Transcript Mo and W Siderophores

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, 67087 Strasbourg Cedex 02, France
Nam Nguyen
What is Nitrogen Fixation?
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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-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.
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
Mo-Nitrogenase
Enzyme
Nitrogen fixing bacteria
Catechol
Siderophores
W ion
Fe ion
Mo ion
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.
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.
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
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 = 0.42
± 0.05).
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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.
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At [W] = 10−5 M, which is toxic, the protochelin
concentration, but not that of DHBA, increases by a factor
of 5.
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).
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
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:
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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
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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
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Two hours after W addition, the production of protochelin by the bacteria increased
dramatically up
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When there was enough protochelin in the medium to bind all the tungstate, the
W Detoxification by Siderophores
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The sensitivity to W toxicity should depend on the
organism’s ability to synthesize and release catechol
siderophores.
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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
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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)
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.
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.
References
1) Adamakis I-DS, Panteris E, Eleftheriou EP. Tungsten Toxicity in Plants. Plants. 2012;1(2):82-99. doi:10.3390/plants1020082.
2) 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
2008 42 (7), 2408-2413 DOI: 10.1021/es702651f