Nucleic Acids Research, 1992, Vol. 20, No. 9 2265-2270
© 1992
MOLECULAR BIOLOGY |
Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik Jahnstraße 29, D-6900 Heidelberg, Germany
*To whom correspondence should be addressed at Max-Planck-Institut für medizinische Forschung, Schriesheimer Straße 101, D-6802 Ladenburg, Germany
Received February 4, 1992. Revised March 31, 1992. Accepted March 31, 1992.
The suicide plasmid pfdA3l-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coil strains by electroporation. This vector carries the bacteriophage fd replication origin, a ß-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 pfdA3l-Tn5 yielded more than 104 transposition events per µg DNA. We have produced and characterized transposon mutants of E.amyiovora 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.