Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR ANALYSIS OF VIRULENCE DETERMINANTS OF SELECT BACTERIA IN FISH DISEASES

Location: Aquatic Animal Health Research

Title: Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids

Authors
item Russo, Riccardo
item Panangala, Victor -
item Wood, Raphael -
item Klesius, Phillip

Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 2, 2009
Publication Date: September 2, 2009
Repository URL: http://hdl.handle.net/10113/36104
Citation: Russo, R., Panangala, V.S., Wood, R.R., Klesius, P.H. 2009. Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids. FEMS Microbiology Letters. 298:105-110.

Interpretive Summary: Genetic manipulation of Edwardsiella ictaluri is imperative for study of host-pathogen interactions at the molecular level. Previous attempts to transform E. ictaluri by uptake of naked DNA have been impeded, perhaps due to suboptimal conditions. In this study we report the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures: [conventional chemical chloride (CaCl2) and one-step (polyethylene glycol, dimethyl sulfoxide and magnesium sulfate) protocols]. We used three different plasmids with different permutations and the highest transformation efficiency was achieved by electroporation compared to the other two methods. Results of this study clearly show that transformation of E. ictaluri by electroporation can be routinely used for molecular genetic manipulation of this organism and it is a quicker and easier method than transformation performed by conjugation.

Technical Abstract: Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA has apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl2)and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO4) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5 ± 0.2 X 10,000 transformants ng -1 plasmid DNA) than with the CaCl2 (8.1 ± 6.1 X 10-1 transformants ng-1 plasmid) and the 'one-step transformation' protocol (2.5 ± 2.7 transformants ng-1 plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl2 one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.

Last Modified: 7/24/2014
Footer Content Back to Top of Page