bacteria. We conclude that aepH-mediated activation of exoprotein gene expression is a feature common to most strains of E. carotovora.
 ...
PMID:Regulation of the production of extracellular pectinase, cellulase, and protease in the soft rot bacterium Erwinia carotovora subsp. carotovora: evidence that aepH of E. carotovora subsp. carotovora 71 activates gene expression in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Escherichia coli. 794 60

Unlike the bacterial pathogens that typically cause the hypersensitive response (HR) in plants, Erwinia chrysanthemi has a wide host range, rapidly kills and macerates host tissues, and secretes several isozymes of the macerating enzyme pectate lyase (Pel). PelABCE- and Out- (secretion-deficient) mutants were observed to produce a rapid necrosis in tobacco leaves that was indistinguishable from the HR elicited by the narrow-host-range pathogens E. amylovora Ea321 and Pseudomonas syringae pv. syringae 61. E. amylovora Ea321 hrp genes were used to identify hybridizing cosmids in a cosmid library of E. chrysanthemi EC16 DNA in Escherichia coli. A 16-kb BamHI fragment in one of these cosmids, pCPP2030, hybridized with E. amylovora hrp genes and was mutagenized with Tn10mini-kan. The mutations were introduced into the PelABCE- mutant CUCPB5006 by marker exchange. Two of the resultant hrp::Tn10mini-kan mutations were found to abolish the ability of CUCPB5006 to cause any necrosis in tobacco leaves unless complemented with pCPP2030. These two mutations were also marker-exchanged into the genome of wild-type strain AC4150. Analysis of DNA sequences flanking the hrp-2::Tn10mini-kan insertion revealed the mutated gene to be similar to a gene in E. amylovora Ea321 hrp complementation group VIII and to P. s. pv. syringae 61 hrpX. Neither of the hrp::Tn10mini-kan mutations affected the production or secretion of pectic enzymes by AC4150 or CUCPB5006. However, the hrp mutations reduced the ability of both AC4150 and CUCPB5006 to incite successful infections in witloof chicory leaves.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Plant Microbe Interact
PMID:Erwinia chrysanthemi hrp genes and their involvement in soft rot pathogenesis and elicitation of the hypersensitive response. 794 26


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