chad saltikov and collaborators microbiology of arsenic redox transformations molecular...
TRANSCRIPT
![Page 1: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/1.jpg)
Chad Saltikov and collaboratorsChad Saltikov and collaborators
Microbiology of arsenic redox transformations
Microbiology of arsenic redox transformations
Molecular GeneticsEnvironment
Public Health
UCSC
![Page 2: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/2.jpg)
Microbial redox transformations of arsenicMicrobial redox transformations of arsenic
Sediment
Aqueous
Arsenate ReducingBacterium
As(III)Arsenite Oxidizer
Arsenic Contamination
![Page 3: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/3.jpg)
Phylogenetic relationship of arsenic metabolizing microbes
Phylogenetic relationship of arsenic metabolizing microbes
![Page 4: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/4.jpg)
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
![Page 5: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/5.jpg)
Oxidases and reductases are in a large family of proteinsOxidases and reductases are in a large family of proteins
![Page 6: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/6.jpg)
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
![Page 7: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/7.jpg)
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
![Page 8: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/8.jpg)
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)
![Page 9: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/9.jpg)
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
![Page 10: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/10.jpg)
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
![Page 11: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/11.jpg)
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
![Page 12: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/12.jpg)
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
![Page 13: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/13.jpg)
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
![Page 14: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/14.jpg)
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
![Page 15: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/15.jpg)
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.
![Page 16: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/16.jpg)
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
![Page 17: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/17.jpg)
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
![Page 18: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/18.jpg)
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)
![Page 19: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/19.jpg)
Secondary structure prediction of CymASecondary structure prediction of CymA
Inner Membrane
![Page 20: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/20.jpg)
Model for microbial arsenate reductionModel for microbial arsenate reduction
DetoxificationRespiration
![Page 21: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/21.jpg)
Environmental significance of arsenate respiratory reduction
Environmental significance of arsenate respiratory reduction
![Page 22: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/22.jpg)
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
![Page 23: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/23.jpg)
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
![Page 24: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/24.jpg)
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
![Page 25: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/25.jpg)
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.
![Page 26: Chad Saltikov and collaborators Microbiology of arsenic redox transformations Molecular GeneticsEnvironment Public Health UCSC](https://reader035.vdocuments.net/reader035/viewer/2022062222/56649dca5503460f94ac10fd/html5/thumbnails/26.jpg)
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