The Siderophore Metabolome of Azotobacter vinelandii

Abstract

In this study, we performed a detailed characterization of the siderophore metabolome, or "chelome," of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.

FIG 1

Schematic of the LC-MS analysis workflow in this study. For untargeted siderophore profiling (“chelomics”), sample preparation and measurement on a high-resolution LC-MS system were followed by mining of the LC-MS data for characteristic Fe isotope patterns associated with Fe complexes and the presence of associated Fe-free ligand species using ChelomEx software (18). MS/MS molecular networks of identified putative free siderophores were created to group structurally related species and assign mass differences between related species to sum formula differences. Identification of new siderophores was assisted by comparison of molecule and fragment masses to a database of known siderophore structures. Finally, manual reconstruction of MS/MS spectra allowed the assignment of chemical structures to several new siderophore structures. In some cases, the structure assignment was informed by additional NMR spectra of isolated compounds. Quantification of identified siderophores was performed on a single quadrupole LC-MS by direct injection of filtered spent media without prior solid-phase extraction (SPE).

FIG 2

Nonribosomal peptide synthetase (NRPS) and NRPS-independent gene clusters in A. vinelandii strain CA (GenBank accession number CP005094.1). Genes necessary for production of specific siderophores are indicated by superscripts: superscript 1 indicates catechols (24); superscript 2 indicates catechols (25); superscript 3 indicates azotobactin (25). Annotated functions of genes as well as a more complete list of azotobactin-related biosynthetic genes can be found in Table S1 in the supplemental material.

FIG 3

Base peak chromatogram (BPC) for the high-resolution LC-MS analysis of the A. vinelandii spent medium (black). Overlying the BPC are extracted ion chromatograms of the known siderophores from A. vinelandii (green). Siderophores that have not been reported before from A. vinelandii include the hydrophilic vibrioferrin (red) and a large number of new siderophores (orange), which were found to be related to the known siderophores produced by the bacterium.

FIG 4

(A) MS/MS molecular network of siderophores produced by A. vinelandii. Each node represents a separate siderophore (adducts, dimers, etc., have been removed) that was required to be present in biological and analytical replicates. The thickness of the edge represents the degree of relatedness between the MS/MS spectra of two species. The known siderophores from A. vinelandii are indicated as black circles, and vibrioferrin is indicated as a yellow circle. Three separate clusters can be recognized and include vibrioferrin, catechol siderophores, and azotobactins. The software Cytoscape was used for visualization. (B, C, D) Nodes in the three clusters shown in more detail. For these networks, nodes were manually arranged, and only selected edges are shown for clarity of presentation. The ring color around the nodes represents the peak area for each species, and the number represents the corresponding m/z value (rounded to one digit). The exact mass difference between two nodes was assigned to chemical sum formulas as indicated. Structures of new siderophores are based on reconstruction of MS/MS spectra and additional UV-vis and NMR spectroscopic data (see the text). The table in panel D shows the MS/MS fragmentation of azotobactin-related compounds. Arrows indicate MS/MS fragments corresponding to the B (arrows to the left) and Y (arrows to the right) fragment ions; λ_max is the absorption maximum of each compound in the UV-vis spectrum. Note that all siderophore species in the azotobactin cluster were doubly charged. ChrA, azotobactin chromophore; Febn, ferribactin; OHFebn, hydroxyl-ferribactin; ChrP, pyoverdine chromophore; 2HChrP, dihydropyoverdine; Ser, serine; Glu, glutamate; Hse, homoserine; Gly, glycine; OHAsp, hydroxyl aspartate; Cit, citrulline; AcOHOrn, acylhydroxyornithine; Hsl, homoserine lactone; MeHse, methylhomoserine; nd, not determined.

FIG 5

Concentration of notable siderophores from A. vinelandii under diazotrophic growth with different sources and availability of Fe. Growth was monitored by optical density at 620 nm (OD₆₂₀). * and ** indicate new A. vinelandii siderophores identified in this study. Double asterisks represent azotobactin derivatives based on MS/MS fragmentation patterns as shown in Fig. 3. Relative standard deviations were <3.5% for the vibrioferrins and the major catechol siderophores based on replicate analyses of a representative spent medium “standard.” The remaining siderophores were measured with slightly larger standard deviations (<10% for siderophore concentrations above 0.5 μM and <20% for lower concentrations).

FIG 6

Occurrence of vibrioferrin biosynthetic genes (pvs) in bacteria from diverse phyla, environments, and lifestyles. Information on gene loci is displayed below each arrow. GenBank accession numbers for genomes shown (in order) are BA000032.2, CP006265.1, CP005094.1, CP010415.1, CP003057.1, CP000744.1, CP006664.1, CP000316.1, AY305378.1, CP001114.1, and BA000030.3. *, aldolase has been shown to be citrate synthase in Staphylococcus production of staphyloferrin B (60).

FIG 7

Proposed biosynthesis for A. vinelandii siderophores. (A) Mining of the A. vinelandii strain CA genome data suggests that two NRPS clusters are involved in catechol siderophore production. (B) Proposed biosynthesis pathway for monocatechol siderophores. (C) Proposed biosynthesis for bis- and tris-catechol siderophores. Bold text indicates previously characterized catechols. Dashed lines indicate pathways for derivatives identified in this study (Fig. 4).