1

Manganese Uptake in Pseudomonas putida

AmyFrontline Mentor: Dr. Sung-Woo Lee

Senior Mentor: Dr. Brad Tebo

2Importance 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 (Mn-SOD, etc.)

• Mn oxides are extremely strong oxidants

Manganese Cycle of Oxidation

3Importance of Siderophores

• Iron chelating (binding) compounds produced by bacteria and fungi in iron limited environments

• Iron commonly found as Fe3+ (insoluble oxides and hydroxides)

Siderophore Process

4Role 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

Structure of Pyoverdine(PVD) complexed with Fe

Fe

5Importance of Siderophores

Siderophore

Siderophore receptor site on cell

Outside the Cell

Inside the Cell

Fe3+

6Importance of SiderophoresSiderophore receptor site on cell

Outside the Cell

Fe3+

Fe3+Inside the Cell

7Importance of SiderophoresSiderophore receptor site on cell

Fe3+

Fe3+

Siderophore

Outside the Cell

Fe3+

Inside the Cell

8Importance of Siderophores

Mn3+

Siderophore

Outside the Cell

Mn3+

Inside the Cell

Mn3+

Siderophore receptor site on cell

Hypothesis: PVD production would facilitate Mn uptake.

9Will siderophores enhance Mn uptake in cells?

PVD (-) – No PVD productionPVD (+) – PVD produced, “wild type” equivalentNo Cells – Serves as a control for the experiment

PVD (-) PVD (+) No Cells

PVD (-)

PVD (+) No Cells

10Comparison of Mn Uptake

Revised Hypothesis: PVD production does not facilitate Mn uptake. Inability to produce PVD encourages Mn uptake.

11Mn Uptake as a Function of [Fe]

Increase[Fe]

Decrease PVD

production

Hypothesis:Increase

Mn uptake

Hypothesis:Increasing [Fe] would increase Mn uptake in PVD (+)

12Mn 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

13Mn Uptake as a Function of [PVD]

14Mn Uptake as a Function of [PVD]

Mn3+

No Uptake

Hypothesis:Adding PVDs back into PVd(-) samples would inhibit Mn uptake.

15Mn 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

16Mn Uptake in Oxidizers/Non-Oxidizers

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

method

17Summary

• 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

18Future 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?

19Thank 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?