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 nonribosomal peptide synthetase-independent siderophore synthetase (NIS) family, and FslC, a pyridoxal-phosphate-dependent decarboxylase. To discern the steps in the biosynthetic pathway, we tested F. tularensis strain LVS and its ΔfslA and ΔfslC 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-14C] ornithine, but not from L-[1-14C] ornithine, was efficiently incorporated into rhizoferrin by LVS. Although neither the ΔfslA nor the ΔfslC mutant produced rhizoferrin, a putative siderophore intermediate labeled by both [U-14C] ornithine and [1-14C] ornithine was secreted by the ΔfslC 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 ΔfslC 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.

The reduced genome of the Francisella tularensis live vaccine strain (LVS) encodes two iron acquisition systems essential for optimal growth and virulence.

Bacterial pathogens require multiple iron-specific acquisition systems for survival within the iron-limiting environment of the host. Francisella tularensis is a virulent intracellular pathogen that can replicate in multiple cell-types. To study the interrelationship of iron acquisition capability and virulence potential of this organism, we generated single and double deletion mutants within the ferrous iron (feo) and ferric-siderophore (fsl) uptake systems of the live vaccine strain (LVS). The Feo system was disrupted by a partial deletion of the feoB gene (ΔfeoB'), which led to a growth defect on iron-limited modified Muller Hinton agar plates. 55Fe uptake assays verified that the ΔfeoB' mutant had lost the capacity for ferrous iron uptake but was still competent for 55Fe-siderophore-mediated ferric iron acquisition. Neither the ΔfeoB' nor the siderophore-deficient ΔfslA mutant was defective for replication within J774A.1 murine macrophage-like cells, thus demonstrating the ability of LVS to survive using either ferrous or ferric sources of intracellular iron. A LVS ΔfslA ΔfeoB' mutant defective for both ferrous iron uptake and siderophore production was isolated in the presence of exogenous F. tularensis siderophore. In contrast to the single deletion mutants, the ΔfslA ΔfeoB' mutant was unable to replicate within J774A.1 cells and was attenuated in virulence following intraperitoneal infection of C57BL/6 mice. These studies demonstrate that the siderophore and feoB-mediated ferrous uptake systems are the only significant iron acquisition systems in LVS and that they operate independently. While one system can compensate for loss of the other, both are required for optimal growth and virulence.