Characterization of genes involved in phaseolotoxin production and its thermal regulation

Abstract

Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight of bean, produces phaseolotoxin, a nonspecific chlorosis-inducing toxin. The wild-type strain of P.s. pv. phaseolicola G50-1 produces phaseolotoxin at 18°C but not at 28°C. When a derivative of a previously reported genomic clone isolated from G50-1 which complements only EMS and UV (but not Tn5) mutants is mobilized into these mutants, as well as in the wild type strain, the transconjugants produce toxin at both 18°C and 28°C. Further characterization of the insert in this clone showed that a 485 bp fragment containing motifs characteristic of DNA binding sites, when present in multiple copies in the transconjugant, relieves repression of phaseolotoxin production at 28°C. Results of gel retardation assays showed that a protein(s) present in extracts of cells grown at 28°C binds specifically to the 485 bp fragment and to a 260 bp subfragment that contains these DNA binding motifs. We suggest that thermoregulation of phaseolotoxin production at 28°C by the wild type and perhaps by the Tox- mutant at both temperatures, involves a regulatory protein(s) that represses transcription of one or more phaseolotoxin structural genes, and that the DNA binding site(s) in the fortuitously cloned fragment derepresses toxin synthesis by titrating this protein. We previously showed that a genomic cosmid clone, pHK120, containing a 24 kb fragment of DNA from the wild-type strain of the pathogen restores toxin production to all EMS, UV and Tn5 Tox- mutants. Tn5 mutagenesis of pHK120, marker exchange of pHK120::Tn5 in the wild-type strain and pair-complementation analysis revealed that a minimum of 8 genes (A through H) are clustered in the insert of pHK 120.