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GENOME-WIDE EXAMINATION OF RADIATION STRESS
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Xiaoyun Qiu

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6/01 c1/CIRC/DateDuep65-p, 1 5

GENOME-WIDE EXAMINATION OF RADIATION STRESS RESPONSE IN
SHEWANELLA ONEIDENSIS MR-l

By

Xiaoyun Qiu

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

2004

ABSTRACT

GENOME-WIDE EXAMINATION OF RADIATION STRESS RESPONSE IN
SHEWANELLA ONEIDENSIS MR-l

By

Xiaoyun Qiu

Shewanella oneidensis MR-l, a Gamma proteobacterium, is notable in the
terminal electron acceptors it uses including some toxic metals and radionuclides. Thus it
has a great potential for bioremediation. However, MR-l is uniformly sensitive to UVC,
UVB, UVA, natural solar radiation as well as ionizing radiation. I delineated the genomic
response of Shewanella oneidensis MR-l to five radiation stresses. A total of 4.2-, 3.9-,
8.1-, 28.0-, and 5.9% of the MR-l genome showed differential expression following
UVC, UVB, UVA, natural solar radiation, and ionizing radiation exposure at a dose that
yields about 20% survival rate, respectively. The gene expression profile of MR-l in
response to ionizing radiation is more similar to that of UVC, which is characterized by a
strong induction of the SOS response and of many prophage related genes, plus some
oxidative stress response. Genomic response to UVB is a combination of the UVC and
UVA patterns, which represents a shift from shorter wavelength of UVR-induced direct
DNA damage and activation of prophages to longer wavelength of UVR-induced global
photo-oxidative damages. I observed the traditional UVA-induced stress responses in
MR-I such as induction of antioxidant enzymes and proteins, sequestration of the
transition metals and activation of the degradative pathways, however, the induction of

heavy metal and multidrug efflux pumps is a previously unknown phenotype for this

stress. Consistent with natural solar UV radiation composition, genomic response to solar
radiation is more similar to that of UVA but with more genes induced for detoxification.
In addition, the number of differentially expressed genes from most functional categories
increased greatly compared to either UVB or UVA or their sum. This unique gene
expression profile indicates that natural solar radiation impacts biological processes in a
much more complex way than previously thought.

Quantitative real time reverse transcription PCR (Q RT-PCR) assays were carried
out in parallel for controls and irradiated samples for 16 selected genes that are involved
in DNA recombination repair, nucleotide excision repair (NER), defending against
oxidative stress, encoding heavy metal and multidrug efi'lux pumps, putative regulatory
genes, transport genes, and metabolic genes. A good correlation was obtained between
array-based transcriptional analysis and Q RT-PCR assays. I further demonstrated that
mutagenic repair, photoreactivation and NER are functional in MR-l although the
expression of NER component genes is not damage inducible. Activation of prophages
and DNA damage appear the major lethal factors in MR-l following short wavelength
UVR (UV C and UVB) and ionizing radiation exposure whereas global photo-oxidative
damage contributes greatly to its UVA and solar radiation sensitivity. In addition,
alteration in gene regulation, e. g. loss the damage inducibility of some DNA repair genes,
perhaps as the consequence of lack of natural selection, may contribute to its high

radiation sensitivity in general.

Copyright by
Xiaoyun Qiu
2004

T 0 my son Evan Thomas Q Carter

ACKNOWLEDGMENTS

I would like to express my deepest gratitude to my advisor, Dr. James M. Tiedje,
for his guidance and support throughout my Ph.D study. I have learned a lot by his
example and was inspired to pursue scientific excellence. I am especially grateful for his
encouragement when I struggled with research works and the challenge of being a mom.
Because of him, I gained confidence in both. Thanks to all the members of Tiedje’s lab,
for their invaluable help and discussions during the past five years. Special thanks to
Verénica Gri’mtzig and Claribel Cruz, for their priceless friendship which I will treasure
for ever. Thanks to the CME staff, Lisa Pline, Pat Englehart and Nikki Mulvaney, for
their great assistance and support.

I thank the members of my guidance committee: Drs. George Sundin, Robert
Hausinger, Michael Thomashow, Syed Hashsham, and J izhong Zhou, for their critical
questions, encouragement, and valuable comments and suggestions throughout this
research. Special thanks to Dr. George Sundin, for his detailed discussion and advice,
especially on UV radiation work. I would like to thank Drs. Alex Vesilenko and Michael
Daly, for their assistance and critical discussion and comments on ionizing radiation
work; and Drs. Benli Chai and James Cole, for their help on computational search of the
SOS box.

I would like to say special thanks to professors Xingfang Qiu and Jinlun Xue at
F udan University. Without their help and encouragement, I would never have been able
to accomplish this. They opened the door of biology and showed me the wonder of being

a biologist when I was a freshman. Ever since then, they always stand besides me and

vi

cheer for the achievements I have accomplished. I am very grateful for everything they
did for me.

I can never thank my parents Dihua Liu and Shaodun Qiu enough. Without their
love, support and encouragement, I would never have been able to complete my dream. I
would like to thank my sister Ping Qiu and her family, Qiling Wang and Shichang Wang,
and my brother Jian Qiu and his family, Yijia Qiu and Xiangiu J ia, for their love, support,
and taking care of our parents ever since I started the college. I also would like to thank
my family-in law, Nancy Nichols, James and Kathe Carter, and Brian, Lisa and Liam
Carter, for their love, support and help during this long journey. It is such a blessing to
have them in my life. I owe the greatest gratitude to my husband, Chris Carter, for his
love, understanding and long term support. I am very grateful for the sacrifices he made
to help me pursue my dream. Finally, I would like to thank my son Evan Carter, for
bringing me great joy during this tough time. I am looking forward to exploring the world

with him together.

vii

PREFACE

The research work on UV radiation presented in this dissertation was sampled at Dr.
George W. Sundin’s lab at Department of Plant Pathology, Michigan State University.
The research work on ionizing radiation was sampled at Dr. Michael Daly’s lab at
Uniformed Services University of the Health Sciences at Bethesda, Maryland. The
fluorescent images presented in this dissertation were taken by Shirley Owens, from
Center for Advanced Microscopy at Michigan State University. The transmission
electron microscopy images presented in this dissertation were taken by Alicia Pastor,
from Center for Advanced Microscopy at Michigan State University. The whole genome
microarray of Shewanella oneidensis MR-l was generated at Dr. Jizhong Zhou’s lab at

Oak Ridge National Laboratory, Oak Ridge, Tennessee.

viii

TABLE OF CONTENTS

 

LIST OF TABLES .................................................................................. xi

LIST OF FIGURES ................................................................................ xiii

LIST OF SUPPLEMENTAL TABLES ......................................................... xv

LIST OF SUPPLEMENTAL FIGURES ....................................................... xvi

CHAPTER 1.

INTRODUCTION AND RATIONALE 1
Radiation and radiation induced biological effects in bacteria ........................ 2
My research objectives ..................................................................... 6
My experimental approaches .............................................................. 13
References ................................................................................... 22

CHAPTER 2.

SURVIVAL OF SHEWANELLA ONEIDENSIS MR-l

AFTER UV RADIATION EXPOSURE ......................................................... 29
Abstract ..................................................................................... 30
Introduction ................................................................................. 31
Materials and methods .................................................................... 33
Results ........................................................................................ 40
Discussion .................................................................................. 51
References .................................................................................. 58

CHAPTER 3.

COMPARATIVE ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES IN
SHEWANELLA ONEIDENSIS MR-l FOLLOWING EXPOSURE TO UVC, UVB

 

AND UVA RADIATION .......................................................................... 63
Abstract ..................................................................................... 64
Introduction .................................................................................. 65
Materials and methods .................................................................... 67
Results ........................................................................................ 73
Discussion .................................................................................. 90
References .................................................................................... 95

CHAPTER 4.

GENOME-WIDE EXAMINATION OF NATURAL SOLAR RADIATION

RESPONSE IN SHEWANELLA ONEIDENSIS MR-l ..................................... 100
Abstract .................................................................................... 101
Introduction ............................................................................... 102
Materials and methods .................................................................. 104

ix

Results and discussion ................................................................... 106

 

References ................................................................................. 125
CHAPTER 5.
TRANSCRIPTONE ANALYSIS OF IONIZING RADIATION RESPONSE IN
SHEWANELLA ONEIDENSIS MR-l ........................................................ 127
Abstract .................................................................................... 128
Introduction ............................................................................... 129
Results and discussion .................................................................. 131
References ................................................................................. 142
CHAPTER 6.
SUMMARY AND FUTURE PERSPECTIVE ................................................ 145
Comparison of transcriptional profiles of MR-l in response to five
radiation stress conditions ............................................................... 146
Comparison of transcriptional profiles in response to UVC between E. coli
MG1655 and S. oneidensis MR-l ...................................................... 152
Comparison of transcriptional profiles in response to ionizing radiation
between D. radiodurans R1 and S. oneidensis MR-l ............................... 153
Future perspective ........................................................................ 154
References ................................................................................. 1 57
APPENDIX A. SUPPLEMENTAL TABLES ................................................ 159
APPENDIX B. SUPPLEMENTAL FIGURES ............................................... 205

Table 1.1

Table 2.1

Table 2.2

Table 2.3

Table 2.4

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Table 3.5

Table 4.1

Table 4.2

Table 4.3

Table 5.1

Table 5.2

LIST OF TABLES

Comparison of major DNA repair genes between E. coli K-12 and S.
oneidensis MR-l .......................................................................... 7
Bacterial strains, plasmids and primers used in the study ....................... 38
D-values (J m‘z) and slope of survival curves from various bacterial

Strains .................................................................................. 41
Examples of putative E. coIi-like SOS box in S. oneidensis MR-l ............ 49
E. coli-like SOS box in strains that are phylogenetically close

to S. oneidensis MR-l ................................................................ 56
Gene and corresponding primers in Q RT-PCR analysis ........................ 72
Common up-regulated genes in response to UVC, UVB and

UVA radiation ......................................................................... 78
The expression level of selected DNA damage repair genes

quantified by Q RT-PCR before and after UVC irradiation .................... 80
The number of induced prophage-related genes following UVC,

UVB and UVA exposure ............................................................ 82
The relative expression of selected genes following UVA

exposure quantified by microarray hybridization and Q RT-PCR ............. 89

Induction of DNA damage genes, the SOS response and genes
involved in defending against oxidative stress in MR-l after
exposure to natural solar radiation .................................................. 114

Relative expression of biosynthesis genes and biosynthesis related
genes in MR-l after exposure to natural solar radiation ........................ 121

Comparison of relative gene expression between microarray analysis
and Q RT-PCR assay ................................................................... 124

Induction of prophage-related genes by ionizing radiation .................... 138

Comparison of relative gene expression between microarray analysis
and Q RT-PCR assay ................................................................ 141

xi

Table 6.1 Summary of stress response following irradiation .............................. 147

Table 6.2 Comparison of genome and the induction of DNA repair genes
among S. oneidensis MR-l , E. coli MG1655 and D. radiodurans R1 ....... 153

xii

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 3.1

Figure 3.2

Figure 3.3

Figure 4.1

Figure 4.2

Figure 4.3

LIST OF FIGURES

Survival of E. coli K12 and Shewanella strains after
UVC irradiation ..................................................................... 42

Survival of S. oneidensis MR-l after UVA irradiation ......................... 43

Survival of S. oneidensis MR-l following photoreactivation
or in the dark after UVC, UVB and solar light irradiation ..................... 45

Analysis of MDR in S. oneidensis MR-l following
UVC and UVB exposure .......................................................... 46

Relative gene expression of NER component genes uvrA, uvrB
and uer at 5 min, 20 min and 60 min after UVC, UVB and
UVA irradiation ..................................................................... 48

Complementary analysis of uvrA of S. oneidensis MR-l
in P. aeruginosa UV11079 (uvrA deficient) alter UVC irradiation .......... 51

The global gene expression trends of MR-l in response to

UVC, UVB and UVA (A) during 3 1h recovery period after

exposure and venn diagram of up-regulated genes in response

to UVC, UVB and UVA irradiation (B) ......................................... 74

Distribution of the differentially expressed genes in various functional
categories following UVC, UVB and UVA exposure ......................... 76

SYBR Green I staining MR-l, MR-l treated with UVC and TEM
images for phage isolated from UVC irradiated MR-l ........................ 83

The global gene expression trend of MR-l in response to solar

radiation during a 1h recovery period after exposure (A) and

comparison of differential expression following UVB, UVA and

solar radiation exposure (B) ...................................................... 108

Comparison of the distribution of the up-regulated (A) and
down-regulated genes (B) in various functional categories
following UVB, UVA and solar radiation exposure ........................... 110

Venn diagram of up-regulated genes (A) and down-regulated
genes (B) in response to UVB, UVA and natural solar radiation ........... 112

xiii

Figure 4.4

Figure 5.1

Figure 5.2

Figure 5.3

Figure 6.1

Figure 6.2

Hierarchical cluster analyses of differentially expressed energy
metabolic genes in response to natural solar radiation ....................... 118

The global gene expression trend in response to ionizing radiation
during a 1h recovery period after gamma ray exposure ..................... 132

Distribution of differentially expressed genes in various functional
categories following gamma ray exposure .................................... 134

Venn diagram of up-regulated genes in response to
ionizing radiation, UVC and UVA .............................................. 135

Functional distribution of up-regulated genes in response to ionizing
radiation, UVC, UVB, UVA and natural solar radiation ..................... 148

Functional distribution of down-regulated genes in response to ionizing
radiation, UVC, UVB, UVA and natural solar radiation ..................... 150

xiv

Supplemental Table 3.1

Supplemental Table 3.2

Supplemental Table 3.3

Supplemental Table 3.4

Supplemental Table 4.]

Supplemental Table 4.2

Supplemental Table 4.3

Supplemental Table 5.1

Supplemental Table 5.2

LIST OF SUPPLEMENTAL TABLES

The relative expression and K-means analysis of
up-regulated genes in MR-l following UVC exposure

The relative expression and K-means analysis of
up-regulated genes in MR-l following UVA exposure

The relative expression and K-means analysis of
up-regulated genes in MR-l following UVB exposure

Comparison and the relative expression of hypothetical
genes following UVC, UVB and UVA exposure .......

The relative expression of other stress-related genes
following solar radiation exposure .......................

Cluster analysis and the relative expression of
metabolic genes in MR-l following solar

radiation exposure ...........................................

The relative expression of biosynthesis genes in MR-l

following solar radiation exposure ........................

The relative gene expression of up-regulated gene in

MR-l following gamma ray exposure ....................

The relative expression of genes involved in defending
against oxidative stress in MR-l following gamma ray
exposure ........................................................................

XV

...... 160

...... 164

...... 173

...... 178

....... 183

...... 185

...... 189

...... 195

........ 203

LIST OF SUPPLEMENTAL FIGURES

Supplemental Figure 3.1 K-means analysis of up-regulated genes in response
to UVC (A), UVB (B) and UVA (C) ............................ 206

Supplemental Figure 3.2 Venn diagram of up-regulated hypothetical genes
in response to UVC, UVB and UVA ........................... 211

xvi

CHAPTER 1

INTRODUCTION AND RATIONALE

Radiation and radiation induced biological effects in bacteria.

Ultraviolation radiation (UV R) is probably the most common physical agent that
damages DNA. Living organisms have had to cope with the genotoxic effects of solar
UV radiation since the beginning of biological evolution on the earth. The UV radiation
spectrum is commonly divided into three wavelength bands designated UVC (100-290
nm), UVB (290-320 nm) and UVA (320-400 nm). Solar UV radiation reaching earth’s
surface consists mainly of UVA (about 95%) and a small portion of UVB (about 5%) due
to ozone filtration. However, unattenuated UV radiation prior to the accumulation of
oxygen in the earth’s atmosphere may have served as an important constraint during the
evolution of terrestrial life.

Since the maximum absorption of DNA is at 260 nm, exposure to both UVB and
UVC will induce the formation of a variety of photoproducts, resulting in various adverse
biological effects. Cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6-4)
photoproduct ((6-4) PD) are two major DNA photoproducts in bacteria. CPD is produced
from the formation of a four-membered ring structure resulting from saturation of the 5,6
double bonds of the two adjacent pyrimidines (Setlow 1966). The yield of CPD is
influenced by nucleotide composition as well as the sequence context (Gordon et al. 1982;
Michell et al. 1992). The (6-4) PD is produced by a covalent linkage between the 06
position of one pyrimidine and the C4 position of the adjacent pyrimidine. Irradiation of
(6-4) PD with 313 nm light leads to the formation of the Dewar isomer (Taylor and Cohrs
1987), which is a significant DNA photoproduct after solar light exposure (Perdiz et a1.
2000). Other UVR induced DNA photoproducts include purine lesions, pyrimidine

hydrates, and thymine glycol. Thymine glycol is also one of the major forms of DNA

base damage induced by ionizing radiation (Demple and Linn 1982; Fisher and Johns
1976; Gasparro and Fresco 1986; Porschke 1973; Setlow 1992; Varghese 1970). UV
radiation also causes cross links and strand breaks. The frequency of stand breaks and
DNA-protein cross-links is dramatically increased by longer wavelength UVR irradiation
(Tyrrell 1991).

UV radiation can damage DNA indirectly through photosensitization, in which
sensitizer molecules in the cell absorb the photons of UV radiation and transfer the
energy to the base in the DNA. Thus long wavelength UV radiation (UV A) can also
induce the formation of CPD (Perdiz et a1. 2000). The sensitizer molecules can also
transfer the energy to oxygen, resulting in highly reactive oxygen species (ROS), which
can cause damage to a variety of molecules as well as physiological processes in the cell
(Eisenstark 1987; Eisenstark 1989).

Similar to UV radiation, ionizing radiation has been a source of naturally
occurring physical damage to the DNA of living organism since the beginning of life.
Ionizing radiation has high energy and great penetrating ability, thus it can directly
damage the cell by depositing energy randomly to any cellular components
(Frankenberg-Schwager 1990; Goodhead 1989; Hutchinson 1985). Damage to DNA
bases has been extensively studied in vitro. Many products and short-lived intermediates
have been characterized (Teoule 1987). Ionizing radiation can also induce protein-DNA
cross links, sugar damage and strand breaks, of which double strand breaks (D835) is the
most lethal effect (Hutchinson 1985; Ilikis 1991; Ward 1988; Ward 1990).

Ionizing radiation can also induce the formation of ROS through the radiolysis of

water, which has been suggested to be a major potential source of indirect damage to

DNA (Riley 1994; Ward 1990). It has been estimated that more than 80% of the energy
of ionizing radiation deposited in the cell results in the abstraction of electrons from
water. Superoxide radical will be produced when oxygen is present. Thus, similar to long
wavelength UV radiation, ionizing radiation can induce oxidative stress in living
organism.

Knowledge of DNA damage repair is primarily from Escherichia coli and
Deinococcus radiodurans, an extremely radiation resistant bacterium. Several DNA
repair pathways, e.g photoreactivation, base excision repair (BER), nucleotide excision
repair (NER), mismatch repair, recombination repair, and the SOS response have been
demonstrated to be important in repairing radiation-induced damage. Recently, Levin-
Zaidman et al. proposed that an ATP-dependent ligase mediated a non-homologous end-
joining pathway (NHEJ) as well as the presence of an unusual ring-like nucleoid
conformation may facilitate the repair of DSBs in D. radiodurans (Levin-Zaidman et a1.
2003). However, this explanation has been challenged by the research in Daly’s lab,
which indicates that high intracellular Mn/F e ratio is the essential factor in D.
radiodurans that contributes to the high radiation resistance (Daly et a1. 2004).

Shewanella oneidensis MR-l, a Gamma proteobacterium, was originally isolated
from the sediment of Oneida Lake, NewYork State (Myers et a1. 1988). Extensive studies
have been carried out on this bacterium due to its respiratory versatility: it can reduce a
variety of compounds including some toxic metals and radionuclides (Liu et al. 2002;
Middleton et a1. 2003). Thus, it has great potential for bioremediation of inorganic
pollutants. However, I found that MR-l is unifome sensitive to UVC, UVB, UVA, solar

light and ionizing radiation. This extreme radiation sensitivity could not be simply

explained from the MR-l genome content. A total of 2.8% of MR—l genome is implicated
in DNA replication, repair and recombination, which is comparable to that of E. coli K12
(2.7%) and D. radiodurans R1 (3.1%) (Blattner et al. 1997; Heidelberg et al. 2002; White
et al. 1999). Compared to E. coli, MR-l has most of the DNA repair pathways including
photoreactivation; NER; BER; methyl directed mismatch repair; recombination repair
and the SOS response, although a few E. coli DNA repair genes are not present on MR-l

genome (Table 1.1). Regarding defense against oxidative stress, MR-l has a putative
OxyR ($01382) and the sigma factor RpoS, but it does not have the SoxR and SoxS

regulators, which play a very important role in defending against superoxide radical
induced oxidative damage in E. coli. As a respiratory generalist, MR-l has 39 c-type
cytochromes, which is much higher than E. coli (7) (Heidelberg et al. 2002).

Cytochromes, along with other components of respiratory chain such as flavins, quinones,
are potential photoreceptors for long wavelength UVR (UV A and UVB). In addition,
Daly et al. showed that in contrast to D. radiodurans, MR-l has a high intracellular ratio
of Fe/Mn (Daly et al. 2004).

Why is MR-l so sensitive to radiation? What are the crucial factors that
contribute to this extreme radiation sensitivity in MR-l? Is it because of a lack of certain
important DNA repair genes or is it because the cell is rich in photoreceptors and rich in
Fe containing proteins? Furthermore, what are the important traits in determining
bacterial radiation resistance or sensitivity? Do evolution and natural selection have any

impact on bacterial radiation resistance or sensitivity and if so, in what manner?

My research objectives.
Objective 1. Functional analysis of putative DNA damage repair pathways and DNA
damage tolerance in S. oneidensis MR-l.

Genome annotation indicates that MR-l has a suite of DNA damage repair genes
and damage tolerance systems (Table 1.1). However, since MR-l has likely been
screened from solar light for millions of years due to its sediment habitat, it is important
to examine whether those putative genes are functional. The data obtained here will
provide us a clue whether natural selection and evolution has an impact on the high UVR
sensitivity in MR-l.

My research focus centers on three repair pathways: photoreactivation, NER and
mutagenic DNA repair (MDR). Photoreactivation in bacteria involves a single enzyme
called photolyase (phrB), which binds to CPDs, and in the presence of light (300-500 nm),
reverses the dimer to its component monomers. NER is present from bacteria to humans
and plays a critical role in protecting cells from a variety of DNA-damaging agents since
it can recognize a broad range of DNA lesions including ionizing radiation induced
purine damage, active oxygen species induced base loss and UV induced pyrimidine
dimers. UmuDC-mediated MDR functions in translesion synthesis enabling bypass of
DNA lesions that would normally block replication by DNA polymerase III. Translesion
DNA synthesis provides the cell with an additional mechanism of survival, however, this
process is accompanied by an elevation of the cellular mutation rate. The increase in
cellular mutation frequency can be assayed by examining the increase in the occurrence

of spontaneous mutants following irradiation.

 

 

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Objective 2. Comparison of transcriptional profiles of MR-l following UVC, UVB
and UVA exposure.

Historically, the investigation of UV radiation damage to DNA marks the
beginning of the study of the repair and tolerance of DNA damage in bacteria. Since UV
radiation at 254 nm is readily available from an ordinary germicidal lamp and it can
efficiently induce DNA lesions, it has been used in most studies even though it has minor
biological relevance. Previous studies had demonstrated that there was a significant
difference between far UV (UVC) and near UV (UVB and UVA) induced lethal,
mutagenic and physiological effects on bacteria (Eisenstark 1987; Eisenstark 1989;
Jagger 1983; Webb 1977). However, there are still many unanswered questions and
controversies left behind. With the concern of ozone depletion in the stratosphere, more
studies have been focused on the biological effects of solar UVB on plants or
phototrophic bacteria or the impact of solar UVB on ecosystems such as carbon and
energy flow. There is no study so far emphasizing the understanding of the molecular
basis of the difference in UVC, UVB and UVA induced damage in bacteria.

Upon DNA damage, many prokaryotes elicit the SOS response, which embodies
the pleiotropic response when the cell is in stress. Many proteins induced as part of this
process are involved in DNA replication, repair and control of cell division. Recently,
Courcelle et al. compared the gene expression profile of E. coli M01655 (a derivative of
E. coli K12) and the IexA deficient mutant following UVC exposure and revealed that
more than 30 genes are subject to SOS regulation (Courcelle et al. 2001). This study
demonstrated that microarray-based gene expression profiling is a powerful tool in

understanding the global stress response in bacteria. Comparison of transcriptional

10

profiles of MR-l following UVC, UVB and UVA exposure will allow us to compare the
global response to three different wavelengths of UVR in MR-l, and thus improve our
understanding on the molecular basis of what are the common and the different damages
induced by UVC, UVB and UVA. In addition, since the gene expression profile of E. coli
following UVC exposure is available (Courcelle et al. 2001), comparison between E. coli
and MR-l will give us an opportunity to identifying the important factors that contribute
to the difference in the UVC resistance and sensitivity between E. coli and S. oneidensis
MR-l.
Objective 3. Examination of transcriptional profiles of MR—l following natural solar
radiation exposure.

Natural solar radiation contains about 3% UV radiation (wavelengths less than
400 nm), 37% visible light (wavelengths between 400-780 nm), and 60% infrared light
(wavelengths longer than 780 nm). The deleterious effect of solar radiation is thought to
be primarily caused by solar UV radiation (Diffey 1991), thus knowledge of the solar
radiation induced biological effects comes primarily from the studies using either UVB or
UVA as radiation sources. Is solar radiation induced biological effects a simple sum of
UVB and UVA effects? S. oneidensis MR-l is extremely sensitive to solar radiation.
More than 80% of the cells die after exposure to the Michigan summer sun light for about
10 to 15 min. What are the primary lethal factors in MR-l following solar radiation
exposure, the DNA damage or the global photo-oxidative damage? By comparing the
gene expression profiles of MR-l among samples irradiated by solar radiation, UVA and
UVB, I hope to gain an understanding of the molecular basis of what are the deleterious

effects in MR-l following natural solar radiation exposure. The knowledge obtained also

11

contributes to our understanding of what are the natural solar radiation induced biological
effects in bacteria in general, a topic in its infacy.

Objective 4. Examination of transcriptional profiles of MR-l following ionizing
radiation exposure.

The high sensitivity of S. oneidensis MR-l to ionizing radiation could be
problematic in bioremediation of radionuclides wastes, e. g. U and Te. Understanding the
causes of this high sensitivity will provide us the knowledge for management of
bioremediation. Ionizing radiation induced biological effects have been studied
extensively in D. radiodurans. Until now, however, there is no conclusive explanation as
to what are the major factors that contribute to the extreme radiation resistance in D.
radiodurans. MR-l could be an excellent model from the other end of the radiation
resistance system for comparison. Recently, Liu et al. described the gene expression
profile of D. radiodurans R1 following exposure to 15 kGy of gamma ray (Liu et al.
2003). Comparison of gene expression profiles between D. radiodurans and S. oneidensis
will provide us insights into the molecular basis of what are the important factors that
contribute to radiation resistance and sensitivity in bacteria. In addition, since ionizing
radiation can damage DNA directly as well as induce oxidative stress, comparison of
gene expression profiles of MR-l among gamma ray, UVC and UVA will delineate the
commonality and differences in biological effects induced by ionizing radiation and UV
radiation, and thus provide clues on what are the major lethal factors in MR-l following

gamma ray exposure.

12

My experimental approaches.

Microarray based transcriptional analysis is a high throughput method, which
allows analyzing thousands of genes in parallel, thus it is a powerful tool in investigating
the global stress response in bacteria. Quantitative real time reverse transcription PCR (Q
RT-PCR) based transcriptional analysis is a sensitive and quantitative method, which is
powerful in detection and quantification of low abundant genes or transcripts. Ever since
the introduction of microarray technology, there has been a tremendous discussion and
rapid changes in array fabrication, array hybridization, data normalization and data
analysis. For Q RT-PCR, issues regarding data normalization and quantification have
been raised and discussed extensively. I will briefly review what I have learned about the
technologies and the rationale of my choices for microarray hybridization, data
normalization and analysis as well as the choice for internal controls in Q RT—PCR
analysis.

DNA microarray technology.

DNA microarrays are basically a high-throughput format of a dot blot. Currently
there are two major types: one is the oligonucleotide-based array and the other is the PCR
product-based array. Oligonucleotides either can be synthesized insitu using
photolithography as developed primarily by Affymetrix Inc (Santa Clara, CA) or first
synthesized in a conventional way and then deposited on a glass surface (e.g. Operon
Technologies). The PCR product-based microarray generally involves designing primers
for each gene; PCR amplification and purification of the PCR products followed by

spotting of the purified PCR products onto a glass surface. I used the PCR-products based

l3

microarrays because they were the only Shewanella arrays available at the time and their
performance and general acceptance were well established.

A DNA microarray experiment consists of array fabrication, probe preparation,
hybridization and data analysis. A variety of methods have been developed for probe
labeling (DeRisi et al. 1997; Gill et al. 2002; Hegde et al. 2000; Richmond et al. 1999). In
the gene expression experiment, cDNA can be labeled by either directly incorporating
fluorescent dye (Cy5 or Cy3) labeled nucleotides (DeRisi et al. 1997) into cDNA during
the reverse transcription step or by a two-step labeling using aminoally-dUTP (Hegde et
a1. 2002), in which primary aliphatic amino groups are first incorporated during cDNA
synthesis, and in the second step, the monofunctional N-hydroxylsuccinimide-activated
fluorescent dye is coupled to cDNA by chemical reaction with the amino functional
groups. Due to the increased labeling efficiency in this method, less starting material is
needed (e.g. 2 pg of total RNA) than direct incorporation (10-20 pg of total RNA). In
addition, since the substrate for the reverse transcription is identical for all samples, the
two-step labeling can reduce the dye bias during incorporation. The hybridization and
wash conditions have to be optimized to minimize the cross-hybridization. In general,
array format, the GC content of the genome and nature of dye used for probe labeling are
the important factors to consider for optimization of hybridization and washing
conditions. I used aminoally-dUTP for labeling simply because it requires less amount of
total RNA. This is particulary valuable for me because, due to the experimental limitation,
e.g. volume and cell density for the UVR treatments, I had difficulty in obtaining the
large quantity of total RNA. I was able to optimize the hybridization and washing

conditions for my system based on the protocol developed by Hegde et al. (2000). I had

14

demonstrated that my experimental condition was stringent enough for detection of
differentially expressed genes.

There are several systematic variables in a DNA microarray experiment that can
affect the measurement of mRNA levels, which include the inherent errors from sample
handling, slide to slide variation, difference in labeling or hybridization efficiency and
variation during the image analysis. Normalization is a process to minimize these
variations and establish a common base for comparison. A variety of methods have been
described for normalization (Dozmorov et al. 2004; Faller et al. 2003; Hoffmann et al.
2002; Kepler et al. 2002; Park et al. 2003; Smyth and Speed 2003; Yang et al. 2002;
Yang et al. 2001; Yoon et al. 2004). Intensity dependent normalization (often called non-
linear or LOWESS normalization) is a technique that is used to eliminate dye-related
artifacts in two-color experiments that cause the CyS/Cy3 ratio to be affected by the total
intensity of the spot. This normalization process attempts to correct for artifacts caused
by non-linear rates of dye incorporation as well as inconsistence in the relative
fluorescence intensity between some red and green dyes (Yang et al. 2002). Since I used
the two dyes (Cy5 and Cy3) for probe labeling and there are more than 4000 genes on the
array, based on recommendation from GeneSpring user manual, I used LOWESS method
for normalization.

Cluster analysis is often employed to group genes with a similar expression
pattern in a microarray based experiment. In an unsupervised mode, cluster analysis uses
algorithms to arrange genes according to similarity in their expression pattern without
applying predefined classes. In the supervised mode, the task is to construct a set of

classification rules which assigns predefined classes to given expression profiles (Brazma

15

and Vilo 2000). Current major clustering methods include hierarchical clustering (Eisen
et al. 1998), self-organizing maps (SOM) (Tamayo et al. 1999), K-means clustering
(Tavazoie et al. 1999) and principle component analysis (Alter et al. 2000). Hierarchical
clustering algorithms can be divided into two types: agglomerative and divisive. The
agglomerative method is a bottom-up approach, where the algorithm starts with 11
separate clusters and successively combines clusters until only one is left. The divisive
method, in contrast, is a top-down approach starting with one cluster and successively
splitting clusters to produce others. The algorithm used to form the clusters must be
defined. The two widely used algorithms are single linkage, which is also called nearest
neighbor, and average linkage. A distance matrix must be calculated before the clustering
is performed. The two most commonly used distance measurements are the Euclidean
distance and the Pearson correlation coefficient. In non-hierarchical cluster analysis the
data are divided into a given cluster number. The most common one is K-means, which
identifies K points that function as cluster centers. Each data point is then assigned to one
of these centers in a way that minimizes the sum of the distance between all points and
their centers. Thus, the goal of K-means is to produce groups of genes with a high degree
of similarity within each group and a low degree of similarity between groups. K-means
is particular useful to identify unique classes of genes that are up- or down-regulated in a
time dependent manner. SOM is similar to the K-means approach, but it has a
geometrical configuration and the number of nodes predefines the configuration. Thus
SOM illustrates the relationship between the groups by arranging them in a two-
dimensional map in addition to dividing genes into groups based on their expression

pattern. Since I was examining the gene expression in a time dependent manner, e. g. at 5,

16

20 and 60 min after irradiation, I used K-means for data analysis. The cluster number for
K-means analysis was determined by pre-analyzing the data using an un-supervised
hierarchical cluster method.

Since the development of microarray technology, it has been applied rapidly and
widely in many other research areas including single nucleotide polymorphism and
mutation detection (Gerry et al. 1999; Hacia et al. 1999), sequencing (Behr et al. 1999;
Cheung et al. 1998), genetic linkage analysis and population genetics (Chakravarti 1999;
Cheung et al. 1998; Gentalen and Chee 1999), comparative genomics (Behr et al. 1999;
Murray et al. 2001), phylogenetic analysis (Kakinuma et al. 2003; Polz et al. 2003;
Reyes-Lopez et al. 2003) as well as in environmental microbiology (Cho and Tiedje 2001;
Cho and Tiedje 2002; Loy et al. 2002; Peplies et al. 2004; Small et al. 2001). Microarray
technology marks a revolution in biology, and promotes biological research from gene
level to genome level.

Real time PCR technology.

Real time PCR captures the sensitivity of PCR methodology and allows the
quantification of target genes in a real-time manner by detecting the fluorescence that is
either directly or indirectly associated with the accumulation of the newly amplified DNA.
Currently fluorescence detection can be achieved by using either a double-stranded DNA
binding dye such as SYBR green or with FRET-based probes such as Taqman 5’
nuclease-sensitive probes or DNA binding probes (Ponchel et al. 2003; Walker 2002).
When irradiated by UV light, SYBR green emits a fluorescent signal if it is intercalated
into double-stranded DNA. The fluorescence emitted by the dye increases proportionally

with the amount of amplified DNA. However, SYBR green is unable to discriminate '

17

between target DNA and non-specific amplification. Thus, highly specific PCR primers
are required. The specificity of PCR amplification can be checked by generating a
melting temperature (Tm) curve after a PCR run (Ririe et al. 1997). The Tm is the
temperature at which 50% of the double stranded DNA separates. The Tm value is
dependent on the length and the nucleotide composition of the amplicon. A single peak at
the corresponding Tm of the amplicon indicates a specific amplification, whereas
additional peaks or broad peaks indicate the presence and the significance of non-specific
amplification.

Quantification by real time PCR can be absolute or relative. Absolute
quantification determines the PCR template copy number by relating the detection signal
to a standard curve. During the PCR amplification process, fluorescence values are
recorded during every cycle and represent the amount of amplified DNA at that point.
The more template present at the beginning of the reaction, the fewer number of cycles it
takes to reach a point in which the fluorescence signal is first recorded as statistically
significant above background (Gibson et al. 1996). This point is defined as the Ct, and
will always occur during the exponential phase of amplification, thus the quantification is
not affected by any reaction components becoming limited as occurs in the plateau phase.
The quantity of template DNA can be obtained by interpolation of its Ct value versus a
linear standard curve of Ct value obtained from a serially diluted standard solution. Since
it is crucial to have the same amplification efficiency of target DNA as with standard
DNA, a DNA fragment that contains the target DNA is usually used for constructing the
standard curve. In general, the standard curves are linear over more than five orders of

magnitude. Relative quantification describe the changes in nucleic acid level between

18

target DNA and a reference sample by comparing their Ct values directly without
referring to a standard curve. Thus the fold change is more important in this case.
Quantification of mRNA by real time PCR involves two steps. The mRNA is
first converted to cDNA by reverse transcription reaction, and the cDNA is used as
template for PCR amplification. An internal control gene is usually required for
normalizing the difference in reverse transcription efficiency. A gene that does not
exhibit a change in expression at the condition examined may serve as an internal control.
Common internal control genes are house keeping genes, e.g. glyceraldehyde-3-
phosphate dehydrogenase (GAPDH), albumin, B-actin, y-actin, and ribosomal rrn genes
(Thellin et al. 1999). However, the use of ribosomal genes as the internal control is a
concern due to their high abundance in the cell. For choice of a housing keeping gene, it
is important to evaluate its suitability first under the experimental condition examined
(Schmittgen and Zakrajsek 2000; Savli et al. 2003). Recently, Vandesompele et al.
(2002) recommended a normalization strategy to obtain an accurate RT-PCR expression
profiling by geometric averaging of at least three internal control genes. However, it is
not very practical since a tremendous number of reactions are added for each PCR run.
To obtain an absolute quantification of mRNA, the efficiency of reverse
transcription for the gene that is used to construct the standard curve has to be considered.
The RNA standard curve can be obtained by sub-cloning the amplicon behind a T7 or
SP6 RNA polymerase promoter and the sense RNA transcript is in—vitro transcribed.
Alternatively, the sense-strand oligodeoxynucleotides of up to 100 nt can be used for
construction of the RNA standard curve (Bustin 2000). The absolute quantification is

time-consuming, however, and not practical for a high throughput format since it requires

19

the construction of an absolute standard curve for each individual gene. Various relative
quantification methods have been developed. The standard curve method is similar to
absolute quantification; however, the standard can be any nucleic acid as long as its
concentration and length of amplicon are known. This standard curve is only used for
calibration of Ct of each target RNA (cDNA). The comparative Ct method detects the
relative gene expression with the formula 2'AACt (Livak and Schmittgen 2001). This
formula is based on the assumption that the amplification efficiencies of the target genes
and the internal control gene are the same, which is not true in most cases. A new
comparative Ct method has been proposed by Liu and Saint (2002) by simulating the
kinetics of real time PCR using experimentally determined parameters. The new method
has demonstrated improved the accuracy of quantification.

The candidate internal control genes for my experiments may come from those
house keeping genes that express constantly under my experimental conditions. The
relative abundance of the internal control is another factor to consider. I chose IdhA
(lactate dehydrogenase) because its expression level remains unchanged in MR-l afier
irradiation. In addition, the basal level of MM is close to most of genes I planned to
quantify, which allowed me to use the same concentration of cDNA for PCR
amplification and yielded a better quantification (less errors introduced by dilutions). To
compare, I also used the 16S ribosomal gene as an internal control in all analysis. Since I
was interested in the absolute abundance of NER component genes uvrA, uvrB and uer
and key DNA damage repair gene recA in MR-l before and after UVC irradiation, I

constructed the standard curves for each of them. For other genes that were used for

20

validation of microarray analysis, their cDNA copies were interperated by using the

standard curve of recA.

21

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28

CHAPTER 2
SURVIVAL OF SHEWAN ELLA ONEIDENSIS MR-l AFTER UV RADIATION

EXPOSURE

Xiaoyun Qiu, George W. Sundin, Benli Chai and James M. Tiedje (2004)

(Applied and Environmental Microbiology 70: 6435-6443)

29

Abstract

We systematically investigated the physiological response as well as DNA
damage repair and damage tolerance in Shewanella oneidensis MR-l following UVC,
UVB, UVA and solar light exposure. MR-l showed the highest UVC sensitivity among
Shewanella strains examined, with D37 and D10 values of 5.6- and 16.5% of Escherichia
coli K12. Stationary cells did not show an increased UVA resistance compared to
exponential phase cells, instead, they were more sensitive at high UVA dose. UVA
irradiated MR-I survived better on TSA than LB plates regardless the growth stage. A
20% survival rate of MR-l was observed following doses of 3.3 J rrr'2 of UVC, 568 J rn’2
of UVB, 25 kJ rn'2 of UVA and 558 J m'2 of solar UVB respectively. Photoreactivation
conferred an increased survival rate to MR-l as much as 177-365 fold, 11- 23 fold and 3-
10 fold following UVC, UVB and solar light irradiation, respectively. A significant UV
mutability to rifampin resistance was detected in both UVC and UVB treated samples
with'the mutation frequency in the range Of 10 '5 to 106. Unlike in E. coli, the expression
of the nucleotide excision repair (NER) component genes uvrA, uvrB and uer was not
damage inducible in MR-l. Complementation of Pseudomonas aeruginosa UA11079
(uvrA') with uvrA of MR-l increased the UVC survival of this strain more than three
orders of magnitude. Loss of damage inducibility of the NER system appears to
contribute to the high sensitivity of this bacterium to UVR as well as other DNA-

damaging agents.

30

Introduction

Solar ultraviolet radiation (UVR) is lethal and potentially mutagenic to all
organisms at species-specific levels. The stratospheric ozone layer absorbs UVC (<290
nrn) effectively, however, both UVA (320 to 400 nm) and UVB (290 to 320nm)
wavelengths penetrate to the earth’s surface. UVR-induced damage is greatly dependent
on the sources of radiation and the time of exposure. Photons of UVB and UVC
wavelengths cause direct DNA damage by inducing the formation of DNA photoproducts
such as cyclobutyl pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidinone ((6-4)
PD) (37). The accumulation of DNA photoproducts can be lethal through the blockage of
DNA replication and transcription. UVA can cause photodamage to a variety of
molecules as well as physiological processes directly or indirectly by inducing the
production of reactive oxygen species (ROS) (5, 6, 18, 53). Distinct differences between
Far- (UV C) and Near-UV (UVA and UVB) damage have been observed in both bacteria
and bacteriophages (6).

Bacteria are particularly vulnerable to UVR damage due to their small size and
unicellular structure. Thus, the possession of mechanisms to repair UVR-induced damage
as well as other sheltering strategies, e.g. pigments and sunscreen molecules, are essential
contributors to the ecological fitness of organisms that are regularly exposed to solar
UVR. Several mechanisms have evolved in bacteria to repair or tolerate UVR-induced
DNA damage. Photoreactivation and nucleotide excision repair (NER) are the two
primary mechanisms that accurately repair UVR-damaged DNA whereas mutagenic
DNA repair (MDR) is a determinant that increases damage tolerance (11). In addition,

many of the genes involved in DNA damage repair are inducible through the SOS

31

response (29). Approximately 30 genes were reported to belong to the SOS regulon of E.
coli. (4, 8).

Photoreactivation in bacteria involves a single enzyme called photolyase, which
binds to CPDs and, in the presence of light (300-500 nm), reverses the dimer to its
component monomers (26). CPD photolyases are widely but also sporadically distributed
among Bacteria, Archaea and eukaryotes (56). NER is present from bacteria to humans
and plays a critical role in protecting cells from a variety of DNA-damaging agents since
it can recognize a broad range of DNA lesions including ionizing radiation induced
purine damage, active oxygen species induced base loss and UV induced pyrimidine
dimers (41). During the repair process, NER component enzymes hydrolyze two
phosphodiester bonds, one on either side of the lesion, to generate an oligonucleotide
carrying the damage. The excised oligonucleotide is released from the duplex, and the
resulting gap is filled and ligated (28, 42, 43). In E. coli, the NER component genes uvrA,
uvrB, and uer are subject to SOS regulation (4, 10, 22, 23, 45). UmuDC-mediated MDR
functions in translesion synthesis enabling bypass of DNA lesions that would normally
block replication by DNA polymerase III (46, 50, 51). Translesion DNA synthesis
provides the cell with an additional mechanism of survival, although the process is
accompanied by an elevation of the cellular mutation rate (46, 55). Expression of the
umuDC operon is regulated by the SOS response in many bacteria (46).

Shewanella oneidensis MR-l, a Gamma proteobacterium, was originally isolated
from the sediment of Oneida Lake, NewYork State (35). Extensive studies have been
carried out on this bacterium due to its respiratory versatility: it can reduce a variety of

compounds including toxic metals and radionuclides (30, 31). This unique feature offers

32

potential for bioremediation by immobilization of soluble metal species at contaminated
sites. To succeed, MR-l has to tolerate toxic levels of pollutants, and exposure to
ionizing or solar radiation. Recently, the genome sequence of MR-l has been completed
(15). It consists of a 4,969,803-bp chromosome with 4,758 predicted ORFs and a
161,613-bp plasmid with 173 ORFs. Three prophages, lambdaSo (51,857 bp), MuSol (34,
551bp) and MuSoZ (35,666 bp) are present on MR-l chromosome (15). Compared to
Escherichia coli K12, MR-l has most of genes in repairing DNA damages and defending
oxidative stress. Knowledge on bacterial UV resistance and repairing mechanisms is
predominantly from Escherichia coli. Limited knowledge on molecular and physiological
responses to UVR is available for environmentally relevant bacteria. Here, we report the
responses of MR-l following UVC, UVB, UVA and natural sunlight exposure. We found
that MR-l was uniformly sensitive to all wavelengths of UVR. We also evaluated the
contribution of photoreactivation, nucleotide excision repair and mutagenic repair to the
survival of MR-l following UVR exposures. An inefficiently expressed NER system in

MR-l appears to contribute to its high sensitivity to both UVB and UVC.

Materials and methods

Bacterial strains, plasmids, and culture conditions. The bacterial strains,
plasmids, and PCR primers used in this study are listed in Table 2.1. E. coli and
Pseudomonas aeruginosa strains were grown in Lmia-Bertani medium (pH 7.2) at 37 °C.
All Shewanella strains were grown at 30 °C in tryptic soy broth except S. algae, which
was grown in a modified marine broth (5 g peptone, 2 g yeast and 17 g sea salts in 1 liter,

pH 7.2). For gene expression experiments, S. oneidensis MR-l was grown in Davis

33

medium (Difco) supplemented with 15 mM of lactic acid. Ampicillin (100 pg ml'l) was
used to grow E. coli carrying plasmids pJJK20, pJB321, pXQOl and pXQO3 whereas
carbenicillin (200 pg ml'l) was used to grow P. aeruginosa carrying the plasmids
described above.

Molecular techniques. Genomic and plasmid DNA isolation, restriction
digestion, gel purification, ligation and transformation were performed using standard
techniques (40). PCR primers (Table 2.1) were designed using the Primer 3 program
(http://www.broad.mit.edu/cgi-bin/primer/primer3.cgi/) and synthesized at the Genomic
Technology Center of Michigan State University.

UV irradiation, photoreactivation and MDR assays. UVA, UVB, and UVC
assays were performed using previously described methods (47, 49). The UVA, UVB,
and UVC sources used were XX-15L, XX-15M, and XX-15 lamps (UV P Products; San
Gabriel, Calif), respectively. The energy output of each lamp was monitored with a UV-
X radiometer (UVP Products) fitted with the appropriate sensor. The UVB lamp was
filtered through cellulose diacetate (Kodacel; Eastman Kodak; Rochester, NY) to
eliminate stray UVC wavelengths. During irradiation, cell suspensions were mixed
continuously to avoid shading effects. In experiments comparing the UVA sensitivity at
different physiological stages, cells grown in Davis medium to exponential phase were
used directly for UVA treatment whereas stationary phase cells were diluted with Davis
medium to an OD600 of about 0.2 (the density at mid-exponential phase in Davis
medium). Photoreactivation assays and MDR assays were conducted as described

previously (24, 25).

34

Solar radiation sensitivity assays. Solar radiation sensitivity assays were
conducted by exposing cell suspensions to ambient solar radiation. The suspensions were
maintained in sterile boxes constructed of 64 mm thick Acrolyte OP-4 plastic
(Professional Plastics, Austin, TX). The Acrolyte OP-4 plastic transmits greater than
90% of the total radiation throughout the UVA and UVB wavelengths (Acrolyte OP-4
technical data sheet; Cyro Industries, Arlington, NJ). Replicate boxes were maintained
on ice on a rocking platform during the exposures. Solar UVB radiation was measured
with a UVB detector (SED240/UVB-1/W) attached to an IL-17OO research radiometer
(International Light, Newburyport, Mass). UVB radiation was measured every second,
and the readings were integrated over the exposure period yielding a quantitative output
in J m'z. At appropriate time points, the boxes were temporarily shaded fiom sunlight
exposure, and two samples (5 ml) were taken. One sample was plated in the dark and the
other was plated following a photoreactivation treatment as described previously (25).

Transcriptional analysis NER using a cDNA microarray. S. oneidensis MR-l
whole genome DNA arrays were produced by Liyou Wu and J izhong Zhou at Oak Ridge
National Laboratory (Oak Ridge, TN). Mid-exponential phase cells (80 ml) grown in
Davis medium were split to two parts, one was used for UVR treatments and the other
was used as controls. The exposure doses were 3.3 J m'2 for UVC, 568 J m'2 for UVB and
25 K1 rn'2 for UVA, which yielded about 20% survival rate. After irradiation, cells were
transferred to a 100 ml flask, and incubated at 30 °C on a shaker (200 rpm). An aliquot of
cells (12 ml) was transferred to a centrifuge tube after 5, 20 and 60 min of incubation,
concentrated by centrifugation at 4 °C. The cell pellet was resuspended in 2 ml of

supernatant and mixed with 4 ml of bacterial RNA protection reagent (Qiagen, Valencia,

35

CA). The cell suspension was kept at room temperature until all the samples were
collected (within 2 h). Cells were then pelleted and stored at -80°C until RNA extraction.
Controls were treated in the same way except the UVR irradiation. Total RNA was
isolated using a Qiagen RNeasy mini Kit (Qiagen), digested with RNase-free DNaseI
(Invitrogen, Carlsbad, CA ) at 25°C for 30 min, extracted with phenol, phenol:
chloroform (1:1), and chloroform, and stored in ethanol at -80°C until use. Both PCR and
gel electrophoresis were used to confirm the complete digestion of any contaminating
DNA. We confrrrned both RNA purity and quality by the 260 nm to 280 nm absorbance
ratio and gel electrophoresis before the reverse transcription reaction. Prehybridization
and RNA labeling were performed as described by Schroeder et al (44) with a 2:3 ratio of
5-(3-aminoallyl)-dUTP and dTI'P. Hybridization and washing were carried out as
described by Hegde et al (14). The array was scanned using Axon 4000B scanner (Axon
Instruments, Inc. Union City, CA). The data were imported into GeneSpring (Silicon
Genetics, Redwood City, CA) for analysis. Data were normalized both per chip and per
gene (Lowess method). Spots with less than 55% of pixels great than background plus
28D were not include in data analysis (34).

Functional analysis of uvrA in P. aeruginosa strain UA11079. The uvrA gene
(SO4030, Gene Bank accession No: NP_719560) from S. oneidensis MR-l was amplified
from 50 ng of genomic DNA using primers uvrA NdeI 5’ and WM BamHI 3’ (Table 2.1),
and cloned into pJJK20 (25), creating plasmid pXQOl (Tablel). A 3.6-kb Sphl and
BamHI fragment from pXQOl containing the 0.75-kb umuDC promoter from E. coli and
the 2.85-kb uvrA gene from MR-l was cloned into pJB321, creating pXQ03 (Table 2.1).

pXQ03 was transferred from E. coli DHlOB to P. aeruginosa UA11079 by tri-parental

36

mating as described by Kim and Sundin (25). The survival after UVC exposure was

assayed as described above.

37

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Results

UVC sensitivity in Shewanella strains. The sensitivity to UVC of five
Shewanella strains from different natural habitats was examined. Escherichia coli strain
K12 was used as the control (Figure 2.1). Both S. algae and S. oneidensis strain MR-4
were more tolerant to UVC radiation whereas S. putrefaciens 200 and S. oneidensis
strains DLM-7 and MR-l were more sensitive to UVC radiation. To compare the degrees
of resistance of the five strains with UVC treatments, we calculated the D37 and D10
values from the regression line of the exponential slope of each survival curve (Table 2.2).
S. algae showed the highest UVC resistance with a D37 of 4.0 J In2 and a D10 of 8.2 J m’z,
respectively, which was about 74.1 and 79.6 % of that for E. coli K12. S. oneidensis strain
MR-l showed the highest UVC sensitivity with a D37 of 0.3 J m'2 and a D10 of 1.7 J m'z,
respectively, which was about 5.6 and 16.5 % of E. coli K12 values (Table 2.2). The
UVC resistance and sensitivity within the Shewanella genus correlated well with the
radiation exposure in the habitat from which they were isolated. Both MR-l and DLM-7,
isolated from lake sediment, and S. putrefaciens 200, isolated from crude oil pipeline,
were from habitats with limited solar radiation exposure, whereas S. algae, isolated from
the surface of a red alga, and S. oneidensis MR-4, isolated from the top 5 m of the Black

Sea, were from habitats with more solar radiation exposure (Table 2.1).

40

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0.0001 1 i 1 I
0 5 10 15 20

we dose (J m ‘2)

 

Figure. 2.1. Survival of E. coli K12 and Shewanella strains after UVC irradiation. (I): E. coli K12; (O ): S.
algae; (D): S. putrefaciens 200; (A )2 S. oneidensis MR—4; (o): S. oneidensis DLM-7; (A): S. oneidensis
MR-l. Plates used for measuring CFU are LB for E. coli K12; marine agar for S. algae and TSA for others.
Each datum represents the mean (:t: the standard error of the mean) from three replicates.

UVA sensitivity in S. oneidensis MR-l. Sensitivity to UVA has been reported to
depend greatly on the physiological conditions of the cell. Exponential cells were more
sensitive to near-UV damage than stationary cells due to their active DNA replication (6),
while the stationary phase triggers numerous protective pathways as well as enzymatic
activities expected to confer some degree of UVA resistance to cells (6, 7, 32). Since
UVA induces oxidative damage to cells, the survival rate is greatly dependent on the

medium used to recover after irradiation. MR-l survived much better on TSA plates than

42

LB plates for both exponential and stationary cells (Figure 2.2). No significant difference
in UVA sensitivity was observed between exponential cells and stationary cells at lower
UVA doses. At high UVA dose, exponential cells were slightly more resistant to UVA.

The difference in survival rate on LB plates was more than 10-fold at the dose of 30 U m‘

2 (Figure 2.2).

100

10‘

Percent survival

 

0.1 :

 

 

0.01 , j . , . ,
0 1o 20 30
UVA dose (kJ m'z)

Figure 2.2. Survival of S. oneidensis MR—l after UVA irradiation. Both log— and stationary phase MR-l
grown in Davis medium were irradiated with various UVA doses and plated on both LB and TSA plates to
measure CFU. (A ): exponential, LB plates; (A): exponential, TSA plates; (I): stationary phase, LB plates;
([1): stationary phase, TSA plates. Each datum represents the mean (is the standard error of the mean) from
at least three replicates.

Contribution of photoreactivation to survival of S. oneidensis strain MR-l
after UVR exposure. Annotated photolyase (512 aa) in S. oneidensis MR-l shares 44%
identity to that of E. coli K12 (472 aa). The amino-terminus contains the conserved

domain for binding light harvesting cofactor and the carboxyl terminus contains the

43

conserved FAD binding domain of DNA photolyase. The tryptophans at enzyme active
sites (W306 in E. coli K12) and the one involved in substrate Pyr<>Pyr specific binding
(W277 in E. coli K12) are conserved in the photolyase of MR-l (W342 and W312 in
MR-l, respectively), which may indicate a similar catalyzing mechanism with that of E.
coli K12 (26). Photoreactivation conferred a significantly increased survival rate to S.
oneidensis MR-l in both UVB and UVC irradiated cells: as much as 177- and 365-fold
after irradiation at UVC doses of 12 and 15 J m'2 (Figure 2.3A) and 11- to 23-fold after
irradiation at UVB doses of 774 to 1032 J m'2 (Figure 2.38). For solar light irradiated
cells, further incubation under visible light for 1h increased the survival rate 3- and 10-
fold at solar UVB doses of 640 and 800 J .m'2 (Figure 2.3C) compared to those plated in

the dark immediately after treatments.

44

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MDR activity in S. oneidensis MR-l. The umuDC operon in S. oneidensis MR-l
is located on the mega plasmid. The by-product of MDR, an increase in cellular mutation
frequency, can be assayed by examining the increase in the occurrence of spontaneous

mutants following irradiation. We examined the occurrence of Rif mutants in both UVC

and UVB treated samples (Figure 2.4). The overall frequency was slightly higher in UVC
treated samples (Figure 2.4A) than those in UVB treated samples (Figure 2.43) over the
UV dose-range used in this study. A mutation frequency as high as 6.6 x 10'6 was
observed at 16.5 J m'2 of UVC (Figure 2.4A). This result indicates that MDR-mediated

translesion synthesis is fimctional in S. oneidensis MR-l.

 

 

 

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O- 200« 100‘
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0 5101s 20 0 50010001500
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Figure 2.4. Analysis MDR in S. oneidensis MR-l following UVC (A) and UVB (B) exposure. The number

of spontaneous mutations conferring RitI (rifampin resistance) in the absence of UVR irradiation has been
subtracted. LB plates were used for measuring CFU. Each datum represents the mean (:t the standard error
of the mean) from three replicates.

46

Expression of NER component genes after UVR exposure. Expression of NER
component genes (uvrA, uvrB and uer) after UVC, UVB and UVA irradiation were
examined using a microarray that contains about 95% of MR-l open reading frames. In
contrast to NER system of E. coli, we did not observe any induction in any of the three
UVR treatments at any of the incubation times. The ratio of irradiated sample to control
(unirradiated sample) was in the range of 0.9-1.2 (Figure 2.5). To confirm that the uvr
genes are truly transcribed, we designed the gene specific primers that targeted both
amino terminal and carboxyl terminal fragments of uvrA, uvrB and 14er (Table 2.1).
Positive amplicons were detected in all UVR irradiated cells as well as in the controls
(unirradiated samples) by RT-PCR (data not shown). This result indicates that, unlike in
E. coli K12, the uvrA, uvrB and uer of MR-l were not damage inducible. In agreement
with above observation, we were unable to identify any E. coli-like SOS box near the
translation region (-200 to +40) (8, 27) for all three genes examined whereas putative
LexA binding sites were found for recA, lexA, umuDC and dinP, a homolog of umuC,

with a relative low HI value (Table 2.3).

47

Ratios of gene expression levels between samples and controls

 

 

Genes

Figure 2.5. Relative gene expression of NER component genes uvrA, uvrB and uer at 5 min (filled with
dots), 20 min (filled with stripes) and 60 min (filled with lines) afier UVC (A), UVB (B) and UVA (C)
irradiation. Ratios were UVR irradiated samples to unirradiated samples at the same time points. Each
datum is the mean (:t the standard error of the mean) of eight to twelve of data points from three biological
replicates and two technical replicates.

48

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49

Functional analysis UvrA of S. oneidensis MR-l. To confirm that the NER
system of MR-l is truly functional, we attempted to complement Pseudomonas
aeruginosa UA11079 (uvrA') (Table 2.1) with uvrA of MR-l. Since we were not sure if
the promoter of uvrA from MR-l was functional in P. aeruginosa, we used the umuDC
promoter from E. coli, which has been demonstrated to be functional in P. aeruginosa
(24), for the expression of uvrA from MR-l. Complementation increased the UVC
survival of the mutant more than three orders of magnitude, but not to the level of the
wild-type PAOl strain (Figure 2.6). The D37 (0.43 J m'z) and Dl0 (2.90 J m'z) values of
the complemented strain were about 11.5- and 42.8% of that for PAOl (D37: 3.73 J m'2
and D10: 6.78 J m'z). Nonetheless, this result demonstrates that UvrA from MR-l is
functional in repairing UVC induced damage although the efficiency is not as high as

with UvrA from PAOl.

50

100 4

0 1 d
. .
.
4
.

Percent survival

0.01 J,

 

 

 

0,001 a ...................

UVC dose (J m")

Figure 2.6 Complementary analysis of uvrA of S. oneidensis MR-l in P. aeruginosa UV11079 (uvrA
deficient) alter UVC irradiation. Figure shows the survival of P. aeruginosa UA11079 (A ), P. aeruginosa
UA11079 complemented with uvrA of MR-l(o) and P. aeruginosa PAOl (O) afier UVC irradiation. LB
plates were used for measuring CFU. Each datum represents the mean (i: the standard error of the mean)
fi'om three replicates.

Discussion

We evaluated the phenotypic responses important to all relevant wavelength
groups of UVR and solar UVR in the environmentally relevant bacterium S. oneidensis
MR-l. An analysis of the MR-l genome (NC_004347 and NC_004349) indicated that
this organism possesses genes that could encode major DNA repair systems including
nucleotide excision repair and recombinational repair, and that MR-l also encodes a
photolyase enzyme and a plasmid-borne mutagenic DNA repair determinant. Regarding

UVA survival, MR-l contains several genes encoding proteins relevant to the removal of

51

reactive oxygen species such as catalase ($00725, 804405, 8017712), superoxide
dismutase ($02881), and proteins of the organic hydroperoxide resistance (0hr) family
($00976, $03409) et al. The potential use in bioremediation, the availability of the
genome sequence, and the phylogenetic relationship of S. oneidensis to other well-
characterized organisms, suggest that this strain is an effective model for physiological
and genetic studies of UV and ionizing radiation effects on an environmental bacterium.
While the UVC resistance and sensitivity within the Shewanella genus correlated
well with the radiation exposure in the habitat from which they were isolated, the uniform
sensitivity of S. oneidensis MR-l to UVA, UVB, UVC and solar UVR may or may not be
a result of lack of UVR exposure. For example, bacteria regarded as tolerant or resistant
to UVR have been recovered from solar—radiation exposed habitats including aquatic
habitats and the plant phyllosphere (17, 21, 47), but little or no correlation was observed
between UVR resistance and the natural levels of solar radiation exposure (12). ‘Great
variability in sensitivity to UVR was observed from marine bacterial isolates (2, 21).
UVR-tolerant organisms with active photoreactivation mechanisms were prevalent from
deep subsurface bacteria which have been screened from solar radiation for more than
one million years (1). Thus, the habitat of isolation is not always an indicator of the UVR
sensitivity of an organism. The uniform UVR sensitivity of MR-l , however, could not be
explained by gene content either. MR-l possesses most of important repair pathways and
determinants compared to phylogenetically related E. coli, and has even more DNA
repair genes than D. radiodurans, a radiation extremely resistant bacterium

(http://www.usuhs.mi1/pat/deinococcus/FrontPage_DR_Web_work/Pages/DNA_repair/d

52

na_repair__pathways.htm). However, the resistance to UVC of D. radiodurans is more
than three magnitudes higher than that of MR-l (12, 33).

The sensitivity to DNA-damaging UVC and UVB wavelengths in MR-l could be
offset by photoreactivation. The contribution of photoreactivation to MR-l survival is
very similar to that observed in other bacteria (25, 56). Photoreactivation makes a larger
contribution to survival following irradiation with UVB or UVC wavelengths in vitro
compared to the increase in survival following exposure to solar UVR. This result is
probably due to the additional lethal effects of UVA wavelengths present in solar UVR
but also has implications for physiological studies aimed at determining the ecological
importance of photoreactivation in microbial communities (20). Dramatic difference in
survival rate between LB and TSA plates following UVA exposure indicates potential
membrane damage caused by UVA (18, 53). We also observed additional decrease in
survival rate when the irradiated MR-l was plated on old LB plates (relative dry).
Sensitivity to UVA radiation in MR-l was also dose dependent. At lower doses, the
survival of exponential phase and stationary phase cells was similar whereas exponential
phase cells were more resistant to higher radiation doses. This result agrees with findings
in studies using 4-thiouridine mutants that showed mutants possessing more DNA
replication forks (similar to exponential growth cells) are more resistant to high UVA
doses than are wild-type bacteria (19). This could explain the dramatic change in UVA
induced photodamage at lower and higher UVA doses.

The plasmid-encoded MDR determinant umuDCs0 contributed to UVR-induced
mutability in MR-l, but the contribution of this determinant to UVR survival is unclear.

Although most MDR determinants transiently increase the mutation rate of cells

53

following UVC irradiation, the contribution of these determinants to increased cell
survival is only apparent in some cases. For example, the MDR determinant rulAB
confers tolerance to UVC wavelengths in P. syringae (48), but deletion of MDR
determinants such as umuDC and samAB from E. coli and Salmonella typhimurium,
respectively, does not affect their cellular UVC sensitivity (36, 55).

Our investigation on the sensitivity to DNA damaging UVC and UVB
wavelengths centered on the NER system of MR-l. This system is probably functional as
organisms harboring mutations in NER component genes (e.g. uvrA, uvrB) are typically
exquisitely sensitive to UVC (41). Indeed, we confirmed the functionality of UvrA
through the ability of this protein to complement the UvrA defect in P. aeruginosa
UA11079 (Figure 2.6). Loss of the damage inducibility of the NER system in MR-l may
contribute to the UVR sensitivity of this organism. For example, in E. coli, the
expression of the uvrA, uvrB, and :4er genes is significantly induced following DNA
damage. However, in P. aeruginosa, an organism that is more sensitive to UVC than E.
coli, both uvrA and uvrB are not DNA damage inducible although this bacterium
possesses an SOS-like system (38, 39). In MR-l, we observed strong SOS induction
following UVB or UVC exposure which included increases in transcript levels of lexA,
recA as well as the umuDC operon (unpublished data). The gene expression level of uvrA,
uvrB and uer, however, remained constant following DNA damage.

We next examined the regulation of NER component genes among five organisms
that are phylogenetically related to S. oneidensis, including E. coli, Haemophilus
influenzae, Pasteurella multocida, Pseudomonas aeruginosa, and Vibrio cholerae (15).

Since LexA and RecA are highly conserved among these bacteria, it is reasonable to

54

hypothesize that a similar mechanism is present in the regulation of the SOS response.
Using the E. coli SOS box consensus sequence and three SOS box searching patterns (8),
we searched for putative SOS box near to a putative translation start codon (-200 to +40)
of either uvrA, uvrB or 14er gene in five organisms. As expected, an SOS box was
identified for all three genes in E. coli (Table 2.4). In Vibrio cholerae, a strong putative
SOS box was identified upstream of the uvrA gene, but no putative SOS box was
identified upstream of both uvrB and 14er (Table 2.4). Relatively strong putative SOS
boxes were identified upstream of both uvrA and uer but not uvrB in both Haemophilus
influenzae and Pasteurella multocida (Table 2.4). Similar to MR-l, no putative SOS box
was identified upstream of uvrA, uvrB or uer in P. aeruginosa PAOl (Table 2.4). In
agreement with their UVC sensitivity, both S. oneidensis MR-l and P. aeruginosa PAOl
lost the damage inducibility of the NER system. Alternatively, the functional efficiency
of the UvrABCD complex in NER may be diminished in both P. aeruginosa PAOl and S.
oneidensis MR-l. Further work is needed to understand the evolution and maintenance

of NER in these organisms.

55

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It is very well known that UVR can induce prophage into lytic cycle. Kidambi
reported that UVB can activate D3 prophage in Pseudomonas aeruginosa in a RecA
dependent manner (16). The novel Shewanella phage lambdaSo shares syntenic regions
with Pseudomonas aeruginosa D3 and enterobacteria HK022 (15). Whether or not
activation of prophage(s) on MR-l genome contributes to its high sensitivity to UVR
needs be investigated.

Despite possessing the relevant repertoire of oxidative damage repair genes, the
results of our study indicate that S. oneidensis MR-l is one of the most UVA-sensitive
organisms known. Genome analysis showed that MR-l has more c-type cytochromes
than many organisms including E. coli, V. cholerae, and P. aeruginosa (15).
Cytochromes, along with flavins, quinones, are potential chromophores for UVA (5, 18,
53). Whether or not the high cytochrome content of MR-l contributes to its high UVA
sensitivity needs detailed investigation. As expected, MR-l is also highly sensitive to
ionizing radiation (Michael Daly, personal communication). The radiation sensitivity of
MR-l may pose potential problems for environmental uses of this strain or its indigenous
relatives in bioremediation of toxic metals or radionuclides since a variety of DNA-
damaging agents as well as ionizing radiation may be present at contaminated sites.
Relatively little is known of the interrelationship of genetic systems and mechanisms
involved in repairing cellular damage caused by UVR and ionizing radiation in organisms
other than D. radiodurans. MR-l is an excellent model to compare and understand the
cellular function and regulation in response to various radiation stresses. This knowledge
will contribute greatly to our fundamental understanding of the important traits in

determining bacterial radiation resistance.

57

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62

CHAPTER 3
COMPARATIVE ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES IN
SHEWANELLA ONEIDENSIS MR-l FOLLOWING EXPOSURE TO UVC, UVB

AND UVA RADIATION

63

Abstract

I delineated the cellular response of Shewanella oneidensis MR-l to ultraviolet
radiation damage by analyzing the transcriptional profile following UVC (254 nm), UVB
(290-320 nm) and UVA (320-400 nm) irradiation at a dose that yields 20% survival rate,
respectively. About 8% of the MR-l genome was differentially expressed in response to
UVA whereas only about 4% of the genome showed differential expression after UVC or
UVB exposure. The response to UVA was immediate with most genes showing induction
at 5 min. In contrast, the response to UVC was relatively slow with most genes showing

induction at 60 min. Two induction peaks were observed after UVB exposure, at 5 min
and at 60 min. Almost 70% of UVB-induced genes were up-regulated in the UVC
treatment whereas only about 40% of UVB-induced genes were up-regulated in the UVA
treatment. Although the SOS response was observed in all three treatments, the induction
was more robust in response to short-wavelength UVR (UV B and UVC). Similarly, more
prophage-related genes were induced by short-wavelength UVR. MR-l showed an active
detoxification mechanism in response to UVA, which included the induction of
antioxidant enzymes and iron sequestering proteins to scavenge reactive oxygen species.
The activation of prophages by UVC and UVB and the induction of multing and heavy
metal efflux pumps and production of toxins following UVB and UVA irradiation

I'liglllight the differences in response to stress induced by different wavelengths of UVR.

64

Introduction

The deleterious effect of ultraviolet radiation (UVR) is highly dependent on the
wavelength of radiation. DNA is the major chromophore following exposure to short-
wavelength UVR. Both UVC (< 290 nm) and UVB (290-320 nm) can induce the
formation of cyclobutyl pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidinone ((6-
4) PD) photoproducts, which are mutagenic and lethal to bacteria if unrepaired (36).
Damage induced by long wavelength UVR is more complex since a variety of non-DNA
photoreceptors with Kmax in the range of 290-400 nm are present in the cell (11, 21). In
addition, both UVB and UVA can produce reactive oxygen species (ROS), causing
oxidative damage to a variety of molecules in the cell (10, 11).

Bacteria have evolved various mechanisms to cope with UVR-induced damage.
In Escherichia coli, both photoreactivation and nucleotide excision repair (NER) are
highly efficient in removing CPDs (23, 40) whereas recA-mediated recombination repair
can bypass CPDs during DNA replication, thus improving DNA damage tolerance (14,
27). The LexA-RecA mediated SOS response is a global response to DNA damage
involving the induction of more than 30 unlinked genes, many of which are involved in
DNA replication and repair and in the control of cell division (8, 24). E. coli also
possesses a variety of glycosylases to repair oxidative DNA damage through the base
excision repair (BER) pathway (14). In addition, several regulatory genes are involved in
protecting cells from oxidative stress. For example, OxyR, a LysR family protein, can be
converted into a transcriptional activator by the formation of a disulfide bond between
two reactive cysteine residues (52), activating the transcription of genes involved in

peroxide metabolism and protection (katG, ahpC, ahpF, dps), in redox balance (gar, grxA,

65

ter) and genes encoding regulators such as fur and oxyS (42). The E. coli SoxRS
regulon provides defense against oxidative damage caused by superoxide anions.
Regulation of the soxRS regulon occurs by two-step transcriptional activation. First,
SoxR is oxidized and becomes an active form, which can stimulate the transcription of
soxS. SoxS in turn activates transcription of target genes by binding to their promoter
region (34, 49). More than 10 genes including nfo (endonuclease IV) and sodA (Mn-
superoxide dismutase) belong to the SoxRS regulon (2). Sigma factor 38 (rpoS) is
another important regulator in E. coli in response to oxidative stress (20). Some genes
that are under control of OxyR are also regulated by RpoS (12). Similar oxidative stress
regulators have been identified in many other bacteria as well as pathogenic bacteria (7, 9,
30, 35, 39, 45).

Although extensive studies have focused on distinguishing different genes and
regulons in response to far UV (UV C) and near UV (UV B and UVA), global genetic
information remains limited due to the complexity of UVR-induced damage and
limitations in the technologies used in the past studies. Microarray technology, however,
allowed me to systematically investigate the global transcriptional response to different
UVR wavelengths and hence enhance our understanding of the effects of global damage
induced by different wavelengths of UVR.

Shewanella oneidensis MR-l, an environmental Gamma Proteobacterium, can
reduce a variety of compounds including toxic metals and radionuclides (26, 29).
Previous data indicated that MR-l is highly sensitive to all wavelengths of UVR, solar
UV and ionizing radiation (38). However, this sensitivity could not be explained by its

genome content. Similar to E. coli, which is more radiation resistant, MR-l encodes the

66

major DNA damage repair and damage tolerance systems including SOS response,
recombination repair, mutagenic repair, nucleotide excision repair, mismatch repair and a
DNA photolyase (17). MR-l also encodes a suite of genes potentially involved in
protection from UVA-induced oxidative stress including moS and a homolog of OxyR
(801328). For scavenging ROS, MR-l has genes encoding for catalase (katB),
catalase/peroxidase (katG-l and katG-2), organic hydroperoxide resistance protein (0hr),
alkyl hydroperoxide reductase (ahpC and ahpF), and a Dps protein (dps). For repair of
oxidative DNA damage, the MR-l genome contains putative genes of tag, ung, mutM,
mutY, mutT, nth and xthA that are important in removing the damaged bases (17).

S. oneidensis MR-l, with its response to UVR previously characterized (38), and
full genome sequence known, represents an excellent candidate bacterium for a
comprehensive analysis of genomic response to varied UVR wavelengths. Here, I
examined the global gene expression profiles in response to UVC (254 nm), UVB (290-
320 nm) and UVA (320-400 nm) in MR-l using a whole genome microarray containing
approximately 95% of total ORFs. My results indicate there are similarities in genomic
response between MR-l and E. coli; however, there are distinct differences which may
contribute to the increased UVR sensitivity of MR-l. In addition, induction of multidrug
and heavy metal efflux pumps and production of toxins following UVA irradiation

highlights previously unknown phenotypes for this stress.

Materials and methods
S. oneidensis MR-l whole genome cDNA array. The S. oneidensis MR-l

whole genome cDNA arrays containing about 95% of total S. oneidensis MR-l ORFs

67

were produced at Oak Ridge National laboratory (15). In brief, a total of 4197 PCR
amplicons and 451 Oligonucleotides were deposited onto Corning Ultra GAPS slides
(Corning, Corning, NY) using a Microgrid II arrayer (Matrix, Hudson, NH) with 16 (4 x
4) SMP2.5 pins (Telechem, Inc., Sunnyvale, CA). The arrays were printed with two
replicates, each containing a 4 x 4 subgrid with the spot distance of 210 microns and the
spot size of 140-180 microns. A total of 276 control spots including black (no DNA
deposited) and 10 different Arabadopis genes (Strategene, La Jolla, CA) and 4 genomic
DNA at each subgrid were also included on the array. After cross-linking the DNA to the
surface of array by UV (250 m1) using a Stratagene Stratalinker (Strategene), arrays were
stored in a desiccator.

Microarray hybridization and data analysis. Gene expression profiling
experiments were performed as described previously (38). Briefly, MR-l was grown in
Davis medium with 15 mM lactate as carbon source until OD500 reached 0.2-0.3. The
culture was split into two portions. One was used for UVR irradiation (3.3 J m'2 for UVC,
568 J m'2 for UVB, and 25 kJ m'2 for UVA) and the other one was used as control. After
irradiation, both samples and controls were incubated at 30°C on a shaker. Cells were
collected at 5 min, 20 min and 60 min of incubation for RNA extraction. Both UVC and
UVB samples were collected in a dark room to avoid photoreactivation. Prehybridization
and RNA labeling were performed as described by Schroeder et al. (41) with a 2:3 ratio
of 5-(3-aminoallyl)-dUTP and dTTP. Hybridization and washing were carried out as
described by Hegde et al. (16). At each time point of each treatment, six hybridizations
from three biological replicates and two technical replicates (dye-swap) were performed.

GENESPRING 6.0 software (Silicon Genetics, Redwood City, CA) was used to analyze

68

all microarray hybridization data. Only those spots with more than 55% of pixels greater
than background plus 2SD (standard diveration) in either the cy5 or cy3 channel were
used for analysis (28). Data were normalized both per chip and per gene (Lowess
method). Those genes that showed a statistically significant change in gene expression
(P<0.05) and a > 2-fold change in magnitude were regarded as significant. The number of
clusters for K-means analysis was determined by pre-analyzing data using hierarchical
cluster analysis in GeneSpring.

Quantitative real time reverse transcription PCR (Q RT-PCR). Q RT-PCR
analysis were performed for 12 selected genes (Table 3.1) using the same RNA samples
as used for microarray analysis. Two micrograms of total RNA from each sample was
converted to cDNA in the same condition as used for the microarray experiment except
that dTTP instead of a mixture of aa-dUTP and dTTP was used. Afier hydrolyzing total
RNA, total cDNA was purified using Qiagen PCR purification kit (Qiagen, Valencia, CA)
and quantified using a spectrophotometer. Gene specific primers (Table 3.1) were
designed using Primer Express® 1.0 software (Applied Biosystems, Foster City, CA). All
amplicons were in the range of 90-100 bp. The specificity was first checked by blasting
the primer sequences against the MR-l genome. Both primer and template concentration
for each gene were optimized in 1X SYBR Master Mixture (Applied Biosystems) using
an ABI 7900HT (Applied Biosystems) Sequence Detection System (Table 3.1). The
reaction specificity was further confirmed by examining the dissociation curves after
each PCR run. Standard curves for recA, uvrA, uvrB and uer were constructed using
purified PCR products. Since I am interested in absolute quantification of NER

component genes, copies of uvrA, uvrB and uer in each sample were interpolated from

69

their corresponding standard curve. For other genes, copies were calculated from the
standard curve for recA gene. Both 16S rm gene and MM were used as internal controls
to normalize the difference in reverse transcription efficiency (43). Duplicate runs were
performed for each sample.

Staining phages with SYBR Green I. MR-l was grown in TSB medium until
OD600 reached 1.0. One ml of culture was collected by centtifirging at 8,000 rpm for 3
min, washed once in 1 ml of saline buffer (0.85% NaCl) and resuspended in 10 ml of
saline buffer. The cell suspension was exposed to 3.3 J m'2 of UVC, after which 1 ml of
cell suspension was transferred into 1 ml of 2X TSB medium. The culture was incubated
at 30 °C on a shaker for 5 h in the dark. The control was performed in the same way
except for the UVC irradiation. After incubation, 100 pl of each culture (UV C-irradiated
sample and the control) were treated with 250 U of DNaseI (sigma D7291) and 250 U of
RNaseA (Sigma R4642) in a final volume of 1 m1 at 25 °C for 30 min to remove free
nucleic acids (3). Sample fixation and staining with SYBR Green I were performed as
described by Noble et al. (33). Bacteriophage Lambda strain W60 (ATCC 97537) was
used as a positive control. The samples were observed using a Zeiss LSM-Pascal (Carl
Zeiss, Germany) microscope with a plan-aprochromat 63X oil objective (N. A. = 1.4).
The phage were viewed at an excitation wavelength of 488 nm. The images were viewed
using Zeiss software Laser Scanning Microscope LSM 5 Pascal Version 3.2 SP2 (Carl
Zeiss).

Examining phages by transmission electron microscopy (T EM). MR-l was
grown in TSB and irradiated by UVC or UVB as described above for SYBR Green I

staining. Afier 5 h incubation in the dark, 0.8 ml of chloroform was added to 20 ml of

70

UVR-irradiated MR-l culture and continued incubation for 15 min to lyse the cells. Both
RNaseA and DNaseI was added to a final concentration of 2 ug/ml, respectively, and
incubated at room temperature for 30 min to remove MR-l genomic DNA and RNA. The
proteins in the solution were precipitated by adding NaCl to a final concentration of 1.0
M and incubated on ice for 30 min. The cell debris was removed by centrifugation at
5000 rpm for 10 min at 4°C. The supernatant was collected for TEM examination. Phage
were fixed with glutaraldehyde at a final concentration of 1%. Five microliter of fixed
phage suspension was added directly onto a Formvar and carbon-coated electron
microscopy grid. The grid was stained for 30 s with uranyl acetate (2%) and phage were
examined with a JEOL 100CX (Japan) transmission electron microscope at an

accelerating voltage of 100 kV. Images in this dissertation are presented in color.

71

 

 

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Results

Global gene expression trends. 1 observed distinct gene expression trends in
MR-l following UVC and UVA exposure. The response to UVA was fast: the maximal
differentiation appeared at 5 min with a total of 239 induced genes and 92 repressed
genes (Figure 3.1A). In contrast, the response to UVC was much slower: the greatest
differentiation occurred at 60 min with 128 induced genes and 51 repressed genes (Figure
3.1A). The UVB-induced gene expression trend appeared to be a combination of the
“UVA-pattern” and the “UVC-pattem”: two response peaks were observed at 5 min
(“UVA-pattern”) and 60 min (“UVC-pattern”), respectively (Figure 3.1A). The genetic
response to UVA was more global with approximately 8% of the genome showing
differential expression whereas only about 4% of the genome was differentially
expressed after UVC or UVB exposure (Figure 3.2). In all three treatments, a greater
number of genes were induced than repressed (Figure 3.2). Almost 70% of UVB-induced
genes were up-regulated in the UVC treatment whereas only about 40% of UVB-induced
genes were up-regulated in the UVA treatment (Figure 3.1B), which indicates that the
UVB-induced stress response in MR-l is more similar to that of UVC. A total of 31
genes were induced by all three UVR wavelengths, which have been clustered into three
groups (Table 3.2). Cluster 1 (16 genes) contained SOS responding genes, a site specific
recombinase gene and seven hypothetical and conserved hypothetical genes. Cluster II
(11 genes) contained genes involved in replication of prophages, transposition, and eight
hypothetical and conserved hypothetical genes. Cluster III (4 genes) contained an ISSolZ

transposase and three conserved hypothetical genes (Table 3.2).

73

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Gene expression profile following UVC irradiation. Based on the TIGR
annotation (l 7), one hundred and thirty four up-regulated genes in response to UVC were
grouped into 11 functional categories, of which both “hypothetical proteins” (41.8%) and
“conserved hypothetical proteins” (23.1%) were dominant (Figure 3.2, UVC). Other large
groups included “other categories” (13.4 %), which mainly are prophage-related genes
and transposases, “DNA metabolism” (8.2%), “protein fate” (3%) and “unknown
function” (3%). Three major clusters were revealed using K-means analysis
(Supplemental Figure 3.1, UVC). The first cluster (39 genes) represented immediate-
responding genes: the induction was observed at 5 min as well as at 20 and 60 min
(Supplemental Figure 3.1, UVC, 1; Supplemental Table 3.1, cluster I). The second cluster
(42 genes) represented intermediate-responding genes: the induction was observed at 20
and 60 min (Supplemental Figure 3.1, UVC, II; Supplemental Table 3.1, cluster II). The
third cluster (52 genes) was late-responding genes: no induction was observed until at 60
min (Supplemental Figure 3.1, UVC, III; Supplemental Table 3.1, cluster III).

Seventy-three down-regulated genes in response to UVC were grouped into 16
functional categories (Figure 3.2, UVC). Besides “hypothetical proteins” (20.5%) and
“conserved hypothetical proteins” (13.7%), a large number of repressed genes belonged
to “energy metabolism” (15.1%), “transport and binding proteins” (13.7%), “regulatory
function” (6.8%), “biosynthesis” (5.5%) and “cell envelope” (5.5%) categories. Since my
focus is primarily on those genes that are induced, which will probably encode proteins
that are most directly involved in DNA repair and detoxification to overcome the cellular

damage, I did not analyze the down-regulated genes in detail.

75

300 - 1D Hypothetical proteins

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UVC UVB UVA

Figure 3.2. Distribution of the differentially expressed genes in various functional categories following
UVC, UVB and UVA exposure. The total number of induced (Up) and repressed (Down) genes were 134
and 73 for UVC, 171 and 23 for UVB and 284 and 1 17 for UVA, respectively (This image is presented in
color).

Induction of DNA damage repair genes after UVC exposure. DNA is the
major target of UVC, thus DNA. damage is the main mutagenic and lethal effect induced
by UVC. Correspondingly, a strong induction of recA and IexA was observed following
UVC exposure, which indicated the induction of the SOS response in MR-l (Table 3.2).
In addition, I observed a similar strong induction of recN, recX, topB, dinP and the

umuDC operon. Induction of the umuDC operon correlated very well with my prior

76

observation of increased mutability in MR-l following UVC exposure (38). Although no
induction was observed for ruvAB, a weak induction of recG (2-2.5 fold), which encodes
a specific helicase, was observed. This result suggests that recombination repair is
functional in MR-l (Supplemental Table 3.1).

My previous work suggests that the NER component genes of MR-l may be
expressed constitutively at a relative low level (38). I attempted to quantify the
expression levels of uvrA, uvrB and uer in both UVC-irradiated and non-irradiated
samples by Q RT-PCR. recA was used as a positive control (induced) and radC was used
as a negative control (non-induced). A consistent result was observed by Q RT-PCR
except the induction fold for recA measured by microarray hybridization was lower
compared to Q RT-PCR assay (Table 3.2 and Table 3.3). This result is consistent with a
previous report on validating cDNA microarray data by Q RT-PCR (50). A better
correlation was obtained using ldhA as internal control (R2: 0.9478) than 16S rrn gene (R2:
0.7394). uvrA was present in about 500 copies and both uvrB and uer in about 200
copies in 500 pg of total cDNA. The basal expression level of all three genes was lower
than that of recA, which averaged about 2500 copies in 500 pg of total cDNA in MR-l

prior to UVC irradiation (Table 3.3).

77

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80

Induction of prophage-related genes by UVR. It is well known that short-
wavelength UVR can induce the lytic cycle of lysogenic bacteriophage. I observed the
induction of a great number of prophage-related genes in MR-l after UVC exposure with
the largest percentage (74.7%) of genes induced from the LambdaSo genome (Table 3.4).
In addition, induction of “early genes” which are involved in LambdaSo replication and
transcription was observed from 5-20 min whereas induction of “later genes” which
encode phage structural proteins was observed only at 60 min. A similar expression
pattern was observed for prophage MuSol, but not for MuSoZ. A total of 15 genes
(SOO643-SOO652 and 800674-800678) were induced from the MuSol genome. The
activation of genes responsible for transposition and a positive regulator of later
transcription (SOO643-SOO652) indicated a potential activation of phage MuSol. Indeed,
gene products of SOO674-SOO678 are structural proteins of Mu. A total of 16 genes
(802653-802668) were induced from the MuSoZ genome, which include genes
responsible for transposition and a positive regulator of later transcription. However, no
genes encoding structural proteins of phage MuSoZ were induced (Supplemental Table
3.1).

The effect of UVB on phage gene induction was comparable to that of UVC for
all three MR-l prophages (Table 3.4). In contrast, UVA exposure induced the expression
of few genes including only 11 of 75 genes of LambdaSo, and 1 of 42 and 2 of 53 genes

in MuSol and MuSoZ, respectively (Table 3.4).

81

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Gene expression data strongly suggested that UVC may induce the lytic cycle of
LambdaSo in MR-l (Table 3.4). Using SYBR Green I staining, I observed phage
particles in the cultures exposed to UVC (Figure 3.38), but not in control cultures (Figure
3.3A). In addition, cells exposed to UVC were greatly enlarged (Figure 3.3B) compared
to the control samples (Figure 3.3A). This observation is consistent with the previous
observation that inhibition of cell division is a consequence of the UVC-induced stress
response in many bacteria (18). In a suspension from UVC irradiated MR-l cells, I
observed phage with a head and a tail structure by TEM (Figure 3.3C). Similar phage

particles were seen in the UVB irradiated samples (data not shown).

 

Figure 3.3. SYBR Green I staining MR-l (A), MR-l treated with UVC (B) and TEM images for phage
isolated from UVC irradiated MR-l (C). The scan zoom was 4.0 for images A and B.

83

Gene expression profile following UVA irradiation. Unlike the UVC gene
expression profile, two hundred and eighty four up-regulated genes were distributed in 16
functional categories more evenly (Figure 3.2, UVA). The top six large groups were
“conserved hypothetical proteins” (19.7%); “hypothetical proteins” (15.5%);
“biosynthesis” (11.6%); “unknown functions” (10.2%); “transport and binding proteins”
(8.8%) and “cellular process” (7.8%) (Figure 3.2, UVA). Compared to the UVC
transcriptional profile, genes in “DNA metabolism” and “other categories” were reduced
from 8.2- and 13.4% to 4.2- and 1.8%, respectively, whereas genes in “regulatory
function”, “signal transduction”, “transcription” and “metabolism” categories showed a
slight increase in percentage (Figure 3.2, UVA). One hundred and seventeen down-
regulated genes in response to UVA were grouped into 15 functional categories. Similar
to UVC expression profile, the largest four functional groups were “energy metabolism”
(21.4%), “transport and binding proteins” (17.9%), “hypothetical proteins” (16.2%) and
“conserved hypothetical proteins” (12.8%) (Figure 3.2, UVA).

As expected, genes involved in repairing DNA damage were induced in MR-l
following UVA irradiation (Table 3.2). Induction of key genes of the SOS regulon was
less substantial compared to UVC. In addition, I observed a strong induction (20 fold) of
phrB, which encodes a DNA photolyase mediating photoreactivation, and a weak
induction of mutL (2.1 fold) which encodes a component of DNA mismatch repair.

Scavenging of UVA-induced reactive oxygen species in MR-l. The removal of
reactive molecules that result from photo-oxidation is a challenge faced by organisms in
coping with UVA-induced stress. The induction of antioxidant enzymes and proteins is a

common strategy in bacteria to scavenge ROS. In S. oneidensis MR-l, I observed at 5

84

min the induction of genes encoding a catalase/peroxidase HPI ($00725: 3.8 fold), alkyl
hydroperoxide reductase subunit C ($00958: 4.3 fold), a cytochrome c551 peroxidase
($02178: 2.8 fold), an organic hydroperoxide resistance protein ($00976: 8.7 fold) and a
putative glutathione peroxidase ($01563: 4.7 fold) (Supplemental Figure 3.1, UVA, II;
Supplemental Table 3.2, cluster II). In addition, I observed the strong induction of
$01773 (8.0 fold), which encodes a catalase related protein and $03349 (11.9 fold),
which encodes a second putative glutathione peroxidase. Induction of these two ORFs
occurred at 5 min and lasted until 20 min (Supplemental Figure 3.1, UVA, III;
Supplemental Table 3.2, cluster III). Although MR-l possesses a katB ($01070) and
another katG ($04405), no induction of either gene was observed. Since the overall
hybridization signals of katB were lower than most of the spots on the array, Q RT—PCR
was performed. No induction of katB following UVA exposure was observed (Table 3.5).

The intracellular iron pool plays an important role in near UVR induced damage.
First, iron-containing proteins may act as chromophores, becoming excited and thereby
damaged directly (10, 21). Ferrous iron can catalyze the formation of hydroxyl radicals
through the Fenton reaction, influencing the generation of ROS following UVA
irradiation (19, 37). Hence, regulation of iron uptake and metabolism and iron
sequestration are important protection mechanisms against UVA—induced oxidative
damage. Indeed, I observed the induction of several iron sequestering proteins such as
$01158 (ferritin-like Dps protein: 3.6 fold), bcp (bacterioferritin comigratory protein: 7.0
fold), and hemH (ferrochelatase: 10.4 fold), which encodes the enzyme that inserts iron
into protoporphrin IX to make heme. Correspondingly, genes involved in iron up-take

were strongly repressed at 5 min after irradiation (S03669-$03675: 0.25-, 0.37-, 0.27-,

85

0.38-, 0.46- and 0.30 fold) (Supplemental Figure 3.1, UVA, V; Supplemental Table 3.2,
cluster V). Also, the expression of $04077, which encodes a putative TonB dependent
receptor, was repressed more than 3-fold during the 1 h recovery period. The expression
of $03669 (hugA), $03670 (tonBl) and $03671 (ebel) increased slightly at 20 min
(2.3, 2.2 and 2.3 fold, respectively), which may indicate the requirement of iron for the
synthesis of new proteins in MR-l following UVA irradiation (Supplemental Table 3.2).

Induction of toxin and toxin secretion related genes after UVA exposure. The
MR-l genome contains a putative pore-forming RTX (repeats in toxin) toxin operon
($04146-$04149) and a gene cluster ($04317-S04319) that is related to RTX
production and secretion. MR-l also contains a gene encoding a putative hemolysin
($01354). Hemolysin can bind to and lyse mammalian cell membranes and, at low
concentration, perturb cell signal transduction causing the release of inflammatory
mediators (44, 47, 48). I observed the induction of $04149, which encodes a RTX (2.0
fold) and $04148 (4.9 fold), which encodes a HlyD family secretion protein involved in
secretion of toxin and $01354 (2.6 fold) (Supplemental Table 3.2).

Secretion of RTX toxins requires three gene products in E. coli: HlyB, HlyD and
TolC. Both HlyB and HlyD are inner membrane proteins, functioning as an ATPase
(I-Ile) and an adaptor (Hle), whereas TolC is an outer membrane exit duct protein (4,
13, 46). This tripartite machinery transports toxins directly across the entire cell envelope.
Interestingly, MR-l is highly redundant in thD. There are a total of 17 ORFs encoding
Hle family proteins, of which six ($01881, $01925, $03483, $04015, $04327 and
$04693) are located closely with genes coding for RND (the resistance-nodulation-cell

division) antiporter Ach/Ach/Ach family protein. High induction was observed in

86

MR-l following UVA irradiation for $01925 (5.4 fold) and $04327 (10.0 fold)
(Supplemental Table 3.2).

Induction of multidrug and heavy metal efflux pumps after UVA exposure.
Similar to HlyD, MR-l is also highly redundant (nine copies) in genes encoding
Ach/Ach/Ach family proteins (17), and has a gene ($04328) encoding a truncated
Ach/Ach/Ach family protein (629 a) due to an authentic frameshifi. In E. coli,
AcrAB-TolC is a major, constitutively-expressed, multidrug efilux pump that provides
resistance to structurally unrelated noxious molecules (1, 32). Ach functions as an
antiporter which uses proton flux as the source of energy whereas AcrA functions as an
adaptor and TolC works in the same way as it does in type I secretion pathway (HlyBD-
TolC) (1, 51). Strong induction of $01923 (7.8 fold), $01924 (10.2 fold) and $04328
(10.2 fold) were observed afier exposure to UVA (Supplemental Figure 3.1, UVA, II;
Supplemental Table 3.2, cluster II). In addition, $00525, which encodes an EmrB/QacA
family protein, showed a 4.5-fold induction. EmrB of E. coli is an integral membrane
translocase which mediates drug extrusion (25). MR-l also carries a chromosome-borne
($04597 and $04598) and a plasmid-bome ($0A0153 and $0A0154) heavy metal
efflux pump. Both $04598 and $0A0153 encode a Cch family protein, which is a
cation/proton antiporter of the RND family protein, whereas both $04597and $0A0154
encode a putative heavy metal efflux membrane fusion protein (M. Romine pers. mm).
Cch along with Cch, a membrane fusion protein and Cch, an outer membrane protein,
confers resistance to cobalt, zinc and cadmium ions (31). Strong induction (6.0-7.0 fold)

was observed for all four ORFs afier UVA exposure (Supplemental Figure 3.1, UVA, III;

87

Supplemental Table 3.2, cluster 111). These data suggest that heavy metal and multidrug
efflux pumps may function as a method of detoxification in UVA-irradiated MR-l cells.

Due to the high similarities among 0RFs encoding the same or similar products, I
may have observed cross-hybridization in my microarray-based gene expression
experiments. To validate my observations, I designed gene specific primers for four
genes ($01923, $01924, $04328 and SOA0154) that encode heavy metal and multidrug
efflux pumps described above (Table 3.1). I also included 0hr (highly induced), recA
(moderately induced) and radC (no induction) in Q RT-PCR analysis for validation and
comparison. Consistent results were observed by the Q RT-PCR assay (Table 3.5). Again,
a better correlation was obtained using ldhA as internal control (R2: 0.8953) than 16$ rm
genes (R2: 0.8). I also confirmed less induction of recA than with UVC and no induction
of radC in UVA-irradiated samples (Table 3.5).

Induction of other stress related genes following UVA irradiation. Other
stress related genes that were induced by UVA exposure included those involved in cell
motility ($01989: 5.5 fold; $03247: 2.8 fold; $03248: 6.1 fold; $03282: 2.4 fold;
$03241: 2.1 fold), in cell signaling ($04170: 13.3 fold) and in producing antibiotic
resistance ($04299: 2.8 fold; $00837: 2.] fold). I also observed a slight induction of
some heat shock and chaperone proteins such as HslU ($04160: 2.1 fold), Hth

($02016: 2.1 fold) and DnaK ($01126: 2.2 fold) (Supplemental Table 3.2).

88

Table 3.5. The relative expression of selected genes following UVA exposure quantified
by microarray hybridization and Q RT-PCR

 

 

 

Gene firs %20 aT60
Array Q RT-PCR Array Q RT-PCR Array Q RT-PCR

recA 3.5 :1: 0.5 4.4 :t 1.8 3.6 i 0.6 4.5 i 0.7 2.3 i 0.2 2.6 i 0.3
radC 0.8 i 0.3 1.4 i 0.5 0.8 :t 0.2 1.5 i 0.3 1.4 i: 0.5 1.2 :1: 0.3
0hr 8.7 i 4.3 49.4 i 22.5 1.5 i 0.6 2.9 i 0.8 0.8 i 0.6 0.8 :1: 0.1
$01923 7.8 :1: 4.7 21.0 :t 7.1 2.8 :t 0.5 5.8 :1: 0.4 0.9 i 0.3 1.0 i 0.1
katB 0.9 i 0.4 1.9 i 0.4 1.1 i 0.8 1.2 :t 0.2 0.9 i 0.2 1.2 i 0.3
$01924 10.2i3.6 15.9i3.4 3.1 10.6 68:23 1.1 21:05 1.3 i0.2
$04328 10.2 i 5.1 16.4 i 8.0 3.0 :1: 1.3 5.8 i1.5 0.9 :1: 0.1 1.0 i 0.1
$0A0154 6.5 :32 11.5:49 3.2: 1.3 7.3 $5.5 1.6i0.8 1.2:t0.6

 

a'1'5, T20 and T60 are the ratios of UVA-irradiated samples to the controls at 5 min, 20 min and 60 min,
respectively. The data reported here were normalized using ldhA as internal control (43). SD was calculated
from six data points which included three independent biological samples and two technical replicates for
each biological sample. Strandard curve for recA was used to calculate the cDNA copies.

Gene expression profile following UVB irradiation. The number of functional
categories of up-regulated genes in response to UVB irradiation (14) was more than that
of UVC (11), but less than that of UVA (16). Similar to the UVC transcriptional profile,
both “hypothetical proteins” (35.1%) and “conserved hypothetical proteins” (29.2%)
were dominant. In addition, the number of genes in “DNA metabolism” and “other
categories” decreased slightly whereas the number of genes in “cellular processes”,
“transporter and binding proteins” and “regulatory function” increased slightly compared
to the UVC profile (Figure 3.2, UVB). Those changes indicated a shift in response to
damage induced by short wavelength UVR to long wavelength UVR: from direct DNA
damage and activation of prophages to global photo-oxidative damage.

Genes induced by UVB could be roughly divided into “UVC-pattern” genes and
“UVA-pattern” genes. “UVC-pattem” genes were mainly distributed in cluster I, III and
IV whereas “UVA-pattern” genes were mainly distributed in the cluster II (Supplemental
Figure 3.1, UVB; Supplemental Table 3.3). A strong $0$ induction was observed

following UVB irradiation, which indicated that, similar to UVC, photons at UVB

89

wavelengths can cause direct DNA damage in MR-l (Table 3.2). Similar to UVA, I
observed the induction of genes encoding for an antioxidant enzyme ($03349: 9.3 fold),
iron sequestration ($03348: 10.7 fold), multidrug efilux pumps ($04328: 4.9 fold), and
production of toxin and resistance traits ($04170: 6.9 fold and $04327: 4.3 fold)
although the number of induced genes in each category was less than that for UVA
(Supplemental Table 3.3). This result confirmed my previous observation that the UVB

induced stress response was more similar to that of UVC (3 8).

Discussion

Possession of efficient DNA repair capacity is essential for the survival of all
organisms following UVR irradiation. Although strong induction of several genes that are
subject to SOS regulation was observed in MR-l, some DNA damage repair genes were
not damage-inducible by UVR. For example, expression of the NER component genes
uvrA, uvrB and uer, and the E. coli $0S regulon genes vaB and recF were not
induced in any of my experimental conditions. Similarly, genes involved in response to
oxidative DNA damage such as nfo, xthA and mutM that are damage-inducible in E. coli
(6, 20, 22), were not induced in MR-l following UVA exposure. Although MR-l
encodes most DNA damage repair genes in common with E. coli, several genes that have
demonstrated importance in DNA repair in E. coli are absent from the MR-l genome. For
example, both din] and dinD, which are highly induced following UVC irradiation in E.
coli (8); both nfo and nfi, which are important genes in BER in E. coli (14) and sodA,
which encodes an inducible superoxide dismutase (MnSOD), are not present in the MR-l

genome. In addition, genes involved in very short patch mismatch repair, e.g. vsr, which

90

encodes a DNA mismatch endonuclease and genes involved in the adaptive response, e. g.
alkA, which encodes a 3-methyl-adenine DNA glycosylase II, are not present on the MR-
1 genome (17). Thus alteration in gene regulation and lack of certain genes may
contribute to the difference in radiation resistance between E. coli and S. oneidensis (3 8).

The cellular response to oxidative stress is important since ROS can damage a
variety of molecules in the cell including DNA, membrane lipids and proteins. ROS can
be produced by the incomplete reduction of oxygen during respiration, by exposure to
radiation or to oxidation-reduction (redox) active drugs or by release from macrophages
in response to bacterial invasion. The induction of more than 280 genes following UVA
irradiation indicated MR-l possesses an active regulation network in response to
oxidative stress. The gene product of $01328 has 34% identity with OxyR of E. coli at
the amino acid level. The conservation of the two cysteine residues (Cy5-203 and Cys-
212 in MR-l) that are required for activation of OxyR in E. coli (Cys-199 and Cys-208 in
E. coli) (52) may suggest a similar regulatory mechanism in MR-l. However, no putative
soxR or soxS is found in the MR-l genome, although there are ten transcriptional
regulators of the MerR family, to which SoxR belongs and five transcriptional regulators
of the Ara/Xyls family, to which SoxS belongs. I observed the induction of two
transcriptional regulators of Ara/Xyls family proteins ($01762: 4.8 fold and $00317: 2.4
fold) following UVA irradiation. The potential roles of these regulators in response to
oxidative stress in S. oneidensis MR-l needs further investigation.

Many sequenced bacterial genomes harbor prophages or phage-like elements,
which have been implicated in pathogenesis and in shaping bacterial as well as viral

genomes (5). Although the genome of MuSol (SOO641-SOO683) is interrupted by the

91

insertion elements ISSod1-3, MuSoZ ($02652-SOZ704) is almost intact (17). My data
strongly suggested that all three prophages in the MR-l genome were potentially active
following UVR irradiation. The activation of prophages in MR-l can be the major lethal
factor following short wavelength UVR exposure. I previously demonstrated that there is
a difference in UVC sensitivity within S. oneidensis strains and among other species of
the Shewanella genus (38). No MR-l type prophages were identified in any other
Shewanella strains that have been sequenced including S. sp. PV-4, S. denim'ficans
08220, S. frigidimarina NCIMB 400 and S. putrefaciens CN-32 (K. Konstantinidis pers.
comm.). All of those strains showed higher UVC resistance than MR-l (data not shown).
These observations suggest that activation of prophages in MR-l contributes greatly to its
high UVC sensitivity.

Approximately 40% of annotated ORFs in MR-l belong to either conserved
hypothetical protein (871 ORFs) or hypothetical protein (1161 ORFs). A total of 181 of
those ORFs were induced under my experimental conditions, among which 18 were
induced in response to all three wavelength groups of UVR, 61 were induced in response
to both UVB and UVC, and 19 were induced in response to both UVB and UVA
(Supplemental Figure 3.2). There are 8, 12 and 64 ORFs induced specifically by UVC,
UVB or UVA irradiation, respectively (Supplemental Figure 3.2; Supplemental Table
3.4). The potential biological function of these ORFs can be inferred based on their
induction pattern among the three treatments. For example, eighteen of hypothetical and
conserved hypothetical proteins that were induced in all three treatments were grouped
either with IS elements, indicating their potential function in transposition, or with the

SOS regulon, indicating their potential function in DNA damage repair or cell division,

92

or with prophage genes, indicating their potential functions in prophage replication,
transcription or transposition and maturation (Table 3.2). One of particular interesting
hypothetical genes is $04604, which is located in the same operon as IexA. Strong
induction of this gene was observed in MR-l following UVC (12.5 fold), UVB (20.0 fold)
and UVA (6.9 fold) exposure. The new annotation indicated that the product of this ORF
is similar to SulA of E. coli (M. Romine, pers. comm.), which is an inhibitor of cell
division in E. coli. sulA is subject to the SOS regulation (8). Induction of $04604 after
UVC exposure correlated well with my observation of the occurrence of filamentous cells
of MR-l (Figure 3.38). Detailed information on each hypothetical or conserved
hypothetical ORF is summarized in Supplemental Table 3.4. Such results provide clues
about the potential biological function of conserved hypothetical and hypothetical
proteins. In conjunction with other approaches, we will gain a better understanding of
those genes in MR-l as well as in other organisms.

This study systematically investigated and compared the genomic response to the
three important wavelength groups of UV radiation: UVC, UVB and UVA. Living
organisms have to face the toxic insults from solar UV radiation since life began on the
earth. Although current solar UV radiation reaching to earth’s surface contains mainly
UVA (95%) and UVB (5%) and no UVC due to ozone filtration, unattenuated UV
radiation prior to accumulation of oxygen in the earth’s atmosphere would have provided
strong selective pressures for the evolution of mechanisms in DNA repair and damage
tolerance. Continued selection for UVA resistance is obvious, but selection for UVC
resistance may occur from the exposure to UVB, from other DNA damaging agents or

from nonstatic environments. Alteration in gene regulation and loss of certain genes

93

important in DNA repair as well as in defending against oxidative stress appears to have
occurred in S. oneidensis MR—l, an organism not known so far to live currently in

environments exposed to solar light.

94

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CHAPTER 4
GENOME-WIDE EXAMINATION OF NATURAL SOLAR RADIATION

RESPONSE IN SHEWANELLA ONEIDENSIS MR-l

100

Abstract

I delineated the cellular response of Shewanella oneidensis MR-l to natural solar
radiation by analyzing the transcriptional profile following exposure to ambient solar
light at a dose which yields about 20% survival rate. More than one thousand genes
showed significant differential expression (P<0.01) of at least a two-fold change in
magnitude during a 1 h recovery period. This genomic response is much greater than that
observed after exposure to UVB or UVA, of which a total of 195 and 403 genes showed
differential expression, respectively. I observed the induction of DNA damage repair
genes, the 808 response as well as a detoxification strategy previously observed for
UVA irradiation. Few prophage-related genes were induced by solar radiation, however,
in contrast to what was observed following UVB or UVC irradiation. Overall, the cellular
response to solar radiation in MR-l was more similar to that of UVA than that of UVB,
but with more genes involved in detoxification induced compared to either UVB or UVA
or their sum. Hence oxidative stress appeared to contribute greatly to the solar radiation
induced cytotoxic effects in MR-l. The number of differentially expressed genes from
most functional categories (15 up-regulated and 19 down-regulated) increased compared
to either of UVB or UVA or their sum, which indicates that natural sunlight impacts

biological processes in a much more complex way than previously thought.

101

Introduction

The deleterious effects of solar light on biological systems are thought to be due
primarily to solar ultraviolet radiation (UVR) (Diffey 1991). Solar UVR reaching earth’s
surface contains mainly (95%) of UVA (320 to 400 nm) and a small portion (about 5%)
of UVB (290 to 320 nm), which is collectively called near UV. UV radiation with
wavelengths less than 290 nm (far UV) is attenuated by the stratospheric ozone layer.
The biological effects induced by near UV are much more complex than for far UV due
to the complex damage processes (Jagger 1983; Webb 1977). First, near UVR damages
the cell through a process called “photosensitization”, in which DNA along with many
other molecules (photoreceptors or chromophores) can absorb the photons in wavelengths
of 290-400 nm, being damaged directly or transfer the energy to other molecules, causing
secondary damage to the cell. Typical photoreceptors include amino acids such as
tryptophan and cysteine; components in respiratory chain such as porphyrins, flavins and
quinones (Jagger 1983), and components of protein synthesis machinery such as tRNA
(Eisenstark 1987). Near UV can also induce the formation of reactive oxygen species
(ROS) including hydrogen peroxide, superoxide anion, hydroxyl radical and singlet
oxygen (Eisenstark 1989), which can damage a variety of cell components as well as
physiological processes. Absorption of near-UV photons by DNA results in formation of
characteristic lesions including pyrimidine dimers (CPD), pyrimidine pyrimidone (6-4)
photoproducts ((6-4) PD) as well as other minor photoproducts such as thymine glycols
and pyrimidine hydrates (Friedberg et al. 1995). The Dewar isomer of (6-4) PD is a
significant photoproduct after solar light exposure (Perdiz et al. 2000). In the presence of

ROS, more DNA lesions are generated due to oxidative DNA damage including single

102

strand breaks, double strand breaks, DNA cross links and DNA protein cross links
(Tyrrell 1991).

Solar UV radiation is perhaps one of the most mutagenic agents to life on earth.
Unattenuated UV radiation prior to accmnulation of oxygen in the earth’s atmosphere
would have provided strong selective pressures for the evolution of mechanisms for DNA
repair and damage tolerance. Tolerance to solar UVR may arise from different
mechanisms such as physical screening of the radiation by near UV radiation absorbing
compounds; by interference with the action of deleterious photoproducts, e.g. quenching
of singlet oxygen by carotenoid pigments; and repair of DNA damage (Jagger 1983).
Bacteria are particularly vulnerable to UVR damage due to their small size and
unicellular structure. Thus, the possession of mechanisms to repair UVR-induced damage
as well as other sheltering strategies are essential contributors to the ecological fitness of
organisms that are regularly exposed to solar UVR.

Shewanella oneidensis MR-l, a facultative anaerobic Gamma proteobacterium,
possesses remarkable respiratory versatility and is widely distributed in nature, with
aquatic environments and sediments as its primary habitat (V enkateswaran et al. 1999).
My previous study demonstrated that this bacterium is extremely sensitive to solar light
(Qiu et al. 2004). For example, more than 80% of cells died afier exposure to Michigan
summer sun light for 10 to 15 min. Genomic responses of MR-l to each component of
solar radiation (UV B and UVA) have been characterized. DNA damage and activation of
prophages in MR-l are the major lethal factors induced by UVB whereas global photo-
oxidative damage is the primary lethal factor induced by UVA. MR-l possesses active

detoxifying mechanisms including the activation of antioxidant enzymes and proteins;

103

production of toxins, and activation of multidrug and heavy metal efflux pumps. Here I
reported my examination of genomic response of MR—l to natural solar light.
Surprisingly, the genomic response to solar light is much more global than to either UVB
or UVA. Approximately 10% of the genome was up-regulated and 18% of the genome
was down-regulated. This unique gene expression profile suggests that natural solar

radiation induced biological effects is much more complex than previously thought.

Materials and methods

S. oneidensis MR-l whole genome cDNA array. 8. oneidensis MR-l whole
genome cDNA arrays containing about 95% of total ORFs were produced at Oak Ridge
National laboratory (Gao et a1. 2004). In brief, a total of 4,197 PCR amplicons and 451
oligonucleotides were deposited onto Corning Ultra GAPS slides (Corning, Corning, NY)
using a Microgrid II arrayer (Matrix, Hudson, NH) with 16 (4 x 4) SMP2.5 pins
(Telechem, Inc., Sunnyvale, CA). The arrays were printed with two replicates, each
containing a 4 x 4 subgrid with the spot distance of 210 microns and the spot diameter of
140-180 microns. A total of 276 control spots including black (no DNA deposited) and
10 different Arabadopis genes (Strategene, La Jolla, CA) and four genomic DNA at each
subgrid were also included on the array. After UV-crosslinking (250 mJ) using a
$trata1inker($trategene), arrays were stored in a desiccator.

Microarray hybridization and data analysis. MR-l was grown in Davis
medium with 15 mM lactate as the carbon source until the OD500 reached 0.2-0.3 at
which time the culture was split into two samples. One sample was used for ambient solar
light irradiation and the other was used as a control. Solar irradiation was performed as

described previously (Qiu et al. 2004). Briefly, 50 m1 cell suspensions were transferred

104

into sterile boxes constructed of Acrolyte OP-4 plastic (Professional Plastics, Austin,
TX), which transmits greater than 90% of the total radiation throughout the UVA and
UVB wavelengths (Acrolyte OP-4 technical data sheet; Cyro Industries, Arlington, NJ).
Two boxes were maintained on ice on a rocking platform during the exposures with
control samples covered with aluminum foil. Solar UVB radiation was measured with a
UVB detector ($ED240/UVB-1/W) attached to an IL-1700 research radiometer
(International Light, Newburyport, Mass). After exposure to 558 J In2 of solar UVB,
which yields about a 20% survival rate for MR-l (Qiu et a1. 2004), both irradiated and
control samples were shaded and placed in a shaker at 30°C.

Cells were collected after 5 min, 20 min and 60 min of incubation for RNA
extraction. Prehybridization and RNA labeling were performed as described by
Schroeder (Schroeder et a1. 2002) with a 2:3 ratio of 5-(3-aminoallyl)-dUTP and dTTP.
Hybridization and washing were carried out as described by Hegde et al. (Hegde et al.
2000). At each time point of each treatment, eight hybridizations from four biological
replicates and two technical replicates (dye-swap) were performed. GENESPRING 6.0
software (Silicon Genetics, Redwood City, CA) was used to analyze all microarray
hybridization data. Only those spots with more than 80% of pixels greater than
background plus 28D in either cy5 or cy3 channel were used for analysis. Data were
normalized both per chip and per gene (Lowess method). Those genes that showed a
statistically significant change in gene expression (P<0.01) and a > 2-fold change in
magnitude were regarded as significant.

Quantitative real time reverse transcription PCR (Q RT-PCR). Q RT-PCR

analysis was performed for 12 selected genes (Table 4.3) as described previously. Briefly,

105

two microgram of total RNA fi'om each sample was converted to cDNA in the same
condition as used for the microarray experiment except that dTTP instead of aa-dUTP
and dTTP mixture was used. After hydrolyzing total RNA, total cDNA was purified
using Qiagen PCR purification kit (Qiagen, Valencia, CA) and quantified using a
spectrophotometer. Gene specific primers were designed using Primer Express® 1.0
software (Applied Biosystems, Foster City, CA). All amplicons were in the range of 90-
100 bp. The specificity was first checked by blasting the primer sequences against the
MR-l genome. Both primer and template concentration for each gene were optimized in
1X SYBR Master Mixture (Applied Biosystems) using an ABI 7900HT Sequence
Detection System (Applied Biosystems). The reaction specificity was further confirmed
by dissociation curves after each PCR run. A standard curve for recA was constructed
using purified PCR product. Since the uvrB gene of MR-l was not damage inducible
(Qiu et al. 2004), it was used as an internal control to normalize the difference in reverse
transcription efficiency (Thellin et al. 1999). Duplicate runs were performed for each

sample. Images in this dissertation are presented in color.

Results and discussion

Global gene expression trend in response to solar radiation. The genomic
response of MR-l to natural solar radiation was examined during a 1 h recovery period
following solar light exposure using a microarray containing almost 95% of the predicted
open reading frames (ORFs). A total of 595 (276 induced and 319 repressed) at 5 min,
973 (399 induced and 574 repressed) at 20 min and 645 (254 induced and 391 repressed)

at 60 min showed significant differential expression (P<0.01) (Figure 4.1A). Of the total

106

differentially expressed genes, about 73-, 60- and 57% showed less than 3-fold change in
gene expression at 5min, 20 min and 60min, respectively (Figure 4.1A). Approximately
10% of the genome (553 genes) was induced whereas about 18% of the genome (884
genes) was repressed during the 1 h recovery period, which is the most extensive
response among all the radiation stress responses examined previously. In addition, more
genes were repressed than induced, which is in contrast to the global expression trend of
either UVB or UVA or their sum (Figure 4.1B). This result suggests that solar radiation
induced stress response in MR-l is much more complex than that of UVB or of UVA.
More cellular processes appear impacted by solar radiation exposure in MR-l although

most of genes responded in a subtle way.

107

A 600

400

M
O
0

Number of genes
0

-200

-400

-600

 

-800

Time (min)

800 —
600 4

400-

F‘l Fl

.-E . .__, |_J
3

 

-200 —
-400 -
-600 a
-800 -
-1000 a

Number of genes

 

 

 

Treatment

Figure 4.1. A: the global gene expression trend in response to solar radiation during a l h recovery period
after exposure. Gray bars indicate the number of differentially expressed genes with change greater than 3
fold; B: comparison of differential expression following UVB (l), UVA (2) and solar radiation (4)
exposure. 3 is the sum of UVB and UVA responses. Plus number indicates the number of induced genes
and minus number indicates the number of repressed genes.

Functional distribution of differentially expressed genes in response to solar
radiation. Based on the TIGR annotation (Heidelberg et a1. 2002), five hundred and fifty
three up-regulated genes after solar radiation exposure were distributed into 19 functional

groups (Figure 4.2A). Besides “hypothetical proteins” and “conserved hypothetical

108

proteins”, the five largest groups are “energy metabolism” (60 genes), “unknown
function” (56 genes), “cellular process” (39 genes), “biosynthesis” (34 genes) and
“transport and binding proteins” (34 genes), which is very similar to that of UVA (Figure
4.2A, group 7, 18, 3, 1 and 17). Since solar UV radiation contains both UVA and UVB, I
compared the solar light induced differential expression profile to the sum of UVB and
UVA responses in MR-l. More genes in 15 functional groups were induced, among
which “energy metabolism” increased most (47 genes) (Figure 4.2A, group 7). Four
functional categories showed a decrease in the number of up-regulated genes following
solar light exposure, of which genes from “other categories” dropped most extensively.
Only one prophage-related gene was induced following solar light exposure (Figure 4.2A,
group 10). This result suggests that, unlike UVB, solar radiation appeared unable to
activate the prophages in MR-l at the dose examined.

A total of 884 genes were repressed in MR-l following solar radiation exposure,
which is much higher than for UVB (23 genes) or UVA (117 genes). The number of
repressed genes in all 19 functional categories increased (Figure 4.2B), of which the
greatest increase was observed for “conserved hypothetical proteins” (128 genes),
“hypothetical protein” (84 genes), “protein synthesis” (78 genes), “biosynthesis” (65
genes) and “transport and binding proteins” (65 genes) (Figure 4.2B, group 9, 19, 12, 1

and 17).

109

Figure 4.2. Comparison of the distribution of the up-regulated (A) and down-regulated (B) genes in various
functional categories following UVB (filled with lines), UVA (filled with dots) and solar radiation (empty
bars) exposure. The solid black column represents the sum of the differentially expressed genes following
UVB and UVA exposure. The functional category of each number stands for: 1: biosynthesis; 2: cell
envelope; 3: cellular processes; 4: central intermediary metabolism; 5: disrupted reading frame; 6: DNA
metabolism; 7: energy metabolism; 8: fatty acid and phospholipid metabolism; 9: conserved hypothetical
proteins; 10: other categories; 11: protein fate; 12: protein synthesis; 13: purines, pyrimidines, nucleosides,
and nucleotides; l4: regulatory functions; 15: signal transduction; 16: transcription; 17: transport and
binding proteins; 18: unknown function; 19: hypothetical proteins.

110

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I observed some common cellular responses to solar radiation, UVB and UVA,
e.g. induction of DNA repair genes; however, the functional distribution of differentially
expressed genes in response to solar radiation is more similar to that of UVA than UVB.
This is not surprising since the majority of solar UV (95%) is in the UVA wavelength
range. Indeed, about 28% of solar radiation induced genes were induced by UVA alone
whereas less than 10% of solar radiation induced genes were induced by UVB alone
(Figure 4.3A). The great number of unique genes (390 induced and 788 repressed) that
showed differential expression only following solar radiation exposure indicates the other
portion of solar radiation, e. g. visible and infrared light, may impact cellular processes in

MR-l (Figure 4.3).

Solar light B Solar light

788

Figure 4.3. Venn diagram of up-regulated genes (A) and down-regulated genes (B) in response to
UVA, UVB and natural solar radiation.

112

Common cellular response to UVB, UVA and solar radiation. Similar to both
UVB and UVA, I observed the induction of several DNA damage repair genes as well as
the 808 response in MR-l following solar radiation exposure although the induction was
less substantial compared to either UVB or UVA (Table 4.1). Two hypothetical genes
(803367 and 804605) and four conserved hypothetical genes (801757, 802603,
802604 and 804604), whose response clustered with DNA damage genes in a previous
study (Chapter 3), showed a slight induction following solar radiation exposure (Table
4.1). These data support our previous report on the potential biological function of those
hypothetical ORFs (Supplemental Table 3.4). Induction of phrB correlated well with our
previous observation of increased survival rate in solar radiation irradiated MR-l after
photoreactivation (Qiu et al. 2004). Similar to UVA induced stress response, I not only
observed the induction of genes involved in scavenging reactive oxygen species, in
sequestering iron, in degradative pathways as well as genes encoding multidrug and
heavy metal efflux pumps, but more genes in each group were induced (T able 4.1). In
addition, more genes involved in general stress response, e.g. heat shock proteins, and in
cell motility were induced following solar radiation exposure (Supplemental Table 4.1). I
also observed the induction of several genes for production of toxin and antibiotic
resistance as I did for UVA (Table 4.1). Strong induction of antioxidant proteins and
enzymes suggested that solar radiation can induce the formation of reactive oxygen
species at the dose examined. Hence, global photo-oxidative damage is the most
important cytotoxic effect induced by solar radiation in MR-l. Similar to UVA, I

observed active detoxification mechanisms in MR-l following solar radiation exposure,

113

 

 

 

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115

which included the activation of antioxidant proteins and enzymes, fine tuning of internal
iron concentration, activation of multidrug and heavy metal efflux pumps as well as
global cellular regulation to overcome solar radiation induced cytotoxic effects.
Response of energy metabolic genes. As discussed above, genes in “energy
metabolism” represent a large portion of total differentially expressed genes following
solar radiation exposure (Figure 4.2). Three distinct response patterns were revealed by
hierarchical cluster analysis (Figure 4.4). The first cluster contained genes that were
induced at least at one time point examined. I observed a strong induction for genes
encoding the components of glyoxylate bypass (aceB and aceA) and for genes involved
in anaerobic respiration (napG, napA, napD and dmsB-l) (Figure 4.4; Supplemental
Table 4.2). In addition, I observed induction of genes involved in degradation of amino
acids including phenylalanine, valine, histidine, serine, leucine and methionine and of
genes involved in electron transport such as cytochrome c, cytochrome b, iron-sulfur
cluster-binding protein, electron transfer flavoprotein-ubiquinone oxidoreductase and
ferredoxin-NADP reductase (Supplemental Table 4.2). The second cluster contains genes
that were induced slightly at 20 min, but repressed at 60 min (Figure 4.4). Genes in this
group included those involved in anaerobic respiration (fdrCAB), fermentation (ath)
and electron transport (hyaB and hoxK) (Figure 4.4 and Supplemental Table 4.2). The
third cluster contains genes that were repressed at least at one time point examined. I
observed a strong repression of genes encoding NADqubiquinone oxidoreductase
(801103-801108), enzymes in the TCA cycle (801927-801929: succinate

dehydrogenase; SOl930-SOI931: 2—oxoglutarate dehydrogenase and 801932-801933:

116

succinyl-CoA synthase) as well as in glycolysis (tpiA, gapA-3 and ppsA) and for genes
encoding the ATP synthase (SO4746-SO4754). (Figure 4.4; Supplemental Table 4.2).
Regulation of metabolic gene expression suggests a fine tuning in MR-l to meet
the energy requirement for recovering from solar radiation induced stress. Repression of
several TCA cycle genes and glycolysis genes and ATP synthase genes indicates a
transient inhibition of energy production, which correlates well with the inhibition of cell
division due to solar radiation induced DNA damage. Interestingly, MR-l carries two
operons (SOO902-SOO907 and SO] 103-$01108) encoding NADH: ubiquinone
oxidoreductase, which is a membrane complex of six subunits (nqrA, nqu, nqu, nqu,
nqrE and nqu) that accommodates a 2Fe-ZS center and several flavins. This enzyme
oxidizes NADH and reduces ubiquinone and uses the energy of this redox reaction to
translocate sodium across the cell membrane. The first operon (800902-800907) showed
a slight induction at 20 min and a repression at 60 min whereas the second operon
(801103-801108) was repressed at 20 min and returned to basal expression at 60 min.
The difference in expression of these two operons may indicate changes in production of
energy following solar radiation exposure since this complex is the entry point of
electrons into the respiratory chain. A shifi from aerobic respiration to anaerobic
respiration and activation of the glyoxylate shunt not only reduces the energy production,
but also minimizes chances of generating additional ROS in the cell due to aerobic
respiration. A similar observation has been reported for Deinococcus radiodurans during

the early recovery stage after exposure to ionizing radiation (Liu et al. 2003).

117

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metabolic genes in response to natural solar radiation. The distance was calculated
using standard correlation. The number indicates the relative expression level for
the selected genes in each cluster. 803980 encodes a cytochrome c552 nitrite
reductase; $04483 encodes a putative cytochrome b; $04484 encodes a
cytochrome c—type protein Shp and 804485 encodes a diheme cytochrome c (This
figure is presented in color).

118

Response of biosynthesis genes. As a consequence of reduction in energy
production, I observed a reduction in biosynthesis of amino acids, proteins, cofactors,
prosthetic groups and carriers following solar radiation exposure (Figure 4.2). Among
153 down-regulated biosynthesis genes, strong repression was observed for genes
involved in biosynthesis of amino acids in the aspartate family, e.g. 304054-804056, in
the glutamate family, e.g. 800275-800279 and in biosynthesis of heme, porphyrin, and
cobalamin, e.g. hemH—l and hemB—2 (Table 4.2 and Supplemental Table 4.3). A total of
78 genes involved in protein synthesis were repressed, which includes 48 genes encoding
ribosomal proteins, nine genes encoding translation factors and others involved in either
tRNA aminoacylation or tRNA and rRNA base modification (Supplemental Table 4.3).
However, I also observed the induction of more than 30 biosynthesis genes following
solar radiation exposure, of which strong induction was seen for the tryptophan operon
(SO3019-SO3024) and for iscS, which encodes a cysteine desulfurase involved in
assembling iron-sulfur clusters for protein (Table 4.2). Tryptophan is a chromophore for
near UV radiation (UVB and UVA) and undergoes a biochemical alternation that yields
hydrogen peroxide as a photoproduct (McCormick 1976). The induction of the
tryptophan operon may be a consequence of the photo-damage to tryptophan in MR-l
during solar radiation exposure. I also observed a strong induction for hemH-Z ($03348),
which encodes a ferrochelatase that inserts iron into protoporphrin IX to make heme. The
induction of this gene was observed in MR-l following UVB and UVA irradiation, which
implicates a potential role in defending against oxidative stress. Interestingly, MR-l
carries another copy of hemH (302019: hemH-l). However, the expression of hemH-l

remained unchanged until 60 min, when it was repressed more than 3-fold (Table 4.2).

119

The different response of hemH-l and hemH-2 may indicate that there is a difference in

their physiological role.

120

 

 

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Validation of gene expression profile by Q RT-PCR. Q RT-PCR analysis was
carried out for 12 selected genes, which included highly induced genes (0hr, katB and
SOA0154), moderately induced genes (recA, 801924 and 804328), slightly induced
gene (801923 ), highly repressed gene (803671) and genes with no change in expression
level (radC, ter and 801762). I previously demonstrated that nucleotide excision repair
component genes uvrA, uvrB and uer were not damage inducible (Qiu et al. 2004), thus
uvrB was used as internal control to normalize the difference in reverse transcription
efficiency. A good correlation (R2: 0.7955) was obtained between microarray analysis
and Q RT-PCR analysis (Table 4.3). The induction fold measured by microarray
hybridization for highly induced genes (0hr, katB and SOA0154) were lower than that
measured by Q RT-PCR (Table 4.3). This trend is consistent with a previous report on
validation of cDNA array by Q RT-PCR analysis (Y uen et al. 2002). The Q RT-PCR
assay confirmed that, similar to UVA, multidrug and heavy metal efi‘lux pumps were
activated by solar radiation. However, unlike UVA, there was no induction of
transcriptional regulator 801762 but a strong induction of katB following solar radiation
exposure, which may indicate the difference between UVA- and natural solar radiation-
induced oxidative damage in MR-l.

Solar UV radiation, especially UVB has been the focus of solar radiation induced
biological effects because of its potential lethal and mutagenic effects. As expected, I
observed the induction of the SOS response in MR-l following exposure to 558 J m'2 of
solar UVB. However, more genes involved in detoxification were induced compared to
either UVA or UVB or their sum, which may indicate that natural solar radiation induced

oxidative damages are much more global and complex than that by UVA or UVB. This

123

could be due to the complexity of natural solar UV radiation. The great genomic response
to solar light indicates that in addition to the cytotoxic effects induced by solar UV

radiation, visible and infrared light may also impact cellular processes in a subtle way.

Table 4.3. Comparison of relative gene expression between microarray analysis and Q

 

 

 

RT—PCR assay
Gene 21.1.5 3170 aT60
Array Q RT-PCR Array Q RT-PCR Array Q RT-PCR

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SOA0154 2.1 i 0.6 2.0 i 0.6 20.0 i 6.9 70.6 i 13.5 4.2 i 2.7 2.4 i 1.6
801923 1.9 i 0.6 1.0 i 0.5 3.3 i 3.5 6.5 :1: 1.4 2.01: 0.4 4.2 i 0.2
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recA 2.3 i 0.4 1.9 i 0.3 2.1 i 0.3 2.4 i 0.4 4.4 i 0.4 3.4 i 0.4
803671 1.0 i 0.2 0.4 :t 0.1 0.3 i 0.3 0.1 i 0.0 0.3 i: 0.2 0.1 :t 0.1
radC 1.3 i 0.2 0.8 i 0.0 0.6 i 0.1 2.2 i 1.4 0.7 :1: 0.1 1.9 :1: 0.2
ter 0.6 i: 0.1 0.3 i 0.1 1.6 i 0.3 1.6 i 0.7 1.9 i 0.4 1.5 i: 0.2
$01762 1.2102 1.1102 1.7i0.3 1.7i0.1 1.4103 0.9i0.l

 

a1‘5, T20 and T60 are the ratios of solar radiation-irradiated samples to the controls at 5 min, 20 min and 60
min, respectively. The data reported here was normalized using uvrB as internal control (T hellin et al.
1999). SD was calculated from eight data points which included four independent biological samples and
two technical replicates for each biological sample. The strandard curve for recA was used to calculate the
cDNA copies.

124

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10.

Diffey, B. L. 1991. Solar Ultraviolet radiation effects on biological systems.
Physics in Medicine and Biology 36: 299-328.

Eisenstark, A. 1987. Mutagenic and lethal effects of near-ultraviolet radiation
(290-400 nm) on bacteria and phage. Environ. M01. Mutagen. 10:317-337.

Eisenstark, A. 1989. Bacterial genes involved in response to near-ultraviolet
radiation. Adv. Genet. 26:99-147.

Friedberg, E. C., G. C. Walker, and W. Siede. 1995. DNA repair and
mutagenesis. ASM Press, Washington, D. C.

Gao, H., Y. Wang, X. Liu, T. Yan, L. Wu, E. Alm, A. Arkin, D. K. Thompson,
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Hegde, P., R. Qi, K. Abernathy, C. Gay, S. Dharap, R. Gaspard, J.
Earlehughes, E. Snesrud, N. Lee, and J. Quackenbush. 2000. A concise guide
to cDNA microarray analysis. Biotechniques. 29:548-562.

Heidelberg, J. F., I. T. Paulsen, K. E. Nelson, R. J. Gaidos, W. C. Nelson, T.
D. Read, J. A. Eisen, R. Seshadri, N. Ward, B. Methe, R. A. Clayton, T.
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126

CHAPTER 5
TRAN SCRIPT OME ANALYSIS OF IONIZING RADIATION RESPONSE IN

SHEWANELLA ONEIDENSIS MR-l

127

Abstract

I observed the induction of 237 genes and repression of 52 genes in Shewanella
oneidensis MR-l during a l h recovery period after gamma ray exposure. The genomic
response of MR-l to ionizing radiation is very similar to UVC, which included a strong
SOS induction and the activation of prophages. I also observed the induction of genes
encoding antioxidant enzymes and proteins, however, no induction was observed for
genes encoding multidrug or heavy metal efflux pumps or for genes involved in
production of toxin and antibiotic resistance as I observed for UVA. The different
detoxifying strategies used by MR-l indicate a distinct difference between oxidative

stress induced by UVA and by ionizing radiation.

128

Introduction

Ionizing radiation is potentially lethal and mutagenic to all organisms. The
cellular response to ionizing radiation is complex due to the complexity of the damage.
Ionizing radiation can randomly damage all cellular components through direct deposit of
radiation energy into target molecules. Meanwhile, it causes damage indirectly by
generating a variety of reactive species, e.g. through radiolysis of water, which further
damages a variety of molecules as well as biological processes in the cell (Rilay 1994;
Tolence 1987).

The cytotoxic and mutagenic effects induced by ionizing radiation are thought to
be primarily the result of DNA damage, which includes single strand breaks (883),
double strand breaks (DSB), base modification, abasic sites and sugar modification
(Goodhead 1994; Teoule 1987). Bacteria have evolved several mechanisms to remove
gamma ray induced DNA lesions and restore the integrity of the genome. RecA mediated
homologous recombinational repair is very important because it can repair DSBs, which
are considered the most lethal effect due to the difficulty of their repair. Base excision
repair (BER), which is involved in the sequential action of a DNA glycosylase and an AP
(apurinic/apyrimidinic) endonuclease followed by repair synthesis of DNA and DNA
ligation (F riedberg et al. 1995) is important in protecting DNA from oxidative damage. In
addition, LexA—RecA mediated SOS response, a global cellular response to DNA damage
involving the induction of more than 30 unlinked genes, nucleotide excision repair (NER),
which is efficient in removing clustered DNA damage, and mismatch repair, particularly
very short patch repair and MutY dependent mismatch repair play very important roles in

bacterial survival following gamma ray irradiation (F riedberg et al. 1995; Teoule 1987).

129

Extensive studies have focused on Deinococcus radiodurans, an extremely radiation
resistant bacterium. A RecA-independent, single stranded DNA annealing repair pathway
was reported to be important in D. radiodurans to recover from a low dose of gamma ray
exposure (Daly et al. 1996). Recently, Levin-Zaidman et al. proposed that an ATP-
dependent ligase mediated non-homologous end-joining pathway (NI-IEJ) as well as the
presence of unusual ring-like nucleoid conformation may facilitate the repair of DSBs in
D. radiodurans (Levin-Zaidman et al. 2003).

The ionizing radiation-induced reactive oxygen species (ROS) include
superoxide (02"), hydrogen peroxide (HzOz), hydroxyl radical (HO'), peroxyl (R00)
and alkoxyl (R0), of which the hydroxyl radical is the most reactive species and
therefore the most hazardous. Bacteria have involved various mechanisms to defend
against oxidative stress. For example, elimination of ROS by antioxidant enzymes,
disruption of perpetuating chain reaction by cellular thiols or other antioxidants,
sequestration of transition metals otherwise available for Fenton-like reactions and
coordination of cellular processes such as inhibition of cell division and activation of
degradative processes to replace damaged molecules (Riley 1994).

Shewanella oneidensis MR-l, an environmental Gamma proteobacterium, can
reduce a variety of compounds including toxic metals and radionuclides (Myers and
Nealson 1988; Middleton et al. 2003). Hence, it has great potential in use for
bioremediation. Previous study indicates that MR-l is sensitive to UV radiation and
ionizing radiation. The molecular basis of this high sensitivity has been investigated for
UV radiation. Here, I examined the genomic response to ionizing radiation in S.

oneidensis MR-l using a microarray containing almost 95% of total ORFs. By comparing

130

the major DNA repair pathways and mechanisms used to defend against oxidative stress
induced by gamma ray to E. coli and to D. radiodurans, I hope to gain a better
understanding of what are the important traits in determining the bacterial radiation

resistance and sensitivity.

Results and discussion

Survival of S. oneidensis MR-l after ionizing radiation exposure. MR-l was
grown in Davis medium with 15 mM lactate as carbon source to an OD600 of about 0.2.
The survival rate following gamma ray exposure was determined using a 60C0 gamma
cell irradiation unit (J. L. Shepherd and Associates, San Fernando, CA). The CPU was
determined on LB plates. A 20% survival rate was obtained at a dose of 40 Gy. The D10
and D37 of MR-l was 49.7 and 33.1 Gy, respectively, which are more than two orders of
magnitude lower than for D. radiodurans (Moseley 1983) and about 5 fold less than for E.
coli (Smith 1976). This result is consistent with the high UV sensitivity of MR-l reported
previously (Qiu et al. 2004).

Gene expression trend in response to ionizing radiation. The gene expression
profile was examined at S, 20 and 60 min after irradiation with 40 Gy of gamma rays
using a microarray containing approximately 95% of total ORFs. RNA extraction,
labeling and hybridization were carried out as described previously (Qiu et al. 2004).
Three independent culture and irradiation treatments were performed and served as
biological replicates. At each time point, two technical replicates in hybridization (Dye-
swap) were carried out for each biological replicate. Genes that showed a statistically

significant change in gene expression (P<0.05) and a > 2-fold change in magnitude were

131

regarded as significant. The total number of differentially expressed genes increased
during the 1 h recovery period. After 5 and 20 min, only 50-60 genes were differentially
expressed whereas at 60 min, a total of 210 genes were induced and 47 genes were
repressed (Figure 5.1). About 6% of the genome responded to ionizing radiation stress,
which is higher than for UVC (4%) but lower than for UVA (8%). The gene expression
trend following gamma ray exposure was very similar to that observed for UVC except
that the increase in the total ntunber of differentially expressed genes was slower from 5

min to 20 min for ionizing radiation.

250 .
200 s
150 a

100‘

 

50‘ %— v

-50 - -\A

-100 I I I T r I

Number ofgenes

 

 

Time (min)

Figure 5.1. The global gene expression trend in response to ionizing radiation during a 1h recovering period
after gamma ray exposure. Diamond (0) represents up-regulated genes and triangle (A) represents down-
regulated genes. The positive number of Y axis represents the number of up-regulated genes where as the
negative number indicates the number of down-regulated genes.

132

Global gene expression profile in response to ionizing radiation. Based on the
TIGR annotation (Heidelberg et al. 2002), the total of 237 up-regulated genes were
distributed into 16 functional categories (Figure 5.2). Similar to the global gene
expression profile of UVC, the four largest groups were “hypothetical proteins” (37.6%),
“conserved hypothetical proteins” (20.7%), “other categories” (11.0%), which mainly are
prophage-related genes and transposases and “DNA metabolism” (8.0%). Unlike the
UVC gene expression profile, more genes categorized in “cellular processes”, “energy
metabolism” and “transport and binding proteins” were induced, which is a characteristic
of the UVA gene expression profile. Consistent with this trend, about 48% of the up-
regulated genes in response to ionizing radiation were induced by UVC whereas only
about 17% of the up-regulated genes in response to ionizing radiation were activated by
UVA (Figure 5.3). Similar to UVC, ionizing radiation can cause DNA damage as well as
activate of prophages in MR-l. In addition, ionizing radiation can cause oxidative stress
in MR-l as I observed for UVA previously (Chapter 3).

A total of 52 down-regulated genes were distributed into 13 functional categories
(Figure 5.2). Similar to both UVC and UVA gene expression profiles, a great number of
genes in the “hypothetical proteins” (19.2%), “conserved hypothetical proteins” (15.4%)
and “energy metabolism” (13.5%) categories were repressed. However, more genes in
“biosynthesis” (19.2%) were repressed than either UVC (5.5%) or UVA (3.4%) (Chapter
3), which was a characteristic of the gene expression profile in response to gamma rays in

MR-l.

133

h.

 

250 ‘ El hypothetical protein

 

[1 Unknown function

ll Transport and binding proteins
I Transcription
I Signal transduction

I Reg ulatory functions

. I Purines, pyrimidines, nucleosides, and nucleotides

i I Protein synthesis

 

 

 

13 Protein fate
I Other categories

I conserved domain protein

Number ofgenes

l
l
l
l
l
l
l
l
l
. l
l l
l l
l l
i E Fatty acid and phospholipid metabolism i
i I Energy metabolism ‘
1 I DNA metabolism 1
l I Disrupted reading frame i
1 El Central intermediary metabolism
1 1:] Cellular processes
l

I Cell envelope

 

‘ I Biosynthesis

hduced Repressed

Figure 5.2. Distribution of the differentially expressed genes in various functional categories following
gamma ray exposure. The total number of induced genes is 237 and of repressed genes is 52 (This figure is
presented in color).

134

Gamma ray

V

Figure 5.3. Venn diagram of up-regulated genes in response to ionizing radiation, UVC and UVA. The
number represents the number of genes in each group.

UVA
UVC

DNA damage repair capacity in S. oneidensis MR-l. About 2.8% of the MR-l
genome is implicated in DNA replication, recombination and repair, which is comparable
to that of E. coli (2.7%) (Blattner et al. 1997) and D. radiodurans (3.1%) (Makarova et al.
2001; White et a1. 1999). Our previous studies indicated that photoreactivation, NER and
the SOS response including mutagenic repair were functional in MR-l (Qiu et al. 2004).
Both RecBCD and RecF recombinational repair pathways are present in MR-l (Table
1.1). In addition, MR-l carries a NAD-dependent DNA ligase (ligA) and an ATP-
dependent DNA ligase (802204), a complete methyl-directed mismatch repair pathway
(mutS, mutL, mutl-I) and genes (murM, mutY and mutT) that are important in preventing
mutation due to the oxidized base 8-oxoG (Heidelberg et al. 2002). However, compared
to E. coli, a few genes involved in DNA repair are absent in MR-l such as genes
encoding the components of adaptive response (aIkA), for the components of very short
patch mismatch repair such as dcm and vsr, and for the components of the RecE mediated
recombinational repair such as recE, recT and ms. No putative genes of din], dinD or

dinJ, which belong to the SOS regulon in E. coli were found in MR-l. In addition,

135

several genes (mug, nfo, nfi, nei and xseB) involved in base excision repair in E. coli are
absent from the MR-l genome (Table1.1).

Induction of DNA damage repair genes after ionizing radiation exposure.
About 13.9% of genes in the DNA replication, recombination and repair category were
induced in MR-l during the 1 h recovery period following gamma ray irradiation
(Supplemental Table 6.1). Similar to cellular response to UVC, a strong SOS induction
was observed (Supplemental Table 6.1), which included the induction of recA (8.8 fold),
IexA (12.7 fold), recN (13.7 fold), recX (8.0 fold), dinP (9.8 fold), topB (7.7 fold), umuD
(15.6 fold), umuC (8.0 fold) and recG (2.4 fold). However, several genes that did not
show any induction following UVC exposure were up-regulated afier gamma ray
irradiation such as polB (4.6 fold), polA (3.3 fold), mutH (2.4 fold), dinG (3.3 fold) and
$00690 (4.4 fold), which encodes a type 11 DNA modification methyltransferase. I also
observed a slight induction of several genes that were involved in DNA replication such
as dnaG (2.1 fold), dnaB (2.6 fold) and $01817 (2.2 fold), which encodes a replication
protein n. In addition, a slight induction of 800393 (2.5 fold), which encodes a DNA
binding protein Fis (Factor for inversion stimulation), was observed at 60 min. Fis is a
site-specific DNA binding protein that can bend DNA and is involved in many site
specific recombinations (Johnson et al. 1987). F is can also act as a transcriptional
activator, regulating the expression of many genes, including its own (F inkel and Johnson
1992). This result indicates that cellular response to gamma ray induced DNA damage is
more complex and global than to UVC. It appeared that both SOS response and

recombinational repair played important roles in repairing gamma ray induced DNA

136

damage. In contrast, I did not observe any induction of phrB, which may suggest that
photoreactivation plays a minor role in repairing gamma ray induced DNA damage.

Induction of prophage-related genes by ionizing radiation. There are three
prophages in the MR-l genome (Heilderberg et al. 2002). Our previous study indicated
that UVC can activate the lytic cycle of prophages, which contributes greatly to the high
UVC sensitivity of MR-l. More prophage related genes were induced following exposure
to gamma ray in MR-l. A total of 64, 26 and 28 ORFs of prophage LambdaSo, MuSol
and MuSoZ were up-regulated, respectively (Table 5.1), which indicates that, similar to
UVC, ionizing radiation can activate the lytic cycles of prophages in MR-l.

Putative genes involved in defending against oxidative stress in MR-l. MR-l
carries genes encoding for catalase, F e-containing superoxide dismutase (F eSOD),
glutathione peroxidase, glutathione S-transferase, organic hydroperoxide resistance
protein and a Dps protein, which plays an important role in sequestration of iron and
stabilizing DNA (Ilari et al. 2002). In addition, MR-l carries genes encoding ferritin, an
iron storage protein and antioxidant which is potentially important in defending against
oxidative stress (Supplemental Table 5.2). MR-l also carries a complete glutaredoxin
system and a complete thioredoxin system (Supplemental Table 5.2), which are known
hydrogen donors for ribonucleotide reductase (RNase), the essential enzyme for
deoxyribonuclcotide and DNA biosynthesis (Prieto-Alarno et al. 2000). Regarding
regulatory genes, MR-l has a putative oxyR (S01328), but no homologs of SoxRS are

found.

137

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138

Gamma ray induced oxidative stress response in MR-l. Several genes that are
directly involved in scavenging ROS were up-regulated in MR-l during a 1 h recovery
period following gamma ray irradiation, which included katB (12.6 fold), katG-l (5.5
fold), ahpFC (3.9- 11.0 fold), ccpA (3.8 fold), dps (3.0 fold) and gst (3.6 fold)
(Supplemental Table 5.2). In addition, 801762, a putative transcriptional regulator that
was induced almost 5-fold in MR-l following UVA exposure, showed an induction of
3.6 fold at 5 min. Both trxA and ter were induced slightly at 60 min (Supplemental
Table 5.2). I also observed induction of some genes that are involved in degradative
process such as 802964 (25.0 fold), which encodes a ClpP protease, SO] 1 15 (4.0 fold),
which encodes an aminoacyl-histidine dipeptidase, 804162 (3.8 fold), which encodes an
ATP-dependent protease HslV, 803577 (2.9 fold), which encodes a ClpB protein and
SO4699 (2.9 fold), which encodes an oligopeptidase. This result indicates that MR-l
actively coordinated various biological processes to overcome gamma ray induced
oxidative stress.

Although both UVA and ionizing radiation can induce the formation of ROS, of
which hydroxyl radical is the major oxygen species of concern (Perk et al. 1990), I
observed a distinct difference between the responses to oxidative stress induced by the
two. For example, I did not observe any induction of genes encoding glutathione
peroxidase (801563 and 801773) following gamma ray irradiation: those were highly
up-regulated in response to UVA. A similar case was observed for ohr, which encodes an
organic hydroperoxide resistance protein. I did not observe any induction of genes
encoding multidrug and heavy metal efflux pumps, genes involved in production and

secretion of toxin and production of antibiotic resistance, and genes involved in cell

139

motility, all of which were induced in MR-l following UVA exposure. The cellular
response to gamma ray induced oxidative stress was much less global than for UVA.
MR-l appears using a different detoxifying strategy to defend against oxidative stress
induced by gamma ray from what I observed previously for UVA-induced oxidative
stress (Chapter 3).

Validation of the gene expression profile by quantitative real time reverse
transcription PCR (Q RT-PCR). Q RT-PCR analysis was carried out for seven selected
genes, which included highly induced genes (recA and katB), moderately induced genes
(801762), slightly induced genes (ter), slightly repressed gene (801490) and genes
with no change in expression level (801924 and SOA0154) as described previously
(Chapter 3). MM was used as internal control to normalize the difference in reverse
transcription efficiency. A high correlation (R2: 0.9115) was obtained between
microarray analysis and Q RT-PCR analysis (Table 5.2). The induction fold measured by
microarray hybridization for highly induced genes (recA and katB) were lower than that
measued by Q RT-PCR. This trend is consistent with previous report (Yuen et al. 2002)
and our previous analysis (Chapter3 and Chapter 4). Q RT-PCR assay confirmed that
there was no induction of multidrug (SOl924) or heavy metal (SOA0154) efflux pump in

MR-l afier exposure to gamma ray.

140

Table 5.2. Comparison of relative gene expression between microarray analysis and Q

 

 

 

RT-PCR assay
Gene ar5 a‘rzo ar60
Array 9 RT-PCR Array Q RT-PCR Array Q RT-PCR
recA 7.0 i 1.4 14.2 i 5.8 9.0 :l: 2.2 15.0 i 3.1 6.8 :t 2.3 13.0 :1: 2.4
katB 14.9 :t 9.6 236.5 i 130.9 1.9 i 1.2 2.7 :t 0.7 1.5 i 0.9 2.1 i 0.5
801762 4.2 :t 2.4 9.4 :t 8.3 3.7 $1.6 5.0 :t 4.0 3.4 :t 2.1 3.7 $1.8
ter 2.1 i 0.8 3.1 :l: 1.7 1.7 i 0.2 2.3 i 0.5 2.4:1: 0.4 4.3 i: 1.1
801924 l.5:t0.3 2.3i0.9 1.1i0.1 1.4i0.5 1.1:l:0.2 1.1102
SOA0154 O.8:l:0.l l.0i0.2 1.1 :l:0.3 13:06 1.1 :tO.4 1.1i0.2
801490 0.8 i 0.1 0.8 i 0.1 0.9 :l: 0.3 0.8 i 0.2 0.5 i 0.2 0.6 i 0.2

 

aTS, T20 and T60 are the ratios of ionizing radiation-irradiated samples to the controls at 5 min, 20 min and
60 min, respectively. The data reported here was normalized using ldhA as internal control (Thellin et al.
1999). SD was calculated from six data points which included three independent biological samples and
two technical replicates for each biological sample. The strandard curve for recA was used to calculate the
cDNA copies. Primers are designed as described previously (Chapter 3) for quantification of genes 301762
(F: 5’CCAAATCCCGTGGTGTACG3’; R: 5’CGCGCAGTAAGCGATTATCC3’), :er (F:
5’CAGCCCCCATCGAGCTC3’; R: 5’CACCAACTGGCCCAAAAATC3’) and 801490 (F:
5’TAAAGGTATCGCCCTGGTGG3’; R: 5’CACTAACGGCAGTTGTGGCTTAG3’).

141

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Makarova, E. V. Koomin, and M. J. Daly. 2003. Transcriptome dynamic of
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USA 100:4191-4196.

Makarova, K. S., L. Aravind, Y. 1. Wolf, R. L. Tatusov, K. W. Minton, E. V.
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H. Qin, L. Jiang, W. Pamphile, M. Crosby, M. Shen, J. J. Vamathevan, P. Lam,
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Daly, K. W. Minton, R. D. Fleischmann, K. A. Ketchum, K. E. Nelson, S.
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144

CHAPTER6

SUMMARY AND FUTURE PERSPECTIVE

145

h

Comparison of transcriptional profiles of MR-l in response to five radiation stress
conditions

I delineated the genomic response of Shewanella oneidensis MR-l to five
environmentally relevant stress conditions: UVC, UVB, UVA, solar radiation and
ionizing radiation at doses that yielded 20% survival rates. A total of 4.2-, 3.9-, 8.1-,
280-, and 5.9% of the MR-l genome showed differential expression following UVC,
UVB, UVA, natural solar light, and ionizing radiation exposure, respectively (Table 6.1).
In all five treatments, both conserved hypothetical and hypothetical are the dominant
gene groups that are differentially expressed (Figure 6.1 and Figure 6.2). The gene
expression profile of MR-l in response to ionizing radiation is more similar to that of
UVC, which is characterized by a strong induction of the SOS response and of many
prophage related genes, but with some oxidative stress response (Table 6.1). Genomic
response to UVB is a combination of the UVC and UVA responses, which supports
previous reports that photons of UVB wavelengths can damage DNA directly as well as
can induce the formation of ROS species (He and Hader 2002). However, induction of
heavy metal and multidrug efflux pumps in MR-l following UVA exposure is a
previously unknown phenotype for this stress although the traditional UVA-induced
stress responses occur in MR-l such as induction of antioxidant proteins and enzymes
and activation of genes involved in degradative pathways. Consistent with natural solar
UVR composition (95% of UVA and 5% of UVB), the genomic response of MR-l to
natural solar light is more similar to that of UVA but with more genes involved in
detoxification induced (Table 6.1). In addition, the number of differentially expressed

genes from most functional categories increased compared to either UVB or UVA or

146

 

their sum. “DNA metabolism” (similar to COGs L group which contains genes involved
in DNA replication, repair and recombination) is the only category where the number of
induced genes remained the same compared to UVA whereas “other category”, which
contains mainly transposases and prophage-related genes, is the only category where the
number of up-regulated genes decreased compared to UVA. In addition, more genes were
repressed than induced following solar light exposure, which is in contrast to any of the
other four treatments (Figure 6.1 and Figure 6.2). The unique transcriptional profile of
MR-l in response to solar radiation suggests that natural solar light impacts MR-l in a
complicated way rather than a simple sum of UVA and UVB responses. Currently, the
cellular response of MR-l to solar light is under investigation by proteomics at The
Environmental Molecular Sciences Laboratory at Pacific Northwest National Laboratory
using the accurate mass tag (AMTs) technology as described previously for D.
radiodurans (Lipton et al. 2002). The comparison of the transcriptional and proteomic
profiles will enhance our understanding of this complicated biological process in MR-l,
and especially aid in the understanding of the importance of the large number of
hypothetical genes in this process.

Table 6.1. Summary of stress response in MR-l following irradiation

 

 

Stress DNA damage Oxidative stress Induction of prophages Genome
genes resmnse
UVC a+++ - ++ 4.2%
UVB +++ + + 3.9%
UVA ++ ++ _ 8.0%
Solar radiation + +++ _ 28.0%
Gamma ray +++ + +++ 5.9%

 

aNumber of + indicates the relative degree of stress response observed in MR-l, which was based on the
number of induced genes and their induction fold.

147

Figure 6.1. Functional distribution of up-regulated genes in response to ionizing radiation, UVC, UVB,
UVA and natural solar radiation (This figure is presented in color).

148

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Figure 6.2. Functional distribution of down-regulated genes in response to ionizing radiation, UVC, UVB,
UVA and natural solar radiation (This figure is presented in color).

150

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151

Comparison of transcriptional profiles in response to UVC between E. coli and S.
oneidensis MR-l

Genomic response to UVC has been investigated in E. coli strain MG1655 (a K-
12 strain having been cured of the temperate bacteriophage lambda and F plasmid) by
Courcelle et al. (2001) under experimental conditions similar to mine. Although I
observed similar responses to those found with MG1655 such as the induction of the SOS
regulon, heat shock genes and transporter genes, I also observed a distinct difference in
response to UVC stress. Among 134 UVC-induced genes in MR-l, 15 are located in the
genome of prophage MuSol, 16 are located in the genome of prophage MuS02 and 56
are located in the genome of prophage LambdaSo, which accounts for 65% of total up-
regulated genes in MR-l. In E. coli MG1655, however, among 165 up-regulated genes,
only 6 are related to prophage (3.6%) (Courcelle et al. 2001).

Although the percentage of the genome dedicated to DNA replication, repair and
recombination is very similar between S. oneidensis MR-l (2.8%) and E. coli MG1655
(2.7%), the number of UVC induced genes is much higher in MG1655 than in MR-l
(Table 6.2). I only observed the induction for 8 out of 137 genes (5.8%) implicated in
“DNA replication, repair and recombination” in MR-l following UVC exposure whereas
18 out of 115 genes (15.7%) showed induction in M61655. These data suggest that many
DNA repair genes in MR-l are losing their damage-inducibility. Alteration in some DNA
repair gene content and regulation may be the consequence of lack of natural selection for

this response in MR-l.

152

Table 6.2 Comparison of genome and the induction of DNA repair genes among S.
oneidensis MR-l, E. coli MG1655 and D. radiodurans R1.

 

 

Strain Totoal Genes in 0065 L Induced by UVC Induced by gamma
ORF group (% of total) (% of L group) ray (% of L group)
as, oneidensis MR-l 493] 137 (2.8%) 8 (5.8%) 19 (13.9%)
”E. coli M61655 4288 115 (2.7%) d18(15.7%) NA
CD. Radiodurans R1 3187 100 (3.1%) NA ‘22 (22%)

 

aaccording to Heidelberg et al. 2002; 1according to Blattner et al. 1997; caccording to White et al. 1999. 3
according to Courcelle et al. 2001; caccording to Liu et al. 2003.

Comparison of transcriptional profiles in response to ionizing radiation between D.
radiodurans and S. oneidensis MR-l

The genomic response to ionizing radiation has been reported in the extremely
radiation resistant strain Deincoccus radiodurans R1 during the 24 h of recovery period
after exposure to 15 KGy of gamma rays (Liu et al. 2003). More than 40% of its genome
showed differential expression with 832 genes induced and 451 genes repressed. This
genomic response is much more global than in MR-l. However, the experimental
condition for D. radiodurans R1 differed greatly from mine, a point to remember when
comparing the transcriptional profiles between D. radiodurans R1 and MR-l. First, D.
radiodurans R1 was grown in nutrient-rich medium (TGY) before irradiation whereas
MR-l was grown in Davis medium; afier irradiation, the D. radiodurans R1 were diluted
20 fold using fresh TGY medium whereas MR-l was returned to the shaker without
changing the culture medium; the genomic response of D. radiodurans R1 was examined
during a 24 h recovery period whereas the genomic response of MR-l was examined

during a 1 h recovery period; The D. radiodurans R1 experiment used a sample before

153

 

irradiation (T0) as the control whereas MR-l experiment used a parellal control at each
corresponding time point (Qiu et al. 2004).

To minimize the complication in comparison, I only looked at those genes that are
annotated with a function in the DNA replication, repair and recombination category.
Twenty two percent of genes implicated in “DNA replication, repair and recombination”
showed induction in D. radiodurans R1 following irradiation with 15 KGy of gamma ray
whereas only 19 out of 137 genes were induced in MR-l (13.9%) (Table 6.2). Similar to
E. coli, the NER component genes uvrA, uvrB and uer were induced in D. radiodurans
R1 (Liu et al. 2003). This comparison supports my previous speculation that loss of DNA
damage inducibility of some DNA repair genes may contribute to the high radiation
sensitivity in MR-l.

Similar to the UVC-induced gene expression profile, almost 50% of gamma ray-
induced genes in MR—l are prophage related genes. A total of 64, 26 and 28 genes were
induced from genomes of LambdaSo, MuSol and MuSoZ, respectively. In contrast, only

five phage-related genes showed induction in D. radiodurans R1 (Liu et al. 2003).

Future perspective

S. oneidensis MR-l is one of most radiation sensitive bacteria now known.
Activation of prophages in MR-l by short wavelength UV radiation (UV C and UVB) and
ionizing radiation appears to be the major lethal factor. Based on gene expression, the
phage particles I observed by TEM is more like LambdaSo since the highest expression
percentage was from the LambdaSo genome. Further identification and characterization

of the phage particles may provide us with useful information. For example, will this

154

phage(s) infect other Shewanella strains? If it is or one of them is the LambdaSo, how
much of LambdaSo is different from E. coli Lambda phage? What are the function of
those 70% of unknown proteins (hypothetical and conserved hypothetical proteins) in the
phage genome(s)? Prophages are found in most of sequenced bacteria. The impact of
prophages on shaping of both bacterial and viral genomes is becoming more widely
recognized (Canchaya et a1. 2004). Currently, sequencing of other Shewanella strains is
ongoing. Preliminary data suggest that there are no MR-l like prophages present on the
genomes of S. sp. PV-4, S. denitrificans 08220, S. fiigidimarina NCIMB 400 and S.
putrefaciens CN-32 (K. Konstantinidis pers. comm). Thus, those strains are good
candidates to address above questions.

The technology I used in this study could not resolve whether one or two or even
all three prophages were activated under my experimental conditions. By deleting the
prophage genome, one should be able to find which prophage(s) were actually being
activated in my experimental conditions. For example, if both LambdaSo and MuSol
were deleted and the phage particles were still seen in the UVC irradiated sample, then
one could conclude that prophage Mu 802 can be activated by UVC.

I observed a good correlation between the UVC survival and natural solar
radiation exposure among Shewanella strains tested for their UVC sensitivity, which may
suggest that the lack of natural selection has impacted the DNA repair and damage
tolerance mechanism in MR-l. But, what specifically is this impact? Comparison of E.
coli K12 and S. oneidensis MR-l genomes indicates that there is an alteration in DNA
repair genes content as well as regulation in MR-l. For example, only a subset of the E.

coli SOS regulon is damage inducible in MR-l. Similar cases were observed for OxyR,

155

which are very important in defending against oxidative stress in E. coli. Thus
identification of those regulons will enhance our understanding of which genes in MR-l
might evolve faster. Furthermore, examination of those regulons in other strains that have
similar evolution history as MR-l, e. g. strain DLM7, will provide additional evidence on
whether alteration in gene content and regulation is the consequence of natural selection.
Interestingly, the survival curves of MR-l and DLM-7 are very similar (Figure 2.1).

By harboring prophages, MR-l is extremely sensitive to UV radiation, which
hinders the study of DNA repair and damage tolerance mechanisms. Isolation of a
derivative strain of MR-l that is cured of prophages would provide a valuable strain for
such studies. The comparison of DNA repair and damage tolerance mechanisms between
the MR-l derivative strain (no prophages strain) with other strains for which one knows
their natural habitat (evolution history) will be more comparable than using MR-l . If one
constantly see similar traits in other non-radiation exposed strains as I saw in MR-l, it
would be strong evidence that loss of genes and/or gene regulation is underway in
lineages no longer exposed to these stresses.

Microarray based transcriptional profiling has become a powerful tool to examine
the coordinate cellular response to perturbations. However, since the microarray method
measures the steady state of mRNA, I was unable to see the dynamic picture following
perturbation such as the stability of mRNA, changes of RNA synthesis and degradation
rates, etc. No one should ever neglect the regulations that occurrs at those steps. Thus
complementary approaches should always be used based upon the questions to be

addressed.

156

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158

APPENDIX A

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APPENDIX B

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