Shewanella oneidensisにおけるcAMP/CRP制御系の役割の解明

29

1

1.1

Shewanella

Shewanella

(Venkateswaran et al. 1999,

Hau and Gralnick 2007, Fredrickson et al. 2008, Rodionov et al. 2011) Shewanella

(

[DMSO] [TMAO] )

(Fe, Mn, Co, V )

(Nealson et al.1994, Liu et al. 2002, Hau et al. 2008, Carpentier et al. 2003) (Flynn et al. 2014)

(Hau and Gralnick 2007, Murphy and Saltikov 2009)

Shewanella ( )

(

; bioelectrochemical systems; BES)

Shewanella ( )

Shewanella

Shewanella PAS

(Fredrickson et al. 2008) Shewanella

Shewanella

1.2

Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 Shewanella

MR-1

(Myers and Nealson 1988) (Heidelberg et al. 2002)

(extracellular electron transfer

pathway; EET )

(Ross et al. 2011) (Figure 1) MR-1 BES

1.3

MR-1

MR-1 EET (CymA,

STC/FccA, OmcA, MtrA, MtrB MtrC) (Shi et al. 2007) (Figure 1)

CymA CymA

(STC/FccA) Mtr

(OmcA, MtrA, MtrB, MtrC) OmcA MtrC

(Shi et al. 2006) MR-1 (Lower et al. 2007, Xiong et al. 2006) (Figure 2A)

(Marsili et al. 2008, Von et al. 2008) (Figure 2B) MR-1

(Pirbadian et al. 2014) (Figure 2C) 2

OmcA

(Coursolle et al. 2010) MtrC

(Mitchell et al. 2012)

1.4

mtr

MR-1 EET (OmcA, MtrA, MtrB, MtrC)

(omcA, mtrC, mtrA, mtrB; mtr ) MR-1

(Figure 3) mtr

(Myers and Myers 1997, Beliaev et al. 2005, Teal et al. 2006, Rosenbaum et al.

2012, Pirbadian et al. 2014) mtr cyclic AMP

(cAMP) receptor protein (CRP) cAMP (

, CyaC) (Saffarini et al. 2003, Charania et al. 2009)

in silico omcA CRP

cAMP MR-1 mtr

1.5

CRP/cAMP

CRP/cAMP CRP/cAMP

Escherichia coli

(Ullmann and Monod

1968, Zubay et al. 1970) MR-1 CRP

(Saffarini et al. 2003, Charania et al. 2009) CRP

cAMP CRP

cAMP E. coli cAMP

(Makman and Sutherland 1965)

Figure 2. MR-1 A. B. C. OM: PP: IM: Mox: Mred; CymA MtrB MtrA MtrC OmcA OmcA e -CymA MtrB MtrA MtrC OmcA OmcA e -Mred Mox MtrB MtrA MtrC OmcA MtrB MtrA MtrC OmcA MtrB MtrA MtrC OmcA Mt rB Mt rA Mt rC O m cA MtrB Mt rA MtrC OmcA Mt rB Mt rA Mt rC Om_cA Mt rB Mt rA Mt rC O m cA MtrB MtrA MtrC OmcA

CymA CymA CymA CymA

Figure 3. mtr

omcA

mtrC

_mtrA

mtrB

mtrF

2

mtr

2.1

2.2

2.2.1

2 (Escherichia coli) LB (Luria-Bertani) 37ºC

WM6026 100 µg/ml

(DAP) BL21 (DE3) 2 × yeast

extrac-triptone (2 × YT) 37ºC 30ºC S. oneidensis LB

(Lactate medium; LM) (15 mM DL- , 9 mM (NH4)2SO4, 5.7 mM

K2HPO4, 3.3 mM KH2PO4, 5.0 g L-1 yeast extract, 30 mM HEPES-NaOH (pH 7.4)) 30ºC

600 nm DU800 spectrophotometer (Backman)

100 ml LM 300 mL OD600 = 0.05 S. oneidensis 180 rpm (OD600 > 2.0) 10 mM LM 80 ml 100 mL S. oneidensis OD600 = 0.01 30ºC (OD600 > 0.25) 100 µg/ml (Am), 50 µg/ml (Km), 15 µg/ml

(Gm) 1.5% Bacto agar (Difco)

2.2.2

RNA

S. oneidensis LM

5 ml 30 ml 5 4ºC 7,000 rpm

RNA Trizol (Invitrogen)

1 ml Trizol 0.2 ml

RNA RNeasy Mini kit (Qiagen) RNase-Free DNase set (Qiagen)

250 µl RNA 250 µl Buffer RLT (1% ) 250 µl

100% RNeasy Mini kit 350 µl

Buffer RW1 80 µl DNase RNA

DNA 350 µl Buffer RW1 500 µl Buffer

RPE 50 µl RNase-Free H2O RNA RNA Bioanalyzer

(Agilent Technologies)

2.2.3

5′ rapid amplification of cDNA ends

(5′-RACE) PCR 2

MR-1

10 µg total RNA SuperScriptIII reverse transcriptase (Invitrogen) IR800

PE-mtrC-100 (Table 2) complementally DNA (cDNA)

10 µg RNA, 770 µM dNTP, 4 pmol PE-mtrC-100 13 µl 65ºC

5 1 4 µl 5 × First-Stand buffer, 5 mM DTT,

40 U RNase OUT Recombinant RNase Inhibitor (Invitorgen), 200 U SuperScriptIII reverse

transciptase 20 µl

: 25ºC (5 ), 50ºC (60 ), 70 ºC (15

) cDNA /

cDNA

Li-Cor4200 automated DNA sequencer (Li-Cor)

5¢-RACE MR-1

total RNA 1.0 µg SMATer RACE cDNA amplification kit (Clontech)

cDNA omcA-RACE-out mtrC-RACE-out (Table 2)

cDNA 5¢ SMATer RACE cDNA Amplification kit

cDNA

omcA-RACE-out mtrC-RACE-out

omcA-RACE-in mtrC-RACE-in (Table 2) kit Universal Primer A mix

PCR PCR QIAquick PCR Purification kit (Qiagen)

PCR PCR 5 Buffer PB kit

750 µl Buffer PE 50 µl Buffer EB DNA PCR

T-vector pMD19 (TaKaRa) JM109

Am LB 2 ml 1 13,000 rpm

250 µl Buffer P1 (RNaseA ) 250 µl Buffer P2

350 µl Buffer N3 10 13,000 rpm

kit Buffer PB Buffer

PE 50 µl Buffer EB

5′

2.2.4

PCR (RT-PCR)

total RNA SuperScriptIII reverse transcriptase (Invitrogen) Rnadom Primers

(Invitrogen) cDNA 5.0 µg RNA, 770 µM dNTP, 250 µg

Random Primers (Invitrogen) 13 µl 65ºC 5

1 4 µl 5 × First-Stand buffer, 5 mM DTT, 40 U RNase OUT Recombinant

RNase Inhibitor (Invitorgen), 200 U SuperScriptIII reverse transciptase

20 µl : 25ºC (5 )

50ºC (60 ) 70ºC (15 )

cDNA DNA TaKaRa Ex Taq Polymerase (TaKaRa)

(Table 2) PCR PCR 5 µl 2.0%

σ ChemiDoc XRS (Bio Rad)

2.2.5

RT-PCR (qRT-PCR)

(16S rRNA , omcA, mtrC) DNA MR-1

total DNA PCR

PCR QIAEX II Gel Extraction kit (Qiagen)

1.5 ml

3 Buffer QX1 10 µl QIAEX II

QIAEX II DNA Buffer QX1 Buffer PE 20 µl TE

DNA Total RNA LM

15 ng total RNA, 1.3 µl 50 mM Mn(OAc)2, 7.5 µl LightCycler RNA Master SYBR

Green I (Roche), 0.15 µM (Table 2) LightCycler 1.5

instrument (Roche) qRT-PCR

61ºC (20 ), 95ºC (30 ), ; 95ºC (5 ), 60ºC (5

), 72ºC (25 ) 45 , 95ºC (0 ), 65ºC (15 ), 95ºC (0 ),

40ºC (30 ) PCR mRNA

3

2.2.6

MR-1 crp pSMV-10 2

(750 bp )

(750 bp ) DNA Table 2

Phusion High-Fidelity DNA polymerase (New England Biolabs) PCR

DNA pSMV-10 SpeI pSMV-crp WM6026 MR-1 (single-crossover ) Km LB LB 17 10% (w/v) LB Km (double-corssover ) PCR

2.2.7

LacZ

QIAquick PCR Purification kit (Qiagen) 2.2.3

PCR pMElacZ EcoRI HindIII EcoRI BamHI

pMElacZ

2.2.8

5 Micropulser (Bio Rad) 180 kV

500 µl LB 1.5 mL

30ºC 2 100 µl Gm LB

2.2.9

LM

OD600 = 0.1 0.5 ml 0.5 ml

Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 2 mM KCl, 200 µl MgSO4, 5 mM

2-) 10 µl 0.1%SDS 15 µl 0.2 ml 4 mg/ml

2- -β-D- (ONPG)

0.5 ml 1M Na2CO3 4 mg/ml ONPG

1M Na2CO3 12,000 × g, 2

420 nm

-Miller unit (Miller, 1972) 3

Miller unit = (1000 × Abs420) / ( (ml) × OD600 × ( ))

2.2.10

His

CRP

N His-tag-CRP MR-1 total DNA

crp_NdeI_F crp_BamHI_R (Table 2) PCR crp

PCR QIAquick PCR Purification kit (Qiagen) 2.2.3

PCR N His pET-28a (+)

(Novagen) NdeI BamHI His-tag-CRP

(pET-crp; Table 1) E. coli BL21 (DE3) 300 mL

100 ml Km 2 × YT OD600 = 0.05 30ºC

180 rpm OD600 = 0.5–0.8

isopropyl-β-D-galactopyranoside (IPTG) 0.1 mM His-tag-CRP

3 50 ml

300 µl IMAC wash buffer (Bio-Nobile) 40 µl 360 µL

IMAC wash buffer 10 400 µl

(Misonix) QuickPick IMAC Metal Affinity

kit for proteins (Bio-Nobile) His-tag-CRP His-tag-CRP

Micro BCA Protein Assay kit (Thermo Fisher Science)

2.2.11

Cy3

DNA

Cy3 DNA 5′ Cy3 (Table

2) PCR PCR

2.2.12

(EMSA)

2 nM Cy3 DNA 0–200 µg His-tag-CRP (10 mM

Tris-HCl (pH 7.6), 0.5 mM EDTA (pH 8.0), 100 mM KCl, 50 µg/ml , 50

µg/ml poly (deoxyinosinic-deoxycytidylic) acid [poly (dI-dC) ], 0–50 µM cAMP, 10%

) 20 µl 30 12.5%

0.5 × Tris-borate-EDTA

150 V 30 Typhoon FLA 9000 (GE Healthcare)

2.2.13

DNA

5′ DNA DNA- (Table

2) 2 nmol

(10 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1 mM EDTA)

100 ml 65ºC 15

4 DNA

Streptavidin Sepharose High Performance (GE Healthcare) (20 mM HEPES

(pH 7.4), 100 mM KCl, 0.5 mM EDTA (pH 8.0), 0.5 mM (DTT), 50 µg/ml

BSA, 10% ) 3

50% 100 µl 50% 100 µl

2 DNA 900 µl 2

DNA 3

1 ml 1 × Blocking Reagent (Roche)

16 DNA

3 100 µl

2.2.14

MR-1 10 mM LM

, ( ; 5 ml, ; 50 ml) 7,000 g, 5 4ºC

300 µl IMAC wash buffer (Bio-Nobile)

IMAC wash buffer 10 (Misonix) 10,000 g

Micro BCA Protein Assay kit (Thermo Fisher

10 µl (200 µg ) 100 µl DNA 900 µl

2 3

25 µl 2 × SDS-Buffer 65ºC 15

20 µl

12.5% (E-T12.5L, ATTO) 20 µl

SDS-PAGE buffer (AE-1410 EzRun, ATTO) 200 mA

AE-1360 EzStrain Silver (ATTO)

500 ml 100 ml (40 ml 50 ml 10 ml 1 ml S-1 ) 10 100 ml 30% 10 100 ml 10 100 ml (100 ml 1 ml S-2 ) 5 30 100 ml (100 ml 0.5 ml S-3 0.5 ml S-4 ) 30 100 ml 100 ml (100 ml 1 ml ) 10 100 ml 2 ChemiDoc XRS (BIORAD)

2.3

2.3.1

RNA RT-PCR (Figure 1) mtrF omcA

(Figure 1B, lane 1) omcA-mtrC mtrC-mtrB

omcA mtrB (Figure 1B, lane 3,

4).

2.3.2

omcA mtrC (transcription start site;

TSS) (Figure 2) (Shao et al. 2014, Beliaev et al. 2001). Shao

et al. (2014) 5′ end sequencing omcA

93 bp (TSSomcA; Figure 2A) Beliaev

et al. (2001) 5′-RACE mtrC ATG 119 bp TSS

Beliaev 10 mM MR-1 5′-RACE mtr TSS TSSomcA, TSSmtrC (Figure 2) mtr TSS TSSmtrC (Figure 3) mtrA mtrB 5′-RACE TSSmtrC

2.3.2

2.3.3

(ES)] (Figure 5) PomcA

(Wang et al. 2013) 15 mM 10 mM , 50 mM PomcA PmtrC ML (Figure 5A, B) 10 mM ES 50 mM ES ES PomcA PmtrC PomcA PmtrC PomcA ES PomcA

2.3.4

pMEomcA-104, pMEmtrC-42, pMEmtrC-100, pMEmtrC-144) MR-1

(Figure 6) 10 mM

LacZ

PomcA 5′ TSSomcA

-104 -54

(Figure 6A) -104 -54 PomcA

-150 -104 2 -150 -104 PomcA PomcA 5′ (Figure 7) 5′

(Table 1; pMEomcA-69, pMEomcA-84, pMEomcA-95, pMEomcA-116,

pMEomcA-127) MR-1

PomcA PomcA

(Figure 7) -104 -116 PomcA (5′-AAAAGGAACTCAGATGATGCTGTT-3′; TSSomcA -90 -113 ) (Figure 2A) PmtrC 5′ TSSmtrC -100 -42 (Figure 6B) -100 -42 PmtrC -203 -144, -144 -100 PmtrC TSSmtrC -203 -100 PmtrC

2.3.5

∆crp

omcA

mtrC

CRP (Gao et al. 2010) PomcA

PmtrC CRP ∆crp (WT) (Figure 6) ∆crp (Saffarini et al. 2003) ∆crp ∆crp ∆crp omcA, mtrC ATG pMEomcA-150 pMEmtrC-203 WT ∆crp omcA mtrC qRT-PCR WT (Figure 3) PomcA PmtrC CRP

2.3.6

P

P

CRP

(electrophoretic mobility shift assay; EMSA) (Figure 8)

PomcA CRP TSSomcA -87 -35 (PBomcA1; Figure 8A)

PBomcA1 DNA CRP PBomcA2

(Figure 8B) PomcA CRP

CRP cAMP DNA

cAMP CRP PBomcA1 (Figure 8B)

CRP PomcA cAMP

PmtrC CRP CRP DNA

TSSmtrC -143 +117 (PBmtrC1; Figure 8A) -4 +117 (PBmtrC2; Figure 8A)

TSSmtrC PBmtrC1 PBmtrC2 2 DNA CRP CRP PBmtrC1 CRP PBmtrC2 (Figure 8C) cAMP DNA CRP PBmtrC1 cAMP (Figure 8C) CRP PomcA PmtrC CRP PomcA PmtrC cAMP PomcA PmtrC CRP

2.3.7

DNA

(5′-AAAAGGAACTCAGATGATGCTGTT-3′; TSSomcA -90_-113) (Figure 2A)

PomcA PomcA

DNA

DNA CRP -54 -84

(PAomcA1) -85 -116 (PAomcA2) CRP

(PAomcA3) 3

(Figure 9A) DNA MR-1

SDS-PAGE (Figure 9B)

PAomcA3 20-23 kDa

(Figure 9B) PAomcA3 MR-1

MALDI TOF-MS PomcA PAomcA3 CRP PAomcA1 PAomcA2

2.4

(Saffarini et al. 2003, Yang et al. 2008) mtr

mtr 2

CRP mtr CRP

CRP

cAMP DNA (Brückner

et al. 2002, Stülke et al. 1999) S. oneidensis MR-1 CRP mtr

(Charania et al. 2009) Shewanella

CRP E. coli S. oneidensis MR-1

cAMP

qRT-PCR ∆crp PomcA PmtrC

WT (Figure 4, Figure 6) TSSomcA

TSSmtrC 5′ PomcA PmtrC EMSA CRP CRP PomcA PmtrC CRP E. coli K12 CRP cAMP DNA DNA CRP

(Kolb et al. 1993, Botsford and Harman 1992) CRP -10 box -35 box

(5′-TGTGA-6N-TCACAA-3′) RNA

(Kolb et al. 1993, Botsford and Harman 1992, Busby and Ebright

1997) TSSomcA TSSmtrC TSS -61.5 -60.5

CRP class I CRP (Joung

et al. 1993, Tebbutt et al. 2002) PomcA PmtrC -35 box

-10 box E. coli (5′-TATAAT-3′; Kolb et al. 1993, Raibaud and

Schwartz 1984) E. coli CRP -35 box S. oneidensis MR-1 CRP E. coli MR-1 cAMP (Saffarini et al. 2003) MR-1 cAMP E. coli cAMP S. oneidensis MR-1 cAMP PomcA PmtrC CRP cAMP 10 mM PomcA PmtrC (Figure 5) 10 mM 15 mM PomcA PmtrC (Figure 5) mtr 50 mM PomcA PmtrC mtr (Wang et al. 2013) PomcA PmtrC 50 mM 10 mM (Figure 5) PomcA PmtrC (Figure 5) mtr

(Beliaev et al. 2005, Teal et al. 2006) PmtrC

(Figure 5B) PmtrC 50 mM PmtrC PomcA (Figure 5A) PomcA PmtrC PomcA PmtrC CRP PomcA PmtrC PomcA TSSomcA -116 -104 5′ TSSomcA -116 CRP 39 E. coli CytR CRP

PomcA (Kristensen et al. 1997,

Valentin-Hansen et al. 1996) Shewanella

omcA CRP (Figure 9) CRP CRP DNA 5′ PmtrC mtrC 5′ PmtrC (Figure 6B) AT E. coli AT H-NS AT PmtrC mtr Figure 1A PomcA PmtrC CRP PomcA (Figure 9) Shewanella EET

PomcA PmtrC (Figure 4 ) Shewanella PAS

mtr

MR-1 OM-cyt

Figure 2. omcA (A) mtrC (B) TSSomcA TSSmtrC (+1) omcA

mtrC TSS

CRP -10 -35 GeSTer program (Mitra et al. 2011)

Figure 5. - PomcA (A) PmtrC (B)

( ) 10 mM ( ) 50 mM ( ) pMEomcA-150

pMEmtrC-203 MR-1 LacZ (ML)

(LL), (ES) 3

(pMElacZ)

MR-1 LacZ 3 Miller units

Table 1.

Strain or plasmid Relevant characteristic Source or _reference

E. coli strains

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] Yanisch-Perro_{n et al. 1985} 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 strains

MR-1 Wild type ATCC

∆crp SO_0624 (crp) disrupted This study

∆cpdA SO_3901 (cpdA) disrupted This study

∆cyaC SO_1329 (cyaC) disrupted This study

∆cpdA∆crp SO_3901 (cpdA), SO_0624 (crp) disrupted This study ∆cpdA∆cyaABC SO_3901 (cpdA), SO_4312 (cyaA), SO_3778 (cyaB), SO_1329

(cyaC) disrupted

This study

∆ldhA SO_ (ldhA) disrupted Nakagawa et

al. 2015

Plasmids

pET-28(a) Expression vector, T7 promoter Novagen pET-crp pET-28(a) containing N terminal His-tag-crp This study pSMV-10 9.1 kb mobilizable suicide vector; oriR6K, mobRP4, sacB, Kmr_,

Gmr Chad Saltikov, _{California Inst.}

of Tech. pSMV-crp 1.6 kb fusion PCR fragment containing ∆crp cloned into the SpeI

site of pSMV-10

This study

pSMV-cpdA 1.6 kb fusion PCR fragment containing ∆cpdA cloned into the SpeI site of pSMV-10

This study

pSMV-cyaA 1.6 kb fusion PCR fragment containing ∆cyaA cloned into the SpeI site of pSMV-10

This study

pSMV-cyaB 1.6 kb fusion PCR fragment containing ∆cyaB cloned into the SpeI site of pSMV-10

This study

pSMV-cyaC 1.6 kb fusion PCR fragment containing ∆cyaC cloned into the SpeI site of pSMV-10

This study

pMElacZ pME4510 derivative, lacZ Gmr _{Endoh et al.} 2003

Table 1. ( )

Strain or plasmid Relevant characteristic Source or _reference pMEmtrC-203 pMElacZ containing region from -203 to +116 relative to TSSmtrC This study pMElldP+1 pMElacZ containing region from +1 to +192 relative to TSSlldP This study pMElldP-60 pMElacZ containing region from -60 to +192 relative to TSSlldP This study pMElldP-182 pMElacZ containing region from -182 to +192 relative to TSSlldP This study pMElldP-541 pMElacZ containing region from -541 to +192 relative to TSSlldP This study pBBR1MCS-2 Broad-host-range vector, lacZ promoter, Kmr _{Kovach et al.}

1995

pBBR1MCS-5 Broad-host-range vector, lacZ promoter, Gmr _{Kovach et al.} 1995

pBBRcrp pBBR1MCS-2 containing crp This study

pBBRdld pBBR1MCS-2 containing dld This study

pBBRcyaC pBBR1MCS-2 containing cyaC This study

Table 2.

Primer Sequence (5′–3′) For use

cpdA_FO CGTTTCTACTTTTATTGGGC cpdA disruption

cpdA_5_OUT GATCACTAGTGATTCATCACAGGCACAAGG cpdA disruption

cpdA_5_IN AACTTTGGTACCGATCACTTTCAGCACATTGAATACCC cpdA disruption

cpdA_3_IN GTGATCGGTACCAAAGTTGGTTATTAACGCGAATGACC cpdA disruption

cpdA_3_OUT GATCACTAGTCCTAAATGGTTAGGTCGGG cpdA disruption

cpdA_RO GCTTCATCGACACTGTTATC cpdA disruption

cyaA_FO CTAATAATGCTTTAACGCGC cyaA disruption

cyaA_5_OUT GATCACTAGTGAGATAAAGCTGGCGATACG cyaA disruption

cyaA_5_IN GGTAGCGTTGCTCACGCTATCCATCCTAGAACACAACAC cyaA disruption

cyaA_3_IN AGCGTGAGCAACGCTACCTTTTAAGCGGGAGCTACCTC cyaA disruption

cyaA_3_OUT GATCACTAGTGTACAGTGGCGTACCATAGG cyaA disruption

cyaA_RO CGCATTATTAAAGGCATGCC cyaA disruption

cyaB_FO GATCTTAAGGCGATCTCAGG cyaB disruption

cyaB_5_OUT GATCACTAGTACTGCAGATCCACGATATCC cyaB disruption

cyaB_5_IN CTGAGCCACGATTGAGTTATTCATCAACAATCTCCATTTTTAC cyaB disruption

cyaB_3_IN AACTCAATCGTGGCTCAGCAATAATCGCTTTGCTGGG cyaB disruption

cyaB_3_OUT GGGATCACTAGTCGTAATCGCCACCATTCC cyaB disruption

cyaB_RO AACCTTATTTGCACAGGTCG cyaB disruption

cyaC_FO CCCCTCAAAATCTGAGCG cyaC disruption

cyaC_5_OUT GATCACTAGTGCGCTAATATGATGGCAGG cyaC disruption

cyaC_5_IN AGCGCCGTTGGTACCTGATGCTCTCACAACGGGTTTTG cyaC disruption

cyaC_3_IN TCAGGTACCAACGGCGCTCCTTTATAAGACTCACAACAGG cyaC disruption

cyaC_3_OUT GATCACTAGTCGGTGAAGAAGTCGTACAG cyaC disruption

cyaC_RO CAGTTGATTGAACGTGACC cyaC disruption

crp_5_OUT GGACTAGTGGTCACGTTTAATCAACTGGCC crp disruption

crp_5_IN GGTAGCGTTGCTCACGCTCAGAGCCATGTCGATGTTCCTCG crp disruption

crp_3_IN AGCGTGAGCAACGCTACCCGTTAAGTTAGACTTCAGCTTG crp disruption

crp_3_OUT GGACTAGTGGGTTTACCTAAATAATCATCGG crp disruption

crp_RO GGCCATGGGCACGATGAATAAGTGC crp disruption

dld_F_BamHI CGCGGATCCACCATCTTGTTCGAAGTCAC dld complementation

dld_F_XbaI CTAGTCTAGACTTCTAAAGAAAAACGGGGC dld complementation

qRT-16S-F AGCGCAACCCCTATCCTTAT qRT-PCR for 16S rRNA

gene

qRT-16S-R CGTAAGGGCCATGATGACTT qRT-PCR for 16S rRNA

gene

qRT-omcA-F GGATACGGCGTTGAAGATGT qRT-PCR for omcA

qRT-omcA-R TGGTATCCGTTCCATTCCAT qRT-PCR for omcA

Table 2. ( )

Primer Sequence (5′–3′) For use

qRT-mtrC-R GCTTCGTTAGTGGCGAAAAC qRT-PCR for mtrC qRT-lldP-F GGCGTTTCATGCAAGTC qRT-PCR for lldP qRT-lldP-R ATCGACGCAAGACCTGC qRT-PCR for lldP qRT-dld-F CATCGGCACTCAACTTCTCA qRT-PCR for dld qRT-dld-R CGCAGGTATCAATCACATCG qRT-PCR for dld qRT-lldF-F CGACCTACAGCGCCTTCTAC qRT-PCR for lldF qRT-lldF-R AGTGTTAAGGCAGCCACCAC qRT-PCR for lldF

qRT-ldhA-F GACCCCTACCCTAATCCAGC qRT-PCR for ldhA

qRTldhA-R GACCCCTACCCTAATCCAGC qRT-PCR for ldhA

crp_NdeI_F CATGCATATGATGGCTCTGATTGGTAAGCC pET-crp

crp_BamHI_R GATCGGATCCTTAACGGGTACCATATACCAC pET-crp

PE-mtrC-100 CCATCACTACCATCATTGCC Primer extension

RT-omcA-mtrF-F TAACAATGGCACCTTCACCA RT-PCR RT-omcA-mtrF-R TCGCCACCTTTATGGATAGC RT-PCR RT-omcA-F CCATCAGGCGTTGATAACCT RT-PCR RT-omcA-R ATACCCAAATTACGGCACCA RT-PCR RT-omcA-mtrC-F GAAAGCCCACGAAAGTGAAG RT-PCR RT-omcA-mtrC-R CCTTCTACGTGGCAAGAAGC RT-PCR RT-mtrCAB-F AAGAAGAAAGGCGCATTGAA RT-PCR RT-mtrCAB-F GAGTGCGGTAAACCAACGAT RT-PCR RT_lldP_F CGCCGCCGTGTTTATTTGGC RT-PCR RT_lldP_R GGATTGCTTAATCGCAGGGC RT-PCR RT_lldP-dld_F CGCCACTCAAAGCTTGGC RT-PCR RT_lldP-dld_R CCAATAACGGCCGCGCC RT-PCR RT_dld_F CCGTTTTGCTTGGTCTACCG RT-PCR RT_dld_R GCCCTTTACGCATCGCCC RT-PCR RT_dld-lldE_F GAGCCCAAGATGCAAGCG RT-PCR RT_dld-lldE_R CGGCTCCTTCATCGACGGC RT-PCR RT_lldEF_F CAGCCTTGCTACGCCAAGCG RT-PCR RT_lldEF_R GGTTAGCGCCCATATCGGC RT-PCR RT_lldFG_F CTAAGGCTGGCGAGTCCG RT-PCR RT_lldFG_R GCAGTGCCGCAAGGCCGC RT-PCR

omcA_R+93 GTAGAAGCTTGATATTTCCCTGCAATAGTTTTAATCA HindIII, LacZ reporter

assay

omcA_F-54 GATGGAATTCTTACCCGCTTAAAGTGACTG EcoRI,LacZ reporter assay

omcA_F-69 GTACGAATTCTCAGATCTCAATCACATTACC EcoRI, LacZ reporter assay

omcA_F-84 GTACGAATTCCCTCAAAAGAAATAGTCAG EcoRI, LacZ reporter assay

omcA_F-95 GTACGAATTCGCTGTTATCTACCTCAAAAG EcoRI, LacZ reporter assay

Table 2. ( )

Primer Sequence (5′–3′) For use

omcA_F-116 GTACGAATTCTATAAAAGGAACTCAGATGATGC EcoRI, LacZ reporter assay

omcA_F-127 GTACGAATTCGCAACTGCCAATATAAAAGG EcoRI, LacZ reporter assay

omcA_F-150 GATCGAATTCATGATGCAGGCCCAAAAG EcoRI, LacZ reporter assay

mtrC_R+117 GATGAAGCTTTTTTCCCTGCATAGGTTTGG HindIII, LacZ reporter

assay

mtrC_F-42 CTCGGGATCCGCTTAGAAGATTTTAACGGCATGT BamHI, LacZ reporter

assay

mtrC_F-100 TGACGGATCCCCTTGTGGTTTAACTACCTCTTTAGAA BamHI, LacZ reporter

assay

mtrC_F-144 CTACGGATCCTCCACCTCACTATCTGTTGTTTTTTTGCTCTC BamHI, LacZ reporter

assay

mtrC_F-203 CTACGGATCCAATTATCTGAATCGAGAGACGAAA BamHI, LacZ reporter

assay

lldP_+192_R GATCGTCGACGCTTGCCTCTACATAAAGTGG SalI, LacZ reporter assay

lldP_+1_F GATCGAATTCTCATACAGCAGCCAAAATG EcoRI, LacZ reporter assay

lldP_-60_F GATCGAATTCCATGTTTTTTAAAAAAATATTTG EcoRI, LacZ reporter assay

lldP_-182_F GATCGAATTCGGATCAGTTAATTGCAATAC EcoRI, LacZ reporter assay

lldP_-360_F GATCGAATTCCAAAGCGTGACCAGAATCAC EcoRI, LacZ reporter assay

lldP_-541_F GATCGAATTCGCCGTATCGCAGCAAAAGCC EcoRI, LacZ reporter assay

omcA_RACE_out CCGCGGTCAGCTTCTGTTTCTCCCAC 5 -RACE PCR

omcA_RACE_in CGGGAGTTAATTGCGCAATACCAAATCGC 5 -RACE PCR

mtrC_RACE_out CGCCCCTTCCGGTATTAATTGCAGTGC 5 -RACE PCR

mtrC_RACE_in GCGAGACCAATCACTGGCATGTCGGC 5 -RACE PCR

lldP_race_out GCGCCGAAGATGATGGTTAAGGGGG 5 -RACE PCR

lldP_race_in ACAACGGATGCGGCCAATAGAGTGG 5 -RACE PCR

omcA-50_F CCCGCTTAAAGTGACTGATAAATACC 5 -Cy3, PBomcA2

omcA-87_F CTACCTCAAAAGAAATAGTCAG 5 -Cy3, PBomcA1

omcA+13_R GGAATTAGATCCCACCTGTAAGGC 5 -Cy3, PBomcA2

omcA-35_R CAGTCACTTTAAGCGGGTAATG 5 -Cy3, PBomcA1

mtrC-4_F CCTTGGGGAATTCTATTTCC 5 -Cy3, PBmtrC2

mtrC-143_F TCCACCTCACTATCTGTTGTTTTTTTGCTCTC 5 -Cy3, PBmtrC1

mtrC+117_R TTTTCCCTGCATAGGTTTGG 5 -Cy3, PBmtrC1,

PBmtrC2

lldP_EMSA_F GGATCAGTTAATTGCAATAC 5 -Cy3, PBlldP1, PBlldP2

lldP_EMSA_R_1 TCAGATAGTTATAAAAAAGG PBlldP1

lldP_EMSA_R_2 TGTCACTTAAAGAGTGGGG PBlldP2

lldP_EMSA-84-35_F ATATACCCCACTCTTTAAGTGACACCGATCACAGTTA AGAAAATCCCATG

Table 2. ( )

Primer Sequence (5′–3′) For use

lldP_EMSA-84-35_ R CATGGGATTTTCTTAACTGTGATCGGTGTCACTTAAA GAGTGGGGTATAT PBlldP3 lldP_EMSA-84-35m _F ATATACCCCACTCTTTACCCCCCACCGAGGGGGGTT AAGAAAATCCCATG 5′-Cy3, PBlldP3m lldP_EMSA-84-35m _R CATGGGATTTTCTTAACCCCCCTCGGTGGGGGGTAA AGAGTGGGGTATAT PBlldP3m

ldhA_EMSA_F TTTGTGCAATAGATGATAAAGCTCACTCTTTTTACGGGGTAA 5′-Cy3, PBldhA

ldhA_EMSA_R TTACCCCGTAAAAAGAGTGAGCTTTATCATCTATTGCACAAA PBldhA

ldhA_EMSA_mut_F TTTGTGCAATAGACCCCCAAGCGGGGGCTTTTTACGGGGTAA 5′-Cy3, PBldhAm

ldhA_EMSA_mut_R TTACCCCGTAAAAAGCCCCCGCTTGGGGGTCTATTGCACAAA PBldhAm

PA_omcA_-85_-116 _F

TATAAAAGGAACTCAGATGATGCTGTTATCTA 5′-biotin, PAomcA1

PA_omcA_-85_-116 _R

TAGATAACAGCATCATCTGAGTTCCTTTTATA PAomcA1

PA_omcA_-54_-84_ F

CCTCAAAAGAAATAGTCAGATCTCAATCACA 5′-biotin, PAomcA2

PA_omcA_-54_-84_ R TGTGATTGAGATCTGACTATTTCTTTTGAGG PAomcA2 PA_omcA_-54_-116 _F TATAAAAGGAACTCAGATGATGCTGTTATCTACCTCAAAAG AAATAGTCAGATCTCAATCACA 5′-biotin, PAomcA3 PA_omcA_-54_-116 _R TGTGATTGAGATCTGACTATTTCTTTTGAGGTAGATAACAG CATCATCTGAGTTCCTTTTATA PAomcA3

qRT-pta-F TAGGTGCCCTGCTGCTTACT qRT-PCR for pta

qRT-pta-R GAGGTTTGCCAAGTGTTGGT qRT-PCR for pta

qRT-pflB-F CCCAATGGTTGTGGGTAAAC qRT-PCR for pflB

qRT-pflB-R AGTGATTGGGTCTGCTTTGG qRT-PCR for pflB

qRT-pykA-F CAAAATCGTCACCACACTGG qRT-PCR for pykA

qRT-pykA-R TGAGTAGCGCGTTTGAGATG qRT-PCR for pykA

qRT-metK-F ATGCCAGCAACGAAACTGAC qRT-PCR for metK

qRT-metK-R ACTTGGCTTTTCGCATCTGG qRT-PCR for metK

qRT-hisC-F AGCAAAATTGTCGCCAGTCG qRT-PCR for hisC

qRT-hisC-R AAAGGTTTGCGCGCTAATGG qRT-PCR for hisC

qRT-acs-F TAATCCAAAGGGCGTATTGC qRT-PCR for acs

qRT-acs-R AACCTACATCGGCCGTACAC qRT-PCR for acs

qRT-metR-F TCAGCTAAGGGTTGGGATTG qRT-PCR for metR

qRT-metR-R TTCTAGGGCATTGAGCCGAGT qRT-PCR for metR

qRT-metE-F GAAGGTGTGGGCTTTACCAA qRT-PCR for metE

Table 2. ( )

Primer Sequence (5′–3′) For use

ScpdA_F_EcoRI GGCCGGAATTCTGGCAGTCAACAACGTTTGG S. oneidensis cpdA

complementation

ScpdA_R_BamHI GGCGCGGATCCTTCCTCAAGCTGCGGTCATT S. oneidensis cpdA

complementation

EcpdA_F_KpnI GGGGGGTACCGCGATTCCGGTTTATTAGCG E. coli cpdA

complementation

EcpdA_R_BamHI GGGGGGGATCCCGAGCTGTTGAAACCGTG E. coli cpdA

complementation

cyaC_F_EcoRI GGGGGAATTCCTATAAACTACGGCATCCTG cyaC constitutive

expression

cyaC_R_SacI GGGGGAGCTCTGAGTATTTTGCCTGTTGTG cyaC constitutive

expression

crp_F_EcoRI GGGGGAATTCTTTCGTTTTAATCAATCGAGG crp constitutive expression

3

CRP

D

-3.1

3.2

3.2.1

BL21 (DE3) LB 2 × yeast extrac-triptone (2 × YT) 30ºC 37ºC

S. oneidensis 10 mM DL- D- L (9 mM (NH4)2SO4, 5.7 mM K2HPO4, 3.3 mM KH2PO4, 30 mM HEPES-NaOH (pH 7.4)) (DL-LMM, D-LMM, L-LMM, PMM ) 5 ml 30 mL S. oneidensis OD600 = 0.05 180 rpm 5 ml 13 mL 30 mM S. oneidensis OD600 = 0.01 OD600 Miniphoto518R (Taitech) 100 µg/ml

Am, 50 µg/ml Km, 15 µg/ml Gm 1.5% Bacto agar (Difco)

3.2.2

S. oneidensis DL-LMM 1 ml

0.2 µm D-/L

(F-kit D-/L-lactate, JK ) 340 nm

Abs340 1200lab (Hitachi) 96 90 µl H2O, 100 µl

5 Abs340 (E0) 2 µl Solution 4

30 Abs340 (E1) 2 µl Solution 5

30 Abs340 (E2) E1 E0 E2 E1

D- L- 3

3.2.3

RNA

RNA DL-LMM S. oneidensis 2.2.2

RNA Bioanalyzer (Agilent Technologies)

3.2.4

qRT-PCR

(16S rRNA, lldP, dld, lldF, ldhA) DNA MR-1 total DNA PCR

(2.2.5 ) Total RNA DL-LMM

(2.2.2 ) mRNA

(16S rRNA) 3

3.2.5

crp

dld

crp dld (pBBRcrp, pBBRdld) crp

dld Phusion High-Fidelity DNA polymerase (New England Biolabs)

crp_F_EcoRI, crp_R_BamHI dld_F_BamHI, dld_R_XbaI ( 2 Table 2)

PCR PCR QIAquick PCR Purification kit (Qiagen)

(2.2.3 ) dld pBBR1MCS-2 EcoRI BamHI BamHI XbaI (pBBRcrp, pBBRdld) crp dld

3.2.6

3.2.7

RT-PCR

MR-1 total RNA 5.0 µg SuperScriptIII reverse transcriptase (Invitrogen) Rnadom Primers

(Invitrogen) cDNA cDNA 2

DNA 2.2.4

3.2.8

LacZ

lldP lacZ

(lldP_+1_F lldP_-541_F) (lldP_+192_R) ( 2 Table 2) PCR

PCR QIAquick PCR Purification kit (Qiagen) 2.2.3

PCR pMElacZ EcoRI SalI

MR-1 ∆crp

3.2.9

3.2.10

His

CRP

His-tag-CRP pET-crp ( 2 Table 1)

BL21 (DE3) 300 mL 100 ml Km 2 × YT

OD600 = 0.05 30ºC 180 rpm OD600 = 0.5–0.8

Isopropyl-β-D-galactopyranoside (IPTG) 0.1 mM

His-tag-CRP 3 50 ml

His-tag-CRP QuickPick IMAC Metal Affinity kit for proteins (Bio-Nobile)

His-tag-CRP Micro BCA Protein Assay kit (Thermo Fisher Science)

3.2.11

EMSA

2 nM Cy3 DNA 0–200 µg His-tag-CRP (10 mM Tris-HCl

(pH 7.6), 0.5 mM EDTA (pH 8.0), 100 mM KCl, 50 µg/ml , 50 µg/ml poly

(deoxyinosinic-deoxycytidylic) acid [poly (di-dc) ], 0 - 50 µM cAMP, 10% )

20 µl 30 12.5%

0.5 × Tris-borate-EDTA

3.3

3.3.1

CRP

-3.3.2

-

CRP

D-LDH (Dld LdhA) LdhA NADH D

-D-LDH (Pinchuk et al. 2009) Dld D

-D-LDH (Pinchuk et al. 2009,

Brutinel and Gralnick, 2012) ∆crp D- dld

MR-1 dld lldP L-LDH lldEFG (LDH ) (Figure 4A) CRP RT-PCR qRT-PCR WT ∆crp MR-1 DL-LMM RNA RT-PCR lldP–dld

(Figure 4B, lane 2) mRNA

lldE–lldF (Figure 4B, lane 5) lldF–lldG (Figure 4B, lane 6)

dld-lldE (Figure 4B, lane 4)

LDH 5 lldP dld lldEFG 2

lldP dld lldF qRT-PCR ∆crp WT lldP dld 20% CRP lldP–dld lldF WT ∆crp CRP lldEFG ∆crp lldP dld TMAO (Figure 5) CRP

3.3.3

crp

3.3.4

lldP-dld

(Brutinel and Gralnick 2012) lldP

lldP-dld lldEFG

lldP-dld D

lldP–dld lldP–dld lldP 5 -RACE lldP 192 bp (TSSlldP) (Figure 8) 70 (-10 box, -35 box) CRP (Gao et al. 2010) CRP lldP–dld lldP-dld lacZ β-pMElacZ lacZ 5′ lldP

( 2 Table 1; pMElldP+1, pMElldP-60, pMElldP-182,

pMElldP-541) MR-1 DL-LMM CRP pMElldP-182 pMElldP-541 (Figure 9) TSSlldP 60 bp 182 bp lldP-dld ∆crp (Figure 9) CRP

3.3.6

CRP

CRP (PBlldP2; Figure 10A) 2 Cy3 DNA

(PBlldP3m; Figure 10A) EMSA

CRP PBlldP3 PBlldP3m (Figure

10B) CRP lldP-dld

3.4

Shewanella

(Serres

and

oneidensis MR-1 MR-1 DL -Shewanella MR-1 D -D-LDH dld CRP CRP MR-1 dld CRP Corynebacterium glutamicum CRP/Fnr GlxR L-LDH ldhA narKGHJI cAMP

(Kohl et al. 2008) cAMP

NADH LDH (iLDH)

(Garvie 1980, Jiang et al. 2014) C. glutamicum D

-D-iLDH (Dym et al. 2000, Kato et al.

2010) MR-1 D- D-iLDH (Dld) (Pinchuk

et al. 2009) CymA

( DMSO

) (Beliaev et al. 2001, Myers and Myers 2002, Gralnick et al. 2006, Shi et al.

2006, Hartshorne et al. 2009, Schuetz et al. 2009, Fonseca et al. 2013) D

L-LDH lldEFG CRP

(Figure 4C) lldEFG L- LlpR

(Pinchuk et al. 2009, Brutinel and Gralnick 2012)

(Figure 7) lldEFG L

Figure 3. crp (pBBR1MCS-2) MR-1 (

) ∆crp ( ) crp (pBBRcrp) ∆crp (

) 50 µg/ml Km D-LMM

Figure S1. Growth of crp-complemented ∆crp on D-lactate. WT harboring the control vector

pBBR1MCS-5 (closed circle), ∆crp harboring pBBR1MCS-5 (open circle), and ∆crp harboring pBBRcrp (open squire) were aerobically grown in MM containing 10 mM D-lactate. Error bars represent standard deviations calculated from at least three independent experiments.

Figure 5. TMAO WT ∆crp lldP dld qRT-PCR WT ∆crp 16S rRNA 0 0.2 0.4 0.6 0.8 1 1.2 1.4 lldP dld

Relative mRNA levels

Figure 6. ∆crp(pBBRdld) D-LMM A. B. TMAO

Figure S3. Growth of dld-complemented ∆crp on D-lactate under TMAO-reducing conditions. WT harboring the control vector pBBR1MCS-2 (closed circle), ∆crp harboring pBBR1MCS-2 (open circle), and ∆crp harboring pBBRdld (open squire) were anaerobically grown in MM containing 30 mM D-lactate and 30 mM TMAO. Error bars represent standard deviations calculated from at least three independent experiments.

Figure 9. β- LacZ WT ( ) ∆crp ( ) DL-LMM CRP 0 50 100 150 Miller units WT ∆crp lldP TSS (+1) pMElldP-541 pMElldP-182 pMElldP+1

Figure 10. EMSA. A. EMSA DNA (PBlldP1, PBlldP2, PBlldP3, PBlldP3m)

CRP B. PBlldP1, PBlldP2, PBlldP3

PBlldP3m CRP CRP cAMP (+) (-)

DNA CRP

fmicb-08-00869 May 13, 2017 Time: 16:27 # 8

Kasai et al. CRP RegulatesD-Lactate Oxidation

FIGURE 9 | Electrophoretic mobility shift assay (EMSA) using CRP and upstream regions of lldP. (A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of N-his-CRP protein samples. Protein samples (5 µg) were analyzed on 12.5% SDS-polyacrylamide gels. Lane 1, Escherichia coli BL21 (DE3) (pET-crp) crude extract; lane 2, purified N-his-CRP; lane 3, molecular weight marker.(B) DNA fragments used as probes. Positions of 50_{and 3}0_{ends of}

the fragments relative to TSSlldP(+1) are shown. White boxes represent the putative CRP-binding motif. The mutated sequences in PBlldP3m are shown in bold.

(C) Binding of CRP to each probe. DNA-binding reactions were performed in the presence (+) or absence (–) of CRP, cAMP, and specific competitor (2 µM unlabeled PBlldP3 probe).

DISCUSSION

Like many other anaerobic respiratory bacteria, Shewanella spp. preferentially catabolize low-molecular-weight organic

acids (Serres and Riley, 2006; Fredrickson et al., 2008),

and studies have used lactate (mostly racemic DL-lactate) as

a substrate for cultivating S. oneidensis MR-1. Despite the importance of lactate as a catabolic substrate for Shewanella spp., however, little is known about how these bacteria regulate catabolic pathways for this compound. Here, we demonstrated that CRP plays a critical role in the transcriptional

regulation of the dld gene encoding D-LDH in MR-1. As

CRP is also involved in the expression of many anaerobic

respiratory genes (Saffarini et al., 2003; Kouzuma et al.,

2015), we suggest that MR-1 uses this transcriptional regulator

to coordinately regulate D-lactate metabolism and anaerobic

respiration. A previous study reported that, in Corynebacterium glutamicum, a CRP/Fnr-type global transcriptional regulator, GlxR, binds to promoter regions of the ldhA gene encoding

a fermentative L-LDH and the narKGHJI operon encoding

nitrate respiratory enzymes in a cAMP-dependent manner (Kohl et al., 2008). It is therefore likely that a broad range

of bacteria utilizes cAMP-dependent regulatory mechanisms for the coordinated expression of catabolic and respiratory pathways.

In many bacteria, lactate is oxidized to pyruvate by

NADH-independent LDHs (iLDHs) (Garvie, 1980;Jiang et al.,

2014). For example, in E. coli and C. glutamicum, D-lactate

is catabolized through membrane-bound D-iLDHs that utilize

membrane-associated quinones as electron-accepting cofactors (Dym et al., 2000; Kato et al., 2010). S. oneidensis MR-1 also

oxidizes D-lactate by D-iLDH (Dld) (Pinchuk et al., 2009),

and is thereby likely to reduce membrane quinones. In this strain, electrons accumulated in the membrane quinone pool are transferred to anaerobic electron acceptors, such as fumarate, DMSO, and metal oxides, via an inner membrane-anchored cytochrome, CymA, and periplasm- and outer membrane-localized proteins, such as FccA, DmsABEF, MtrCAB, and

OmcA (Beliaev et al., 2001; Myers and Myers, 2002; Gralnick

et al., 2006; Shi et al., 2006; Hartshorne et al., 2009; Schuetz et al., 2009; Fonseca et al., 2012). It is therefore suggested

that D-lactate oxidation in MR-1 is metabolically linked

to anaerobic respiratory pathways via membrane-associated quinones.

Frontiers in Microbiology | www.frontiersin.org 8 May 2017 | Volume 8 | Article 869

fmicb-08-00869 May 13, 2017 Time: 16:27 # 8

Kasai et al. CRP RegulatesD-Lactate Oxidation

FIGURE 9 | Electrophoretic mobility shift assay (EMSA) using CRP and upstream regions of lldP. (A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of N-his-CRP protein samples. Protein samples (5 µg) were analyzed on 12.5% SDS-polyacrylamide gels. Lane 1, Escherichia coli BL21 (DE3) (pET-crp) crude extract; lane 2, purified N-his-CRP; lane 3, molecular weight marker.(B) DNA fragments used as probes. Positions of 50_{and 3}0_{ends of}

the fragments relative to TSSlldP(+1) are shown. White boxes represent the putative CRP-binding motif. The mutated sequences in PBlldP3m are shown in bold.

(C) Binding of CRP to each probe. DNA-binding reactions were performed in the presence (+) or absence (–) of CRP, cAMP, and specific competitor (2 µM unlabeled PBlldP3 probe).

DISCUSSION

Like many other anaerobic respiratory bacteria, Shewanella spp. preferentially catabolize low-molecular-weight organic acids (Serres and Riley, 2006; Fredrickson et al., 2008), and studies have used lactate (mostly racemic DL-lactate) as a substrate for cultivating S. oneidensis MR-1. Despite the importance of lactate as a catabolic substrate for Shewanella spp., however, little is known about how these bacteria regulate catabolic pathways for this compound. Here, we demonstrated that CRP plays a critical role in the transcriptional regulation of the dld gene encoding D-LDH in MR-1. As

CRP is also involved in the expression of many anaerobic respiratory genes (Saffarini et al., 2003; Kouzuma et al., 2015), we suggest that MR-1 uses this transcriptional regulator to coordinately regulate D-lactate metabolism and anaerobic respiration. A previous study reported that, in Corynebacterium glutamicum, a CRP/Fnr-type global transcriptional regulator, GlxR, binds to promoter regions of the ldhA gene encoding a fermentative L-LDH and the narKGHJI operon encoding

nitrate respiratory enzymes in a cAMP-dependent manner (Kohl et al., 2008). It is therefore likely that a broad range

of bacteria utilizes cAMP-dependent regulatory mechanisms for the coordinated expression of catabolic and respiratory pathways.

In many bacteria, lactate is oxidized to pyruvate by NADH-independent LDHs (iLDHs) (Garvie, 1980;Jiang et al., 2014). For example, in E. coli and C. glutamicum,D-lactate is catabolized through membrane-boundD-iLDHs that utilize membrane-associated quinones as electron-accepting cofactors (Dym et al., 2000;Kato et al., 2010). S. oneidensis MR-1 also oxidizes D-lactate by D-iLDH (Dld) (Pinchuk et al., 2009), and is thereby likely to reduce membrane quinones. In this strain, electrons accumulated in the membrane quinone pool are transferred to anaerobic electron acceptors, such as fumarate, DMSO, and metal oxides, via an inner membrane-anchored cytochrome, CymA, and periplasm- and outer membrane-localized proteins, such as FccA, DmsABEF, MtrCAB, and OmcA (Beliaev et al., 2001;Myers and Myers, 2002;Gralnick et al., 2006;Shi et al., 2006;Hartshorne et al., 2009;Schuetz et al., 2009; Fonseca et al., 2012). It is therefore suggested that D-lactate oxidation in MR-1 is metabolically linked

to anaerobic respiratory pathways via membrane-associated quinones.

Frontiers in Microbiology | www.frontiersin.org 8 May 2017 | Volume 8 | Article 869

A

7

7.1

CRP

(Brückner and Titgemeyer 2002, Stülke and

Hillen 1999) E. coli K-12 CRP cAMP

cAMP

(phosphotransferase system; PTS) PTS

PTS PTS

Cya cAMP

cAMP (Green et al. 2014)

Shewanella CRP

Shewanella sp. ANA-3 CRP

(Murphy and Saltikov 2009) S. oneidensis MR-1

CRP Fe(III) Mn(IV) DMSO

(mtr, fccA, nap, dms )

(Saffarini et al. 2003, Charania et al. 2009)

CRP CRP MR-1 cAMP/CRP 2 MR-1 Mtr mtr CRP 3 MR-1 CRP D- (Dld) (dld) Dld D -D -MR-1 D- CRP 5 CRP cAMP CpdA CpdA (∆cpdA) SAM ∆cpdA

Beliaev AS, Klingeman DM, Klappenbach JA, Wu L, Romine MF, Tiedje JM et al. (2005). Global transcriptome analysis of Shewanella oneidensis MR-1 exposed to different terminal electron acceptors. J Bacteriol. 187(20):7138-7145.

Beliaev AS, Saffarini DA, McLaughlin JL, Hunnicutt D. (2001). MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1. Mol Microbiol. 39(3):722-730.

Botsford JL and Harman JG. (1992). Cyclic AMP in prokaryotes. Microbiol Rev. 56(1):100-122. Brückner R and Titgemeyer F. (2002) Carbon catabolite repression in bacteria: choice of the carbon source and autoregulatory limitation of sugar utilization. FEMS Microbiol Lett. 209(2):141-148.

Brutinel ED, Gralnick JA. (2012). Preferential utilization of D-lactate by Shewanella oneidensis.

Appl Environ Microbiol. 78(23):8474-8476.

Busby S and Ebright RH. (1997) Transcription activation at class II CAP-dependent promoters. Mol Microbiol. 23(5):853-859.

Carpentier W, Sandra K, De Smet I, Brigé A, De Smet L, Van Beeumen J. (2003). Microbial reduction and precipitation of vanadium by Shewanella oneidensis. Appl Environ Microbiol. 69:3636-3639.

Charania MA, Brockman KL, Zhang Y, Banerjee A, Pinchuk GE, Fredrickson JK et al. (2009). Involvement of a membrane-bound class III adenylate cyclase in regulation of anaerobic respiration in Shewanella oneidensis MR-1. J Bacteriol. 191(13):4298-4306.

Coursolle D, Baron DB, Bond DR, Gralnick JA. (2010). The Mtr respiratory pathway is essential for reducing flavins and electrodes in Shewanella oneidensis. J Bacteriol. 192(2):467-474. doi: 10.1128/JB.00925-09.

Dym O, Pratt EA, Ho C, Eisenberg D. (2000). The crystal structure of D-lactate dehydrogenase, a

Flynn TM, O'Loughlin EJ, Mishra B, DiChristina TJ, Kemner KM. (2014). Sulfur-mediated electron shuttling during bacterial iron reduction. Science. 344:1039-1042. doi: 10.1126/science.1252066. Fonseca BM, Paquete CM, Neto SE, Pacheco I, Soares CM, Louro RO. (2013). Mind the gap: cytochrome interactions reveal electron pathways across the periplasm of Shewanella oneidensis MR-1. Biochem J. 449(1):101-108.

Fredrickson JK, Romine MF, Beliaev AS, Auchtung JM, Driscoll ME, Gardner TS, et al. (2008). Towards environmental systems biology of Shewanella. Nat Rev Microbiol. 6:592-603. doi: 10.1038/nrmicro1947.

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