A suite of citrate-derived siderophores from a marine Vibrio species isolated following the Deepwater Horizon oil spill

Nearly all microbes require iron for growth. The low concentration of iron found in the ocean makes iron acquisition a particularly difficult task. In response to these low iron conditions, many bacteria produce low-molecular-weight iron-binding molecules called siderophores to aid in iron uptake. We report herein the isolation and structural characterization of a suite of amphiphilic siderophores called the ochrobactins-OH, which are produced by a Vibrio species isolated from the Gulf of Mexico after the 2010 Deepwater Horizon oil spill. The citrate-based ochrobactins-OH are derivatives of aerobactin, replacing the acetyl groups with fatty acid appendages ranging in size from C8 to C12, and are distinctly different from the ochrobactins in that the fatty acid appendages are hydroxylated rather than unsaturated. The discovery of the marine amphiphilic ochrobactin-OH suite of siderophores increases the geographic and phylogenetic diversity of siderophore-producing bacteria.     

Identification of new members within suites of amphiphilic marine siderophores

Marine bacterial isolates Vibrio sp. HC0601C5 and Halomonas meridiana str. HC4321C1 were isolated off the coast of southern California and were found to produce an expanded suite of previously identified amphiphilic siderophores. Specifically two new members of the amphibactin family, amphibactins S and T, which have a C14:1 ω-7 fatty acid and a saturated C12 fatty acid, respectively, were produced by Vibrio sp. HC0601C5. These siderophores are produced in addition to a number of previously described amphibactins and are excreted into the culture supernatant. Two new members of the aquachelin family of siderophores, aquachelins I and J, which have an hydroxylated C12 fatty acid and a saturated C10 fatty acid, respectively, were produced by Halomonas meridiana str. HC4321C1. These four new siderophores are more hydrophilic than their previously reported relatives, aquachelins A–D and the amphibactin suite of siderophores.     

Siderophores of Marinobacter aquaeolei: petrobactin and its sulfonated derivatives

Siderophores are low molecular weight, high-affinity iron(III) ligands, produced by bacteria to solubilize and promote iron uptake under low iron conditions. Two prominent structural features characterize the majority of the marine siderophores discovered so far: (1) a predominance of suites of amphiphilic siderophores composed of an iron(III)-binding headgroup that is appended by one or two of a series of fatty acids and (2) a prevalence of siderophores that contain α-hydroxycarboxylic acid moieties (e.g., β-hydroxyaspartic acid or citric acid) which are photoreactive when coordinated to Fe(III). Variation of the fatty acid chain length affects the relative amphiphilicity within a suite of siderophores. Catecholate sulfonation is another structural variation that would affect the hydrophilicity of a siderophore. In addition to a review of the marine amphiphilic siderophores, we report the production of petrobactin disulfonate by Marinobacter aquaeolei VT8. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

SIDEROPHORE‐INDEPENDENT IRON UPTAKE BY IRON‐LIMITED CELLS OF THE CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition by iron‐limited cyanobacteria is typically considered to be mediated mainly by siderophores, iron‐chelating molecules released by iron‐limited cyanobacteria into the environment. In this set of experiments, iron uptake by iron‐limited cells of the cyanobacterium Anabaena flos‐aquae (L.) Bory was investigated in cells resuspended in siderophore‐free medium. Removal of siderophores decreased iron‐uptake rates by ～60% compared to siderophore‐replete conditions; however, substantial rates of iron uptake remained. In the absence of siderophores, Fe(III) uptake was much more rapid from a weaker synthetic chelator [N‐(2‐hydroxyethyl)ethylenediamine‐N,N′,N′‐triacetic acid (HEDTA); log K_cond = 28.64 for Fe(III)HEDTA(OH)^?] than from a very strong chelator [N,N′‐bis(2‐hydroxybenzyl)‐ethylenediamine‐N,N′‐diacetic acid (HBED); log K_cond = 31.40 for Fe(III)HBED^?], and increasing chelator:Fe(III) ratios decreased the Fe(III)‐uptake rate; these results were evident in both short‐term (4 h; absence of siderophores) and long‐term (116 h; presence of siderophores) experiments. However, free (nonchelated) Fe(III) provided the most rapid iron uptake in siderophore‐free conditions. The results of the short‐term experiments are consistent with an Fe(III)‐binding/uptake mechanism associated with the cyanobacterial outer membrane that operates independently of extracellular siderophores. Iron uptake was inhibited by temperature‐shock treatments of the cells and by metabolically compromising the cells with diphenyleneiodonium; this finding indicates that the process is dependent on active metabolism to operate and is not simply a passive Fe(III)‐binding mechanism. Overall, these results point to an important, siderophore‐independent iron‐acquisition mechanism by iron‐limited cyanobacterial cells.     

Identification of Novel Decenoic Acids in Heated Butter

Novel decenoic acids such as (E)-4-decenoic acid and (E)- and (Z)-5-,6-decenoic acid were detected as minor components in heated butter using GC and GC/MS. The formation mechanism of these novel decenoic acids is discussed on the basis of the result of the reaction of δ-decalactone with active clay in a model experiment.     

Trace metal complexation by the triscatecholate siderophore protochelin: structure and stability

Although siderophores are generally viewed as biological iron uptake agents, recent evidence has shown that they may play significant roles in the biogeochemical cycling and biological uptake of other metals. One such siderophore that is produced by A. vinelandii is the triscatecholate protochelin. In this study, we probe the solution chemistry of protochelin and its complexes with environmentally relevant trace metals to better understand its effect on metal uptake and cycling. Protochelin exhibits low solubility below pH 7.5 and degrades gradually in solution. Electrochemical measurements of protochelin and metal–protochelin complexes reveal a ligand half-wave potential of 200 mV. The Fe(III)Proto³⁻ complex exhibits a salicylate shift in coordination mode at circumneutral to acidic pH. Coordination of Mn(II) by protochelin above pH 8.0 promotes gradual air oxidation of the metal center to Mn(III), which accelerates at higher pH values. The Mn(III)Proto³⁻ complex was found to have a stability constant of log β₁₁₀ = 41.6. Structural parameters derived from spectroscopic measurements and quantum mechanical calculations provide insights into the stability of the Fe(III)Proto³⁻, Fe(III)H₃Proto, and Mn(III)Proto³⁻ complexes. Complexation of Co(II) by protochelin results in redox cycling of Co, accompanied by accelerated degradation of the ligand at all solution pH values. These results are discussed in terms of the role of catecholate siderophores in environmental trace metal cycling and intracellular metal release.     

Structural and thermodynamical properties of CuII amyloid-β16/28 complexes associated with Alzheimer’s disease

The aggregation of the peptide amyloid-β (Aβ) to form amyloid plaques is a key event in Alzheimer’s disease. It has been shown that Cu^II can bind to soluble Aβ and influence its aggregation properties. Three histidines and the N-terminal amine have been proposed to be involved in its coordination. Here, for the first time, we show isothermal titration calorimetry (ITC) measurements of the Cu^II binding to Aβ16 and Aβ28, models of the soluble Aβ. Moreover, different spectroscopic methods were applied. The studies revealed new insights into these Cu^II–Aβ complexes: (1) ITC showed two Cu^II binding sites, with an apparent K _d of 10⁻⁷ and 10⁻⁵ M, respectively; (2) the high-affinity site has a smaller enthalpic contribution but a larger entropic contribution than the low-affinity binding site; (3) azide did not bind to Cu^II in the higher-affinity binding site, suggesting the absence of a weak, labile ligand; (4) azide could bind to the Cu^II in the low-affinity binding site in Aβ28 but not in Aβ16; (5) ¹H-NMR suggests that the carboxylate of aspartic acid in position 1 is involved in the ligation to Cu^II in the high-affinity binding site; (6) the pK _a of 11.3 of tyrosine in position 10 was not influenced by the binding of 2 equivalents of Cu^II.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Amino acid variability in the peptide composition of a suite of amphiphilic peptide siderophores from an open ocean Vibrio species

In response to iron-depleted aerobic conditions, bacteria often secrete low molecular weight, high-affinity iron(III)-complexing ligands, siderophores, to solubilize and sequester iron(III). Many marine siderophores are amphiphilic and are produced in suites, wherein each member within a particular suite has the same iron(III)-binding polar head group which is appended by one or two fatty acids of differing length, degree of unsaturation, and degree of hydroxylation, establishing the suite composition. We report the isolation and structural characterization of a suite of siderophores from marine bacterial isolate Vibrio sp. Nt1. On the basis of structural analysis, this suite of siderophores, the moanachelins, is amphiphilic and composed of two N-acetyl-N-hydroxy-d-ornithines, one N-acetyl-N-hydroxy-l-ornithine, and either a glycine or an l-alanine, appended with various saturated and unsaturated fatty acid tails. The variation in the small side-chain amino acid is the first occurrence of variation in the peptidic head group structure of a set of siderophores produced by a single bacterium.     

Marine amphiphilic siderophores: marinobactin structure, uptake, and microbial partitioning

Marinobactins A-E are a suite of amphiphilic siderophores which have a common peptidic head group that coordinates Fe(III), and a fatty acid which varies in length and saturation. As a result of the amphiphilic properties of these siderophores it is difficult to study siderophore-mediated uptake of iron, because the amphiphilic siderophores partition indiscriminately in microbial and other membranes. An alternative method to distinguish amphiphilic siderophore partitioning versus siderophore-mediated active uptake for Fe(III)-marinobactin E has been developed. In addition, a new member of the marinobactin family of siderophores is also reported, marinobactin F, which has a C(18) fatty acid with one double bond and which is substantially more hydrophobic that marinobactins A-E.     

Isolation and Characterization of an Fe(III)-Chelating Compound Produced by Pseudomonas syringae

The phytopathogenic bacterium Pseudomonas syringae produces a fluorescent pigment when it is grown in iron-deficient media. This pigment forms a very stable Fe(III) complex that was purified in this form by using a novel procedure based on ultrafiltration and column chromatography. The Fe(III) complex has a molecular weight of 1,100 and contains 1 mol of Fe(III). The pigment is composed of an amino acid moiety with three threonines, three serines, one lysine, δ-N-hydroxyornithine, and a quinoline-type fluorescent chromophore. These features and its stability constant (in the range of 10³²) suggest that the fluorescent pigment of P. syringae is related to the siderophores produced by another Pseudomonas species.     

Amonabactin: a family of novel siderophores from a pathogenic bacterium

 Four peptide-based bis-catecholate siderophores, collectively known as the amonabactins, have been isolated from Aeromonas hydrophila. They have been fully characterized: tandem mass spectroscopy established the sequence of the amino acid components, chiral gas chromatographic mass spectra established the amino acid chirality, and two-dimensional NMR techniques determined the full connectivity and structure. Each of the amonabactins was synthesized and the synthetic material was compared to the natural product as a final proof of structure. These siderophores are bis-catecholates with the backbone composed of either tri- or tetrapeptides in the sequence (gly)-(L)-lys-(L)-lys-(D)-aro, where glycine is the optional amino acid attached to the N^ε of the N terminus lysine and aro is either tryptophan or phenylalanine. The ligand units, 2,3-dihydroxybenzamide groups, are attached to the N^ε amine of the C terminus lysine and either to glycine, if present, or to N^ε amines of the N terminus lysine. Each of the amonabactins supports growth of the organism under low-iron conditions in vitro and in serum. The architecture of these siderophores includes an inverted aromatic amino acid and unusual linkages which should prevent enzymatic hydrolysis of the peptide backbones. This, along with their ability to successfully compete for iron in serum, suggests a role in the pathogenicity of the organism. This paper is number 62 in the series Coordination Chemistry of Microbial Ion Transport. Details of the previous paper are given in [1]. Received: 6 June 1997 / Accepted: 9 September 1997     

The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity

In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity of Arabidopsis thaliana Glx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV–vis, EPR, and ¹H NMR spectroscopies. Glx2-2-containing fractions that were purple, yellow, or colorless were separated during purification, and the differently colored fractions were found to contain different amounts of Fe and Zn(II). Spectroscopic analyses of the discrete fractions provided evidence for Fe(II), Fe(III), Fe(III)–Zn(II), and antiferromagnetically coupled Fe(II)–Fe(III) centers distributed among the discrete Glx2-2-containing fractions. The individual steady-state kinetic constants varied among the fractionated species, depending on the number and type of metal ion present. Intriguingly, however, the catalytic efficiency constant, k _cat/K _m, was invariant among the fractions. The value of k _cat/K _m governs the catalytic rate at low, physiological substrate concentrations. We suggest that the independence of k _cat/K _m on the precise makeup of the active-site metal center is evolutionarily related to the lack of selectivity for either Fe versus Zn(II) or Fe(II) versus Fe(III), in one or more metal binding sites.     

Bacterial siderophores: the solution stoichiometry and coordination of the Fe(III) complexes of pyochelin and related compounds

Pyochelin, its analog 3′′-nor-NH-pyochelin, and the related methyl hydroxamate, 2-(2′-hydroxyphenyl)-4,5-dihydrothiazol-4-carboxylic acid methoxymethyl amide, have been prepared together with their Fe(III) complexes. The solution stoichiometry and the coordination of the three Fe(III) complexes in methanol or buffered (pH∼2) 50:50 (v/v) methanol–water mixtures were determined using various spectroscopic methods: UV–vis absorption, X-ray absorption, extended X-ray absorption fine structure and electron paramagnetic resonance. All three systems showed both a 1:1 and 2:1 ligand–Fe(III) stoichiometry, but presented different coordination properties. Conditional formation constants (pH∼2) were determined for both the 1:1 and 2:1 complexes in all three systems. Computation of the coordination-conformational energies by semiempirical methods indicated that the coordination in the case of the 2:1 complexes of pyochelin–Fe(III) and 3′′-nor-NH-pyochelin–Fe(III) was asymmetrical, with one molecule of pyochelin (or 3′′-nor-NH-pyochelin) tetradentately coordinated (O1, N1, N2 and O3) to the Fe(III), and the second molecule bound bidentately (O1, N1 or N2, O3), to complete the octahedral geometry. In contrast, two molecules of the methyl hydroxamate each provided a set of tridentate ligand atoms in the formation of the 2:1 ligand–Fe(III) complex. These results are consistent with the role of pyochelin in the uptake of iron by the FptA receptor in the outer membrane of Pseudomonas aeruginosa and in several gram-negative bacteria.     

Effects of charged peptides on electron transfer from [Fe(CN)6]4– to cytochrome c or plastocyanin

 Interactions of charged peptides, such as aspartic acid peptides (Aspptds) and lysine peptides (Lysptds), with cytochrome c (cyt c) or plastocyanin (PC) have been studied by measuring electron transfer between [Fe(CN)₆]^4– and cyt c or PC in the presence of these peptides. Aspptds, up to penta-aspartic acid, served as competitive inhibitors of electron transfer from [Fe(CN)₆]^4– to oxidized cyt c, while Lysptds, up to penta-lysine, promoted electron transfer from [Fe(CN)₆]^4– to oxidized PC. The electron transfer inhibitory effects of Aspptds are explained as competitive inhibition due to neutralization of the positively charged amino acid residues at the surface of cyt c by electrostatic interactions, whereas the electron transfer promoting effects of Lysptds may be due to formation of PC·Lysptd or Lysptd·[Fe(CN)₆]^4– complexes subsequently forming an electron transferring complex, PC·Lysptd·[Fe(CN)₆]^4–, without repulsion of the negative charges. The inhibitory effect of Aspptds and promotional effect of Lysptds became significant as the net charge or concentration of the peptides increased. The promotional effects of Lysptds decreased as the net charge of the PC negative patch was decreased by mutagenesis. Thus, charged peptides may serve as a probe for investigation of the molecular recognition character of proteins. Received: 19 May 1998 / Accepted: 27 July 1998     

Organization of biosynthetic gene cluster for avermectin in Streptomyces avermitilis: analysis of enzymatic domains in four polyketide synthases

The analysis of the incorporation of ¹³C-labeled precursors into avermectins indicates that the avermectin aglycons are synthesized by head-to-tail condensation of various acyl groups, which is similar to the biosynthesis of other polyketides. Polyketide synthases (PKS) use the appropriate CoA ester as a primer and add acetate units from malonyl-CoA and propionate units from methylmalonyl-CoA to assemble the polyketides. Avermectin aglycons are formed by addition to the starter unit (2-methylbutyrate or isobutyrate) of 12 acyl condensations in the order P–A–A–A–A–P–P–A–P–A–P–A (P, propionyl; A, acetyl). Within the 90-kb gene cluster for avermectin biosynthesis, the central 65-kb segment was found to be required for aglycon biosynthesis by phenotypic analysis of strains containing deletion or insertion mutations in this region. A complete sequence analysis of the 65-kb segment indicated that this segment encodes avermectin PKS. The avermectin PKS genes are organized into two converging blocks of ORFs. From the results of sequencing analysis, a feature of the two regions, aveA1/aveA2 and avea3/aveA4, is that they encode four kinds of large multifunctional polypeptides containing 55 domains which possess putative fatty acid synthase-like activities. The avermectin PKS (AVES 1–4) appear to contain two, three, or four modules. AVES 1 and 2 contain two and four modules, respectively, whereas AVES 3 and AVES 4 each contains three modules. The 12 modules correspond to the 12 cycles required for synthesis of the avermectin aglycon. Journal of Industrial Microbiology & Biotechnology (2001) 27, 170–176. Received 21 September 1999/ Accepted in revised form 14 September 2000     

Self-hydroxylation of taurine/α-ketoglutarate dioxygenase: evidence for more than one oxygen activation mechanism

2-Aminoethanesulfonic acid (taurine)/α-ketoglutarate (αKG) dioxygenase (TauD) is a mononuclear non-heme iron enzyme that catalyzes the hydroxylation of taurine to generate sulfite and aminoacetaldehyde in the presence of O₂, αKG, and Fe(II). Fe(II)TauD complexed with αKG or succinate, the decarboxylated product of αKG, reacts with O₂ in the absence of prime substrate to generate 550- and 720-nm chromophores, respectively, that are interconvertible by the addition or removal of bound bicarbonate and have resonance Raman features characteristic of an Fe(III)–catecholate complex. Mutagenesis studies suggest that both reactions result in the self-hydroxylation of the active-site residue Tyr73, and liquid chromatography nano-spray mass spectrometry/mass spectrometry evidence corroborates this result for the succinate reaction. Furthermore, isotope-labeling resonance Raman studies demonstrate that the oxygen atom incorporated into the tyrosyl residue derives from H₂ ¹⁸O and ¹⁸O₂ for the αKG and succinate reactions, respectively, suggesting distinct mechanistic pathways. Whereas the αKG-dependent hydroxylation likely proceeds via an Fe(IV)=O intermediate that is known to be generated during substrate hydroxylation, we propose Fe(III)–OOH (or Fe(V)=O) as the oxygenating species in the succinate-dependent reaction. These results demonstrate the two oxygenating mechanisms available to enzymes with a 2-His-1-carboxylate triad, depending on whether the electron source donates one or two electrons.     

Coupled biogeochemical cycling of iron and manganese as mediated by microbial siderophores

Siderophores, biogenic chelating agents that facilitate Fe(III) uptake through the formation of strong complexes, also form strong complexes with Mn(III) and exhibit high reactivity with Mn (hydr)oxides, suggesting a pathway by which Mn may disrupt Fe uptake. In this review, we evaluate the major biogeochemical mechanisms by which Fe and Mn may interact through reactions with microbial siderophores: competition for a limited pool of siderophores, sorption of siderophores and metal–siderophore complexes to mineral surfaces, and competitive metal-siderophore complex formation through parallel mineral dissolution pathways. This rich interweaving of chemical processes gives rise to an intricate tapestry of interactions, particularly in respect to the biogeochemical cycling of Fe and Mn in marine ecosystems.     

<Emphasis Type="Italic">“Pseudoalteromonas januaria”</Emphasis> SUT 11 as the Source of Rare Lipodepsipeptides

The marine bacterium “Pseudoalteromonas januaria” SUT 11 isolated from a seawater sample produced the rare cell-bound cyclic lipodepsipeptides A/A′, B/B′, and C/C′. The matrix-assisted laser desorption/ionization mass spectra indicated that one bromine atom presented in the peptides B/B′ and C/C′, whereas the component A/A′ contained no bromine atom. The acyldepsipeptides A/A′–C/C′ have an identical amino acid sequence, Thr-Val-Asn-Asn-Leu/allo-Ile, but differed in C-terminal amino acid and acyl moieties. Peptides A–C have Leu as a C-terminal amino acid, whereas peptides A′-C′ have allo-Ile. Acyl moieties in peptides A/A′, B/B′, and C/C′ have been found to consist of 11-(4′-hydroxyphenyl)-undeca-2,4,6,8,10-pentaenic acid, 9-(3′-bromo-4′-hydroxyphenyl)-nona-2,4,6,8-tetraenic acid, and 11-(3′-bromo-4′-hydroxyphenyl)-undeca-2,4,6,8,10-pentaenic acid, respectively. The structure of a main pair of peptides B/B′ with molecular masses 843/845 Da has been determined by means of ultraviolet, infrared, and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that tandem nano-electrospray ionization mass spectrometry is a very efficient way for the fast and sensitive investigation of lipopeptides A/A′ and C/C′ with molecular masses 791 and 869/871 Da, respectively, which have been isolated in small amounts.     

Purification and characterization of rhodobactin: a mixed ligand siderophore from Rhodococcus rhodochrous strain OFS

The siderophore produced by Rhodococcus rhodochrous strain OFS, rhodobactin, was isolated from iron-deficient cultures and purified by a combination of XAD-7 absorptive/partition resin column and semi-preparative HPLC. The siderophore structure was characterized using 1D and 2D ¹H, ¹³C and ¹⁵N NMR techniques (DQFCOSY, TOCSY, NOESY, HSQC and LR-HSQC) and was confirmed using ESI-MS and MS/MS experiments. The structural characterization revealed that the siderophore, rhodobactin, is a mixed ligand hexadentate siderophore with two catecholate and one hydroxamate moieties for iron chelation. We further investigated the effects of Fe concentrations on siderophore production and found that Fe limiting conditions (Fe concentrations from 0.1 μM to 2.0 μM) facilitated siderophore excretion. Our interests lie in the role that siderophores may have in binding metals at mixed contamination sites (containing metals/radionuclides and organics). Given the broad metabolic capacity of this microbe and its Fe scavenging ability, R. rhodochrous OFS may have a competitive advantage over other organisms employed in bioremediation. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence for rapid microscale bacterial redox cycling of iron in circumneutral environments

The potential for microscale bacterial Fe redox cycling was investigated in microcosms containing ferrihydrite-coated sand and a coculture of a lithotrophic Fe(II)-oxidizing bacterium (strain TW2) and a dissimilatory Fe(III)-reducing bacterium (Shewanella alga strain BrY). The Fe(II)-oxidizing organism was isolated from freshwater wetland surface sediments which are characterized by steep gradients of dissolved O₂ and high concentrations of dissolved and solid-phase Fe(II) within mm of the sediment–water interface, and which support comparable numbers (10⁵–10⁶ mL⁻¹) of culturable Fe(II)-oxidizing and Fe(III)-reducing reducing. The coculture systems showed minimal Fe(III) oxide accumulation at the sand-water interface, despite intensive O₂ input from the atmosphere and measurable dissolved O₂ to a depth of 2 mm below the sand–water interface. In contrast, a distinct layer of oxide precipitates formed in systems containing Fe(III)-reducing bacteria alone. Examination of materials from the cocultures by fluorescence in situ hybridization indicated close physical juxtapositioning of Fe(II)-oxidizing and Fe(III)-reducing bacteria in the upper few mm of sand. Our results indicate that Fe(II)-oxidizing bacteria have the potential to enhance the coupling of Fe(II) oxidation and Fe(III) reduction at redox interfaces, thereby promoting rapid microscale cycling of Fe. This revised version was published online in August 2006 with corrections to the Cover Date.