The Stability of Lytic Sulfolobus Viruses 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. i 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. ii iii 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. iv 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 v 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 vi 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. vii 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. viii 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). 1 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