Morris E. Jones , Christine M. Fennessey*, Thomas J. DiChristina*, Martial Taillefert

1. Voltammetric Technique for Rapid Screening of Microbial Iron(III) Reduction by Shewanella oneidensis strain MR-1 Morris E. Jones, Christine M. Fennessey*, Thomas J. DiChristina*, Martial Taillefert EAS GSS 2006 NSF – Biogeosciences Program * Dept of Biology Photo by Ken Nealson

2. Why study iron reducing bacteria (FeRB)? • Carbon cycling • anaerobic iron vs sulfate • Bioremediation • radionuclides • Mineral stability • iron surface chemistry • Metal corrosion • petroleum pipeline • One of the first respiratory processes on earth

3. Dissimilatory microbial iron reduction • At circumneutral pH Fe3+ mostly found as a solid (Stumm and Morgan 1996) • Two common oxidation states, Fe3+ and Fe2+ • Fe2+ rapidly oxidized in the presence of oxygen (Millero, et.al. 1987) • The iron reductase has not been found (DiChristina, et.al. 2005) • How are FeRB able to use FeOx as a Terminal Electron Acceptor • Four hypotheses for microbial iron reduction • Direct contact (Meyers and Meyers 1993) • Nanowires (Lovley, et.al. 2005) • Electron shuttles (Lovely et.al. 1996) • Ligand promoted dissolution (Nevin et.al. 2002)

4. Potential iron reduction pathways Dichristina, 2005 Lovley, 2005

5. 100 m diameter Au wire Pt Counter Hg-Au Working Ag/AgCl Reference What we see with voltammetry • Square wave voltammetry • Scan potential • Measure current • Under anaerobic conditions, with FeOx as TEA, Org-Fe(III) is produced • Environment • Laboratory • Conventional screening techniques only detect Fe(II) reduction product Arnold, 1988 Satilla River sediment core Org-Fe(III) Fe(II) Fe(II) FeS Org-Fe(III)

6. Research plan • Use voltammetry as a screening tool for iron reduction activity • Create mutants from wild type S.oneidensis • Random vs targeted mutants • Screen for iron reducing activity • Org-Fe(III) • Fe(II) • Possible intermediates • Locate genes • Identify proteins

7. Fe(III) Wild-type S. oneidensis MR-1 X EMS ethane methyl sulfonate Random single nucleotide mutation Voltammetric screening for iron reduction deficiency Fe(III) X Clone Bank Mobilize wild-type gene clone bank into mutants Voltammetric screening for iron reduction activity Fe(III) Mutation and Complementation

8. Voltammetric screening array Pt Counter Hg-Au Working Ag/AgCl Reference • 40 mM FeOx • Westlake media • Single colonies • Anaerobic 24 hrs • Eight electrodes • 13.5 min per row • 3 hrs per tray • Anaerobic • voltammetry • Scan potential • Measure current • Org-Fe(III) • Fe(II)

9. Possible screening outcomes • Conventional screening techniques only detect Fe(II) reduction product • No Peaks • Ligand knocked out • Single pathway • No Org-Fe(III) • Ligand knocked out • Iron still reduced • No Fe(II) • Ligand remains • Reductase knocked out • Intermediates • Cysteine Cystine

10. Validation using known organisms • Most screen positive for Org-Fe(III) • As expected Tc18, T121, vibrio, Tc9 show deficiency • Validates technique for screening = 40 mM FeOx added = No FeOx added

11. 2 1 Example of preliminary results To date, 600 mutants have been screened. All positive for Org-Fe(III) and Fe(II)

12. Conclusions • Shewanellaoneidensis produces soluble org-Fe(III) during iron reduction • Unlike other screening techniques, voltammetry can screen for org-Fe(III), Fe(II), and intermediates produced during iron reduction • Possible to screen large numbers of mutants rapidly, making random mutagenesis more feasible • Are we there yet? How much longer?

13. questions