Specificity of monoclonal antibodies to strains of Dickeya sp. that cause bacterial heart rot of pineapple.

During a severe outbreak of bacterial heart rot that occurred in pineapple plantations on Oahu, Hawaii, in 2003 and years following, 43 bacterial strains were isolated from diseased plants or irrigation water and identified as Erwinia chrysanthemi (now Dickeya sp.) by phenotypic, molecular, and pathogenicity assays. Rep-PCR fingerprint patterns grouped strains from pineapple plants and irrigation water into five genotypes (A-E) that differed from representatives of other Dickeya species, Pectobacterium carotovorum and other enteric saprophytes isolated from pineapple. Monoclonal antibodies produced following immunization of mice with virulent type C Dickeya sp. showed only two specificities. MAb Pine-1 (2D11G1, IgG1 with kappa light chain) reacted to all 43 pineapple/water strains and some reference strains (D. dianthicola, D. chrysanthemi, D. paradisiaca, some D. dadantii, and uncharacterized Dickeya sp.) but did not react to reference strains of D. dieffenbachiae, D. zeae, or one of the two Malaysian pineapple strains. MAb Pine-2 (2A7F2, IgG3 with kappa light chain) reacted to all type B, C, and D strains but not to any A or E strains or any reference strains except Dickeya sp. isolated from Malaysian pineapple. Pathogenicity tests showed that type C strains were more aggressive than type A strains when inoculated during cool months. Therefore, MAb Pine-2 distinguishes the more virulent type C strains from less virulent type A pineapple strains and type E water strains. MAbs with these two specificities enable development of rapid diagnostic tests that will distinguish the systemic heart rot pathogen from opportunistic bacteria associated with rotted tissues. Use of the two MAbs in field assays also permits the monitoring of a known subpopulation and provides additional decision tools for disease containment and management practices.

The flagellar sigma factor fliA is required for Dickeya dadantii virulence.

The genome sequence of the Enterobacteriaceae phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi) revealed homologs of genes required for a complete flagellar secretion system and one flagellin gene. We found that D. dadantii was able to swim and swarm but that ability to swarm was dependent upon both growth media and temperature. Mutation of the D. dadantii fliA gene was pleiotropic, with the alternate sigma factor required for flagella production and development of disease symptoms but not bacterial growth in Nicotiana benthamiana leaves. The flagellar sigma factor was also required for multiple bacterial phenotypes, including biofilm formation in culture, bacterial adherence to plant tissue, and full expression of pectate lyase activity (but not cellulase or protease activity). Surprisingly, mutation of fliA resulted in the increased expression of avrL (a gene of unknown function in D. dadantii) and two pectate lyase gene homologs, pelX and ABF-0019391. Because FliA is a key contributor to virulence in D. dadantii, it is a new target for disease control.

Biogenesis of Fe/S proteins and pathogenicity: IscR plays a key role in allowing Erwinia chrysanthemi to adapt to hostile conditions.

The Erwinia chrysanthemi genome is predicted to encode three systems, Nif, Isc and Suf, known to assist Fe/S cluster biogenesis and the CsdAE cysteine desulphurase. Single iscU, hscA and fdx mutants were found sensitive to paraquat and exhibited reduced virulence on both chicory leaves and Arabidopsis thaliana. Depletion of the whole Isc system led to a pleiotropic phenotype, including sensitivity to both paraquat and 2,2'-dipyridyl, auxotrophies for branched-chain amino acids, thiamine, nicotinic acid, and drastic alteration in virulence. IscR was able to suppress all of the phenotypes listed above in a sufC-dependent manner while depletion of the Isc system led to IscR-dependent activation of the suf operon. No virulence defects were found associated with csdA or nifS mutations. Surprisingly, we found that the sufC mutant was virulent against A. thaliana, whereas its virulence had been found altered in Saintpaulia. Collectively, these results lead us to propose that E. chrysanthemi possess the Fe/S biogenesis strategy suited to the physico-chemical conditions encountered in its host upon infection. In this view, the IscR regulator, which controls both Isc and Suf, is predicted to play a major role in the ability of E. chrysanthemi to colonize a wide array of different plants.

Differential role of ferritins in iron metabolism and virulence of the plant-pathogenic bacterium Erwinia chrysanthemi 3937.

During infection, the phytopathogenic enterobacterium Erwinia chrysanthemi has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. Previous studies have shown that a tight control of the bacterial intracellular iron content is necessary for full virulence. The E. chrysanthemi genome possesses two loci that could be devoted to iron storage: the bfr gene, encoding a heme-containing bacterioferritin, and the ftnA gene, coding for a paradigmatic ferritin. To assess the role of these proteins in the physiology of this pathogen, we constructed ferritin-deficient mutants by reverse genetics. Unlike the bfr mutant, the ftnA mutant had increased sensitivity to iron deficiency and to redox stress conditions. Interestingly, the bfr ftnA mutant displayed an intermediate phenotype for sensitivity to these stresses. Whole-cell analysis by Mössbauer spectroscopy showed that the main iron storage protein is FtnA and that there is an increase in the ferrous iron/ferric iron ratio in the ftnA and bfr ftnA mutants. We found that ftnA gene expression is positively controlled by iron and the transcriptional repressor Fur via the small antisense RNA RyhB. bfr gene expression is induced at the stationary phase of growth. The sigmaS transcriptional factor is necessary for this control. Pathogenicity tests showed that FtnA and the Bfr contribute differentially to the virulence of E. chrysanthemi depending on the host, indicating the importance of a perfect control of iron homeostasis in this bacterial species during infection.

Erwinia chrysanthemi requires a second iron transport route dependent of the siderophore achromobactin for extracellular growth and plant infection.

Full virulence of the pectinolytic enterobacterium Erwinia chrysanthemi strain 3937 depends on the production in planta of the catechol-type siderophore chrysobactin. Under iron-limited conditions, E. chrysanthemi synthesizes a second siderophore called achromobactin belonging to the hydroxy/carboxylate class of siderophore. In this study, we cloned and functionally characterized a 13 kb long operon comprising seven genes required for the biosynthesis (acs) and extracellular release (yhcA) of achromobactin, as well as the gene encoding the specific outer membrane receptor for its ferric complex (acr). The promoter of this operon was negatively regulated by iron. In a fur null mutant, transcriptional fusions to the acsD and acsA genes were constitutively expressed. Band shift assays showed that the purified E. chrysanthemi Fur repressor protein specifically binds in vitro to the promoter region of the acsF gene confirming that the metalloregulation of the achromobactin operon is achieved directly by Fur. The temporal production of achromobactin in iron-depleted bacterial cultures was determined: achromobactin is produced before chrysobactin and its production decreases as that of chrysobactin increases. Pathogenicity tests performed on African violets showed that achromobactin production contributes to the virulence of E. chrysanthemi. Thus, during infection, synthesis of these two different siderophores allows E. chrysanthemi cells to cope with the fluctuations of iron availability encountered within plant tissues. Interestingly, iron transport mediated by achromobactin or a closely related siderophore probably exists in other phytopathogenic bacterial species such as Pseudomonas syringae.

The Erwinia chrysanthemi EC16 hrp/hrc gene cluster encodes an active Hrp type III secretion system that is flanked by virulence genes functionally unrelated to the Hrp system.

Erwinia chrysanthemi is a host-promiscuous plant pathogen that possesses a type III secretion system (TTSS) similar to that of the host-specific pathogens E. amylovora and Pseudomonas syringae. The regions flanking the TTSS-encoding hrp/hrc gene clusters in the latter pathogens encode various TTSS-secreted proteins. DNA sequencing of the complete E. chrysanthemi hrp/hrc gene cluster and approximately 12 kb of the flanking regions (beyond the previously characterized hecA adhesin gene in the left flank) revealed that the E. chrysanthemi TTSS genes were syntenic and similar (>50% amino-acid identity) with their E. amylovora orthologs. However, the hrp/hrc cluster was interrupted by a cluster of four genes, only one of which, a homolog of lytic transglycosylases, is implicated in TTSS functions. Furthermore, the regions flanking the hrp/hrc cluster lacked genes that were likely to encode TTSS substrates. Instead, some of the genes in these regions predict ABC transporters and methyl-accepting chemotaxis proteins that could have alternative roles in virulence. Mutations affecting all of the genes in the regions flanking or interrupting the hrp/hrc cluster were constructed in E. chrysanthemi CUCPB5047, a mutant whose reduced pectolytic capacity can enhance the phenotype of minor virulence factors. Mutants were screened in witloof chicory leaves and then in potato tubers and Nicotiana clevelandii seedlings. Mu dII1734 insertion in one gene, designated virA, resulted in strongly reduced virulence in all three tests. virA is immediately downstream of hecA, has an unusually low G+C content of 38%, and predicts an unknown protein of 111 amino acids. The E. chrysanthemi TTSS was shown to be active by its ability to translocate AvrPto-Cya (a P. syringae TTSS effector fused to an adenylate cyclase reporter that is active in the presence of eukaryote calmodulin) into N. benthamiana leaf cells. However, VirA(1-61)-Cya was not translocated into plant cells, and virA expression was not affected by mutations in E. chrysanthemi Hrp regulator genes hrpL and hrpS. Thus, the 44-kb region of the E. chrysanthemi EC16 genome that is centered on the hrplhrc cluster encodes a potpourri of virulence factors, but none of these appear to be a TTSS effector.

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.

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.

Coupling of iron assimilation and pectinolysis in Erwinia chrysanthemi 3937.

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.

HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings.

Erwinia chrysanthemi is representative of a broad class of bacterial pathogens that are capable of inducing necrosis in plants. The E. chrysanthemi EC16 hecA gene predicts a 3,850-aa member of the Bordetella pertussis filamentous hemagglutinin family of adhesins. A hecATn7 mutant was reduced in virulence on Nicotiana clevelandii seedlings after inoculation without wounding. Epifluorescence and confocal laser-scanning microscopy observations of hecA and wild-type cells expressing the green fluorescent protein revealed that the mutant is reduced in its ability to attach and then form aggregates on leaves and to cause an aggregate-associated killing of epidermal cells. Cell killing also depended on production of the major pectate lyase isozymes and the type II, but not the type III, secretion pathway in E. chrysanthemi. HecA homologs were found in bacterial pathogens of plants and animals and appear to be unique to pathogens and universal in necrogenic plant pathogens. Phylogenetic comparison of the conserved two-partner secretion domains in the proteins and the 16S rRNA sequences in respective bacteria revealed the two datasets to be fundamentally incongruent, suggesting horizontal acquisition of these genes. Furthermore, hecA and its two homologs in Yersinia pestis had a G+C content that was 10% higher than that of their genomes and similar to that of plant pathogenic Ralstonia, Xylella, and Pseudomonas spp. Our data suggest that filamentous hemagglutinin-like adhesins are broadly important virulence factors in both plant and animal pathogens.

hrp genes of Erwinia chrysanthemi 3937 are important virulence factors.

We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN delta pel (delta pel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.

SoxR-dependent response to oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase.

Erwinia chrysanthemi causes soft-rot disease in a great variety of plants. In addition to the depolymerizing activity of plant cell wall-degrading enzymes, iron acquisition and resistance to oxidative stress contribute greatly to the virulence of this pathogen. Here, we studied the pin10 locus originally thought to encode new virulence factors. The sequence analysis revealed six open reading frames that were homologous to the Escherichia coli sufA, sufB, sufC, sufD, sufS and sufE genes. Sequence similarity searching predicted that (i) SufA, SufB, SufD, SufS and SufE proteins are involved in iron metabolism and possibly in Fe-S cluster assembly; and (ii) SufC is an ATPase of an ABC transporter. The reverse transcription-polymerase chain reaction procedure showed that the sufABCDSE genes constitute an operon. Expression of a sufB:uidA fusion was found to be induced in iron-deficient growth conditions and to be repressed by the iron-sensing Fur repressor. Each of the six suf genes was inactivated by the insertion of a cassette generating a non-polar mutation. The intracellular iron level in the sufA, sufB, sufC, sufS and sufE mutants was higher than in the wild type, as assessed by increased sensitivity to the iron-activated antibiotic streptonigrin. In addition, inactivation of sufC and sufD led to increased sensitivity to paraquat. Virulence tests showed that sufA and sufC mutants exhibited reduced ability to cause maceration of chicory leaves, whereas a functional sufC gene was necessary for the bacteria to cause systemic invasion of Saintpaulia ionantha. The E. coli sufC homologue was inactivated by reverse genetic. This mutation was found to modify the soxR-dependent induction of soxS gene expression. We discuss the possibility that SufC is a versatile ATPase that can associate either with the other Suf proteins to form a Fe-S cluster-assembling machinery or with membrane proteins encoded elsewhere in the chromosome to form an Fe-S ABC exporter. Overall, these results stress the importance of the connection between iron metabolism and oxidative stress during the early steps of infection by E. chrysanthemi.

Erwinia chrysanthemi strains cause death of human gastrointestinal cells in culture and express an intimin-like protein.

The bacterium Erwinia chrysanthemi is a model plant pathogen, responsible for causing cell death in plant tissue. Cell-wall depolymerizing enzymes and avirulence proteins essential for parasitism by this bacterium utilize dedicated type II and type III secretion systems, respectively. Although E. chrysanthemi is not recognized as a mammalian pathogen, we have observed that the bacterium can adhere to, cause an oxidative stress response in and kill cultured human adenocarcinoma cells. These bacteria express a surface protein that bears immunological identity to intimin, a protein required for full virulence of enterohemorrhagic and enteropathogenic Escherichia coli. A type III secretion mutant of E. chrysanthemi was observed to have a significantly lower capability of causing death than the wild-type strain in parallel cultures of human colon adenocarcinoma cells. These observations suggest that E. chrysanthemi has the potential to parasitize mammalian hosts as well as plants.

Evidence against a direct antimicrobial role of H2O2 in the infection of plants by Erwinia chrysanthemi.

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.

Reciprocal secretion of proteins by the bacterial type III machines of plant and animal pathogens suggests universal recognition of mRNA targeting signals.

Bacterial pathogens of both animals and plants use type III secretion machines to inject virulence proteins into host cells. Although many components of the secretion machinery are conserved among different bacterial species, the substrates for their type III pathways are not. The Yersinia type III machinery recognizes some secretion substrates via a signal that is encoded within the first 15 codons of yop mRNA. These signals can be altered by frameshift mutations without affecting secretion of the encoded polypeptides, suggesting a mechanism whereby translation of yop mRNA is coupled to the translocation of newly synthesized polypeptide. We report that the type III machinery of Erwinia chrysanthemi cloned in Escherichia coli recognizes the secretion signals of yopE and yopQ. Pseudomonas syringae AvrB and AvrPto, two proteins exported by the recombinant Erwinia machine, can also be secreted by the Yersinia type III pathway. Mapping AvrPto sequences sufficient for the secretion of reporter fusions in Yersinia revealed the presence of an mRNA secretion signal. We propose that 11 conserved components of type III secretion machines may recognize signals that couple mRNA translation to polypeptide secretion.

Iron regulation and pathogenicity in Erwinia chrysanthemi 3937: role of the Fur repressor protein.

Low iron availability is a triggering signal for coordinated expression of the genes encoding pectate lyases PelB, PelC, PelD, and PelE, and chrysobactin iron transport functions, which are two main determinants of phytopathogenicity of the Erwinia chrysanthemi strain 3937. The possible implication of the ferric uptake regulation (Fur) protein in this process was investigated. The E. chrysanthemi fur gene was cloned by functional complementation of an Escherichia coli fur mutant and sequenced. The 444-bp open reading frame identified was found to code for a protein highly similar to the E. coli Fur regulator. An E. chrysanthemi fur null mutant was constructed by reverse genetics. This mutant showed altered growth capacity and reduced pathogenicity on African violets. In a fur background, transcriptional lacZ fusions to genes belonging to the E. chrysanthemi high affinity iron transport systems were constitutively expressed. Transcription of the pelA, pelD, and pelE genes was analyzed, using fusions to the uidA reporter gene. Iron availability and a fur mutation did not influence the expression of pelA. In the presence of iron, pelD and pelE transcription levels were higher in the fur mutant than in the parental strain. Furthermore, iron deficiency stimulated the expression of both fusions in the fur mutant. These findings indicate that, in E. chrysanthemi 3937, (i) Fur negatively controls iron transport and genes encoding PelD and PelE, and (ii) additional factor(s) mediate iron regulation of the pel genes.

The pir gene of Erwinia chrysanthemi EC16 regulates hyperinduction of pectate lyase virulence genes in response to plant signals.

The plant pathogenic bacterium Erwinia chrysanthemi secretes pectate lyase proteins that are important virulence factors attacking the cell walls of plant hosts. Bacterial production of these enzymes is induced by the substrate polypectate-Na (NaPP) and further stimulated by the presence of plant extracts. The bacterial regulator responsible for induction by plant extracts was identified and purified by using a DNA-binding assay with the promoter region of pelE that encodes a major pectate lyase. A novel bacterial protein, called Pir, was isolated that produced a specific gel shift of the pelE promoter DNA, and the corresponding pir gene was cloned and sequenced. The Pir protein contains 272 amino acids with a molecular mass of 30 kDa and appears to function as a dimer. A homology search indicates that Pir belongs to the IclR family of transcriptional regulators. Pir bound to a 35-bp DNA sequence in the promoter region of pelE. This site overlaps that of a previously described negative regulator, KdgR. Gel shift experiments showed that the binding of either Pir or KdgR interfered with binding of the other protein.

Processing of the pectate lyase PelI by extracellular proteases of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi causes soft rot on various plants. The maceration of plant tissues is mainly due to the action of endopectate lyases. The E. chrysanthemi strain 3937 produces eight endopectate lyases (PelA, PelB, PelC, PelD, PelE, PelI, PelL and PelZ) that are secreted by the Out pathway. The necrotic response elicited by the wild-type E. chrysanthemi strain on tobacco leaves is due to an extracellular protein secreted by the Out machinery. Purification of the active factor revealed that it corresponds to a pectate lyase presenting immunological cross-reaction with PelI. Analysis of pelI and out mutants indicated that the necrosis-inducing pectate lyase results from a post-translational modification of PelI occurring extracellularly both in culture media and in planta. This modification consists of the cleavage of 97 N-terminal amino acids by the extracellular proteases of E. chrysanthemi. The enzymatic properties of the maturated form, PelI-3, are not, or only weakly, modified. However, this maturation gives rise to a small size and basic form that is active as a defence elicitor in plants.

The exuT gene of Erwinia chrysanthemi EC16: nucleotide sequence, expression, localization, and relevance of the gene product.

Galacturonic acid (GalUA) is a major component of pectin and polygalacturonic acid in the plant cell wall. In the phytopathogen Erwinia chrysanthemi, the uptake of molecules derived from degradation of these polymers is an important early step in the events preceding induction of pectinases, ultimately leading to plant tissue maceration. Uptake systems for GalUA and dimers of GalUA have been described and shown to be inducible in E. chrysanthemi. The GalUA uptake gene (exuT) was cloned and sequenced. Nucleotide sequence analysis identified an open reading frame encoding a 345-amino-acid polypeptide with a calculated mass of 37,825 Da. This polypeptide is predicted to be an integral membrane protein based on its high nonpolar amino acid content and hydropathic profile. Localization studies with the labeled polypeptide in the T7-RNA polymerase system also suggest that ExuT is a membrane protein. This evidence is further supported by the observation of hybrid ExuT-PhoA proteins in the bacterial cytoplasmic membrane following immunoblot analysis. Northern (RNA) analysis indicated that the gene is inducible in the presence of the monomer, GalUA. A targeted mutation in the exuT gene affected the utilization of GalUA as a role carbon source for growth. Maceration of potato tuber tissue by this mutant was delayed and reduced, when compared with the parental strain EC16.

The role of iron in plant host-pathogen interactions.

Iron is unlikely to be readily available in plant tissues for invading microorganisms. Soft rot, caused by Erwinia chrysanthemi strain 3937 on African violets, is a valuable model for studying the role of iron and its ligands in plant-pathogen interactions. These studies could lead to the development of new control strategies against microbial infections of plants.

Erwinia chrysanthemi harpinEch: an elicitor of the hypersensitive response that contributes to soft-rot pathogenesis.

Mutants of the soft-rot pathogen Erwinia chrysanthemi EC16 that are deficient in the production of the pectate lyase isozymes PelABCE can elicit the hypersensitive response (HR) in tobacco leaves. The hrpNEch gene was identified in a collection of cosmids carrying E. chrysanthemi hrp genes by its hybridization with the Erwinia amylovora hrpNEa gene. hrpNEch appears to be in a monocistronic operon, and it encodes a predicted protein of 340 amino acids that is glycine-rich, lacking in cysteine, and highly similar to HrpNEa in its C-terminal half. Escherichia coli DH5 alpha cells expressing hrpNEch from the lac promoter of pBluescript II accumulated HrpNEch in inclusion bodies. The protein was readily purified from cell lysates carrying these inclusion bodies by solubilization in 4.5 M guanidine-HCl and reprecipitation upon dialysis against dilute buffer. HrpNEch suspensions elicited a typical HR in tobacco leaves, and elicitor activity was heat-stable. Tn5-gusA1 mutations were introduced into the cloned hrpNEch and then marker-exchanged into the genomes of E. chrysanthemi strains AC4150 (wild type), CUCPB5006 (delta pelABCE), and CUCPB5030 (delta pelABCE outD::TnphoA). hrpNEch::Tn5-gusA1 mutations in CUCPB5006 abolished the ability of the bacterium to elicit the HR in tobacco leaves unless complemented with an hrpNEch subclone. An hrpNEch::Tn5-gusA1 mutation also reduced the ability of AC4150 to incite infections in witloof chicory leaves, but it did not reduce the size of lesions that did develop. Purified HrpNEch and E. chrysanthemi strains CUCPB5006 and CUCPB5030 elicited HR-like necrosis in leaves of tomato, pepper, African violet, petunia, and pelargonium, whereas hrpNEch mutants did not.(ABSTRACT TRUNCATED AT 250 WORDS)