Genetic, structural, and functional diversity of low and high-affinity siderophores in strains of nitrogen fixing Azotobacter chroococcum.

To increase iron (Fe) bioavailability in surface soils, microbes secrete siderophores, chelators with widely varying Fe affinities. Strains of the soil bacterium Azotobacter chroococcum (AC), plant-growth promoting rhizobacteria used as agricultural inoculants, require high Fe concentrations for aerobic respiration and nitrogen fixation. Recently, A. chroococcum str. NCIMB 8003 was shown to synthesize three siderophore classes: (1) vibrioferrin, a low-affinity α-hydroxy carboxylate (pFe = 18.4), (2) amphibactins, high-affinity tris-hydroxamates, and (3) crochelin A, a high-affinity siderophore with mixed Fe-chelating groups (pFe = 23.9). The relevance and specific functions of these siderophores in AC strains remain unclear. We analyzed the genome and siderophores of a second AC strain, A. chroococcum str. B3, and found that it also produces vibrioferrin and amphibactins, but not crochelin A. Genome comparisons indicate that vibrioferrin production is a vertically inherited, conserved strategy for Fe uptake in A. chroococcum and other species of Azotobacter. Amphibactin and crochelin biosynthesis reflects a more complex evolutionary history, shaped by vertical gene transfer, gene gain and loss through recombination at a genomic hotspot. We found conserved patterns of low vs. high-affinity siderophore production across strains: the low-affinity vibrioferrin was produced by mildly Fe limited cultures. As cells became more severely Fe starved, vibrioferrin production decreased in favor of high-affinity amphibactins (str. B3, NCIMB 8003) and crochelin A (str. NCIMB 8003). Our results show the evolution of low and high-affinity siderophore families and conserved patterns for their production in response to Fe bioavailability in a common soil diazotroph.