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Two major virulence determinants of the plant-pathogenic enterobacterium Erwinia chrysanthemi strain 3937 are the production of pectate lyase enzymes that degrade plant cell walls and expression of two high-affinity iron uptake systems mediated by two structurally unrelated siderophores, chrysobactin and achromobactin. Low iron availability is a signal that triggers transcription of the genes encoding pectate lyases PelD and PelE as well as that of genes involved in iron transport. This metalloregulation is mediated by the transcriptional repressor Fur. In this study, we analyzed the molecular mechanisms of this control. We purified the Erwinia chrysanthemi Fur protein. Band shift assays showed that Fur specifically binds in vitro to the regulatory regions of the genes encoding the ferrichrysobactin outer membrane receptor Fct and the pectate lyases PelD and PelE. We identified the Fur-binding sites of these promoter regions by performing DNase I footprinting experiments. From these data, we propose that Fur could inhibit the activation of the pelD and pelE genes by the cAMP receptor protein CRP according to an anti-activation mechanism. To identify other possible effectors involved in this control, we screened a bank of insertion mutants for an increase in transcriptional activity of pelD and fct genes in response to iron limitation. We isolated a mutant affected in the kdgK gene encoding the 2-keto-3-deoxygluconate (KDG) kinase, an enzyme involved in pectin catabolism. The growth of this mutant in the presence of pectic compounds led to a constitutive expression of iron transport genes as well as complete derepression of the pectinolysis genes. This effect was caused by intracellular accumulation of KDG. However, the derepression of iron transport genes by KDG does not involve the KdgR regulator of pectinolysis genes, which uses KDG as inducer. Thus, in Erwinia chrysanthemi, iron depletion or presence of KDG induces transcription of the genes involved in iron assimilation and pectinolysis. These important pathogenicity functions are coregulated by responding to common signals encountered in planta.
Mol Plant Microbe Interact 2002 Nov
PMID:Coupling of iron assimilation and pectinolysis in Erwinia chrysanthemi 3937. 1242 24

The production of pectinase, the major virulence determinant of soft-rot Erwinia species, is controlled by many regulatory factors. We focused on the major regulatory proteins, KdgR, CRP, Pir, and PecS, characterized mainly in E. chrysanthemi, and tested for their presence and function in the control of pectate lyase (Pel) and polygalacturonase (Peh) production in E. carotovora subsp. carotovora. Homologues of kdgR and crp but not of pir and pecS were detected by Southern blot analyses in E. carotovora subsp. carotovora. In fact, KdgR and CRP homologues of E. carotovora subsp. carotovora had high amino acid identities to those of E. chrysanthemi, including a complete match of the hypothetical helix-turn-helix DNA-binding motif. However, in Western blot analyses using anti-Pir (E. chrysanthemi) antibodies, a cross-reacting protein was present in both Erwinia species, although Pel production in E. carotovora subsp. carotovora was not further stimulated by adding plant extract into the medium containing PGA (polygalacturonic acid) in which hyperinduction by Pir has been reported in E. chrysanthemi EC16. When plasmids that contained each of these regulatory genes from E. chrysanthemi were introduced into E. carotovora subsp. carotovora, Pel production was controlled as predicted from their roles in E. chrysanthemi, except for PecS. PecS exerted a positive control in E. carotovora subsp. carotovora, in contrast to a negative control in E. chrysanthemi. DNA-binding assays demonstrated that KdgR, CRP, Pir, and PecS of E. chrysanthemi and KdgR and CRP homologues of E. carotovora subsp. carotovora could bind to the promoter regions of pel-1, pel-3, and peh of E. carotovora subsp. carotovora. Taken together, KdgR and CRP homologues of E. carotovora subsp. carotovora may regulate Pel and Peh production as in E. chrysanthemi. However, the presence of Pir and PecS homologues in E. carotovora subsp. carotovora was not identified in this study, though these proteins of E. chrysanthemi were functional on the promoter regions of the pectinase genes of E. carotovora subsp. carotovora.
Mol Plant Microbe Interact 2003 Mar
PMID:Comparative study of regulatory mechanisms for pectinase production by Erwinia carotovora subsp. carotovora and Erwinia chrysanthemi. 1265 Apr 54

Identifying parasitism genes encoding proteins secreted from a nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Meloidogyne incognita parasitism genes were cloned by microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. Of 2,452 cDNA clones sequenced, deduced protein sequences of 185 cDNAs had a signal peptide for secretion and, thus, could have a role in root-knot nematode parasitism of plants. High-throughput in situ hybridization with cDNA clones encoding signal peptides resulted in probes of 37 unique clones specifically hybridizing to transcripts accumulating within the subventral (13 clones) or dorsal (24 clones) esophageal gland cells of M. incognita. In BLASTP analyses, 73% of the predicted proteins were novel proteins. Those with similarities to known proteins included a pectate lyase, acid phosphatase, and hypothetical proteins from other organisms. Our cell-specific analysis of genes encoding secretory proteins provided, for the first time, a profile of putative parasitism genes expressed in the M. incognita esophageal gland cells throughout the parasitic cycle.
Mol Plant Microbe Interact 2003 May
PMID:A profile of putative parasitism genes expressed in the esophageal gland cells of the root-knot nematode Meloidogyne incognita. 1274 7

Two distinct cDNA clones showing sequence homology to higher-plant pectate lyase (Pel) genes were isolated from ripening banana fruits. The transcripts were detected only in fruit tissue and both were strongly ripening-related. Yeast transformation with the most highly expressed Pel clone produced a recombinant protein with pectate lyase activity, demonstrating that this sequence was likely to encode a pectate lyase protein in planta. An assay developed for measuring the action of the endogenous enzyme from banana pulp tissue revealed a significant increase in calcium-dependent pectate lyase activity during ripening. The enhanced levels of enzyme activity corresponded with an increase in soluble polyuronides from banana pulp.
Plant Mol Biol 2003 Apr
PMID:Pectate lyase gene expression and enzyme activity in ripening banana fruit. 1277 45

Resolution of the 3D structures and IgE epitopes of allergens may identify common or conserved features of allergens. Jun a 1, the predominant allergen in mountain cedar pollen, was chosen as a model for identifying common structural and functional features among a group of plant allergens. In this study, synthetic, overlapping peptides of Jun a 1 and sera from patients allergic to mountain cedar pollen were used to identify linear epitopes. A 3D model of Jun a 1 was produced using the Bacillus subtiles pectate lyase (PL) as a template and validated with biophysical measurements. This allowed mappings of four IgE binding sites on Jun a 1. Two of the epitopes mapped to turns or loops on the surface of the model structure. The other two epitopes mapped to the beta-sheet region, homologous to the catalytic site of PL. This region of Jun a 1 is highly conserved in the group 1 allergens from other cedar trees as well as microbial PLs. The finding that two out of three major IgE epitopes map to highly conserved catalytic regions of group 1 cedar allergens may help to explain the high degree of cross-reactivity between cedar pollen allergens and might represent a pattern of reactivity common to other allergens with catalytic activity.
Mol Immunol 2003 Dec
PMID:Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1 map to the pectate lyase catalytic site. 1456 74

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.
Mol Plant Microbe Interact 2004 Feb
PMID:Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system. 1496 32

A negative correlation was observed between the aggressiveness of several Erwinia chrysanthemi strains on potato tuber and their osmotic tolerance. The disruption of the ousA gene encoding the major osmoprotectant uptake system highly enhanced bacterial virulence on potato tubers. The ousA disruption also increased the maceration efficiency on potato tubers under anaerobic conditions. In the absence of oxygen, pectate lyase (Pel) production was significantly higher in the tissue macerated with the ousA- strain than with the wild type. Oxygen content is significantly different between infected and healthy tissues; therefore, ousA may be a contributory factor in the infection progression within the host. In minimal medium, ousA disruption enhanced Pel production and pelE expression only under micro-aerobiosis conditions. The effect on Pel was reversed by reintroduction of the ousA gene. The osmoprotectectants glycine betaine, proline betaine, and pipecolic acid are known to be taken up via OusA and to have an inhibitory effect on Pel production. However, their effects on Pel activity were not (glycine betaine) or only weakly (proline and pipecolic acid) affected by ousA disruption. Furthermore, no correlation was observed between their effects on Pel activities and their osmoprotection efficacies. The results demonstrate a relationship between E. chrysanthemi pathogenicity factors and the activity of ousA under low oxygen status. The evidence indicates that ousA and osmoprotectant effects on Pel are not linked to osmoregulation and that complex regulations exist between Pel production, ousA, and osmoprotection via compounds liberated during the plant infection.
Mol Plant Microbe Interact 2005 Feb
PMID:Mutations of ousA alter the virulence of Erwinia chrysanthemi. 1572 84

Erwinia chrysanthemi strain 3937 is a necrotrophic bacterial plant pathogen. Pectinolytic enzymes and, in particular, pectate lyases play a key role in soft rot symptoms; however, the efficient colonization of plants by E. chrysanthemi requires additional factors. These factors include HrpN (harpin), a heat-stable, glycine-rich hydrophilic protein, which is secreted by the type III secretion system. We investigated the expression of hrpN in E. chrysanthemi 3937 in various environmental conditions and different regulatory backgrounds. Using lacZ fusions, hrpN expression was markedly influenced by the carbon source, osmolarity, growth phase, and growth substrate. hrpN was repressed when pectinolysis started and negatively regulated by the repressors of pectate lyase synthesis, PecS and PecT. Primer extension data and in vitro DNA-protein interaction experiments support a model whereby PecS represses hrpN expression by binding to the hrpN regulatory region and inhibiting transcript elongation. The results suggest coordinated regulation of HrpN and pectate lyases by PecS and PecT. A putative model of the synthesis of these two virulence factors in E. chrysanthemi during pathogenesis is presented.
Mol Plant Microbe Interact 2005 Nov
PMID:PecS and PecT coregulate the synthesis of HrpN and pectate lyases, two virulence determinants in Erwinia chrysanthemi 3937. 1635 55

Two pectate lyase genes (Bx-pel-1 and Bx-pel-2) were cloned from the pine wood nematode, Bursaphelenchus xylophilus. The deduced amino acid sequences of these pectate lyases are most similar to polysaccharide lyase family 3 proteins. Recombinant BxPEL1 showed highest activity on polygalacturonic acid and lower activity on more highly methylated pectin. Recombinant BxPEL1 demonstrated full dependency on Ca2+ for activity and optimal activity at 55 degrees C and pH 8 to 10 like other pectate lyases of polysaccharide lyase family 3. The protein sequences have predicted signal peptides at their N-termini and the genes are expressed solely in the esophageal gland cells of the nematode, indicating that the pectate lyases could be secreted into plant tissues to help feeding and migration in the tree. This study suggests that pectate lyases are widely distributed in plant-parasitic nematodes and play an important role in plant-nematode interactions.
Mol Plant Microbe Interact 2006 Mar
PMID:Cloning and characterization of pectate lyases expressed in the esophageal gland of the pine wood nematode Bursaphelenchus xylophilus. 1657 Jun 58

In Erwinia chrysanthemi (EC16) the clustered pelA and pelE genes encode an acidic (pI 4.2) and a basic (pI 10.0) pectate lyase (Pel), respectively. The pelA gene has been isolated on a 1.2 kb restriction fragment and the direction of transcription determined. DNA hybridization analysis showed that the pelE sequence shares DNA homology with pelA but not with pelB or pelC, two genes encoding other Pel species in EC16. Since Pel A and Pel E enzymes showed little similarity in terms of catalytic properties, it is proposed that pelA and pelE are duplicates which have highly diverged.
Mol Gen Genet 1987 Oct
PMID:Genetic analysis of the pelA-pelE cluster encoding the acidic and basic pectate lyases in Erwinia chrysanthemi EC16. 1719 15


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