Peter Bellemann

Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization

Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.

Visualization of capsule formation by Erwinia amylovora and assays to determine amylovoran synthesis

Exopolysaccharide (EPS) synthesis by Erwinia amylovora depends on environmental and genetic predispositions. To measure the amount of the acidic EPS amylovoran synthesized by E. amylovora cell cultures, a turbidity assay using cetylpyridinium salt was developed. The EPS produced by bacteria grown on solid media was additionally characterized by its water content. The amylovoran capsules were visualized in situ by staining with fluorescein isothiocyanate (FITC)-labelled lectin from Abrus precatorius, which reacts with the galactose residue of the EPS side chain. The staining and the turbidity assays were applied to suspension cell cultures or to cells from colonies and did not require any purification steps. Lectin staining was superior to electron microscopic (EM) techniques for visualization of capsules. For EM, the capsule was stabilized with polycationic ferritin. In contrast to lectin staining, only a small fraction of the cells was found to be EPS-coated in the EM assay. An increase in capsulation and in amylovoran production was found in conjunction with mutations in a ribosomal protein conferring resistance to streptomycin. Furthermore, the presence of sorbitol in the growth environment resulted in high synthesis of amylovoran. Cells in the stationary growth phase continued to produce amylovoran. Apparently, the strong dependence of the fireblight pathogen on capsules requires the capacity for EPS synthesis in all growth stages in order to escape plant defence reactions.

Identification of Erwinia amylovora by growth morphology on agar containing copper sulfate and by capsule staining with lectin

Erwinia amylovora strains formed yellow colonies on minimal agar medium MM2 containing asparagine and copper sulfate (MM2Cu), in contrast to a white morphology on minimal agar without copper salt. Additionally, the colonies were mucoid to various extents. The yellow color was characteristic for the fire blight pathogen, including strains from raspberry and from other unusual host plants, and was used to establish a novel plating technique for identification of E. amylovora. The new identification method was especially superior to semi-selective media with sucrose when natural levan-deficient strains were assayed. No growth of E. amylovora was observed for the similar medium MM1 containing 2 mM CuSO4, due to its low content of as paragine. Identification by colony morphology on MM2 agar with copper was confirmed by staining the bacterial capsules with FITC-labeled lectin from Abrus precatorious, a compound which has a high affinity for galactose residues, the main sugar in the capsular exopolysaccharide amylovoran of E. amylovora. Other plant-associated bacteria usually did not produce the typical colony morphology of E. amylovora on MM2 agar with copper. Furthermore, those cells were not stained with the Abrus lectin. Capsule staining was also observed for weakly mucoid strains of E. amylovora, but not for strains with mutations affecting amylovoran synthesis. The secretion of fluorescent compounds by Pseudomonas syringae pathovars and even growth of any other bacterial colonies adjacent to E. amylovora could interfere with the formation of typical yellow colonies on MM2Cu, which could be white in case of dense plating. After screening for white colonies on LB agar, E. amylovora was identified in extracts from Cotoneaster leaves and in bark from apple trees with fire blight symptoms by its yellow growth pattern on MM2Cu agar and by capsule staining. The proposed selective medium gives a clear signal, is easy to prepare, does not contain dyes or any compounds toxic to humans, and can also detect E. amylovora strains deficient in levan synthesis.

Genetic transfer of amylovoran and stewartan synthesis between Erwinia amylovora and Erwinia stewartii.

DNA fragments with ams genes of Erwinia amylovora and cps genes of Erwinia stewartii were transferred to exopolysaccharide (EPS)-deficient mutants of the other species. The resulting EPSs were characterized by sensitivity to EPS-dependent bacteriophages, staining with amylovoran-specific fluorescein-isothiocyanate-labelled lectin and chemical techniques, such as determination of the sugar composition and methylation analysis in order to distinguish between amylovoran and stewartan. Degradation by the stewartan-dependent phage phi-K9 was used to detect stewartan production, and staining with a lectin from Abrus precatorius detected amylovoran capsules. The patterns of sugar linkages were determined by methylation analysis. Stewartan contained a significantly higher glucose to galactose ratio than amylovoran and produced a characteristic signal from 6-linked glucose residues. By these criteria, most E. stewartii cps mutants displayed exclusively amylovoran synthesis when complemented with the E. stewartii cps genes produced stewartan. The complementation to an EPS-positive phenotype may require most genes of the ams or the cps operon. An exception was an E. stewartii cpsK mutant that made predominantly stewartan when complemented with the ams cosmid. IR spectra showed that both amylovoran and stewartan were acylated when synthetized in E. amylovora, but not in E. stewartii. The amylovoran-producing E. stewartii merodiploids regained virulence to corn seedlings when mucoidy was restored by the ams cluster, but the stewartan-producing E. amylovora ams-/cps+ strains were weakly virulent on pear slices and avirulent on apple seedlings.

Mutual adaptation of bacteriophage fd, pfd plasmids and their host strains.

The synthetic plasmid pfdC1 with the replication origin of phage fd and fd gene 2 grows autonomously in E. coli cells. DNA sequencing revealed several mutations compared to the fd genome causing reduced expression of viral gene 2 protein, which can be toxic for the host cell. Another adaptation was noticed for E. coli strains with a copy of fd gene 2 on the F-episome and a pfdA-plasmid with a minimal fd replication origin, when maintained at 42 degrees C. The carrier cells adjusted their cellular metabolism to these stress conditions, whereas replication functions of the plasmid or expression of fd gene 2 on the F-episome were not changed. The filamentous bacteriophages tend to reduce their genome size into miniphages, which was also observed for phages with an antibiotic resistance gene. Bacteriophages with a transposon insertion in the viral gene 2 had a tendency to restore the mutated gene by exchange with the functional gene 2 carried in recA-host cells. Mobilization of pfd-plasmids with RP4 transfer functions was reduced due to interference of replication and transfer in the rolling circle mode. The vectors used in these studies can also be applied as cloning vectors, which are compatible with many other plasmid vectors.

Exopolysaccharides in the Interaction of the Fire-Blight Pathogen Erwinia Amylovora with its Host Cells

The fireblight pathogen Erwinia amylovora depends on synthesis of exopolysaccharides in order to prevent defense reactions of the host plants. Mutants in genes for capsular polysaccharides, for levan surcrase and in the galactose metabolism were created by mutagenesis with transposon Tn5. A deficiency for levan sucrase did not affect virulence on pears. Mutants in synthesis of acidic exopolysaccharides (EPS) were apathogenic. The gal-mutant induced phytoalexins and callose production in plant cells. Both defense reactions were suppressed by dihydrophenylalanine, a phenylalanine analog secreted by some E. amylovora strains. This compound is synthesized in the shikimic acid pathway of the bacteria and is inhibitory in plants for synthesis of aromatic amino acids. The other mutants without EPS-synthesis were genetically and physically characterized. The insertions were mapped in a gene cluster, which is controlled by regulatory genes like the cloned and sequenced rcsA-gene.