Self-Assembling Amphiphilic Siderophores from Marine Bacteria

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Science  18 Feb 2000:
Vol. 287, Issue 5456, pp. 1245-1247
DOI: 10.1126/science.287.5456.1245


Most aerobic bacteria secrete siderophores to facilitate iron acquisition. Two families of siderophores were isolated from strains belonging to two different genera of marine bacteria. The aquachelins, from Halomonas aquamarina strain DS40M3, and the marinobactins, from Marinobacter sp. strains DS40M6 and DS40M8, each contain a unique peptidic head group that coordinates iron(III) and an appendage of one of a series of fatty acid moieties. These siderophores have low critical micelle concentrations (CMCs). In the absence of iron, the marinobactins are present as micelles at concentrations exceeding their CMC; upon addition of iron(III), the micelles undergo a spontaneous phase change to form vesicles. These observations suggest that unique iron acquisition mechanisms may have evolved in marine bacteria.

Low iron concentrations in surface seawater [typically from 20 pM to 1 nM (1)] limit primary production by phytoplankton in regions characterized by high concentrations of nitrate and other nutrients but low concentrations of chlorophyll (HNLC, high nitrate low chlorophyll) (2). In addition to phytoplankton and cyanobacteria, heterotrophic bacteria make up an important class of microorganisms in the ocean that are also limited by low iron levels in HNLC regions (3–5). Heterotrophic bacteria constitute up to half of the total particulate organic carbon in ocean waters (4), and in some regions, such as the subarctic Pacific, heterotrophic bacteria can even contain higher cellular concentrations of iron than phytoplankton (5). Heterotrophic bacteria thus compete successfully for iron against phytoplankton and cyanophytes and play a substantial role in the biogeochemical cycling of iron in the ocean. However, little is known about the molecular mechanisms used by marine bacteria, in particular, and other marine microorganisms, in general, to sequester iron. Marine bacteria are known to produce siderophores (6–8), which are low–molecular weight compounds secreted to scavenge Fe(III) from the environment and to facilitate its uptake into microbial cells. We report herein the structures and properties of a class of self-assembling amphiphilic siderophores produced by marine bacteria. Two families of siderophores, produced by two different genera of bacteria, each contain a unique peptidic head group that coordinates Fe(III) and one of a series of fatty acid tails.

Three strains, designated DS40M3, DS40M6, and DS40M8, were isolated from the same sample of ocean water, which had been collected at a depth of 40 m over the continental slope in the eastern equatorial Atlantic (7). The aquachelin siderophores (Fig. 1), produced by Halomonas aquamarina DS40M3 (Fig. 2), and the marinobactin siderophores (Fig. 1), produced by Marinobacter species strains DS40M6 and DS40M8 (Fig. 2), were isolated and purified from the supernatant of bacterial cultures, as previously described (7). The amino acid composition of the aquachelins and marinobactins, including the enantiomeric configuration, was determined with Marfey's reagent [N-a-(2,4-dinitro-5-fluorophenyl)-l-alaninamide] (9). The amino acid sequence was established by tandem mass spectrometry (Fig. 1) and confirmed by nuclear magnetic resonance (NMR) spectroscopy (10). The position of the d- andl-amino acids was determined from amino acid analysis of partially hydrolyzed peptide fragments generated from the native siderophore (11). Elucidation of the fatty acid moieties involved gas chromatography–mass spectrometry comparison to standard methyl ester derivatives, ozonolysis to establish the position of the double bond, and NMR to elucidate the configuration of the double bond (10). The connectivity of diaminobutyric acid and β-hydroxyaspartic acid in the marinobactin ring was determined by NMR (10).

Figure 1

The structures of the marinobactins and aquachelins. The vertical lines through the structures show the mass-to-charge ratio values for the y and b fragments obtained for marinobactin E and aquachelin D by tandem mass spectrometry. The “y” and “b” nomenclature refers to the charge when retained by the COOH-terminal fragment or the NH2-terminal fragment of the peptide, respectively (24).

Figure 2

Phylogenetic tree of marine proteobacteria based on maximum parsimony analysis of 16S rRNA gene sequences (25). The tree is one of eight best trees obtained by the branch and bound search algorithm. The eight trees vary only in the arrangement within the genera Marinobacter andHalomonas. Branch lengths are proportional to number of changes, with a transversion cost of two. Bootstrap support of greater than 70% is indicated next to nodes. The position of the outgroup (alpha proteobacterium Agrobacterium tumefaciens) is indicated by an arrow. In addition to aquachelin D and marinobactin E, the structures of aerobactin (7), vulnibactin (8), and anguibactin (26) are shown next to the marine bacterium from which they are produced.

The only terrestrial siderophores that bear a structural resemblance to marinobactins and aquachelins are the mycobactins and exochelins produced by mycobacteria, such as Mycobacterium tuberculosis, which also usually contain a fatty acid tail (12, 13). The exochelins and mycobactins share a common hydrophilic core that coordinates Fe(III), but they differ in the substitution and chain length of the fatty acid. The hydrophilic exochelins, which are secreted into the growth medium, have short alkyl side chains (C2-C8) and a terminal methylester moiety (12), whereas the mycobactins, which remain associated with the bacterium, have longer fatty acid groups (C16-C21) (13). The mycobacteria acquire iron by a shuttle system in which the secreted exochelins transport ferric ion to the membrane-associated mycobactins (12). However, it is clear that the iron acquisition process of mycobacteria differs fundamentally from that of H. aquamarina and the Marinobacter species. The marinobactins, aquachelins, and mycobactins have long fatty acid moieties, but only the marinobactins and aquachelins are secreted into the growth medium, and they are surprisingly hydrophilic. In addition, we find no evidence in the marine bacteria for cell membrane–associated siderophores, further illustrating the difference in iron uptake strategies of these bacteria.

The unique feature of the marinobactin and aquachelin siderophores, with their polar peptidic head groups and hydrophobic fatty acid tails, is their amphiphilic, surface-active nature, which leads to the formation of self-assembled structures. The iron-free marinobactins are characterized by unusually low critical micelle concentrations (CMC) ranging from 25 μM for marinobactin E to 150 μM for the shorter fatty acid chain lengths (14). The ferrated marinobactins also behave as surface-active agents, with slightly higher CMC values (15). Dynamic light-scattering results on a 0.2 mM solution of [Fe(III)–marinobactin E] indicate the presence of spherical particles ranging in diameter from 140 to 180 nm (16), whereas the corresponding iron-free form of marinobactin E did not scatter light, suggesting that it is limited to micellar assembly. Similar results were obtained for aquachelins A and D. Cryoelectron microscopy on a 2 mM solution of the [Fe(III)–marinobactins D] complex demonstrated the formation of polydisperse spherical vesicles ranging in size from 50 to 200 nm (Fig. 3). These vesicles spontaneously self-assemble in solution upon addition of Fe(III) to the desferri form of the siderophore. The tendency of the free ligand to form micelles is not surprising, given that single-chain surfactants generally form micelles as a result of the large polar head group compared with the nonpolar tail, promoting a conically shaped molecule. As predicted, the transition from micelle to vesicle upon coordination of ferric ion is consistent with a more cylindrically shaped molecule (17).

Figure 3

Cryoelectron micrograph of Fe(III)–marinobactin D siderophore vesicles in aqueous solution. Representative vesicles are indicated by arrows. Samples were prepared for imaging by the method of Bellare et al. (27). The vesicles shown here result from 2 mM Fe(III)–marinobactins D in 100 mM tris-HCl (pH 8.0); the sample was rapidly frozen from a temperature of 25°C (28). The ratio of marinobactin D2 to D1 was about 3:1.

The transformation from micelle to vesicle upon Fe(III) coordination (Fig. 4) is the first example, to our knowledge, of such a metal-induced phase change in a biologically produced compound. The presence of this metal-induced switch raises questions about the physiological role for this transformation. One potential physiological advantage of a micelle or vesicle structure, as opposed to a monomolecular species, might be protection from proteolytic cleavage, as has been recently demonstrated for synthetic lipopeptides (18). In fact, proteases and other degradative enzymes are widely dispersed in ocean water and even reside on the surface of pelagic marine bacteria (19). Although we would not expect micelles or vesicles to form in the low dissolved organic carbon and iron conditions of seawater, appropriate conditions might exist in particles or other microenvironments. These assemblies and the general lipophilic nature of the siderophores could represent a specific adaptation, perhaps for concentration of iron or for increasing the residence time in such habitats.

Figure 4

Schematic representation of the phase change from a micellar assembly of the marinobactins in the absence of Fe(III) to vesicle formation upon coordination of Fe(III) to the siderophore. The relative head group size between micelle and vesicle is not drawn to scale.

The aquachelins produced by H. aquamarina strain DS40M3 and the marinobactins produced by Marinobacterstrains DS40M6 and DS40M8 are remarkably similar in that each has a peptidic head group that coordinates Fe(III) and a fatty acid tail, which confers the lipophilic properties to this class of siderophores. It is quite striking that these siderophores, whose distinctive properties hint at the possibility of a novel iron acquisition mechanism, are made by strains from two different genera within the gamma proteobacteria. Whether the structural strategy represented by these siderophores constitutes a specific adaptation to the seawater environment and whether it is widespread among marine bacteria are important questions for further studies.

  • * To whom correspondence should be addressed. E-mail: Butler{at}


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