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 Photo by Ken Nealson * Dept of Biology

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 Fe 3+ mostly found as a solid (Stumm and Morgan 1996) Two common oxidation states, Fe 3+ and Fe 2+ Fe 2+ 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. 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 Pt Counter Hg-Au Working Ag/AgCl Reference 100  m diameter Au wire Org-Fe(III) Fe(II) Org-Fe(III) Fe(II) FeS Satilla River sediment core Arnold, 1988

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

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

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 ABCDEFGH MR-118184728272324MR-1 Tc92413126341Tc18 CN-32323052511219B31 38 S42516317131T121 MR-1R55121020123120MetB GspD6190521152103PsrA Amaz.73231536123114Nrfa/MtrC U14813242213392200R MetC923917641310vibrio MR-110225216274193MR-1 WL1100010000WL Mn12 Mn = 40 mM FeOx added = No FeOx added

11. Example of preliminary results 2 1 A-BC-DABCDEFGHE-FG-H MR1 17 363012303311 MR1 WL 01111010 M1M291110146897M25M26 M3M41914 15830129M27M28 M5M6311821279301712M29M30 M7M8241318286241011M31M32 M9M103223274112331415M33M34 M11M122918303410311312M35M36 M13M143727384114432617M37M38 M15M164635445217574018M39M40 M17M183020384225433615M41M42 M19M203924465030484716M43M44 To date, 600 mutants have been screened. All positive for Org-Fe(III) and Fe(II)

12. Conclusions Shewanella oneidensis 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