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Catechol Siderophores Control Tungsten Uptake and Toxicity in the Nitrogen-Fixing Bacterium Azotobacter vinelandii Thomas Wichard†, Jean-Philippe Bellenger†,

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Presentation on theme: "Catechol Siderophores Control Tungsten Uptake and Toxicity in the Nitrogen-Fixing Bacterium Azotobacter vinelandii Thomas Wichard†, Jean-Philippe Bellenger†,"— Presentation transcript:

1 Catechol Siderophores Control Tungsten Uptake and Toxicity in the Nitrogen-Fixing Bacterium Azotobacter vinelandii Thomas Wichard†, Jean-Philippe Bellenger†, Aurélie Loison‡ and Anne M. L. Kraepiel*§ Department of Geosciences and Chemistry Department, Princeton Environmental Institute, Guyot Hall, Princeton University, Princeton, New Jersey 08544, and UMR 7512 (CNRS-ULP), ECPM, 25 Rue Becquerel, Strasbourg Cedex 02, France Nam Nguyen

2 What is Nitrogen Fixation?
The availability of fixed Nitrogen is scarce due to the unreactive nature of N2 molecule. To make nitrogen more bioavailable to living organisms, nitrogen-fixing bacteria transform atmospheric nitrogen into fixed nitrogen that can be used by plants. Nitrogen fixation is a process in which nitrogen (N2) in the Earth's atmosphere is converted into ammonia (NH3). Nitrogen-fixing bacteria, microorganisms capable of transforming atmospheric nitrogen into fixed nitrogen (inorganic compounds usable by plants). More than 90 percent of all nitrogen fixation is effected by these organisms, which thus play an important role in the nitrogen cycle.

3 Nitrogen-fixing bacteria: Azotobacter vinelandii
Azotobacter vinelandii is gram-negative diazotroph that can fix nitrogen into usable form such as ammonia while grown aerobically. It is a genetically tractable system that is used to study nitrogen fixation. These bacteria are easily cultured and grown.

4 What are siderophores? Siderophores are small organic molecules that bind ferric iron Fe (3+) with high affinity due to their multidentate ligand character. In addition to siderophore production under low iron condition, outer-membrane receptor proteins are produced that facilitate the transfer of iron into the bacterium. When sufficient levels of Fe have been acquired by microorganisms, the biosynthesis of siderophores and their outer membrane receptor proteins is stopped Siderophores are small, high-affinity iron chelating. Siderophores are small organic molecules that bind ferric iron Fe (3+) with high affinity due to their multidentate ligand character.

5 Siderophores

6 Nitrogen fixing bacteria
Mo-Nitrogenase Enzyme Catechol Siderophores W ion Fe ion Mo ion

7 Main points W is toxic to Nitrogen-fixing bacteria.
Catechol siderophores (produced by A. vinelandii) modulate the relative uptake of metals. (particularly protochelin, azotochelin and DHBA) Binding of metals by CS allows bacteria to take up Fe and Mo CS increase rapidly at high [W]. CS take up Mo more rapidly than W. Mutant deficient of CS have high [W] than the wild type.

8 Introduction One of the most important uses of Mo is in the enzyme nitrogenase, which reduces N2(g) into ammonium and supplies new nitrogen to Earth’s ecosystems. A number of N2-fixing bacteria, including (A. vinelandii), use a high-affinity transport system for Mo uptake. The transport system encoded by the mod operon thus does not differentiate between MoO42− and WO42− , which is dangerous because W incorporated into the nitrogenase can turn off the enzyme. In plants, W has primarily been used as an inhibitor of the molybdoenzymes, since it antagonizes molybdenum (Mo) for the Mo-cofactor (MoCo) of these enzymes. However, recent advances indicate that, beyond Mo-enzyme inhibition, W has toxic attributes similar with those of other heavy metals. These include hindering of seedling growth, reduction of root and shoot biomass, ultrastructural malformations of cell components, aberration of cell cycle, disruption of the cytoskeleton and deregulation of gene expression related with programmed cell death

9 Growth Rates and Cellular Quotas of W and Mo in A.V
Figure 1. (A) Growth rates of wild type A. vinelandii (strain OP) as a function of [W] at various Mo concentrations. (B) Molar cellular quotas of W, Mo, and Fe normalized to phosphorus (P) in strain OP as a function of [W] ([Mo] = 10−8 M, [Fe] = 5 × 10−6 M, and OD = 0.6 ± 0.1). - Tungstate is more toxic at low molybdate concentration. - The growth rate of strain OP remains at its maximum for tungstate concentrations below 1 µM; higher tungstate concentrations result in decreasing growth rates - For a Mo concentration of 10−8 M, the decrease in growth rates at high [W] corresponds to an increase in the cellular W quotas - It is the accumulation of W, not a change in the cellular concentration of Mo or Fe due to W interference, that causes the decrease in growth rates

10 Catechol Production Figure 2. Quantification of total concentrations of protochelin (hatched), azotochelin (gray), and DHBA (black) released into the growth medium of Fe-sufficient cultures of strain OP (wild type) as a function of [W] ([Mo] = 10−8 M, [Fe] = 5 × 10−6 M, and OD = ± 0.05). In the concentration range [W] = 10−8 to 10−6 M, where the bacteria grow at their maximum rate, DHBA and protochelin concentrations remain constant. At [W] = 10−5 M, which is toxic, the protochelin concentration, but not that of DHBA, increases by a factor of 5.

11 Complexation of W and Fe by catechol siderophores
Figure 3. Complexation of W and Fe by catechol siderophores in the growth medium of strain OP (wild type, [Mo] = 10−8 M, [Fe] = 5 × 10−6 M, [W] = 8 × 10−6, and OD = 0.5). HPLC chromatograms of extracts from the growth medium and metal analysis of collected fractions by ICP-MS. Circles and triangles indicate concentrations of metal complexes in the growth medium calculated on the basis of metal concentrations in HPLC fractions. (1, DHBA; 2, iron−protochelin; 3, tungsten−protochelin; 4, protochelin; IS, internal standard).

12 Short-term Uptake of W and Mo
Figure 4. Short-term uptake of tungsten (open symbols) and molybdenum (closed symbols) by strain OP (wild type). Open symbols and downward triangles: bacterial cells preconditioned at [98Mo] = 2 × 10−8 M and [W] = 10−7 M, harvested and resuspended into fresh medium for measurement of Mo and W bacterial uptake. The resuspension medium contains [95Mo] = 2 × 10−8 M, [W] = 10−7 M and an excess (=10−6 M) of ligand. Ligand = azotochelin (Az) or protochelin (Pro). Upward triangles: same as downward triangles but no W was added to the preconditioning and resuspension media. One representative experiment is shown. A. vinelandii cells grown at low [Mo] and high [W] were collected and resuspended into fresh medium with an excess of protochelin (10−6 M) to complex all Mo (2 × 10−8 M) and W (10−7 M). Even though Mo is present at a concentration five times lower than W, it is taken up at least 10 time faster, demonstrating the preferential uptake of molybdenum−protochelin over tungsten−protochelin

13 Catechol Release after W Addition
Figure 5. Growth, catechol production, and metal accumulation in strain OP (wild type) grown at [Fe] = 5 × 10−6 M and [Mo] = 10−7 M in response to W addition (A). Cell density of the culture (diamonds) and release of protochelin (circles) and azotochelin (squares) by the bacteria into the medium before and after W addition ([W] = 5 × 10−6 M, 25.5 h after inoculation, dotted line). Cross-hatched areas indicate protochelin concentrations higher than [W] =5 × 10−6 M. (B) Production rate of protochelin based on its concentration in the growth medium. (C) Accumulation of Fe (closed triangles), Mo (open circles), and W (open triangles) in the bacteria: addition of a toxic concentration of W (5 × 10−6 M) to a culture of A. vinelandii that had been growing exponentially in the absence of tungsten the growth rate of the culture decreased abruptly and, for about 2 h, protochelin excretion stopped completely. Over the same time interval, W was taken up very rapidly Two hours after W addition, the production of protochelin by the bacteria increased dramatically up When there was enough protochelin in the medium to bind all the tungstate, the accumulation of W in the cells stopped almost completely, even though 90% of the W originally added was still in solution

14 W Detoxification by Siderophores
The sensitivity to W toxicity should depend on the organism’s ability to synthesize and release catechol siderophores. The intracellular W quotas increase with [W] in all three strains, and the W quotas of the mutant strains are markedly higher than that of strain OP for [W] > 1 × 10−7 M (Figure 6B). These data are consistent with the observation that OP (unlike F196 and P100) releases large amounts of protochelin Mo quotas of the wild type and the mutants were not affected by azotochelin addition; they represent an essentially complete uptake of Mo from the medium, indicating efficient Mo uptake in the presence of siderophores (OP and F 196) or in their absence (P100)

15 Conclusions When exposed to toxic levels of W, the bacteria respond quickly by producing large amounts of protochelin. The production of catechol siderophores thus provides the bacteria with a means to take up preferentially the metals it needs, Fe and Mo (and V), and not W which is toxic. This was confirmed by demonstrating the high sensitivity to W toxicity of catechol-deficient mutants and the detoxification brought about by the addition of azotochelin. The binding of metals by catechol siderophores excreted into the medium provides the bacteria with a precise tool to control metal acquisition. The uptake of toxic metals, such as W, is repressed to very low rates compared to that of essential metals, such as Fe and Mo.

16 Insights From this article, we can see the role of siderophore in biological function: metal binding to take up needed metal. Nitrogen-fixing microbes are free-living and fix nitrogen for their own benefits. The crucial role of Molybdenum nitrogenase in nitrogen fixation. Mo nitrogenase is the most efficient catalyst for N2 reduction, then V-nitrogenase, then Fe-nitrogenase because it evolves large amount of H2 gas and produces much less ammonia.

17 References Adamakis I-DS, Panteris E, Eleftheriou EP. Tungsten Toxicity in Plants. Plants. 2012;1(2): doi: /plants Catechol Siderophores Control Tungsten Uptake and Toxicity in the Nitrogen-Fixing Bacterium Azotobacter vinelandii ;Thomas Wichard, Jean-Philippe Bellenger, Aurélie Loison, and Anne M. L. Kraepiel; Environmental Science & Technology (7), DOI: /es702651f


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