Evaluation and Optimization of a DNA Transfer System for Edwardsiella ictaluri

Abstract

To develop DNA transfer and mutagenesis procedures for Edwardsiella ictaluri, optimal conditions for exchange between a recipient strain of E. ictaluri and a donor strain of Escherichia coli were experimentally determined by the use of a mobilizable transposon carried on a suicide vector. In general, transfer and subsequent mutant production were optimal when the ratio of cells of E. ictaluri to donor cells was 4:1 to 10:1 and was satisfactory out to a ratio of 45:1. Initial cell concentrations also were important; the number of mutants produced approximately doubled for each log increase in initial donor and recipient cell numbers at a recipient-to-donor ratio of 31:1. The most important determinant of mutant production was temperature, primarily because of the difference in optimal growth temperatures for the donor E. coli and the recipient E. ictaluri. Optimal mutant production occurred at 24°C, a considerable reduction occurred at 28°C, and very poor to zero production was seen at 33°C. The majority of the mutants produced were in single loci, with a low frequency of plasmid integration events that could be detected by establishing resistance to ampicillin. This system can be used to efficiently produce large numbers of stable, isogenic mutants in E. ictaluri as well as other fish pathogens and can also be adapted for use in complementation studies and for gene transfer by allelic exchange.