Manganese Uptake in Pseudomonas putida

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Manganese Uptake in Pseudomonas putida
Amy Frontline Mentor Dr. Sung-Woo Lee Senior
Mentor Dr. Brad Tebo
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Importance of Manganese

• 2nd most abundant transition metal in Earths
  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 (Mn-SOD, etc.)
• Mn oxides are extremely strong oxidants

Manganese Cycle of Oxidation
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Importance of Siderophores
Siderophore Process

• Iron chelating (binding) compounds produced by
  bacteria and fungi in iron limited environments
• Iron commonly found as Fe3 (insoluble oxides and
  hydroxides)

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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
Siderophore receptor site on cell
Inside the Cell
Fe3
Siderophore
Outside the Cell
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Importance of Siderophores
Siderophore receptor site on cell
Inside the Cell
Fe3
Fe3
Outside the Cell
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Importance of Siderophores
Siderophore receptor site on cell
Fe3
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Importance of Siderophores
Siderophore receptor site on cell
Inside the Cell
Mn3
Siderophore
Hypothesis PVD production would facilitate Mn
uptake.
Mn3
Mn3
Outside the Cell
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Will siderophores enhance Mn uptake in cells?
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
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Comparison of Mn Uptake
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
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)
• Results
• No effect on PVD ()
• PVD (-) has elevated uptake when Fe increases

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Mn Uptake as a Function of PVD
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Mn Uptake as a Function of PVD
Mn3
No Uptake
Hypothesis Adding PVDs back into PVd(-) samples
would inhibit Mn uptake.
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Mn Uptake as a Function of PVD

• Added increasing amounts of PVD to PVD (-)
• Results
• Increased PVD showed decreased Mn uptake
• Supports hypothesis that PVDs complex Mn and
  inhibit uptake

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Mn Uptake in Oxidizers/Non-Oxidizers

• From these results, no significant difference
• Further investigation using a more specific method

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Summary

• 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

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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!

• 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

Questions?