Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation ofErwinia amylovoraandEscherichia colicells

Abstract

The suicide plasmid pfdA31-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coli strains by electorporation. This vector carries the bacteriophage fd replication origin, a beta-lactamase gene and the transposon Tn5. For propagation the plasmid depends on host cells producing fd gene-2 protein. Electroporation of E.amylovora or E.coli cells with plasmid pfdA31-Tn5 yielded more than 10(4) transposition events per micrograms DNA. We have produced and characterized transposon mutants of E.amylovora affecting either galactose metabolism or the synthesis of the phytotoxin (L)-2,5-dihydrophenylalanine. A Tn5-insertion in a gene, involved in exopolysaccharide synthesis of E.amylovora strain Ea7/74, was subcloned into vector pfdA31 and used to mutagenize E.amylovora strain Ea1/79 by site-directed recombination.

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

“…These methods circumvented complex vector systems like plasmids with phage Mu (Steinberger & Beer, 1988 ;Barny et al, 1990) or phage A as Tn5 donor, which was also barely efficient in our hands for Tn5 mutagenesis in E. amylovora strains containing a plasmid with the lamB gene. Most recently, electroporation with small Tn5-carrying suicide plasmids has provided another approach for efficient transposon mutagenesis of E, amylovora (Metzger et al, 1992). Many of the Tn5 mutants obtained were initially variable on successive screenings for the production of virulence symptoms and could revert to pathogenic strains after passaging them on plates.…”

Genetics of galactose metabolism of Erwinia amylovora and its influence on polysaccharide synthesis and virulence of the fire blight pathogen

“…DNA was transferred into E. amylovora by mating or by electroporation. For Tn5 mutagenesis of E. amylovora, the suicide plasmid pfdA31-Tn5 was introduced into strain Eal/79Sm by electroporation (34). For replication, this plasmid relies on the phage fd gene 2 protein, which is absent in normal cells.…”

Expression of firefly luciferase gene in Erwinia amylovora

“…Purified plasmid pCAL-luc1 was transferred into E. amylovora by electroporation using Gene Pulser II (BIO-RAD), as described by Metzger et al (22). In brief, bacteria were grown in LB broth at 28°C to a density of OD 590 = 0.5-0.8.…”

“…We transformed E. amylovora with the purified pCALluc1 by electroporation (22) and screened recombinant clones. The transformed bacterial cells were then cultured in different concentrations of IPTG (0.1, 1, 2, 5 mmol/L) and at different times (1, 2, 3 h, overnight) to evaluate the optimal luciferase expression.…”