IDENTIFICATION OF AN ERWINIA AMYLOVORA GENE ENCODING CATALASE

Catalases, enzymes that degrade hydrogen peroxide (H₂O₂), function as virulence factors for some bacteria pathogenic to animals.
These enzymes are important for bacterial survival during the host immune response, which includes generation of H₂O₂ by immune cells.
Thus, catalase-deficient strains of such bacteria are greatly reduced in their virulence and require larger doses of inoculum to elicit disease than those able to degrade H₂O₂ (e.g.
Franzon et al., 1990). In fact, plant cells have been reported to produce H₂O₂ at a level comparable to that produced by activated animal immune cells (Legendre et al., 1993).

Because H₂O₂ is a component of the plant defense response, the ability to detoxify H₂O₂ may facilitate the establishment of plant pathogenic bacteria in host plants.
This could occur by avoiding the generation of other active oxygen molecules (e.g. hydroxyl radical) during the plant defense response, and by interfering with the potential functions of H₂O₂ in cell-wall strengthening and signal transduction (Tenhaken et al., 1995).

We recently have cloned a gene encoding catalase, katE, from Erwinia amylovora to test the hypothesis that the ability to degrade H₂O₂ is important to the pathogenic capability of the fire blight pathogen.
The cloned gene will be disrupted and the disrupted allele will be introduced to E. amylovora Ea321 by marker-exchange.
The pathogenicity of these near isogenic strains (E. amylovora Ea321 and its catalase-deficient derivative) then will be compared by inoculating fire blight hosts.

Members of a previously constructed strain Ea321 genomic library, cloned into pCPP9, a vector carrying a spectinomycin resistance marker (Bauer, 1990), were screened for catalase activity by functional complementation of the Escherichia coli catalase-deficient strain UM255 (Mulvey et al., 1988), a catalase-deficient derivative of E. coli CSH57a.
After introducing the genetic library into UM255 by triparental mating, transconjugants were transferred to Luria agar plates containing 50 μg/ml. spectinomycin.
Bacteria were grown overnight at 37°C; plates then were flooded with 3% H₂O₂ solution, and colonies were screened for bubbles.
Colonies exhibiting bubbles (O₂ gas), an indication of catalase activity, were selected, single-colony purified, and rescreened.
Six independent cosmid clones, of approximately 4,000 transconjugant colonies, bestowed bubbling to E. coli UM255 in the presence of H₂O₂. EcoRI restriction digests demonstrated that all six clones shared an approximately 7 kb DNA restriction fragment.
Two cosmids, designated pCPP1177 and pCPP1201, were selected for further study.

pCPP1177 was subjected to random transposon mutagenesis with Tn 10minikan (Way et al., 1984). Insertions that abolished the ability of pCPP1177 to bestow the bubbling phenotype to E. coli UM2, a catalase-deficient derivative of E. coli CSH7 (Loewen et al., 1985), were identified by screening as above for the loss of bubbling.

Two mutagenized derivatives of pCPP1177 that do not confer catalase activity to E. coli UM2 were identified.
The corresponding katE alleles in each cosmid were designated katEC75 and kat EC87. Efforts to introduce these disrupted alleles into the

VII International Workshop on Fire Blight

R.J. Laby, S.V. Beer

https://doi.org/10.17660/ActaHortic.1996.411.46