Voltammetric Technique for Rapid Screening of Voltammetric Technique for Rapid Screening of Microbial Iron(III) Reduction by Microbial Iron(III) Reduction by Shewanella oneidensisShewanella oneidensis

strain MR-1strain 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

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

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)

Potential iron reduction pathways

Dichristina, 2005

Lovley, 2005

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

Single colony MR1, 40mM FeOx, 24 hrs

0.000.10

0.200.300.400.50

0.600.700.80

0.901.00

-1.80-1.30-0.80-0.30

E - [V]

I - [u

A]

w/o iron

w/ iron

0.0 -0.5 -1.0 -1.5

0

20

40

60

80

100

120

140

160

I -

[nA

]

E - [V]

Org-Fe(III)

Fe(II)

Org-Fe(III)

Fe(II)

FeS

Satilla River sediment core

Arnold, 1988

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

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)

Fe(III)

Fe(III)

EMS

ethane methyl sulfonate

Mobilize wild-type gene clone bank

into mutants

Voltammetric screening for iron reduction activity

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 CounterHg-Au WorkingAg/AgCl Reference

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

Single colony GspD, 40mM FeOx, 24 hrs

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

-1.80-1.30-0.80-0.30

E - [V]

I - [

uA

]

w/o iron

w/ iron

Validation using known organisms

• Most screen positive for Org-Fe(III)• As expected Tc18, T121, vibrio, Tc9

show deficiency• Validates technique for screening

A B C D E F G H

MR-1 1 8 18 4 7 28 27 23 24 MR-1

Tc9 2 4 1 31 2 6 3 4 1 Tc18

CN-32 3 2 30 5 25 1 12 1 9 B31

38 S 4 25 1 63 1 7 1 3 1 T121

MR-1R 5 5 12 10 20 1 23 1 20 MetB

GspD 6 19 0 52 1 15 2 10 3 PsrA

Amaz. 7 3 23 15 36 1 23 1 14 Nrfa/MtrC

U14 8 13 2 42 2 13 3 9 2 200R

MetC 9 2 39 17 64 1 3 1 0 vibrio

MR-1 10 22 5 21 6 27 4 19 3 MR-1

WL 11 0 0 0 1 0 0 0 0 WL

Mn 12                 Mn

= 40 mM FeOx added

= No FeOx added

Example of preliminary results

• 2

• 1A-B C-D A B C D E F G H E-F G-H

MR1 MR1 17 17 36 30 12 30 33 11 MR1 MR1

WL WL 0 1 1 1 1 0 1 0 WL WL

M1 M2 9 11 10 14 6 8 9 7 M25 M26

M3 M4 19 14 14 15 8 30 12 9 M27 M28

M5 M6 31 18 21 27 9 30 17 12 M29 M30

M7 M8 24 13 18 28 6 24 10 11 M31 M32

M9 M10 32 23 27 41 12 33 14 15 M33 M34

M11 M12 29 18 30 34 10 31 13 12 M35 M36

M13 M14 37 27 38 41 14 43 26 17 M37 M38

M15 M16 46 35 44 52 17 57 40 18 M39 M40

M17 M18 30 20 38 42 25 43 36 15 M41 M42

M19 M20 39 24 46 50 30 48 47 16 M43 M44

To date, 600 mutants have been screened. All positive for Org-Fe(III) and Fe(II)

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?

questions