Arsenic Methylation

192 Pages·2012·1.51 MB·English

by  Angel Wu

University of Wollongong
Research Online
University of Wollongong Thesis Collection University of Wollongong Thesis Collections
2005
A comparative study of arsenic methylation in a
plant, yeast and bacterium
Jiehua Wu
University of Wollongong
Recommended Citation
Wu, Jiehua, A comparative study of arsenic methylation in a plant, yeast and bacterium, Doctor of Philosophy thesis, School of
Biological Sciences, University of Wollongong, 2005. http://ro.uow.edu.au/theses/1914
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A Comparative Study of Arsenic Methylation
in a Plant, Yeast and Bacterium

A thesis submitted in partial fulfilment of
the requirements for the award of the degree

Doctor of Philosophy

From

THE UNIVERSITY OF WOLLONGONG

By

Jiehua Wu, M.Sc.

School of Biological Sciences
2005
Declaration

I, Jiehua Wu, declare that this thesis is submitted in partial
fulfilment of the requirements for the award of Doctor of Philosophy,
in the School of Biological Sciences, University of Wollongong. It
does not include any material previously published by another
person except where due reference is made in the text. The
experimental work described in this thesis is original and wholly my
own work. The document has not been submitted for a degree in any
other academic institution.

Jiehua Wu

II
Acknowledgments
Acknowledgments

In completing this thesis, I can’t keep from thanking many people. First of all, I
convey my thanks to supervisors Associate Prof. Ross McC. Lilley and Dr. Ren
Zhang without whom this project could not have happened. It is impossible to
express the gratitude I feel for their expert guidance and supervision. I am greatly
indebted for their patient, encouragement, support and belief that I could see the
project through to the end.

Special thanks must be expressed to Ross Lilley and his wife Xi Hui. Ross provided
the job opportunity as technical officer, which supported me financially. The
kindness and care from Ross and Xi Hui let me feel my home in Australia.

I extend my thanks to James Petrie. During the research with yeast and bacteria,
James offered me assistance within some experiment processes, touching cell
growth and collection, purification of extracts of cells, and extraction of methylated
products. I found enlightenment in his smart ideas.

I am particularly grateful to the labmates of Ross’ and Ren’s labs for support and
assistance during the long, and sometimes stressful, period of research. In no
particular order, I thank Michael Kelly, Lixia Liu, Chenwei Shu, Yanzhe Wu, You
Wang, Qian Wang, Donna Harmon and Sean Isbester. I have always found these
people to be friendly, helpful and willing to provide advice.

I am grateful to all the members of the School of Biological Sciences for their help
and friendship. Special thanks must be given to Jan Fragiacomo and Mari Dwarte
for their administrative assistance. I often think of Jan after her retirement.

My great thanks to Jin Gao (Geoffrey), a former Ph.D. student of Department of
III
Acknowledgments
Chemistry, although we met mostly on the trains between Sydney and Wollongong
on the way to University, his kindness, intelligence and humour brought me
happiness, enlightened and encouraged me to complete this thesis. But a detestable
sin took his life just four days before the last Christmas day. I lost my best friend,
Geoffrey, forever.

The help and support from my Mother, Father and Brother are always what I rely on,
even if they live far away in China. I am greatly grateful to my family.

Finally, I would like to dedicate this thesis to my dear Mother. I can never forget the
tears in my mother’s eyes at the Shanghai airport when she saw me off for Australia,
an unknown world to us at that time. Her words still linger in my ears.
Unfortunately, she could not wait for my graduation, even seeing me again.
However, now I believe she is looking at me from the heaven, smiling and happy
with my completion of my Ph.D. thesis. Thanks for her blessing.
IV
Table of Contents
TABLE OF CONTENTS

Title Page I
Declaration II
Acknowledgments III
Table of Contents V
Publication out of this thesis so far XII
Keywords and Abbreviations XIII
Abstract XIV

Chapter I. INTRODUCTION: REVIEW OF THE LITERATURE
1
1.1. Arsenic, Arsenical Forms and Structures
1.1.1. Arsenic physical and chemical properties 2
1.1.2. Formation and structures of arsenicals 3
1.1.3. Arsenate and arsenite 4

1.2. Arsenic Species in the Environment
1.2.1. Arsenic distribution 7
1.2.1.1. The arsenic cycle 7
1.2.1.2. Background arsenic in soils 8
1.2.1.3. Arsenic in aquatic systems 10
1.2.1.4. Arsenic mobilization 11
1.2.2. Application of arsenic 15
1.2.3. Pollution of arsenic 17
1.2.3.1. Arsenic in arsenic-contaminated soils 17
1.2.3.2. Arsenic contamination in water 19
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Table of Contents
1.2.3.3. Arsenic contamination in Australia 20
1.2.4. Arsenic bioavailablity and uptake by organisms 23
1.2.5. Toxicity of arsenic to organisms 27

1.3. Natural Resistance and Tolerance Mechanisms for Arsenic
1.3.1. Arsenic transport out of cells and into the vacuole 30
1.3.1.1. ars operons in bacteria 30
1.3.1.2. The families of arsenic-resistance transporters
in yeast 34
1.3.1.3. Arsenic resistance in mammalian cells 36
1.3.1.4. Arsenic transport in plants 36
1.3.2. Arsenic chelation 37
1.3.2.1. Roles of glutathione in chelation 37
1.3.2.2. Roles of arsenic-binding protein in animals 37
1.3.2.3. Roles of peptides in chelation of arsenic in plants
(phytochelation) 39
1.3.3. Arsenic metabolism and organo-arsenical synthesis 42
1.3.3.1. Biomethylation 42
1.3.3.1.1. Methylation of arsenic by bacteria 43
1.3.3.1.2. Methylation of arsenic by yeast and other fungi 45
1.3.3.1.3. Methylation of arsenic by fresh water algae 47
1.3.3.1.4. Methylation of arsenic by mammals 47
1.3.3.1.5. Methylation of arsenic by terrestrial plants 48
1.3.3.2. Organo-arsenical synthesis by marine organisms 49

1.4. Enzymology of Arsenic Biomethylation
1.4.1. Enzymes catalyzing reduction of arsenic compounds 52
1.4.1.1. Arsenate reductase 53
1.4.1.2. Methylarsonate reductase 54
1.4.2. Enzymes catalyzing methyl group transfers to
arsenic compounds 55
VI
Table of Contents
1.4.2.1. Arsenite methyltransferase 55
1.4.2.2. Methylarsinate methyltransferase 56
1.4.2.3. S-adenosyl-methionine:arsenic(III)
methyltransferase 57

1.5. Strategies for Arsenic Bioremediation of Arsenic Pollution
1.5.1. Strategies for the removal of toxic metals by plants 59
Phytoextraction 59
Rhizofiltration 59
Phytostabilization 60
1.5.2. Removal of arsenic from soils 60
1.5.2.1. Remediation of arsenic-contaminated soils 60
1.5.2.2. Phytoremediation of arsenic 62

1.6. Project aims and experimental approach 63

Chapter II. MATERIALS AND METHODS 64

2.1. Materials
2.1.1. Plants and growth conditions 65
2.1.2. Yeast and growth conditions 65
2.1.3. E. coli cells and growth conditions 66
2.1.4. Reagents 67

2.2. Methods
2.2.1. Preparation of cell extracts 68
2.2.1.1. Yeast and E. coli 68
2.2.1.2. Plant tissues 68
2.2.2. Arsenic methyltransferase assay 69
VII
Table of Contents
2.2.3. Extraction and determination of methylated arsenic
compounds 69
2.2.4. Separation of methylated metabolites 70
2.2.5. Preparation and determination of authentic MMA
and DMA 70
2.2.6. Expression and purification of the yeast proteins
YHR209w and YER175c 71
2.2.7. Digestion of the yeast GST fusion proteins 71

Chapter III. METHYLATION OF ARSENIC IN VITRO BY
CELL EXTRACTS FROM BENTGRASS
(AGROSTIS TENUIS) 72

3.1. Introduction 73
3.2. Results
3.2.1. Validation of extraction process 75
3.2.2. Effect of arsenate in the culture media on Agrostis tenuis 76
3.2.2.1. Effect of arsenate concentration in culture Media
on methylation by leaf extracts 76
3.2.2.2. Arsenate tolerance in Agrostis tenuis 77
3.2.2.3. Time-course analysis of Arsenite Methylation, and
separation of products 80
3.2.2.4. Methylation of arsenate 82
3.2.2.5. Methylation by roots 85
3.3. Discussion 86

Chapter IV. INDUCTION OF ARSENIC METHYLATION IN
THE YEAST SACCHAROMYCES CEREVISIAE
AND THE BACTERIUM E. COLI 90

4.1. Introduction 91
4.2. Results
4.2.1. Effect of arsenate and arsenite concentration in culture
media on the growth of S.cerevisiae strains 93

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