Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Summary

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Abstract

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D₂O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Introduction

Electrochemical techniques to directly characterize a redox protein in an intact bacterial cell have recently emerged since the discovery of metal-reducing microbial strains, such as S. oneidensis MR-1 or Geobacter sulfurreducens PCA, which have outer membrane c-type cytochrome complexes (OM c-Cyts) exposed to the cell exterior^1,2,3,4,5. The OM c-Cyts mediate electron transport from the respiratory chain to solid substrates located extracellularly. This transport is referred to as....

Protocol

1. Formation of a Monolayer Biofilm of S. oneidensis MR-1 on an ITO Electrode (Figure 1)

NOTE: To prevent the contamination of the electrochemical reactor with other microbes, all the media, implements, and components of the electrochemical reactor should be sterilized in advance. When using S. oneidensis MR-1 cells and constructing the electrochemical reactors, all the procedures should be conducted on a clean bench.

1. Cultivation of S. oneidensis MR-1 cells
   NOTE: A monolayer biofilm of S. oneidensis MR-1 was formed on an ITO electrode following the conditio....

Results

After 25 h of potential application at +0.4 V (versus SHE), a monolayer biofilm was formed on the working electrode of ITO glass, which was previously confirmed by either a scanning electron microscopy or a confocal microscopy⁴. The representative time course of current production from the S. oneidensis MR-1 during the formation of a monolayer biofilm is shown in Figure 2. Although the current alters in every measurement, the .......

Discussion

Our whole-cell electrochemical assay has several technical advantages compared with protein electrochemistry. While protein purification requires multi-step time-consuming procedures, our whole-cell method takes one day of self-organized biofilm formation after cell culture. To achieve a stable interaction between OM c-Cyts and the electrode, we need only sterilization and cleaning of the electrode surface; it does not require electrode modification for organizing the orientation of proteins⁴

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass cylinder	N/A	N/A	Custom-made, used as the electrochemical reactor
PTFE cover and base	N/A	N/A	Custom-made, used as a cover and a foundation of the electrochemical reactor
Buthyl rubber	N/A	N/A	Custom-made, inserted between each component of electrochemical reactor
Septa	GL Science	3007-16101	Used as an injection port of electrochemical reactor
Indium tin-doped oxide (ITO) electrode	GEOMATEC	No.0001	Used as a working electrode, 5Ω/sq
Ag/AgCl KCl saturated electrode	HOKUTO DENKO	HX-R5	Used as a reference electrode, Φ0.30mm
Platinum wire	The Nilaco Cooporation	PT-351325	Used as a counter electrode
Luria-Bertani (LB) Broth, Miller	Becton, Dichkinson and Company	244620	Medium for precultivation of S. oneidensis MR-1
Bacto agar	Becton, Dichkinson and Company	214010
Anthraquinone-1-sulfonate (α-AQS)	TCI	A1428
Flavin mononucleotide (FMN)	Wako	184-00831
NaHCO3	Wako	191-01305	Used for defined medium (DM)
CaCl2 · 2H2O	Wako	031-00435	Used for DM
NH4Cl	Wako	011-03015	Used for DM
MgCl2 · 6H2O	Wako	135-00165	Used for DM
NaCl	Wako	191-01665	Used for DM
2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)	DOJINDO	346-08235	Used for DM
Sodium Lactate Solution	Wako	195-02305
Bacto Yeast Extract	Becton, Dichkinson and Company	212750
Deuterium oxide (D, 99.9%)	Cambridge Isotope Laboratories, Inc.	DLM-4-PK	Additive for kinetic isotope effect experiments
Incubator	TOKYO RIKAKIKAI CO. LTD.	LTI-601SD	Used for precultivation
Shaker	TAITEC	NR-3	Used for precultivation
Autoclave machine	TOMY SEIKO CO. LTD.	LSX-500	Used for sterilization of the electrochemical reactor and the medium
Clean bench	SANYO	MCV-91BNF	Used to prevent the contamination of the electrochemical reactor and the medium with other microbes
Centrifuge separator	Eppendorf	5430R	Rotational speed upto 6000×g is required
Nitrogen gas generator	Puequ CO. LTD.	PNTN-2	Nitrogen gas cylinder can also be used instead of gas generator
UV-vis spectrometer	SHIMADZU	UV-1800	Used for optimization of cell density
Potentiostat	BioLogic	VMP3	Used for biofilm formation and kinetic isotope effect experiments
Thermal water circulator	AS ONE	TR-1A	Used for maintanance of temperature of electrochemcial reactor
Faraday cage	HOKUTO DENKO	HS-201S	Used for electrochemical experiments