The cellular location and effect on nisin immunity of the NisI protein from Lactococcus lactis N8 expressed in Escherichia coli and L. lactis

Abstract Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cm^t-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 × 10³ transformants per μg DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 × 10² transformants per μg pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens.