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Chad Saltikov and collaboratorsChad Saltikov and collaborators
Microbiology of arsenic redox transformations
Microbiology of arsenic redox transformations
Molecular GeneticsEnvironment
Public Health
UCSC
Microbial redox transformations of arsenicMicrobial redox transformations of arsenic
Sediment
Aqueous
Arsenate ReducingBacterium
As(III)Arsenite Oxidizer
Arsenic Contamination
Phylogenetic relationship of arsenic metabolizing microbes
Phylogenetic relationship of arsenic metabolizing microbes
Arsenic oxidation and reduction pathwaysArsenic oxidation and reduction pathways
ArseniteOxidation
ArsenateRespiration
ArsenicDetoxification
Adapted from Silver and Phung 2005. Appl. Env. Micro. 71(2):599-608
Oxidases and reductases are in a large family of proteinsOxidases and reductases are in a large family of proteins
arrA genes from other bacteriaarrA genes from other bacteria
Akaliphilus metalliredigenes: 66%*Geobacter uraniumreducens: 65%*Desulfosporosinus sp. Y5: 66%*
*aa similarities to ArrA of ANA-3
The arsenic island of Shewanella:ANA-3, CN-32, and W3-18-1
The arsenic island of Shewanella:ANA-3, CN-32, and W3-18-1
W3-18-1
CN-32
What environmental conditions trigger arsenate reduction?
ArsC (detoxify)vs.
ArrA (respire)
What environmental conditions trigger arsenate reduction?
ArsC (detoxify)vs.
ArrA (respire)
As(V)
As(III)
Monitor two arsenate reduction pathwaysin our model arsenate reducer
Shewanella sp. ANA-3
Monitor two arsenate reduction pathwaysin our model arsenate reducer
Shewanella sp. ANA-3
(an)aerobic vs. As
Monitor the transcription of arrA and arsC
Saltikov and Newman 2003 PNAS 100(19):10983-10988
Quantify gene specific mRNA:Quantify gene
specific mRNA:
Grow culture to mid log
Extract/Purify RNA
Reverse TranscribeRNA
Quantify gene-specific mRNA
by real time PCR
Standard curve for
y = -3.356x + 19.857
R2 = 0.9982
15
17
19
21
23
25
27
29
-3 -2 -1 0 1
log ng DNA
Threshold Cycle (Ct)0.04 ng
Dynamic expression in various growth phases with As(V) as electron acceptor
Dynamic expression in various growth phases with As(V) as electron acceptor
Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396
How does phosphate influence As(V) respiration and arr/ars expression
How does phosphate influence As(V) respiration and arr/ars expression
As(V)+Pi Fumarate+Pi Expression
Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396
arrA arsC Substrate: expressiona inductionb expressiona inductionb
Oxygen 0.012 ± 0.004 - 0.015 ± 0.005 -
( )As V 0.015 ± 0.002 - 0.10± 0.02 7 ( )As III 0.013 ± 0.002 - 0.37 ± 0.06 25
Fumarate 0.09 ± 0.03 - 0.04 ± 0.01 - ( )As V 0.72 ± 0.09 7.8 0.44 ± 0.08 10.3 ( )As III 0.92 ± 0.06 10.0 0.71 ± 0.04 16.9
Nitrate 0.05 ± 0.02 - 0.04 ± 0.00 - ( )As V 0.08 ± 0.01 - 0.21 ± 0.11 5.1 ( )As III 0.07 ± 0.01 - 0.68 ± 0.12 16.2
TMAO 0.02 ± 0.00 - 0.02 ± 0.00 - ( )As V 0.43 ± 0.17 20.1 0.26 ± 0.09 13.6 (As )III 0.29 ± 0.03 13.5 0.61 ± 0.08 32.7
. a Expression represents the ratio of the relative quantity of arrA or arsCtranscripts to that of the housekeeping gene gyrB.
. b Induction was determined by normalizing the expression value for arrA orarsC to no As condition .s
What about other electron acceptors?What about other electron acceptors?
As(III) = inducerO2 and NO3
- inhibit
Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396
What are the sensitivities of arrA and arsC expression to As?
What are the sensitivities of arrA and arsC expression to As?
Log [arsenite] µM Log [arsenate] µM
∆arrA, ∆arsCWild-typeA. B.
NoAs
NoAs
Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396
Are cytochromes required for arsenate respiration in our model organism?
factoid: Shewanella has 39 reading frames encoding c-type cytochromes
Iron containing proteins similar to heme of a red blood cell.
Are cytochromes required for arsenate respiration in our model organism?
factoid: Shewanella has 39 reading frames encoding c-type cytochromes
Iron containing proteins similar to heme of a red blood cell.
CymA--tetraheme cytochromeis required for respiring arsenateCymA--tetraheme cytochrome
is required for respiring arsenate
UQH2
UQ
periplasm
cytoplasmdehydrogenase
ArrAB
As(V) As(III)
Adapted from Schwalb et al. 2003 Biochemistry 42(31):9491-9497
cymA is required for respiring As(V) in CN-32cymA is required for respiring As(V) in CN-32
Growth on nitrate Growth on arsenate
cymA restores growth on As(V)cymA restores growth on As(V)
0
0.05
0.1
0.15
0.2
0 2 4 6 8 10
ANA-3+pBBR1MCS2
AN-CymA+pBBR1MCS2
AN-CymA+ANpCymA
AN-CymA+CNpCymA
Time (hours)
Secondary structure prediction of CymASecondary structure prediction of CymA
Inner Membrane
Model for microbial arsenate reductionModel for microbial arsenate reduction
DetoxificationRespiration
Environmental significance of arsenate respiratory reduction
Environmental significance of arsenate respiratory reduction
Can arrA be used to monitor and track As(V) reduction?Can arrA be used to monitor and track As(V) reduction?
A. ArrA protein and primer design L. Ladder: 100 bp
• ANA-3 arrA deleted
• Shew. oneidensis MR-1
• Desulfitobacterium dehalogenens
• E. coli
• Pseudomonas chloraphis
• Shewanella sp. ANA-3
• Desulfito. hafniense
• Desulfito. frappieri
• D. strain GBFH
• Wolinella sp.
• Wolinella succinogenes
• Citrobacter sp.
• Bacillus str. E1H
• Bacillus, str. MLS10
• Sulfur. barnesii SES-3
• Shewanella sp. HAR-4
• Chrysiogenes
• OREX-4
• Pyrobac. arsenaticum
16S rDNA primers
arrA primers
16S rDNA primers
B. Detection in various strains
Malasarn et al. 2004 Science 06: 455
Pore water concentrationsof total As, Fe, and Mn inFe/As rich reservoir
Pore water concentrationsof total As, Fe, and Mn inFe/As rich reservoir
*
MnFeAs
Kneebone et al. 2002 ES&T 36(3):381-386
1 2 3 4 5 6 7 8 Blanks
1517
12001000
500
400
300
200
100
New primers for detecting arrA-like genes in As-enriched sediments
New primers for detecting arrA-like genes in As-enriched sediments
Increasing depth in core
PrimerDimmers
ConclusionsConclusions
Two genetic pathways for arsenate reduction– Respiratory by arr and detoxification by ars
Respiration pathway triggered by As:– As(III) > As(V)– Repressed by nitrate and oxygen
The mechanism for arsenate reduction involves other components in the cell.
The arrA gene is a useful marker for As redox– Gene copy number seems to correlate with redox
gradients of As … more work to be done.
Acknowledgments/CollaboratorsAcknowledgments/Collaborators
UC Santa Cruz– Julie Nilsen
Caltech– Prof. Dianne Newman, Prof. Janet Hering, Rich
Wildman
USGS– Dr. Ron Oremland, Dr. Thomas Kulp, Dr. Larry
Miller, Shelly Hoeft