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