Impact of mutations in hemA and hemH genes on pyoverdine production by Pseudomonas fluorescens ATCC17400

A Pseudomonas fluorescens Tn5 mutant, with decreased production of the siderophore pyoverdine, was obtained, with the transposon inserted in the hemA gene coding for glutamyl tRNA reductase, the enzyme that catalyzes the first step of heme biosynthesis. Since this mutant was leaky. a second round of transposition was needed to obtain a second mutant completely auxotrophic for the heme precursor delta -aminolevulinate (ALA). Pyoverdine production by this mutant is ALA-dependent at concentrations above those needed to sustain growth. A transposon mutant in the hemH gene that encodes the enzyme ferrochelatase showing a characteristic red fluorescence upon UV exposure as a result of porphyrins accumulation, was obtained by selecting transconjugants on LB medium containing hemin. The Delta hemH mutant was characterized and the corresponding hemH gene sequenced. Antibodies against P. fluorescens HemH detected the protein both in soluble and membrane fractions of the wild-type and confirmed the absence of the enzyme in the mutant. The Delta hemH mutant failed to produce pyoverdine, but the production of the siderophore was restored by introduction of the Pseudomonas aeruginosa hemH gene in trans. These results indicate that de novo heme biosynthesis is needed for a normal level of siderophore pyoverdine production. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.