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Manganese Uptake in
Pseudomonas putida
Amy
Frontline Mentor: Dr. Sung-Woo Lee
Senior Mentor: Dr. Brad Tebo
Importance of Manganese 2
• 2nd most abundant transition metal
in Earth’s crust
Manganese Cycle of
Oxidation
• 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 (Mn-
SOD, etc.)
• Mn oxides are extremely strong
oxidants
Importance of Siderophores 3
Siderophore Process
• Iron chelating (binding)
compounds produced by
bacteria and fungi in iron
limited environments
• Iron commonly found as
Fe3+ (insoluble oxides and
hydroxides)
Role of Pyoverdine in Pseudomonas putida GB-1 4
• Pseudomonas putida produces
siderophores called pyoverdine
(PVD) which are yellow-green in
color and fluorescent
• PVD forms a stronger complex Fe
with Mn3+ than with Fe3+
• PVD, in an environment with
excess Fe and Mn, will inhibit Structure of Pyoverdine
Mn oxidation in GB-1 (PVD) complexed with Fe
Importance of Siderophores 5
Siderophore receptor site on cell
Inside the Cell
Siderophore
Fe3+
Outside the Cell
Importance of Siderophores 6
Siderophore receptor site on cell
Inside the Cell
Fe3+
Fe 3+
Outside the Cell
Importance of Siderophores 7
Siderophore receptor site on cell
Inside the Cell
Siderophore
Fe3+
Fe 3+
Fe 3+
Outside the Cell
Importance of Siderophores 8
Siderophore receptor site on cell
Inside the Cell
Siderophore
Mn3+
Hypothesis:
PVD production would
Mn3+ facilitate Mn uptake.
Mn3+
Outside the Cell
Will siderophores enhance Mn uptake in cells? 9
PVD (-) PVD (+) No Cells
PVD (+) No Cells
PVD (-)
PVD (-) – No PVD production
PVD (+) – PVD produced, “wild type” equivalent
No Cells – Serves as a control for the experiment
Comparison of Mn Uptake 10
12
Mn in supernatant
10
(μM) 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.
Mn Uptake as a Function of [Fe] 11
Increase Decrease Hypothesis:
PVD Increase
[Fe] production Mn uptake
Hypothesis:
Increasing [Fe] would increase Mn uptake in PVD (+)
Mn Uptake as a Function of [Fe] 12
• Added increasing
50
concentrations of Fe (0, 5, 10,
20 M)
40
Mn Uptake (μM)
Results: 30
PVD (-)
20 PVD (+)
• No effect on PVD (+)
10
• PVD (-) has elevated uptake
when [Fe] increases 0
0 10 20
[Fe] (μM)
Mn Uptake as a Function of [PVD] 13
50
40
Mn Uptake (μM)
30
20
10
0
0 10 20
[Fe] (μM)
Mn Uptake as a Function of [PVD] 14
50
40
Mn Uptake (μM)
30 Mn3+
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] 15
• Added increasing amounts
of PVD to PVD (-)
50
Results: 40
Mn Uptake (μM)
30
• Increased [PVD] showed
decreased Mn uptake 20
10
• Supports hypothesis that
PVDs complex Mn and 0
inhibit uptake 0 50 100
[PVD] (μM)
Mn Uptake in Oxidizers/Non-Oxidizers 16
9
Mn Uptake (μM) 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
Summary 17
• 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 18
• 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?
Thank You! 19
• Dr. Sung-Woo Lee
• Dr. Brad Tebo
• Karen Wegner
• Elizabeth Woody
Questions?
• Dr. Antonio Baptista
• CMOP
• OGI/OHSU
• ASE and Saturday Academy
• Tebo/Haygood/Blackburn Lab
