Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 May 11;20(9):2265–2270. doi: 10.1093/nar/20.9.2265

Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells.

M Metzger 1, P Bellemann 1, T Schwartz 1, K Geider 1
PMCID: PMC312340  PMID: 1317549

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.

Full text

PDF

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bellemann P., Geider K. Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization. J Gen Microbiol. 1992 May;138(5):931–940. doi: 10.1099/00221287-138-5-931. [DOI] [PubMed] [Google Scholar]
  2. Cangelosi G. A., Best E. A., Martinetti G., Nester E. W. Genetic analysis of Agrobacterium. Methods Enzymol. 1991;204:384–397. doi: 10.1016/0076-6879(91)04020-o. [DOI] [PubMed] [Google Scholar]
  3. Dolph P. J., Majerczak D. R., Coplin D. L. Characterization of a gene cluster for exopolysaccharide biosynthesis and virulence in Erwinia stewartii. J Bacteriol. 1988 Feb;170(2):865–871. doi: 10.1128/jb.170.2.865-871.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ellard F. M., Cabello A., Salmond G. P. Bacteriophage lambda-mediated transposon mutagenesis of phytopathogenic and epiphytic Erwinia species is strain dependent. Mol Gen Genet. 1989 Sep;218(3):491–498. doi: 10.1007/BF00332415. [DOI] [PubMed] [Google Scholar]
  6. Falkenstein H., Bellemann P., Walter S., Zeller W., Geider K. Identification of Erwinia amylovora, the Fireblight Pathogen, by Colony Hybridization with DNA from Plasmid pEA29. Appl Environ Microbiol. 1988 Nov;54(11):2798–2802. doi: 10.1128/aem.54.11.2798-2802.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Geider K., Baldes R. Influence of fd gene 2-protein and the viral replication origin on the compatibility of pfd-plasmids. Nucleic Acids Res. 1988 Jul 25;16(14A):6385–6396. doi: 10.1093/nar/16.14.6385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Geider K., Hohmeyer C., Haas R., Meyer T. F. A plasmid cloning system utilizing replication and packaging functions of the filamentous bacteriophage fd. Gene. 1985;33(3):341–349. doi: 10.1016/0378-1119(85)90242-2. [DOI] [PubMed] [Google Scholar]
  9. Gilchrist A., Smit J. Transformation of freshwater and marine caulobacters by electroporation. J Bacteriol. 1991 Jan;173(2):921–925. doi: 10.1128/jb.173.2.921-925.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hinton J. C., Sidebotham J. M., Hyman L. J., Pérombelon M. C., Salmond G. P. Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence. Mol Gen Genet. 1989 May;217(1):141–148. doi: 10.1007/BF00330953. [DOI] [PubMed] [Google Scholar]
  11. Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. doi: 10.1016/0076-6879(79)68021-7. [DOI] [PubMed] [Google Scholar]
  12. Sclafani R. A., Wechsler J. A., Schuster H. The isolation and characterization of escherichia coli dnaB::Tn10 insertion mutations. Mol Gen Genet. 1981;182(1):112–118. doi: 10.1007/BF00422776. [DOI] [PubMed] [Google Scholar]
  13. Simon R., Quandt J., Klipp W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene. 1989 Aug 1;80(1):161–169. doi: 10.1016/0378-1119(89)90262-x. [DOI] [PubMed] [Google Scholar]
  14. Wirth R., Friesenegger A., Fiedler S. Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation. Mol Gen Genet. 1989 Mar;216(1):175–177. doi: 10.1007/BF00332248. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES