Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells.
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.
Metzger
M
M
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.
Bellemann
P
P
Schwartz
T
T
Geider
K
K
D015139:Blotting, Southern; D004251:DNA Transposable Elements; D004558:Electric Stimulation; D004885:Erwinia / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005690:Galactose / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids
D004251:DNA Transposable Elements; D005690:Galactose