RESISTANCE OF Escherichia coli TOWARDS TOXIC
METALLOIDAL OXYANIONS AND gshA, gshB GENES IN THEIR GLUTATHIONE
BIOSYNTHETIC PATHWAY, AND AN EFFORT TO PRODUCE AN
Escherichia coli STRAIN WITH A HIGHER GLUTATHIONE CONTENT
A Thesis
Presented to
The Faculty of the Department of Chemistry
Sam Houston State University
In Partial Fulfillment
of the Requirements for the Degree of
Master of Science
by
Gayan Adikari Appuhamillage
August, 2013
RESISTANCE OF Escherichia coli TOWARDS TOXIC
METALLOIDAL OXYANIONS AND gshA, gshB GENES IN THEIR GLUTATHIONE
BIOSYNTHETIC PATHWAY, AND AN EFFORT TO PRODUCE AN
Escherichia coli STRAIN WITH A HIGHER GLUTATHIONE CONTENT
by
Gayan Adikari Appuhamillage
APPROVED:
Dr. Thomas G. Chasteen
Thesis Director
Dr. Donovan C. Haines
Dr. Richard E. Norman
Approved:
Dr. John B. Pascarella, Dean
College of Sciences
ABSTRACT
Adikari Appuhamillage, Gayan, Resistance of Escherichia coli towards toxic metalloidal
oxyanions and gshA, gshB genes in their glutathione biosynthetic pathway, and an effort
to produce an Escherichia coli strain with a higher glutathione content. Master of Science
(Chemistry), August, 2013, Sam Houston State University, Huntsville, Texas.
Purpose
The purpose of this research was: (1) to determine the reducing power of two E.
coli strains, AG1/pCA24NgshA and AG1/pCA24NgshB, for the toxic oxyanions SeO32-,
SeO42-, and TeO32- in comparison to their wild type E. coli strain AG1 in terms of their
relative ability to produce intracellular glutathione (GSH); and (2) to produce an E. coli
strain having a better reducing power over the above strains, in terms of the production of
intracellular GSH for the bioremediation of toxic metalloidal oxyanions.
Methods
In part (1), using bacterial precultures prepared in the absence and the presence of
isopropyl-β-D-1-thiogalactopyranoside (IPTG), all of the following experimental
methods were performed. Minimum inhibitory concentration measurements were carried
out using 96-microwell plates by the OD600 method and the resazurin dye method. The
specific growth rates were obtained by measuring the slope of the exponential phase of
bacterial growth curves. These experiments were carried out to investigate the relative
toxicity of each of the oxyanions towards each of the E. coli strains used. Intracellular
GSH levels were measured using a colorimetric analysis with 5,5’-dithiobis(2nitrobenzoic acid). Intracellular protein contents were measured with the standard
Bradford assay method in order to estimate intracellular GSH levels present per mass of
protein.
iii
In part (2), polymerase chain reactions, restriction enzyme digestions, gene
ligations, DNA purification, and gel electrophoresis techniques were used in an effort to
achieve the goal.
Findings
In part (1), the relative toxicity of the oxyanions for the three E. coli strains
increased in the order Na2SeO4 < Na2SeO3 < Na2TeO3. Specific growth rates confirmed
that the presence of any of these toxicants was unfavorable for the normal growth of
these E. coli strains.
The presence of higher GSH levels in AG1/pCA24NgshA and AG1/pCA24NgshB
relative to AG1, caused the higher resistance of these two strains towards Na2SeO3 and
Na2SeO4 and proved the involvement of gshA and gshB genes in the glutathione
biosynthetic pathway. Higher IPTG levels caused a decrease of intracellular GSH.
In part (2), the E. coli strain BW 25113 could be used successfully as the source
of the gshA gene for its amplification.
Keywords: Reducing power, Intracellular glutathione, IPTG, Minimum inhibitory, Gene
ligations, Gel electrophoresis, Plasmid pSB1A2.
iv
ACKNOWLEDGEMENTS
First of all I would like to thank Dr. Thomas G. Chasteen, my research supervisor.
Without his guidance and courage, this work would not be possible. Being my research
supervisor, he guided me towards new pathways and applications in Chemistry. Learning
instrumentation from him was the most enjoyable and unforgettable thing that I ever
achieved. He greatly supported me with correct technical writing tools for my thesis
writing as well. Truly he has been an amazing mentor and a very kind hearted person. I
would also like to acknowledge Dr. Donovan C. Haines for his great help for my research
work. The gene manipulation section could have never been achieved without his
excellent guidance. My acknowledgements go also to Dr. Norman, the Chair for his great
support in my research work and thesis writing.
I would also like to thank Rebecca A. Montes, Desiré Lopez, Iris Porter, and
Tharaka Wansapura. Their help and assistance in the laboratory was really unforgettable
and courageous for my work. In addition, I would like to thank the research group of Dr.
Claudio C. Vásquez of the Universidad de Santiago de Chile for sending me the
necessary bacterial cultures and also Mr. Waldo Diaz for his excellent support and
knowledge given to me in the field of microbiology and biochemistry in order to achieve
success in this work.
Lastly, I would like to thank my wife and my parents. Without their support, love
and encouragement I would not be where I am today. This thesis is specifically dedicated
to my loving wife Mrs. Sankalya Ambagaspitiya for her endless love, support and
encouragement, tolerating all the hardships with studies at a University far away from
mine.
v
TABLE OF CONTENTS
Page
ABSTRACT ....................................................................................................................... iii
ACKNOWLEDGEMENTS .................................................................................................v
TABLE OF CONTENTS ................................................................................................... vi
LIST OF TABLES ...............................................................................................................x
LIST OF FIGURES ........................................................................................................... xi
CHAPTER
I
INTRODUCTION ...................................................................................................1
Part (1). The Environmental Importance of Metalloids ...........................................1
Selenium ..................................................................................................................2
Tellurium..................................................................................................................3
Remediation Methods for Metalloids ......................................................................4
The Use of Microorganisms in Bioremediation of Se, Te Metalloids .....................5
Glutathione (GSH) as a Reducing Agent for Bioremediation of Se
and Te.......................................................................................................................7
Some Bioengineered E. coli Strains for Bioremediation of Metalloids...................8
MIC and Bacterial Growth Curve Determinations ..................................................9
Part (2). An Effort to Produce an E. coli Strain With a Higher GSH
Content ...................................................................................................................10
vi
II
MATERIALS AND METHODS ...........................................................................11
Part (1). Determination of the Degree of Resistance of AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains Towards Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants ................................................................................................................11
Determination of the Effect of isopropyl-β-D-1-thiogalactopyranoside (IPTG) on
the Resistance of AG1, AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli
Strains for Na2SeO3, Na2SeO4, and Na2TeO3 Toxicants .......................................16
Determination of the Effect of IPTG on the Specific Growth Rate of AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains.................................16
Determination
of
the
Intracellular
Glutathione
Levels
in
AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains.................................17
Part (2). An Effort to Produce an E. coli Strain With a Higher GSH Content ......20
III
RESULTS ..............................................................................................................27
Part (1). Determination of the Degree of Resistance of AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains Towards Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants ................................................................................................................27
Determination
of
the
Effect
of
IPTG
on
the
Resistance
of
AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains for Na2SeO3,
Na2SeO4, and Na2TeO3 Toxicants .........................................................................40
Determination of the Effect of IPTG on the Specific Growth Rate of AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains.................................42
Determination
of
the
Intracellular
Glutathione
Levels
in
AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains.................................44
vii
Part (2). An Effort to Produce an Escherichia coli Strain With a Higher GSH
Content ...................................................................................................................48
IV DISCUSSION ........................................................................................................51
Part (1). Determination of the Degree of Resistance of AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains Towards Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants ................................................................................................................51
Determination
of
the
Effect
of
IPTG
on
the
Resistance
of
AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains for Na2SeO3,
Na2SeO4, and Na2TeO3 Toxicants .........................................................................58
Determination of the Effect of IPTG on the Specific Growth Rate of AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains.................................58
Determination
of
the
Intracellular
Glutathione
Levels
in
AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains.................................58
Part (2). An Effort to Produce an Escherichia coli Strain With a Higher GSH
Content ...................................................................................................................64
V
CONCLUSIONS....................................................................................................69
Part (1). Determination of the Degree of Resistance of AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains Towards Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants ................................................................................................................69
Determination
of
the
Effect
of
IPTG
on
the
Resistance
of
AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains for Na2SeO3,
Na2SeO4, and Na2TeO3 Toxicants .........................................................................70
viii
Determination of the Effect of IPTG on the Specific Growth Rate of AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains.................................70
Determination
of
the
Intracellular
Glutathione
Levels
in
AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains.................................70
Part (2). An Effort to Produce an Escherichia coli Strain With a Higher GSH
Content ...................................................................................................................72
BIBLIOGRAPHY ..............................................................................................................73
APPENDIX ........................................................................................................................83
VITA ..................................................................................................................................84
ix
LIST OF TABLES
Table
Page
1
Results of MIC for AG1 Strain Using OD600 Measurement Method.. ..................27
2
Results of MIC for AG1/pCA24NgshA Strain Using OD600 Measurement
Method. ..................................................................................................................29
3
Results of MIC for AG1/pCA24NgshB Strain Using OD600 Measurement
Method. ..................................................................................................................31
4
Specific Growth Rate of AG1 in the Absence and the Presence of Toxicants. .....39
5
Specific Growth Rate of AG1/pCA24NgshA in the Absence and the Presence of
Toxicants. ...............................................................................................................39
6
Specific Growth Rate of AG1/pCA24NgshB in the Absence and the Presence of
Toxicants. ...............................................................................................................39
7
MIC of Each Toxicant for AG1 with Increasing Concentrations of IPTG. ...........40
8
MIC of Each Toxicant for AG1/pCA24NgshA with Increasing Concentrations of
IPTG. ......................................................................................................................41
9
MIC of Each Toxicant for AG1/pCA24NgshB with Increasing Concentrations of
IPTG. ......................................................................................................................41
10 Specific Growth Rates of Each Bacterial Strain with Increasing Concentrations of
IPTG. ......................................................................................................................44
11 Intracellular Glutathione Amounts of the Bacterial Strains with Increasing
Concentrations of IPTG. ........................................................................................47
12 Summary of MIC Results. .....................................................................................51
x
LIST OF FIGURES
Figure
Page
1 Overview of Biosynthesis of γ-Glutamylcysteine and Glutathione. ........................7
2
MIC of Na2SeO3 for AG1 Strain Using the Resazurin Dye Method. ....................28
3
MIC of Na2SeO4 for AG1 Strain Using the Resazurin Dye Method. ....................28
4
MIC of Na2TeO3 for AG1 Strain Using the Resazurin Dye Method. ....................28
5
MIC of Na2SeO3 for AG1/pCA24NgshA Strain Using the Resazurin Dye
Method. ..................................................................................................................29
6
MIC of Na2SeO4 for AG1/pCA24NgshA Strain Using the Resazurin Dye
Method. ..................................................................................................................30
7
MIC of Na2TeO3 for AG1/pCA24NgshA Strain Using the Resazurin
Dye Method. ..........................................................................................................30
8
MIC of Na2SeO3 for AG1/pCA24NgshB Strain Using the Resazurin
Dye Method. ..........................................................................................................31
9
MIC of Na2SeO4 for AG1/pCA24NgshB Strain Using the Resazurin
Dye Method. ..........................................................................................................31
10 MIC of Na2TeO3 for AG1/pCA24NgshB Strain Using the Resazurin
Method. ..................................................................................................................32
11 Growth Curve of AG1 in the Absence of Toxicants..............................................32
12 Growth Curve of AG1 in the Presence of Na2SeO3 (15.625 mM). .......................32
13 Growth Curve of AG1 in the Presence of Na2SeO4 (125 mM). ............................33
14 Growth Curve of AG1 in the Presence of Na2TeO3 (0.002 mM). .........................33
xi
15 Comparison of Growth Curves of AG1 in the Absence and the Presence of
Toxicants. ...............................................................................................................33
16 Growth Curve of AG1/pCA24NgshA in the Absence of Toxicants. .....................34
17 Growth Curve of AG1/pCA24NgshA in the Presence of Na2SeO3
(62.5 mM). .............................................................................................................34
18 Growth Curve of AG1/pCA24NgshA in the Presence of Na2SeO4 (500 mM). .....34
19 Growth Curve of AG1/pCA24NgshA in the Presence of Na2TeO3
(0.002 mM). ...........................................................................................................34
20 Comparison of Growth Curves of AG1/pCA24NgshA in the Absence and the
Presence of Toxicants. ...........................................................................................35
21 Growth Curve of AG1/pCA24NgshB in the Absence of Toxicants. .....................36
22 Growth Curve of AG1/pCA24NgshB in the Presence of Na2SeO3
(62.5 mM). .............................................................................................................36
23 Growth Curve of AG1/pCA24NgshB in the Presence of Na2SeO4
(500 mM). ..............................................................................................................37
24 Growth Curve of AG1/pCA24NgshB in the Presence of Na2TeO3
(0.002 mM). ...........................................................................................................37
25 Comparison of Growth Curves of AG1/pCA24NgshB in the Absence and the
Presence of Toxicants. ...........................................................................................38
26 Comparison of Growth Curves of AG1 with Increasing Concentrations of
IPTG. ......................................................................................................................42
27 Comparison of Growth Curves of AG1/pCA24NgshA with Increasing
Concentrations of IPTG. ........................................................................................43
xii
28 Comparison of Growth Curves of AG1/pCA24NgshB with Increasing
Concentrations of IPTG. ........................................................................................43
29 The Calibration Curve for the Determination of Intracellular Glutathione Levels
in the Bacterial Strains. ..........................................................................................45
30 The Calibration Curve for the Determination of Intracellular Protein Content in
the Bacterial Strains. ..............................................................................................46
31 Comparing the Absorbance Values of Intracellular Glutathione Levels of the
Bacterial Strains with Increasing Concentrations of IPTG. ...................................46
32 Microwells Showing the Yellow Product Formed After Adding the
5,5’-dithiobis(2-nitrobenzoic acid) Reagent to E. coli Cells Having
Intracellular GSH. ..................................................................................................47
33 gshA Gene Showing Important Restriction Enzyme Sites. Dark Region: Region of
gshA Which Codes for its Characteristic Enzyme L-Glutamate Cysteine Ligase.
White Regions: Outer Regions of gshA Which do not Code for its Characteristic
Enzyme. .................................................................................................................48
34 The Restriction Map of Biobrick BBa_I715039 Containing the RFP Gene With
the Carrying Plasmid pSB1A2. RBS, Ribosomal Binding Site; Amp, Ampicillin
Resistant Gene. ......................................................................................................49
35 White and Red Colonies After Transformation of the gshA Gene With Plasmid
pSB1A2 into Competent E. coli Cells. ..................................................................49
36 DNA Bands of the Standard Ladder and the Resulting DNA Fragment of the
Sample (after restriction enzyme digestion) on a 1.2% (w/v) Agarose Gel. bp,
base pairs. ...............................................................................................................50
xiii
37 Role of GSH-dependent Reduction Systems in E. coli as a Defense Against
Oxidative Stress. Disulfide Bond Reduction by GSH Together With Glutaredoxin
is Shown. ................................................................................................................53
38 Metabolism of Selenium in E coli. ........................................................................54
39 Illustration of Reduction of TeO32- in an E. coli Cell and Also Mechanisms of
TeO32- Toxicity. ROS, Reactive Oxygen Species; Ovals Represent Proteins and/or
Enzymes. ................................................................................................................56
40 Microwells Showing the Red Color of Elemental Se0 Formed by the Reduction of
SeO42- and SeO32- by Intracellular GSH of E. coli.................................................57
41 PT5-lac Hybrid Promoter. .........................................................................................60
42 A Linear Representation of the pCA24N Circular Plasmid (A). Location of the
gshA gene of the AG1/pCA24NgshA Strain in the pCA24N Plasmid (B). Location
of the gshB Gene of the AG1/pCA24NgshB Strain in the pCA24N Plasmid (C).
Ori, Origin of Replication; Cat, Chloramphenicol Resistant Gene. ......................61
43 The Correctly Ligated Product (ligated gshA gene with the carrying plasmid
pSB1A2). ...............................................................................................................65
xiv
1
CHAPTER I
INTRODUCTION
Part (1). The Environmental Importance of Metalloids
A metalloid is a chemical element with properties that are in between those of
metals and nonmetals. Among the metalloids in the periodic table, arsenic (As) in group
15, and selenium (Se) and tellurium (Te), which belong to group 16, are the most
important ones in environmental analysis due to their toxic effects at higher accumulation
levels (Emsley, 2011; Greenwood and Earnshaw, 1997).
Several anthropogenic activities lead to accumulation of these metalloids in the
environment. Mining, agriculture, metallurgy, the combustion of fossil fuels, faulty waste
disposal and military operations have released enormous amounts of toxic heavy metals
and metalloids into the environment (Wernick and Themelis, 1998; Wijnhoven et al.,
2007). Although the presence of trace levels of some of these metalloids (e.g. Se) is
useful for biological functions, excess amounts cause environmental pollution and
adverse health effects in both flora and fauna (Emsley, 2011). The type of chemical
species present is an important factor which determines the toxicity of a particular metal
or a metalloid in the biota (Donard and Caruso, 1998). Cleaning up the environment by
removing these persistent and hazardous contaminants needs approaches that allow for a
precise restoration of polluted sites (Kotrba et al., 2009). This research study focuses on
the metalloids Se and Te.
2
Selenium
Se was first discovered and named by J.J. Berzelius in 1817 and is the 66th most
abundant element in the earth’s crust with an average concentration of 50 ppb
(Greenwood and Earnshaw, 1997). Six naturally occurring isotopes of Se have been
identified with different relative abundances (%). These are 80Se, 49.61%; 78Se, 23.77%;
76
Se, 9.37%;
82
Se, 8.73%;
77
Se, 7.63%;
74
Se, 0.89%. Se is most commonly found in
soluble forms in the environment as the oxyanions selenite (SeO32-) and selenate (SeO42-).
Se is found also in fossil fuels, shales, alkaline soils and as a constituent in over 40
minerals including ferroselite, challomenite, schmeiderite, crooksite, clausthalite, etc.
(Stolz and Oremland, 1999; CRC Handbook, 2012). The main source of commercial Se is
the anode slime deposited during the electrolytic refining of copper (CRC Handbook,
2012). Se is also recovered from the sludge accumulated in sulfuric acid plants and from
electrostatic precipitator dust collected during the processing of copper and lead
(Greenwood and Earnshaw, 1997).
A wide spectrum of industrial applications of Se exists. Se is used in the glass
industry and in the production of photocells, solar cells, rectifiers, photocopiers, exposure
meters for photography, and also as an additive to stainless steel (Emsley, 2011; CRC
Handbook, 2012).
Toxicity of Se
Even though Se is an essential micronutrient to mammals at low concentrations
(Allan et al., 1999; Rayman, 2000; Schwarz and Foltz, 1957; Stadtman, 1974; Stadtman,
1977), it becomes toxic at more elevated levels (Lemire et al., 2012; Kuo and Jiang,
2008; Fordyce, 2005; Adams and Johnson, 1981). Se has a very narrow range between
3
dietary deficiency (< 40 µg per day) and toxic levels (> 400 µg per day), making it
necessary to carefully control the intake of Se by humans and other animals (Fordyce,
2005). Se toxicity (selenium poisoning) can result in a condition called selenosis and this
can be acute (short-term) or chronic (long-term). Acute toxicity is typically caused by
occupational
exposure
in
the
industrial
sector
to
microelectronics,
Xerox,
semiconductors, and optoelectronics (Pedrero and Madrid, 2009) while chronic toxicity
occurs due to the presence of high levels of Se in food and water (Pedrero and Madrid,
2009).
Symptoms of selenosis (Lemire et al., 2012; Pedrero and Madrid, 2009) include
gastrointestinal upsets (nausea, vomiting, abdominal pain, diarrhea), hair loss, white
blotchy nails, garlic breath odor, fatigue, irritability, mild nerve damage, hyperreflexia,
tremor, peripheral neuropathy, and lack of mental alertness. Extreme cases of selenosis
can result in cirrhosis of the liver, pulmonary edema (an abnormal buildup of fluid in the
lungs, which leads to swelling), thrombocytopenia (low blood platelets), thyroid
problems, and death.
Tellurium
Te is another metalloid of interest here. It was first discovered by Franz-Joseph
Müller von Reichenstein in 1782 and was named by Martin Heinrich Klaproth in 1798.
Te is the 72nd most abundant element in the earth’s crust and the eight stable isotopes of
Te include
122
130
Te, 34.08%;
128
Te, 31.74%;
126
Te, 18.84%;
125
Te, 7.07%;
124
Te, 4.74%;
Te, 2.55%; 123Te, 0.89%; 120Te, 0.09%. Tellurite (TeO32-) and tellurate ([TeO2(OH)4]2-)
are common oxyanions of Te. The TeO32- anion is the most toxic form for most
organisms (Emsley, 2011). Tellurate is poorly soluble in water. Minerals which contain
4
Te include calaverite (AuTe2) and sylvanite (AgAuTe4). Te is commercially extracted as
a by-product of copper and lead production. Several industrial applications of Te include
the use of the metalloid in alloys, in steel and copper to improve machinability, in solar
panels, as a semiconductor material, in rubber vulcanization, in CdTe nanoparticles
(Monras et al., 2012) and in the production of glass fibers to increase optical refraction.
Toxicity of Te
Ingestion of Te in even small amounts by mammals causes dreadful smelling
breath and appalling body odor because of enteric bacterial production of dimethyl
telluride (Emsley, 2011). Effects of inhalation on humans include drowsiness, dry mouth,
metal taste, headache, garlic odor and nausea. Short term exposure effects include eye
and respiratory tract irritation, adverse effects on the liver and the central nervous system,
abdominal pain, constipation and vomiting.
Remediation Methods for Metalloids
Remediation is the stopping or minimizing the environmental damage done by
released toxicants. There are several conventional techniques to achieve this for toxic
metals or metalloids. These include chemical methods in wastewater treatment plants
(e.g. coagulation with ferric salts) and some physico-chemical methods to treat
toxicants/waste in soil, such as chemical extraction with acids/chelating agents,
electrolysis and size separation techniques (Kotrba et al., 2009). In general, these
conventional methods have some disadvantages which include high acquisition and
operating costs, lower efficiency at low metalloid concentrations and significant
alteration in physico chemical properties of soil/water (Kotrba et al., 2009). These
5
disadvantages can lead to implementation of more environmental friendly, cost effective,
novel techniques like bioremediation, the use of microorganisms (Zhang and
Frankenberger, 2006; Zhang et al., 2004; Soudi et al., 2009; ) or plant species (Witters et
al., 2012; Kotrba et al., 2009)
to remove or neutralize contaminants from the
environment. It has been suggested that biological processes for treating toxic effluents
are more cost effective than chemical and physical methods due to their efficiency (Paul
et al., 2005; de Souza et al., 2002).
The Use of Microorganisms in Bioremediation of Se, Te Metalloids
Bioremediation is the use of biological agents such as microorganisms or plant
species to remove or neutralize contaminants from the environment. Microorganisms like
bacteria and fungi are used for the bioremediation of Se and Te, owing to their ability to
convert toxic, water soluble forms of Se and Te to less soluble and less toxic forms. As
part of biodegradation, microbes resistant to metalloids can lessen the metalloidcontaining compound’s toxicity by converting the toxic forms to either elemental or
methylated forms which are relatively less toxic (Soudi et al., 2009; Maiers et al., 1988;
Van Fleet-Stalder and Chasteen, 1998; Basnayake et al., 2001; Swearingen et al., 2004;
Burra et al., 2010).
Many microorganisms tolerate selenium levels ranging from 5.0 µg L-1 (0.063
µM) to 2.0 × 103 µg L-1 (25 µM) by oxidation-reduction reactions and by conversion of
inorganic to organic forms especially by methylation (Chasteen and Bentley, 2003).
Almost all metalloid-resistant microbes carry out some form of metalloid reduction and
many also carry out methylation (Doran, 1982; Chasteen and Bentley, 2003; Burra et al.,
6
2009). By means of bioremediation, microbes affect the global cycling of Se (Maiers et
al., 1988; Wilber, 1980).
Research
investigations
have
involved
extensive
genetic
studies
of
microorganisms for their resistance towards tellurite, some resistance being
chromosomal, others involving plasmids (Chasteen and Bentley, 2003). Studies have
used Bacillus sp. strain STG-83 (Soudi et al., 2009), Escherichia coli (Guzzo and Dubow,
2000; Dyllick-Brenzinger et al., 2000; Kormutakova et al., 2000), Pseudomonas
aeruginosa, Agrobacterium tumefaciens, Erwinia carotovora (Trutko et al., 2000;
Mougel et al., 2001), Bacillus stearothermophilus (Vásquez et al., 2001), and
photosynthetic bacteria (Karls et al., 1998). Seven of the eight so called promiscuous
plasmids which were used to distribute tellurite-resistance determinants (TeR) to different
strains of Escherichia coli (E. coli) had the ability to convert tellurite (added to the
culture at a concentration of 50 ppm) to black Te0, and it was suggested that such strains
had “the potential of being exploited for the bioremediation of the oxyanion tellurite from
liquid waste” (Chasteen and Bentley, 2003). Hence, these research studies have shown
the capability of using bioengineered microorganisms for the bioremediation of tellurite.
7
Glutathione (GSH) as a Reducing Agent for Bioremediation of Se and Te
During some of the previous studies based on using metalloid-resistant bacteria as
a bioremediation strategy (Zhang and Frankenberger, 2006; Zhang et al., 2004; Cantafio
et al., 1996; Frankenberger and Arshad, 2001; Adams and Pickett, 1998; Monrás et al.,
2012), GSH, which is found inside several bacterial species (Fahey et al., 1978) has been
identified as a reducing agent for toxic oxyanions such as SeO32-, SeO42-, and TeO32-.
GSH can reduce the toxicity of the above chemical species by producing insoluble
elemental Se and Te which can be removed from the medium through filtration. A
metabolic pathway has been found inside some E. coli strains which can produce GSH
inside the microorganism (Suzuki et al., 1999). It has been revealed that the genes gshA
which encodes L-glutamate cysteine ligase enzyme (GshA) and gshB which encodes
glutathione synthetase enzyme (GshB) are involved in this GSH biosynthetic pathway as
in Figure 1 (Kim et al., 2008).
Figure 1. Overview of Biosynthesis of γ-Glutamylcysteine and Glutathione.
Both genes gshA and gshB are involved in the biosynthesis of GSH in E. coli.
Moreover, L-glutamate cysteine ligase enzyme (GshA), which is encoded by the gshA
gene, catalyzes the rate-limiting step in GSH biosynthesis (Murata and Kimura, 1982).
Hence, these factors prove the importance of GSH and the GSH biosynthetic pathway in
E. coli for the bioremediation of Se and Te by E. coli.
8
Some Bioengineered E. coli Strains for Bioremediation of Metalloids
In an attempt to make use of the reducing ability of GSH for the bioremediation
of metalloids, the two genes (gshA and gshB) encoding the principle enzymes involved in
GSH biosynthesis were bioengineered to overexpress in E. coli (in order to produce
higher GSH levels) and produced AG1/pCA24NgshA and AG1/pCA24NgshB strains,
respectively, from the wild type AG1 (Murata and Kimura, 1982; Gushima et al., 1984).
The wild type AG1 strain has gshA and gshB genes in its chromosome while the mutants
AG1/pCA24NgshA and AG1/pCA24NgshB contain additional copies of gshA and gshB,
respectively, in a separate plasmid pCA24N. In a very recent study, these three E. coli
strains have been used and compared for their relative ability to synthesize fluorescent
CdTe nanoparticles in vivo (Monrás et al., 2012).
Our primary goal was to determine whether the bioengineered bacterial strains
(AG1/pCA24NgshA and AG1/pCA24NgshB) actually have a better reducing power for
the oxyanions SeO32-, SeO42-, and TeO32- in comparison to the AG1 wild type and, if so,
whether that better reducing power confers more bacterial resistance to these toxic
anions.
The goal was achieved through metalloid minimum inhibitory concentration
(MIC) measurements (Islam et al., 2008; Primm and Franzblau, 2007; Di Gregorio et al.,
2006; Harrison et al., 2005), bacterial growth curve determinations (Burra et al., 2009),
and measurements of intracellular GSH (Allen et al., 2000; Abegg et al., 2012; Biswas et
al., 2007).
9
MIC and Bacterial Growth Curve Determinations
The minimum inhibitory concentration (MIC) is the lowest toxicant concentration
at which the growth of bacteria is halted or inhibited after a predetermined time. MIC has
been widely used as a measure of relative toxicity of certain toxicants/antibiotics for
certain bacterial species (Islam et al., 2008; Primm and Franzblau, 2007; Di Gregorio et
al., 2006; Harrison et al., 2005).
A typical bacterial growth curve (a plot of optical density measured at a given
wavelength vs. time) consists of four basic phases: lag phase, exponential phase (log
phase), stationary phase, and death phase. In the lag phase, the individual bacterial cells
are maturing and not yet dividing. During the exponential phase, the number of new
bacteria appearing per unit time is proportional to the present population. Exponential
growth can’t continue indefinitely, however, because the medium is soon depleted of
nutrients and enriched with wastes. The number of new cells created is limited by the
growth factor and as a result the rate of cell growth matches the rate of cell death. This is
the stationary phase and is represented as a horizontal linear part of the curve. During the
death phase, bacteria run out of nutrients and die.
The exponential phase is important since the slope of the exponential phase of a
typical bacterial growth curve provides the specific growth rate which is characteristic of
a particular bacterium in a given growth medium at a given temperature. Through a series
of growth curve experiments, Burra et al. (2009) found that TeO32- was more toxic to the
metalloid-resistant bacterium LHVE than [TeO2(OH)4]2- and both the Te-containing
compounds were much more toxic than SeO32-, SeO42- and selenocyanate (SeCN-1).
10
The research reported here was designed to investigate the relative resistance of
the three E. coli strains (AG1, AG1/pCA24NgshA and AG1/pCA24NgshB) towards the
SeO32-, SeO42-, and TeO32- oxyanions of Se and Te in order to identify the bioremediation
potential of these strains on these toxic metalloidal oxyanions. Also, the bioengineered
strains, AG1/pCA24NgshA and AG1/pCA24NgshB, opened the gate to investigate the
roles played by the genes gshA and gshB in the GSH biosynthetic pathway and also the
importance of controlling the conditions (e.g. levels of inducers like isopropyl-β-D-1thiogalactopyranoside, IPTG) for the production of GSH.
Part (2). An Effort to Produce an E. coli Strain With a Higher GSH Content
In order to improve the bacterial potential of reducing the toxic oxyanions SeO32-,
SeO42-, and TeO32- by intracellular GSH, an effort was taken to increase the copy number
of the gshA gene in E. coli. Due to the fact that the gshA gene codes for the enzyme
which catalyzes the rate-limiting step in GSH biosynthesis (Murata and Kimura, 1982),
the goal was to obtain an E. coli strain with a better ability to reduce GSH for the
bioremediation of the above toxicants especially for TeO32-.
This was initiated with an E. coli strain (BW 25113) which contained the
chromosomal gshA gene. Using this chromosomal gshA gene as the template, followed
by a series of polymerase chain reactions (PCR), the gshA gene was amplified, digested
with restriction enzymes, and ligated into a suitable plasmid pSB1A2 which contained a
biobrick (BBa_I715039) including the RFP gene coding for a red fluorescent protein
(RFP). After transformation of this ligated product into competent E. coli cells, DNA
purification, restriction enzyme digestion, and gel electrophoresis were performed to
ensure a successful ligation of the gshA gene.
11
CHAPTER II
MATERIALS AND METHODS
Part (1). Determination of the Degree of Resistance of AG1, AG1/pCA24NgshA, and
AG1/pCA24NgshB E. coli Strains Towards Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants
Reagents
Reagents used in these experiments include: BactoTM tryptone (Becton Dickson,
Sparks, MD, USA), yeast extract (EMD Chemicals, Gibbstown, NJ, USA), tris HCl
(BDH Chemicals, West Chester, PA, USA), sodium selenite, 5,5’-dithiobis(2nitrobenzoic acid), K2HPO4, KH2PO4, and L-glutathione reduced (Sigma-Aldrich, St.
Louis, MO, USA), sodium selenate (Alfa Aesar, Ward Hill, MA, USA), sodium tellurite
(Sigma-Aldrich, St. Louis, MO, USA), agar, IPTG, Bradford reagent, bovine serum
albumin (Amresco, Solon, OH, USA), chloramphenicol (Avocado Research Chemicals
Ltd, Heysham, Lancs, UK), and resazurin sodium salt (Alfa Aesar, Ward Hill, MA,
USA).
Other equipment and instruments
Other equipment used in these experiments include: RiOs 3 water purification
system from Millipore (Billerica, MA, USA) for water deionization, culture shaker
(VWR International, LLC, Radnor, PA, USA), and digital sonicator (Misonix,
Farmingdale, NY, USA).
12
Instruments used include: Varioskan Flash- Thermo Scientific SkanIt multimode
plate reader (Thermo Fisher Scientific, Vantaa, Finland) and autoclave -Tuttnauer-2540 E
(Heidolph Brinkmann, LLC, Elk Grove Village, IL 60007, USA).
Bacterial strains
Bacterial strains used were: AG1, AG1/pCA24NgshA, and AG1/pCA24NgshB E.
coli strains (provided by Claudio Vásquez, Universidad de Santiago de Chile).
(A) Determination of MIC
The minimum inhibitory concentration (MIC) value of each of the toxicants
Na2SeO3,
Na2SeO4,
and
Na2TeO3
for
the
three
bacterial
species
AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB was determined using the 96 micro-well
plate technique (Islam et al., 2008; Primm and Franzblau, 2007; Di Gregorio et al., 2006;
Harrison et al., 2005). Moreover, MIC values were determined separately by two basic
techniques, by the determination of optical density (OD) at 600 nm using the Varioskan
Flash multimode plate reader and using resazurin dye.
Preparing bacterial pre-cultures
Ten µL of prepared chloramphenicol solution (5 mg/ mL) were added into 990 µL
of freshly prepared Luria-Bertani (LB) medium in three separate sterile Eppendorf tubes
(1.5 mL) to make sure only strains expressing the antibiotic-resistant plasmid grew. A
single colony of AG1, AG1/pCA24NgshA, and AG1/pCA24NgshB from an agar plate
was transferred into each tube separately under sterile conditions. These Eppendorf tubes
containing the bacterial solutions were then closed and incubated at 37 °C in a shaker for
18 h.
13
Preparing 1:100 bacterial inoculums
After 18 h of incubation, the OD600 was measured for each pre-culture. When the
measured OD600 was between 0.4 and 0.5, the pre-cultures was diluted to 0.005 OD600
with fresh LB up to a total adjusted volume of 10 mL in three separate sterile tubes (15
mL). Each were closed and incubated further at 37 °C in a shaker until the culture OD600
was between 0.1 and 0.2.
Loading of 96-microwell plate with each toxicant
Freshly prepared LB medium (150 µL) was added to each well in a row of 12
wells of a sterile 96-microwell plate. The highest toxicant (Na2SeO3) concentration was
prepared in the first well by adding 150 µL of a Na2SeO3 stock solution (2000 mM)
prepared using fresh LB as the solvent. Two-fold dilutions were performed across the
plate by pipetting out 150 µL of the toxicant solution from the first well after mixing 10
times and adding to the next well and continuing this up to the 12th well in the row where
the final 150 µL solution was discarded. Triplicate solutions were prepared for each of
the toxicant concentration and this whole procedure was repeated with Na2SeO4 and
Na2TeO3 toxicants as well. Separate microwell plates were prepared in this manner for
the three different bacterial strains.
Mixing bacterial strains with the toxicants
After the OD600 of the incubated bacterial strains was measured to be between 0.1
and 0.2, 10 µL from one of the bacterial solutions was added to all microwells. The same
procedure was followed with the other two bacterial strains. Controls were prepared for
each bacterial strain by adding 10 µL of the bacterial solution into a microwell containing
150 µL of fresh LB. Microwells containing 160 µL of only fresh LB were the blanks.
14
Determining MIC with the OD600 measurement method
These plates were closed with lids and incubated at 37 °C in a shaker for 24 h.
Finally after 24 h, OD600 in each microwell was measured using the Varioskan Flash
multimode plate reader. The OD600 values in the wells in which no bacterial growth was
present were similar to the OD600 values of the blank. The OD600 values in the wells
where bacterial growth was present were similar to the OD600 values of the control. The
MIC for each toxicant for each strain was identified as the toxicant concentration in the
microwell immediately before a microwell with an OD600 equal to that strain’s control
well.
Determining MIC using the resazurin dye method
The above prepared plates were closed with lids and incubated at 37 °C in a
shaker for 24 h. Ten µL of resazurin sodium salt (6.75 mg/ mL) was added to each
microwell (Sarker et al., 2007). After another 24 h of incubation at 37 °C in a shaker, the
plates were checked for color change. Live bacterial cells were identified by a pink color,
and the retention of a blue color (the unreduced resazurin color) meant that the bacterial
growth was severely inhibited due to the presence of the added toxicant. The MIC for
each toxicant for each strain was identified as the toxicant concentration in the blue
colored microwell immediately before the first microwell that showed pink color after 24
h.
15
(B) Determination of the Specific Growth Rate
After determining the MIC value of each toxicant (Na2SeO3, Na2SeO4, and
Na2TeO3) for AG1, AG1/pCA24NgshA, and AG1/pCA24NgshB, the effect of each
toxicant on the specific growth rate of each of these bacterial species was determined.
Half of the MIC as determined above was used as the toxicant concentration for growth
curve experiments for each strain.
Preparation of the bacterial pre-cultures and preparation of the 1:100 inocula were
carried out as before. Each of these bacterial cultures was incubated further at 37 °C in a
shaker until the OD600 was ~0.1. One hundred and fifty µL from one of these bacterial
cultures was added to each of three separate microwells of a sterile 96- microwell plate.
A volume of 50 µL from each the three toxicants (having a concentration of half of the
MIC) was added separately to those wells. The procedure was performed in triplicate for
each toxicant in order to test the reproducibility of the results. In the same manner, the
procedure was carried out for the other two bacterial strains.
Controls were prepared by adding 50 µL of fresh LB into microwells containing
150 µL of each particular bacterial culture. Blanks were the microwells containing only
200 µL of fresh LB. These microwell plates were covered and the OD600 was measured at
37 °C in each well, just after the introduction of the samples and every 15 min for 15 h
while shaking. The specific growth rate was identified as the slope of the exponential
phase in each bacterial growth curve for a plot of ln OD v. time (Burra et al., 2009).
16
Determination of the Effect of isopropyl-β-D-1-thiogalactopyranoside (IPTG) on the
Resistance of AG1, AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains for
Na2SeO3, Na2SeO4, and Na2TeO3 Toxicants
Determination of MIC
Bacterial pre-cultures were prepared by adding IPTG after the addition of 10 µL
of the chloramphenicol solution (5 mg/ mL) into 990 µL of freshly prepared LB medium
in three separate sterile Eppendorf tubes (1.5 mL) to have final IPTG concentrations of
0.05, 0.1, 0.2, 0.4, 0.8 and 1.0 mM separately for each bacterial strain. The rest of the
same procedure was repeated and the MIC values were determined separately by the two
techniques (described above).
Determination of the Effect of IPTG on the Specific Growth Rate of AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains
Bacterial pre-cultures were prepared in the same way as above with IPTG. The
same procedure was carried out as before to determine the specific growth rate but
without the addition of any toxicant.
17
Determination of the Intracellular Glutathione Levels in AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains
Determination of the intracellular glutathione levels in the absence of IPTG
Pre-cultures of each of the bacterial species (AG1, AG1/pCA24NgshA, and
AG1/pCA24NgshB) were prepared as before. After 24 h of incubation at 37 °C in a
shaker, each culture was centrifuged at 10,000 rpm for 15 min and the pellets were
collected. These pellets were dissolved in tris HCl (0.1 M) buffer of pH 8.0 separately.
Two mL of each of the resulting suspensions were transferred into separate tubes (15 mL)
followed by pulsed sonication for two 1 min cycles at an amplitude of 1 on an ice bath in
order to break bacterial cell walls. Afterwards, each of the resulting suspensions was
again centrifuged at 10,000 rpm for 15 min. The supernatants were collected and the
pellets which contain cell debris were discarded. Fifty μL from each of the supernatants
were saved for the protein determination test which is described later.
Into a 725 μL volume from each of the above unsaved supernatants, 50 μL of
5,5’-dithiobis(2-nitrobenzoic acid) solution (6 mg/mL of 0.1 M phosphate buffer, pH 8.0)
was added and mixed well (Allen et al., 2000; Abegg et al., 2012; Rahman et al., 2007).
Each of the resulting yellow-colored solutions was then divided into three similar
volumes (200 μL) to make triplicate solutions for each bacterium (AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB). These were then added into wells of a
sterile 96-microwell plate. Finally, this plate was incubated at 37 °C for 2 min and
absorbance was measured at 412 nm (Rahman et al., 2007) with the Varioskan Flash
multimode plate reader. A412 values were recorded at 10 min time intervals while
incubating at 37 °C until the absorbance reached a constant value.
18
Determination of the intracellular glutathione levels in the presence of IPTG
Bacterial pre-cultures were prepared by adding IPTG after the addition of 10 µL
of the chloramphenicol solution (5 mg/ mL) into 990 µL of freshly prepared LB medium
in three separate sterile Eppendorf tubes (1.5 mL) to have final IPTG concentrations of
0.05, 0.1, 0.2, 0.4, 0.8 and 1.0 mM separately for each bacterial strain. Subsequent
procedures were the same as above.
Standard glutathione solutions were prepared by using 0.1 M tris HCl buffer of
pH 8.0 as the solvent, to have concentrations of 5.0, 11, 22, 44, and 88 µM. The total
volume of each standard solution was 725 µL. Fifty µL of the prepared 5,5’-dithiobis(2nitrobenzoic acid) solution (6 mg/mL of 0.1 M phosphate buffer, pH 8.0) were added to
each above standard glutathione solution (Biswas et al., 2007). Each of the resulting
yellow solutions was then divided into 200 µL volumes and added to wells of a sterile
96-microwell plate. This plate was then incubated at 37 °C for 2 min and the absorbance
was measured at 412 nm at 10 min time intervals while incubating at 37 °C until the
values were constant. This calibration was used to determine the unknown concentrations
of glutathione in the samples.
Determination of the protein content of the bacterial strains
To each of the 50 µL of the saved supernatants (after the centrifugation at 10,000
rpm for 15 min) during the glutathione determination step, 1.0 mL of Bradford reagent
was added in sterile Eppendorf tubes (1.5 mL) separately. Each of the resulting blue
solutions was then divided into 200 µL volumes and added to wells of a sterile 96microwell plate. After 5 min, the absorbance of these solutions was recorded at 595 nm
(Noble and Bailey, 2009).
19
A calibration curve was prepared as follows. Standard solutions of bovine serum
albumin (BSA) protein were prepared with a prepared stock solution (1 mg/ mL) and
Bradford reagent (Noble and Bailey, 2009) to have BSA concentrations of 2, 4, 10, 15
and 20 µg/ mL. Each of the resulting blue solutions was then divided into three 200 µL
volumes, added to a sterile 96-microwell plate and after 5 min, the absorbance in each
well was recorded at 595 nm. The calibration was used to determine the unknown
concentrations of protein in the samples.
20
Part (2). An Effort to Produce an E. coli Strain With a Higher GSH Content
Reagents
Reagents used in this experiment include: ampicillin (50 mg/ mL), phenol/
chloroform/iso-amyl alcohol (25:24:1) solution, chloroform/iso-amyl alcohol (24:1)
solution (Amresco, Solon, OH, USA), gshA-1f primer, gshA-1r primer, T4 DNA ligase
(Sigma-Aldrich, St. Louis, MO, USA), Pfu turbo 10x PCR buffer, pfu polymerase
enzyme (National Institute of Health, Bethesda, Maryland, USA), NE Buffer 4, T4 10x
DNA ligase reaction buffer, purified BSA (100x, 10 mg/ mL), restriction enzymes PstI
(20,000 U/ mL), XbaI (20,000 U/ mL), and EcoRI (20,000 U/ mL), dNTPs (New England
BioLabs, Ipswich, MA, USA), agarose (Mallinckrodt Baker Inc., Phillipsburg, NJ, USA),
6x DNA loading buffer, DNA reference ladders (Promega, Madison, WI, USA), and
ethidium bromide solution of 10 mg/ mL (EMD Millipore, Billerica, MA, USA). Super
Optimal Broth- Catabolite repression (SOC) medium, NaOH (0.2 M) containing 1%
sodium dodecyl sulfate (SDS), ethanol (95%), aqueous ethanol (70%), sodium acetate
solution (pH 5.2), and buffer solutions (1x TE, 1x GTE, 1x TAE, 1x potassium acetate of
pH 5.0) were prepared according to the standard protocols (Sambrook and Russel, 2000).
Other equipment and instruments
Other equipment used include: PCR machine (Eppendorf, Hauppauge, NY, USA),
gel chambers (FisherBiotech, Pittsburgh, PA, USA), UV transilluminator (Upland, CA,
USA), and minicentrifuge (Fisher Scientific, Pittsburgh, PA, USA).
21
Bacterial strains
Bacterial strains used were: BW 25113 (provided by Claudio Vásquez,
Universidad de Santiago de Chile) strain and competent E. coli cells.
(A) Multiplication of the gshA gene using PCR
Preparation of PCR template
Twenty µL of ultra-pure water (type 01) were added into a sterile PCR tube. A
single colony of BW 25113 strain from an agar plate was introduced into this tube under
sterile conditions. This was capped and heated at 95 °C for 20 min in a thermocycler in
order to make the DNA inside the bacterial cells accessible for the PCR experiments.
Preparation of primer solutions
Primers were designed to amplify the gshA gene in a biobrick format and were
ordered from Sigma-Aldrich, St. Louis, MO, USA. The nucleotide sequence of the gshA1f primer was 5’-GTTTCTTCGAATTCGCGGCCGCTTCTAGTTGATCCCGGACGTA
TCAC-3’ and that of gshA-1r was 5’-GTTTCTTCCTGCAGCGGCCGCTACTAGTATT
ATCAGGCGTGTTTTTCCAG-3’. One hundred µM solutions of the forward primer
gshA-1f (30.8 nmol) and the reverse primer gshA-1r (25.6 nmol) were prepared with
ultra-pure water (type 01) using 309 µL and 257 µL volumes respectively. Solutions (15
µM) were prepared from each of these primers by adding 85 µL of ultra-pure water (type
01) to 15 µL of each of the prepared primer solutions (100 µM).
22
Carrying out PCR reaction
To a sterile PCR tube, 36 µL of ultra-pure water (type 01), 5 µL of Pfu turbo 10x
PCR buffer, 1 µL of dNTP solution, 5 µL of the prepared PCR template, 1 µL from the
prepared 15 µM gshA-1f primer solution, 1 µL from the prepared 15 µM gshA-1r primer
solution, and 1 µL of Pfu polymerase enzyme were added in the given order.
Immediately after the addition of the Pfu polymerase enzyme, the PCR tube was capped
and all the solutions were collected at the bottom of the tube through spinning for 10 s
using a minicentrifuge. The resulting PCR tube was placed in the PCR machine and the
PCR was allowed to occur for 2 h inside the tube (95 °C for 2.5 min, 65 °C for 30 s, and
72 °C for 1 min per 1 PCR cycle).
(B) Restriction enzyme digestions
Restriction enzyme digestions were performed on the resulting PCR product and
on a biobrick (BBa_I715039) which contained a RFP gene in a plasmid pSB1A2 before
the ligation of the gshA gene as follows. Into a sterile PCR tube, 40 µL of ultra-pure
water (type 01), 0.5 µL of 100x BSA solution (10 mg/ mL), 5 µL of NE Buffer 4, 1 µL of
PstI restriction enzyme (20,000 U/ mL), 1 µL of XbaI restriction enzyme (20,000 U/
mL), and 2.5 µL of the PCR product were added in the given order. To another PCR tube,
the same solutions were added in the same order except the last component was replaced
with 2.5 µL of the RFP containing plasmid DNA. These two tubes were capped, and all
the solutions were collected at the bottom of the tube through spinning for 10 s using a
minicentrifuge. The resulting tubes were subjected to thermocycler incubation under the
program ‘brickcut2’ for an incubation period at 37 °C for 30 min and then at 80 °C for 20
min.
23
(C) Ligation of the gshA gene into the plasmid pSB1A2
To a sterile PCR tube, 11 µL of ultra-pure water (type 01), 2 µL of the digested
PCR product, 2 µL of the digested plasmid solution, 2 µL of T4 10x DNA ligase reaction
buffer, and 1 µL of T4 DNA ligase enzyme were added in the given order. The tube was
capped and all the solutions were collected at the bottom of the tube through spinning for
10 s using a minicentrifuge. The resulting solution was incubated at room temperature
overnight.
(D) Transformation of the ligated product into competent E. coli cells
Chemically competent E. coli cells that had the ability to take up extracellular
DNA from its environment were obtained from a -80 °C freezer and were kept in ice for
thawing. Two µL of the above ligated product were transferred into a microcentrifuge
tube (1.5 mL) containing 50 µL of the competent E. coli cells. The tube was gently mixed
and was incubated for 30 min on ice. Then the cells were subjected to heat shock by
removing the tube from ice and immediately submerging it in a 42 °C water bath for 30 s
while agitating slightly. The tube was returned to ice and was incubated for 2 min. Again
it was removed from ice and 250 µL of sterile SOC broth medium was added. The
mixture was incubated for 1 h at 37 °C on a shaker. One hundred µL of the resulting cell
suspension was pipetted out to the surface of a prepared agar plate (ampicillin added as
the antibiotic). The cell suspension was plated using a bacterial spreader to an even
distribution and the plate was incubated for 24 h at 37 °C. The resulting bacterial plate
was observed for normal white-colored (ligated with the gshA gene in place of the RFP
gene) and red-colored (ligated with the RFP gene) colonies.
24
(E) Purification of the transformed DNA for gel electrophoresis to test the success of
ligation
a. Cell lysis
Twenty five mL of an overnight culture of some of the above white colonies were
added into a sterile conical tube (50 mL). The tube was centrifuged at 3500 rpm for 10
min to pellet the cells. The supernatant was decanted and 2 mL of 1x TE buffer was
added to the cell pellet. The pellet was resuspended in the buffer by vortexing vigorously.
A 20 mL volume of the buffer was then added and vortexing was also carried out to wash
the cells of the culture media. The suspension was centrifuged again at 3500 rpm for 10
min to pellet the cells. The wash solution was then discarded with the supernatant and 2
mL of 1x GTE buffer was added to the conical tube. The cells were then resuspended
again by vigorous vortexing and was placed on ice. Four mL of freshly prepared NaOH
(0.2 M) containing 1% SDS was added to the tube. The suspension was mixed well.
b. Renaturation
Three mL of ice cold 1x potassium acetate buffer (pH 5.0) was added to the above
resulting suspension and it was mixed well followed by incubating on ice for 5 min. Then
the mixture was centrifuged for 20 min at 8000 rpm.
c. DNA extraction
Two mL from the upper clear aqueous layer containing the DNA from the above
final step was transferred into a new conical tube (15 mL). Two mL of phenol:
chloroform: iso-amyl alcohol (25:24:1) solution was added into the tube which contained
DNA. The tube was inverted gently to mix the aqueous and organic phases until a cloudy
emulsion formed. The tube was centrifuged at 10,000 rpm for 10 min to separate the
25
phases. The aqueous phase containing the DNA was transferred into a new conical tube
(15 mL). The extraction and centrifugation were done two more times following the
above same procedure. The final aqueous layer was also transferred into a new conical
tube (15 mL). Two mL of chloroform: iso-amyl alcohol (24:1) solution was added to the
conical tube to remove any traces of phenol from the aqueous layer containing the DNA
and the solution was mixed well. The tube was again centrifuged at 10,000 rpm for 10
min to separate the phases. The aqueous phase containing the DNA was transferred into a
new conical tube (15 mL).
d. DNA precipitation
One hundred and fifty µL of sodium acetate (3 M, pH 5.2) was added to the
conical tube containing the DNA and was mixed well. A volume of 3.3 mL of ice cold
ethanol (95%) was added to the tube and was mixed well once again. The resulting
suspension was again centrifuged at 10,000 rpm for 10 min to pellet the plasmid DNA
from the solution. The supernatant was removed and 1 mL of aqueous ethanol (70%) was
added to the pellet to remove any traces of sodium acetate. Ethanol was removed through
pipetting and evaporation from the pellet for 2 min. One hundred and fifty µL of ultrapure water (type 01) was added to the tube and the solution was mixed well to redissolve
the DNA. This purified DNA sample was stored at 4 °C for subsequent experiments.
26
e. DNA concentration and purity measurements
The DNA concentration (as ng/µL of solution) and purity measurements (as an
absorbance ratio of DNA (at 260 nm) to the protein content (at 280 nm)) were taken
using microdrop-plate technique and the Varioskan Flash multimode plate reader. One
µL of the DNA sample was used every time to put onto the microdrop-plate.
f. Restriction enzyme digestion using EcoRI
Two and half µL of purified DNA sample was transferred to a PCR tube followed
by addition of 41 µL of ultra-pure water (type 01). Five µL of NE Buffer 4 was added
followed by 0.5 µL of 100x BSA solution (10 mg/ mL) and 1 µL of EcoRI enzyme
(20,000 U/ mL). The tube was capped and all the solutions were collected at the bottom
of the tube through spinning for 10 s using a minicentrifuge. The tube was incubated at
37 °C for 1 h in the PCR machine.
g. Gel electrophoresis
Twenty µL of the above digested product was transferred into a microcentrifuge
tube (1.5 mL). Four µL of 6x DNA loading buffer was then added and the solution was
mixed well. Five µL of a DNA reference standard and 20 µL of the above prepared
digested DNA solution were added into each well of a freshly prepared 1.2% (w/v)
agarose gel (0.72 g of agarose in 60 mL of 1x TAE buffer). The voltage was set to 90 V
and the bands were allowed to run for 25 min. The gel was removed from the chamber,
placed in a 200 mL volume of an ethidium bromide solution (0.5 µg/ mL) and was
stained on a shaker for 45 min. The resulting gel was removed from the staining solution
and was placed on a UV transilluminator for band observation under 365 nm UV light.
27
CHAPTER III
RESULTS
Part (1). Determination of the Degree of Resistance of AG1, AG1/pCA24NgshA, and
AG1/pCA24NgshB E. coli Strains Towards Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants
Determination of MIC
Cultures of AG1, the wild type E.coli strain, were examined to determine
minimum inhibitory concentrations. Toxicants consisting of both water soluble seleniumand one tellurium- containing oxyanions (SeO32-, SeO42-, and TeO32-) were used. MIC
values generated via OD600 measurement method are summarized in Table 1 and
generated via the resazurin dye method in images in Figures 2, 3, and 4. As others have
found for wild type microorganisms in general, for this strain tellurite is much more toxic
than either selenate or selenite (Nies, 1999; Turner et al., 2001), and these two different
techniques compare well.
Table 1. Results of MIC for AG1 Strain Using OD600 Measurement Method.
Toxicant
MIC/ mM
Na2SeO3
31.25
Na2SeO4
250
Na2TeO3
4.00 × 10-3
28
Determining MIC using the resazurin dye method:
Na2SeO3 concentration/ mM
31.25
15.63
7.81
3.91
1.95
0.98
0.49
Figure 2. MIC of Na2SeO3 for AG1 Strain Using the Resazurin Dye Method.
Na2SeO4 concentration/ mM
250
125
62.50
31.25
15.63
7.81
3.91
Figure 3. MIC of Na2SeO4 for AG1 Strain Using the Resazurin Dye Method.
Na2TeO3 concentration/ mM (×10-4)
40
20
10
5
2.50
1.25
0.62
Figure 4. MIC of Na2TeO3 for AG1 Strain Using the Resazurin Dye Method.
29
Cultures of the AG1/pCA24NgshA strain were also examined to determine the
minimum inhibitory concentrations. The same toxicants SeO32-, SeO42-, and TeO32- were
used. MIC values generated via OD600 measurement method are summarized in Table 2
and generated via the resazurin dye method in images in Figures 5, 6, and 7. For this
strain also, tellurite is much more toxic than either selenate or selenite and these two
different techniques compare well.
Table 2. Results of MIC for AG1/pCA24NgshA Strain Using OD600 Measurement
Method.
Toxicant
MIC/ mM
Na2SeO3
125
Na2SeO4
1000
Na2TeO3
4.00 × 10-3
Determining MIC using the resazurin dye method:
Na2SeO3 concentration/ mM
125
62.50
31.25
15.63
7.81
3.91
1.95
Figure 5. MIC of Na2SeO3 for AG1/pCA24NgshA Strain Using the Resazurin Dye
Method.
30
Na2SeO4 concentration/ mM
1000
500
250
125
62.50
31.25
15.63
Figure 6. MIC of Na2SeO4 for AG1/pCA24NgshA Strain Using the Resazurin Dye
Method.
Na2TeO3 concentration/ mM (×10-4)
40
20
10
5
2.50
1.25
0.62
Figure 7. MIC of Na2TeO3 for AG1/pCA24NgshA Strain Using the Resazurin Dye
Method.
Cultures of the AG1/pCA24NgshB strain were also examined to determine the
minimum inhibitory concentrations with the same toxicants SeO32-, SeO42-, and TeO32-.
MIC values generated via OD600 measurement method are summarized in Table 3 and
generated via the resazurin dye method in images in Figures 8, 9, and 10. For this strain
also, tellurite is much more toxic than either selenate or selenite and once again the two
different techniques compare well.
31
Table 3. Results of MIC for AG1/pCA24NgshB Strain Using OD600 Measurement
Method.
Toxicant
MIC/ mM
Na2SeO3
125
Na2SeO4
1000
Na2TeO3
4.00 × 10-3
Determining MIC using the resazurin dye method:
Na2SeO3 concentration/ mM
125
62.50
31.25
15.63
7.81
3.91
1.95
Figure 8. MIC of Na2SeO3 for AG1/pCA24NgshB Strain Using the Resazurin Dye
Method.
Na2SeO4 concentration/ mM
1000
500
250
125
62.50
31.25
15.63
Figure 9. MIC of Na2SeO4 for AG1/pCA24NgshB Strain Using the Resazurin Dye
Method.
32
Na2TeO3 concentration/ mM (×10-4)
40
20
10
5
2.50
1.25
0.62
Figure 10. MIC of Na2TeO3 for AG1/pCA24NgshB Strain Using the Resazurin Method.
Determination of the Specific Growth Rate
Growth curve analysis
Specific growth rate analysis was carried out for AG1, the wild type E. coli strain
in order to uncover the effect of the toxicants on the specific growth rate of this strain. In
order to obtain a growth curve, one half of the MIC that was determined in the previous
section for each toxicant for this strain, was used as the toxicant concentration in each
occasion here. Controls were prepared without the toxicant in each occasion. The results
are shown in Figures 11, 12, 13, 14, and 15. The slope of the exponential phase of the
growth curve slightly decreases in the presence of the toxicants in comparison with the
0.000
-0.500
-1.000
-1.500
-2.000
-2.500
-3.000
ln OD600
ln OD600
control. This is observed in Figure 15.
control (AG1)
0
500
Time (minutes)
1000
0.000
-0.500
-1.000
-1.500
-2.000
-2.500
-3.000
with selenite
(15.625 mM)
0
500
Time (minutes)
1000
Figure 11. Growth Curve of AG1 in the
Figure 12. Growth Curve of AG1 in the
Absence of Toxicants.
Presence of Na2SeO3 (15.625 mM).
-2.500
0.000
-0.500
-1.000
-1.500
-2.000
-2.500
-3.000
-2.550
with selenate
(125 mM)
0
500
Time (minutes)
ln OD600
ln OD600
33
with tellurite
(0.002 mM)
-2.600
-2.650
-2.700
0
1000
100
200
Time (minutes)
300
Figure 13. Growth Curve of AG1 in the
Figure 14. Growth Curve of AG1 in the
Presence of Na2SeO4 (125 mM).
Presence of Na2TeO3 (0.002 mM).
0.000
-0.500
ln OD600
-1.000
Control (AG1)
-1.500
with Selenite (15.625 mM)
-2.000
with selenate (125 mM)
-2.500
with tellurite (0.002 mM)
-3.000
0
200
400
600
800
1000
Time (minutes)
Figure 15. Comparison of Growth Curves of AG1 in the Absence and the Presence of
Toxicants.
34
Cultures of AG1/pCA24NgshA were used for the specific growth rate analysis for
the same reason as before, following the same procedure. The results are shown in
Figures 16, 17, 18, 19, and 20. The slope of the exponential phase of the growth curve
slightly decreases in the presence of the toxicants in comparison with the control. This is
0.000
-0.500
-1.000
-1.500
-2.000
-2.500
-3.000
control (AG1/pCA24NgshA)
0
500
Time (minutes)
ln OD600
ln OD600
illustrated in Figure 20.
0.000
-0.500
-1.000
-1.500
-2.000
-2.500
-3.000
with selenite (62.5 mM)
0
1000
500
Time (minutes)
1000
Figure 16. Growth Curve of
Figure 17. Growth Curve of
AG1/pCA24NgshA in the Absence of
AG1/pCA24NgshA in the Presence of
Toxicants.
Na2SeO3 (62.5 mM).
-2.300
-0.500
with selenate
(500 mM)
ln OD600
-1.000
-1.500
-2.400
with tellurite
(0.002 mM)
ln OD600
0.000
-2.500
-2.000
-2.500
-2.600
0
500
1000
Time (minutes)
0
100
200
300
Time (minutes)
Figure 18. Growth Curve of
Figure 19. Growth Curve of
AG1/pCA24NgshA in the Presence of
AG1/pCA24NgshA
Na2SeO4 (500 mM).
Na2TeO3 (0.002 mM).
in the Presence of
35
0.000
-0.500
control (AG1/pCA24NgshA)
ln OD600
-1.000
with selenite (62.5 mM)
-1.500
with selenate (500 mM)
-2.000
with tellurite (0.002 mM)
-2.500
-3.000
0
200
400
600
800
1000
Time (minutes)
Figure 20. Comparison of Growth Curves of AG1/pCA24NgshA in the Absence and
the Presence of Toxicants.
Cultures of AG1/pCA24NgshB were also used for the specific growth rate
analysis for the same reason as before, following the same procedure. The results are
shown in Figures 21, 22, 23, 24, and 25. Once again the slope of the exponential phase of
the growth curve slightly decreases in the presence of the toxicants in comparison with
the control. This is illustrated in Figure 25.
ln OD600
36
0.000
-0.500
-1.000
-1.500
-2.000
-2.500
-3.000
control (AG1/pCA24NgshB)
0
500
1000
Time (minutes)
Figure 21. Growth Curve of AG1/pCA24NgshB
ln OD600
in the Absence of Toxicants.
0.000
-0.500
-1.000
-1.500
-2.000
-2.500
-3.000
with selenite
(62.5 mM)
0
500
1000
Time (minutes)
Figure 22. Growth Curve of AG1/pCA24NgshB
in the Presence of Na2SeO3 (62.5 mM).
37
0.000
ln OD600
-0.500
with selenate
(500 mM)
-1.000
-1.500
-2.000
-2.500
-3.000
0
500
1000
Time (minutes)
Figure 23. Growth Curve of AG1/pCA24NgshB
ln OD600
in the Presence of Na2SeO4 (500 mM).
-2.200
-2.250
-2.300
-2.350
-2.400
-2.450
-2.500
-2.550
-2.600
with tellurite
(0.002 mM)
0
100
200
300
Time (minutes)
Figure 24. Growth Curve of AG1/pCA24NgshB
in the Presence of Na2TeO3 (0.002 mM).
38
0.000
-0.500
-1.000
ln OD600
control (AG1/pCA24NgshB)
-1.500
with selenite (62.5 mM)
-2.000
with selenate (500 mM)
-2.500
with tellurite (0.002 mM)
-3.000
0
200
400
600
800
1000
Time (minutes)
Figure 25. Comparison of Growth Curves of AG1/pCA24NgshB in the Absence and the
Presence of Toxicants.
Specific growth rate analysis
The specific growth rate values were obtained as the slope of the exponential
phase of each bacterial growth curve and those values are summarized below for the AG1
strain in Table 4, for the AG1/pCA24NgshA strain in Table 5, and for the
AG1/pCA24NgshB in Table 6. In each strain, the specific growth rates slightly decrease
in the presence of the toxicants when compared with the control. The lowest specific
growth rate was observed in the presence of Na2TeO3 in each strain.
39
Table 4. Specific Growth Rates of AG1 in the Absence and the Presence of Toxicants.
Condition
Specific growth rate/ min-1
control (no toxicant)
0.0093 ± 0.0002
with Na2SeO3 (15.625 mM)
0.0077 ± 0.0002
with Na2SeO4 (125 mM)
0.0080 ± 0.0001
with Na2TeO3 (0.002 mM)
0.0006 ± 0.0001
Table 5. Specific Growth Rate of AG1/pCA24NgshA in the Absence and the Presence of
Toxicants.
Condition
Specific growth rate/ min-1
control (no toxicant)
0.0092 ± 0.0006
with Na2SeO3 (62.5 mM)
0.0044 ± 0.0002
with Na2SeO4 (500 mM)
0.0028 ± 0.0002
with Na2TeO3 (0.002 mM)
0.0011 ± 0.0001
Table 6. Specific Growth Rate of AG1/pCA24NgshB in the Absence and the Presence of
Toxicants.
Condition
Specific growth rate/ min-1
control (no toxicant)
0.0095 ± 0.0004
with Na2SeO3 (62.5 mM)
0.0047 ± 0.0001
with Na2SeO4 (500 mM)
0.0037 ± 0.0003
with Na2TeO3 (0.002 mM)
0.0015 ± 0.0001
40
Determination of the Effect of IPTG on the Resistance of AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains for Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants
The effect of IPTG on the MIC of each of the toxicants for each E. coli strain was
examined. The same IPTG concentration series were used in each occasion. The controls
were prepared with samples where no IPTG was added and the MIC values were
obtained by both the OD600 method and the resazurin dye method. The results are shown
as a summary for the AG1 strain in Table 7, the AG1/pCA24NgshA strain in Table 8, and
the AG1/pCA24NgshB strain in Table 9. As the overall result, a decrease in the MIC
values was observed upon increasing the IPTG concentrations for the three strains with
each toxicant.
MIC results from OD600 measurement method and the resazurin dye method:
Table 7. MIC of Each Toxicant for AG1 with Increasing Concentrations of IPTG.
IPTG
concentration/ mM
0
(control)
0.05
0.1
0.2
0.4
0.8
1.0
MIC of Na2SeO3/
mM
MIC of Na2SeO4/
mM
MIC of Na2TeO3/
mM
31.25
31.25
31.25
31.25
31.25
15.63
15.63
250
250
250
250
250
125
125
4.00 × 10-3
4.00 × 10-3
4.00 × 10-3
4.00 × 10-3
4.00 × 10-3
2.00 × 10-3
2.00 × 10-3
41
Table 8. MIC of Each Toxicant for AG1/pCA24NgshA with Increasing Concentrations of
IPTG.
IPTG
concentration/ mM
0
(control)
0.05
0.1
0.2
0.4
0.8
1.0
MIC of Na2SeO3/
mM
MIC of Na2SeO4/
mM
MIC of Na2TeO3/
mM
125
125
62.50
62.50
62.50
62.50
31.25
1000
1000
1000
1000
1000
500
500
4.00 × 10-3
4.00 × 10-3
1.60 × 10-2
1.60 × 10-2
8.00 × 10-3
4.00 × 10-3
4.00 × 10-3
Table 9. MIC of Each Toxicant for AG1/pCA24NgshB with Increasing Concentrations of
IPTG.
IPTG
concentration/ mM
0
(control)
0.05
0.1
0.2
0.4
0.8
1.0
MIC of Na2SeO3/
mM
MIC of Na2SeO4/
mM
MIC of Na2TeO3/
mM
125
125
62.50
62.50
62.50
31.25
31.25
1000
1000
1000
1000
1000
500
500
4.00 × 10-3
4.00 × 10-3
1.60 × 10-2
8.00 × 10-3
8.00 × 10-3
4.00 × 10-3
4.00 × 10-3
42
Determination of the Effect of IPTG on the Specific Growth Rate of AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains
Growth curves
This section of the procedure was carried out in order to test any possible effect(s)
on the specific growth rate of the E. coli strains upon addition of IPTG in the absence of
any toxicant. Controls were prepared without adding any IPTG. The resulting growth
curves are shown in Figure 26 for the AG1 strain, Figure 27 for the
AG1/pCA24NgshAstrain, and Figure 28 for the AG1/pCA24NgshB strain. A slight
decrease of the slope of the exponential phase (with respect to the controls) of each
growth curve was observed upon increasing IPTG concentrations for each E. coli strain.
The specific growth rate in the presence of each IPTG concentration was obtained as
before for each strain and the results are summarized in Table 10. The overall result was
a slight decrease of the specific growth rates upon increasing IPTG concentrations for
each strain.
0.000
-0.500
control (AG1, no IPTG)
ln OD600
-1.000
with IPTG (0.05 mM)
-1.500
with IPTG (0.1 mM)
-2.000
with IPTG (0.2 mM)
with IPTG (0.4 mM)
-2.500
with IPTG (0.8 mM)
-3.000
with IPTG (1.0 mM)
-3.500
0
200
400
600
800
1000
Time (minutes)
Figure 26. Comparison of Growth Curves of AG1 with Increasing Concentrations of
IPTG.
43
0.000
ln OD600
-0.500
-1.000
control (AG1/pCA24NgshA,
no IPTG)
with IPTG (0.05 mM)
-1.500
with IPTG (0.1 mM)
-2.000
with IPTG (0.2 mM)
-2.500
with IPTG (0.4 mM)
with IPTG (0.8 mM)
-3.000
with IPTG (1.0 mM)
-3.500
0
200
400
600
800
1000
Time (minutes)
Figure 27. Comparison of Growth Curves of AG1/pCA24NgshA with Increasing
Concentrations of IPTG.
0.000
control (AG1/pCA24NgshB,
no IPTG)
with IPTG (0.05 mM)
-0.500
ln OD600
-1.000
with IPTG (0.1 mM)
-1.500
with IPTG (0.2 mM)
-2.000
with IPTG (0.4 mM)
-2.500
with IPTG (0.8 mM)
-3.000
with IPTG (1.0 mM)
-3.500
0
200
400
600
800
1000
Time (minutes)
Figure 28. Comparison of Growth Curves of AG1/pCA24NgshB with Increasing
Concentrations of IPTG.
44
Table 10. Specific Growth Rates of Each Bacterial Strain with Increasing Concentrations
of IPTG.
IPTG
concentration/ mM
Specific growth
rate/ min-1
of AG1
Specific growth
rate/ min-1
of
Specific growth
rate/ min-1
of
AG1/pCA24NgshA
AG1/pCA24NgshB
0
(control)
0.05
0.0093 ± 0.0002
0.0092 ± 0.0006
0.0095 ± 0.0004
0.0083 ± 0.0002
0.0092 ± 0.0006
0.0094 ± 0.0006
0.1
0.0085 ± 0.0002
0.0091 ± 0.0005
0.0093 ± 0.0003
0.2
0.0082 ± 0.0002
0.0092 ± 0.0004
0.0093 ± 0.0005
0.4
0.0084 ± 0.0001
0.0090 ± 0.0008
0.0093 ± 0.0003
0.8
0.0073 ± 0.0003
0.0076 ± 0.0003
0.0086 ± 0.0004
1.0
0.0072 ± 0.0002
0.0074 ± 0.0003
0.0080 ± 0.0005
Determination of the Intracellular Glutathione Levels in AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains
The three strains were examined for their intracellular GSH levels both in the
absence and the presence of IPTG. Intracellular GSH levels were measured using a
colorimetric analysis with 5,5’-dithiobis(2-nitrobenzoic acid). Intracellular protein
contents were also measured with the standard Bradford assay method in order to
estimate intracellular GSH levels present per mass of protein. Calibration curves for the
determination of intracellular GSH and protein contents are shown in Figure 29 and 30,
respectively, while Figure 31 illustrates the comparison of the absorbance values of
intracellular GSH levels of the three bacterial strains both in the absence and the presence
45
of IPTG. Figure 32 shows the yellow product formed after adding the 5,5’-dithiobis(2nitrobenzoic acid) reagent to E. coli cells having intracellular GSH. According to the
results shown in Figure 31, at zero IPTG concentrations, absorbance values at 412 nm are
much
higher
for
the
two
bioengineered
species
AG1/pCA24NgshA
and
AG1/pCA24NgshB than that of AG1 the wild type. And also, as an overall pattern,
absorbance at 412 nm increases with increasing IPTG concentrations up to a limit, but
then decreases. Table 11 summarizes intracellular GSH contents in each E. coli strain vs.
IPTG concentrations. With respect to the controls, intracellular GSH levels slightly
Absorbance at 412 nm
decrease with increasing IPTG for the three E. coli strains.
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
y = 0.0099x - 0.0064
R² = 0.9996
0
20
40
60
80
100
Glutathione concentration (µM)
Figure 29. The Calibration Curve for the Determination of Intracellular Glutathione Levels
in the Bacterial Strains.
46
Absorbance at 595 nm
0.450
y = 20.876x + 0.0015
R² = 0.9989
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0
0.005
0.01
0.015
0.02
0.025
BSA concentration (mg/ mL)
Figure 30. The Calibration Curve for the Determination of Intracellular Protein Content
in the Bacterial Strains.
1.000
Absorbance at 412 nm
0.950
0.900
0.850
AG1
0.800
AG1/pCA24NgshA
0.750
AG1/pCA24NgshB
0.700
0.650
0.600
0
0.2
0.4
0.6
0.8
1
1.2
IPTG concentration (mM)
Figure 31. Comparing the Absorbance Values of Intracellular Glutathione Levels of the
Bacterial Strains with Increasing Concentrations of IPTG.
47
Figure 32 . Microwells Showing the Yellow Product Formed After Adding the
5,5’-dithiobis(2-nitrobenzoic acid) Reagent to E. coli Cells Having Intracellular GSH.
Table 11. Intracellular Glutathione Amounts of the Bacterial Strains with Increasing
Concentrations of IPTG.
IPTG
concentration/
mM
glutathione content
(µmol/ mg protein)
of AG1
glutathione content
(µmol/ mg protein)
of AG1/pCA24NgshA
glutathione content
(µmol/ mg protein)
of AG1/pCA24NgshB
0
(control)
0.05
10.9 ± 0.3
15.2 ± 0.2
14.8 ± 0.2
10.6 ± 0.2
15.2 ± 0.3
14.3 ± 0.5
0.1
10.5 ± 0.2
14.9 ± 0.2
14.4 ± 0.6
0.2
10.1 ± 0.3
14.4 ± 0.3
13.8 ± 0.3
0.4
9.2 ± 0.1
12.9 ± 0.6
12.4 ± 0.4
0.8
7.9 ± 0.3
11.0 ± 0.3
10.7 ± 0.2
1.0
7.0 ± 0.2
10.1 ± 0.4
9.8 ± 0.1
48
Part (2). An Effort to Produce an Escherichia coli Strain With a Higher GSH
Content
During this section of the research, the gshA gene was targeted to be incorporated
into E. coli to have multiple copies of the gene. Here, the gshA gene was amplified using
the chromosomal gshA gene of the E. coli species BW 25113 as the template, using a
series of PCR reactions. The PCR primers used were gshA-1f and gshA-1r. The DNA
sequence of gshA-1f is 5’-gtttcttcgaattcgcggccgcttctagttgatcccggacgtatcac-3’ and of
gshA-1r it is 5’-gtttcttcctgcagcggccgctactagtattatcaggcgtgtttttccag-3’. A restriction map of
the amplified gshA gene (Murata and Kimura, 1982) after PCR, is shown in Figure 33.
Figure 33. gshA Gene Showing Important Restriction Enzyme Sites. Dark Region:
Region of gshA Which Codes for its Characteristic Enzyme L-Glutamate Cysteine
Ligase. White Regions: Outer Regions of gshA Which do not Code for its Characteristic
Enzyme.
After the digestion with restriction enzymes XbaI and PstI, these PCR products
(gshA genes) were subjected to ligation in a suitable plasmid pSB1A2 (2079 bp in size).
Here, gshA, the gene of interest, was targeted to ligate in the plasmid pSB1A2 which
contained a biobrick (BBa_I715039) including a gene coding for a red fluorescent protein
(RFP). The restriction map of this biobrick (932 bp in size) carrying plasmid is shown in
Figure 34.
49
Figure 34. The Restriction Map of Biobrick BBa_I715039 Containing the RFP Gene
With the Carrying Plasmid pSB1A2. RBS, Ribosomal Binding Site; Amp, Ampicillin
Resistant Gene.
After the transformation of the ligated product into competent E. coli cells, the
resulting colonies were observed as in Figure 35. A majority of normal white colonies
was observed with a few red colonies.
few red
colonies
more white
colonies
Figure 35. White and Red Colonies After Transformation of the gshA Gene With Plasmid
pSB1A2 into Competent E. coli Cells.
50
Afterwards,
DNA
purification,
restriction
enzyme
digestion,
and
gel
electrophoresis were performed to further determine the success of the ligation. Figure 36
illustrates the resulting DNA fragment on a 1.2% (w/v) agarose gel. A prominent single
DNA fragment was observed in the sample with a size of ~ 4000 bp in comparison with
the standard DNA bands.
bp
bands of the
standard
DNA
ladder
the resulting DNA
10,000
3000
fragment of the
2000
1500
sample
1000
2
750
2500
250/253
Figure 36. DNA Bands of the Standard Ladder and the Resulting DNA Fragment of the
Sample (after restriction enzyme digestion) on a 1.2% (w/v) Agarose Gel. bp, base pairs.
51
CHAPTER IV
DISCUSSION
Part (1). Determination of the Degree of Resistance of AG1, AG1/pCA24NgshA, and
AG1/pCA24NgshB E. coli Strains Towards Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants
MIC results
Both measurement methods, the OD600 method and the resazurin dye method,
have produced the same MIC value for a given toxicant in a particular E. coli strain used.
This is clearly shown in the comparison of the MIC results in Table 1 with Figure 2, 3,
and 4 for the AG1 strain,
Table 2 with Figure 5, 6, and 7 for the AG1/pCA24NgshA
strain, and Table 3 with Figure 8, 9, 10 for the AG1/pCA24NgshB strain. The overall
results are tabulated in Table 12.
Table 12. Summary of MIC Results.
Overall results of the determined MIC (mM) values
Toxicant
For AG1
For AG1/pCA24NgshA
For AG1/pCA24NgshB
Na2SeO3
31.25
125
125
Na2SeO4
250
1000
1000
Na2TeO3
4.00× 10-3
4.00× 10-3
4.00× 10-3
A higher MIC value for a given toxicant indicates the need of a larger toxicant
concentration to inhibit bacterial growth. In other words, a higher MIC value for a given
52
toxicant represents a higher resistance of the particular bacterium for that toxicant.
Hence, the above results show that both bioengineered E. coli strains have a relatively
higher resistance to Na2SeO3 and Na2SeO4 than that of AG1 the wild type. Moreover
there is low resistance to Na2TeO3 for all the strains. It is also clear that the resistance to
Na2SeO4 is higher than that of Na2SeO3 for the three strains. Therefore, based on the
results the toxicity of the tested metalloidal oxyanions increases as Na2SeO4 < Na2SeO3 <
Na2TeO3 for the three E. coli strains used.
Specific growth rate results
The results suggest that the presence of these toxic metalloidal oxyanions
represses the growth of the E. coli strains and the situation is worst with Na2TeO3.
The results can be interpreted in detail as follows. The E. coli strains used in the
research differ from each other mainly in the potential to produce intracellular GSH.
GSH, being the most abundant low molecular weight thiol compound in aerobic bacterial
cells (Suzuki et al., 1999), is responsible for maintaining the reduced environment of E.
coli cytosol against oxidative stress as a protective mechanism (Carmel-Harel and Storz,
2000). GSH is a target in selenium and tellurite toxicity in E. coli (Turner et al., 2001).
GSH reduces the higher oxidation numbers of Se in the toxic oxyanions Na2SeO3 and
Na2SeO4 into non-toxic elemental Se0 (Turner et al., 1998) and that of Te in the toxic
oxyanion Na2TeO3 into non-toxic elemental Te0 (Turner et al., 2001). In terms of the
reaction kinetics, Na2TeO3 rapidly oxidizes GSH (Turner et al., 2001) while the reduction
of Na2SeO3 by GSH is relatively slower followed by Na2SeO4 (Turner et al., 1998).
Glutathione disulfide (GSSG), which is formed upon oxidation of GSH by these
toxicants, is reduced by glutathione reductase at the expense of NADPH (Turner et al.,
53
1998). GSH-dependent reduction systems (Meister and Anderson, 1983; Spector et al.,
1988; Carmel-Harel and Storz, 2000) in E. coli therefore usually defend against oxidative
stress (Figure 37).
Figure 37. Role of GSH-dependent Reduction Systems in E. coli as a Defense Against
Oxidative Stress. Disulfide Bond Reduction by GSH Together With Glutaredoxin is
Shown.
It is useful to examine the fate of each of the toxic oxyanion Na2SeO3, Na2SeO4,
and Na2TeO3 in the presence of GSH inside E. coli cells. Selenate and selenite can enter
an E. coli cell through the sulfate permease system (Heider and Böck, 1993; Kredich,
1996; LaRossa, 1996; Turner et al., 1998). Once inside the cell, selenite reacts with GSH
following the Painter reaction (Painter, 1941) to form GS-Se-SG (selenodiglutathione),
which is further reduced to GS-SeH (glutathioselenol). Even further reduction of this
compound leads to HSe- (hydrogen selenide) and to Se0 (Turner et al., 1998). In contrast,
selenate, once inside the E. coli cell, reacts relatively slowly with GSH and forms selenite
which subsequently undergoes the above set of reactions (Turner et al., 1998). The most
important thing to mention here is, that as a consequence of the reaction of selenite with
GSH, H2O2 (hydrogen peroxide) and O2- (superoxide) are formed (Kramer and Ames,
1988) inside the cell which create oxidative stress to the E. coli cell. Some of the adverse
effects of this oxidative stress include DNA damage and mutations, lipid peroxidation,
54
disassembly of iron-sulfur clusters, disulfide bond formation, and protein oxidation
(Carmel-Harel and Storz, 2000; Chasteen et al., 2009). This explains, at least in part, how
the toxicity of selenite in E. coli occurs (Carmel-Harel and Storz, 2000). All the above
mentioned reactions are shown below in Figure 38 (Turner et al., 1998).
Figure 38. Metabolism of Selenium in E coli.
The fate of tellurite in the presence of GSH in E. coli is of special importance.
TeO32- is highly toxic for most bacteria at concentrations as low as 1 µg mL-1 (Taylor,
1999). This factor is even more significant when compared with other metals and
metalloids such as selenium, chromium, iron, mercury, cadmium, and copper, which
become toxic at concentrations 100-fold or even 1000-fold higher than that of TeO32(Nies, 1999). TeO32- has been found to be more toxic to living organisms than elemental
tellurium (Te0) and tellurate ([TeO2(OH)4]2-), with its toxicity being related to its activity
as a strong oxidant (Soudi et al., 2009). It is also known that Te oxyanions interact with
cellular thiols (RSH), and it has been found that GSH is one of the most important targets
55
of TeO32- in E. coli (Turner et al., 2001). Based on the similarity between Se and Te
chemistry, it was postulated that GSH can reduce TeO32- to Te0 (Turner et al., 2001) and
this occurs kinetically more rapidly than the reduction of selenite or selenate by GSH.
Moreover, SeO32- is reduced more rapidly than SeO42- even before uptake in some cases
(Milne, 1998). Slow and limited reduction of SeO42- has been observed for E. coli (Fels
and Cheldelin, 1949).
It is important to detail how TeO32- becomes toxic to E. coli cells. To exert its
toxicity, TeO32- must enter the target cell, most probably through the phosphate entry
route (Elías et al., 2012). Part of the incoming TeO32- is reduced by nitrate reductase, and
in addition, reduced thiols such as GSH, and enzymes like catalase, dihydrolipoamide
dehydrogenase, and other unspecific reductases can reduce TeO32- (Chasteen et al.,
2009). However, TeO32- reduction to Te0 generates O2- and other reactive oxygen species
(ROS) like H2O2. Increasing ROS levels trigger oxidative stress, and consequently,
cellular damage occurs. Moreover, the produced O2- damages [Fe-S] centers in proteins
and enzymes (aconitase and fumarase), initiates membrane lipid peroxidation and protein
oxidation. The Fe released during the damage of [Fe-S] centers can generate hydroxyl
radical (OH) through Fenton or Haber-Weiss reactions and the generated OH causes
macromolecular damage, especially to DNA (Chasteen et al., 2009). A list of these
reactions is shown in Figure 39.
56
Figure 39. Illustration of Reduction of TeO32- in an E. coli Cell and Also Mechanisms of
TeO32- Toxicity. ROS, Reactive Oxygen Species; Ovals Represent Proteins and/or
Enzymes.
GSH is not the only thiol oxidized by TeO32-. Other low molecular weight and
high molecular weight thiols are affected but at a slower rate than that of GSH (Turner et
al., 2001). GSH is the initial thiol compound that reacts with TeO32- within the cytoplasm
of the E. coli cell and once this key thiol metabolite is exhausted, TeO32- begins to react
with other RSH compounds (Turner et al., 2001). Usually, the levels of enzymes and
metabolites of thiol redox biochemistry are elevated in bacteria following oxidative stress
(Christman et al., 1985) which results in a decrease of GSH levels upon reaction with the
toxicants such as Na2SeO3, Na2SeO4, and Na2TeO3 (as discussed above). These
components would be overwhelmed by TeO32--mediated thiol oxidation resulting in the
inability of the cell to cope with any oxidative stress. These activities, in part, may
account for the higher level of TeO32- toxicity in E. coli. Therefore, the observed trend of
the relative toxicity of the toxic oxyanions Na2SeO3, Na2SeO4, and Na2TeO3 (in the order
57
of Na2SeO4 < Na2SeO3 < Na2TeO3) for the E. coli strains used can be rationalized in this
way.
The stationary phase was not observed in the bacterial growth curves which were
obtained in the presence of the toxicants Na2SeO4 and Na2SeO3. Instead, after the
exponential phase, the ln OD600 values tend to increase slightly with time (Figure 15, 20,
and 25). This is due to the formation of red Se0 (Figure 40) as the reduction product of
Na2SeO4 and Na2SeO3; however, selenite produced more Se0 under the same conditions.
On the other hand, the growth curves obtained in the presence of Na2TeO3 achieve the
death phase relatively early and all the preceding phases (lag, exponential (log), and
stationary) are compressed (Figure 15, 20, and 25). This is basically due to the highly
unfavorable growth conditions that result in the presence of TeO32-oxyanion.
Figure 40. Microwells Showing the Red Color of Elemental Se0 Formed by the Reduction
of SeO42- and SeO32- by Intracellular GSH of E. coli.
58
Determination of the Effect of IPTG on the Resistance of AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains for Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants
IPTG is frequently used as an inducer of the lac operon for physiological work.
The purpose of using IPTG in this research work was to find out whether IPTG can be
used to enhance the production of intracellular GSH levels inside the bacterial strains
providing a higher resistance to the metalloidal oxyanions. The results show a slightly
lower resistance of these three E. coli species for the three toxicants with increasing IPTG
levels. The possible reasons are discussed in the last section of part (1) in the discussion.
Determination of the Effect of IPTG on the Specific Growth Rate of AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains
The results demonstrate that the bacterial strains experience a repression of their
growth in the presence of higher levels of IPTG even without the presence of any of the
toxicants Na2SeO3, Na2SeO4, and Na2TeO3. The rationalization is given in detail in the
last section of part (1) in the discussion.
Determination of the Intracellular Glutathione Levels in AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains
GSH, being the primary thiol of the thiol-mediated reduction system of the E. coli
cell, as discussed earlier, is a major protective agent of the cell from oxidative stress.
GSH can modulate the activities of the E. coli OxyR, SoxR, and SoxS transcription
factors. OxyR regulates the adaptive response to H2O2 while SoxR and SoxS regulate the
adaptive response to O2-(Carmel-Harel and Storz, 2000). H2O2 and O2- are reactive
59
oxygen species which are produced as a result of reactions of Na2SeO3, Na2SeO4, and
Na2TeO3 with GSH (discussed before). Since these (H2O2, O2-) are responsible for
cellular damaging events, it is clear that GSH acts to protect the E. coli cell from these
toxicants. Moreover, GSH detoxifies H2O2 by reacting with it, forming GS-SG (oxidized
form of GSH) and H2O with the catalytic aid from glutathione peroxidase (Fahey et al.,
1978). Therefore, this may be, in part, the reason for observing a relatively higher
resistance for both Na2SeO3 and Na2SeO4 toxicants in the bioengineered species
AG1/pCA24NgshA and AG1/pCA24NgshB with respect to the wild type AG1 during the
MIC experiments. This also suggests that the production of GSH levels in these
bioengineered strains are higher than that of AG1 the wild type. But as discussed earlier,
TeO32- bacterial toxicity is much higher and it is more difficult to control by GSH. This
can possibly be the reason for observing the same MIC value for Na2TeO3 for all the
three E. coli species.
Very important conclusions can be drawn from the results of this section on the
determination of the intracellular glutathione levels in the three E. coli strains. Since the
absorbance at 412 nm is directly proportional to the GSH concentration, it is clear that the
bioengineered strains produce more intracellular GSH than that of the wild type.
Therefore the incorporation of multiple gene copies of gshA in the AG1/pCA24NgshA
strain and gshB in the AG1/pCA24NgshB strain has been successful in providing these
cells the ability to produce higher intracellular GSH than that of the wild type AG1 where
only one of each of these genes is present.
Another noticeable trend: when increasing IPTG concentrations, absorbance at
412 nm (denoting the presence of GSH) increases up to around 0.2 mM IPTG
60
concentration in both AG1/pCA24NgshA and AG1/pCA24NgshB and then decreases. In
AG1, absorbance at 412 nm increases very slightly up to around 0.1 mM IPTG and then
decreases. This means that with IPTG concentrations, intracellular GSH content increases
until it reaches a maximum value and then decreases in these E. coli strains.
The bioengineered species AG1/pCA24NgshA and AG1/pCA24NgshB have been
constructed by incorporating multiple copies of an additional plasmid pCA24N (Monrás
et al., 2012; Kitagawa et al., 2005). In the strain AG1/pCA24NgshA, the gene gshA has
been ligated into pCA24N to contain multiple copies (20-50) of the gene; and the gene
gshB has been ligated into pCA24N to contain multiple copies (20-50) in the
AG1/pCA24NgshB strain. In each of these strains the pCA24N plasmid contains a PT5-lac
hybrid promoter (Kitagawa et al., 2005) which functions to express the genes gshA and
gshB. This PT5-lac hybrid promoter has two basic regions, a promoter region from a T5
phage virus and a lac operator region (Figure 41).
Figure 41. PT5-lac Hybrid Promoter.
In the AG1/pCA24NgshA strain, the gshA gene is ligated after this lac operator
region and in the AG1/pCA24NgshB strain, the gshB gene is ligated after the lac operator
(Figure 42 B, C).
61
Figure 42. A Linear Representation of the pCA24N Circular Plasmid (A). Location of the
gshA Gene of the AG1/pCA24NgshA Strain in the pCA24N Plasmid (B). Location of the
gshB Gene of the AG1/pCA24NgshB Strain in the pCA24N Plasmid (C). Ori, Origin of
Replication; Cat, Chloramphenicol Resistant Gene.
In addition, the pCA24N plasmid contains the lacIq gene (Figure 42 A, B, C)
which produces higher levels of lac repressor molecules which usually function to inhibit
the expression of the lac operon genes by binding to the lac operator (Kitagawa et al.,
2005). Since the genes gshA and gshB (Figure 42 B, C) have replaced the usual lac
operon genes lacZ, lacY, and lacA, these lac repressor molecules reduce the expression of
these gshA and gshB genes by binding to the lac operator region of the PT5-lac hybrid
promoter. These E. coli strains (AG1, AG1/pCA24NgshA, and AG1/pCA24NgshB) have
RNA polymerases in their cytosols. These bind to the promoter region of the PT5-lac
hybrid promoter which triggers the expression of the gshA and gshB genes. The binding
of the lac repressor molecules to the lac operator region (as mentioned above) interferes
with the binding of RNA polymerases to the promoter region of the PT5-lac hybrid
promoter and consequently the expression of the genes gshA and gshB occurs at very low
levels. But, IPTG, when present, binds to these lac repressor molecules and inactivates
them and thereby promotes the expression of gshA and gshB genes. Since these genes are
62
involved in the biosynthesis of intracellular GSH in E. coli (as mentioned in the
introduction), higher GSH levels can be expected in the presence of IPTG. There is
neither a gshA gene nor a gshB gene present right after the PT5-lac hybrid promoter in
AG1, the wild type strain. Hence the presence of IPTG should not affect the production
of intracellular GSH in AG1 via the plasmid. But according to the results in Figure 31,
GSH levels increased slightly even in AG1 at slightly higher IPTG concentrations. It was
the same pattern with both AG1/pCA24NgshA and AG1/pCA24NgshB strains.
Therefore, the presence of IPTG has caused an increase in GSH levels but not in a
plasmid dependent manner. With that said, these cultures were not normalized here for
cell density so the early apparent GSH increase, as seen in Figure 31, might be the result
of the slight effect of IPTG on cell growth instead of GSH production.
The reason for observing GSH levels in these strains with no added IPTG can be
explained as follows. The PT5-lac hybrid promoters in these E. coli strains are not fully
repressed by the lac repressors. They usually contribute for leaky expression of the gshA
and gshB genes in the AG1/pCA24NgshA strain and the AG1/pCA24NgshB strain
respectively. Therefore even in the absence of IPTG, these plasmid genes are ‘on’
slightly. Genomic chromosomal expression of the two genes gshA and gshB also occurs
in these two strains. Therefore these two E. coli strains produce more intracellular GSH
than that of AG1, where only the genomic chromosomal expression of the two genes
gshA and gshB occurs.
It is very important to figure out the reason for the decreasing trend of GSH with
higher IPTG concentrations (Table 11). The three E. coli strains have the usual lac
operon in their genomic chromosomes and also the lacI gene. The lac operon is a
63
functioning unit of genomic DNA which controls the lactose metabolism inside the cell
through a regulation of the expression of three structural genes lacZ, lacY, and lacA. The
lacI gene encodes the lactose repressor which binds very tightly to the lac operator and
thereby blocks the expression of the above structural genes in the absence of lactose.
IPTG, when present, binds to the lac repressor and inactivates it, thereby inducing the
expression of those structural genes. These structural genes lacZ, lacY, and lacA code for
specific enzymes/proteins. Therefore, higher levels of IPTG force the cell to produce
more and more intracellular proteins which can lead to metabolic and oxidative stress
conditions. GSH, being the major cellular thiol, acts against this oxidative stress as
discussed earlier. It is very possible that this is the reason for observing lower levels of
GSH in the presence of higher IPTG levels (Table 11) as IPTG-induced oxidants
presumably begin to consume GSH. Both factors, the increase of the cellular protein
content and also the consumption of GSH to act against the oxidative stress created by
increasing IPTG concentrations, may have acted to cause lower intracellular GSH
contents at increasing IPTG concentrations. The presence of lower levels of GSH thereby
might have reasonably caused lower resistance (lower MIC values) to the toxicants
especially Na2SeO3, Na2SeO4 at increasing IPTG concentrations (Table 7, 8, 9). These
unfavorable conditions, metabolic stress and oxidative stress may have caused lower
specific growth rates observed at increasing IPTG concentrations (Table 10).
64
Part (2). An Effort to Produce an Escherichia coli Strain With a Higher GSH
Content
As mentioned in the introduction, an effort was undertaken to increase the copy
number of the gshA gene in E. coli in order to improve the bacterial potential of reducing
the toxic oxyanions Na2SeO3, Na2SeO4, and especially Na2TeO3 by increasing
intracellular GSH. Owing to the fact that the bioengineered E. coli species
AG1/pCA24NgshA and AG1/pCA24NgshB showed a much lower resistance to Na2TeO3
with respect to the other two toxicants, the experiments described in this section were
carried out with the goal of inducing better reducing power in E. coli via gene
manipulation. The observed results after the transformation of the ligated products into
the competent E. coli cells (Figure 35) can be interpreted as follows.
Due to the possibility of using XbaI and PstI as the restriction enzymes to cut both
gshA the gene of interest (Figure 33), and the biobrick containing the RFP gene (Figure
34), those two enzymes were used for the restriction enzyme digestion process. Since
there were many copies of gshA as the result of PCR, subsequent ligation followed by the
transformation into competent E. coli cells should have ended up with many colonies
having the correctly ligated product represented by normal white colonies. These white
colonies have gshA, the gene of interest in place of the RFP gene. The red colonies
observed were due to the ligation of the expressible RFP gene (since it contains the
promoter and the RBS region of the biobrick BBa_I715039 which make it a functional
gene) with the Plasmid pSB1A2, coding a red fluorescent protein. The biobrick
BBa_I715039 was chosen as a marker to ease the process of selecting the wanted
colonies from the unwanted colonies. Figure 35 illustrates the results of this section.
65
After the transformation of the ligated products into E. coli cells, subsequent
DNA purification, restriction enzyme digestion, and gel electrophoresis were performed
to further confirm a successful ligation of the gshA gene. Here the restriction enzyme
digestion was carried out with EcoRI restriction enzyme. The correctly ligated normal
white colonies were used here and the final gel electrophoresis results were compared
with a standard DNA ladder on a 1.2% (w/v) agarose gel (Figure 36). The results can be
interpreted as follows.
The correctly ligated product (ligated gshA gene with the carrying plasmid
pSB1A2) has two EcoRI sites, one in the coding region of the gshA gene and the other in
the plasmid pSB1A2 (Figure 43). Hence the outcome of a successful EcoRI digestion
should be two pieces of DNA bands with different sizes.
Figure 43. The Correctly Ligated Product (ligated gshA gene with the carrying plasmid
pSB1A2).
The size of the plasmid pSB1A2 is 2079 bp (without the gshA gene) and the size
of the gshA gene with the added regions from the primers is 1579 bp. Therefore the total
size of the ligated product is the sum of these two individual pieces, which is 3658 bp.
66
Therefore the larger size band is expected to be 3459 bp since it is now lacking the piece
of DNA of gshA which is from one EcoRI site to the other and this little piece is 199 bp.
But according to the results shown in Figure 36, only one DNA fragment was observed
which was ~ 4000 bp in size. There was no indication of the 199 bp fragment. Due to the
fact that this was always the result achieved after repetition of the steps, there may not
had been two EcoRI sites as expected in the ligated product which was subjected to
EcoRI restriction digestions. Therefore, most probably this ligated product was not the
expected one which means that an appropriate ligation may not had occurred between the
two XbaI ends and the two PstI ends of the gshA gene and the plasmid pSB1A2. The
possible reasoning can be explained as follows.
The base sequence of the used forward primer of the gshA gene (gshA-1f) is 5’gtttcttcgaattcgcggccgcttctagttgatcccggacgtatcac-3’. The restriction enzyme site XbaI was
targeted to be incorporated into the gshA gene through this gshA-1f primer and the correct
recognition site of the XbaI is 5’-…TCTAGA…-3’. In the gshA-1f primer, it appeared as
5’-…TCTAG…-3’ (5’-gtttcttcgaattcgcggccgcttctagttgatcccggacgtatcac-3’) due to an
error made in designing the primer. Consequently, in the gshA gene, the XbaI site may
not have been present as expected and that can be the reason for not achieving the
correctly ligated product with the plasmid pSB1A2 as described above. The observed
DNA fragment (which was ~ 4000 bp) may be a result of two self-ligated plasmid
(pSB1A2) fragments. This self-ligated fragment of 4158 bp (each pSB1A2 part is 2079
bp in size) may have given rise to this larger fragment (~ 4000 bp) after the deletion of
the smaller fragment (18 bp in size) between the two EcoRI sites present, upon digestion
with EcoRI restriction enzyme. This very small fragment may not have been separated
67
and identified well in a 1.2% (w/v) agarose gel, but may have been defused in the gel
matrix. With this said, the white colonies that were used for the gel electrophoresis were
not actually the expected ones. Therefore, the actual reason for observing more of the
white colonies over the red ones was because of the higher number of colonies resulted
from bacterial transformations without including the biobrick coding the RFP protein in
the plasmid pSB1A2 (i.e. with self-ligated plasmids pSB1A2) and also due to the
possibility of having colonies (still white) without any foreign DNA transformations.
Work on these experiments was stopped at this point; however, future work needs
to be carried out in the following manner. A different gshA-1f primer should be tested
which can incorporate a XbaI site successfully to the gshA gene in order to achieve a
correctly ligated gshA gene with the plasmid pSB1A2 by using XbaI and PstI restriction
enzymes as described. The EcoRI site in the coding region of the gshA gene would have
to be deleted through site-directed mutagenesis in order to minimize deletion problems of
the coding regions of gshA when using EcoRI in future analyzes. Moreover, a suitable
promoter region and a RBS region should be ligated into the cloned gshA gene for its
active expression. After a successful completion of the plasmid design with an
expressible gshA gene inside E. coli cells, these cells now having a higher copy number
of the gshA gene should be used with the same toxicants, toxicity measurements, specific
growth rate measurements, and intracellular GSH content measurements which were
carried out for the three E. coli strains AG1, AG1/pCA24NgshA, and AG1/pCA24NgshB
as before, in order to compare the results. Another suggestion is to design an E. coli strain
which has multiple copies of both gshA and gshB genes. Since both of these genes are
68
involved in the GSH biosynthetic pathway of E. coli, this can also be a competitive
outcome for the bioremediation of toxic metalloidal oxyanions.
69
CHAPTER V
CONCLUSIONS
Part (1). Determination of the Degree of Resistance of AG1, AG1/pCA24NgshA, and
AG1/pCA24NgshB E. coli Strains Towards Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants
From MIC results
The resistance towards the oxyanions Na2SeO3 and Na2SeO4 is higher in the
bioengineered E. coli strains AG1/pCA24NgshA and AG1/pCA24NgshB than that of
AG1 the wild type.
The toxicity of Na2TeO3 is the highest among all the oxyanions tested for the
three E. coli strains.
The relative toxicity of the oxyanions tested for the three E. coli strains increases
in the order Na2SeO4 < Na2SeO3 < Na2TeO3.
From specific growth rate results
The specific growth rate of each E. coli strain decreases in the presence of the
toxicants when compared to the controls which confirms that the presence of any of
these toxicants is unfavorable for the normal growth of these tested E. coli species.
The presence of TeO32- is highly unfavorable for the normal growth of these
tested E. coli species.
70
Determination of the Effect of IPTG on the Resistance of AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains for Na2SeO3, Na2SeO4, and Na2TeO3
Toxicants
The resistance of AG1, AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli
strains for Na2SeO3, Na2SeO4, and Na2TeO3 toxicants slightly decreases at higher IPTG
concentrations.
Determination of the Effect of IPTG on the Specific Growth Rate of AG1,
AG1/pCA24NgshA, and AG1/pCA24NgshB E. coli Strains
The specific growth rates of AG1, AG1/pCA24NgshA, and AG1/pCA24NgshB E.
coli strains slightly decrease at higher IPTG concentrations.
Determination of the Intracellular Glutathione Levels in AG1, AG1/pCA24NgshA,
and AG1/pCA24NgshB E. coli Strains
In the absence of IPTG, the intracellular glutathione levels show the highest value
in AG1/pCA24NgshA followed by AG1/pCA24NgshB and AG1.
This also confirms that both the genes gshA and gshB are involved in the
glutathione biosynthetic pathway of these E. coli strains.
The presence of higher glutathione levels confers a higher resistance to both
Na2SeO3 and Na2SeO4 toxicants for these two bioengineered species AG1/pCA24NgshA
and AG1/pCA24NgshB as compared to AG1.
71
Increasing the IPTG concentration slightly, helps increase intracellular GSH but
not in a plasmid (pCA24N) dependent manner. Higher IPTG levels lead to a decreased
production of GSH in all of the three E. coli strains.
Increasing IPTG concentrations caused the production of more and more
intracellular proteins and thereby increases cellular oxidative stress which is also
indicated by unfavorable growth conditions at high IPTG levels in all of the E. coli
strains used.
Intracellular GSH acts against presumed oxidative stress induced by the highest
levels of IPTG, and consequently the bacterial resistance to metalloidal oxyanions
provided by glutathione is reduced in all of the three E. coli strains used at the highest
levels of IPTG.
Intracellular GSH levels present in any of the three E. coli strains tested were not
able to provide an adequate resistance towards Na2TeO3 when using changes in MIC as a
measure of resistance. Hence, the bioengineered species AG1/pCA24NgshA and
AG1/pCA24NgshB could be successfully used for the bioremediation of toxicants
Na2SeO3 and Na2SeO4 but not for Na2TeO3.
If IPTG is used as an inducer of intracellular GSH with these bioengineered E.
coli strains for this bioremediation process, the IPTG levels should be controlled to avoid
unfavorable oxidative stress conditions that lower GSH levels.
72
Part (2). An Effort to Produce an Escherichia coli Strain With a Higher GSH
Content
The chromosomal gshA gene in the E. coli strain BW 25113 can be used as a
template for the amplification of this gene through PCR reactions.
The plasmid pSB1A2 which contains the biobrick (BBa_I715039) including the
RFP gene coding for a red fluorescent protein (RFP) can be used successfully to ligate
the gshA gene.
A different gshA-1f primer should be tested which can incorporate an XbaI site
successfully to the gshA gene in order to achieve a correctly ligated gshA gene with the
plasmid pSB1A2 by using XbaI and PstI restriction enzymes as described.
Future work needs to be carried out in order to estimate the relative capability to
bioremediate the toxic oxyanions especially Na2TeO3, by this E. coli strain in comparison
with the bioengineered E. coli species AG1/pCA24NgshA and AG1/pCA24NgshB.
73
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83
APPENDIX
Chemical Abstract Service Registry Numbers
Compound
CAS Number
Agar
9002-18-0
5, 5’-dithiobis(2-nitrobenzoic acid)
69-78-3
Isopropyl-β-D-1-thiogalactopyranoside
367-93-1
L-glutathione reduced
70-18-8
Resazurin sodium salt
62758-13-8
Sodium selenate
13410-01-0
Sodium selenite
10102-18-8
Sodium tellurite
10102-20-2
Tryptone
73049-73-7
Yeast extract
8013-01-2
84
VITA
Gayan Adikari Appuhamillage was born on February 7, 1984 in Colombo, Sri
Lanka, to Thilakarathna Adikari Appuhamillage and Pathmalatha Jayasooriya
Arachchilage. He is the only child in his family. After completion of high school studies
at Ananda College, Colombo, he began working on his undergraduate studies at
University of Kelaniya, Sri Lanka in April, 2005. He followed a four year Chemistry
special degree at University of Kelaniya. He has been the co-author of a research article
in analytical chemistry that had been published in the Sri Lanka Association of
Advancement of Science (SLAAS). With the goal of becoming a chemist, he graduated
with his B.S. in Chemistry with a second class upper division honors in October, 2009.
His dream of pursuing a post graduate degree came alive as he was offered a scholarship
as a graduate teaching assistant at Sam Houston State University, USA in June, 2011.
There, he began working on his Master’s degree in Chemistry in September, 2011.
Throughout his graduate career he carried out research with Dr. Thomas G. Chasteen in
Bioanalytical Chemistry. During his graduate studies, he orally presented his Master’s
degree research at the 2013 University Wide Graduate Research Exchange Program.