電気化学活性細菌におけるエネルギー代謝機構の解明

2019

4.2.11. His-tag ArcA ... 48

4.2.12. Cy3 ... 48

4.2.13. Electrophoresis mobility shift assay (EMSA) ... 49

5.2.11. Electrophoresis mobility shift assay (EMSA) ... 79

5.3.

... 79

5.4.

... 83

... 87

6.

... 97

6.1.

... 97

6.2.

... 97

6.2.9. Electrophoresis mobility shift assay (EMSA) ... 99

1.

1.1.

1.2.

(electrochemically active bacteria; EAB) 2

(Rabaey and Rozendal, 2010)3 2

2 EAB (Kracke et al., 2015)3

electrotroph 2 chemotroph phototroph 3 Acidithiobacillus Desulfovibrio 2 (Deng et al., 2018)3 (Nakamura et al., 2010)2 3 2 (Reysenbach and Shock, 2002)2 3 2 2 “ 3

1.3.

(bioelectrochemical systems; BES) 3 BES Fig. 1-1

3BES ( )

( ) 3 2

2

(EAB ) 3

2

(Rabaey and Rozendal, 2010)3BES 2EAB 3

2 3 2 2 3EAB 2 BES 3

1.4.

(Moscoviz et al., 2016)3 Fig. 1-2 3

2 2

(Förster et al., 2017)3 2 2

4 3

3 2BES

2

3 2

(Förster et al., 2017; Wu et al., 2019a, 2019b) (Fig. 1-2)3Förster

(2017) 2 2 3 Wu (2019a, 2019b) 2 2 1.7 3 2 3 2 2 EAB 2

Δ 10 µA/cm2 (Förster et al., 2017)3

(Sturm-richter et al., 2015)2 3 2 EAB 2EAB ′ 3 2 EAB 2 3 2 2 2 ′ EAB 3

1.5. Shewanella oneidensis MR-1

(Myers and Nealson, 1988)3MR-1 “

2

(Rodionov et al., 2010)3 2 2 2

2 2 2 (DMSO 2

(Nealson and Saffarini, 1994)3Shewanella

3 2 3 MR-1 2 (Heidelberg et al., 2002)2 2 2 3 2 2 (Tefft and Teravest, 2019)3 2MR-1 3

1.6. S. oneidensis MR-1

MR-1 Fig. 1-3 (Hirose et al.,

2019a)3MR-1 glucose uniporter (Glf) glucokinase (Glk)

2 3 2 N- 2 6-3MR-1 Entner-Doudoroff (ED) 2 Embden-Meyerhof-Parnas (EMP) 1 ATP 1 3 3

Δ NADH 2acetyl-CoA pyruvate dehydrogenase

(PDH) 2 Δ 2acetyl-CoA pyruvate-formate lyase (PFL) 3PFL 2 formate dehydrogenase (FDH) CO2 3MR-1 3 FDH (fdnGHI2fdhABC-12fdhABC-2) 2 3 2 Δ 2 Δ 3 2 2NADH lactate

dehydrogenase (LdhA) D- (Kasai et al.

2019)3 2

3

MR-1 N- 2 (D- L- )

3D- L

2 D- 3 2L- 2LldEFG

3 2 L

-3

Acetyl-CoA Δ Δ 3 Δ

acetyl-CoA TCA 2NADH

3 2 Δ ( Δ ) acetyl-CoA

2 MR-1 × ATP 3

Δ ATP ( )

(Hunt et al., 2010)3 2 Δ 2TCA

NADH ′ isocitrate dehydrogenase (Icd) 2-oxoglutarate

dehydrogenase (Suc) 2 TCA 3 2

MR-1 NADH NADH (NAD+ _{) 2} 3 3MR-1 8 (UQ8) 7 (MK7) 2 Δ UQ8 (+0.11 V vs. ; 2 )2 Δ MK7 (-0.074 V) 3 Δ UQ8 PetA 2 c 2 3 PetA

” 2ATP synthase ATP 3 2 Δ

2CymA MK7 2

3 FccA 2

3 2NapB

2 NapA 3

NapB NrfA 3DMSO

DmsE DmsABF 2DMSO

3 / FccA/CctA 2

MtrCAB/OmcA 3 (TMAO)

2CymA (TorC) 2TMAO

TorA 3

MR-1 Δ ( Δ )

-2 Δ

(Kasai et al., 2019)3 2MR-1 NADH dehydrogenase FDH

Δ 2

2

3 2MR-1 2 2

hydrogenase

(Meshulam-simon et al., 2007)3MR-1 hydrogenase Hya Hyd

2 3 2 Δ 3 2 hydrogenase 3

1.7. MR-1

FccA/CctA 2 (Myers and Myers,

2000)3MtrCAB OmcA 2MtrA

(Hartshorne et al., 2009)3MtrB β × 2

(Beliaev and Saffarini, 1998)3MtrC OmcA

2MtrC OmcA = 2:1 (Shi et al., 2006)3FccA

CctA 2CymA Mtr

(Sturm et al., 2015)3 2FccA 2

CymA (Myers and Myers, 2000)3

( ) 2 “

(Firer-Sherwood et al., 2008)3 2MR-1 3

protein (CRP) (Kasai et al., 2015)3CRP

× × 2MR-1

(Saffarini et al., 2003)3

2 Δ MetR

× × CRP (Mogi et al.,

unpublished data)3 DNA

2MR-1

3 2MR-1 2

(Gorby et al., 2006; Pirbadian et al., 2014)3 MtrCAB/OmcA

2

(Pirbadian et al., 2014)3

1.8.

“ 2

3 “

sensor kinase 2DNA 2

response regulator 3 sensor kinase (Hpt)

3 2

4 3 “

2 3

2Arc system 3

γ-2S. oneidensis2Escherichia coli2Serratia marcescens2Klebsiella pneumoniae2Vibrio choleae2Salmonella Typhimurium Haemophilus

influenzae (Hirose et al., 2019b)3Arc system E. coli ( )

2sensor kinase PAS

(Bekker et al., 2010)3

Arc system ArcB/ArcA 3 2

Δ sensor kinase ArcB 2

ArcB 3 response regulator

ArcA 2 ArcA (ArcA-P)

3 2 Δ 2ArcB

2ArcA-P 2ArcA DNA 3DNA ArcA

2 ArcA “ ′

(Park et al., 2013)3 Arc system

2 (Park

et al., 2013)3

2MR-1 Arc system sensor kinase ArcS2 HptA2

response regulator ArcA (Lassak et al., 2010)3

2HptA ArcS ArcA 2

3MR-1 ArcA ArcA 2 DNA

” (Gao et al., 2008)3 2 sensor kinase 2

MR-1 ArcS PAS 2 Chache sensor

2 ArcB (Lassak et al., 2013)3

2ArcS ArcB 3

2 2 Fnr (fumarate nitrate

reductase regulator) (Gunsalus and Park, 1994)3

“ DNA 2 3

2DNA 3 2

3 2Fnr Δ 2

′ (Myers et al., 2013)3MR-1 Fnr

EtrA (electron transport regulator A) 2 ArcA ′

(Cruz-García et al., 2011)3

1.9.

2S. oneidensis MR-1 4 2

2

(Grobbler et al., 2018, 2014; Kitayama et al., 2017; Nakagawa et al., 2015)3 2

EAB 2MR-1 Shewanella decolorationis S12 2

Desulfovibrio ferrophilus IS5 Geobacter sulfurreducens 2

(Deng and Okamoto, 2018; Li et al., 2019; Lian

et al., 2016)3 2EAB 2

× 3

Fig. 1-3 MR-1 (Hirose et al., 2019a)3GlcNAc: N-acetylglucosamine; GlcNAc-6P: N-acetylglucosamine-6-phosphate; KDG-6P: 2-keto-3-deoxygluconate 6-phosphate; PEP: phosphoenolpyruvate; Q: quinone; QH2: quinol; NagP: N-acetylglucosamine transporter; NagK: N-acetylglucosamine kinase; NagA: N-acetylglucosamine-6-phosphate deacetylase; NagB: glucosamine/fructose-6-phosphate aminotransferase; Fbp: fructose-1,6-bisphosphatase; Fba: fructose-bisphosphate aldolase; TpiA: triosephosphate isomerase; Pgi: glucose-6-phosphate isomerase; Zwf: glucose-6-phosphate 1-dehydrogenase; Edd: phosphogluconate dehydratase; Eda: 2-dehydro-3-deoxyphosphogluconate aldolase/ (4S)-4-hydroxy-2-oxoglutarate aldolase; PpsA: pyruvate, water dikinase; PckA: phosphoenolpyruvate carboxykinase; Pdh: pyruvate dehydrogenase; GltA: citrate synthase; Acn: aconitate hydratase; Icd: isocitrate dehydrogenase; SucAB: 2-oxoglutarate dehydrogenase; SucCD: succinyl-CoA synthetase; Sdh: succinate dehydrogenase/fumarate reductase; Fum: fumarate reductase; Mdh: malate dehydrogenase; AceB: malate synthase; AceA: isocitrate lyase; Pta: phosphate acetyltransferase; AckA: acetate kinase; Acs: acetyl-CoA synthetase; Fdh: formate dehydrogenase; Dld: d-lactate dehydrogenase, quinone dependent; Lld: l-lactate dehydrogenase; LdhA: d-lactate dehydrogenase, NAD dependent; Glf: glucose uniporter; GalP: galactose:H+ symporter; Glk: glucokinase; Xks1: xylulokinase; Xyl2: xylitol dehydrogenase; Xyl1: NAD(P)H-dependent xylose reductase repressor.

Glucose _external Glucose-6P Gluconate-6P Pyruvate Acetyl-CoA Lactate Formate CO₂ Acetyl-P Acetate Xylulose-5P Xylose Xylitol Xylulose GlcNAc _external GlcNAc-6P GlcN-6P Fructose-6P Fructose-1,6P Glyceraldehyde-3P GlcNAc _internal KDG-6P PEP Glucose _internal Xyl1 Xks1 NagP NagK NagA Eda Pgl, Zwf Glk Acs AckA Pta GltA Oxaloacetate Malate Fumarate Succinate Succinyl-CoA Citrate Isocitrate 2-oxogulutarate Glyoxylate Acn AceA Icd SucAB SucCD Sdh Fum Mdh PckA _PykA PpsA Edd Fba, TpiA Pgi Chitin Pfl*† Pdh Fdh Dld Lld NagB Chitinase AceB LdhA ATP

ADP ADPATP

Fig. 1-4 MR-1 (Hirose et al., 2019b)3LDH, lactate dehydrogenase; FDH, formate dehydrogenase; NDH, NADH dehydrogenase; Q, quinone; QH2, quinole; OM, outer

membrane; IM, inner membrane.

4.

4.1.

2EAB 4 2

2 2

(Bosch et al., 2014; Grobbler et al., 2014; Li et al., 2019; Lian et al., 2016)3 EAB

2 × 3 4 2EAB 3 MR-1 3

4.2.

4.2.1.

Appendix 1 3E. coli S. oneidensis MR-1

LB 3 Δ 5 ml LB 2 100 ml 50 ml LB 2 40 mM 3 10 mM (PMM; 9 mM (NH4)2SO4, 5.7 mM K2HPO4, 3.3 mM KH2PO4, 30 mM HEPES-NaOH (pH7.4)) 2 5 ml PMM 30 ml 2S. oneidensis MR-1 OD600 0.05 2 3 30°C2180 rpm 2 3 8 ml PMM 13 ml 2 20 mM 2.5 mM 2S. oneidensis MR-1 OD600 = 0.01 2 3 5 min 2 “ Δ 230°C 3OD600

mini photo 518R photometer ( ) 21 h 3

(MnO2) 2100 ml PMM 80 ml 2

20 mM MnO2 2MR-1 OD600 = 0.01 2

3 10 min “ Δ 224 h 1 ml

2MnO2 3MnO2 Kouzuma et al. (2012) 2leucoberblin

blue 3

4.2.2.

Appendix 2 3

DISMIC-25HP; Advantec) 1.5 ml 2 3

B-PER bacterial protein extraction reagent (Thermo Fisher Scientific)

3 1 ml 3 ml 2 15min 3

— 2 1.5ml 2 3

Micro BCA protein assay kit (Thermo Fisher Scientific) 2 3

4.2.5.

2 2 (HPLC) (Agilent 1100

series) 3 1 ml 2

(0.2 µm pore size, DISMIC-25HP; Advantec) 3HPLC

Zorbax column (SB-Aq, 4.6 by 150 mm; Agilent)2 10%

20 mM potassium phosphate buffer (pH 2) 3 , 1 ml min-1; , 5µl;

, 25°C; , UV 210 nm 3 2

3

4.2.6. RNA

Total RNA Trizol (Invitrogen) 2RNeasy Mini kit RNase-free

DNase Set (QIAGEN) 3 RNA Agilent 2100 Bioanalyzer RNA

pico Chips (Agilent) 2 3

4.2.7. qRT-PCR

PCR_(qRT-PCR) LightCycler 1.5 instrument LightCycler SYBR Green I (Roche) 3PCR 15 ng total RNA21.3 µl 50 mM Mn(OAc)22

7.5 µl RNA Master SYBR Green I20.15 µM 3

Appendix File 2 3nuoI (SO_1014)2SO_09392SO_45092atpG (SO_4748)2

dld (SO_1521)2SO_15382SO_4360216S rRNA PCR

3 16S rRNA 3

4.2.8. DNA

×

S. oneidensis MR-1 ×

FairPlay III Labeling kit (Agilent) 3 cDNA

× 2WT n = 52∆arcS n = 6 ( ×

; n = 32 × ; n = 2) 3

GeneSpring GX version 11.5 (Agilent Technologies) 3 Student’s T-test Benjamini-Hochberg false discovery rate (BH-FDR) 3 Δ fold change (FC) 1 2.0 0.5 (|log2FC| 1 1.0)2P < 0.05

3DNA × 6 (SO_09392

dld2SO_15382SO_43602SO_45092atpG ) qRT-PCR ′ 36

qRT-PCR 3 Cluster 3.0 Treeview software

3

4.2.9. NADH/NAD

NADH/NAD+ _NAD+_{/NADH assay kit (Colorimetric; Abcam)}

3 × 600 µl Extraction buffer 2 Lysing

Matrix E tube (MPB) 3Fast prep FP120 (MPB) Speed: 4 40 sec

3— (15000 rpm, 4°C, 10 min) 2 10 kD spin column (Abcam) 3 3

4.2.10.

WT 150 ml OD600 1.0 2

+0.5 V –0.1 V 2 3

Bekker et al. (2007)2Grammel et al. (2008) 3

Δ 2 3 3 ml (60%) : buffer (10 mM) 3 ml 3 2 3 ml 3 — ( ) 3 2 3 Δ 2 × 2- (4:1, v/v) 120 µl 2 3

MR-1 UQ8 MK7 (Venkateswaran et al. 1999)2

3 2Coenzyme Q8 (AVT) -7

(WAKO) 3 HPLC (Prominence series, shimadzu) 2 L-

3 ° × UV (2 2MK7; 248 nm UQ8; 290 nm) 2 (λmax) (RT) 3

4.2.11. His-tag

ArcA

total DNA PCR 2arcA DNA 3PCR QIA

quick PCR purification kit (QIAGEN) 3 2BamHI NdeI

2 DNA mini Elute reaction clean up kit (Qiagen)

3 DNA BamHI NdeI pET-28a (+) (Novagen)

3 (pET-28_arcA) BL21(DE3) (Novagen)

3 BL21(DE3)(pET-28_arcA) Km 2×YT

237°C2180 rpm OD600 0.7 3300 ml

Km 2×YT 100 ml 2OD600 0.05 3

30 C2180 rpm OD600 0.7 2Isopropyl-β-D-galactopyranoside

(IPTG) 500 µM 225°C2180 rpm 3 50 ml

2— × 3 × IMAC wash buffer

(Bio-Nobile) 300 µl 2 3 buffer 10 2

Misonix 2— 3

His-tag ArcA Quickpick IMAC metal affinity kit

(Bio-Nobile) 2 3 His-tag ArcA

SDS-PAGE 2 3 micro BCA

protein assay kit (Thermo Fisher Scientific) 2 3

4.2.12. Cy3

Appendix 2 3SO_00112sucA (SO_1930)2nuoA

(SO_1021)2atpI (SO_4754) DNA 3Cy3

DNA PCR 3PCR DNA QIAXII

gel extraction kit (QIAGEN) 3Nano drop (ND-10002 )

4.2.13. Electrophoresis mobility shift assay (EMSA)

EMSA Gao et al. (2008) 3 His-tag

ArcA 2× (200 mM Tris/HCl (pH 7)220 mM MgCl22250 mM KCl2

100 mM (Sigma)) 1:1 1 h

His-tag ArcA 3DNA 100 mM Tris/HCl (pH7.4)210 mM MgCl22

20 mM KCl22 mM DTT20.2 µg/ml poly (dI-dC)210% 22 nM Cy3 DNA

2 His-tag ArcA (0, 100, 200, 300 ng) 12 µl 2

(Takara) 15°C21 h 3 × 1 h

12.5 % 3

0.5×Tris-borate-EDTA (TBE) buffer 2200 V 3 Typhoon

FLA 9000 (FUJIFILM2GE healthcare) 2 3

4.2.14. ×

nuoA lacZ 3nuoA MR-1

total DNA PCR 3 nuoA 5

100 bp 3 Appendix 2

3PCR QIA quick PCR purification kit (Qiagen) 2

3 PCR × pMElacZ (Endoh et al., 2003)

EcoRI BamHI 3 pMElacZ Antactic phosphatase (New

England Biolabs) 3pMElacZ PCR

2pMElacZ 3

(pMElacZ-R) 2 ”

3

4.2.15. ×

S. oneidensis MR-1 × Kasai et al. (2015)

3× 3

MR-1 LB 5 ml 22 ml — (16000 g21 min24°C) 3 ×

300 mM 2 2 100 µl 300 mM 3

50 µl 1-3 µg

Gene Pulser cuvette, gap: 0.1 cm (Bio Rad)

2

20 min

3

Micropulser (Bio Rad)

180 kV

4 sec

3

2

500 µl LB

µg/ml) × × 3 ×

30

4.2.16.

mM Na2HPO4240 mM NaH2PO4 2H2O210 mM KCl2MgSO4 7H2O250 mM

2-) 500 µl2 15 µl 0.1% SDS 10 µl 215 sec

35 min 2200 µl 2- -β- (ONPG)

3 2stop solution (1 M Na2CO3)

β 3OPNG stop solution 3

— (12,000 × g22 min24°C) 2 300 µl × 2

× ° (SH-12002 ) 420 nm (Abs420)

3β- ° Miller unit = (1000×Abs420) / ( (min) ×

(ml) × OD600) 3 3 3

4.3.

4.3.1.

MR-1

S. oneidensis MR-1 ( ; WT)

218 ml 2

+0.5 V (high potential; HP)2+0.2 V (middle potential; MP) 0 V (low potential;

LP) 3 2

(Fig. 4-1A)3 2 2

(data not shown)3 2 (Fig. 4-1B)3 2 (

) 2 (Fig.

4-1C)3 MR-1

4.3.2.

(Fig. 4-4)3 Table 4-1 3 D-lactate

dehydrogenase (dld)2NADH dehydrogenase (nuo) F0F1-ATP synthase (atp) 2

HP Δ LP Δ

3 2

2 3

MR-1 Δ ( Δ ) ×

ATP 2 (NADH dehydrogenase F0F1-ATP synthase)

(Hunt et al., 2010)3 NADH

(pyruvate dehydrogenase (PDH)2NADH dehydrogenase succinate dehydrogenase)

2 pyruvate-formate lyase (PFL) formate

dehydrogenase (FDH) CO2 (Pinchuk et al., 2011)3

2 × 2HP Δ pyruvate dehydrogenase (aceF)2succinate

dehydrogenase (sucAB)2nuo2 atp 2

MR-1 NADH 2 ATP

3 2FDH 3 2fdnGHI (SO_0101-SO_0103)

fdhABC2 (SO_4513-SO_4515) HP Δ 2fdhABC1 (SO_4509-SO_4511) LP Δ

3 2MR-1 FDH 3 2 FDH – 3

4.3.3.

(Fig. 1-4) (Simon et al., 2008)3 2Geobacter CymA 2

(Levar et al., 2017)3 cymA ′

dehydrogenase 3 Δ

NADH dehydrogenase 3MR-1 Nuo2Ndh2Nqr1

Nqr2 4 NADH dehydrogenase 2 NADH dehydrogenase

(∆NDH) 2HP2MP LP Δ (Fig. 4-5)3 2∆NDH HP Δ 2MP LP Δ 3 HP Δ NADH dehydrogenase ′ 2 NADH 3 2 ∆PDH ∆PFL (Fig. 4-5) HP Δ 80% NADH ( ) 2HP Δ ∆NDH 3 2HP Δ ∆NDH 2 3 2∆NDH NADH 2HP Δ NADH 2 3 2HP2MP LP Δ WT ∆NDH NADH/NAD+ _{(Fig. 4-6)3 2∆NDH WT} HP Δ NADH/NAD+ _{2∆NDH NADH} 3∆NDH NADH MP LP Δ 2NADH/NAD+ _{(Fig. 4-6)3 2MP Δ} ∆NDH WT (Fig. 4-5)2HP Δ MP Δ NADH/NAD+ 3 2 2MR-1 2 NADH 3

4.3.4. NADH dehydrogenase

′

(Fig. 4-7A)3 2 Δ 0 V (LP) Δ 2NADH

3 2∆NDH (Fig. 4-7B) MnO2 (Fig.

2 NADH 3 2MR-1 2 3

4.3.5. Arc system

2HP Δ NADH:ubiquinone oxidoreductase (nuo)

2HP Δ UQ8 3 2 2 UQ 3 UQ8 2HP Δ LP Δ UQ8 UQH2 UQ 3 2HP Δ LP Δ UQ8 2 (Fig. 4-8)3 2 3 2 2 HP Δ UQ 3 2Arc system ′ 3MR-1

Arc system sensor kinase (ArcS)2 (HptA)2response regulator (ArcA)

3 ArcS ∆arcS

2 (+0.5 V vs. –0.1 V)

2Arc system ′ 3 ∆arcS

Fig. 4-2 Appendix 4

′ (Fig. 4-2)3

2WT 322 2305 ∆arcS

(Fig. 4-9A)3 2105 ∆arcS

2

3 2WT ∆arcS mean-average (MA) plot

2∆arcS (fold change) (Fig. 4-9B)3

2Arc system 3

Arc system 2qRT-PCR

WT ∆arcS nuoI (Nuo ) HP LP

Δ 3 2WT HP Δ nuoI

2∆arcS HP2LP Δ WT nuoI 2

Δ (Fig. 4-10)3 2Arc system nuoI

′ 3

2

nuoI (Fig. 4-11)3 2arcS

3 2MR-1

Arc system 2

3 2 2WT

∆arcS 17 (Fig. 4-9A)3

Arc system 2 D-lactate dehydrogenase

dld 3 cyclic-AMP receptor protein (CRP)

(Kasai et al., 2017)3 2CRP (mtrCAB ) 2dld Arc system CRP ′ 3 qRT-PCR 2 nuo Arc system ′ 3 2 Arc system 3

nuoA DNA ArcA electrophoresis mobility shift

assay (EMSA) 3 2 ArcA nuoA

DNA (Fig. 4-12)2nuo (nuoA–N )

ArcA 3ArcA

DNA 2 DNA ArcA

2 “ ArcA

3 2 Arc system

atp suc ArcA (Fig. 4-13)3

Arc system 3

ArcA nuo 2nuoA

lacZ × 3EMSA nuoA 5

100 bp DNA ” 2× (pMElacZ) )

- ° (lacZ) 3 WT ∆arcA

2β- ° (LacZ) (Fig. 4-14A B)3 2

(+1) –112 (Fig. 4-14A) 2WT ∆arcA

LacZ (Fig. 4-14B)3 2–112 –63 2

∆arcA LacZ 2WT 3

2ArcA ′ nuoA –63 2–63 –112

2 (Fig. 4-14A)3EMSA (Fig. 4-12)

nuoA ArcA 2 3 (Table 4-1) (Fig. 4-5)2∆NDH (Fig. 4-7) 2ArcA (LP Δ ) 2nuoA (PnuoA) 3 2–273 2 2–112

ArcA LacZ 2∆arcA

(HP) Δ Δ 2 Δ PDH Nuo NADH (Fig. 4-15)3Nuo

2 2 H+_/e

-2 (Efremov et al., 2010)3Nuo CymA

2 NADH 3 3 (Fig. 4-1B) (Table 4-1) 2HP Δ “ acetyl-CoA 2TCA 3 2MR-1 2 (Fig. 4-15)3 2 3 2 2Arc system 3 2MnO2 NADH (Fig. 4-7C)3 2MnO2 2 ( 2 ) 3MR-1 2

(Nealson and Saffarini, 1994)3MR-1

Arc system 2 2

3 2MR-1 Shewanella Nuo

2 Nqr 3

MR-2 Shewanella ′

2Nuo Arc system MR-1

3 2Shewanella NADH dehydrogenase

– 3

(Table 4-1) 2 2 Arc

system 3

cyclic AMP (cAMP) receptor protein (CRP)

2Arc system ′ (Kasai et al., 2015)3

2MnO2 4

3

MR-1 Arc system

(Gao et al., 2008)3 TCA ′

Arc system 2MR-1

Arc system (Gao et al., 2008)3 2

× (Table 4-1) 2TCA 2-oxogulutarate dehydrogenase

suc Arc system Δ 3 2MR-1

Δ suc 2 TCA

(Tang et al., 2007)3 2EMSA (Fig. 4-13) suc Arc system

2MR-1 TCA 2

3 TCA

3

Arc system TCA ′ 2nuo

(Bongaerts et al., 1995; Lin, 1996; Park et al., 2013)3 2

3 2MR-1 nuo Arc

system (Fig. 4-12)3 2

Arc system

3 (Table 4-1) qRT-PCR (Fig. 4-10)2

∆NDH (Fig. 4-5) (Fig. 4-7) 2 Nuo

2

3 2lacZ × (Fig. 4-14B) 2ArcA

nuoA 2PnuoA “

3 2ArcS Δ

HptA ArcA 2 ArcA

“ nuoA 3 2

∆arcS nuo (Fig. 4-10 4-11)2 ArcA

nuo 3

2ArcA ( )

3ArcS

(Lassak et al., 2013)2∆arcS ArcA 2 nuo

3

3 2 Δ

UQ (Fig. 4-8)3

2 - 3 2UQ

MK 3

2 2 Mtr

CymA MK (McMillan et al., 2012)3CymA UQ

Table 4-1 ( )

Process Locus tag Gene Annotation Log2 FC*

Lactate and pyruvate oxidation

SO_1521 dld Respiratory FAD-dependent D-lactate dehydrogenase

2.32

SO_0425 aceF Dihydrolipoamide acetyltransferase 1.52

Formate oxidation SO_0101 fdnG Nitrate-inducible formate dehydrogenase molybdopterin-binding subunit

2.97 SO_0102 fdnH Nitrate-inducible formate dehydrogenase

iron-sulfur subunit

3.33 SO_0103 fdnI Nitrate-inducible formate dehydrogenase

cytochrome b subunit

2.89 SO_4509 fdhA Formate dehydrogenase molybdopterin-binding

subunit

–1.43

SO_4510 fdhB Formate dehydrogenase fes subunit –1.08

SO_4511 fdhC Formate dehydrogenase cytochrome b subunit –1.00 SO_4513 fdhA Fnr-inducilble formate dehydrogenase

molybdopterin-binding subunit

1.96 SO_4515 fdhC Fnr-inducible formate dehydrogenase

cytochrome b subunit

1.94

TCA cycle SO_1930 sucA 2-Oxoglutarate dehydrogenase complex

dehydrogenase E1 component

1.75 SO_1931 sucB 2-Oxoglutarate dehydrogenase complex

succinyl-CoA:dihydrolipoate S-succinyltransferase E2 component

1.77

SO_1933 sucD Succinyl-CoA synthase alpha subunit 1.66 NADH oxidation SO_1010 nuoM NADH-ubiquinone oxidoreductase subunit M 2.85 SO_1012 nuoK NADH-ubiquinone oxidoreductase subunit K 2.68 SO_1013 nuoJ NADH-ubiquinone oxidoreductase subunit J 2.58 SO_1014 nuoI NADH-ubiquinone oxidoreductase subunit I 2.46 SO_1015 nuoH NADH-ubiquinone oxidoreductase subunit H 2.89 SO_1016 nuoG NADH-ubiquinone oxidoreductase subunit G 2.61 SO_1017 nuoF NADH-ubiquinone oxidoreductase subunit F 2.46 SO_1018 nuoE NADH-ubiquinone oxidoreductase subunit E 1.78 SO_1019 nuoCD NADH-ubiquinone oxidoreductase subunit CD 1.90

ATP synthesis SO_4746 atpC ATP synthase F1 epsilon subunit 2.19

SO_4747 atpD ATP synthase F1 beta subunit 2.22

SO_4748 atpG ATP synthase F1 gamma subunit 2.50

SO_4749 atpA ATP synthase F1 alpha subunit 2.43

SO_4750 atpH ATP synthase F1 delta subunit 1.94

SO_4751 atpF ATP synthase F0 B subunit 1.85

SO_4752 atpE ATP synthase F0 C subunit 1.71

SO_4753 atpB ATP synthase F0 A subunit 1.37

*_Log

Fig. 4-4 (COG) 3

0 1 2 3 4 5 6 7

Energy production and conversion [C] Cell cycle control, cell division, chromosome partitioning [D] Amino acid transport and metabolism [E] Nucleotide transport and metabolism [F] Carbohydrate transport and metabolism [G] Coenzyme transport and metabolism [H] Lipid transport and metabolism [I] Translation, ribosomal structure and biogenesis [J] Transcription [K] Replication, recombination and repair [L] Cell wall/membrane/envelope biogenesis [M] Cell motility [N] Posttranslational modification, protein turnover, chaperones [O] Inorganic ion transport and metabolism [P] Secondary metabolites biosynthesis, transport and catabolism [Q] General function prediction only [R] Function unknown [S] Signal transduction mechanisms [T] Intracellular trafficking, secretion, and vesicular transport [U] Defense mechanisms [V]

Fig. 4-8 3WT +0.5 V –0.1 V

2 (UQ-8red) (UQ-8ox) 3

Fig. 4-9 Arc system ′ 3(A) WT ∆arcS 3(B) × mean-average 3 305 17 105 ΔarcS WT

A

B

A: log2 signal intensity

WT

∆arcS ΔarcS

Fig. 4-10 nuoI 3WT ∆arcS +0.5 V –0.1 V 2qRT-PCR nuoI 3 3 3 (P < 0.05) 3 0 0.5 1 1.5 2

WT_high WT_low ΔarcS_high ΔarcS_low

Fig. 4-11 3

WT ∆arcS

HP LP HP LP

Normalized signal intensity

Fig. 4-12 EMSA nuoA ArcA (ArcA-P) 3

Competitor DNA 2DNA ” 3

ArcA-P (ng)

nuoA

Fig. 4-13 EMSA sucA atpI ArcA-P 3

SO_00112 nuoA ” 3

ArcA-P – + – + – + – +

Fig. 4-15 Δ Δ MR-1 3Fdh: formate

dehydrogenase2Nuo: NADH dehydrogenase2 c (FccA

Abe, H., Doi, Y., Fukushima, T., Eya, H., 1994. Biosynthesis from gluconate of a random copolyester consisting of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by

Pseudomonas sp. 61-3. Int. J. Biol. Macromol. 16, 115–119.

https://doi.org/10.1016/0141-8130(94)90036-1

Bekker, M., Alexeeva, S., Laan, W., Sawers, G., De Mattos, J.T., Hellingwerf, K., 2010. The ArcBA two-component system of Escherichia coli is regulated by the redox state of both the ubiquinone and the menaquinone pool. J. Bacteriol. 192, 746–754. https://doi.org/10.1128/JB.01156-09 Beliaev, A.S., Saffarini, D.A., 1998. Shewanella putrefaciens mtrB encodes an outer membrane

protein required for Fe(III) and Mn(IV) reduction. J. Bacteriol. 180, 6292–6297.

Bongaerts, J., Zoske, S., Weidner, U., Linden, G., 1995. Transcriptional regulation of the proton translocating NADH dehydrogenase (nuoA‐N) of Escherichia coli by electron acceptors, electron donors and gene regulators. Mol. Microbiol. 16, 521–534.

https://doi.org/10.1111/j.1365-2958.1995.tb02416.x

Bosch, J., Lee, K.Y., Hong, S.F., Harnisch, F., Schröder, U., Meckenstock, R.U., 2014. Metabolic efficiency of Geobacter sulfurreducens growing on anodes with different redox potentials. Curr.

Microbiol. 68, 763–768. https://doi.org/10.1007/s00284-014-0539-2

Bretschger, O., Obraztsova, A., Sturm, C.A., In, S.C., Gorby, Y.A., Reed, S.B., Culley, D.E., Reardon, C.L., Barua, S., Romine, M.F., Zhou, J., Beliaev, A.S., Bouhenni, R., Saffarini, D., Mansfeld, F., Kim, B.H., Fredrickson, J.K., Nealson, K.H., 2007. Current production and metal oxide

reduction by Shewanella oneidensis MR-1 wild type and mutants. Appl. Environ. Microbiol. 73, 7003–7012. https://doi.org/10.1128/AEM.01087-07

Call, D.F., Wagner, R.C., Logan, B.E., 2009. Hydrogen production by Geobacter species and a mixed consortium in a microbial electrolysis cell. Appl. Environ. Microbiol. 75, 7579–7587.

https://doi.org/10.1128/AEM.01760-09

Claassens, N.J., Sousa, D.Z., Dos Santos, V.A.P.M., De Vos, W.M., Van Der Oost, J., 2016. Harnessing the power of microbial autotrophy. Nat. Rev. Microbiol. 14, 692–706. https://doi.org/10.1038/nrmicro.2016.130

Cruz-García, C., Murray, A.E., Rodrigues, J.L.M., Gralnick, J. a, McCue, L.A., Romine, M.F., Löffler, F.E., Tiedje, J.M., 2011. Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in

Shewanella oneidensis MR-1. BMC Microbiol. 11, 64. https://doi.org/10.1186/1471-2180-11-64

https://doi.org/10.1126/sciadv.aao5682

Deng, X., Okamoto, A., 2018. Electrode potential dependency of single-cell activity identifies the energetics of slow microbial electron uptake process. Front. Microbiol. 9, 1–8.

https://doi.org/10.3389/fmicb.2018.02744

Dibrov, P., Dibrov, E., Pierce, G.N., 2018. Na + -NQR ( Na + -translocating NADH(: ubiquinone oxidoreductase ) as a novel target for antibiotic. FEMS Microbiol. Rev. 41.5,653–671. https://doi.org/10.1093/femsre/fux032

Duhl, K. L.; Tefft, N. M.; TerAvest, M.A., 2018. Shewanella oneidensis MR-1 utilizes both sodium- and proton-pumping NADH dehydrogenases during aerobic growth. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.00415-18

Efremov, R.G., Baradaran, R., Sazanov, L. a, 2010. The architecture of respiratory complex I. Nature 465, 441–445. https://doi.org/10.1038/nature09066

Endoh, T., Habe, H., Yoshida, T., Nojiri, H., Omori, T., 2003. A CysB-regulated and σ54-dependent regulator, SfnR, is essential for dimethyl sulfone metabolism of Pseudomonas putida strain DS1.

Microbiology 149, 991–1000. https://doi.org/10.1099/mic.0.26031-0

Firer-Sherwood, M., Pulcu, G.S., Elliott, S.J., 2008. Electrochemical interrogations of the Mtr

cytochromes from Shewanella: Opening a potential window. J. Biol. Inorg. Chem. 13, 849–854. https://doi.org/10.1007/s00775-008-0398-z

Förster, A.H., Beblawy, S., Golitsch, F., Gescher, J., 2017. Biotechnology for Biofuels Electrode ‑ assisted acetoin production in a metabolically engineered Escherichia coli strain. Biotechnol.

Biofuels 1–11. https://doi.org/10.1186/s13068-017-0745-9

Gao, H., Wang, X., Yang, Z.K., Chen, J., Liang, Y., Chen, H., Palzkill, T., Zhou, J., 2010.

Physiological roles of ArcA, Crp, and EtrA and their interactive control on aerobic and anaerobic respiration in Shewanella oneidensis. PLoS One 5, e15295.

https://doi.org/10.1371/journal.pone.0015295

Gao, H., Wang, X., Yang, Z.K., Palzkill, T., Zhou, J., 2008. Probing regulon of ArcA in Shewanella

oneidensis MR-1 by integrated genomic analyses. BMC Genomics 9, 42.

https://doi.org/10.1186/1471-2164-9-42

Gorby, Y.A., Yanina, S., Mclean, J.S., Rosso, K.M., Moyles, D., Dohnalkova, A., Beveridge, T.J., Chang, I.S., Kim, B.H., Kim, K.S., Culley, D.E., Reed, S.B., Romine, M.F., Saffarini, D.A., Hill, E.A., Shi, L., Elias, D.A., Kennedy, D.W., Pinchuk, G., Watanabe, K., Ishii, S., Logan, B., Nealson, K.H., Fredrickson, J.K., 2006. Electrically conductive bacterial nanowires produced by

11358-11363. https://doi.org/10.1073/pnas.0604517103

Grobbler, C., Virdis, B., Nouwens, A., Harnisch, F., Rabaey, K., Bond, P.L., 2018. Effect of the anode potential on the physiology and proteome of Shewanella oneidensis MR-1. Bioelectrochemistry 119, 172–179. https://doi.org/10.1016/j.bioelechem.2017.10.001

Grobbler, C., Virdis, B., Nouwens, A., Harnisch, F., Rabaey, K., Bond, P.L., 2014. Use of SWATH mass spectrometry for quantitative proteomic investigation of Shewanella oneidensis MR-1 biofilms grown on graphite cloth electrodes. Syst. Appl. Microbiol. 1–5.

https://doi.org/10.1016/j.syapm.2014.11.007

Gunsalus, R.P., Park, S.J., 1994. Aerobic-anaerobic gene regulation in Escherichia coli: control by the ArcAB and Fnr regulons. Res. Microbiol. 145, 437–450.

https://doi.org/10.1016/0923-2508(94)90092-2

Hartshorne, R.S., Reardon, C.L., Ross, D., Nuester, J., Clarke, T.A., Gates, A.J., Mills, P.C., Fredrickson, J.K., Zachara, J.M., Shi, L., Beliaev, A.S., Marshall, M.J., Tien, M., Brantley, S., Butt, J.N., Richardson, D.J., 2009. Characterization of an electron conduit between bacteria and the extracellular environment. Proc. Natl. Acad. Sci. 106, 22169–22174.

https://doi.org/10.1073/pnas.0900086106

Heidelberg, J.F., Paulsen, I.T., Nelson, K.E., Gaidos, E.J., Nelson, W.C., Read, T.D., Eisen, J.A., Seshadri, R., Ward, N., Methe, B., Clayton, R.A., Meyer, T., Tsapin, A., Scott, J., Beanan, M., Brinkac, L., Daugherty, S., DeBoy, R.T., Dodson, R.J., Durkin, A.S., Haft, D.H., Kolonay, J.F., Madupu, R., Peterson, J.D., Umayam, L.A., White, O., Wolf, A.M., Vamathevan, J., Weidman, J., Impraim, M., Lee, K., Berry, K., Lee, C., Mueller, J., Khouri, H., Gill, J., Utterback, T.R., McDonald, L.A., Feldblyum, T. V., Smith, H.O., Venter, J.C., Nealson, K.H., Fraser, C.M., 2002. Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis. Nat.

Biotechnol. 20, 1118–1123. https://doi.org/10.1038/nbt749

Hirose, A., Kasai, T., Koga, R., Suzuki, Y., Kouzuma, A., Watanabe, K., 2019a. Understanding and engineering electrochemically active bacteria for sustainable biotechnology. Bioresour.

Bioprocess. 1–15. https://doi.org/10.1186/s40643-019-0245-9

Hirose, A., Kouzuma, A., Watanabe, K., 2019b. Towards development of electrogenetics using electrochemically active bacteria. Biotechnol. Adv.

https://doi.org/10.1016/j.biotechadv.2019.02.007

Hunt, K.A., Flynn, J.M., Naranjo, B., Shikhare, I.D., Gralnick, J.A., 2010. Substrate-level

phosphorylation is the primary source of energy conservation during anaerobic respiration of

https://doi.org/10.1128/JB.00090-10

Ishii, M., Takishita, S., Iwasaki, T., Peerapornpisal, Y., Yoshino, J., Kodama, T., Igarashi, Y., 2000. Purification and Characterization of Membrane-bound Hydrogenase from Hydrogenobacter

thermophilus Strain TK-6, an Obligately Autotrophic, Thermophilic, Hydrogen-oxidizing

Bacterium. Biosci. Biotechnol. Biochem. 64, 492–502. https://doi.org/10.1271/bbb.64.492 Ishii, S., Kosaka, T., Hori, K., Hotta, Y., Watanabe, K., 2005. Coaggregation facilitates interspecies

hydrogen transfer between Pelotomaculum thermopropionicum and Methanothermobacter

thermautotrophicus. Appl. Environ. Microbiol. 71, 7838–7845.

https://doi.org/10.1128/AEM.71.12.7838-7845.2005

Ishii, T., Kawaichi, S., Nakagawa, H., Hashimoto, K., Nakamura, R., 2015. From

chemolithoautotrophs to electrolithoautotrophs: CO2 fixation by Fe(II)-oxidizing bacteria coupled with direct uptake of electrons from solid electron sources. Front. Microbiol. 6, 1–9. https://doi.org/10.3389/fmicb.2015.00994

Jones, S.W., Fast, A.G., Carlson, E.D., Wiedel, C.A., Au, J., Antoniewicz, M.R., Papoutsakis, E.T., Tracy, B.P., 2016. CO2 fixation by anaerobic non-photosynthetic mixotrophy for improved carbon conversion. Nat. Commun. 7, 12800. https://doi.org/10.1038/ncomms12800

Kane, A.L., Brutinel, E.D., Joo, H., Maysonet, R., VanDrisse, C.M., Kotloski, N.J., Gralnick, J.A., 2016. Formate metabolism in Shewanella oneidensis generates proton motive force and prevents growth without an electron acceptor. J. Bacteriol. 198, 1337–1346.

https://doi.org/10.1128/JB.00927-15

Karthikeyan, R., Singh, R., Bose, A., 2019. Microbial electron uptake in microbial electrosynthesis(: a mini ‑ review. J. Ind. Microbiol. Biotechnol. https://doi.org/10.1007/s10295-019-02166-6 Kasai, T., Kouzuma, A., Nojiri, H., Watanabe, K., 2015. Transcriptional mechanisms for differential

expression of outer membrane cytochrome genes omcA and mtrC in Shewanella oneidensis MR-1. BMC Microbiol. 15, 68. https://doi.org/10.1186/s12866-015-0406-8

Kasai, T., Kouzuma, A., Watanabe, K., 2017. CRP regulates D-lactate oxidation in Shewanella

oneidensis MR-1. Front. Microbiol. 8, 1–11. https://doi.org/10.3389/fmicb.2017.00869

Kasai, T., Suzuki, Y., Kouzuma, A., Watanabe, K., 2019. Roles of D -Lactate Dehydrogenases in the Anaerobic Growth of Shewanella oneidensis MR-1 on Sugars. Appl. Environ. Microbiol. 85, 1– 11. https://doi.org/10.1128/AEM.02668-18

Kimura, Z.I., Okabe, S., 2013. Acetate oxidation by syntrophic association between Geobacter

sulfurreducens and a hydrogen-utilizing exoelectrogen. ISME J. 7, 1472–1482.

Kitayama, M., Koga, R., Kasai, T., Kouzuma, A., Watanabe, K., 2017. Structures, compositions, and activities of live Shewanella biofilms formed on graphite electrodes in electrochemical flow cells.

Appl. Environ. Microbiol. 83, 1–11. https://doi.org/10.1128/AEM.00903-17

Kouzuma, A., Hashimoto, K., Watanabe, K., 2012. Roles of siderophore in manganese-oxide reduction by Shewanella oneidensis MR-1. FEMS Microbiol. Lett. 326, 91–98.

https://doi.org/10.1111/j.1574-6968.2011.02444.x

Kracke, F., Vassilev, I., Kr??mer, J.O., 2015. Microbial electron transport and energy conservation - The foundation for optimizing bioelectrochemical systems. Front. Microbiol. 6, 1–18.

https://doi.org/10.3389/fmicb.2015.00575

Lassak, J., Bubendorfer, S., Thormann, K.M., 2013. Domain analysis of ArcS, the hybrid sensor kinase of the Shewanella oneidensis MR-1 Arc two-component system, reveals functional differentiation of its two receiver domains. J. Bacteriol. 195, 482–92.

https://doi.org/10.1128/JB.01715-12

Lassak, J., Henche, A.-L., Binnenkade, L., Thormann, K.M., 2010. ArcS, the cognate sensor kinase in an atypical Arc system of Shewanella oneidensis MR-1. Appl. Environ. Microbiol. 76, 3263–74. https://doi.org/10.1128/AEM.00512-10

Le Laz, S., Kpebe, A., Lorquin, J., Brugna, M., Rousset, M., 2014. H2-dependent azoreduction by

Shewanella oneidensis MR-1: Involvement of secreted flavins and both [Ni-Fe] and [Fe-Fe]

hydrogenases. Appl. Microbiol. Biotechnol. 98, 2699–2707. https://doi.org/10.1007/s00253-013-5208-z

Levar, C.E., Hoffman, C.L., Dunshee, A.J., Toner, B.M., Bond, D.R., 2017. Redox potential as a master variable controlling pathways of metal reduction by Geobacter sulfurreducens. ISME J. 11, 741–752. https://doi.org/10.1038/ismej.2016.146

Li, D.B., Li, J., Liu, D.F., Ma, X., Cheng, L., Li, W.W., Qian, C., Mu, Y., Yu, H.Q., 2019. Potential regulates metabolism and extracellular respiration of electroactive Geobacter biofilm. Biotechnol.

Bioeng. 116, 961–971. https://doi.org/10.1002/bit.26928

Lian, Y., Yang, Y., Guo, J., Wang, Y., Li, X., Fang, Y., Gan, L., Xu, M., 2016. Electron acceptor redox potential globally regulates transcriptomic profiling in Shewanella decolorationis S12. Sci.

Rep. 6, 1–9. https://doi.org/10.1038/srep31143

Light, S.H., Su, L., Rivera-lugo, R., Cornejo, J.A., Louie, A., Iavarone, A.T., 2018. A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria. Nature.

https://doi.org/10.1038/s41586-018-0498-z

protein of Escherichia coli(: characterization of DNA binding at target promoters. J. Bacteriol. 178, 6238–6249.https://doi.org/10.1128/jb.178.21.6238-6249.1996

Liu, C., Gorby, Y.A., Zachara, J.M., Fredrickson, J.K., Brown, C.F., 2002. Reduction kinetics of Fe(III), Co(III), U(VI), Cr(VI), and Tc(VII) in cultures of dissimilatory metal-reducing bacteria. Biotechnol. Bioeng. 80, 637–649. https://doi.org/10.1002/bit.10430

Logan, B.E., 2009. Exoelectrogenic bacteria that power microbial fuel cells. Nat. Rev. Microbiol. 7, 375–381. https://doi.org/10.1038/nrmicro2113

Madsen, C.S., TerAvest, M.A., 2019. NADH dehydrogenases contribute to extracellular electron transfer by Shewanella oneidensis MR-1 in bioelectrochemical systems. bioRxiv 1–15. https://doi.org/http://dx.doi.org/10.1101/657668

Marshall, M.J., Plymale, A.E., Kennedy, D.W., Shi, L., Wang, Z., Reed, S.B., Dohnalkova, A.C., Simonson, C.J., Liu, C., Saffarini, D.A., Romine, M.F., Zachara, J.M., Beliaev, A.S., Fredrickson, J.K., 2008. Hydrogenase- and outer membrane c-type cytochrome-facilitated reduction of technetium(VII) by Shewanella oneidensis MR-1. Environ. Microbiol. 10, 125–136. https://doi.org/10.1111/j.1462-2920.2007.01438.x

McMillan, D.G.G., Marritt, S.J., Butt, J.N., Jeuken, L.J.C., 2012. Menaquinone-7 is specific cofactor in tetraheme quinol dehydrogenase CymA. J. Biol. Chem. 287, 14215–14225.

https://doi.org/10.1074/jbc.M112.348813

Melo, A.M.P., Bandeiras, T.M., Teixeira, M., 2004. New Insights into Type II NAD ( P ) H(: Quinone Oxidoreductases. Microbiol. Mol. Biol. Rev. 68, 603–616.

https://doi.org/10.1128/MMBR.68.4.603

Meshulam-simon, G., Behrens, S., Choo, A.D., Spormann, A.M., 2007. Hydrogen Metabolism in

Shewanella oneidensis MR-1. Appl. Environ. Microbiol. 73, 1153–1165.

https://doi.org/10.1128/AEM.01588-06

Moscoviz, R., Toledo-Alarcón, J., Trably, E., Bernet, N., 2016. Electro-Fermentation: How To Drive Fermentation Using Electrochemical Systems. Trends Biotechnol. 34, 856–865.

https://doi.org/10.1016/j.tibtech.2016.04.009

Myers, C.R., Nealson, K.H., 1988. Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor. Science. 240, 1319–1321.

https://doi.org/10.1126/science.240.4857.1319

Myers, J.M., Myers, C.R., 2000. Role of the tetraheme cytochrome CymA in anaerobic electron transport in cells of Shewanella putrefaciens MR-1 with normal levels of menaquinone. J.

Myers, K.S., Yan, H., Ong, I.M., Chung, D., Liang, K., Tran, F., Keleş, S., Landick, R., Kiley, P.J., 2013. Genome-scale Analysis of Escherichia coli FNR Reveals Complex Features of

Transcription Factor Binding. PLoS Genet. 9, 11–13. https://doi.org/10.1371/journal.pgen.1003565

Nakagawa, G., Kouzuma, A., Hirose, A., Kasai, T., Yoshida, G., Watanabe, K., 2015. Metabolic Characteristics of a Glucose-Utilizing Shewanella oneidensis Strain Grown under

Electrode-Respiring Conditions. PLoS One 10, e0138813. https://doi.org/10.1371/journal.pone.0138813

Nakamura, R., Takashima, T., Kato, S., Takai, K., Yamamoto, M., Hashimoto, K., 2010. Electrical current generation across a black smoker chimney. Angew. Chemie - Int. Ed. 49, 7692–7694. https://doi.org/10.1002/anie.201003311

Nealson, K.H., Saffarini, D., 1994. Iron and manganese in anaerobic respiration: environmental significance, physiology, and regulation. Annu. Rev. Microbiol. 48, 311–343.

https://doi.org/10.1146/annurev.micro.48.1.311

Pankratova, G., Szypulska, E., Pankratov, D., Leech, D., Gorton, L., 2019. Electron Transfer between the Gram-Positive Enterococcus faecalis Bacterium and Electrode Surface through Osmium Redox Polymers. ChemElectroChem 6, 110–113. https://doi.org/10.1002/celc.201800683 Park, D.M., Akhtar, M.S., Ansari, A.Z., Landick, R., Kiley, P.J., 2013. The bacterial response

regulator ArcA uses a diverse binding site architecture to regulate carbon oxidation globally.

PLoS Genet. 9, e1003839. https://doi.org/10.1371/journal.pgen.1003839

Pinchuk, G.E., Geydebrekht, O. V, Hill, E. a, Reed, J.L., Konopka, A.E., Beliaev, A.S., Fredrickson, J.K., 2011. Pyruvate and lactate metabolism by Shewanella oneidensis MR-1 under fermentation, oxygen limitation, and fumarate respiration conditions. Appl. Environ. Microbiol. 77, 8234–40. https://doi.org/10.1128/AEM.05382-11

Pinchuk, G.E., Hill, E. a., Geydebrekht, O. V., de Ingeniis, J., Zhang, X., Osterman, A., Scott, J.H., Reed, S.B., Romine, M.F., Konopka, A.E., Beliaev, A.S., Fredrickson, J.K., Reed, J.L., 2010. Constraint-based model of Shewanella oneidensis MR-1 metabolism: A tool for data analysis and hypothesis generation. PLoS Comput. Biol. 6, 1–8.

https://doi.org/10.1371/journal.pcbi.1000822

Pirbadian, S., Barchinger, S.E., Man, K., Suk, H., Jangir, Y., Bouhenni, R.A., 2014. Shewanella

oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular

Pradella, S., Hippe, H., Stackebrandt, E., 1998. Macrorestriction analysis of Desulfurella acetivorans and Desulfurella multipotens. FEMS Microbiol. Lett. 159, 137–144.

https://doi.org/10.1016/S0378-1097(97)00561-2

Rabaey, K., Rozendal, R.A., 2010. Microbial electrosynthesis - Revisiting the electrical route for microbial production. Nat. Rev. Microbiol. 8, 706–716. https://doi.org/10.1038/nrmicro2422 Reysenbach, A.L., Shock, E., 2002. Merging genomes with geochemistry in hydrothermal ecosystems.

Science. 296, 1077–1082. https://doi.org/10.1126/science.1072483

Rodionov, D.A., Yang, C., Li, X., Rodionova, I.A., Wang, Y., Obraztsova, A.Y., Zagnitko, O.P., Overbeek, R., Romine, M.F., Reed, S., Fredrickson, J.K., Nealson, K.H., Osterman, A.L., 2010. Genomic encyclopedia of sugar utilization pathways in the Shewanella genus. BMC Genomics 11. https://doi.org/10.1186/1471-2164-11-494

Ross, D.E., Flynn, J.M., Baron, D.B., Gralnick, J.A., Bond, D.R., 2011. Towards Electrosynthesis in

Shewanella(: Energetics of Reversing the Mtr Pathway for Reductive Metabolism. PLoS One 6.

https://doi.org/10.1371/journal.pone.0016649

Rowe, A.R., Rajeev, P., Jain, A., Pirbadian, S., Okamoto, A., Gralnick, J.A., El-Naggar, M.Y., Nealson, K.H., 2018. Tracking electron uptake from a cathode into Shewanella cells: Implications for energy acquisition from solid-substrate electron donors. MBio 9, 1–19. https://doi.org/10.1128/mBio.02203-17

Saffarini, D. a, Schultz, R., Beliaev, A., 2003. Involvement of Cyclic AMP ( cAMP ) and cAMP Receptor Protein in Anaerobic Respiration of Shewanella oneidensis. J. Bacteriol. 185, 3668– 3671. https://doi.org/10.1128/JB.185.12.3668

Sharma, P., Stagge, S., Bekker, M., Bettenbrock, K., Hellingwerf, K.J., 2013. Kinase Activity of ArcB from Escherichia coli Is Subject to Regulation by Both Ubiquinone and Demethylmenaquinone.

PLoS One 8. https://doi.org/10.1371/journal.pone.0075412

Shi, L., Chen, B., Wang, Z., Elias, D.A., Mayer, M.U., Gorby, Y.A., Ni, S., Lower, B.H., Kennedy, D.W., Wunschel, D.S., Mottaz, H.M., Marshall, M.J., Hill, E.A., Beliaev, A.S., Zachara, J.M., Fredrickson, J.K., Squier, T.C., 2006. Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella

oneidensis MR-1. J. Bacteriol. 188, 4705–4714. https://doi.org/10.1128/JB.01966-05

Simon, J., van Spanning, R.J.M., Richardson, D.J., 2008. The organisation of proton motive and non-proton motive redox loops in prokaryotic respiratory systems. Biochim. Biophys. Acta -

Bioenerg. 1777, 1480–1490. https://doi.org/10.1016/j.bbabio.2008.09.008

Physiological Role of the Proton-Translocating NADH:Quinone Oxidoreductase (Complex I) Across Bacteria. MBio 6, e00389-15-. https://doi.org/10.1128/mBio.00389-15

Sturm-richter, K., Golitsch, F., Sturm, G., Kipf, E., Dittrich, A., Beblawy, S., Kerzenmacher, S., Gescher, J., 2015. Unbalanced fermentation of glycerol in Escherichia coli via heterologous production of an electron transport chain and electrode interaction in microbial electrochemical cells. Bioresour. Technol. 186, 89–96. https://doi.org/10.1016/j.biortech.2015.02.116

Sturm, G., Richter, K., Doetsch, A., Heide, H., Louro, R.O., Gescher, J., 2015. A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime. ISME J. 9, 1802–1811. https://doi.org/10.1038/ismej.2014.264

Tang, Y.J., Hwang, J.S., Wemmer, D.E., Keasling, J.D., 2007. Shewanella oneidensis MR-1 fluxome under various oxygen conditions. Appl. Environ. Microbiol. 73, 718–29.

https://doi.org/10.1128/AEM.01532-06

Tefft, N.M., Teravest, M.A., 2019. Reversing an Extracellular Electron Transfer Pathway for Electrode-Driven Acetoin Reduction. ACS Synth. Biol.

https://doi.org/10.1021/acssynbio.8b00498

Teran-Melo, J.L., Peña-Sandoval, G.R., Silva-Jimenez, H., Rodriguez, C., Alvarez, A.F., Georgellis, D., 2018. Routes of phosphoryl-group transfer during signal transmission and signal decay in the dimeric sensor histidine kinase ArcB. J. Biol. Chem. 293, jbc.RA118.003910.

https://doi.org/10.1074/jbc.RA118.003910

Tran, Q.H., Bongaerts, J., Vlad, D., Unden, G., 1997. Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications. Eur. J. Biochem. 244, 155–160.

https://doi.org/10.1111/j.1432-1033.1997.00155.x

Venkateswaran, K., Moser, D.P., Dollhopf, M.E., Lies, D.P., Saffarini, D. a, MacGregor, B.J.,

Ringelberg, D.B., White, D.C., Nishijima, M., Sano, H., Burghardt, J., Stackebrandt, E., Nealson, K.H., 1999. Polyphasic taxonomy of the genus Shewanella and description of Shewanella

oneidensis sp. nov. Int. J. Syst. Bacteriol. 49, 705–724.

https://doi.org/10.1099/00207713-49-2-705

Wu, Z., Wang, J., Liu, J., Wang, Y., Bi, C., Zhang, X., 2019a. Engineering an electroactive

Escherichia coli for the microbial electrosynthesis of succinate from glucose and CO2. Microb. Cell Fact. 18, 1–14. https://doi.org/10.1186/s12934-019-1067-3

(1) Nakagawa, G., Kouzuma, A., Hirose, A., Kasai, T., Yoshida, G., & Watanabe, K. (2015). Metabolic characteristics of a glucose-utilizing Shewanella oneidensis strain grown under electrode-respiring conditions. PloS one, 10(9), e0138813.

(2) Hirose, A., Kasai, T., Aoki, M., Umemura, T., Watanabe, K., & Kouzuma, A. (2018). Electrochemically active bacteria sense electrode potentials for regulating catabolic pathways. Nature communications, 9(1), 1-10.

2

(1) Kouzuma A, Kasai T, Hirose A, & Watanabe K (2015). Catabolic and regulatory systems in Shewanella oneidensis MR-1 involved in electricity generation in microbial fuel cells.

Frontiers in microbiology, 6, 609. (2) 2 2 . . 47:74-79. (3) 2 2 . . 58:33-38 (4) 2 2 . 12 . . CMC . (5) 2 2 2 . Shewanella . .

(6) Hirose, A., Kouzuma, A., & Watanabe, K. (2019). Towards development of

electrogenetics using electrochemically active bacteria. Biotechnology advances, 37(6), 107351.

(7) Hirose, A., Kasai, T., Koga, R., Suzuki, Y., Kouzuma, A., & Watanabe, K. (2019). Understanding and engineering electrochemically active bacteria for sustainable biotechnology. Bioresources and Bioprocessing, 6(1), 10.

(8) Inohana Y, Matsumoto A, Nagoya M, Hirose A, Kouzuma A, Watanabe K. Rice paddy-field microbial fuel cells: fundamental and recent progress. Bio-electrochemical systems: A sustainable platform for fuels and chemicals. Springer/nature. In press.

(1) ○Hirose A., Kouzuma A., Watanabe K. Molecular mechanisms for sensing and responding to electrode potentials in Shewanella oneidensis MR-1. Bio micro world 2015, Barcelona, Spain, October, 2015.

(2) ○Hirose A., Kouzuma A., Aoki M., Umemura T., Watanabe K. Electrode

(3) Hirose A., Kasai T., Kouzuma A. and ○Watanabe K. Shewanella senses electrode potential for catabolic regulation. The 3rd European Meeting of the International Society for Microbial Electrochemistry and Technology, Rome, Italy, September, 2016.

(4) Hirose A., Kasai T., Kouzuma A. and ○Watanabe K. Shewanella senses electrode potential for catabolic regulation. The 3rd Asian-Pacific Meeting of the International Society for Microbial Electrochemistry and Technology, Busan, South Korea, August,

2016. .

(5) ○Kouzuma A., Hirose A., Mogi H., Kasai T., and Watanabe K. A novel mode of regulation for electrochemical activities of Shewanella oneidensis MR-1. International Society for Microbial Electrochemistry and Technology, Okinawa, Japan, October, 2019.

(1) ○ . Shewanella oneidensis

. 2015 , , 2015 3 .

(2) ○Hirose A., Kouzuma A. and Watanabe K. Molecular mechanisms for sensing and responding to electrode potentials in Shewanella oneidensis MR-1.

2015 , , 2015 10 . (3) ○ . Shewanella oneidensis MR-1 . 2016 , , 2016 3 . (4) ○ . Shewanella oneidensis MR-1 . 2016 , , 2016 6 . (5) ○ . . 15 , , 2016 11 . (6) ○ . Shewanella oneidensis MR-1 . 2017 2017 3 . (7) ○ . Shewanella oneidensis MR-1 . 2017, , 2017 8 .

MR-1 . 2018 2018 3 .

(10) ○ . Shewanella oneidensis MR-1 NADH

Appendix

Appendix 1

Strain or plasmid Relevant characteristic Source of reference

Escherichia coli

DH5α F-, φ 80dlacZ∆M15, ∆(lacZYA-argF)U169, deoR, recA1,

endA1, hsdR17(rK-, mK+), phoA, supE44, λ-, thi-1,

gyrA96, relA1

Takara JM109 recA1. endAl, gyrA96, thi. hsdR17, supE44, relA1, λ-,

∆(lac-proAB), [F’, traD36, proAB, lacIq Z∆M15]

Takara

JM109λpir JM109 lysogenized with λpir Penfold and

Pemberton, 1992 WM6026 lacIq, rrnB3, DElacZ4787, hsdR514, DE(araBAD)567,

E(rhaBAD)568, rph-1, att-lambda::pAE12-del(oriR6K-cat::frt5), DE(endA)::frt, uidA(delMluI)::pir(wt), attHK::pJK1006-del1/2 (deloriR6K-cat::frt5, deltrfA::frt) William Metcalf, University of Illinois

BL21 DE3 F– ompT hsdR17 (rB– mB+) gal dcm(DE3) F–, ompT,

hsdSB (rB– mB–), gal(λcI 857, ind1, sam7, nin5,

lacUV5-T7gene1), dcm(DE3)

Novagen

S. oneidensis strain

MR-1 Wild type ATCC

ΔPFL SO_2912 pflB disruption This study

ΔPDH SO_0424 aceE disruption This study

ΔNDH SO_1017 nuoF , SO_3517 ndh , SO_0907 nqrF-1 ,

SO_1108 nqrF-2 disruption

This study

ΔarcS SO_0577 arcS disruption This study

ΔarcA SO_3988 arcA disruption This study

ΔhyaB SO_2098 hyaB disruption This study

ΔhydA SO_3920 hydA disruption This study

ΔhydAΔhyaB SO_2098 hyaB , SO_3920 hydA disruption This study

Δatp SO_4746 atpC to SO_4754 atpI disruption This study

ΔubiA SO_0468 (ubiA) disruption This study

ΔmenA SO_1910 (menA) disruption This study

ΔnuoF SO_1017 nuoF This study

Δndh SO_3517 ndh This study

ΔnqrF-1 SO_0907 nqrF-1 This study

ΔnqrF-2 SO_1108 nqrF-2 This study

ΔnuoFΔndh SO_1017 nuoF , SO_3517 ndh disruption This study

ΔnuoFΔNDHΔubiA SO_1017 (nuoF), SO_3517 (ndh), SO_0468 (ubiA) disruption

This study

ΔnuoFΔNDHΔmenA SO_1017 (nuoF), SO_3517 (ndh), SO_1910 menA

disruption

This study

NUO SO_3517 ndh , SO_0907 nqrF-1 , SO_1108 nqrF-2

disruption

This study

NDH SO_1017 nuoF , SO_0907 nqrF-1 , SO_1108

nqrF-2 disruption

This study

NQR1 SO_1017 nuoF , SO_3517 ndh , SO_1108 nqrF-2

disruption

This study

NQR2 SO_1017 nuoF , SO_3517 ndh , SO_0907 nqrF-1

disruption

This study

ΔcymA SO_4591 (cymA) disruption Bretschger et al.

Appendix 1

Strain or plasmid Relevant characteristic Source of reference

(omcA(mtrC SO_1779 (omcA), SO_1778 (mtrC) disruptionn Bretschger et al. 2007

ΔetrA SO_2356 (etrA) disruption This study

Plasmid

pET-28 a Expression vector, T7 promoter Novagen

pET-arcA pET-28(a) containing N terminal His-tag-arcA This study

pSMV-10 9.1 kb mobilizable suicide vector; oriR6K, mobRP4,

sacB, Kmr, Gmr

Chad Saltikov, California Inst. of

Tech. pSMV-pfl 1.6 kb fusion PCR fragment containing ∆pfl cloned into

the SpeI site of pSMV-10

This study pSMV-aceE 1.6 kb fusion PCR fragment containing ∆aceE cloned

into the SpeI site of pSMV-10

This study pSMV-nuoF 1.6 kb fusion PCR fragment containing ∆nuoF cloned

into the SpeI site of pSMV-10

This study pSMV-ndh 1.6 kb fusion PCR fragment containing ∆ndh cloned into

the SpeI site of pSMV-10

This study pSMV-nqrF-1 1.6 kb fusion PCR fragment containing ∆nqrF-1 cloned

into the SpeI site of pSMV-10

This study pSMV-nqrF-2 1.6 kb fusion PCR fragment containing ∆nqrF-2 cloned

into the SpeI site of pSMV-10

This study pSMV-arcS 1.6 kb fusion PCR fragment containing ∆arcS cloned

into the SpeI site of pSMV-10

This study pSMV-arcA 1.6 kb fusion PCR fragment containing ∆arcA cloned

into the SpeI site of pSMV-10

This study pSMV-atp 1.6 kb fusion PCR fragment containing ∆atp cloned into

the SpeI site of pSMV-10

This study pSMV-hyaB 1.6 kb fusion PCR fragment containing ∆hyaB cloned

into the SpeI site of pSMV-10

This study pSMV-hydA 1.6 kb fusion PCR fragment containing ∆hydA cloned

into the SpeI site of pSMV-10

This study pSMV-ubiA 1.6 kb fusion PCR fragment containing ∆ubiA cloned

into the SpeI site of pSMV-10

This study pSMV-menA 1.6 kb fusion PCR fragment containing ∆menA cloned

into the SpeI site of pSMV-10

This study pSMV-etrA 1.6 kb fusion PCR fragment containing ∆etrA cloned into

the SpeI site of pSMV-10

This study

pMElacZ pME4510 derivative, lacZ Gmr Endoh et al. 2003

pME-PnuoA pMElacZ containing nuoA upstream region This study

pME-PnuoA_TSS-7 pMElacZ containing region from -7 to +163 relative to

TSSnuoA

This study pME-PnuoA_TSS-63 pMElacZ containing region from -63 to +163 relative to

TSSnuoA

This study pME-PnuoA_TSS-112 pMElacZ containing region from -112 to +163 relative

to TSSnuoA

This study pME-PnuoA_TSS-273 pMElacZ containing region from -273 to +163 relative

to TSSnuoA

This study pME-PnuoA_TSS-361 pMElacZ containing region from -361 to +163 relative

to TSSnuoA

This study pME-PnuoA_TSS-407 pMElacZ containing region from -407 to +163relative

to TSSnuoA

This study

pME-Pndh pMElacZ containing ndh upstream region This study

pME-Pnqr1 pMElacZ containing nqr1 upstream region This study

Appendix 1

Strain or plasmid Relevant characteristic Source of reference

pME-PyedY pMElacZ containing yedY SO_2042 upstream

region

This study

pME-PSO_0939 pMElacZ containing SO0939 upstream region This study

pME-PfeoA pMElacZ containing feoA b3408 upstream region This study

pME-PhyaA pMElacZ containing E. coli JM109 hyaA b0972

upstream region

This study

pME-PgadE pMElacZ containing E. coli JM109 gadE b3512

upstream region

This study pME-Pnqr1-ABS pMElacZ containing nqr1 upstream region deleted

5'-UTR

This study pME_Plac pMElacZ containing E. coli JM109 lacZ upstream

region

Appendix 2

Primer Sequence 5' to 3' Modification, for use

qRT-nuoI-F TTTCGAGATGGGCGAGTATC qRT-PCR for nuoI SO_1014 qRT-nuoI-R CGCTCATGCGATAGAAGTTG qRT-PCR for nuoI SO_1014

qRT-dld-F CATCGGCACTCAACTTCTCA qRT-PCR for dld SO_1521

qRT-dld-R CGCAGGTATCAATCACATCG qRT-PCR for dld SO_1521

qRT-SO_4509-F CAAGCCGTTTTGATCAAGGT qRT-PCR for SO_4509

qRT-SO_4509-R ATCCTTCTGTGCGATCTTGG qRT-PCR for SO_4509

qRT-SO_0939-F AATCGACGCCAAGCATTAAC qRT-PCR for SO_0939

qRT-SO_0939-R TTTTCCCCGAGTGCTAATTG qRT-PCR for SO_0939

qRT-atpG-F CGTGAAAAGCTGGAAAGAGC qRT-PCR for atpG SO_4748 qRT-atpG-R TGCTTGTGCCGATACTTGTC qRT-PCR for atpG SO_4748

qRT-SO_1538-F ACCTCAGTGATCTTGGCCTC qRT-PCR for SO_1538

qRT-SO_1538-R CGCCTTCTTCAATCACAGCA qRT-PCR for SO_1538

qRT-SO_4360-F CGTTCATCACACCCGCTAAC qRT-PCR for SO_4360

qRT-SO_4360-R CGCATGGCAGGTATAACAGG qRT-PCR for SO_4360

qRT-nuoF-F ATTTAGCAACGCTCGAGCAG qRT-PCR for nuoF

qRT-nuoF-R ATTTAATGGCGCTGGCAAGC qRT-PCR for nuoF

qRT-ndh-F TGCTTGCTTTAGGTGGTGTC qRT-PCR for ndh qRT-ndh-R AGGGCATCCAAGAGCTTTTG qRT-PCR for ndh qRT-nqrF-1-F AGCCGCTGAGAATGACAACT qRT-PCR for nqrF-1 qRT-nqrF-1-R ACCGAGGAGTTCATGATTGG qRT-PCR for nqrF-1 qRT-nqrF-2-F GCGCTTACTCGATGGCTAAC qRT-PCR for nqrF-2 qRT-nqrF-2-R ACCGAATGGACCAGAAATTG qRT-PCR for nqrF-2 Overexpression arcA_NdeI_F CGCCATATGCAAAATCCGCACATTCTGATCG ArcA SO_3988 overexpression, NdeI arcA_BamHI_R CGCGGATCCTTAGTCTTCTAAGTTACCGCAG AAACG ArcA SO_3988 overexpression, BamHI EMSA nuoA-gelshift-F GCAATACATTGGCAACCA

EMSA for nuoA SO_1021 upstream region

Cy3-nuoA-gelshift-R

GGTCACGATTAAGTTTCATCC

EMSA for nuoA SO_1021 upstream region, 5'-Cy3 SO_0011-gelshift-F

GGTATAATCGGGGAGTTTTTA

EMSA for SO_0011 upstream region

Cy3-SO_0011-gelshift-R TTCTCTGACATATTATTCTCTC

EMSA for SO_0011 upstream region, 5'-Cy3

sucA-gelshift-F

GCGAGCTGTGTAATGCAAGAA

EMSA for sucA (SO_1930) upstream region

Cy3-SO_

sucA-gelshift-R TGATGCCTTGGTGCATTTCTA

EMSA for sucA (SO_1930) upstream region, 5'-Cy3

atpI-gelshift-F

TGGCTGAATTAGACGGAATTC

EMSA for atpI SO_4754 upstream region

Cy3-atpI-gelshift-R TACTCAACTCATCTTCTCCGC

EMSA for atpI SO_4754 upstream region, 5'-Cy3

fdnG-gelshift-F GTGAGTTCGGCCCAAATAGAG

EMSA for fdnG SO_0101 upstream region

Appendix 2

Primer Sequence 5' to 3' Modification, for use

feoA_gelshift-R CGCGGATCCAGGTGCCTACTTGTTTCT

EMSA for feoA (b3408) upsteam region, 5'-Cy3

hyaA_gelshift-R CGCGGATCCATCGCACGTCTCTCCTCC

EMSA for hyaA (b0972) upstream region, 5'-Cy3

gadE_gelshift-R CGCGGATCCAACTTGCTCCTTAGCCGT

EMSA for gadE (b3512) upstream region, 5'-Cy3 Gene disruption

arcS_FO AAAGATGATGCCTTGGCTGG arcS SO_0577 disruption arcS_5O-SpeI GATGACTAGTGCTACACAAGAACGATGTG

SpeI, arcS SO_0577 disruption

arcS_5I

CTGATCGGTGCAAAAGTTGTCGATCTGCAAT

ACGTG arcS SO_0577 disruption

arcS_3I

AACTTTTGCACCGATCAGCACCGTTAATTGTT

CGCC arcS SO_0577 disruption

arcS_3O-SpeI TAGCACTAGTCGACATGAATGTACCGTCAG

SpeI, arcS SO_0577 disruption

arcS_RO TTGATACCCATCCTCTGGCA arcS SO_0577 disruption arcS_5-linker CTGATCGGTGCAAAAGTT arcS SO_0577 disruption arcS_3-linker AACTTTTGCACCGATCAG arcS SO_0577 disruption pflB_5O AGCGTGATGCTATTCACAGGG pflB SO_2912 disruption pflB_5-linker GGTCACACCAGCACCTGA pflB SO_2912 disruption pflB_3O GGCGTCTTAGTACACCGC pflB SO_2912 disruption pflB_3-linker TCAGGTGCTGGTGTGACC pflB SO_2912 disruption pflB_FO GCTAGCTGTGATGCAGAG pflB SO_2912 disruption pflB_RO CCATGGGCATACATTGCC pflB SO_2912 disruption pflB_5O-SpeI-IN GAAGGTAGACTAGTATCGTCGTTCCGTGCCT G SpeI, pflB SO_2912 disruption pflB_3O-SpeI-IN GAAGGTAGACTAGTCACCGTCGAGTTGATAG C SpeI, pflB SO_2912 disruption pflB_5I GGTCACACCAGCACCTGAAGATTTCCAATCA

CCAGG pflB SO_2912 disruption

pflBB_3I

TCAGGTGCTGGTGTGACCCAGCAAGATGTGA

TCACG pflB SO_2912 disruption

aceE_5O CTGATGTGATCATGGCGC aceE SO_0424 disruption aceE_5-linker GGTGTTAGCCACTGAAGC aceE SO_0424 disruption aceE_3O GCACTGATCATATCGCCC aceE SO_0424 disruption aceE_3-linker GCTTCAGTGGCTAACACC aceE SO_0424 disruption aceE_FO CGCCAAATGGCCTATAGC aceE SO_0424 disruption aceE_RO CAACGACAGCAGTTTGCC aceE SO_0424 disruption aceE_5-O-SpeI-IN

GAAGGTAGACTAGTAGCAAATGATCCTCGAG G

SpeI, aceE SO_0424 disruption

aceE_3-O-SpeI-IN

GAAGGTAGACTAGTCGATCACATCGACATTG C

SpeI, aceE SO_0424 disruption

aceE_5I GGTGTTAGCCACTGAAGCGGATCTACGTCTTGTAGC aceE SO_0424 disruption aceE_3I

GCTTCAGTGGCTAACACCGATCAATCCACAG

TACGC aceE SO_0424 disruption

nuoF_3O-SpeI-IN GAAGGTAGACTAGTCAAACTGCATGTCCCAC

T

SpeI, nuoF SO_1017 disruption

Appendix 2

Primer Sequence 5' to 3' Modification, for use

nuoF_5O CTTTGCTCACCTGCAACA nuoF SO_1017 disruption nuoF_FO GTTACTACCTGACCAGCG nuoF SO_1017 disruption nuoF_RO GCCGTGGAAACGGTTTTC nuoF SO_1017 disruption nuoF_3I GGTTCAGTGCAGGCTGGTGATCCAGCCCAAT

CTGCT nuoF SO_1017 disruption

nuoF_5I ACCAGCCTGCACTGAACCGAGAACTTTGCTC

TGTGG nuoF SO_1017 disruption

nuoF_5-linker ACCAGCCTGCACTGAACC nuoF SO_1017 disruption nuoF_3-linker GGTTCAGTGCAGGCTGGT nuoF SO_1017 disruption ndh_3-linker GCTGGTTCAGTGACCGCT ndh SO_3517 disruption ndh_5-linker AGCGGTCACTGAACCAGC ndh SO_3517 disruption ndh_FO TAGCGCCTTCATTTTCGG ndh SO_3517 disruption ndh_RO TGAGCTCGTTATTCACCC ndh SO_3517 disruption ndh_3-O ACCGCCACTTTCAGAAGATCC ndh SO_3517 disruption ndh_5-O GCGCTAACAATGTGTAAACGG ndh SO_3517 disruption ndh_3I GCTGGTTCAGTGACCGCTGGCCAAAGCTGAA

ATTAC ndh SO_3517 disruption

ndh_5I AGCGGTCACTGAACCAGCCACTATTCGCTTA

GTAGC ndh SO_3517 disruption

ndh_3O-SpeI GAAGGTAGACTAGTGCGAGGGGATTATAGAG_C SpeI, ndh SO_3517

disruption

ndh_5-O-SpeI GAAGGTAGACTAGTATCGCATCAAGCCAAAC

G

SpeI, ndh SO_3517 disruption

nqrF-1_3-linker GGTGCTGTGTCAACCGCT nqrF-1 SO_0907 disruption

nqrF-1_5-linker AGCGGTTGACACAGCACC nqrF-1 SO_0907 disruption

nqrF-1_FO TGTGCGTGAGTTGTTAGG nqrF-1 SO_0907 disruption

nqrF-1_RO AAGAATGCCACGGTTAGC nqrF-1 SO_0907 disruption

nqrF-1_3O GTAGTGAGATCAATGCGGCAC nqrF-1 SO_0907 disruption

nqrF-1_5O GGCTGGTACTTACCTAACGAG nqrF-1 SO_0907 disruption nqrF-1_3I GGTGCTGTGTCAACCGCTCGGTGATTAAGAT GCTCG nqrF-1 SO_0907 disruption nqrF-1_5I AGCGGTTGACACAGCACCATGCCTATGCCTA TTGCC nqrF-1 SO_0907 disruption nqrF-1_3O-SpeI GAAGGTAGACTAGTCCTAACTACAACACAGG G SpeI, nqrF-1 SO_0907 disruption nqrF-1_5O-SpeI GAAGGTAGACTAGTGCATTAACGTGATCCAG C SpeI, nqrF-1 SO_0907 disruption

nqrF-2_3-linker AACTCAATCGTGGCTCAG nqrF-2 SO_1108 disruption

nqrF-2_5-linker CTGAGCCACGATTGAGTT nqrF-2 SO_1108 disruption

nqrF-2_FO GGATGGTATTGGTAACGG nqrF-2 SO_1108 disruption