Cloning and characterization of genes conferring copper resistance in epiphytic ice nucleation active Pseudomonas syringae strains

Many epiphytic Pseudomonas syringae strains obtained from asymptomatic host and nonhost plants are resistant to high concentrations of copper ions. A genomic DNA library of one such strain, AL513, was constructed in the broad host-range cosmid cloning vector pLAFR3. A cosmid designated pCOPR1.1 that conferred near wild-type levels of copper resistance when conjugated into the copper-sensitive (Cu-s) P. syringae strain AL487 was identified. A 6.5-kb PstI fragment in pCOPR1.1 conferred this copper resistance. Insertional inactivation mutagenesis of this cosmid with the reporter transposon Tn3-Spice indicated that a region of approximately 5.5 kb, denoted copJ, is required for copper resistance. Transcriptional activity of copJ::Tn3-Spice gene fusions inserted into the P. syringae AL513 genome by marker exchange mutagenesis were induced within 30-45 min when as little as 0.6 mu g of Cu2+ per milliliter was added to culture media. Zinc ions are toxic to this bacterial strain at concentrations of more than 10 mu g/ml in culture media; however, zinc induced copJ at concentrations less than 1 mu g/ml. Increases in the proportion of cells in the population that tolerated high doses of copper were correlated with increased transcriptional activity of copJ. In planta induction times of copJ were approximately sixfold longer than those observed in vitro.