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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 electrophoretic transformation of Edwardsiella ictaluri using three different plasmids

Authors
item Russo, Riccardo
item Panangala, Victor
item Wood, Ryan - AUBURN UNIVESITY
item Klesius, Phillip

Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 17, 2009
Publication Date: July 6, 2009
Citation: Russo, R., Panangala, V.S., Wood, R.R., Klesius, P.H. 2009. Chemical and electrophoretic transformation of Edwardsiella ictaluri using three different plasmids. FEMS Microbiology Letters. 298:105-110.

Interpretive Summary: Edwardsiella ictaluri, the etiologic agent of enteric septicemia in channel catfish (ESC) has been recognized as an important bacterial pathogen with a potential to cause appreciable economic loss to the aquaculture industry. Molecular genetic manipulation of this bacterium is important from the perspective of determining virulence factors, invasive pathways, and mechanisms of host-pathogen interactions. In literature has been reported that E. ictaluri can be successfully transformed with foreign DNA only by conjugation, transduction and transformation. In this study, we describe for the first time successful transformation of seven strains of E. ictaluri using two different chemical procedures [conventional calcium chloride and “one-step” (polyethylene glycol, dimethyl sulfoxide, magnesium sulphate)] and electroporation. Seven strains of E. ictaluri were transformed with three different plasmids: pZsGreen, pUC18 and pET-30a(+). The plasmids were stably maintained in E. ictaluri grown in presence of antibiotic for 12 or more passages. Our studies clearly reflect that E. ictaluri was successfully transformed and that electroporation is an efficient procedure for transformation of this species. Furthermore, transformation of E. ictaluri by calcium chloride or electroporation is a quicker and easier method than transformation performed by conjugation or transduction.

Technical Abstract: Edwardsiella ictaluri the cause of enteric septicemia in channel catfish (Ictalurus punctatus) (ESC), is a bacterial pathogen having a considerable economic impact on the cultured catfish industry. Molecular genetic manipulation of this bacterium is important from the perspective of determining virulence factors, invasive pathways, and mechanisms of host-pathogen interactions. We report here the first successful transformation of seven strains of E. ictaluri using two different chemical procedures [conventional calcium chloride and “one-step” (polyethylene glycol, dimethyl sulfoxide, magnesium sulphate)] and electroporation. Seven strains of E. ictaluri were transformed using DNA from three different plasmids (pZsGreen, pUC18, pET-30a(+)). The highest transformation efficiency was achieved by electroporation (7.0 ± 0.4 x 10 X 4 transforments per ng of plasmid DNA) compared to calcium chloride-mediated transformation (2.0 ± 0.2 transforments per ng of plasmid DNA) or the “one-step” protocol (9.0 ± 0.3 transforments per ng of plasmid DNA). The plasmids were stably maintained in E. ictaluri grown in presence of antibiotic for 12 or more passages. Optimal conditions for electroporation included a field strength of 12.5 kV cm-1, a time constant of 4 msec, capacitance setting of 25 µF, using a 0.2 cm (gap) electroporation cuvette. This optimized system will enable successful transformation of E. ictaluri for molecular genetic manipulation of this organism for determination of virulence traits and other host-pathogen interaction studies.

Last Modified: 4/21/2014
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