Identification of a chromosomal region required for biosynthesis of the phytotoxin coronatine by Pseudomonas syringae pv. tomato

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Abstract
Pseudomonas syringae pv. tomato produces the chlorosis-inducing phytotoxin coronatine. Five of 3700 (0.13%) kanamycin-resistant mutants generated by random Tn5 mutagenesis were unable to synthesize this toxin. Clone pEC18, isolated from a cosmid pLAFR1 library of wild-type P. syringae pv. tomato DC3000 genomic DNA, complemented four of the mutants. Restriction enzyme analysis of pEC18 and corresponding clones from the four mutants indicated that the Tn5 insertion sites of these four mutants spanned a 19-kb region of DC3000 genomic DNA. Complementation tests with subclones of pEC18 confirmed the relative locations of the Tn5 insertions. Because pEC18 did not complement all five mutants or confer the ability to produce toxin on nonproducing P. syringae pathovars, sequences outside this cloned region must also be involved in toxin synthesis. As demonstrated by Southern blot analysis, this cloned region was not on the 68-kb indigenous plasmid of DC3000. The only P. syringae pathovars with DNA homologous to sequences within the coronatine gene cluster were the coronatine producers P. syringae pv. tomato, P. syringae pv. atropurpurea, and P. syringae pv. glycinea. Since the hybridization patterns of these toxin producers were identical, this locus is highly conserved and appears crucial to the synthesis of coronatine.Key words: coronatine, phytotoxin mutants, Pseudomonas syringae pv. tomato.