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Environment. Molecular Genetics. Arsenic. Public Health. Microbiology of arsenic redox transformations. UCSC. Chad Saltikov and collaborators. Arsenic Contamination. Arsenite Oxidizer. As(III). Arsenate Reducing Bacterium. Microbial redox transformations of arsenic. Aqueous.

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Environment

Molecular Genetics

Arsenic

Public Health

Microbiology of arsenic redox transformations

UCSC

Chad Saltikov and collaborators


Arsenic Contamination

Arsenite Oxidizer

As(III)

Arsenate Reducing

Bacterium

Microbial redox transformations of arsenic

Aqueous

Sediment


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 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:

Grow culture to mid log

Extract/Purify RNA

Reverse Transcribe

RNA

Quantify gene-

specific mRNA

by real time PCR

0.04 ng


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

Fumarate+Pi

Expression

Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396


What about other electron acceptors?

As(III) = inducer

O2 and NO3- inhibit

Saltikov et al. J. Bacteriology 2005. 187 (21): 7390-7396


No

As

No

As

What are the sensitivities of arrA and arsC expression to As?

A.

B.

Wild-type

∆arrA, ∆arsC

Log [arsenite] µM

Log [arsenate] µM

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 cytochromesIron containing proteins similar to heme of a red blood cell.


CymA--tetraheme cytochromeis 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)


Secondary structure prediction of CymA

Inner Membrane


Model for microbial arsenate reduction

Respiration

Detoxification


Environmental significance of arsenate respiratory 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

B. Detection in various strains

arrA primers

16S rDNA primers

16S rDNA primers

Malasarn et al. 2004 Science 06: 455

Can arrA be used to monitor and track As(V) reduction?


*

Pore water concentrationsof total As, Fe, and Mn inFe/As rich reservoir

Mn

Fe

As

Kneebone et al. 2002 ES&T 36(3):381-386


1 2 3 4 5 6 7 8 Blanks

New primers for detecting arrA-like genes in As-enriched sediments

Increasing depth in core

1517

1200

1000

500

400

300

Primer

Dimmers

200

100


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


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