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.

Crochelins: Siderophores with an Unprecedented Iron‐Chelating Moiety from the Nitrogen‐Fixing Bacterium Azotobacter chroococcum

“…Ab ioinformatic search in the recently sequenced genome of A. chroococcum [5] reveals three siderophore gene clusters,p ossibly leading to the production of vibrioferrin (Figure 1a), amphibactin (Figure 1b), and an unknown metallophore ( Figure 1c). Tw oo ft hese were in the vibrioferrin [10] and amphibactin [11] class of siderophores,i n line with our bioinformatic analyses ( Figure 1; Supporting Information, Table S1). Identifiable siderophoreb iosynthetic gene clusters in Azotobacter chroococcum.…”

“…[6][7][8] Despite its importance and widespread use in agriculture,h owever,i ts secondary metabolome is largely unknown. [9,10] In this method, the bacterium is cultured in iron-limited and iron-replete media, and the resulting supernatants are subjected to solid-phase extraction, highresolution (HR)-HPLC-MS,a nd HR-MS/MS.T he data are then computationally assessed, using the mass isotopes of iron, to provide ap icture of all the siderophores that the organism can biosynthesize. Ab ioinformatic search in the recently sequenced genome of A. chroococcum [5] reveals three siderophore gene clusters,p ossibly leading to the production of vibrioferrin (Figure 1a), amphibactin (Figure 1b), and an unknown metallophore ( Figure 1c).…”

“…Structures of siderophores have not yet been reported from A. chroococcum,a nd how this bacterium gains access to iron, which it requires to generate high concentrations of nitrogenases, remains to be determined. [9,10] Ther esults of this workflow applied to A. chroococcum allowed us to identify three distinct groups of iron-binding metabolites (Figure 2; Supporting Information, Figure S1). To gain insights into the products of these gene clusters,weemployed our recently reported workflow for uncovering the chelome of A. chroococcum,t hat is,t he secondary metabolites that it Figure 1.…”

Structural Characterization of Natural Nickel and Copper Binding Ligands along the US GEOTRACES Eastern Pacific Zonal Transect

“…However, LC-ICPMS does not provide information on the chemical identity of ligands. Chromatographic separation combined with electrospray ionization mass spectral characterization (ESIMS) provides molecular weights for metal-containing compounds based on peak coherence between chromatographic features that match the isotopic fine structure of metals such as Fe, Cu and Ni (Mounicou et al, 2009;Baars et al, 2014Baars et al, , 2015Boiteau and Repeta, 2015). Finally, high mass accuracy Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS) can achieve resolving power and mass accuracy sufficient to assign elemental formulas to metal containing compounds (McKenna et al, 2014;Waska et al, 2015).…”

Mass spectrometry and associated technologies delineate the advantageously biomedical capacity of siderophores in different pathogenic contexts

“…). For example, crochelins, a new structural class of siderophores, was found in Azotobacter chrococcum culture supernatants with an LC‐MS/MS method (Baars et al ., ; ) (Fig. ).…”