Structure and Membrane Affinity of a Suite of Amphiphilic Siderophores Produced by a Marine Bacterium

Structure and membrane affinity of a suite of amphiphilic siderophores produced by a marine bacterium Jennifer S. Martinez*, Jayme N. Carter-Franklin*, Elizabeth L. Mann†, Jessica D. Martin*, Margo G. Haygood†, and Alison Butler*‡ *Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510; and †Marine Biology Research Division and Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093-0202 Edited by Jack Halpern, The University of Chicago, Chicago, IL, and approved January 31, 2002 (received for review December 6, 2002) Iron concentrations in the ocean are low enough to limit the The relative scarcity of iron in the oceanic environment, growth of marine microorganisms, which raises questions about where the diffusion of siderophores may be of concern, raises the molecular mechanisms these organisms use to acquire iron. questions about the types of siderophores that marine bacte- Marine bacteria have been shown to produce siderophores to ria produce. Recently, we reported two suites of amphiphilic facilitate iron(III) uptake. We describe the structures of a suite peptide siderophores, the marinobactins and aquachelins, of amphiphilic siderophores, named the amphibactins, which which are produced by distinct genera of marine bacteria are produced by a nearshore isolate, ␥ Proteobacterium, Vibrio (Fig. 1) (16). The marinobactins, produced by the Marino- sp. R-10. Each amphibactin has the same Tris-hydroxamate- bacter sp. strain DS40M6, are characterized by a 6-aa peptide containing peptidic headgroup composed of three ornithine resi- headgroup that coordinates iron through two hydroxamate dues and one serine residue but differs in the acyl appendage, groups and a ␤-hydroxy-aspartic acid residue. The N terminus which ranges from C-14 to C-18 and varies in the degree of sat- contains an appendage of one of a series of fatty acid resi- uration and hydroxylation. Although amphiphilic siderophores dues, ranging from C-12 to C-16. The aquachelins, produced are relatively rare, cell-associated amphiphilic siderophores are by Halomonas aquamarina, are characterized by a 7-aa pep- even less common. We find that the amphibactins are cell- tide with N-terminal appendages ranging from C-12 to C-14 associated siderophores. As a result of the variation in the na- fatty acids. The suite of fatty acids found in marinobactins ture of the fatty acid appendage and the cellular location of the and aquachelins comprises both saturated and unsaturated amphibactins, the membrane partitioning of these siderophores alkyl chains containing one double bond in the cis configura- was investigated. The physiological mixture of amphibactins had tion. The marinobactins have been shown to partition into ؋ 3 ؋ 2 ؊1 a range of membrane affinities (3.8 10 to 8.3 10 M ) that semisynthetic vesicles (23), indicating that these amphiphilic are larger overall than other amphiphilic siderophores, likely peptides could potentially associate with bacterial membranes accounting for their cell association. This cell association is likely as a means of preventing siderophore diffusion. However, an important defense against siderophore diffusion in the under our growth and isolation conditions, both the marino- oceanic environment. The phylogenetic affiliation of Vibrio sp. bactins and aquachelins are primarily observed in the growth R-10 is discussed, as well as the observed predominance of am- medium. Thus, although the marinobactins and aquachelins phiphilic siderophores produced by marine bacteria in contrast to those produced by terrestrial bacteria. can associate with biological membranes, they are also quite water-soluble. Hundreds of siderophores have been structurally character- defining characteristic of vast regions of the world’s ized, yet few are amphiphilic (24). Other amphiphilic sid- Aoceans is the surprisingly low level of iron in surface erophores include the ornibactins, produced by Burkholderia waters, in contrast to the abundance of iron in most terres- (Pseudomonas) cepacia; corrugatin, produced by Pseudomonas trial environments. Iron is an essential nutrient for the growth corrugata; acinetoferrin, produced by Acinetobacter haemolyti- of microorganisms (1) and an important bioactive metal in cus; rhizobactin 1021, produced by Sinorhizobium meliloti; and seawater. The low level of iron is now known to limit primary the carboxymycobactins, produced by mycobacteria, all of production by autotrophic microorganisms in ocean regions which are produced and secreted into the growth medium by that are replete in major nutrients such as nitrate, phosphate, terrestrial bacteria. With the exception of the ornibactins and and silicate (2–8). Results from four separate mesoscale addi- ⅐ carboxymycobactins, these are all single siderophores as op- tions of iron (1–2 nM, FeSO4 7H2O) to large patches of ocean water (e.g., Ϸ70–100 km2) in the equatorial Pacific (2, 7, 8) posed to a suite of siderophores that vary in the nature of the fatty acid tail (25–30). Mycobacteria, as well as other high and the Southern Ocean (5, 6) show that primary production ϩ by photosynthetic microorganisms increased markedly in re- G C Gram-positive bacteria, also produce the only struc- sponse to iron. Bacterial production can also be either di- turally characterized family of siderophores to date that are rectly or indirectly stimulated by the addition of iron (9–11), associated with the bacterial membrane and are not secreted although in some situations this occurs only after carbon limi- into the growth medium. The mycobactins (Fig. 1) produced tation has been relieved (12). In any case, bacteria have rela- by mycobacteria are a suite of lipidic siderophores that differ tively high Fe:C ratios and clearly play a role in biogeochemi- cal cycling of iron (9). This paper was submitted directly (Track II) to the PNAS office. To acquire iron, many aerobic bacteria, including marine Abbreviations: DMPC, dimyristoylphosphatidylcholine; SSU, small subunit; RDP, Ribosomal species (13–20), produce siderophores. These low molecular Database Project. weight compounds chelate iron(III) with high affinity and are Data deposition: The sequences reported in this paper have been reported in the GenBank synthesized in response to low iron concentrations (21, 22). database (accession nos. AY217768 Ochrobactrum SP18; AY217769 Agrobacterium SP25; Siderophores function in the receptor-mediated transport of AY217770 Vibrio R-10, 5Ј end of the SSU rRNA gene; AY217771 Vibrio R-10, 3Ј end; iron into microbial cells, as well as in the extracellular chela- AY217772 Vibrio BLI-41; and AY217773 Pseudoalteromonas luteoviolacea strain S2. tion of iron(III). ‡To whom correspondence should be addressed. E-mail: [email protected]. 3754–3759 ͉ PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0637444100 Downloaded by guest on September 26, 2021 SPECIAL FEATURE Fig. 1. Structures of the amphiphilic marinobactins and aquachelins produced by Marinobacter sp. strain DS40M6 and H. aquamarina strain DS40M3, respectively, and the lipophilic mycobactins produced by mycobacteria (16, 25). primarily in fatty acid chain length (C-9 to C-21) or minor erophores normally associated with the cell pellets could, on modifications in the head group (25, 28). Mycobactins coordi- rare occasions, be found in the supernatant as well. These nate iron(III) through both oxygen atoms of the hydroxamate secreted siderophores were isolated from the supernatant by groups and the nitrogen and oxygen of the 2-hydroxyphenyl- batch adsorption onto Amberlite XAD-2 resin (Supelco) and oxazoline moiety. The formobactins, nocobactins, and elution with methanol as previously described (17). Siderophore- amamistatins, isolated from Nocardia asteroides, Rhodococcus containing fractions, corresponding to elution with 100% bronchialis, Rhodococcus rubropertinctus, and Rhodococcus methanol, were pooled, dried in vacuo, and applied to a C4 CHEMISTRY terrae, are nearly identical to the mycobactin siderophores in reversed-phase HPLC column (Vydac, Hesperia, CA). The both head group and hydrocarbon tail and are also found siderophores were purified by using a gradient of 100͞0 within the bacterial cellular membrane (31–35). (% A͞B) to 0͞100 (% A͞B) over 37 min, and the program We describe a suite of amphiphilic siderophores produced was held an additional 10 min at 0͞100 (% A͞B), after which by a marine Gram-negative bacterium (Vibrio sp. R-10). the gradient was reversed, returning to 100͞0(%A͞B) over ϭ These siderophores, named the amphibactins, are defined by 10 min [A 99.95% ddH2O (Nanopure) and 0.05% triflu- ϭ a short peptide headgroup and one of a series of saturated, oroacetic acid (TFA); B 19.95% ddH2O, 0.05% TFA, and unsaturated, or hydroxylated fatty acid appendages ranging 80% methanol]. The absorbance of the eluent was monitored from C-14 to C-18. The amphibactins differ from the mari- at 215 nm. nobactins and aquachelins (Figs. 1 and 2) by a smaller head group (four vs. six and seven amino acids, respectively) and Structure Determination. The masses and amino acid connectiv- generally longer fatty acid tails. Like the mycobactins, this ity of the amphibactins were determined by electrospray MS suite of amphiphilic siderophores is cell-associated. Because and tandem MS with instrumentation similar to that de- siderophores are virulence factors for many pathogenic bacte- scribed previously (16, 17). The

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