Citryl Ornithine Is an Intermediate in a Three-Step Biosynthetic Pathway for Rhizoferrin in Francisella .

The Gram-negative bacterium Francisella tularensis secretes the siderophore rhizoferrin to scavenge necessary iron from the environment. Rhizoferrin, also produced by a variety of fungi and bacteria, comprises two citrate molecules linked by amide bonds to a central putrescine (diaminobutane) moiety. Genetic analysis has determined that rhizoferrin production in F. tularensis requires two enzymes: FslA, a siderophore synthetase of the non-ribosomal peptide synthetase-independent siderophore synthetase (NIS) family and a pyridoxal-phosphate dependent decarboxylase, FslC. To discern the steps in the biosynthetic pathway, we tested cultures of F. tularensis strain LVS and its Δ fslA and Δ fslC mutants for the ability to incorporate potential precursors into rhizoferrin. Unlike putrescine supplementation, supplementation with ornithine greatly enhanced siderophore production by LVS. Radioactivity from [L-U-C] ornithine, but not from [1-C] ornithine, was efficiently incorporated into rhizoferrin by LVS. Although neither the Δ fslA nor the Δ fslC mutant produced rhizoferrin, a putative siderophore intermediate labeled by both [U-C] ornithine and [1-C] ornithine was secreted by the Δ fslC mutant. Rhizoferrin was identified by liquid chromatography and mass spectrometry in LVS culture supernatants, while citryl-ornithine was detected as the siderophore intermediate in the culture supernatant of the Δ fslC mutant. Our findings support a three-step pathway for rhizoferrin production in Francisella; unlike the fungus Rhizopus delemar, where putrescine functions as a primary precursor for rhizoferrin, biosynthesis in Francisella preferentially starts with ornithine as the substrate for FslA-mediated condensation with citrate. Decarboxylation of this citryl ornithine intermediate by FslC is necessary for a second condensation reaction with citrate to produce rhizoferrin.