Chelation, uptake, and intracellular binding of trace metals

Extra-cellular iron siderophores: structure and regulation

We know from electrochemical measurements that dissolved iron in surface sea water is complexed by small, strong chelators. Laboratory studies show that some marine bacteria release siderophores — low molecular weight, strong, specific iron-complexing agents. These siderophores are probably either the strong chelators revealed by electrochemical measurements, or their break-down products.

Cebic's first objective is to characterize the chelating agents from important marine bacteria in order to determine how much of the iron that gets complexed in seawater is complexed by siderophores and their break-down products. The first step is to collect samples of the two most important types of siderophore-producing microorganisms: a photoautotrophic cyanophyte (Synechococcus), and a series of heterotrophic bacteria (the DS40M series, including D. aquamarina and a species of Marinobacter). The organisms will be isolated from samples of water collected in iron-limited ocean regions, since organisms in those regions are the most likely to produce siderophores in abundance. When suitable organisms have been isolated, we will employ established HPLC techniques to isolate siderophores from laboratory cultures. These siderophores will be characterized structurally, then used both to develop field-friendly analytical techniques and to study mechanisms of degradation and regulation. Details follow.

Structural characterization: Chiral amino acid analysis elucidates the amino acid composition of the isolated samples, while mass spectral techniques (ESI, tandem ESI, etc) provide information on molecular weight and amino acid sequence. One- and two-dimensional NMR (1H, 13C) elucidate the structure of other portions of the siderophores, and confirm results of other structural characterization techniques.

Development of field analytical techniques. While exquisitely sensitive, existing field techniques for organically bound metals are unable to resolve the nature of ligands. To detect siderophores in sea water we need extremely sensitive and highly specific techniques that can resolve subtle differences in a siderophore's structure. This means devising preconcentration protocols for use in the field, based on extraction techniques used for culture samples. It also means developing chromatographic techniques capable of revealing small structural differences in the ligands. Preliminary work suggests that both tasks are feasible.

Degradation studies. Simple laboratory studies of the microbial and photochemical degradation of siderophores in both free and iron-bound forms can tell us how long these compounds survive in seawater, and also help identify siderophore breakdown products, which (instead of, or in addition to, the chelators themselves) may be the organic ligands that complex the bulk of iron in the ocean.

Regulation. By measuring siderophore production under a range of metal concentrations in laboratory conditions we will study how, and to what extent, the environmental concentration of iron effects the production of siderophores. This work will be done in conjunction with the next project: Siderophore uptake.

Siderophore uptake

Here, we aim to identify the receptors responsible for transporting the characterized siderophore(s) from the outside of a cell to the inside, to determine the chemical specificity of the receptors, to elucidate the kinetics of siderophore uptake, and to develop markers for field studies.

As a first step, we need to gain control over siderophore receptor production. So we will mutate our model siderophore-producing organisms until we see signs that the mutation has affected the production of receptor proteins. "Receptor mutants" are easily recognized because they over-produce siderophore relative to the wild-type strain: without receptor proteins, the cell is starved for iron so it produces more siderophores in a futile effort to take in more iron.

Once we've succeeded in creating receptor mutants, we can compare the relevant parts of the DNA of the mutant and wild-type organisms in order to locate the mutated gene — the gene responsible for the production of receptor proteins. A similar procedure will allow us to locate the gene for siderophore production.

We can also use standard methods like protein electrophoresis to identify the actual receptor protein: this protein will be present in the wild-type organism, but missing from the mutant.

Once we know the DNA sequence that codes for the receptor protein, we can clone the gene and express it in a host under laboratory conditions. We can do the same thing with the gene for siderophore production. By making the protein and siderophore in excess and putting them together in simplified systems with radio-labeled iron, we can study the chemistry of the interaction under controlled conditions.

Iron uptake by phytoplankton: extra-cellular iron reductase

The bacteria that produce siderophores and other chelating agents make iron more abundant, but the siderophores they produce present a challenge to other microbiota, especially eukaryotic phytoplankton, which must disassemble the chelate-metal complexes to get the iron they need. From what we know of iron transport in a number of representative species of microalgae, organic iron complexes cannot cross the cell membrane of eukaryotic phytoplankton. Iron acquisition in these organisms depends on an adequate supply of dissolved inorganic species - iron that isn't bound to biological chelating agents. But ambient concentrations of inorganic iron species are so low, and the undissolved iron particulates so inert, that the supply of available inorganic iron is insufficient to meet their requirements.

Yeast and some higher plants reduce iron bound to organic complexes outside the cell, then take in the reduced iron. Cebic scientists have discovered that oceanic phytoplankton have a similar technology.

Cebic's goal is to figure out how they do it. Specifically, our objectives are:

• to isolate and characterize the membrane-bound iron reductase of microalgae
• to determine its chemical specificity - which species of organic iron can it capture and reduce?
• to study its physiological regulation - how does iron reductase production respond to the presence of iron in the environment?
• to develop tools for determining its abundance and activity in nature.

Laboratory experiments show that Thalassiosira oceanica, a small, centric diatom, reduces iron bound to a variety of ligands, including the fungal siderophore desferrioxamine B (DFOB) and some siderophores produced by marine bacteria. We know how to culture this organism and we understand its iron requirements fairly well, so we will use it as a model system.

In principle the reduction of iron-chelator complexes can be measured by detecting increases in the concentration of uncomplexed iron. This can be done, e.g., using an easily detected standard chelating agent as a label: before iron can form complexes with this label chelator, it must first be reduced.

The technology for doing this exists, but until recently it has lacked the sensitivity needed to detect the sub-nanomolar concentrations of uncomplexed iron that would be necessary to detect the activity of these natural reductase enzymes. But Cebic scientists have recently modified an established flow-injection analysis technique to provide the needed sensitivity. This should allow them to determine if iron-chelator-complex reduction occurs in natural samples. The specificity of this natural reduction technology can also be tested, by combining the model system with a variety of chelated-iron compounds, including the siderophores purified in project a-1.

Early results have revealed an unexpected link between iron and nitrogen. Iron reductase activity decreases not only as the external iron concentration increases, but also as nitrate is supplied. We believe that the nitrate effect arises due to competition between nitrate and iron for a common reductant. We intend to test this hypothesis.

Iron uptake by eukaryotic phytoplankton: transport through the cell membrane

How does iron get from the outside of a eukaryotic-phytoplankton cell to the inside? Very likely, the extra-cellular reduction of the iron-chelator complexes described in section a-3 is the first step in the process. This can be tested by inhibiting the production or activity of the reductase enzyme and then measuring the amount of iron inside the cell. If the reduction of iron-chelator complexes is an essential step in iron uptake, then inhibiting the reductase enzyme will suppress it.

Beyond this first step we know very little about how iron is taken up by phytoplankton. Our working hypothesis is that the process is similar to iron uptake in yeast. Iron transport in the yeast Saccharomyces cervisiae involves a reductase and two other proteins: a copper-containing oxidase binds Fe(II) liberated by the reductase, oxidizing it to Fe(III) while it is being taken in by a permease enzyme on the cell membrane. Cebic's preliminary physiological and kinetic data on iron uptake in eukaryotic phytoplankton are consistent with a similar molecular mechanism.

Two approaches will be taken to studying iron transport in diatoms. First, we will generate iron-reductase mutants and transport-mutants (using techniques similar to those discussed in the section on siderophore uptake, above) to study these processes (reduction and transport) in isolation. Second, we will compare the relevant genes in Thalassiosira oceanica to those in yeast, seeking genetic similarities that might indicate common uptake machinery. A putative Fe(II) permease from Arabidopsis has been identified using similar methodology.

Intracellular binding: ferritins in marine microbes

In the laboratory, the iron taken up by microorganisms is often stored in a ferritin, presumably for use in the biosynthesis of iron proteins, and for protection from oxidative damage due to free intracellular iron. Ferritins are known to occur in many microorganisms, but at present, we have no information on the existence or role of ferritins in marine environments.

In this project, Cebic scientists intend to determine whether ferritins are present in marine microbes, both prokaryotes and eukaryotes, whether they differ between the organisms that thrive in iron-poor and iron-rich environments, and whether their synthesis is triggered by natural or human-induced iron inputs.

Scientists are examining laboratory cultures of organisms used in other projects (both new isolates and those already in culture) for the presence and diversity of ferritin. Some ferritin gene sequences are known. The parts of the ferritin gene sequence least likely to vary among different forms of ferritin are used to devise primers for PCR amplification of the putative ferritin genes. This test gives a positive result whenever ferritin genes are present in a particular organism. Cultures that yield positive results are used to characterize newly discovered ferritins: if the gene sequences for these ferritins differ from known forms, these differences are noted and examined to elucidate the range of structural diversity among ferritins.

Using primers derived from laboratory studies, we probe for the presence of ferritin genes in natural and iron-amended field samples. Automated DNA sequencing of ferritin amplicons are used to determine the diversity of ferritin genes. Subgroups of bacterial, animal, and plant ferritins are enumerated, using a quantitative PCR technique to determine the abundance of each type of ferritin. We can determine what organisms are present in the sample, and in what proportions, via a parallel 16S rRNA analysis of environmental samples. Analyzing natural and iron-amended samples in this helps us to determine the community response to iron addition. The ferritin gene sequence from reference organisms identified by 16S analyses assist in establishing what organisms are involved in iron storage under varying environmental conditions.

We are also attempting to detect the actual presence of ferritin (not just the ferritin gene) in natural samples, either by testing for the gene expression (i.e., the presence of mRNA) or direct (spectroscopic or immunological) detection of the protein and stored iron.

Metal binding by phytochelatins

In plants, several trace metals are known to become bound, either for detoxification or for storage, by small polypeptides known as phytochelatins. Unlike ferritin, we already have data showing the presence of phytochelatins in natural marine samples. Phytochelatins are always present in laboratory cultures of microalgae, and their concentration increases in response to minute additions of the trace metals cadmium, zinc, and copper. Field studies suggest that relatively high levels of cadmium and copper are responsible for elevated phytochelatin concentrations in organisms in coastal waters.

We know phytochelatins exist in nature, but we do not know what metals are actually bound by them. We also don't know whether this binding strictly serves a detoxification purpose, or if phytochelatin-bound metals are used later to build proteins.

As is the case with ferritins, a method to quantify the extent of phytochelatin binding of a particular metal in field samples would be valuable for assessing the deficiency or sufficiency of particular metals in marine ecosystems.

We are developing analytical methods for the in vivo detection of phytochelatin binding of various metals (Cd, Cu, Co, Ni and Zn), and applying these methods to both cultured and natural samples to determine what metals are bound to phytochelatin and whether these metals are eventually utilized biochemically.

To minimize the handling of the samples (and the attending artifacts and difficulties), Cebic is employing X-ray absorption spectroscopy (XAS). XAS is a powerful tool for determining the chemical speciation of mixtures. Its strength lies in the fact that it detects all forms of the element under study, in all physical states. Because of the difference in bonding energy, the K-edge absorption spectra of metals ligated to sulfur are shifted with respect to spectra of the same metals ligated to oxygen or nitrogen. We have found that even quite subtle changes in ligation have quite distinct effects on XAS edge spectra.

The first step is to measure K-edge spectra of metal-phytochelatin complexes in both chemically synthesized samples and samples purified from culture. We should then be able to apply this method directly to filtered (not purified) samples from cultures and from natural seawater, provided spectra with reasonable signal to noise ratio can be obtained.

Once we have successfully applied XAS to the study of phytochelatins, we will apply it to the study, in natural samples, of the metal centers of various enzymes from the other Cebic projects, and the toxic complexes trace metals form inside cells when not sequestered by phytochelatins.

Next project: Enzymes and biogeochemical cycles
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