CHARACTERIZATION OF A GENOMIC REGION REQUIRED FOR PRODUCTION OF THE ANTIBIOTIC PYOLUTEORIN BY THE BIOLOGICAL-CONTROL AGENT PSEUDOMONAS-FLUORESCENS PF-5

A 21-kb region required for the biosynthesis of the polyketide antibio tic pyoluteorin by the biological control agent Pseudomonas fluorescen s Pf-5 was identified and cloned. Seven previously isolated mutants de ficient in pyoluteorin production (Plt(-)) had Tn5 insertions spanning the 21-kb region, Sequences flanking Tn5 inserts were cloned from gen omic DNA of three Plt(-) mutants and used as probes to identify wild-t ype alleles of the pit loci from a genomic library of Pf-5. Five cosmi ds containing overlapping regions of genomic DNA hybridized to one or more of the probes. One cosmid, pJEL1938, contained the entire 21-kb r egion and, when introduced into a Plt(-) mutant, partially restored py oluteorin production. To study the expression of the genes required fo r pyoluteorin biosynthesis, the transposon Tn3-nice, which contains a promoterless ice nucleation gene (inaZ) and a type I neomycin phosphot ransferase gene, was introduced into the genomic pit region of Pf-5. C arbon sources that influenced pyoluteorin production by Pf-5 had paral lel effects on ice nucleation activity of Pf-5 containing a genomic pl t::Tn3-nice fusion, indicating that inaZ was transcribed from a promot er of the pit region, Cells of Pf-5 containing a genomic plt::Tn3-nice fusion expressed ice nucleation activity on cotton and cucumber seeds planted in field soil. The expression of plt genes by Pf-5 in the cuc umber spermosphere was delayed in comparison with expression in the co tton spermosphere, This study demonstrates that genes required for pyo luteorin production were expressed in situ by the biological control b acterium.