MUTANTS OF ERWINIA AMYLOVORA ALTERED IN PATHOGENICITY BY TRANSPOSON MUTAGENESIS

Mutants with altered pathogenic capabilities are needed to investigate the molecular basis of pathogenicity of Erwinia amylovora. The technique of transposon mutagenesis was used to generate non-pathogenic insertional mutants of E. amylovora. Transposon insertion results in a polar mutation in which the continuity of the mutated gene is disrupted and the expression of downstream genes in the same operon is abolished.
A mutant phenotype is expected to be the consequence of a single transposon insertion because additional insertions are rare due to the expression of a transposition repressor encoded by the transposon itself.

The conjugative plasmid pJB4JI was used as the vector of transposon Tn5 for mutagenesis of E. amylovora. It was originally constructed as a suicide vehicle for use in Rhizobium sp., and it contains Tn5 inserted into genes of bacteriophage Mu.
Because the plasmid cannot replicate stably, loss of Tn5 occurs in subsequent generations unless transposition occurs from the plasmid into genomic DNA of the host cell.
Therefore, most transconjugants selected based on the antibiotic (kanamycin) resistance of the transposon should contain Tn5 insertions in their genomes.
Since Tn5 inserts in a random manner, some insertions are likely to be in genes involved in pathogenicity; these can be identified through pathogenicity tests.

The transposon vector pJB4JI proved suitable for mutagenesis of E. amylovora only to a limited extent.
The plasmid was stably maintained in more than 50% of the transconjugants, which necessitated screening of a large number of transconjugants.
In addition, when genomic Southern blots of DNA from mutants were probed with ³²P-Tn5, several mutants with multiple Tn5 insertions were discerned.
When identical blots were probed with ³²P-Mu, coinsertion of Mu genes with Tn5 was apparent in some mutants.
However, mainly single-site mutations are desired for genetic studies.

A mutagenesis system was developed that has a better chance for the development of single-site mutations.
A derivative of the bacteriophage lambda, b₂₂₁cI₈₅₇rex::Tn5, has similar properties as pJB4J1, without the complications caused by Mu.
It can bind to the lambda receptor of Escherichia coli and deliver Tn5 into the bacterial cell.
The lambda derivative cannot integrate into the recipient’s genome due to a mutation.
The lambda receptor protein is a product of

IV International Workshop on Fire Blight

E.M. Steinberger, S.V. Beer

https://doi.org/10.17660/ActaHortic.1987.217.25