Siderophore Biosynthesis Research Articles

Eukaryotic Acquisition of a Bacterial Operon

Operons are a hallmark of bacterial genomes, where they allow concerted expression of functionally related genes as single polycistronic transcripts. They are rare in eukaryotes, where each gene usually drives expression of its own independent messenger RNAs. Here, we report the horizontal operon transfer of a siderophore biosynthesis pathway from relatives of Escherichia coli into a group of budding yeast taxa. We further show that the co-linearly arranged secondary metabolism genes are expressed, exhibit eukaryotic transcriptional features, and enable the sequestration and uptake of iron. After transfer, several genetic changes occurred during subsequent evolution, including the gain of new transcription start sites that were sometimes within protein-coding sequences, acquisition of polyadenylation sites, structural rearrangements, and integration of eukaryotic genes into the cluster. We conclude that the genes were likely acquired as a unit, modified for eukaryotic gene expression, and maintained by selection to adapt to the highly competitive, iron-limited environment.

Metabolome and transcriptome-wide effects of the carbon storage regulator A in enteropathogenic Escherichia coli

The carbon storage regulator A (CsrA) is a conserved global regulatory system known to control central carbon pathways, biofilm formation, motility, and pathogenicity. The aim of this study was to characterize changes in major metabolic pathways induced by CsrA in human enteropathogenic Escherichia coli (EPEC) grown under virulence factor-inducing conditions. For this purpose, the metabolomes and transcriptomes of EPEC and an isogenic ∆csrA mutant derivative were analyzed by untargeted mass spectrometry and RNA sequencing, respectively. Of the 159 metabolites identified from untargeted GC/MS and LC/MS data, 97 were significantly (fold change ≥ 1.5; corrected p-value ≤ 0.05) regulated between the knockout and the wildtype strain. A lack of csrA led to an accumulation of fructose-6-phosphate (F6P) and glycogen synthesis pathway products, whereas metabolites in lower glycolysis and the citric acid cycle were downregulated. Associated pathways from the citric acid cycle like aromatic amino acid and siderophore biosynthesis were also negatively influenced. The nucleoside salvage pathways were featured by an accumulation of nucleosides and nucleobases, and a downregulation of nucleotides. In addition, a pronounced downregulation of lyso-lipid metabolites was observed. A drastic change in the morphology in the form of vesicle-like structures of the ∆csrA knockout strain was visible by electron microscopy. Colanic acid synthesis genes were strongly (up to 50 fold) upregulated, and the abundance of colanic acid was 3 fold increased according to a colorimetric assay. The findings expand the scope of pathways affected by the csrA regulon and emphasize its importance as a global regulator.

Siderophore Detection Using Chrome Azurol S and Cross-Feeding Assays.

More than 500 siderophores that bind ferric iron have been characterized and grouped by type based on their chemical structure. The chrome azurol S (CAS) assay is a universal colorimetric method that detects siderophores independent of their structure. In this assay, siderophores scavenge iron from an Fe-CAS-hexadecyltrimethylammonium bromide complex, and subsequent release of the CAS dye results in a color change from blue to orange. Solution-based experiments with CAS result in a quantitative measure of siderophore production, while an observable color change on CAS agar plates can be performed for qualitative detection of siderophores. Cross-feeding assays are another useful method to detect and characterize siderophores produced by bacteria. Under iron-limiting conditions, cross-feeding assays test the ability of an indicator strain to grow when supplied with a specific siderophore (from a test strain) to which it has a cognate receptor required for import into the cell. The cross-feeding assay can be tested with a variety of wild-type strains, siderophore biosynthesis mutants, and siderophore receptor mutants.

Kinetic Analyses of the Siderophore Biosynthesis Inhibitor Salicyl-AMS and Analogues as MbtA Inhibitors and Antimycobacterial Agents.

There is a paramount need for expanding the drug armamentarium to counter the growing problem of drug-resistant tuberculosis. Salicyl-AMS, an inhibitor of salicylic acid adenylation enzymes, is a first-in-class antibacterial lead compound for the development of tuberculosis drugs targeting the biosynthesis of salicylic-acid-derived siderophores. In this study, we determined the Ki of salicyl-AMS for inhibition of the salicylic acid adenylation enzyme MbtA from Mycobacterium tuberculosis (MbtAtb), designed and synthesized two new salicyl-AMS analogues to probe structure-activity relationships (SAR), and characterized these two analogues alongside salicyl-AMS and six previously reported analogues in biochemical and cell-based studies. The biochemical studies included determination of kinetic parameters ( Kiapp, konapp, koff, and tR) and analysis of the mechanism of inhibition. For these studies, we optimized production and purification of recombinant MbtAtb, for which Km and kcat values were determined, and used the enzyme in conjunction with an MbtAtb-optimized, continuous, spectrophotometric assay for MbtA activity and inhibition. The cell-based studies provided an assessment of the antimycobacterial activity and postantibiotic effect of the nine MbtAtb inhibitors. The antimycobacterial properties were evaluated using a strain of nonpathogenic, fast-growing Mycobacterium smegmatis that was genetically engineered for MbtAtb-dependent susceptibility to MbtA inhibitors. This convenient model system greatly facilitated the cell-based studies by bypassing the methodological complexities associated with the use of pathogenic, slow-growing M. tuberculosis. Collectively, these studies provide new information on the mechanism of inhibition of MbtAtb by salicyl-AMS and eight analogues, afford new SAR insights for these inhibitors, and highlight several suitable candidates for future preclinical evaluation.

Iron Acquisition in Mycobacterium tuberculosis.

The highly contagious disease tuberculosis (TB) is caused by the bacterium Mycobacterium tuberculosis (Mtb), which has been evolving drug resistance at an alarming rate. Like all human pathogens, Mtb requires iron for growth and virulence. Consequently, Mtb iron transport is an emerging drug target. However, the development of anti-TB drugs aimed at these metabolic pathways has been restricted by the dearth of information on Mtb iron acquisition. In this Review, we describe the multiple strategies utilized by Mtb to acquire ferric iron and heme iron. Mtb iron uptake is a complex process, requiring biosynthesis and subsequent export of Mtb siderophores, followed by ferric iron scavenging and ferric-siderophore import into Mtb. Additionally, Mtb possesses two possible heme uptake pathways and an Mtb-specific mechanism of heme degradation that yields iron and novel heme-degradation products. We conclude with perspectives for potential therapeutics that could directly target Mtb heme and iron uptake machineries. We also highlight how hijacking Mtb heme and iron acquisition pathways for drug import may facilitate drug transport through the notoriously impregnable Mtb cell wall.

Legonoxamines A-B, two new hydroxamate siderophores from the soil bacterium, Streptomyces sp. MA37

Two new siderophores belonging to the hydroxamate class, Legonoxamine A (1) and B (2) have been isolated from the soil bacterium, Streptomyces sp. MA37, together with one known compound, desferrioxamine B (3). Their structures were elucidated based on spectroscopic methods including 1D, 2D NMR, MS, as well as by comparison with the relevant literatures. To our knowledge, this is the first report describing a siderophore containing the N-hydroxyl phenylacetyl cadaverine (HPAC) moiety in the structure. Based on bioinformatics analysis and previous knowledge of the biosynthesis of the hydroxamate-type siderophore, the biosynthetic gene cluster (lgo) responsible for the production of 1–3 was identified in the annotated genome of the producing strain. The supplementation of phenylacetate and benzoate analogues with meta substitution into the cultures of Streptomyces sp. MA37 resulted in the production of new legonoxamine A derivatives as observed in LC-HR-ESIMS, suggesting that the legonoxamine biosynthetic pathway has a good degree of natural flexibility of accepting unnatural precursors with different functional groups.

Engineering Pseudochelin Production in Myxococcus xanthus.

Myxobacteria utilize the catechol natural products myxochelin A and B in order to maintain their iron homeostasis. Recently, the production of these siderophores, along with a new myxochelin derivative named pseudochelin A, was reported for the marine bacterium Pseudoalteromonas piscicida S2040. The latter derivative features a characteristic imidazoline moiety, which was proposed to originate from an intramolecular condensation reaction of the β-aminoethyl amide group in myxochelin B. To identify the enzyme catalyzing this conversion, we compared the myxochelin regulons of two myxobacterial strains that produce solely myxochelin A and B with those of P. piscicida S2040. This approach revealed a gene exclusive to the myxochelin regulon in P. piscicida S2040, coding for an enzyme of the amidohydrolase superfamily. To prove that this enzyme is indeed responsible for the postulated conversion, the reaction was reconstituted in vitro using a hexahistidine-tagged recombinant protein made in Escherichia coli, with myxochelin B as the substrate. To test the production of pseudochelin A under in vivo conditions, the amidohydrolase gene was cloned into the myxobacterial plasmid pZJY156 and placed under the control of a copper-inducible promoter. The resulting vector was introduced into the myxobacterium Myxococcus xanthus DSM 16526, a native producer of myxochelin A and B. Following induction with copper, the myxobacterial expression strain was found to synthesize small quantities of pseudochelin A. Replacement of the copper-inducible promoter with the constitutive pilA promoter led to increased production levels in M. xanthus, which facilitated the isolation and subsequent structural verification of the heterologously produced compound.IMPORTANCE In this study, an enzyme for imidazoline formation in pseudochelin biosynthesis was identified. Evidence for the involvement of this enzyme in the postulated reaction was obtained after in vitro reconstitution. Furthermore, the function of this enzyme was demonstrated in vivo by transferring the corresponding gene into the bacterium Myxococcus xanthus, which thereby became a producer of pseudochelin A. In addition to clarifying the molecular basis of imidazoline formation in siderophore biosynthesis, we describe the heterologous expression of a gene in a myxobacterium without chromosomal integration. Due to its metabolic proficiency, M. xanthus represents an interesting alternative to established host systems for the reconstitution and manipulation of biosynthetic pathways. Since the plasmid used in this study is easily adaptable for the expression of other enzymes as well, we expand the conventional expression strategy for myxobacteria, which is based on the integration of biosynthetic genes into the host genome.

Effect of iron limitation on the isotopic composition of cellular and released fixed nitrogen in Azotobacter vinelandii

Most biological nitrogen transformations have characteristic kinetic isotope effects used to track these processes in modern and past environments. The isotopic fractionation associated with nitrogen fixation, the only biological source of fixed nitrogen (N), provides a particularly important constraint for studies of nitrogen cycling. Nitrogen fixation using the ‘canonical’ Mo-nitrogenase produces biomass with a δ15N value of ca. −1‰ (vs. atmospheric N2). If the ‘alternative’ V- and Fe-only nitrogenases are used, biomass δ15N can be between −6‰ and −7‰. These biomass values are assumed to be relatively invariant and to reflect the cellular level expressed isotope effect of nitrogen fixation. However, field and laboratory studies report wide ranges of diazotrophic biomass δ15N (from −3.6‰ to +0.5‰ for Mo-based nitrogen fixation). This variation could be partly explained by the release of dissolved organic N (DON) that is isotopically distinct from biomass. The model nitrogen fixer Azotobacter vinelandii secretes siderophores, small molecules that aid in Fe uptake and can comprise >30% of fixed nitrogen. To test whether siderophores (and other released N) can decouple biomass δ15N from the isotope effect of nitrogen fixation we measured the isotopic composition of biomass and released N in Fe-limited A. vinelandii cultures fixing nitrogen with Mo- and V-nitrogenases. We report that biomass δ15N was elevated under Fe limitation with a maximum value of +1.2‰ for Mo-based nitrogen fixation. Regardless of the nitrogenase isozyme used, released nitrogen δ15N was also 2–3‰ lower than biomass δ15N. Siderophore nitrogen was found to have a slightly higher δ15N than the rest of the DON pool but was still produced in large enough concentrations to account for increases in biomass δ15N. The low δ15N of siderophores (relative to biomass) is consistent with what is known from compound specific isotope studies of the amino acids used in siderophore biosynthesis, and indicates that other amino-acid derived siderophores should also have a low δ15N. The implications for studies of nitrogen fixation are discussed.

Ferricrocin, the intracellular siderophore of Trichoderma virens, is involved in growth, conidiation, gliotoxin biosynthesis and induction of systemic resistance in maize

Fungal siderophores are known to be involved in iron acquisition and storage, as well as pathogenicity of mammals and plants. As avirulent plant symbionts, Trichoderma spp. colonize roots and induce resistance responses both locally and systemically. To study the role of intracellular siderophore(s) in Trichoderma-plant interactions, we have obtained mutants in a non-ribosomal peptide synthetase, TvTex10, that was predicted to be involved in intracellular siderophore(s) biosynthesis. This gene has a detectable basal level of expression and is also upregulated under iron-deplete conditions. This is unlike two other siderophore-encoding genes, which are tightly regulated by iron. Disruption of tex10 gene using homologous recombination resulted in mutants with enhanced growth rate, reduced conidiation and hyper-sensitivity to oxidative stress as compared to wildtype strain. The mutants also produced reduced levels of gliotoxin and dimethyl gliotoxin but have enhanced ability to colonize maize seedling roots. The mutants were also impaired in induction of induced systemic resistance (ISR) in maize against the foliar pathogen Cochliobolus heterostrophus.

Multiple siderophores: bug or feature?

It is common for bacteria to produce chemically diverse sets of small Fe-binding molecules called siderophores. Studies of siderophore bioinorganic chemistry have firmly established the role of these molecules in Fe uptake and provided great insight into Fe complexation. However, we still do not fully understand why microbes make so many siderophores. In many cases, the release of small structural variants or siderophore fragments has been ignored, or considered as an inefficiency of siderophore biosynthesis. Yet, in natural settings, microbes live in complex consortia and it has become increasingly clear that the secondary metabolite repertoires of microbes reflect this dynamic environment. Multiple siderophore production may, therefore, provide a window into microbial life in the wild. This minireview focuses on three biochemical routes by which multiple siderophores can be released by the same organism-multiple biosynthetic gene clusters, fragment release, and precursor-directed biosynthesis-and highlights emergent themes related to each. We also emphasize the plurality of reasons for multiple siderophore production, which include enhanced iron uptake via synergistic siderophore use, microbial warfare and cooperation, and non-classical functions such as the use of siderophores to take up metals other than Fe.

An insight into the iron acquisition and homeostasis in Aureobasidium melanogenum HN6.2 strain through genome mining and transcriptome analysis.

Aureobasidium melanogenum HN6.2 is a unique yeast strain who can produce the siderophore of fusigen under iron starvation to guarantee its survival. However, a comprehensive understanding of mechanisms involved in iron acquisition and homeostasis for it is still vacant. In this study, genome sequencing and mining revealed that A. melanogenum HN6.2 strain was the first yeast species that exclusively possessed all the four known mechanisms for the iron acquisition: (i) the siderophore-mediated iron uptake; (ii) reductive iron assimilation; (iii) low-affinity ferrous uptake; and (iv) heme utilization, which suggested its stronger adaptability than Aspergillus fumigatus and Saccharomyces cerevisiae. This HN6.2 strain also employed the vacuolar iron storage for immobilizing the excessive iron to avoid its cellular toxicity. Specially, genome mining indicated that A. melanogenum HN6.2 strain could also synthesize ferricrocin siderophore. Further HPLC and Q-Tof-MS analysis confirmed that the siderophores synthesized by this strain consisted of cyclic fusigen, linear fusigen, ferricrocin, and hydroxyferricrocin and they played parallel roles as both intracellular and extracellular siderophores. Also, the heme utilization for this strain was experimentally verified by the knock-out of heme oxygenase gene. For iron homeostasis, the transcriptome analysis revealed that this strain mainly employed two central regulators of SreA/HapX to tune iron uptake and storage at the transcriptional level. It was also noted that mitogen-activated protein kinase C gene (MpkC) exhibited a transcriptional up-regulation under iron sufficiency, suggesting that it may serve as another factor involved in the repression of siderophore biosynthesis. This is the first genetic blueprint of iron acquisition and homeostasis for A. melanogenum.

The Transcription Factor VdHapX Controls Iron Homeostasis and Is Crucial for Virulence in the Vascular Pathogen Verticillium dahliae.

Iron homeostasis is essential for full virulence and viability in many pathogenic fungi. Here, we showed that the bZip transcription factor VdHapX functions as a key regulator of iron homeostasis for adaptation to iron-depleted and iron-excess conditions and is required for full virulence in the vascular wilt fungus, Verticillium dahliae Deletion of VdHapX impaired mycelial growth and conidiation under both iron starvation and iron sufficiency. Furthermore, disruption of VdHapX led to decreased formation of the long-lived survival structures of V. dahliae, known as microsclerotia. Expression of genes involved in iron utilization pathways and siderophore biosynthesis was misregulated in the ΔVdHapX strain under the iron-depleted condition. Additionally, the ΔVdHapX strain exhibited increased sensitivity to high iron concentrations and H2O2, indicating that VdHapX also contributes to iron or H2O2 detoxification. The ΔVdHapX strain showed a strong reduction in virulence on smoke tree seedlings (Cotinus coggygria) and was delayed in its ability to penetrate plant epidermal tissue.IMPORTANCE This study demonstrated that VdHapX is a conserved protein that mediates adaptation to iron starvation and excesses, affects microsclerotium formation, and is crucial for virulence of V. dahliae.

Eine vielseitige Enzymklasse für die Synthese ringförmiger Siderophore

The macrocyclic hydroxamate siderophore avaroferrin of Shewanella algae inhibits ferric iron dependent swarming motility of Vibrio alginolyticus. Investigating the biosynthesis of avaroferrin and related siderophores demonstrates the importance of the substrate pool for product formation and reveals an unprecedented flexibility in the substrate range of the responsible synthetases. Exploiting the promiscuity of these enzymes allowed to generate a broad spectrum of 15 different ring-size engineered siderophores some of which also inhibit Vibrio’s swarming behavior.

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

Siderophores are chemically diverse small molecules produced by microorganisms for chelation of irons to maintain their survival and govern some important biological functions, especially those cause that infections in hosts. Still, siderophores can offer new insight into a better understanding of the diagnosis and treatments of infectious diseases from the siderophore biosynthesis and regulation perspective. Thus, this review aims to summarize the biomedical value and applicability of siderophores in pathogenic contexts by briefly reviewing mass spectrometry (MS)-based chemical biology and translational applications that involve diagnosis, pathogenesis, and therapeutic discovery for a variety of infectious conditions caused by different pathogens. We highlight the advantages and disadvantages of siderophore discovery and applications in pathogenic contexts. Finally, we propose a panel of new and promising strategy as precision-modification metabolomics method, to rapidly advance the discovery of and translational innovations pertaining to these value compounds in broad biomedical niches. © 2018 Wiley Periodicals, Inc. Mass Spec Rev XX:XX-XX, 2018.

Structural insight into a heme relay from human hemoglobin to SbnI, a regulator of siderophore biosynthesis in S. aureus

Structural insight into a heme relay from human hemoglobin to SbnI, a regulator of siderophore biosynthesis in <i>S. aureus</i>

Structural and functional studies of the metalloregulator Fur identify a promoter-binding mechanism and its\xa0role in Francisella tularensis virulence

Francisella tularensis is a Gram-negative bacterium causing tularaemia. Classified as possible bioterrorism agent, it may be transmitted to humans via animal infection or inhalation leading to severe pneumonia. Its virulence is related to iron homeostasis involving siderophore biosynthesis directly controlled at the transcription level by the ferric uptake regulator Fur, as presented here together with the first crystal structure of the tetrameric F. tularensis Fur in the presence of its physiological cofactor, Fe2+. Through structural, biophysical, biochemical and modelling studies, we show that promoter sequences of F. tularensis containing Fur boxes enable this tetrameric protein to bind them by splitting it into two dimers. Furthermore, the critical role of F. tularensis Fur in virulence and pathogenesis is demonstrated with a fur-deleted mutant showing an attenuated virulence in macrophage-like cells and mice. Together, our study suggests that Fur is an attractive target of new antibiotics that attenuate the virulence of F. tularensis.

β-Hydroxyaspartic acid in siderophores: biosynthesis and reactivity.

A growing number of siderophores are found to contain β-hydroxyaspartic acid (β-OH-Asp) as a functional group for Fe(III) coordination, along with the more common catechol and hydroxamic acid groups. This review covers the structures, biosynthesis, and reactions of peptidic β-OH-Asp siderophores. Hydroxylation of Asp in siderophore biosynthesis is predicted to be carried out either through discrete aspartyl β-hydroxylating enzymes or through hydroxylating domains within non-ribosomal peptide synthetases, both of which display sequence homology to known non-heme iron(II), α-ketoglutarate-dependent dioxygenases. Ferric complexes of β-OH-Asp siderophores are photoreactive, resulting in reduction of Fe(III) and oxidative cleavage of the siderophore to yield distinct types of photoproducts. Probing the photoreactivity of synthetic Fe(III)-α-hydroxycarboxylate clusters yields mechanistic insights into the different photoproducts observed for β-OH-Asp and other α-hydroxycarboxylate siderophore Fe(III) complexes.

Pseudomonas aeruginosa L10: A Hydrocarbon-Degrading, Biosurfactant-Producing, and Plant-Growth-Promoting Endophytic Bacterium Isolated From a Reed (Phragmites australis).

Bacterial endophytes with the capacity to degrade petroleum hydrocarbons and promote plant growth may facilitate phytoremediation for the removal of petroleum hydrocarbons from contaminated soils. A hydrocarbon-degrading, biosurfactant-producing, and plant-growth-promoting endophytic bacterium, Pseudomonas aeruginosa L10, was isolated from the roots of a reed, Phragmites australis, in the Yellow River Delta, Shandong, China. P. aeruginosa L10 efficiently degraded C10–C26 n-alkanes from diesel oil, as well as common polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, phenanthrene, and pyrene. In addition, P. aeruginosa L10 could produce biosurfactant, which was confirmed by the oil spreading method, and surface tension determination of inocula. Moreover, P. aeruginosa L10 had plant growth-stimulating attributes, including siderophore and indole-3-acetic acid (IAA) release, along with 1-aminocyclopropane-1-carboxylic (ACC) deaminase activity. To explore the mechanisms underlying the phenotypic traits of endophytic P. aeruginosa L10, we sequenced its complete genome. From the genome, we identified genes related to petroleum hydrocarbon degradation, such as putative genes encoding monooxygenase, dioxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase. Genome annotation revealed that P. aeruginosa L10 contained a gene cluster involved in the biosynthesis of rhamnolipids, rhlABRI, which should be responsible for the observed biosurfactant activity. We also identified two clusters of genes involved in the biosynthesis of siderophore (pvcABCD and pchABCDREFG). The genome also harbored tryptophan biosynthetic genes (trpAB, trpDC, trpE, trpF, and trpG) that are responsible for IAA synthesis. Moreover, the genome contained the ACC deaminase gene essential for ACC deaminase activity. This study will facilitate applications of endophytic P. aeruginosa L10 to phytoremediation by advancing the understanding of hydrocarbon degradation, biosurfactant synthesis, and mutualistic interactions between endophytes and host plants.

Genome Sequence of Pseudomonas chlororaphis Lzh-T5, a Plant Growth-Promoting Rhizobacterium with Antimicrobial Activity.

ABSTRACTPseudomonas chlororaphis Lzh-T5 is a plant growth-promoting rhizobacterium (PGPR) with antimicrobial activity isolated from tomato rhizosphere in the city of Dezhou, Shandong Province, China. Here, the draft genome sequence of P. chlororaphis Lzh-T5 is reported, and several functional genes related to antifungal antibiotics and siderophore biosynthesis have been found in the genome.

Structural and functional delineation of aerobactin biosynthesis in hypervirulent Klebsiella pneumoniae

Aerobactin, a citryl-hydroxamate siderophore, is produced by a number of pathogenic Gram-negative bacteria to aid in iron assimilation. Interest in this well-known siderophore was reignited by recent investigations suggesting that it plays a key role in mediating the enhanced virulence of a hypervirulent pathotype of Klebsiella pneumoniae (hvKP). In contrast to classical opportunistic strains of K. pneumoniae, hvKP causes serious life-threatening infections in previously healthy individuals in the community. Multiple contemporary reports have confirmed fears that the convergence of multidrug-resistant and hvKP pathotypes has led to the evolution of a highly transmissible, drug-resistant, and virulent "super bug." Despite hvKP harboring four distinct siderophore operons, knocking out production of only aerobactin led to a significant attenuation of virulence. Herein, we continue our structural and functional studies on the biosynthesis of this crucial virulence factor. In vivo heterologous production and in vitro reconstitution of aerobactin biosynthesis from hvKP was carried out, demonstrating the specificity, stereoselectivity, and kinetic throughput of the complete pathway. Additionally, we present a steady-state kinetic analysis and the X-ray crystal structure of the second aerobactin synthetase IucC, as well as describe a surface entropy reduction strategy that was employed for structure determination. Finally, we show solution X-ray scattering data that support a unique dimeric quaternary structure for IucC. These new insights into aerobactin assembly will help inform potential antivirulence strategies and advance our understanding of siderophore biosynthesis.