Identification of an indigenous plasmid carrying a gene for trimethoprim resistance in Pseudomonas syringae pv. glycinea

Summary Pseudomonas syringae pv. glycinea Race 8 strain PgB3 is naturally resistant to trimethoprim (Tp) at concentrations up to 500 g/ml. A genomic library of total PgB3 DNA was constructed by ligating EcoRI-restricted DNA into the EcoRI site of the cosmid vector, pLAFR1, packaging the DNA in vitro into bacteriophage lambda, and transducing E. coli DH1 cells. Of 960 cosmid clones selected for resistance to tetracycline, six were resistant to trimethoprim at 500 g/ml. An insert into pLAFR1 of about 9.4 kb was shown to be consistently present in the tirmethoprim-resistant clones. Southern blot analysis using radioactively labeled insert DNA as probe indicated that the 9.4 kb fragment hybridized only with a 40 kb indigenous plasmid from PgB3 designated pPg2.