The Stability of Lytic Sulfolobus Viruses

Home , Phenotype mixing, Stygiolobus, Sulfolobus

A thesis submitted to the Graduate School of the University of Cincinnati In partial fulfillment of The requirements for the degree of

Master of Sciences

in the Department of Biological Sciences of the College of Arts and Sciences 2017

Khaled S. Gazi B.S. Umm Al-Qura University, 2011 Committee Chair: Dennis W. Grogan, Ph.D.

Abstract

Among the three domains of cellular life, archaea are the least understood, and functional information about archaeal viruses is very limited. For example, it is not known whether many of the viruses that infect hyperthermophilic archaea retain infectivity for long periods of time under the extreme conditions of geothermal environments. To investigate the capability of viruses to Infect under the extreme conditions of geothermal environments. A number of plaque- forming viruses related to Sulfolobus islandicus rod-shaped viruses (SIRVs), isolated from

Yellowstone National Park in a previous study, were evaluated for stability under different stress conditions including high temperature, drying, and extremes of pH. Screening of 34 isolates revealed a 95-fold range of survival with respect to boiling for two hours and 94-fold range with respect to drying for 24 hours. Comparison of 10 viral strains chosen to represent the extremes of this range showed little correlation of stability with respect to different stresses. For example, three viral strains survived boiling but not drying. On the other hand, five strains that survived the drying stress did not survive the boiling temperature, whereas one strain survived both treatments and the last strain showed low survival of both. The basis for these differences has not been identified, but the extent of the variation suggests that multiple properties of each viral isolate combine to determine the biological stability of the virions. Finally, selection of stable viral particles from an unstable strain in regards to high temperature was possible.

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Acknowledgements

First, I would like to thank Dr. Dennis Grogan for giving me this opportunity to work in his lab and for all his advice and guidance the whole time I spent in his lab to become a better scientist. Second, I must thank my committee members Dr. Kinkle and Dr. Hamilton for their advice and opinions. Third, I want to thank the faculty, graduate students, and the employees in the department of biological sciences for the welcoming environment, and my friends for their support. Next, I would like to thank Al-Baha University for the financial support. Finally, I should thank my family in Saudi Arabia for their endless support to continue my education abroad, and a special acknowledgment for the Tidwell family in Cincinnati for their enormous support.

Table of Contents

Title..……………………….……………………………………………………………………………………………i Abstract…...………………………………………………………………………………………………………....ii Acknowledgements……………………..……………………………………………………………………...iv Table of Contents……….……………………………………………………………………………….………..v List of Tables…….……………………………………………………………………………...... vii List of Figures…….…………………………………………………………………………………………….…viii

CHAPTER ONE. Introduction 1

Introduction…………………….…………………………………………………………………………1 Purpose …………………………………………………………….……………………………………..11 Works Cited………………………………………………………………………………………………12

CHAPTER TWO. Natural Variation in The Stability of Sulfolobus Islandicus Rod- Shaped Viruses Isolated from Yellowstone National Park 20

Introduction……………………………………………………………………………………………..20 Purpose…………………………………………………………………………………………………….21 Methods………...…………………………………………………………………………..……………22 Results …………………………………………………………………………………...... 26 Discussion…………………………………………………………………………………………………35 Works Cited………………………………………………………………………………………………42

CHAPTER THREE. Increasing the stability of SIRV virions toward high temperature 46

Introduction……………………………………………………………………………………………..46 Purpose…………………………………………………………………………………………………….48 Methods…………….……………………………………………..………………………………….....49 Results ………………………………...... …………………………………………………………...... 51 Discussion …………………………………………………………………………………………….….62 Works Cited….…………………………………………………………………………………………..56

CHAPTER FOUR. Conclusions 68

Works Cited……………………………………………………………………….……………..……..70

APPENDIX. Evaluation of the sensitivity of Sulfolobus islandicus strains to SIRV strain V60 71

Background and Purpose………………………………………………………………………….71

Methods……………………………………………………………………..……………………………72

Results……………………………………………………………………………………………………..72

Discussions……………………………………………………………………………………………….74

Works Cited……………………………………………………………….……………………………..85

List of Tables

Table 2.1: Survival ratio of SIRV strains challenged by high temperature for 2 hours.

Table 2.2: Values are log (final titer/control titer) challenged by drying for 24 hours at room temperature

Table 2.3: Values are log (final titer/control titer) challenged by 5M urea for an hour at 60℃

Table 2.4: Values are log (final titer/control titer) challenged by guanidinium Cl for an hour at 60℃

Table 2.5: Values are log (final titer/control titer) challenged by chloroform for 30 minutes at 60℃

Table 2.6: Correlations among treatments applied on ten strains of SIRVs isolated from YNP.

Table 3.1: Survivals after the first stage of selection.

Table 3.2: Survivals after the second stage of selection.

Table 3.3: Survivals of selected particles after treated with Ethanol for 36 hours.

Table 3.4: Survivals of the selected strain 40.32G in different extreme conditions.

Table 5.1: Results of YNP S. islandicus strains isolated in 1999.

Table 5.2: Results of YNP S. islandicus strains isolated in 2000.

Table 5.3: Repeated attempts to select resistant to SIRV strain V60.

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List of Figures

Figure 2.1: Average PFU counts of different SIRV strains in pH 1-10 (A, B, C, D, and E).

Figure 2.2: Average PFU of survival ratio of SIRV strains challenged by 18% ethanol.

Figure 2.3: comparing the stability of SIRV strains in 2 hours boiling temperature to 24 hours drying.

Figure 3.1: Survival ratio of SIRV strains challenged by high temperature for 2 hours after each step of selection.

Figure 3.2: Plaque plate of 40.32 & 40.32G viruses separated on a lawn of S. islandicus 16-4 cells.

Figure 3.3: Average absorbance values for the growth of the host cells infected with SIRV 40.32 and 40.32G every 4 hours for 2 days.

Figure 3.4: Average PFU in infected cultures of both strains 40.32 and 40.32G.

Figure 3.5: Survival ratio of 40.32 compared to 40.32G challenged for an hour by 18% ethanol, 5M urea, and 5M guanidinium Cl, in addition to 30 minutes in chloroform, and 24 hours drying.

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CHAPTER ONE

Introduction

Archaea

Archaea are single cell organisms that represent the third domain of cellular life alongside Bacteria and Eukarya (Woese et al., 1990). Initially, archaea were not distinguished from bacteria (Woese, C. R., & Fox, G. E. 1977). However, in the 1970’s a group of scientists confirmed that archaea differ biochemically and physiologically from their bacterial equivalents

(Woese, C. R., & Fox, G. E. 1977), which was further clarified by the rRNA sequencing (Woese et al., 1990). Thus, the existing three domains system replaces the five kingdoms classification

(Whittaker, R. H. 1959). Mainly, some archaea and bacteria share similar shape and morphology and size, but few numbers of the archaea have unique shapes, i.e., Haloquadratum walsbyi with flat and square-shaped cells (Stoeckenius, W. 1981). On the other hand, archaea share some features with eukaryotes, i.e., enzymes involved in transcription and translation (Woese, C. R.,

& Fox, G. E. 1977).

Despite these similarities to bacteria and eukaryotes, archaea also have metabolism system that allow them to thrive and survive in extreme environments, such as hot and acidic springs, and salt lakes (Woese, C. R., & Fox, G. E. 1977). Furthermore, archaea have been found in a wide range of habitats, including the human body (Bang et al., 2015). The domain Archaea branches to four phyla, Crenarchaeota (Woese et al., 1990), Euryarchaeota (Bernardet, A. L., &

Bowman, J. P. 2015), Korarchaeota et al., 2008), and Nanoarchaeota (Wimmer et al., 2002).

Nevertheless, most of the characterized isolate are within the Euryarchaeota and

Crenarchaeota. Crenarchaeota are known predominantly as thermophilic or hyperthermophiles

(Woese et al., 1990). For instance, Pyrolobus fumarii, a Crenarchaeota, was shown grow at 113

℃ (Blochl et al., 1997).

The genus Sulfolobus includes the best-studied representative in the family Sulfolobaceae of

Crenarchaeota phylum (Brock et al., 1972). Sulfolobus species inhabit aquatic acidic and thermal terrestrial environments (pH 2-3 and temperatures of 75-80 ℃). Many Sulfolobus spp. have been isolated from Yellowstone National Park, USA, in addition to several regions around the world, i.e., Italy, El Salvador, Russia, and Japan (Brock et al., 1972; Whitaker et al., 2005;

Suzuki et al., 2002). Sulfolobus is significant because of its enormous contribution in the scientific and industrial fields. For example, in microbial ecology studies, Sulfolobus is one of the main subjects because of its membrane lipids biomarkers (Sturt et al., 2004). Sulfolobus is also became the focus of the molecular mechanisms of DNA replication studies because they have closely related systems for replicating their genomic DNA with eukaryotes, usually in a simplified form in archaea (She et al., 2001; Zang et al., 2005).

In biotechnology, Sulfolobus, because of its natural stability in high temperature, inspired scientists to develop Affitins, which are artificial proteins. Affitins is derived from the DNA binding protein Sac7d, from S. acidocaldarius. What special about this protein is that the amino acids on the binding surface of Sac7d can be randomized and give the Affitin the ability to be directed towards various targets or antigens (Mouratou et al., 2007; Krehenbrink et al., 2008;

Correa et al., 2014; Pacheco et al., 2014). Moreover, Sulfolobus is a target of DNA damage response and genome repairing studies (Grogan, D. W. 2000), because of the extreme

2 conditions they thrive in. In addition to previous benefits, Sulfolobus also a target for studies related to DNA damaging with UV-irradiation and physical stresses (Wood et al., 1997; Fröls et al., 2008, Ajon et al., 2011), and hyperthermophilic microbes DNA instability and spontaneous mutation (Grogan, D. W. 1998; Grogan et al., 2001). Finally, Sulfolobus is important for viral archaea interaction studies because it serves as a host for many of the hyperthermophilic viruses. Including both kinds of infection cycles, the lysogenic, which does not lyse the host cell, such as Sulfolobus spindle-shaped virus (Stedman et al., 1999; Stedman et al., 2003; Palm et al.,

1991), and the lytic, which lyse and kill the host cell, such as Sulfolobus islandicus rod-shaped virus (Zillig et al., 1994; Prangishvili et al., 1999).

Sulfolobus islandicus rod-shaped viruses

Sulfolobus islandicus rod-shaped viruses (SIRVs) belong to the species of the hyperthermophilic family Rudivirdae (Prangishvili et al., 1999). The first report of SIRV was in

1994 when a team of scientists sampled acidic springs in Iceland. The strain was named SIRV originally, (Zillig et al., 1994), then it was renamed as SIRV1 after the discovery of other strains of the same virus (Zillig et al., 1998). The second SIRV had 1.5 kbp larger genome and 70 nm longer capsid than the initial one, so they called it SIRV2 (Zillig et al., 1998; Prangishvili et al.,

1999). So far, the Rudivirdae have been observed in various regions around the world. From hot-springs of the Azores, two new Rudiviruses have been described, Acidianus rod-shaped virus 1 (ARV1) (Vestergaard et al., 2005) and Stygiolobus rod-shaped virus (SRV) (Vestergaard et al., 2008). Moreover, in the hot springs in Los Azufres National Park, Mexico metagenomic

3 investigation identified a new Rudivirus, called Sulfolobus Mexican Rudivirus 1 (SMR1) (Servín-

Garcidueñas 2013). Most recently, many of SIRVs that are genetically different than all the past described viruses represent the Rudivirdae family, have been isolated from Yellowstone

National Park, Wyoming, USA (Fackler 2015; Rice et al., 2001).

SIRV morphology and composition

SIRV has a rigid rod-shaped virion about 23 nm in width and about 900 nm in length, with three hair-like filaments at the ends (Prangishvili et al., 1999; Okutan et al., 2013). The rod has α helix architecture with an estimated helix pitch around 2.3 nm (Mochizuki et al., 2012;

Prangishvili et al., 1999; DiMaio et al., 2015). The main capsid portion is a low-MW protein that coats the genome (dsDNA) and forms the rod; its amino acid sequence is conserved among

SIRVs (Prangishvili et al., 2013). A high-MW protein forms the short fibers at the virion ends; its amino acid sequence varies among SIRVs (Steinmetz et al., 2008; Prangishvili et al., 2013). Both of these capsid proteins are glycosylated (Steinmetz et al., 2008). The genome is a linear double stranded DNA takes the A form DNA (A-DNA) (DiMaio et al., 2015). This capsid protein structure imposes the A form on the SIRV DNA which is important in protecting the viral genome. The

SIRV genome contains about 35.5 kbp; however, the length and sequences differ among the members of the Rudivirdae family (Prangishvili et al., 2013). Genome sequencing of SIRVs has revealed many genes that are considered core genes of the Rudivirdae family (e.g. the dUTPase,

Holliday junction cleaving enzymes (Hjc), and a single-strand specific endonuclease)

(Prangishvili et al., 1998; Birkenbihl et al., 2001; Gardner et al., 2011).

SIRV host organism

The primary host of SIRVs is the acidophilic hyperthermophilic archaeon, Sulfolobus islandicus within the order Sulfolobales of the phylum Crenarchaeota (Jaubert et al., 2013).

However, SIRV2 has the ability also to infect Sulfolobus solfataricus (Okutan et al., 2013; Deng et al., 2014). Sulfolobus genus includes many species along with S. islandicus, such as S. solfataricus, S. acidocaldarius, and many others (Brock et al., 1972; Jaubert et al., 2013). S. islandicus was named after Iceland where it was initially isolated. However, it has not been validly described as a species (Zillig et al., 1994). S. islandicus inhabits hot acidic springs with an optimal growth at pH3 and 80℃ (Guo et al., 2011). S. islandicus has been isolated from many regions on the globe, such as hot springs in Iceland (Zillig et al., 1998), hot springs at the

Mutnovsky Volcano, Kamchatka, Russia (Reno et al., 2009), and hot springs at Yellowstone

National Park, Wyoming, USA (Whitaker et al., 2003).

The two SIRV isolates from Iceland (SIRV1 and SIRV2) have been widely studied and characterized (Prangishvili et al., 1999; Peng et al., 2001; Guillière et al., 2009; Oke et al., 2011;

Prangishvili et al., 2013) and established as a system for studying Archaeal host-virus interactions in the laboratory (Peng et al., 2001; Deng et al., 2014; Guo et al., 2015). One of the unique interactions between the Sulfolobus archaea and Rudivirdae family is their lytic system; which is a different way to open the cell envelope of host and release new viral particles that has not be seen in other virus systems. (Daum et al., 2014). The virus forms virus-associated pyramids (VAPs) which are seven isosceles triangles that appear on the membrane of the host cell and open outward at the end of the infection cycle (Quax et al., 2011). The pyramid

5 structure is formed by the Protein forming Virus-Associated Pyramids/SIRV2_P98 (PVAP) (Daum et al., 2014). The VAP-based release mechanism has not been reported in Bacteria and Eukarya, indicating this may be a feature exclusive to Archaea (Quax et al., 2010).

Why study thermophilic viruses?

Thermophilic archaeal viruses are very good candidates for studying viral biodiversity and evolution, because they are highly diverged compared to bacteriophages. (Weinbauer and

Rassoulzadegan 2004; Uldahl and Peng 2013). In addition, thermophilic archaeal viruses are important in clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins (CRISPR-Cas) immune system studies, especially to prove the limitation of the CRISPR loci maximum sizes, and the selection of the protospacer is random and non-directional

(Bautista et al., 2017; Erdmann et al., 2014). Furthermore, studying archaeal thermophilic virus- host interaction proved that a response from the host can result in extensive variation in the viral genome (Martinez-Alvarez et al., 2017; Prangishvili et al., 2013; Guo et al., 2011;

Prangishvili et al., 1999).

Applications of Thermophilic Viral Enzymes in Biotechnology and Basic Research

Polymerases isolated from the viruses are diverse compared to other kinds of polymerases, especially when it comes to the primary amino acid sequence and biochemical activities. In addition, viral polymerases are functionally separate from their host cells

(Schoenfeld et al., 2010). Thermostability (up to 95℃) is a valuable property for many DNA polymerase applications. Many thermostable DNA polymerases have been described for PCR; these fall into one of two groups of high molecular and biochemical similarity, which are thermostable bacterial Pol I-type enzymes and thermostable archaeal Pol II-type enzymes, but no thermostable viral-type Pol has been described. This makes the hyperthermophilic viruses perfect candidates for studies that focus on discovering viral DNA polymerases that have biotechnological possibilities. For example, a few years ago, (Moser et al., 2012) recovered the sequence of a thermostable DNA polymerase from a viral metagenome, which found to be a powerful RT-PCR enzyme. Moreover, a promising protein-primed DNA polymerase gene recognized from the genome of the Acidianus bottle-shaped virus (ABV). This protein-primed

DNA polymerase helps with PCR genome amplification (Peng et al., 2007). This feature could make the enzyme a good fit for PCR amplification of larger DNA fragments.

Applications of thermophilic viruses in industry

Thermophilic viruses are adapted to extreme environments. Alongside the high temperature, these environments sometimes are accompanied by a number of severe conditions, physical (e.g. radiation or pressure) and geochemical (e.g. salinity or extreme pH).

Therefore, different biomolecules within the virus, especially the nucleic acids and proteins may

7 have adapted to more than one extreme condition. Because of this, thermophilic viruses are good sources for thermostable enzymes that withstand high temperatures and other extreme conditions that exist in the environment. Recently, industrial demand for biocatalysts has increased, as illustrated by viral enzymes used to make chemical transformations of organic compounds (Jayasinghe et al., 1993). Therefore, attention been increased toward organisms that survive in high-temperature environments (van den Burg 2003).

Applications of thermophilic viruses in nanotechnology

Except for recently discovered new giant viruses (Abergel et al., 2015), the virus particle, including the genome, the protecting capsid, and sometimes a covering lipid envelope, is a nanoscale biomolecular unit. In the last few years, bionano-particles including the virus capsid and the envelope or the protein cage, have been the key focus of applications in biotechnology and studies to use these particles as matrix and building bulks for nano- materials (Douglas, T.,

& Young, M. 1998). The coat proteins and the capsids of the viruses have attracted attention because of the self-assembly with atomic exactness naturally (Steinmetz 2010), and many are relatively easy to produce large quantities of particles (Steinmetz et al., 2008). For nanotechnology, the development of the Viral Nano-particle (VNP) has motivated the search for novel VNPs with useful chemical features or natural structures.

For example, Cowpea chlorotic mottle virus has been used as a controlled chemical reaction vessel because of its pH controlled and metal ion-dependent reversible permeability (Douglas,

T., & Young, M. 1999). Cowpea mosaic virus also has been applied for possible applications ranging from electronics to medicine (Ma, L. et al., 2006). Furthermore, the bacteriophage M13

8 have both, rod-shaped architectures and helical arrangements of capsid proteins (CPs), which have proposed as templates for nanowires and batteries (Wang et al., 2002 & Nam et al., 2006).

Presently, the hyperthermophilic viruses are of interest of these VNP studies because of their nano-size, self-assembly and biological viability. They are also relatively stable in extreme conditions, such as high temperatures, especially SIRVs (Steinmetz et al., 2008). One of the earliest studies on VNP was in 1996 by using plant virus vectors to pass expression of alien proteins in plants (Scholthof et al., 1996). In 1997, Tomato Bushy Stunt Virus (TBSV) had been modified to induce specific immune responses in Human Immunodeficiency Virus-1 (HIV-1) by displaying the antigenic peptide sequences (Joelson et al., 1997). After that, the doors have been widely opened for the viruses in the nanotechnology (Manchester, M., & Steinmetz, N. F.

2009; Douglas, T., & Young, M. 2006). One of the main benefits of the VNP is in the pharmacology, particularly, as vaccines and drugs delivery (Plummer, E. M., & Manchester, M.

2011; Ludwig, C., & Wagner, R. 2007; Garcea, R. L., & Gissmann, L. 2004; Singh, R., & Kostarelos,

K. 2009). Up to now, many of the viruses have been engineered to contribute in nanotechnology applications. For example, Cowpea Chlorotic Mottle Virus (CCMV), Brome

Mosaic Virus (BMV), Cucumber Mosaic Virus (CMV), Red Clover Necrotic Mosaic Virus

(RCNMV), Carnation Mottle Virus (CarMV), Cowpea Mosaic Virus (CPMV), Turnip Yellow Mosaic

Virus (TYMV), and Tobacco Mosaic Virus (TMV) have been used as building blocks in the construction of new assemblies and as models to produce new materials (Evans, D. J. 2008;

Strable, E., & Finn, M. G. 2009). In addition, M13 bacteriophage and TMV, used in the fusion of nano-wires of metals (Flynn et al., 2003, Young et al., 2008). Furthermore, CCMV and TMV were used to build protein cages for the synthesis of magnetic nano-materials (Klem et al., 2005;

Young et al., 2008). Moreover, CPMV, CCMV, and bacteriophages MS2 and M13 have used as platforms for analytical imaging (Manchester, M., & Singh, P. 2006).

Purpose

Because of its high thermostability and acid-resistant nature, SIRV has been proposed to be a platform for functionalization for technological and/or medical applications via viral nano- particle applications. The purpose of this thesis was to investigate SIRV strains isolated from

Yellowstone National Park, with a focus on determining limits of their structural integrity. The results of the first project led to further investigation of whether the stability of SIRVs isolates toward different extreme conditions could be increased, and to estimate the limitation of the stability increases.

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CHAPTER TWO

Natural Variation in the Stability of Sulfolobus islandicus Rod-Shaped

Viruses Isolated from Yellowstone National Park

Introduction

Bacteriophages served for a long time as a model for prokaryote viral studies (Dennehy

2009; Turgeon et al., 2014; Emmoth et al., 2017). The relative lack of archaeal viruses was due to the complications associated with culturing them (Oren et al., 1997; Santos et al., 2012) and their archaeal hosts (Snyder et al., 2015). Recently, the archaeal viruses have started to attract attention regarding the development of molecular biology and biotechnology (Schoenfeld et al.,

2010; Moser et al., 2012; Peng et al., 2007). Moreover, the focus on thermophilic archaeal viruses have been elevated because of their benefits in nanotechnology (Douglas, T., & Young,

M. 1998; Steinmetz et al., 2008); especially the Viral Nano-particle (VNP) (Steinmetz et al.,

2008; and as explained in chapter one). So far, more than 100 archaeal viruses have been identified, but only slightly more than half of the archaeal viruses are studied in any details

(Snyder et al., 2015). In the last decade, the Rudivirdae family has become a focus of archaeal viral research (Krupovic et al., 2011; Snyder et al., 2015). Hyperthermophilic Sulfolobus islandicus is the most common host for SIRVs (Jaubert et al., 2013), but these viruses also have been reported to infect the related species Sulfolobus solfataricus (Deng, L. et al., 2014; Okutan et al., 2013). S. islandicus inhabits hot acidic springs and grow optimally at pH3 and 80℃) (Guo et al., 2011).

Purpose

The purpose of this project was to determine the virion stability of existing isolated SIRV strains from Yellowstone National park (YNP) to inactivation. Since SIRVs replicate and persist in a very harsh environment (80℃ & pH=3), virion stability under extreme conditions should be critical for SIRV propagation in nature and, SIRVs capsid should have extra stability to protect the genome.

SIRV virions take the α helix shape which wraps around the A-form DNA genome of the virus.

This protection mechanism is similar to bacterial spores for protecting the DNA (DiMaio et al.,

2015). The main focus of this project was to challenge different SIRV strains to multiple forms of stress and measure the loss of infectivity. The aim was to answer the following questions: i) how stable are the virions with respect to environmental stress, ii) which forms of stress reveal stability differences among SIRV isolates, and iii) does stability against different forms of stress correlate among the different SIRV isolates?

Methods

SIRV strains

All SIRV strains were isolated from several hot springs at Yellowstone National Park in the summers of 2008, 2011 and 2014. 2014 by members of the Grogan research group under research permit YELL-05382. The temperatures among the springs sampled varied from 65-

82℃, and the pH value were within a range of 2.5-3.5. Glass vials were used to collect mud/sediment along with liquid samples from the hot springs. Samples were sealed and kept at room temperature during transport back to the laboratory for culturing, where they were clonally purified and cryopreserved (D. Grogan, personal communication).

Host strain and culture conditions

Sulfolobus islandicus K0016-4 isolated and cryopreserved in 9% DMSO at -80℃ was used in this study as the SIRV host strain. S. islandicus was inoculated on dextrin-tryptone (DT) media plates

(Grogan, D. & Gunsalus, P. 1993) and incubated at 80℃. Samples yielding abundant growth or colony count >2000 were diluted 20-fold, re-plated, and incubated at 80℃.

Virus preparation

To release the viral particles and destroy host cells, SoniFier cell disruptor model W140

(manufactured by Branson Sonic Power Co.) was used to destroy the Sulfolobus cells. Then two rounds of centrifugation were needed. The supernatant was moved to sterilized tubes after each round. The final supernatant was stored in the refrigerator (4℃).

Plaque assay

100μL of a lysate and 50μL of host cells were added to 4mL of molten overlay media containing

0.3 % gel gum (Gelrite, Research Products International; IL, USA) and 0.1g/L glutamic acid

(Thermo Fisher Scientific, USA). The inoculated overlay media were vortexed, poured onto fresh

DT media plates, and allowed to solidify. Plates were incubated at 80℃ until lawn formation on the plate. Plates yielding isolated plaques were counted and used to calculate plaque forming unit (PFU) per 1mL volume.

High temperature treatment

Arbitrarily chosen strains of SIRVs isolated from YNP were challenged by adding 100 µL of a lysate to 900 µL of Sulfolobus dilution (Sdil) buffer (Grogan, D. & Gunsalus, P. 1993) in 2mL

Eppendorf tubes. Tubes were then placed in a water bath at boiling temperature (99-100℃) for

2 hours. After that, surviving PFU were counted by adding 100 µL of the virus and 50 μL of

S. islandicus to 4mL of liquid overlay medium and vortexed. Inoculated overlay media were poured onto DT plates and kept at the room temperature until solidified. Plates were incubated at 80℃ for 72 hours until pure plaques formed, and then PFU was counted. For the control, the same strains were treated the same way except they were kept at room temperature.

After the boiling temperature treatment, ten of the SIRV strains were chosen to represent the

SIRV population isolated from YNP and where ere then challenged with the other treatments

(drying, extreme pH, ethanol, urea, guanidinium, and chloroform).

Drying treatment

1000 µL of the virion suspensions in Sdil buffer were transferred to watch glasses (5 cm diameter) and placed to dry in the incubator (60℃) and left in the room temperature for 24H.

Control strains were kept without drying in watch glasses in Sdil buffer at the room temperature. Surviving PFU was calculated the same way described in the temperature treatment, and the same way was used with all following treatments.

Extreme pH treatment

100 µL of the virion suspensions in Sdil buffer added to 900 µL Sdil, H2SO4 or KOH was included to give the desired pH values (pH1-pH10), then incubated for an hour at 60℃. Then the PFU was calculated as given above after serial dilution.

Ethanol treatment

100 µL of the virus suspension was added to 1000 µL of diluted ethanol to result in final concentrations of 18% & 45% ethanol. Then the mix was kept in 60℃ incubator, and PFU was counted every 12H for three days. The mix (virus and ethanol) were in Eppendorf tubes and sealed to prevent any loss of the ethanol. To calculate the survival PFU, viral suspensions were diluted serially 1:10 in Sdil buffer, so the amount of the ethanol in the sample did not affect the host cells. Controls were mixed with Sdil buffer and treated the same way.

Chloroform challenge

100 µL of the virion suspensions were added to 1000 µL of absolute chloroform in Eppendorf tubes, capped and incubated for 30 minutes at 60℃. The control was treated the same way, but Sdil buffer was used instead of chloroform. Survival PFU was diluted and counted as described above.

Chaotrope treatment

100 µL of the virion suspensions were added to 900 µL 5M urea or 900 µL guanidinium Cl. Both mixes were incubated for an hour at 60℃. The control was treated the same way, but Sdil buffer was used instead of the testing reagents. After that, survival PFU was counted as described earlier.

Results

Randomly chosen strains of SIRVs isolated from YNP were subjected to evaluate their capsid stability in different extreme conditions. Stability was measured by counting the PFU after each treatment and compared to control PFU. The logarithm of (PFU after treatment/ control PFU) indicates the stable and unstable strains.

High temperature

The results show that 13 of the SIRV strains are thermostable, after a high temperature treatment. On the other hand, 21 strains showed a decrease in PFU number under the same condition. Of these, virus strains V3 and 27.5 were the least stable and the number of the PFU decreased greatly (more than 98%) compared to the other thermostable strains V7N100 and

27.11* (see Table 2.1).

Therefore, ten strains were chosen randomly for the additional survival tests. Representing both groups, five are thermostable (V3, V7*, V45, 27.5, and 40.32) and the other five are unstable in high temperature (V36, V55, V65, 27.16, and V60).

Table 2.1: Survival ratio of SIRV strains challenged by high temperature for 2 hours. Rank SIRV isolate average log(survival) 1 V7N100 0.158362 2 27.11* 0.007382

3 14.19i -0.00536 4 27.11x -0.00877 5 V65 -0.01323 6 27.8 -0.02119 7 V7x -0.03218

8 V45N100 -0.05552 9 V60 R tube -0.06215

10 27.2O -0.06695 11 V36 -0.07525 12 V55 -0.09691 13 27.11 -0.10375 14 27.4 -0.1707 15 27.17 -0.23657 16 27.3 -0.31158 17 27.4* -0.32331 18 27.14* -0.37381 19 27.14 -0.41454 20 27.2 -0.52288 21 V65* -0.61083

22 27.12* -0.79588 23 27.12 -1.05959 24 27.16 -1.06695 25 V33N100 -2.07918 26 V45 -2.24988

27 27.16x -2.27875

28 27.15 -2.33244 29 40.32 -2.39794 30 27.13 -2.58503 31 V7* -2.81673

32 V ido -3.17609 33 V3 -3.19382 34 27.5 -3.52288 Survival is expressed as the average log of (PFU after the treatment divided by the control PFU. n= 2) and. Mann Whitney U test. P-value <0.001. H0: The effect of the high temperature on the survival rate of the virions is the same for both SIRV groups.

Drying

The ten strains were dried for 24 hours. This revealed that the ten strains ranged from very stable (27.16, V55, and 40.32) to relatively unstable (V60, V36, and V65; see Table 2.2).

Table 2.2: Values are log (final titer/control titer) challenged by drying for 24 hours at room temperature, averaged over the indicated number of independent trials (n) of SIRV strains. Rank Virus strain average SD (n=3) log(survival)

1 27.16 -0.027 0.23 2 V55 -0.055 0.315 3 40.32 -0.079 0.106

4 27.5 -0.087 0.061 5 V7* -0.21 0.271 6 V3 -0.833 0.015 7 V65 -0.916 0.017 8 V36 -1.479 0.005

9 V45 -1.624 0.001 10 V60 -1.973 0.002

t.test P-value= 0.00039. H0: The effect of the drying on the survival rate of the virions is the same for both SIRV groups, stable and unstable.

Survival as a function of pH

The same ten strains were incubated at a range of pH values for an hour at 60℃. The highest survival generally occurred around pH5 and the lowest survival was seen in alkaline condition.

Strain 27.5 was relatively stable in the different pH values (pH1-pH9); and was the only strain that produced plaques after pH9 incubation, but PFU was not detected after pH10 treatment

(see Figure 2.1).

Figure 2.1: Average PFU counts of different SIRV strains in pH 1-10 (A, B, C, D, and E). The numbers were normalized by dividing the counts of each reading by the highest count of the same pH value and transferred to the logarithm scale. n= 2. For easier comparison, strains with similar pH stability profiles have been paired on the same panel.

Ethanol

In 18% ethanol, most of the strains were very stable, except for V45 & 27.5. These strains were very sensitive to 18% ethanol and the PFU values decreased by factor 250 after 36 for strain

V45 and decreased 31 times after 48 hours for strain 27.5 (see Figure 2.2). In contrast, in 45% ethanol, all the strains were sensitive to this concentration of the ethanol after a short time compared to 18% ethanol.

V45 27.5 V3 27.16 V55 V65 V36 40.32 V7* V60

0.2

-0.2

-0.6

-1

-1.4

-1.8

-2.2 LOG (SURVIVAL RATIO) (SURVIVAL LOG

-2.6

-3 Initial 1 H 12H 24H 36H 48H 60H 72H TIME

Figure 2.2: Average PFU of survival ratio of SIRV strains challenged by 18% ethanol. n= 3.

Urea, guanidinium, and chloroform

Most of the strains were relatively stable when challenged by 5M urea. Only two strains (V65 &

V45) were very sensitive (<0.05% survivors) to urea treatment (see Table 2.3). However, 5M guanidinium Cl had a powerful effect on the SIRVs. Strain V7* succeeded to produce some plaques, but strain V55 was the only stable strain in the guanidinium Cl (see Table 2.4).

Moreover, chloroform had also harsh effect on the SIRVs. Strain V7* was the most stable strain, while strains 27.5 and 40.32 lost nearly all infectivity in this treatment (see Table 2.5).

Table 2.3: Values are log (final titer/control titer) challenged by 5M urea for an hour at 60℃, averaged over the indicated number of independent trials (n) of SIRV strains.

Rank Virus strain average log(survival) SD (n=3) 1 V7* -0.046 0.092 2 40.32 -0.06 0.125

3 27.16 -0.128 0.134 4 27.5 -0.273 0.117 5 V60 -0.412 0.12 6 V55 -0.484 0.175

7 V36 -0.592 0.009 8 V3 -0.664 0.028 9 V65 -3.647 ------10 V45 -4.221 ------

Table 2.4: Values are log (final titer/control titer) challenged by guanidinium Cl for an hour at 60℃, averaged over the indicated number of independent trials (n) of SIRV strains.

Rank Virus strain average log(survival) SD (n=3)

1 40.32 -0.062 0.126

2 V55 -0.377 0.083

3 V65 -0.886 0.019

4 V3 -1.156 0.033

5 V36 -1.251 0.012

6 27.5 -1.258 0.008

7 V7* -1.322 0.063

8 V45 -1.352 0.011

9 27.16 -1.362 0.008

10 V60 -1.405 0.015

Table 2.5: Values are log (final titer/control titer) challenged by chloroform for 30 minutes at 60℃, averaged over the indicated number of independent trials (n) of SIRV strains.

Rank Virus strain average log(survival) SD (n=3)

1 V7* -0.086 0.051

2 27.16 -0.954 0.038

3 V55 -1.037 0.101

4 V65 -1.176 0.057

5 V45 -1.176 0.028

6 V60 -1.190 0.058

7 40.32 -1.225 0.007

8 27.5 -1.276 0.011

9 V3 -1.326 0.025

10 V36 -1.711 0.017

Discussion

In this study, I profiled different SIRV strains isolated from YNP according to their stability and activity in different conditions that are stressful for the capsid proteins. High temperature disrupts hydrogen bonds and non-polar hydrophobic interactions. The bonds are disrupted because heat increases the kinetic energy which causes the molecules to vibrate too fast and aggressively. Some proteins coagulate by high temperature because the protein was interpreted as the result of an increase in β-sheet structure instead of the helical structure

(Mine et al., 1990). Moreover, pH changes disrupt proteins by ionic charges. A form of double replacement reaction occurs where the negative and positive ions in the protein change partners with the negative and positive ions in the new acid or base added. Furthermore, it has been shown that low pH induces partially folded positions in several proteins (Polverini et al.,

2006; Bychkova et al., 1992). Guanidinium and urea are strong protein denaturants that function as chaotropic agents ("Urea," 2017). Guanidinium and urea unfold proteins and turn them into their original polypeptide chains and break down the three-dimensional structure of proteins. In addition to increasing the solubility of hydrophobic molecules, interaction with

Urea solutions could be irreversibly altered, and some proteins may lose their binding function.

Urea denatures the protein through its favorable interactions with nonpolar and peptide backbone (Holthauzen, & Bolen, D. W. 2007). Ethanol based on the increased efficacy in the presence of water, generally cause rapid denaturation of proteins (McDonnell, G., & Russell, A.

D. 1999). Moreover, alcohol causes precipitation of proteins mainly because it significantly lowers the dielectric constant of the aqueous solution (Zellner et al., 2005). Chloroform is one

35 of the main solvents that accelerate the rate of denaturation of proteins by breaking the hydrophobic interactions and replacing them with hydrogen bonds (Asakura et al., 1978).

The initial results show that different strains of SIRVs are very stable in many extreme conditions. However, the stability varies among the strains. Some of the strains were very stable in one treatment but could not survive in another one. For example, when we compare the stability in the boiling temperature to the dry condition, most of the strains survived in one treatment or the other, except for V45 and V55. Strain V45 was very unstable in both treatments, while strain V55 was very stable when treated with high temperature for 2 hours and dried for 24 hours (see Figure 2.3). Statistically, the stability of the SIRV strains did not show any correlation between the two treatments (boiling for two hours and drying for 24 hours) (see Table 2.6). These results show that even when the SIRVs live in an aquatic hyperthermophilic environment, there is a limit for their stability in the high temperature at the same time they can survive in a dry condition too.

24H Drying 2H Boiling

27.5 V3 27.16 V55 V65 V45 V36 40.32 V7* V60 0.5

-0.5

-1

-1.5

-2

-2.5 LOG (SURVIVAL RATIO) (SURVIVAL LOG

-3

-3.5

-4

Figure 2.3: comparing the stability of SIRV strains in 2 hours boiling temperature to 24 hours drying. Values are given above (Table 2.1 & Table 2.2).

Most of the SIRV strains tested were very stable when treated with 18% ethanol at 60℃. Only two strains, V45 and 27.5, had a severe drop in the PFU numbers with time, 36 hours and 48 hours, respectively. On the other hand, in 45% ethanol, the strains could not survive. Strain V55 showed a little stability but did not continue for a long time. The Steinmetz group has done a similar study on SIRV2. The results in this thesis consistent with the results from Steinmetz et al., 2008, who reported that the virion of SIRV2 was stable in 20% ethanol and unstable in 50% ethanol. The results of both studies signify that ethanol at a concentration of 45% and greater reduces the ability of SIRVs to form plaques. However, an earlier study by the Prangishvili group showed that particles of SIRV1 and SIRV2 were very stable when treated with absolute ethanol, but duration of the treatment was not indicated (Prangishvili et al., 1999). Higher concentrations of ethanol (70%) have more viricidal activity as described by Block (Block, S. S.

2001). However, absolute ethanol or more than 95% ethanol have a very little or no antimicrobial effect after 24 Hours (Rotter, M. L. 1996; Harrington, C., & Walker, H. 1903).

The pH treatment results (after normalizing) showed that SIRVs survive best in acidic conditions, but the survival rates vary among the strains. Strain 27.5 succeeded to maintain its stability in a wide range of pH (pH1 – pH9). Most of the strains could not survive above pH 8, and some were stable only at lower pH (strain V60 after pH6). This shows that alkaline condition impaired the ability of SIRVs to survive. Moreover, SIRVs prefer pH5 to pH6 while the host organism S. islandicus prefers pH3. The cytoplasm of Sulfolobus cells has been found to maintain an internal pH around pH5.5 to pH6.5 (Baker-Austin, C., & Dopson, M. 2007; Lübben,

M., & Schäfer, G. 1989). The stability data indicate that SIRVs were reasonably stable in the internal pH of the host (pH5.5 - pH6.5), and at the pH values often found in the environment

38 where SIRVs are found. Moreover, SIRVs have a wider stability range when compared to the bacteriophage T2 and T7, which have optimum range for stability around pH 5-9 and pH 6-8, respectively (Sharp et al., 1946; Kerby et al., 1949).

Urea, guanidinium, and chloroform treatments effect ranged widely. The guanidinium chloride and chloroform were somehow harsh on the SIRVs. Chloroform is one of the strongest solvents for many organics and biomolecules, including the proteins (Watts et al., 2004; Kwak et al.,

2008). The major protein of SIRV2 is glycosylated and associated with the genomic DNA

(Szymczyna et al., 2009; Steinmetz et al., 2008; Prangishvili et al., 2013). Chloroform caused acute damage to the virion particles, as indicated by the drop in the survival numbers of the

SIRVs (see Table 2.5). Guanidinium Cl and urea are strong chaotropes and two of the strongest denaturants of protein folding (Lapanje, S. 1978; Pace, C. N. 1986; Bennion, B. J., & Daggett, V.

2003), and 5M Guanidinium chloride caused severe loss in many of the SIRVs (see Table 2.4).

On the other hand, 5M urea was affective on the SIRVs, but not strong enough to fully denature the capsid protein of the SIRVs. However, most of the SIRV strains were relatively stable when treated with 5M urea (see Table 2.3). The results of this study are similar to another study also showed that SIRV1 and SIRV2 were very stable in 6M urea (Prangishvili et al., 1999).

Furthermore, some of the strains treated with extreme conditions in this study resulted in similar outcomes with SIRV1, and some were totally different. For example, an earlier study showed that SIRV1 were unstable and almost deactivated after only 12 minutes at a temperature of 100℃ but at the same time, it was stable when treated with 6M urea. In this case, SIRV1 is similar to some of the strains included in this study, i.e., strains 27.5, 40.32, and

V7* (Prangishvili et al., 1999).

Overall, there was no correlation of relative stability of virions toward the various treatments evaluated (boiling, drying, urea, guanidinium Cl, chloroform; P-value > 0.05). However, there was one correlation that showed marginal significance P-value = 0.05, using Pearson’s correlation test = 0.63 (see Table 2.6). Thus, examining more SIRV isolates will give a better indication about whether the correlation between survival of drying and of chloroform treatment is significant or not.

Table 2.6: Correlations among treatments applied on ten strains of SIRVs isolated from YNP. Values calculated using Pearson's correlation test.

Treatment Drying Boiling Chloroform Guanidinium Cl Urea

Drying ------0.374 0.631 (0.050) -0348 (0.324) 0.217 (0.28) (0.545)

Boiling -0.374 ------0.491 (0.149) 0.218 (0.544) -0.154 (0.28) (0.669)

Chloroform 0.631 -0.491 ------0.438 (0.204) 0.583 (0.050) (0.149) (0.076)

Guanidinium Cl -0348 0.218 -0.438 (0.204) ------0.230 (0.324) (0.544) (0.522)

Urea 0.217 -0.154 0.583 (0.076) -0.230 (0.522) ------(0.545) (0.669)

The values given represent: cor (P-Value). H0: There is no relationship between the treatments.

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CHAPTER THREE

Increasing the stability of SIRV virions toward high temperature

Introduction

In 1859, Darwin’s theory has been accepted widely in the biology scientific community

(Darwin, C. 1988). This evolutionary selection can result speciation (Howard, D. J., & Berlocher,

S. H. 1998; Hoorn, C., & Wesselingh, F. P. 2010; Cook, O. F. 1908). In addition, ecology has a huge influence on selection and creation of new species of the population (Nosil, P. 2012).

Ecological speciation occurs when individuals interact with the changes in the environment and adjust to the new situation (Rundle, H. D., & Nosil, P. 2005). The new ability passes directly to the next population and results into a new group that is different than the initial population

(Schluter, D. 2001; Feldgarden et al. 2003). What distinguishes the ecological speciation is that this kind of speciation arises by different selections pressures among the various habitats (Nosil,

P. 2012). Because selection in nature often takes a long time, artificial selection has been used in the laboratories for detailed investigations of speciation and evolution (Rice, W. R., &

Hostert, E. E. 1993; Gavrilets, S. 2003). Thus, adaptive laboratory evolution (ALE) has attracted the attention in the evolution studies (Conrad et al. 2011; Portnoy et al. 2011; Nam et al. 2011;

Oud 2012; Çakar et al. 2012).

Microorganisms are the core of the ALE studies because of: (a) they don’t require complicated nutrients, (b) they can be easily cultured in the laboratory, and (c) they can grow very fast and can be cultured for many hundred generations within weeks or months. Viruses also have been

46 investigated for ALE. For example, a population of human immunodeficiency virus type 1 (HIV

1) was selected to be resistant to the drug (3'-thiacytidine inhibitors); this rapid selection of resistant virus has not been seen in the past (Tisdale et al., 1993). Besides, using simple in vitro selection helped with quick identification of important functional structures in the Adeno-

Associated Virus 2 (AAV2) capsid protein (Judd et al., 2011).

Purpose

The purpose of this project is to determine whether the stability of the SIRVs in different extreme conditions can be increased by selection. The approach involved treating the SIRVs with extreme conditions and recovering rare survivors. Thus, in this project, the main goal is to select pure clonal viruses that are more stable than the initial clones. This approach was used to investigate two questions: (a) is there a limit to increase the stability, (b) and is there a relationship between the selected virions and the originals in their ability to survive in different conditions?

Methods

Survival counts

PFU were counted by plaque assay method (described in chapter two). The logarithm of (PFU after treatment/ control PFU) shows the survival, which indicates the stable and unstable strains.

High temperature stability selection

Five SIRV strains (27.16, 40.32, V45, V55, and V36) challenged previously by high temperature were chosen for increasing the stability. A volume of lysate (100 µL) was added to 900 µL Sdil buffer in 2mL Eppendorf tubes, then the tubes were held in a boiling water bath (99-100℃) for

3 hours in the first stage of selection, and for 5 hours in the second stage.

High temperature stress

All the five SIRV strains (27.16, 40.32, V45, V55, and V36), after each selection step were cultured to grow under standard conditions. After that, the strains were challenged by high temperature (99-100℃) for two hours. The controls were treated the same way, but were kept at room temperature and were not challenged by high temperature. PFU were counted for both, controls and challenged viruses.

Ethanol treatment

Two strains (V45 and 27.5) were chosen for this experiment. For each strain, 100 µL of the virus suspension was added to 900 µL of 20% ethanol (to increase the selection) in Eppendorf tubes.

Then incubated at 60℃ for an hour. The viral suspensions were diluted serially 1:10 in Sdil buffer. Survivors were allowed to grow in their normal condition, then challenged again by 18% ethanol. Controls were mixed with Sdil buffer and treated the same way, but no ethanol. After that, PFU were counted.

Reproduction of the viruses

Six samples of both strains (40,32 and 40.32G) were measured for reproduction every four hours for two days at 80℃ and pH=3 (MOI less than 1). The host cells were infected after four hours of incubating. Absorbance was taken using Milton Roy Spectronic 21D

Spectrophotometer (wavelength 600 nm). PFU were counted as described previously.

Results

Selecting increased thermos-stability

Five randomly chosen strains of SIRVs isolated from YNP were challenged by high temperature

(99-100℃) for two hours to measure thermostability. Three were found to be unstable (27.16,

40.32, and V45) and two were stable (V55 and V36). This was followed by the first round of selection, which increased the time of high temperature treatment to three hours (<0.1% survivors were re-cultured and increased PFU by about 3 factors for the next round). Similarly, the second round of selection increased the time to five hours. The idea behind the two stages of high temperature with increasing treatment length is to decrease survival and increase selection. Survivors after each stage were allowed to grow in normal condition for SIRVs then subjected to high temperature for an additional to two hours to measure their thermostability.

Increasing the stability of unstable strains in ethanol

Strains V45 and 27.5 challenged previously by 18% ethanol were unstable and PFU values decreased by factor 250 after 36 for strain V45 and decreased 31 times after 48 hours for strain

27.5. Strains V45 and 27.5 were therefore challenged again with 18% ethanol for 36 hours.

Survivors were allowed to multiply in normal condition then challenged again with 18% ethanol.

Challenging the selected viral particles for extreme conditions

Pure plaques of the strain 40.32G were selected and allowed to grow and produce new virions, then were challenged with urea, guanidinium Cl, chloroform, drying, and 18% ethanol the same way was described in chapter two.

Selection for high temperature stability

After first step of challenging with boiling temperature for three hours, there were no significant changes in the results of the strains stability, compared to the parental strain, after treating the survival with 100℃ for two hours (see Table 3.1). After the second step of selection, and boiling the viral particles for five hours, survivals of strain 40.32 (named 40.32G) were able to thrive and were very stable (Log [survival] = -0.033) after challenging them again with two hours in boiling temperature. Survivors of strains 27.5 and V45 did not show significant stability after the second step. Survivals of strains V55 and V36 were very stable after both steps (see Table 3.2).

Selection for ethanol stability

Survivors of strains V45 and 27.5 after 36 hours in 18% ethanol were relatively rare (Table 3.3).

Survivors of the both strains did not show any altered inactivation compared to the initial strains after 36 hours in 18% ethanol, however (Table 3.3).

40.32G in extreme conditions

The selected strain 40.32G showed similar results to the initial strain 40.32. It was found to be stable after the drying treatment (24 hours) and urea treatment (an hour in 5M urea). On the

52 other hand, it is unstable when treated with 18% ethanol and chloroform for an hour and 30 minutes, respectively. In 5M guanidinium Cl, the selected virions are relatively stable compared to other treatments (see Table 3.4).

Table 3.1: Survivals after the first stage of selection. Values are log (final titer/control titer) challenged by 2 hours at boiling temperature n=1.

Virus strain Log (survival) 40.32G -0.397 27.16 -1.823 V45 -1 V55 -0.118 V36 -0.072

Table 3.2: Survivals after the second stage of selection. Values are log (final titer/control titer) challenged by 2 hours at boiling temperature, averaged over the indicated number of independent trials (n) of SIRV strains.

Virus strain Log (survival) SD (n=3) 40.32G -0.033 0.008 27.16 -1.897 0.283 V45 -2.200 0.074 V55 -0.095 0.034 V36 -0.035 0.031

Table 3.3: Survivals of selected particles after treated with Ethanol for 36 hours. Values are log (final titer/control titer) challenged by 18% ethanol for 36 hours at room temperature.

Virus strain Log (survival) V45 -1.875 27.5 -1.268

Figure 3.1: Survival ratio of SIRV strains challenged by high temperature for 2 hours after each step of selection. Survival is expressed as the average log of (pfu after the treatment divided by the control pfu).

Table 3.4: Survivals of the selected strain 40.32G in different extreme conditions compared to the parental strain 40.32. Values are log (final titer/control titer) challenged by specified treatments and times, averaged over the indicated number of independent trials (n) of SIRV 40.32G & 40.32G.

40.32G 40.32 treatments time temperature Ave SD (n=3) Ave SD (n=3)

18% Ethanol 1H 60℃ -2.298 0.008 -0.863 0.137

5M Urea 1H 60℃ -0.222 0.095 -0.901 0.125

5M Guanidinium Cl 1H 60℃ -0.663 0.115 -0.899 0.126

Chloroform 30 min 60℃ -2.180 0.006 -2.110 0.007

Drying 24 H Room temp. -0.255 0.173 -0.079 0.106

40.34G plaque size

The selected isolate 40.32G formed large pure plaques on DT plates with host Sulfolobus islandicus K0016-4. Average plaque size is 5 mm after 27 hours of incubation, here as the initial strain 40.32 forms plaques about 1 to 2 mm diameter (see Figure 3.2).

Figure 3.2: Plaque plate of 40.32 & 40.32G viruses plated on a lawn of S. islandicus 16-4 cells. Plates were photographed after three days of incubation.

Reproductive rates of original and selected strains

Reproductive rates of strains 40.32 and 40.32G were measured by counting the PFU each 4 hours of infection, accompanying the measurement of host cells growth. The absorbance and PFU counts indicate that the selected strain 40.32G is in some way more virulent. One possibility is that the virions were released from the host earlier than the initial strain 40.32 (after 8H and 12H of infection, respectively). In addition, the final PFU of 40.32G was greater compared to 40.32 the average log of 3.3 vs 0.7 (see Figures 3.3 and 3.4).

Figure 3.3: Average absorbance values for the growth of the host cells infected with SIRV 40.32 and 40.32G every 4 hours for 2 days. The values were normalized by dividing each value by the first value at zero H (40.32= 0.004 and 40.32G= 0.006). n=6 for each strain.

Figure 3.4: Average log of PFU in infected cultures of both strains 40.32 and 40.32G. PFU were counted every 4 hours for 2 days. The culture was infected after 4 hours of incubation. n=6 for each strain.

Figure 3.5: Survival ratio of 40.32 compared to 40.32G challenged for an hour by 18% ethanol, 5M urea, and 5M guanidinium Cl, in addition to 30 minutes in chloroform, and 24 hours drying. Survivals are expressed as the average log of (pfu after the treatment divided by the control pfu). The symbols: (*): P-value = <0.05, and (**): P-value = <0.01

H0: There are no changes in the mean of the virion groups when challenged by different extreme conditions before selection and after selection.

Discussion

In this study, I used high temperature in an attempt to select thermostable variants of

SIRV strains. The results show that the selected virions of SIRV 40.32 (named 40.32G) are very stable at high temperature, while the initial strain was unstable in two hours at 100℃. Because the viral particles were boiled for five hours, this step resulted in a very low PFU (<0.5%), which enriches the particles that are more stable than others. Succeeding in selecting these particles resulted in very stable virions at high temperature (see Figure 3.1).

The selection of ethanol treatment was not successful for the two strains included in the selection experiment. The difficulties in the ethanol selection may be because of specific effect of the ethanol on the protein denaturation. However, even when there were plaques after the selection, these plaques were not stable more than the initial strain.

Strains V55 and V36 did not show any significant difference in stability after selection compared to the initial particles. Moreover, the strain 40.32G did not pass significantly the stability of the other strains that are originally stable in high temperature (see Figure 3.1).

The difference in the plaque size between the selected and the initial isolates is very obvious.

The number of plaques and plaque size on a plate are affected by the agar or gel concentration, temperature of the media incubation, and concentration of the suspension of the host cells

(Ellis, E. L., & Delbrück, M. 1939; Terzaghi, B. E., & Sandine, W. E. 1975; Klaenhammer, T. R., &

Sanozky, R. B. 1985). Thus, these conditions were controlled when analyzing initial and selected

SIRV strains. The selected (40.32G) virion forms larger plaques compared to the originals (5mm and 2mm), respectively. This result indicates that 40.32G has a different phenotype than the

62 parental strain 40.32. Bigger plaques suggest faster productivity and infection rate. A previous study showed that the plaque size of the Newcastle disease virus grown in chick embryo cell culture was related to the virulence of the virus strains. In Newcastle disease virus study, there was a direct positive correlation between the size of the plaques produced by different strains and the virulence rates (Reeve, P., & Poste, G. 1971).

Previous results (Chapter 2) showed a general lack of correlation of stability profiles among

SIRVs. Pearson’s correlation test showed no significant P-values (P-value >0.05). The selected particle 40.32G was challenged by various extreme conditions to test the virion stability. In addition to high temperature, 40.32G is very stable in urea and drying conditions, while it lost its stability in 18% ethanol, guanidinium, and chloroform. However, when comparing the survival rates of 40.32G to 40.32, we can see that there are some significant changes in the survival rates. Especially, the ethanol (P-value = <0.05) and guanidinium (P-value = <0.01) treatments. The initial strain was very stable in the previous two treatments, but the selected strain decreased more than 2.5 times in survivors after the treatments. Strain 40.32G was very unstable when treated with the chloroform as was the initial strain. The results show that the selected strain 40.32G had the advantage to survive in high temperature, but at the same time lost the ability to survive in ethanol and guanidinium Cl, which the initial strain 40.32 can survive (see Figure 3.5).

This can be an example of life-history trade-offs, which are changes in the adaptation in a favor of one phenotype that usually result in loss of other phenotypes. Many studies have been done on the virus life-history trade-offs. For example, phage Φ6 when was selected to resist heat shock, the cost of the adaptation was low reproduction (Dessau et al. 2012). The same cost was shown for the selection of phage T7 to withstand urea (Heineman, R. H., & Brown, S. P. 2012).

In addition, foot-and-mouth disease virus showed smaller plaques to adapt low pH conditions

(Vázquez-Calvo et al. 2014), and West Nile virus showed low fitness at normal pH to stand the low pH (Martín-Acebes, M. A., & Saiz, J. 2011). In addition to bigger plaque size, strain 40.32G also had the advantage of the progeny faster, larger amount of producing new viral particles, and relatively stable in high temperature. On the other hand, it lost the advantage of stability in other conditions, such as ethanol and chloroform.

Works Cited

Çakar, Z. P., Turanlı‐Yıldız, B., Alkım, C., Yılmaz, Ü., & Nielsen, J. (2012). Evolutionary engineering of saccharomyces cerevisiae for improved industrially important properties. FEMS Yeast Research, 12(2), 171-182. doi:10.1111/j.1567-1364.2011.00775.x. Conrad, T. M., Lewis, N. E., & Palsson, B. Ø. (2011). Microbial laboratory evolution in the era of genome‐scale science. Molecular Systems Biology, 7(1), np-n/a. doi:10.1038/msb.2011.42. Cook, O. F. (1908). Evolution without isolation. The American Naturalist, 42(503), 727-731. doi:10.1086/279001. Darwin, C. (1988). On the origin of species, 1859. London: W. Pickering. Dessau, M., Goldhill, D., McBride, R. L., Turner, P. E., & Modis, Y. (2012). Selective pressure causes an RNA virus to trade reproductive fitness for increased structural and thermal stability of a viral enzyme. PLoS Genetics, 8(11), e1003102. doi:10.1371/journal.pgen.1003102. Ellis, E. L., & Delbrück, M. (1939). The growth of bacteriophage. The Journal of general physiology, 22(3), 365-384. Feldgarden, M., Stoebel, D. M., Brisson, D., & Dykhuizen, D. E. (2003). Size doesn't matter: Microbial selection experiments address ecological phenomena. Ecology, 84(7), 1679- 1687. doi:10.1890/0012-9658(2003)084[1679:SDMMSE]2.0.CO;2. Gavrilets, S. (2003). perspective: Models of speciation: What have we learned in 40 years? Evolution, 57(10), 2197-2215. doi:10.1554/02-727. Heineman, R. H., & Brown, S. P. (2012). Experimental evolution of a bacteriophage virus reveals the trajectory of adaptation across a Fecundity/Longevity trade-off. Plos One, 7(10), e46322. doi:10.1371/journal.pone.0046322. Hoorn, C., & Wesselingh, F. P. (2010). Amazonia--landscape and species evolution: A look into the past. Hoboken, NJ;Chichester, UK;: Wiley-Blackwell.

Howard, D. J., & Berlocher, S. H. (1998). Endless forms: Species and speciation. New York: Oxford University Press. Judd, J., Silberg, J., &Suh, J. (2011). Directed evolution of adeno-associated virus: Construction of an enriched random insertion library and artificial selection to identify structure- function relationships. Molecular Therapy, 19, S158. doi:10.1016/S1525-0016(16)36980- 5.

Klaenhammer, T. R., & Sanozky, R. B. (1985). Conjugal transfer from streptococcus lactis ME2 of plasmids encoding phage resistance, nisin resistance and lactose-fermenting ability: Evidence for a high-frequency conjugative plasmid responsible for abortive infection of virulent bacteriophage. Journal of General Microbiology, 131(6), 1531-1541. Martín-Acebes, M. A., & Saiz, J. (2011). A west nile virus mutant with increased resistance to acid-induced inactivation. Journal of General Virology, 92(4), 831-840. doi:10.1099/vir.0.027185-0 Nam, H., Conrad, T. M., & Lewis, N. E. (2011). The role of cellular objectives and selective pressures in metabolic pathway evolution. Current Opinion in Biotechnology, 22(4), 595- 600. doi:10.1016/j.copbio.2011.03.006. Nosil, P. (2012). Ecological speciation. Oxford University Press. ISBN 978-0199587117. Oud, B., Maris, A. J. A., Daran, J., & Pronk, J. T. (2012). Genome‐wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast. FEMS Yeast Research, 12(2), 183-196. doi:10.1111/j.1567-1364.2011.00776.x. Portnoy, V. A., Bezdan, D., & Zengler, K. (2011). Adaptive laboratory evolution — harnessing the power of biology for metabolic engineering. Current Opinion in Biotechnology, 22(4), 590-594. doi:10.1016/j.copbio.2011.03.007. Reeve, P., & Poste, G. (1971). Studies on the cytopathogenicity of Newcastle disease virus: relation between virulence, polykaryocytosis and plaque size. Journal of General Virology, 11(1), 17-24. Rice, W. R., & Hostert, E. E. (1993). Laboratory experiments on speciation: What have we learned in 40 years? Evolution, 47(6), 1637-1653. doi:10.1111/j.1558- 5646.1993.tb01257.x. Rundle, H. D., & Nosil, P. (2005). Ecological speciation. Ecology Letters, 8(3), 336-352. doi:10.1111/j.1461-0248.2004.00715.x. Schluter, D. (2001). Ecology and the origin of species. Trends in ecology & evolution, 16(7), 372- 380. Terzaghi, B. E., & Sandine, W. E. (1975). Improved medium for lactic streptococci and their bacteriophages. Journal of Applied Microbiology, 29(6), 807-813. Tisdale, M., Kemp, S. D., Parry, N. R., & Larder, B. A. (1993). Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase. Proceedings of the National Academy of Sciences of the United States of America, 90(12), 5653-5656. doi:10.1073/pnas.90.12.5653.

Vázquez-Calvo, A., Caridi, F., Sobrino, F., & Martín-Acebes, M. A. (2014). An increase in acid resistance of foot-and-mouth disease virus capsid is mediated by a tyrosine replacement of the VP2 histidine previously associated with VP0 cleavage. Journal of Virology, 88(5), 3039-3042. doi:10.1128/JVI.03222-13.

CHAPTER FOUR

Conclusion

In this thesis, different SIRV strains previously isolated by Grogan lab members were used to evaluate the diversity of the SIRV population in Yellowstone National Park. The results of the stability profiling in chapter two showed that different SIRV particles are stable in many extreme conditions. Different SIRV isolates exhibited distinct profiles of stability toward diverse forms of stress. Strain V55 was stable under most of the conditions tested in this thesis. SIRV

V55 was not the most stable strain in all conditions it was treated with, but it managed to be stable and survive in almost all the conditions. Other strains were stable in one condition or more and unstable in another. Overall, a negligible correlation between the treatments was seen, i.e., the level of resistance to one treatment did not predict the level of resistance to any other. Moreover, the survival rate of most SIRVs was highest near pH5-pH6, which contrasts with Sulfolobus spp. (optimal growth near pH3). The capsid protein of the SIRVs must have very stable interactions in order to allow the virions to thrive in different harsh conditions. Also, the

A-form of DNA, which is the form found in the virion (DiMaio et al. 2015) is known to be more stable than the common B-form DNA (Whelan et al., 2014). Thus, the structure of the A-DNA in the SIRVs also helped to protect the genome in many extreme conditions.

It was possible to select SIRV particles with increased thermostability. Selected virus

40.32G showed a significant increase in the stability at high temperature compared to 40.32

(inactivation average after was about 2.3 factors more than before selection). This change falls under viruses inter-environmental life history trade-offs (Knies et al., 2009; Goldhill, D. H., &

Turner, P. E. 2014). In this project, the comparison of the two strains 40.32 and 40.32G showed that the adaptation to the new condition of high temperature was accompanied by changes in other phenotypes. The selected particles produced significantly bigger plaques, but at the same time, they lost stability in other conditions, i.e., ethanol (the stability before was about 2.2 factors more than after selection) and chloroform (0.6 factors differences). The mechanism of the fitness trade-offs or reproduction/survival trade-offs in viruses is not clear yet (Goldhill, D.

H., & Turner, P. E. 2014).

The outcome of this study has shown that some of the particles also withstand extra harsh settings better than others, such as high temperature for 2 hours, drying for 24 hours, extreme pH for an hour, 18% ethanol for at least three days, 5M urea for an hour, 5M guanidinium Cl for an hour, and chloroform saturated buffer for 30 minutes. Thus, the stability of SIRVs makes them promising subjects for viral Nano-particle studies. Furthermore, SIRVs did not pass the stability range of what is already in nature at high temperature. For example, the selected strain 40.32G showed the same stability range of other originally stable SIRVs in high temperature such as strains V55 and V36. Finally, this thesis showed evidence that there are tradeoffs in the SIRVs to adapt to a new condition (changes in some abilities towards adjusting with a new condition).

Works Cited

DiMaio, F., Yu, X., Rensen, E., Krupovic, M., Prangishvili, D., & Egelman, E. (2015). A virus that infects a hyperthermophile encapsidates A-form DNA. Science, 348(6237), 914-917. doi:10.1126/science.aaa4181. Goldhill, D. H., & Turner, P. E. (2014). The evolution of life history trade-offs in viruses. Current opinion in virology, 8, 79-84. Knies, J. L., Kingsolver, J. G., & Burch, C. L. (2009). Hotter is better and broader: thermal sensitivity of fitness in a population of bacteriophages. The American Naturalist, 173(4), 419-430. Whelan, D. R., Hiscox, T. J., Rood, J. I., Bambery, K. R., McNaughton, D., & Wood, B. R. (2014). Detection of an en masse and reversible B-to A-DNA conformational transition in prokaryotes in response to desiccation. Journal of the Royal Society Interface, 11(97), 20140454.

APPENDIX

Evaluation of the sensitivity of Sulfolobus islandicus strains to SIRV

strain V60

Background and Purpose i) A collection of Sulfolobus strains isolated from hot springs in Italy, Kamchatka,

California, Iceland, and YNP had been tested against SIRVs isolated from YNP, and about half of the strains tested were resistant to killing, with no obvious geographical pattern (D. Grogan, unpublished results). This suggested that sensitivity to SIRVs varies within populations around the world. ii) Laboratory cultures of a sensitive host strain yield SIRV-resistant clones at high frequencies (Fackler 2015). These resistant clones accumulate and overgrow infected cultures that are incubated for long periods of time

The purpose of this project was to investigate how resistance to SIRV varies among

Sulfolobus islandicus. In addition to scoring S. islandicus strains as sensitive vs resistant, sensitive strains were compared with respect the number of resistant variants and the timing of their appearance as colonies.

Methods

Strain originally isolated from Yellowstone National Park, Wyoming in 1999 and 2000. S. islandicus strains were revived on plates of dextrin-tryptone (DT) medium (see the methods in

Chapter two) by Duyen Nguyen. The resulting clones were grown in 2mL liquid DT medium for five days before being tested for V60 resistance.

Twenty microliter of each culture was spotted onto a DT plates containing a soft overlay and sufficient PFU of the V60 virus to yield high MOI (≈8). The overlay plates were incubated, and relative growth was growth was noted at 2,3,6,8 and 10 days. The number of cells in each spot was estimated by resuspending representative spots in dilution buffer, followed by serial dilution and plate counts.

Results

Scoring of 279 strains of S. islandicus isolated from YNP in 1999 and 2000 showed that

184 to be resistant to V60 strain (66%), while 95 of the strains were sensitive (34%). The sensitivity was confirmed by a subsequent cross-streaking test to make sure that these strains are sensitive (see Table 5.1 and 5.2).

Seventy-nine percent (146 strains) of the resistant mutants showed dense growth on the plates after two to three days post infection, while the remaining strains required up to six days to show visible growth after six days. This suggests that both fast- and slow-growing strains can be resistant to SIRV. Eight of the resistant strains revealed small plaques in the spot after three days

72 post-infection, and these plaques become larger during the incubation. Among the 95 strains of

V60- sensitive S. islandicus, four did not produced any resistant colonies after 10 days, while other

91 strains were able to produce such colonies after few days.

Cross-streaking to confirm the resistant strains used V60 and S. islandicus strain K0016-4 used as a positive (sensitive) control (Fackler 2015). Strains which grow evenly and densely across the plate were considered as resistant strains, while strains that did not grow over the virus zone were considered as sensitive strains. Cross- streaking was applied to the fresh cultures, not after infection by the SIRV V60.

The strains that did not produce any colonies resistant to V60 in the initial screening were incubated again with the virus to determine whether longer exposure and culturing could generate resistant cells. After 10 days of incubation in liquid medium, the host cells were spotted on overlay containing SIRV V60, the plates were incubated for 10 days, and observation was taken every day. Twenty percent of the culture was transferred to start a new culture for the next step, and this was repeated for 5 steps (see Table 5.3). After each step, the remaining cultures were frozen at -70℃. Strain K0016-4 was used as a control. The results as summarized as follows:

First and second and third steps: strains 2,302,269,406, and 448 did not have any growth when incubated with the V60, while strain 16-4 showed resistant cells after the 5th day.

In the fourth step: strain number 2 started showing resistant cells after 7 days, besides strain 16-

4 which showed resistant after 5 days. On the other hand, other four strains did not show any growth.

In the fifth step: strain number 2 showed growth after 5 days this time, but surprisingly, strain

61-4 did not show any growth for 10 days (Table 5.3).

Discussion

The results show that the number of Sulfolobus islandicus resistant strains to SIRV V60 is high comparing to sensitive strains. In nature, especially in this harsh environment (high temperature and acidic pH) archaea dominate the area. Therefore, it is initial for them to develop strategies to cope with those challenges. One of them is dealing with the viruses, which limit their growth.The proportion of resistant isolates differed somewhat between the set of strains collected in 1999 (45%) and in 2000 (84%) (see Table 1).

Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) found in bacteria and archaea have been compared to an immune system. The spacer sequences in CRISPRs usually match attacking genetic elements, such as viruses, plasmids, and prophages (Labrie et al., 2010;

Horvath, P., & Barrangou, R 2010). S. islandicus genome showed spacer matches after challenge by Sulfolobus spindle-shaped virus (SSV9) (Bautista et al., 2015). However, S. islandicus resistance to SIRV CRISPR-Cas failed to show any significant match (Fackler 2015).

In the observations summarized above, spots of eight of the resistant strains produced what appeared to be some clear plaques during prolonged incubation. This result suggests that the resistant strains can be infected with new forms of V60 that arise spontaneously. However, attempts to recover PFU by picking these plaques and spotting on lawns of the same (V60- resistant) host cells, or on V60-sensitive (K00 16-4) cells, did not succeed.

Table 5.1: Results of YNP S. islandicus strains isolated in 1999.

Strain number Result* 2 days 3 days 6 days 8 days 10 days 1 R ++ 2 S + ++ ++++ ++++ ++++ 3 S +++ +++ +++ 4 R + + +++ +++ +++ 5 R + ++ 6 R + + 7 S + +++ +++ 8 S + +++ +++ 9 S + ++ ++++ ++++ ++++ 10 S ++ +++ ++++ 11 S + +++ ++++ ++++ 12 S ++ ++ +++ +++ ++++ 13 S + ++ +++ +++ ++++ 14 R +++ +++ ++++ ++++ ++++ 15 S +++ +++ ++++ 16 R ++ +++ ++++ 17 S ++ +++ ++++ ++++ ++++ 18 S + ++ +++ +++ +++ 19 R + ++ +++ ++++ 20 R ++ ++ +++ +++ +++ 21 S ++ +++ +++ ++++ ++++ 22 R + ++ +++ +++ ++++ 23 R + +++ +++ ++++ ++++ 24 S + ++ ++ +++ +++ 25 R + ++ +++ 26 R + + ++ ++ ++++ 27 S ++ ++ +++ 28 R + ++ +++ +++ ++++ 29 S + ++ +++ +++ ++++ 30 R ++ ++ +++ 32 S + ++ +++ +++ ++++ 34 S + +++ +++ ++++ 35 R ++ ++ +++ 36 R + ++ +++ 37 S + ++ +++ ++++ ++++ 38 S ++ ++ +++

39 S + ++ ++ +++ 40 R + ++ ++ +++ 41 S + + +++ +++ ++++ 42 S ++ +++ +++ ++++ 43 R + ++ +++ ++++ 44 R + ++ ++++ ++++ ++++ 45 S + ++ ++++ ++++ ++++ 46 S + +++ ++++ ++++ ++++ 47 S + ++ ++ 48 S + ++ ++++ ++++ ++++ 49 R + ++ +++ +++ ++++ 50 R + ++ +++ ++++ ++++ 51 R + +++ +++ ++++ ++++ 53 S + ++ +++ ++++ 54 S + ++ +++ ++++ ++++ 55 R + +++ +++ ++++ ++++ 58 R + +++ +++ +++ ++++ 59 S + ++ +++ +++ ++++ 60 R + ++ +++ +++ ++++ 61 R ++ 62 S + +++ +++ +++ 64 S + +++ ++++ ++++ ++++ 65 R ++ +++ ++++ ++++ 68 R ++ +++ +++ ++++ ++++ 69 R ++ +++ ++++ ++++ ++++ 70 R + +++ ++++ ++++ ++++ 71 S ++ +++ +++ ++++ ++++ 72 S + ++ ++ +++ +++ 73 R +++ +++ ++++ 74 R + 75 S ++ +++ +++ 78 S +++ ++++ ++++ ++++ 79 S 82 S + ++ ++++ ++++ ++++ 83 S +++ +++ ++++ ++++ ++++ 84 R + ++ ++++ ++++ ++++ 85 S + +++ ++++ ++++ 86 S + + ++ +++ 87 R + +++ +++ ++++

88 R ++ ++ ++ 89 R + ++++ ++++ ++++ 90 S ++ +++ +++ +++ 91 S + ++ +++ +++ 92 S +++ +++ ++++ ++++ ++++ 93 S + + ++ +++ ++++ 94 R + +++ +++ 95 S + + + 96 S + ++ +++ +++ +++ 97 R + ++ +++ +++ +++ 99 S + +++ ++++ ++++ ++++ 100 S + ++ +++ ++++ ++++ 101 R ++ +++ +++ ++++ 102 R + +++ +++ ++++ 103 S + +++ ++++ ++++ ++++ 105 S ++ +++ +++ +++ 107 S ++ +++ +++ +++ 108 S + +++ +++ +++ ++++ 109 R +++ ++++ ++++ ++++ 110 S ++ +++ ++++ +++ 111 S + + ++ ++++ 112 S +++ +++ ++++ ++++ 113 R +++ +++ ++++ ++++ 114 S + +++ ++++ ++++ ++++ 115 R + +++ +++ +++ ++++ 116 S + ++ +++ +++ ++++ 117 R + ++ ++ 119 R + +++ ++++ ++++ ++++ 120 S + + ++ +++ 121 S + +++ +++ +++ ++++ 122 R + +++ ++++ ++++ ++++ 123 S ++ ++ +++ +++ 124 S + +++ +++ ++++ ++++ 125 S + +++ +++ +++ +++ 126 R + ++ +++ 127 S ++ +++ ++++ ++++ ++++ 128 R + +++ ++++ ++++ ++++ 129 S + ++ +++ ++++ ++++ 130 R ++ ++

131 S + +++ ++++ ++++ 132 R + +++ ++++ ++++ 133 S + +++ ++++ ++++ ++++ 134 S ++ +++ ++++ 135 R + +++ ++++ ++++ ++++ 136 R + ++ ++++ ++++ ++++ 138 S + ++ +++ +++ ++++ 139 S ++ +++ 140 R + ++ 141 R + + ++ ++ ++ 142 R + + ++ 143 R + ++ +++ 144 S + ++ ++++ ++++ ++++ 290 S + ++ +++ ++++ ++++

* = this assignment was confirmed by cross-streaking (S = sensitive and R = resistant). + = CFU X10^2 or less ++ = CFU X10^4 +++ = CFU X10^6 ++++ = CFU X10^7 or more. Colony forming unit (CFU) count estimated is the average counting of 9 random S. islandicus strains’ CFU.

Table 5.2: Results of YNP S. islandicus strains isolated in 2000.

Strain number Result* 2 days 3 days 6 days 8 days 10 days 1 S + 2 S 3 R + + 4 R + ++ +++ ++++ ++++ 5 R + ++ ++++ 13 R + 14 S + 15 R + +++ 21 R 22 S + + ++ 23 R + + ++ 24 R + + ++ 31 S + + ++ 34 S + + ++ ++ 35 S 41 R + + ++ +++ 42 R + + +++ 43 S + + ++++ 44 R ++ ++ + ++ +++ 45 R ++ ++ +++ +++ ++++ 51 R ++ ++ +++ +++ +++ 52 R ++ ++ +++ +++ +++ 53 R + ++ +++ ++++ ++++ 54 R ++ ++ +++ ++++ ++++ 55 R ++ ++ +++ ++++ ++++ 61 R + + ++ +++ ++++ 62 R + ++ ++ 64 R + + ++ ++ 65 S + ++ 71 R + ++ +++ ++++ ++++ 72 R + ++ +++ ++++ ++++ 74 R + ++ +++ ++++ ++++ 75 R + ++ +++ ++++ ++++ 86 R + + + ++ 88 R + + ++ ++++ 89 R + + +++ +++ ++++

96 S ++ +++ ++++ 97 S + ++ ++++ 98 S + ++ ++++ 99 R + + ++ ++ ++++ 100 S + ++ ++++ 126 R + ++ +++ Ø +++ Ø ++++ Ø 127 R + ++ +++ +++ ++++ 128 R + ++ +++ Ø +++ Ø ++++ Ø 129 S + ++ ++ 130 R + ++ +++ Ø +++ Ø 151 R + ++ ++++ ++++ 153 R + ++ +++ ++++ ++++ 154 R + +++ +++ ++++ 156 R + ++ +++ ++++ 157 R + ++ '++ +++ 180 R + + ++ ++ ++ 181 R + ++ +++ +++ +++ 182 R + ++ ++ ++ 183 R + ++ +++ +++ ++++ 184 R + +++ ++ ++++ 205 R + ++ Ø ++ Ø ++ Ø ++ Ø 206 R + ++ +++ +++ 208 R + + +++ ++ ++++ 209 S + ++ +++ 210 R + + ++ +++ 211 R + ++ +++ +++ Ø +++ Ø 213 R + + ++ ++++ ++++ 214 S + ++ ++++ 231 R + ++ +++ ++++ ++++ 232 R + +++ ++++ 233 R +++ ++ +++ 234 R + + ++ +++ ++++ 235 R + ++ +++ 245 R + ++ +++ +++ ++++ 246 R ++ Ø +++ Ø +++ Ø 247 S + ++ +++ 248 S + + + 249 R + + + + + 265 R + +++ +++ ++++ ++++

266 R ++ +++ ++ +++ 267 R + + ++ ++ +++ 268 R + + + + + 269 S 271 R + ++ ++ +++ ++++ 273 R + ++ ++ Ø ++ Ø ++++ Ø 274 R + ++ +++ +++ ++++ 275 R + ++ +++ ++++ ++++ 301 R + ++ +++ ++++ ++++ 302 S + + 303 R + ++ +++ ++++ ++++ 304 R + ++ +++ ++++ ++++ 305 R + ++ ++ ++ +++ 317 R + ++ ++ +++ +++ 318 R + +++ ++++ ++++ 319 R + ++ +++ ++++ ++++ 320 R + +++ +++ ++++ 331 R + +++ ++++ ++++ 332 R +++ +++ ++++ 333 R + ++ +++ ++++ ++++ 334 R + ++ +++ +++ ++++ 335 R + ++ +++ ++++ ++++ 345 R + ++ +++ ++++ ++++ 346 R + ++ +++ ++++ ++++ 347 R + ++ +++ ++++ ++++ 348 R + +++ ++++ 349 R + + + + + 360 R + + + ++ ++ 307 R + + ++ ++++ ++++ 308 R + + ++ ++++ ++++ 309 R + ++ +++ ++++ ++++ 310 R + ++ +++ ++++ ++++ 361 R ++ +++ ++++ ++++ 362 R + + ++ +++ 363 R + ++ +++ ++++ ++++ 364 R + ++ +++ ++++ ++++ 386 R + +++ ++++ ++++ 387 R + + +++ ++++ ++++ 388 R + ++ +++ ++++ ++++

389 R ++ ++ +++ ++++ ++++ 406 S 409 R 410 R ++ +++ ++++ 433 R + ++ ++ +++ ++++ 435 R ++ +++ ++++ ++++ ++++ 445 R + ++ ++++ ++++ ++++ 446 R ++ ++++ ++++ 448 S 449 R + ++ +++ ++++ ++++ 465 R + +++ ++++ ++++ ++++ 467 R ++ +++ ++++ ++++ ++++ 468 R ++ +++ ++++ ++++ ++++ 482 R + ++ +++ ++++ ++++ 483 R + + 485 R + ++ +++ ++++ ++++ 505 R + + ++ +++ 507 R + + ++ ++ 521 R + +++ ++++ ++++ ++++ 523 R ++ +++ +++ ++++ ++++ 524 R ++ +++ ++++ ++++ ++++ 561 R ++ +++ ++++ ++++ ++++ 562 R + ++ +++ ++++ ++++ 563 R ++ ++ +++ ++++ ++++ 564 R + ++ +++ ++++ ++++ 579 R + ++ ++++ ++++ ++++ 611 S + + + 613 R + +++ ++++ ++++ ++++ 615 R ++ +++ +++ Ø +++ Ø +++ Ø 640 R + ++ ++ +++ ++++ 641 R + ++ +++ ++++ 642 R ++ +++ +++ ++++ 643 R + ++ ++++ ++++ ++++ 644 R ++ +++ ++++ ++++ ++++ 660 R + +++ ++++ ++++ 535 S + ++ ++++ 536 R + +++ ++++ ++++ ++++

* = this assignment was confirmed by cross-streaking (S = sensitive and R = resistant). Ø = with plaques.

+ = CFU X10^2 or less. ++ = CFU X10^4 +++ = CFU X10^6 ++++ = CFU X10^7 or more. Colony forming unit (CFU) count estimated is the average counting of 9 random S. islandicus strains’ CFU.

Table 5.3: Repeated attempts to select resistant to SIRV strain V60.

Strain # 1st Step 2nd Step 3rd Step 4th Step 5th Step 2 NO NO NO Yes, after 7D Yes, after 5D 302 NO NO NO NO NO 269 NO NO NO NO NO 406 NO NO NO NO NO 488 NO NO NO NO NO Control (16-4) Yes, after 5D Yes, after 5D Yes, after 5D Yes, after 5D NO

D = days of incubation.

Works Cited

Bautista, M., Zhang, C., & Whitaker, R. (2015). Virus-induced dormancy in the archaeon sulfolobus islandicus. Mbio, 6(2) doi:10.1128/mBio.02565-14. Fackler, J.R. (2015), The characterization of lytic viruses infecting the hyperthermophilic Sulfolobus islandicus isolated from Yellowstone National Park, master’s thesis, University of Cincinnati. Horvath, P., & Barrangou, R. (2010). CRISPR/Cas, the immune system of bacteria and archaea. Science, 327(5962), 167-170. doi:10.1126/science.1179555. Labrie, S. J., Samson, J. E., & Moineau, S. (2010). Bacteriophage resistance mechanisms. Nature Reviews Microbiology, 8(5), 317-327. doi:10.1038/nrmicro2315.