IRON REGULATION OF SIDEROPHORE BIOSYNTHESIS AND TRANSPORT IN PSEUDOMONAS-PUTIDA WCS358 - INVOLVEMENT OF A TRANSCRIPTIONAL ACTIVATOR AND OF THE FUR PROTEIN

Pseudobactin 358 is the yellow-green fluorescent siderophore produced by Pseudomonas putida WCS358 in conditions of iron limitation. The gen es encoding for siderophore biosynthesis are iron-regulated at the tra nscriptional level. Previous work has shown that a positive regulator, PfrA, is absolutely required for the activation under iron-limiting c onditions of pseudobactin 358 biosynthesis, In this study we identifie d a set of Tn5 insertion mutants of strain WCS358 which lost the abili ty to activate an iron-regulated siderophore promoter, These mutants n o longer produced pseudobactin 358, Molecular analysis revealed that t hey carried a Tn5 insertion in a gene, designated pfrl (Pseudomonas fe rric regulator), which codes for a protein (Pfrl) of 19.5 kDa. Pfrl co ntains a putative helix-turn-helix motif typical of DNA-binding protei ns and has homology to two DNA-binding transcriptional activators, Fed from Escherichia coli and Pupl from P. putida. The proposed role of P frl in strain WCS358 is an activator protein regulating pseudobactin 3 58 biosynthesis under iron limitation. The pfrl promoter region contai ns a sequence which displays high identity to the Fur-box consensus, T his 19 bp consensus sequence is recognized by Fur, an iron-binding rep ressor protein found in many different bacteria, The E. coli Fur prote in can bind to the pfrl promoter region, indicating that this activato r gene is likely to be iron-regulated by Fur. We also report the ident ification and characterization of the P. putida WCS358 fur gene. The F ur protein of strain WCS358 is structurally and functionally similar t o all other cloned Fur proteins from other bacterial species.