Prof. Alison Butler

Prof. Alison Butler,
UC Santa Barbara.

A ferrisiderophore binding protein in E. coli.

vesicles

Micelles (top) and a vesicle (bottom) formed by marinobactins before and after iron binding, respectively.

Chelation, uptake, and binding of trace metals


Background

    Chelation and uptake

Upon entering sea water, many important trace metals are quickly bound to chelators, organic molecules produced by marine bacteria and possibly some microalgae, to scavenge essential trace metals from the environment and facilitate their uptake by microbial cells.

Iron, zinc, and other trace metals are used by these organisms to build enzymes that catalyze biochemical reactions at key points in the nitrogen and carbon cycles. There is evidence, for example, that in regions of the world's oceans iron scarcity limits primary production, the transformation of carbon from inorganic forms to organic forms. Primary production is the ultimate source of the organic carbon molecules forming the bulk of the tissue of all living organisms. Primary production is nature's most effective way of removing the greenhouse gas CO2 from the atmosphere. So there is good reason to want to know how trace metals are chelated, taken up, and used.

Not much is known about trace metal chelation and uptake. We don't know, for example, how many different kinds of chelators there are. With a few exceptions we don't know what their structure is, or how microbes detect them and take them in. Except for iron chelators we don't know precisely what their function is.

Because its solubility in sea water is low, iron is rare in the open ocean. When iron is scarce, marine bacteria (and possibly some kinds of microalgae) make siderophores, small molecules that like to be bound to iron. Most iron chelators are probably siderophores or their break-down products. Siderophore production and binding is part of a specialized biological machinery that helps these organisms harvest iron. This machinery includes not only the siderophore-producing proteins, but also cell-membrane-bound proteins that enable ingestion of siderophore-complexed iron.

Siderophores effectively increase the solubility of iron in sea water, allowing ecosystems to support more microbes by making more iron available for essential biological processes. But chelated iron is available only to organisms that can take it in.

Most microalgae can't directly ingest siderophore-bound iron, but evolution has endowed them with a different technology for getting the iron they need: reductase enzymes that take apart chelated iron complexes outside the cell. These enzymes help microalgae compete for iron with siderophore-producing bacteria.

Cebic scientists are working on several fronts to understand these processes. In Cebic's first years the focus has been on understanding siderophore structure and function. In the coming years Cebic scientists begin in earnest studying iron-uptake mechanisms in siderophore-producing bacteria and marine microalgae.


    Storage

Trace metals are essential to the physiology of microorganisms, but when cellular concentrations get too high they can be toxic. Iron, for example, can cause oxygen to react destructively with many biomolecules.

So microorganisms have evolved mechanisms for buffering free trace metals: when an organism takes in more metal than it needs it stores the excess in either storage proteins like ferritins or intracellular chelators like phytochelatins and metallothioneins.

Intracellular storage of trace metals is clearly advantageous when there are too many trace metal ions; it also seems to be an advantage later when there are too few. Metal ions stored when abundant may be used to build essential proteins when there aren't enough metals in an organism's immediate environment to support cell growth and reproduction.

In many organisms, including humans, the amount of ferritin is a good indicator of the sufficiency or deficiency of an organism's iron supply. The situation is probably much the same in marine microbes. Once we know how, counting ferritins will probably will be a good way to determine whether iron is abundant or scarce in a particular marine ecosystem, and—hence—whether adding iron is likely to increase the rate of growth of (e.g.) carbon-fixing microbes and the rate of removal of CO2 from the atmosphere.

Scientists have detected iron-binding storage proteins (ferritins and bacterioferritins) in laboratory cultures, but never in aquatic microbes in the field. Cebic is developing techniques to detect, identify, and survey iron-storage proteins in marine microorganisms so that their functions—in organisms and global environment—can be studied in context. Cebic is especially interested in comparing ferritin structure and function in organisms that thrive in iron-rich and iron-poor waters.

Unlike ferritins, phytochelatins, which are known to chelate metals like cadmium, zinc, and copper, have been detected in the field—inside phytoplankton—and their intra-cellular concentration has been found to respond to changes in the amount of trace-metal in the surrounding water. Yet little is known about phytochelatins. We lack the most basic knowledge about their structure. We don't know if they, like ferritins, store trace metals temporarily, or whether they sequester metals permanently to mitigate toxicity. We also don't know the full range of metals that phytochelatins can bind to.

Cebic will study the binding by phytochelatins of metals other than iron, mainly by observing the coordination of metals by various ligands using a cluster of related analytical techniques known collectively as X-ray absorbtion spectroscopy, or XAS.

This work has both general and specific objectives. The general objective is to develop techniques to study metal coordination in important biochemical systems. The specific objective is to learn about the binding of metals by phytochelatins: what metals do they bind? What is the fate of these metals? Are they eventually reused or are they bound forever?


Progress

    Marine siderophores

Alison Butler, Jennifer Martinez, and their colleagues at UC-Santa Barbara, in collaboration with Margo Haygood's research group at the Scripps Institution of Oceanography, have isolated and characterized several siderophores from marine microorganisms. These siderophores—the first marine siderophores to be characterized—are structurally quite different from known terrestrial siderophores. Aquachelins from Halomonas aquamarina DS40M3 and marinobactins from Marinobacter sp. DS40M6 and DS40M8 both have a water-insoluble fatty acid part and a water-soluble peptide part. This unusual structure causes these amphiphilic siderophores to form, in the absence of iron, micelles: clusters of molecules attached together by their fatty-acid tails. Upon binding to Fe(III), micelles come together to form vesicles: spherical shells, hollow in the middle, with many siderophore molecules bound together. This work, published in Science has spawned speculation about what role, if any, the amphiphilic nature of these marine siderophores plays in iron uptake. Do micelles and vesicles perform an essential function, or is this striking behavior merely an interesting accident?

Martinez, J. S., Zhang, G. P., Holt, P. D., Jung, H.-T., Carrano, C. J., Haygood, M. G., Butler, Alison, Self-Assembling Amphiphilic Siderophores from Marine Bacteria. Science 287: 1245-47 (2000)

*        *        *

Scientists sometimes use desferrioxamine B, a siderophore from a terrestrial bacterium, to reduce the amount of iron available to microbes in sea water. This allows scientists to study how marine microbes respond to extremely low concentrations of available iron. It is assumed in these experiments that terrestrial siderophores are different enough from marine siderophores that terrestrial-siderophore-bound iron will be unavailable to marine microbes. And for the most part it seems to be true.

But Martinez, Haygood, and Butler have found that marine and terrestrial siderophores can be very much alike. The scientists isolated and characterised a marine siderophore, desferrioxamine G, that is almost identical to desferrioxamine B. Because a marine bacterium produces desferrioxamine G, it is very likely that it, and maybe some other marine bacteria, will be able to take up and utilize desferrioxamine-B-bound iron. So it may be necessary to reexamine those experiments that assumed that the complexed iron was unavailable to all marine microbes.

Martinez, JS, Haygood, MG, and Butler, Alison. Identification of a natural desferrioxamine siderophore produced by a marine bacterium. Limnol. Oceanogr. 46(2) 420-424 (2001).

*        *        *

    Phthalate uptake

Not all metal chelators are biological; some common human-made pollutants also chelate metals. One example is phthalates, chemicals used in the manufacture of soft-plastic products. Environmental phthalates have become common in recent years, and when present in sea water they scavenge metals that would otherwise be available to microorganisms.

But some microorganisms are able to metabolize phthalates; as environmental phthalates have become more common, these organisms have increased in number. Cebic scientists Huang-Kuang Chang and Gerben Zylstra of Rutgers have studied one such organism, Burkholderia cepacia ATCC 17616, by expressing its OphD gene in E. coli, which normally lacks the ability to take in and metabolize phthalate. The mutated E. coli was able to transport phthalate across its cell membrane, demonstrating that the OphD gene is responsible for phthalate uptake.

However, Chang and Zylstra also found that when the OphD gene was "knocked out" of B. cepacia the organism was still able to take in phthalate. So the OphD phthalate uptake pathway isn't the only one available: B. cepacia has apparently evolved redundant means of phthalate acquisition. Chang and Zylstra's work was published in the Journal of Bacteriology:

Chang, H.-K., and Zylstra, G. J. Characterization of the phthalate permease OphD from Burkholderia cepacia ATCC 17616. J. Bacteriol. 181: 6197-6199 (1999).

*        *        *

Determining the speciation of trace metal ions is important but technically difficult. Measuring total concentration of a metal in a seawater sample does not provide information about the amount of free metal ion, which is most likely the form of the metal ion that is toxic. In this study three different research groups (Bruland & Rue from UC Santa Cruz, Donat & Skrabal from Old Dominion University, and Moffett from Woods Hole) used three methods of competitive ligand equilibrium/adsorptive cathodic stripping voltammetry with different ligands. All of the groups analyzed the same water sample and all groups determined that more than 99.97% of the dissolved copper was chelated with strong copper-binding ligands. The presence of strong chelating ligands lowers the free copper concentration to approximately 1 x 10-13 M, a level thought not to be toxic even to the most metal sensitive organisms.

K.W. Bruland, E.L. Rue, J.R. Donat, S.A. Skrabal, and J.W. Moffett. Intercomparison of voltametric techniques to determine the chemical speciation of dissolved copper in a coastal seawater sample, Analytica Chimica Acta, 405: 99-113 (2000).

*        *        *

Certain toxigenic species of the diatom Pseudo-nitzschia produce the toxin domoic acid, which can harm humans and kill birds and mammals. What is the physiological role of domoic acid? The authors show that domoic acid can bind iron and copper, and they determine the conditional stability constants against select ligands. Given the strength of these stability constants and the concentration of domoic acid in algal blooms, domoic acid is capable of affecting the speciation of copper and iron in the environment, perhaps increasing the bioavailability of iron and diminishing the bioavailability of copper. The authors further find that cultures grown under iron-limiting conditions produced an order of magnitude more domoic acid (or, anyway, of iron-binding chelators with the same conditional binding constant as domoic acid) and cultures grown under elevated copper conditions produced nearly 20 times more copper-binding chelator with a similar conditonal binding constant as domoic acid. This result is again consistent with domoic acid being a metal-regulating molecule. There is evidence that pseudo-nitzschia has a high iron requirement. It has been observed to be selected for in iron addition experiments. Perhaps an initial influx of iron leads to an algal bloom but then as the iron concentration starts to diminish when algae grow, the algae secrete domoic acid.

E.L. Rue and K.W. Bruland. Domoic acid binds iron and copper: a possible role for the toxin produced by the marine diatom Pseudonitschia, Marine Chemistry 76: 127-134 (2001).

*        *        *

The authors collected and filtered a lot of sea water - more than 200 liters for each sample. They determined the molecular weight classes of the adsorbed material, and found that 63 percent of the extracted compounds fell within the range of siderophores (300-1000 Dalton). The isolated compounds had conditional iron-binding affinities similar to purified marine siderophores produced in laboratory cultures and to the ambient iron-binding ligands observed in seawater. Hydroxymate or catecholate functional groups were present in each compound for which iron binding was detected. The collection technique appears to be working and holds the promise that sufficient quantities of siderophores will be collected from natural waters to facilitate structural characterization.

H.M. Macrellis, C.G. Trick, E.L. Rue, G. Smith, K.W. Bruland. Collection and detection of natural iron-binding ligands from seawater, Marine chemistry 76: 175-187 (2001).

*        *        *

Iron siderophores are characterized by their exceptionally high affinities for iron. The high affinities enable the siderophores to bind tightly to iron even in environments where iron concentrations are very low. How, then, is iron made bioavailable? CEBIC scientists have discovered a possible mechanism. Through a series of experiments involving careful structural analysis of reaction products, Barbeau, Rue, Bruland, and Butler identified a chemical reaction involving siderophores that may be important in the environment. In the presence of sunlight, iron(III) aquachelin (a marine siderophore) undergoes a photochemical reaction that results in the chemical transformation of the ligand and the reduction of Fe(III) to Fe(II). The photochemical reaction breaks the ligand apart, producing a peptide component and a fatty acid component. The peptide component can still bind iron but with lower efficiency than the whole ligand. Experiments in which the authors used labeled iron siderophores (Fe59) showed that iron was more bioavailable in the presence of photolyzed ironsiderophores than unphotolyzed iron siderophores, consistent with the hypothesis that photochemical lability of iron from iron siderophore complexes is important in iron cycling in the marine environment.

K. Barbeau, E.L. Rue, K. W. Bruland and A. Butler. Photochemical cycling of iron in the surface ocean mediated by microbial iron(III)-binding ligands, Nature 413: 409-413 (2001)

 Next project: Trace metals, enzymes, & biogeochemical cycles
 Return Home

© 2000 The Princeton Environmental Institute, Princeton, New Jersey. Cebic is an Environmental Molecular Sciences Institute made possible by grants from the National Science Foundation and the U.S. Department of Energy. François M. M. Morel, Director.