Transcript ASE presentation

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Manganese Uptake in
Pseudomonas putida
Amy
Frontline Mentor: Dr. Sung-Woo Lee
Senior Mentor: Dr. Brad Tebo
Importance of Manganese
• 2nd most abundant transition metal
in Earth’s crust
• Three oxidation states of Mn found
in the environment:
– Mn (II)
– Mn (III)
– Mn (IV)
• Soluble forms used as essential
micronutrients
– e.g. cofactors for enzymes (MnSOD, etc.)
• Mn oxides are extremely strong
oxidants
Manganese Cycle of
Oxidation
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Importance of Siderophores
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Siderophore Process
• Iron chelating (binding)
compounds produced by
bacteria and fungi in iron
limited environments
• Iron commonly found as
Fe3+ (insoluble oxides and
hydroxides)
Iron is often
insoluble
(oxides,
hydroxides)
Cells produce
siderophores
Siderophore
binds to
recognition
site on cell
Iron binds to
siderophore
complex
Iron is reduced
(Fe3+ into Fe2+)
Iron is taken
up by the cell.
Role of Pyoverdine in Pseudomonas putida GB-1
• Pseudomonas putida produces
siderophores called pyoverdine
(PVD) which are yellow-green in
color and fluorescent
• PVD forms a stronger complex
with Mn3+ than with Fe3+
• PVD, in an environment with
excess Fe and Mn, will inhibit
Mn oxidation in GB-1
Fe
Structure of Pyoverdine
(PVD) complexed with Fe
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Importance of Siderophores
Inside the Cell
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Siderophore receptor site on cell
Siderophore
Outside the Cell
Fe3+
Importance of Siderophores
Inside the Cell
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Siderophore receptor site on cell
Fe3+
3+
Fe
Outside the Cell
Importance of Siderophores
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Siderophore receptor site on cell
Inside the Cell
Fe3+
Siderophore
3+
Fe
3+
Fe
Outside the Cell
Importance of Siderophores
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Siderophore receptor site on cell
Inside the Cell
Mn3+
Siderophore
Mn3+
Hypothesis:
PVD production would
facilitate Mn uptake.
Mn3+
Outside the Cell
Will siderophores enhance Mn uptake in cells?
PVD (-)
PVD (+)
PVD (+)
No Cells
No Cells
PVD (-)
PVD (-) – No PVD production
PVD (+) – PVD produced, “wild type” equivalent
No Cells – Serves as a control for the experiment
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Comparison of Mn Uptake
Mn in supernatant
(μM)
12
10
8
6
4
2
0
No Cells
PVD (-)
PVD (+)
Revised Hypothesis:
PVD production does not facilitate Mn uptake.
Inability to produce PVD encourages Mn uptake.
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Mn Uptake as a Function of [Fe]
Increase
[Fe]
Decrease
PVD
production
Hypothesis:
Increase
Mn uptake
Hypothesis:
Increasing [Fe] would increase Mn uptake in PVD (+)
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Mn Uptake as a Function of [Fe]
• Added increasing
concentrations of Fe (0, 5, 10,
20 M)
• No effect on PVD (+)
• PVD (-) has elevated uptake
when [Fe] increases
50
Mn Uptake (μM)
Results:
12
40
30
PVD (-)
20
PVD (+)
10
0
0
10
[Fe] (μM)
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Mn Uptake as a Function of [PVD]
Mn Uptake (μM)
50
40
30
20
10
0
0
10
[Fe] (μM)
20
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Mn Uptake as a Function of [PVD]
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Mn Uptake (μM)
50
40
Mn3+
30
20
10
No Uptake
0
0
10
20
[Fe] (μM)
Hypothesis:
Adding PVDs back into PVd(-)
samples would inhibit Mn uptake.
Mn Uptake as a Function of [PVD]
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• Added increasing amounts
of PVD to PVD (-)
Results:
• Increased [PVD] showed
decreased Mn uptake
• Supports hypothesis that
PVDs complex Mn and
inhibit uptake
Mn Uptake (μM)
50
40
30
20
10
0
0
50
[PVD] (μM)
100
Mn Uptake (μM)
Mn Uptake in Oxidizers/Non-Oxidizers
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8
7
6
5
4
3
2
1
0
No cells
GB-1
90-51
• From these results, no significant difference
• Further investigation using a more specific
method
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Summary
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• PVDs do not facilitate Mn uptake
• Mn uptake inhibited by PVDs. Mn uptake increases when [PVD]
decreases.
• Mn uptake in PVD (+) was unaffected by [Fe]. Mn uptake in PVD (-)
increases with [Fe]  5 M
• Mn(II) appears to be the form of Mn taken up by cells, not Mn(III)
– PVD Fe(III) uptake system does not allow Mn(III) to enter cells that way
• Ability to take up Mn not correlated with ability to oxidize Mn
Future Work
• Why is PVD repressing Mn uptake in PVD (+)?
• How does Fe encourage Mn uptake in PVD (-)?
• Will PVD-Mn(III) inhibit Fe uptake?
– If Fe uptake is inhibited, is that why Mn oxidation is inhibited?
• How do cells differentiate between complexed Mn and
complexed Fe?
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Thank You!
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Dr. Sung-Woo Lee
Dr. Brad Tebo
Karen Wegner
Elizabeth Woody
Dr. Antonio Baptista
CMOP
OGI/OHSU
ASE and Saturday Academy
Tebo/Haygood/Blackburn Lab
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Questions?