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![Page 1: Metal reduction pathways important for arsenic mobilization Chad W. Saltikov Assistant Professor UC, Santa Cruz Microbiology and Environmental Toxicology.](https://reader035.fdocuments.in/reader035/viewer/2022062718/56649eaa5503460f94baf27c/html5/thumbnails/1.jpg)
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
![Page 2: Metal reduction pathways important for arsenic mobilization Chad W. Saltikov Assistant Professor UC, Santa Cruz Microbiology and Environmental Toxicology.](https://reader035.fdocuments.in/reader035/viewer/2022062718/56649eaa5503460f94baf27c/html5/thumbnails/2.jpg)
Pore water concentrationsof As and FePore water concentrationsof As and Fe
Suvasis Dixit and Janet HeringCaltech
MCL
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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)
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Metal-Reduction in ShewanellaMetal-Reduction in Shewanella
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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)
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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 -
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Arsenate Respiratory Reductase in Bacteria
Arsenate Respiratory Reductase in Bacteria
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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
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mtrD mtrE mtrComcA mtrA mtrBmtrF
Metal reduction genes in ShewanellaMetal reduction genes in Shewanella
UCSC Genome Browser http://microbes.ucsc.edu
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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.
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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
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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
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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)
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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
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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
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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?
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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
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CymA and As(V) and Fe(III) reductionCymA and As(V) and Fe(III) reduction
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∆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
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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
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Predicted structure of CymAPredicted structure of CymA
CymANrfH
K91K90
K96D97
D89K82
hemes
Lys (K) to Gln (Q) (basic to neutral)Asp (D) (acidic)
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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
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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
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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
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CymA-MQH2 effects on ARRCymA-MQH2 effects on ARR
(Gln, neutral)
(Arg, basic)
(Met, neutral)
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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
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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.
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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
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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
)
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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
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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
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Effects of arsR2 on arrA/arsC gene expressionEffects of arsR2 on arrA/arsC gene expression
arsR1 arsR2
1 kb
ars arr
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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
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As(V) respiration and global regulators
As(V) respiration and global regulators
(fnr)
Growth on 10 mM Arsenate
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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
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Arsenate reduced after 8 hoursArsenate reduced after 8 hours
0.0
0.50
1.0
1.5
2.0
2.5
StrainWT ∆crp
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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)
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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
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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