Desferrioxamine-dependent iron transport in Erwinia amylovora CFBP1430: cloning of the gene encoding the ferrioxamine receptor FoxR

Iron deprivation of Erwinia amylovora CFBP1430, a species causing fire blight on Pomoïdeae, was shown to induce the production of siderophores of the desferrioxamine (dfo) family and two outer membrane polypeptides with apparent molecular weight of about 70 and 80 kDa, respectively. Cyclic dfo E was characterized as the major metabolite. Phage MudII_pR13 insertional mutagenesis and screening on CAS-agar medium yielded three dfo non-producing and one overproducing clones. These clones failed to grow in the presence of the Fe(III) chelator EDDHA and were determined further as dfo and ferrioxamine transport negative mutants, respectively. The transport mutant which appeared to lack the 70 kDa polypeptide in the outer membrane allowed the purification of dfo E. Growth under iron limitation of dfo negative mutants was stimulated with ferrioxamine E and B but not with other ferrisiderophores tested. The host DNA sequence flanking the left terminal part of the MudII_pR13 prophage responsible for the transport mutation was cloned and used to probe a parental gene library by DNA-DNA hybridization. Two recombinant cosmids restoring the transport mutation to normal were identified. Both cosmids also conferred the ability to utilize ferrioxamine B and E as iron sources on a FhuE¹ mutant of Escherichia coli. This correlated with the production of an additional polypeptide of 70 kDa in the outer membrane of E. coli transconjugants, thus confirming that this protein serves the ferrioxamine receptor function (FoxR) in E. amylovora.R. Kachadourian and A. Dellagi have contributed equally to this work.     

Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence

Phage MudIIPR13 insertional mutagenesis of Erwinia amylovora CFBP1430 allowed us to isolate 6900 independent CmR mutants. The frequencies of different auxotrophs in this population indicated that MudIIPR13 had inserted randomly in E. amylovora. Screening of 3500 CmR mutants on (i) apple calli and (ii) pear and apple seedlings led to the isolation of 19 non-pathogenic prototrophic single mutants, four of which expressed a LacZ+ hybrid protein. Expression of the fusion proteins was temperature sensitive. The 19 mutants could be separated into two classes according to their behaviour on tobacco: 13 were unable to elicit the hypersensitive response on tobacco (Hrp-) while six still could (Dsp-). The 19 MudIIPR13 insertions all mapped in the same virulence region. The MudIIPR13 insertions of Hrp- mutants were all clustered on the left part of this region, while the MudIIPR13 insertions of Dsp- mutants were located on the right part. All of the mutants except one, which proved to have a large deletion of the entire virulence region, could be complemented functionally by cosmids from an E. amylovora CFBP1430 genomic library. No hybridization was observed between the cosmid pPV130, which complemented 12 hrp::MudIIPR13 mutations, and the hrp genes from Pseudomonas syringae pv. phaseolicola (Lindgren et al., 1986), P. syringae pv. tomato (N.J. Panopoulos, unpublished data) or P. solanacearum (Boucher et al., 1987). Further analysis of the large virulence region will allow mapping of the border of the virulence region and facilitate the study of the function and regulation of the hrp and dsp genes.     

Expression of firefly luciferase gene in Erwinia amylovora

In this study we describe an ef?cient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the ?re?y luciferase gene of Photinus pyralis, which is further controlled by IPTG‐inducible promoter. Stably transformed E. amylovora cells maintain the same infectivity as the wild‐type strain and, after induction with IPTG, produce luciferase. Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL). The transformed E. amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post‐infection. Our ?ndings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments.     

Bacteriophages of Erwinia amylovora

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and PODOVIRIDAE:     

Bacteriophages of Erwinia amylovora

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae.     

Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.     

Erwinia amylovora Infection of Hawthorn Blossom

Stamens of freshly opened flowers of hawthorn were inoculated with E;. amylovora and the development of blossom infection was monitored by viable bacterial counts and light and electron microscopy. Some bacterial multiplication occurred on the anther surface, over the dehiscence zone and over the junctions ot the anther-wall cells. bacteria invaded the anther loculc, via the ruptured dehiscence zone, and possibly also vid the stomata surrounding the filament insertion. bacteria within the locule multiplied rapidk with estimated doubling-times which were longer than those derived from in vitro data. Pollen grains Irom infected anthers were found to be heavily eontaminated with bacteria. The invasion of anther tissue, with the production of contaminated pollen, may be important epidemiologieally both as a phase of rapid bacterial multiplication and in the insect-mediated spread of this disease.     

Plant nitrate supply regulates Erwinia amylovora virulence gene expression in Arabidopsis

We showed previously that nitrogen (N) limitation decreases Arabidopsis resistance to Erwinia amylovora (Ea). We show that decreased resistance to bacteria in low N is correlated with lower apoplastic reactive oxygen species (ROS) accumulation and lower jasmonic acid (JA) pathway expression. Consistently, pretreatment with methyl jasmonate (Me-JA) increased the resistance of plants grown under low N. In parallel, we show that in planta titres of a nonvirulent type III secretion system (T3SS)-deficient Ea mutant were lower than those of wildtype Ea in low N, as expected, but surprisingly not in high N. This lack of difference in high N was consistent with the low expression of the T3SS-encoding hrp virulence genes by wildtype Ea in plants grown in high N compared to plants grown in low N. This suggests that expressing its virulence factors in planta could be a major limiting factor for Ea in the nonhost Arabidopsis. To test this hypothesis, we preincubated Ea in an inducing medium that triggers expression of hrp genes in vitro, prior to inoculation. This preincubation strongly enhanced Ea titres in planta, independently of the plant N status, and was correlated to a significant repression of JA-dependent genes. Finally, we identify two clusters of metabolites associated with resistance or with susceptibility to Ea. Altogether, our data showed that high susceptibility of Arabidopsis to Ea, under low N or following preincubation in hrp-inducing medium, is correlated with high expression of the Ea hrp genes in planta and low expression of the JA signalling pathway, and is correlated with the accumulation of specific metabolites.     

Ultrastructure of the Cell Wall of Erwinia amylovora

Thin sectioned cells of Erwinia amylovora revealed two electron-dense layers in their walls when fixed at 24 to 27 C and three when fixed at 4 C.     

Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU.     

Expression of bovine lactoferrin cDNA confers resistance to Erwinia amylovora in transgenic pear

The siderophore produced by Erwinia amylovora, the causal agent of fire blight of Maloideae, is one of the virulence factors of this bacterium. The production of siderophores enables E. amylovora to overcome the conditions of iron limitation met in plant tissue, and may also protect the bacteria against active oxygen species produced through the Fenton reaction. In this paper, we have examined the ability of an iron chelator protein, encoded by the bovine lactoferrin gene, to reduce fire blight susceptibility in pear (Pyrus communis L.). Transgenic pear clones expressing this gene controlled by the CaMV35S promoter were produced by Agrobacterium tumefaciens mediated transformation. Transformants were analysed by RT-PCR and western blot to determine lactoferrin expression levels. Most transgenic clones demonstrated significant reduction of susceptibility to fire blight in vitro and in the greenhouse when inoculated by E. amylovora. These transgenic clones also showed a significant reduction of symptoms when inoculated with two other pear bacterial pathogens : Pseudomonas syringae pv. syringae and Agrobacterium tumefaciens. Moreover, we have shown that this increase in bacterial resistance was correlated with an increase in root ferric reductase level activity and leaf iron content. Despite negative effects on the growth of a few clones, our results indicate the potential of lactoferrin gene transformation to protect pear from fire blight through increased iron chelation.     

Identification of Erwinia amylovora genes induced during infection of immature pear tissue

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In this study, we used a modified in vivo expression technology system to identify E. amylovora genes that are activated during infection of immature pear tissue, a process that requires the major pathogenicity factors of this organism. We identified 394 unique pear fruit-induced (pfi) genes on the basis of sequence similarity to known genes and separated them into nine putative function groups including host-microbe interactions (3.8%), stress response (5.3%), regulation (11.9%), cell surface (8.9%), transport (13.5%), mobile elements (1.0%), metabolism (20.3%), nutrient acquisition and synthesis (15.5%), and unknown or hypothetical proteins (19.8%). Known virulence genes, including hrp/hrc components of the type III secretion system, the major effector gene dspE, type II secretion, levansucrase (lsc), and regulators of levansucrase and amylovoran biosynthesis, were upregulated during pear tissue infection. Known virulence factors previously identified in E. (Pectobacterium) carotovora and Pseudomonas syringae were identified for the first time in E. amylovora and included HecA hemagglutinin family adhesion, Peh polygalacturonase, new effector HopPtoC(EA), and membrane-bound lytic murein transglycosylase MltE(EA). An insertional mutation within hopPtoC(EA) did not result in reduced virulence; however, an mltE(EA) knockout mutant was reduced in virulence and growth in immature pears. This study suggests that E. amylovora utilizes a variety of strategies during plant infection and to overcome the stressful and poor nutritional environment of its plant hosts.     

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

Characterization of hns genes from Erwinia amylovora

The small basic histone-like protein H-NS is known for bacteria to attenuate virulence of several animal pathogens. An hns homologue from E. amylovora was identified by complementing an E. coli hns-mutant strain with a cosmid library from E. amylovora. A 1.6 kb EcoRI-fragment complemented the mucoid phenotype and repressed the ss-glucosidase activity of E. coli PD32. The open reading frame encoding an H-NS-like protein of 134 amino acid was later shown to be located on plasmid pEA29 (McGhee and Jones 2000). A chromosomal hns gene was amplified with PCR consensus primers and localized near galU of E. amylovora. E. amylovora mutants were created by insertion of a resistance cassette, and the intact gene was inserted into a high copy number plasmid for constitutive expression. Purified chromosomal H-NS protein preferentially bound to a DNA fragment from the lsc region and bending was predicted for an adjacent fragment with the rlsB-promoter. Levan production was significantly increased by hns mutations. Synthesis of the capsular exopolysaccharide amylovoran and of levan were reduced, when hns from the E. amylovora plasmid was overexpressed. A mutation in chromosomal hns of E. amylovora increased amylovoran synthesis, and both mutations retarded symptom formation on immature pears.     

Gene Transmission Among Strains of Erwinia amylovora

Stable donor strains of Erwinia amylovora were obtained from strain EA178R(1) (harboring an Escherichia coli F'lac) by selection for clones resistant to curing by acridine orange. These donor strains (EA178R(1)-99 and EA178R(1)-111) transfer chromosomal markers (arg, cys, gua, ilv, met, pro, ser, trp); the frequency of the appearance of recombinants prototrophic for Cys, Gua, Met, Ser, and Trp is highest (> 10(-5)), followed by recombinants prototrophic for Arg, Ilv, and Pro (10(-7) to 10(-5)). The results of interrupted matings, as well as the frequency of transmission of various markers, suggest that cys is transferred as an early marker by both donor strains. The Hfr state of these donor strains is rather likely on the basis of the following observations. The donor strains exhibit a relatively efficient and possibly oriented chromosome transfer; the Lac(+) character is not cured by acridine orange in these donor strains; and these donor strains do not transfer F.     

Distribution of Streptomycin Resistant Strains of Erwinia amylovora in Egypt during 1988

A survey of the occurrence of strains of Erwinia amylovora resistant to streptomycin in certain Egyptian pear orchards was earned out during April and May 1988. Twenty-two isolates out of 604 isolates collected from 11 orchards showed resistance to streptomycin. All the streptomycin resistant (Str^r) strains isolated in the present work were resistant to high levels of streptomycin with minimal inhibitory concentrations ranging from 1000 to 3000 μg/ml. The occurrence of Str^r strains in Egypt is still limited and the population of resistant strains was at relatively low level. However, such occurrence of E. amylovora with resistance to streptomycin is a potentially serious situation.