Transcript Molecular biology of respiratory arsenate reduction

Metal reduction pathways important for
arsenic mobilization
Chad W. Saltikov
Assistant Professor
UC, Santa Cruz
Microbiology and Environmental Toxicology
Pore water concentrations
of As and Fe
MCL
Suvasis Dixit and Janet Hering
Caltech
Metal Reduction and Arsenic Mobilization
As(III)
As(V) Reducers
e-
eAs(V)
iron oxide
e-
As(V)
As(V)
Fe(II)
Fe(III) Reducers
As(III) Oxidizers
Metal-Reduction in Shewanella
Toxicity, Fate, and Transport of Arsenic
 Environmental impact of metal-reduction
 Electron transport chains for ARR/Mtr
 Regulation of metal reduction pathways
Fe(III)
As(V)
As(III)
HPO32O2
NO3-
Shewanella sp. strain ANA-3: our model
Substrate
Growth
Arsenate
+
Nitrate
+
Fumarate
+
TMAO
+
Fe(OH)3
+
MnO2
+
Oxygen
+
Thiosulfate
+
DMSO
Saltikov et al. AEM 2003
Arsenate Respiratory Reductase in Bacteria
The 23 kb “Arsenic Island” in
Shewanella species
As(V)
Arsenic
respiration detoxification
UCSC Genome Browser http://microbes.ucsc.edu
Metal reduction genes in Shewanella
mtrD
mtrE
mtrF
omcA
mtrC
mtrA
UCSC Genome Browser http://microbes.ucsc.edu
mtrB
Impacts of metal-reducing bacteria on
arsenic contamination and water quality
Fe(III) vs. As(V) reduction
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Fe(III) vs. As(V) reduction and
As mobilization
Strain
Genotype
Fe(III)
Reduction
As(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 Reduction
In batch cultures
Dissolve Arsenate
Dissolved Iron
400
350
ANA-3
0.8
Dissolved arsenate (µM)
Dissolved Iron(II) (mM)
1
0.6
ARM1 (²arsC, ²arrA)
0.4
0.2
0
FERM1 (²mtrD-mtrB)
FARM1 no cells
0
50
100
150
200
Time (hours)
250
300
350
no cells
FARM1
300
250
ARM1 (²arsC, ²arrA)
200
150
100
ANA-3
50
0
FERM1 (²mtr²omC)
0
50
100
Time (hours)
150
End-point solid phase arsenic chemistry
1000
900
µM arsenic
800
700
600
As(V)
As(III)
500
400
300
200
100
0
ANA-3
FARM
FERM
Strain
ARM-1
No Cell
Arsenic mobilization from ferrihydrite
coated sand: advective flow
263 mg As/kg sand
5500 mg Fe/kg sand
Iron oxide coated sand
24 μL/min
8
7
arsenite µM
6
FERM (no Fe reduction)
5
4
3
WT
2
1
(no As reduction)
ARM
0
0
50
100
150
200
pore volume
Picture from Herbal and Fendorf
no cells
250
Comparison of Fe and As Elution under
advective conditions
Dissolved Fe
Dissolved As
9
160
8
WT
140
Total As (uM)
[dissolved iron] µM
180
120
100
ARM
80
60
40
FERM
20
no
cells
0
0
50
100
150
200
250
7
FERM
6
5
4
WT
3
2
1
0
no
cells
ARM
0
pore volume
3 mM lactate
As(V) on ferrihydrite (3.5 mmol Kg-1)
25 % of the adsorption maximum at pH 7.1
50
1 00
1 50
pore volumes
200
250
Preliminary 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
CymA and ArrAB and Mtr/OmC
Regulation of arrA
Regulation of mtr/omc
CymA and As(V) and Fe(III) reduction
∆cymA causes pleiotropic effects on
respiration pathways
ANA-3
CN-32
MR-1
Substrate
cymA
wt
cymA
wt
cymA
wt
Arsenate
-
+
-
+
-
-
Fumarate
-
+
+
+
-
+
DMSO
-
-
nd
nd
-
+
Nitrate
+
+
+
+
-
+
TMAO
+
+
+/-
+/-
+
+
O2
+
+
+
+
+
+
Thiosulfate
+
+
+
+
+
+
Fe(III)
-
+
-
+
-
+
Mn(IV)
-
+
-
+
-
+
-
no growth
+
growth
Murphy and Saltikov, 2007
Does CymA
interact with
Menaquinone?
Fe(III) Fe(II)
Mtr/Omc
ArrAB
c-heme
CymA
Q
QH2
NADH2
NAD+
4X[FeS]
As(V)
As(III)
Mo
[FeS]
Predicted structure of CymA
NrfH
hemes
D89
K82
CymA
D97
K96
K91
K90
Lys (K) to Gln (Q) (basic to neutral)
Asp (D) (acidic)
Over-expression of CymA in E. coli
Cytoplasmic
Membranes
Western
(anti-V5)
1. CymA - ccm
2. CymA + ccm
3. K90Q + ccm
Heme Stain
1. Ladder
2. No pCYMA
3. CymA - ccm
4. CymA + ccm
5. K90Q + ccm
MQH2-mediated reduction of CymA
Cym A K90Q + ccm
Cym A + ccm
0.05
0.05
0.04
0.04
0.04
0.03
0.03
0.03
0.02
0.02
0.01
0.01
0.05
Relative Abs
No cym A
DT
DMNH2
0.02
0.01
0
500 520 540 560 580 600
0
500 520 540 560 580 600
nm
Dithionite
DMNH2
NaBH4
nm
DMN
DMNH2
2,3 dimethyl 1,4 naphthoquinone(ol)
0
500 520 540 560 580 600
nm
0.3 mg/ml protein
~4% CymA
~600 nM
Does CymA interact with menaquionols?
Relative Fluorescence
CymA + ccm
CymA-K90Q + ccm
7
25
6
40
35
20
15
3
10
1 mg/m l
2 mg/m l
3 mg /ml
30
25
5
4
No CymA
20
15
10
2
5
1
5
0
0
0
0.5
1
1.5
µM HOQNO
2
0
0
0.5
1
1.5
µM HOQNO
2
0
0.5
1
1.5
µM HOQNO
Calculated disassociation Kd is ~90 nM HOQNO
2
CymA-MQH2 effects on ARR
(Gln, neutral)
(Met, neutral)
(Arg, basic)
Next steps for CymA
CymA
Quinone substrate range?
ArrAB reduction by CymA
Mtr/Omc reduction by CymA
Multasking nature of CymA
D97
K96
K91
K90
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.
5.85
6.18
8
9.1
Oxygen
Fe(III) oxide
Fe-Citrate
Arsenate
3.68
2.64
0.0762
0.0265
0.0815
0.0332
0.0358
0.0285
0.0344
0.0241
0.0374
2
0.704
1.84
4
2.73
3.22
4.03
4.6
6
0.0162
Expression (relative to gyrB)
10
8.93
Expression of mtr/omc in ANA-3
0
mtrD
(2672)
mtrD
mtrE
(2673)
mtrE
mtrF
(2674)
mtrF
omcA
(2675)
omcA
mtrC
(2676)
mtrC
mtrA
(2677)
mtrA
mtrB
(2678)
mtrB
Note: Beliaev et al. 2005 showed 2-8 fold decrease in MR-1
arrA expression with other substrates?
10 mM As(V)
No As
w/ 5 mM As(V)
w/ 1 mM As(III)
Expression (arrA/gyrB)
1.5
1
0.5
0
As(V)
Saltikov et al. J. Bact. 2005
O2
Fumarate
Nitrate
TMAO
Possible regulators of arr and mtr/omc
Environmental Condition
Transcription
Factor/Sensor
Arsenite
ArsR √
Arsenate
?
Anaerobic
Fnr , ArcA
Aerobic
cAMP
Nitrate/Nitrite
?
CRP, Cya
NarP, NarQ
Arsenite regulators:
ArsR
helix turn helix, ~12 kDa
DNA binding
Binds As(III), Sb(III), PAO
ArsR
ars
Quantitative Gene Expression
ArsR
arr
As(III)
As(III)
Six arsR like genes in ANA-3
As(III)
Effects of arsR2 on arrA/arsC gene expression
ars
arr
arsR1
arsR2
1 kb
ArsR binds the arr/ars intergenic region
ars
Pars
Parr
ArsR2
ArsR2
~350 bp
5’-Cy3
ArsR2 Binding
Protein+DNA
Free DNA
arr
As(V) respiration and global regulators
Growth on 10 mM Arsenate
(fnr)
CRP and arrA gene expression
Growth
A. arrA Expression
O2
Shift -O2
+
As(V)
Sample:
0hr, 8 hr
B. 16S rRNA Expression
Arsenate reduced after 8 hours
[arsenate] mM
2.5
2.0
1.5
1.0
0.50
0.0
WT
Strain
² crp
ArsR and CRP binding @ arr promoter
ars
Parr
arr
ArsR2
B
Shift (ArsR/CRP)
Shift (CRP)
Shift (ArsR)
Free DNA
Model for arr and mtr/omc regulation
NO3NO2-
Low O2
As(V)
ArrS
ArrR ArrR-P
?
Hpt
NarQ
NarP NarP-P
low O2, ?
Cya
cAMP ATP
ArcA ArcA-P
CRP
cAMP
genes
ArsR
arrAB
+As(III)
-As(III)
mtrDEF omcA mtrCAB
high redox
low redox
Fnr (EtrA)
Thank you!
Acknowledgments:
UCSC:
– Julie Murphy, Carolina Reyes, Kamrun Zargar
Stanford:
– Prof. Scott Fendorf, Kate Tufano
Funding:
– NSF, UC Toxic Substance, CRCC, MBRS, Cota
Robles
~Santa Cruz, California, USA~