An Optimized Method for Efficient Conjugal Transfer in Flavobacterium covae.

The fish pathogen Flavobacterium covae causes significant mortality and there is a need to better understand virulence factors that contribute to its pathogenesis. Few highly virulent F. covae strains may be genetically manipulated with existing conjugal transfer methods, therefore the aim of this study was to increase conjugal transfer efficiency and broaden the range of recipient F. covae strains. Different parameters were evaluated for improvement of F. covae conjugal transfer efficiency using plasmid pCP23, and improved conjugal transfer was observed by altering the Escherichia coli donor strain, media and donor to recipient cell ratios. An optimized method including these changes was compared to a previously published conjugation method, which enabled conjugal transfer of pCP23 into seven of eleven F. covae strains in contrast to only four strains with the previous method. Additionally, the use of the optimized method resulted in orders of magnitude increases in conjugal transfer efficiency for multiple F. covae strains relative to the previous method (p < 0.0001), with ML-17-22A observed to have the greatest conjugation efficiency of 1.2 × 106 transconjugants per 109 recipient cells. This optimized conjugation method will facilitate a better understanding of the molecular mechanisms of pathogenesis in virulent F. covae strains.