Fire Blight Pathogen Erwinia Amylovora Research Articles

Characterization of the amsI gene product as a low molecular weight acid phosphatase controlling exopolysaccharide synthesis of Erwinia amylovora

The ams region, responsible for amylovoran synthesis of the fireblight pathogen Erwinia amylovora, contains the gene amsI encoding a 144 amino acid protein with homology to mammalian low molecular weight acid phosphatases [Bugert and Geider (1995) Mol. Microbiol. 15, 917–933]. A DNA fragment with amsI was cloned under the control of the lac promoter on a high copy number plasmid. The gene product of amsI is about 17 kDa in a protein expression system and had the enzymatic activity of an acid phosphatase. This is the first report about a low molecular weight acid phosphatase activity in prokaryotes. As part of the large ams transcript, expression of amsI was affected by the activator proteins RcsA and RcsB. Overexpression of amsI in E. amylovora caused a strong increase of acid phosphatase activity, but additionally a strong reduction in EPS synthesis, phenotypically similar to a mutation in the gene. The gene product may participate in changes of phosphorylation required for the biosynthesis of EPS such as recycling the lipid carrier diphosphate to the monophosphate form.

The use of luciferase as a reporter for response of plant cells to the fireblight pathogen Erwinia amylovora

We have introduced the gene encoding luciferase from Photinus pyralis into pear and tobacco cells in order to judge the reaction of plant tissue to damaging conditions such as incubation at high temperature or inoculation with a pathogen. The constitutive expression of the luciferase gene via a strong promoter slowly decreased during propagation of the transformed pear cell line. After various stress treatments the resulting luciferase activity and the ATP content of the plant cells were determined by bioluminescence and found to correspond to each other. Inoculation of transformed pear cells with Erwinia amylovora resulted in a continuous decrease of luciferase activity in contrast to tobacco cells, where the enzyme activity was significantly higher in the first period after inoculation with bacteria compared to the untreated control cells. The pattern of the luciferase activity reflected the slow damage of the host-plant cells by E. amylovora and the elevated metabolism of the non-host cells after inoculation with the pathogen.

Assessment of somaclonal variation in apple. I. Resistance to the fire blight pathogen,Erwinia amylovora

SummaryAfter inoculation of glasshouse-grown somaclones regenerated from apple leaf discs (cv. Greensleeves) 33% of 270 showed an increase in resistance to the fire blight pathogen Erwinia amylovora. In contrast only 21% produced less severe symptoms than parental material after inoculation of plants held in culture in vitro. Sixteen somaclones which showed the highest levels of fire blight resistance were tested intensively as both glasshouse-grown plants and as micropropagated plants in culture. In tests conducted with glasshouse-grown plants up to 60% of plants of the most promising somaclones exhibited minimum symptoms after inoculation compared with 4% of ‘Greensleeves’ parental plants. The comparable figures for inoculations of in vitro cultured plants were 70% and 8%. The growth of E. amylovora was reduced in resistant somaclones compared with parental plants. Pretreating leaf discs with virulent E. amylovora cells prior to somaclone regeneration had no effect on the frequency of regenerated somaclones exhibiting increased resistance to the pathogen.

Characterization and influence on virulence of the levansucrase gene from the fireblight pathogen Erwinia amylovora

Levan, a β-2,6-fructofuranan, produced by the fireblight pathogen Erwinia amylovora , is synthesized from sucrose by the extracellular enzyme levansucrase. Levansucrase mutants, created by Tn5 transposon mutagenesis, showed retarded development of necrotic symptoms on inoculated pear seedlings. By complementation experiments the levansucrase gene (lsc) was located in a 1·4 kb PstI-EcoRV chromosomal DNA fragment, isolated from a genomic library of E. amylovora. An open reading frame of 415 amino acids, promotor sequences and a ribosome binding site were identified by DNA sequencing. The cloned lsc gene of E. amylovora was expressed in E. coli from its own promotor. The enzyme accumulated in E. coli in the periplasm and caused an altered colony morphology on sucrose containing solid media. When analysed by SDS-PAGE and isoelectric focusing, the enzyme purified from E. coli was found to be identical with the protein isolated from E. amylovora.

A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii

A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned. Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants. Five complementation groups, essential for amylovoran synthesis and virulence in E. amylovora, were identified and designated ams A-E. The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii. Mucoidy and virulence were restored to E. stewartii mutants in four cps complementation groups by the cloned E. amylovora ams genes. Conversely, the E. stewartii cps gene cluster was able to complement mutations in E. amylovora ams genes. Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different. EPS production and virulence were also restored to E. amylovora amsE and E. stewartii cpsD mutants by clones containing the Rhizobium meliloti exo A gene.

Proferrioxamine siderophores of Erwinia amylovora. A capillary liquid chromatographic/electrospray tandem mass spectrometric study

AbstractOn‐line capillary liquid chromatography–electrospray mass spectrometry and tandem mass spectrometry, in combination with precursor feeding and acetylation studies, were used to identify and characterize the siderophores of the fire blight pathogen Erwinia amylovora. Proferrioxamines D2, E, G1, G2, X1, and X2 were found, with D2 and E being the major siderophores for all strains of E. amylovora studied. These proferrioxamines have previously been observed with other microbial species. In addition, eleven additional hydroxamate compounds were found, nine of which occur naturally and two which were induced by directed fermentation. The novel natural proferrioxamines included three cyclic tetrahydroxamates, designated T1–3. The most abundant of these, T1, consists of four 5‐succinylamino‐1‐hydroxyaminopentane residues. In T2 and T3, one of these residues is substituted by 4‐succinylamino‐1‐hydroxyaminobutane and 3‐succinylamino‐1‐hydroxyaminopropane, respectively. E. amylovora also incorporated 3‐succinylamino‐1‐hydroxyaminopropane into a novel trihydroxamate, which has been designated X7. The novel finding that proferrioxamines may partly be comprised of 1,3‐diaminopropane residues is noteworthy because previous feeding studies with Streptomyces olivaceus Tü 2718 suggested that 1,3‐diaminopropane is biosynthetically not tolerated. A truncated proferrioxamine G1 was also identified, which is referred to as G1t, and, on feeding of diaminobutane, tetrahydroxamates T7 and T8, which are derived from T1 by substitution of two and three diaminopentane with diaminobutane residues. The structures of five other hydroxamates, with molecular masses of 530 (X8), 544 (X9), 622 (T4), 719 (T5) and 733 (T6), are not yet known. The results prove that the biosynthetic capabilities of E. amylovora for proferrioxamine production are flexible. E. amylovora may therefore be a good organism for the fermentative production of novel proferrioxamines that may be useful for reversing or avoiding aluminum and iron intoxications in man, for tumor diagnosis and therapy with radiolabeled antibodies and for use as DNA‐cleaving reagents. On the other hand, the production of all the different proferrioxamines involves common biosynthetic steps, some of which are not used in higher plants or vertebrates. Interference with the biosynthesis of proferrioxamines may therefore provide an opportunity for the development of alternative fire blight control agents.

Levan and levansucrase synthesized by the fireblight pathogen Erwinia amylovora

The fireblight pathogen Erwinia amylovora synthesizes two different types of extracellular polysaccharide (EPS) when cultured in vitro, an acidic and a neutral compound. Neutral EPS was prepared from culture supernatants by cross-flow ultrafiltration and molecular washing, and purified by column chromatography. This material represented most of the crude EPS, which was produced in high-sucrose medium. It was entirely composed of fructose. The signals obtained from the EPS-preparation by 13C-NMR spectroscopy were identical with those of levan from Aerobacter levanicum indicating a β-2,6 linkage of the sugar residues. The neutral component of the EPS was thus identified as levan (β-2,6- d-fructofuranan). In the protein fraction associated with crude EPS, the levan polymerizing enzyme levansucrase (β-2,6- d-fructan: d-glucose 6-fructosyltransferase, EC 2.4.1.10) was detected. The enzyme was also produced at similar levels with several carbon sources by the bacterium. In polyacrylamide gel eletrophoresis its molecular weight under completely denaturating conditions was 46 kDa, when purified to apparent homogeneity by membrane-exchange chromatography. However, in isoelectric focussing, the enzyme preparation produced two bands at pI 4·0 and pI 3·6, respectively. Both bands displayed enzyme activity in an in situ levansucrase assay. Secretion of levansucrase may play an important role for multiplication of E. amylovora in sucrose containing plant tissue.

The 29 kb Plasmid, Common in Strains of Erwinia amylovora, Modulates Development of Fireblight Symptoms

To cure the fireblight pathogen Erwinia amylovora of a 29 kb plasmid (pEA29) that is common in strains of these bacteria, restriction fragments of this plasmid were inserted into plasmids based on replication functions of bacteriophage fd, which cannot replicate in bacteria without expression of viral gene 2-protein. A 4·4 kb PstI fragment including the unique BamHI site of pEA29 allowed pfd plasmids to be propagated in E. amylovora. Furthermore, selection for these plasmids removed the natural 29 kb plasmid, apparently because of plasmid incompatibility. The pfd plasmids with the 4·4 kb insertion had a high tendency to segregate in E. amylovora when grown without selective pressure. Plasmid-free E. amylovora strains were virulent in standard tests on slices of immature pears, but symptom development was delayed compared to the wild-type. When assayed on pear seedlings a deficiency in pathogenicity was observed. Furthermore, strains without pEA29 spread more slowly on a lawn of pear cells than did wild-type strains. Plasmid-free strains of E. amylovora were not auxotrophic, but synthesis of extracellular polysaccharide was altered under certain growth conditions.