Transcript How does the cell regulate arsenate respiration and

Microbiology of arsenic redox
transformations
UCSC
Chad Saltikov and collaborators
Environment
Molecular Genetics
Public Health
Microbial redox transformations of arsenic
Aqueous
Arsenate Reducing
Bacterium
Sediment
Arsenic Contamination
As(III)
Arsenite Oxidizer
Phylogenetic
relationship of
arsenic
metabolizing
microbes
Arsenic oxidation and reduction pathways
Arsenite
Oxidation
Arsenate
Respiration
Arsenic
Detoxification
Adapted from Silver and Phung 2005. Appl. Env. Micro. 71(2):599-608
Oxidases and reductases are in a large family of proteins
arrA 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
CN-32
W3-18-1
What environmental conditions trigger
arsenate reduction?
ArsC (detoxify)
vs.
ArrA (respire)
As(V)
As(III)
Monitor two arsenate reduction pathways
in 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:
Grow culture to mid log
Extract/Purify RNA
Reverse Transcribe
RNA
Quantify genespecific mRNA
by real time PCR
Threshold Cycle (Ct)
Standard curve for
29
y = -3.356x + 19.857
27
R2 = 0.9982
25
23
21
19
0.04 ng
17
15
-3
-2
-1
log ng DNA
0
1
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
As(V)+Pi
Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396
Fumarate+Pi
Expression
What about other electron acceptors?
arrA
arsC
inductionb
-
expressiona
0.015  0.005
0.015  0.002
0.013  0.002
-
0.10 0.02
0.37  0.06
7
25
Fumarate
As(V)
As(III)
0.09  0.03
0.72 ± 0.09
0.92 ± 0.06
7.8
10.0
0.04 ± 0.01
0.44 ± 0.08
0.71 ± 0.04
10.3
16.9
Nitrate
0.05 ± 0.02
-
0.04 ± 0.00
-
As(V)
As(III)
0.08 ± 0.01
0.07 ± 0.01
-
0.21 ± 0.11
0.68 ± 0.12
5.1
16.2
TMAO
As(V)
As(III)
0.02 ± 0.00
0.43 ± 0.17
0.29 ± 0.03
20.1
13.5
0.02 ± 0.00
0.26 ± 0.09
0.61 ± 0.08
13.6
32.7
Substrate:
Oxygen
As(V)
As(III)
expressiona
0.012  0.004
inductionb
As(III) = inducer
O2 and NO3- inhibit
-
a. Expression represents the ratio of the relative quantity of arrA or arsC transcripts to that of the housekeep ing
gene gyrB.
b. Induction was determined by normalizing the expression value for arrA or arsC to no As conditions.
Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396
What are the sensitivities of arrA and arsC
expression to As?
A.
B.
∆arrA, ∆arsC
Wild-type
No
As
No
As
Log [arsenite] µM
Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396
Log [arsenate] µM
Are cytochromes required for arsenate
respiration in our model organism?
factoid: Shewanella has 39 reading frames encoding ctype cytochromes
Iron containing proteins similar to heme of a red blood
cell.
CymA--tetraheme cytochrome
is required for respiring arsenate
periplasm
As(V)
As(III)
ArrAB
UQH2
UQ
dehydrogenase
cytoplasm
Adapted from Schwalb et al. 2003 Biochemistry 42(31):9491-9497
cymA is required for respiring As(V) in CN-32
Growth on nitrate
Growth on arsenate
cymA restores growth on As(V)
0.2
ANA-3+pBBR1MCS2
AN-CymA+pBBR1MCS2
AN-CymA+ANpCymA
Growth (OD 600 nm)
0.15
AN-CymA+CNpCymA
0.1
0.05
0
0
2
4
6
Time (hours)
8
10
Secondary structure prediction of CymA
Inner Membrane
Model for microbial arsenate reduction
Respiration
Detoxification
Environmental significance of arsenate
respiratory reduction
Can arrA be used to monitor and track As(V) reduction?
A. ArrA protein and primer design
B. Detection in various strains
arrA primers
16S rDNA primers
16S rDNA primers
Malasarn et al. 2004 Science 06: 455
L.
Ladder: 100 bp
1.
ANA-3 arrA deleted
2.
Shew. oneidensis MR-1
3.
Desulfitobacterium
dehalogenens
4.
E. coli
5.
Pseudomonas chloraphis
6.
Shewanella sp. ANA-3
7.
Desulfito. hafniense
8.
Desulfito. frappieri
9.
D. strain GBFH
10.
Wolinella sp.
11.
Wolinella succinogenes
12.
Citrobacter sp.
13.
Bacillus str. E1H
14.
Bacillus, str. MLS10
15.
Sulfur. barnesii SES-3
16.
Shewanella sp. HAR-4
17.
Chrysiogenes
18.
OREX-4
19.
Pyrobac. arsenaticum
Pore water concentrations
of total As, Fe, and Mn in
Fe/As rich reservoir
*
Mn
Fe
As
Kneebone et al. 2002 ES&T 36(3):381-386
New primers for detecting arrA-like genes in
As-enriched sediments
Increasing depth in core
1517
1200
1000
1
2
3
4
5
6
7
8
Blanks
500
400
300
200
100
Primer
Dimmers
Conclusions
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/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