Generation and Characterization of Tn 5 Insertion Mutations in Pseudomonas syringae pv. tomato

Abstract

Tn 5 -induced insertion mutations were generated in the Pseudomonas syringae pv. tomato genome by mating this plant pathogen with an Escherichia coli strain carrying the suicide plasmid vector for Tn 5 , pGS9. Km r transconjugants occurred at frequencies ranging from 2 × 10 −7 to 9 × 10 −6 ; approximately 5.5% of these transconjugants were also Cm r , indicating the presence of additional pGS9 DNA sequences. Approximately 1% of the Km r Cm s mutants were auxotrophic. Southern blot analysis revealed that the Tn 5 element had inserted into one unique site on the chromosome for each Km r Cm s transconjugant examined. Physical and genetic tests of Tn 5 -induced auxotrophs showed that Tn 5 mutations in P. syringae pv. tomato were very stable and that secondary transposition of Tn 5 or its insertion sequence IS 50 was a rare event. Nine of 920 Km r Cm s transconjugants screened on tomato seedlings either were avirulent or produced very mild symptoms. Each of the virulence mutants was the result of a unique single-site Tn 5 insertion. Five mutants also failed to induce a hypersensitivity reaction on tobacco.