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Manganese Uptake in Pseudomonas putida

Manganese Uptake in Pseudomonas putida. Amy Frontline Mentor : Dr. Sung-Woo Lee Senior Mentor : Dr. Brad Tebo. 2nd most abundant transition metal in Earth’s crust Three oxidation states of Mn found in the environment: Mn (II) Mn (III) Mn (IV)

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Manganese Uptake in Pseudomonas putida

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

  2. 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 (Mn-SOD, etc.) Mn oxides are extremely strong oxidants Importance of Manganese Manganese Cycle of Oxidation

  3. Iron chelating (binding) compounds produced by bacteria and fungi in iron limited environments Iron commonly found as Fe3+ (insoluble oxides and hydroxides) Importance of Siderophores Siderophore Process

  4. 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

  5. Importance of Siderophores Siderophore receptor site on cell Inside the Cell Fe3+ Siderophore Outside the Cell

  6. Importance of Siderophores Siderophore receptor site on cell Inside the Cell Fe3+ Fe3+ Outside the Cell

  7. Importance of Siderophores Siderophore receptor site on cell Inside the Cell Fe3+ Siderophore Fe3+ Fe3+ Outside the Cell

  8. 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

  9. 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

  10. Comparison of Mn Uptake Revised Hypothesis: PVD production does not facilitate Mn uptake. Inability to produce PVD encourages Mn uptake.

  11. Mn Uptake as a Function of [Fe] Increase [Fe] Decrease PVD production Hypothesis:IncreaseMn uptake Hypothesis: Increasing [Fe] would increase Mn uptake in PVD (+)

  12. Added increasing concentrations of Fe (0, 5, 10, 20 M) Results: No effect on PVD (+) PVD (-) has elevated uptake when [Fe] increases Mn Uptake as a Function of [Fe]

  13. Mn Uptake as a Function of [PVD]

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

  15. 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

  16. Mn Uptake in Oxidizers/Non-Oxidizers • From these results, no significant difference • Further investigation using a more specific method

  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 Summary

  18. 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?

  19. 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?

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