Portland State University PDXScholar Biology Faculty Publications and Presentations Biology 2010 The Isolation of Viruses Infecting Archaea Kenneth M. Stedman Portland State University Kate Porter Mike L. Dyall-Smith Follow this and additional works at: https://pdxscholar.library.pdx.edu/bio_fac Part of the Bacteria Commons, Biology Commons, and the Viruses Commons Let us know how access to this document benefits ou.y Citation Details Stedman, Kenneth M., Kate Porter, and Mike L. Dyall-Smith. "The isolation of viruses infecting Archaea." Manual of aquatic viral ecology. American Society for Limnology and Oceanography (ASLO) (2010): 57-64. This Article is brought to you for free and open access. It has been accepted for inclusion in Biology Faculty Publications and Presentations by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. MANUAL of MAVE Chapter 6, 2010, 57–64 AQUATIC VIRAL ECOLOGY © 2010, by the American Society of Limnology and Oceanography, Inc. The isolation of viruses infecting Archaea Kenneth M. Stedman1, Kate Porter2, and Mike L. Dyall-Smith3 1Department of Biology, Center for Life in Extreme Environments, Portland State University, P.O. Box 751, Portland, OR 97207, USA 2Biota Holdings Limited, 10/585 Blackburn Road, Notting Hill Victoria 3168, Australia 3Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Abstract A mere 50 viruses of Archaea have been reported to date; these have been investigated mostly by adapting methods used to isolate bacteriophages to the unique growth conditions of their archaeal hosts. The most numer- ous are viruses of thermophilic Archaea. These viruses have been discovered by screening enrichment cultures and novel isolates from environmental samples for their ability to form halos of growth inhibition, or by using electron microscopy to screen enrichment cultures for virus-like particles. Direct isolation without enrichment has not yet been successful for viruses of extreme thermophiles. On the other hand, most viruses of extreme halophiles, the second most numerous archaeal viruses, have been isolated directly from hypersaline environ- ments. Detailed methods for the isolation of viruses of extremely thermoacidophilic Archaea and extremely halophilic Archaea are presented in this manuscript. These methods have been extremely effective in isolating novel viruses. However, Archaea comprise much more than extreme thermoacidophiles and extreme halophiles. Therefore a vast pool of archaeal viruses remain to be discovered, isolated, and characterized, particularly among the methanogens and marine Archaea. Some suggestions for expansion of the described methods are discussed. We hope these suggestions will provide an impetus for future work on these and other Archaeal viruses. Introduction: but to date only about 50 viruses have been reported that infect this large and diverse group of organisms (Prangishvili Archaea have been shown by molecular techniques to be et al. 2006). Most archaeal viruses have been isolated from widespread in many ecosystems (e.g., Chaban et al. 2006), either extreme thermoacidophiles or extreme halophiles (Prangishvili et al. 2006; Porter et al. 2007). This work was pioneered by the late Wolfram Zillig, but was not systemati- *Corresponding author: E-mail: [email protected] cally addressed until the work of Prangishvili and Dyall- Acknowledgments: Smith, respectively. Early virus isolates of extreme halophiles Publication costs for the Manual of Aquatic Viral Ecology were pro- vided by the Gordon and Betty Moore Foundation. This document is (haloviruses) were of the head-and-tail type, the same mor- based on work partially supported by the U.S. National Science phology observed in more than 90% of described bacterio- Foundation (NSF) to the Scientific Committee for Oceanographic phages; more recent isolates have included representatives of Research under Grant OCE-0608600. Any opinions, findings, and con- spindle-shaped and spherical morphotypes (Porter et al. clusions or recommendations expressed in this material are those of the 2007; Pietila et al. 2009). In contrast, none of the viruses of authors and do not necessarily reflect the views of the NSF. The authors would like to thank D. Prangishvili for his insights and extremely thermophilic crenarchaea are of the head-and-tail vast knowledge of the isolation of viruses of extremely thermophilic type, but show a fascinating variety of unique morphologies Archaea. KS would like to thank D. Grogan for the suggestion of PEG and genomes, indicating that we have only just begun to 400 as a bath liquid. The authors would also like to thank an anony- appreciate the diversity of archaeal viruses (Prangishvili et al. mous reviewer whose suggestions greatly improved the manuscript. 2006). Research in the Stedman lab is supported by NSF (MCB: 0702020) and NASA (NNX07AJ26G and NNX07AT63A). MDS is grateful to D. Viruses in high temperature acidic environments are sur- Oesterhelt, and the Department of Membrane Biochemistry, MPI, for prisingly low in abundance, commonly 103 per mL, as deter- their continuing support. mined by either nucleic acid staining techniques (Ortmann et ISBN 978-0-9845591-0-7, DOI 10.4319/mave.2010.978-0-9845591-0-7.57 al. 2006; Prangishvili et al. 2006) or direct counting of virus- Suggested citation format: Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. The isolation like particles (VLP) (Ortmann et al. 2006; Prangishvili et al. of viruses infecting Archaea, p. 57–64. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. 2006). The reason for this is unknown. Therefore almost all of 57 Stedman et al. Archaeal virus isolation the viruses isolated from thermoacidophilic Archaea come used for reduction, but with less success; Stedman, unpub- from enrichment cultures of environmental samples. lished). The air in the tube is displaced with CO2 and N2 by the Hypersaline waters are similar to marine ecosystems, with Hungate technique and the tube is stoppered (Hungate et al. high VLP counts, commonly around 108 VLP per mL (Guixa- 1966). A cap is placed on the tube and the assemblage auto- Boixareu et al. 1996; Oren et al. 1997; Diez et al. 2000; Pedros- claved. A gas phase of 160 kPa of CO2 and 1 kPa of H2S has also Alio et al. 2000a; Danovaro et al. 2005; Bettarel et al. 2006). been used successfully (Prangishvili 2006). Despite the high virus levels, low cell growth rates, and fre- Sample collection and transportation for thermoacidophilic quent observations of VLPs inside cells, some studies predict Archaea—Liquid and wet sediment samples are collected from that haloviruses are not major regulating factors of commu- turbid terrestrial hot springs with high temperature >70˚C and nity size (Guixa-Boixareu et al. 1996; Pedros-Alio et al. 2000a; low pH <4. The pH is often tested with pH paper because it is Pedros-Alio et al. 2000b). Although the viral role in microbial less susceptible to temperature changes than most pH elec- population control remains unclear, high virus numbers indi- trodes. Samples are collected in sterile 50-mL conical flasks at cate that they should be readily isolable directly from water the end of an extendible pole with a clamp (see Fig. 1A). After samples but, to date, only about 21 well-described haloviruses most of the sediment is allowed to settle, the pH of the liquid have been reported in the published literature (Pagaling et al. is carefully adjusted to ca. 5.5 with solid CaCO3 by slow addi- 2007; Porter et al. 2007; Pietila et al. 2009). tion and stirring. Once the pH is adjusted the sample is trans- Methanogens are the first-identified and probably best- ferred to a pre-prepared anaerobic tube using a syringe (see characterized members of the Archaea; however, reports of above and Fig. 1A inset). If the resazurin indicator changes to their viruses are surprisingly sparse in the literature, with only pink, drops of H2S-saturated water are added until the sample three different viruses described, one characterized in detail, clears. Samples can be maintained for up to 2 weeks at room and ten viruses or proviruses reported. Viruses of methanogenic temperature before enrichment. Archaea have been isolated from anaerobic sludge digesters Alternative sample collection—If the laboratory is relatively (Meile et al. 1989; Nolling et al. 1993) and found in super- close to the sampling location, water and sediment samples natants of known cultures (Wood et al. 1989). It is unclear are collected as above, but instead of an anaerobic tube, a ster- whether this lack of published viruses is due to the low abun- ile screw-cap vial or centrifuge bottle is completely filled so dance of viruses of Methanogens or insufficient screening. that very little air is present. Samples can then be transported However, a recent bioinformatic analysis of the incomplete at ambient temperature and should be enriched within 8–10 h genome of Methanococcus voltae strain A3 indicated the pres- of collection (Rice et al. 2001). ence of at least two different proviruses (Krupovic and Bamford Enrichment culture for host and virus isolation—Samples col- 2008) and highlights the need for further study of viruses of lected either in anaerobic tubes or filled centrifuge tubes are methanogenic Archaea. diluted 1:50 or 1:100 in Sulfolobus growth medium (Zillig et al. This manuscript gives methods for isolation of viruses of 1994) containing either yeast extract (0.1% w/v) and sucrose the thermoacidophilic archaeon Sulfolobus and close relatives (0.2% w/v) as carbon sources or Tryptone (0.2% w/v) in long- and viruses from hypersaline waters. Methods used for the iso- necked Erlenmeyer flasks (see Fig. 1B inset), and incubated at lation of viruses of other thermophilic Archaea are also dis- 80˚C with shaking (150 rpm) for up to 2 weeks. The salts in cussed.