Isolation and analysis of genes involved in siderophore biosynthesis in plant-growth-stimulating Pseudomonas putida WCS358

The plant-growth-stimulating Pseudomonas putida WCS358 was mutagenized with transposon Tn5. The resulting mutant colony bank was screened for mutants defective in the biosynthesis of the fluorescent siderophore. A total of 28 mutants, divided into six different classes, were isolated that were nonfluorescent or defective in iron acquisition or both. These different types of mutants together with the probable overall structure of the siderophore, i.e., a small peptide chain attached to a fluorescing group, suggest a biosynthetic pathway in which the synthesis of the fluorescing group is preceded by the synthesis of the peptide part. A gene colony bank of P. putida WCS358 was constructed with the broad-host-range cosmid vector pLAFR1. This genomic library, established in Escherichia coli, was mobilized into the 28 individual mutants, screening for transconjugants restored in fluorescence or growth under iron-limiting conditions or both. A total of 13 cosmids were found to complement 13 distinct mutants. The complementation analysis revealed that at least five gene clusters, with a minimum of seven genes, are needed for siderophore biosynthesis. Some of these genes seem to be arranged in an operon-like structure.