Metal reduction pathways important for arsenic mobilization

Metal reduction pathways important for arsenic mobilization

Chad W. Saltikov

Assistant ProfessorUC, Santa Cruz

Microbiology and Environmental Toxicology

Pore water concentrationsof As and FePore water concentrationsof As and Fe

Suvasis Dixit and Janet HeringCaltech

MCL

As(V)iron oxide

Metal Reduction and Arsenic MobilizationMetal Reduction and Arsenic Mobilization

e-As(III) Oxidizers

e-

As(III)As(V) Reducers

e-

Fe(III) Reducers

As(V)Fe(II)

As(V)

Metal-Reduction in ShewanellaMetal-Reduction in Shewanella

Environmental impact of metal-reduction

Electron transport chains for ARR/Mtr Regulation of metal reduction pathways

Toxicity, Fate, and Transport of Arsenic

Toxicity, Fate, and Transport of Arsenic

As(V)As(III)

HPO32-

NO3-O2

Fe(III)

Shewanella sp. strain ANA-3: our model

Shewanella sp. strain ANA-3: our model

Saltikov et al. AEM 2003

Substrate Growth

Arsenate +

Nitrate +

Fumarate +

TMAO +

Fe(OH)3 +

MnO2 +

Oxygen +

Thiosulfate +

DMSO -

Arsenate Respiratory Reductase in Bacteria

Arsenate Respiratory Reductase in Bacteria

The 23 kb “Arsenic Island” in Shewanella species

The 23 kb “Arsenic Island” in Shewanella species

Arsenicdetoxification

Arsenicdetoxification

As(V)respiration

As(V)respiration

UCSC Genome Browser http://microbes.ucsc.edu

mtrD mtrE mtrComcA mtrA mtrBmtrF

Metal reduction genes in ShewanellaMetal reduction genes in Shewanella

UCSC Genome Browser http://microbes.ucsc.edu

Impacts of metal-reducing bacteria on arsenic contamination and water

quality

Impacts of metal-reducing bacteria on arsenic contamination and water

quality

Fe(III) vs. As(V) reduction

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Fe(III) vs. As(V) reduction and As mobilization

Fe(III) vs. As(V) reduction and As mobilization

Strain GenotypeFe(III)

ReductionAs(V)

Reduction

ANA-3 wt + +ARM1

∆arrA, ∆arsC + -

FERM1

∆mtrDEF∆omcA

∆mtrCAB- +

FARM1∆arrA, ∆arsC,

∆mtr/omc- -

•As:HFO= 0.015•500 mg HFO-As(V)•1 mM As(V) total•Lactate 20 mM•0.5 mM Phosphate•10 mM HEPES pH 7•Basalt salts medium

Iron(III) and Arsenate ReductionIn batch cultures

Iron(III) and Arsenate ReductionIn batch cultures

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250 300 350

Dissolved Iron(II) (mM)

Time (hours)

ANA-3

ARM1 (∆arsC, ∆arrA)

FERM1 (∆mtrD-mtrB)

FARM1 no cells

Dissolved Iron Dissolve Arsenate

0

50

100

150

200

250

300

350

400

0 50 100 150

Dissolved arsenate (µM)

Time (hours)

ANA-3

ARM1 (∆arsC, ∆arrA)

FERM1 (∆mtr∆omC)

no cellsFARM1

End-point solid phase arsenic chemistry

End-point solid phase arsenic chemistry

0

100

200

300

400

500

600

700

800

900

1000

ANA-3 FARM FERM ARM-1 No Cell

Strain

µM arsenic

As(V)

As(III)

Arsenic mobilization from ferrihydrite coated sand: advective

flow

Arsenic mobilization from ferrihydrite coated sand: advective

flow

Iron oxide coated sand

0

1

2

3

4

5

6

7

8

0 50 100 150 200 250

pore volume

arsenite µM WT

ARM

FERM

no cells

263 mg As/kg sand5500 mg Fe/kg sand24 μL/min

(no Fe reduction)

(no As reduction)

Picture from Herbal and Fendorf

Comparison of Fe and As Elution under advective conditions

Comparison of Fe and As Elution under advective conditions

0

20

40

60

80

100

120

140

160

180

0 50 100 150 200 250

pore volume

[dissolved iron] µM

0

1

2

3

4

5

6

7

8

9

0 50 100 150 200 250

pore volumes

Total As (uM)

Dissolved Fe Dissolved As

FERM

WT

ARMFERM

WT

ARM

nocells

nocells

3 mM lactate

As(V) on ferrihydrite (3.5 mmol Kg-1)

25 % of the adsorption maximum at pH 7.1

Preliminary ConclusionsPreliminary Conclusions

ArrA reduces solid phase arsenate reduction ∆arrA strain has a problem reducing Fe(III)-

oxide As(III) mobilization highest in ∆mtr/omc strain

How does ArrA access solid-phase As(V)?

Is the “Fe(III) reductase” blocked by As(V)?

Why does uncoupling Fe-reduction increase As release?

Characterizing the metabolic pathways essential for arsenic

mobilization

Characterizing the metabolic pathways essential for arsenic

mobilization

CymA and ArrAB and Mtr/OmC Regulation of arrA Regulation of mtr/omc

CymA and As(V) and Fe(III) reductionCymA and As(V) and Fe(III) reduction

∆cymA causes pleiotropic effects on respiration pathways

∆cymA causes pleiotropic effects on respiration pathways

ANA-3 CN-32 MR-1Substrate cymA wt cymA wt cymA wt

Arsenate - + - + - -

Fumarate - + + + - +

DMSO - - nd nd - +

Nitrate + + + + - +

TMAO + + +/- +/- + +

O2 + + + + + +

Thiosulfate + + + + + +

Fe(III) - + - + - +

Mn(IV) - + - + - +

Murphy and Saltikov, 2007- no growth + growth

Does CymA interact with

Menaquinone?

Does CymA interact with

Menaquinone?

4X[FeS]

As(V) As(III)ArrAB

Mo[FeS]

Mtr/Omc

CymA

Fe(III) Fe(II)

NADH2 NAD+

c-heme

QH2

Q

Predicted structure of CymAPredicted structure of CymA

CymANrfH

K91K90

K96D97

D89K82

hemes

Lys (K) to Gln (Q) (basic to neutral)Asp (D) (acidic)

Over-expression of CymA in E. coliOver-expression of CymA in E. coli

1. CymA - ccm2. CymA + ccm3. K90Q + ccm

1. Ladder2. No pCYMA3. CymA - ccm4. CymA + ccm 5. K90Q + ccm

Western(anti-V5) Heme StainCytoplasmic

Membranes

MQH2-mediated reduction of CymAMQH2-mediated reduction of CymA

CymA + ccm

0

0.01

0.02

0.03

0.04

0.05

500 520 540 560 580 600

CymA K90Q + ccm

0

0.01

0.02

0.03

0.04

0.05

500 520 540 560 580 600

No cymA

0

0.01

0.02

0.03

0.04

0.05

500 520 540 560 580 600

Rela

tive A

bs

nm nm nm

DT

DMNH2

DMNH2

Dithionite

NaBH4DMN DMNH2

2,3 dimethyl 1,4 naphthoquinone(ol)

0.3 mg/ml protein~4% CymA~600 nM

Does CymA interact with menaquionols?

Does CymA interact with menaquionols?

0

1

2

3

4

5

6

7

0 0.5 1 1.5 2

µM HOQNO

0

5

10

15

20

25

0 0.5 1 1.5 2

µM HOQNO

0

5

10

15

20

25

30

35

40

0 0.5 1 1.5 2

1 mg/ml2 mg/ml3 mg /ml

µM HOQNO

Rela

tive F

luore

scence

CymA + ccm CymA-K90Q + ccm No CymA

Calculated disassociation Kd is ~90 nM HOQNO

CymA-MQH2 effects on ARRCymA-MQH2 effects on ARR

(Gln, neutral)

(Arg, basic)

(Met, neutral)

Next steps for CymANext steps for CymA

CymA

K91K90

K96D97

Quinone substrate range? ArrAB reduction by CymA Mtr/Omc reduction by CymA Multasking nature of CymA

How does arrA and mtr/omc gene expression respond to different

environmental conditions?

How does arrA and mtr/omc gene expression respond to different

environmental conditions?

Oxygen should repress both pathways

Arsenate should induce arrA

Fe should induce mtr/omc.

mtrD mtrE mtrComcA mtrA mtrBmtrF

Expression of mtr/omc in ANA-3Expression of mtr/omc in ANA-3

0

2

4

6

8

10

mtrD(2672)

mtrE(2673)

mtrF(2674)

omcA(2675)

mtrC(2676)

mtrA(2677)

mtrB(2678)

OxygenFe(III) oxideFe-CitrateArsenate

Note: Beliaev et al. 2005 showed 2-8 fold decrease in MR-1

arrA expression with other substrates?

arrA expression with other substrates?

Saltikov et al. J. Bact. 2005

0

0.5

1

1.5

As(V) O2 Fumarate Nitrate TMAO

10 mM As(V)No Asw/ 5 mM As(V)w/ 1 mM As(III)

Expression (

arrA/gyrB

)

Possible regulators of arr and mtr/omc

Possible regulators of arr and mtr/omc

Environmental ConditionTranscription Factor/Sensor

Arsenite ArsR √

Arsenate ?

Anaerobic Fnr , ArcA

Aerobic ?

cAMP CRP, Cya

Nitrate/Nitrite NarP, NarQ

Arsenite regulators:Arsenite regulators:

Six arsR like genes in ANA-3

ars arr

ArsR ArsR

As(III)

As(III)As(III)

Quantitative Gene Expression

ArsRhelix turn helix, ~12 kDaDNA bindingBinds As(III), Sb(III), PAO

Effects of arsR2 on arrA/arsC gene expressionEffects of arsR2 on arrA/arsC gene expression

arsR1 arsR2

1 kb

ars arr

ParrPars

ars

ArsR binds the arr/ars intergenic regionArsR binds the arr/ars intergenic region

ArsR2ArsR2

arr

ArsR2 Binding

Free DNAProtein+DNA

~350 bp 5’-Cy3

As(V) respiration and global regulators

As(V) respiration and global regulators

(fnr)

Growth on 10 mM Arsenate

CRP and arrA gene expressionCRP and arrA gene expressionA. arrA Expression

B. 16S rRNA Expression

Growth

O2

Shift -O2

+

As(V)

Sample:

0hr, 8 hr

Arsenate reduced after 8 hoursArsenate reduced after 8 hours

0.0

0.50

1.0

1.5

2.0

2.5

StrainWT ∆crp

Parr

ars

ArsR and CRP binding @ arr promoterArsR and CRP binding @ arr promoter

ArsR2

arr

B

Free DNA

Shift (ArsR)

Shift (ArsR/CRP)Shift (CRP)

Model for arr and mtr/omc regulationModel for arr and mtr/omc regulation

arrAB mtrCABomcAmtrDEF

ATP

cAMP

cAMP

low O2, ?

Cya

ArcA

? NarQ

Hpt

Low O2

NO3-

NO2-

ArcA-P

NarP NarP-P

+As(III)

-As(III)

CRP

Fnr (EtrA)low redox

high redoxArsR

ArrR

ArrS

As(V)

ArrR-P

genes

Thank you!Thank you!

~Santa Cruz, California, USA~

Acknowledgments: UCSC:

– Julie Murphy, Carolina Reyes, Kamrun Zargar

Stanford:– Prof. Scott Fendorf, Kate Tufano

Funding:– NSF, UC Toxic Substance, CRCC, MBRS, Cota

Robles