Fingerprinting aquatic virus communities

To circumvent the limitations of cultivation-based studies of complex microbial communities, molecular fingerprinting techniques such as pulsed field gel electrophoresis (PFGE) and denaturing gradient gel electrophoresis (DGGE) have been used to examine their richness, diversity, and dynamics. PFGE is based on the electrophoretic separation of extremely large DNA, raising the upper size limit from 50 kb (standard agarose separation) to well over 10 Mb. This technique has been used to separate aquatic virus genomes ranging in size from tens to hundreds of kilo base pairs (kb); aquatic virus genomes range from 15 to 630 kb, with the majority between 20 and 80 kb. DGGE, on the other hand, is based on the electrophoretic separation of PCRamplified gene fragments of similar sizes, but differing in base composition or sequence. In this chapter, we provide a brief overview of each of these methods and their application to the study of aquatic viruses. We describe some of the common equipment, reagents, and procedures involved, and conclude by briefly considering some of the strengths and weaknesses of each method. *Corresponding author: E-mail: *ruth.sandaa@bio.uib. no Acknowledgments Publication costs for the Manual of Aquatic Viral Ecology were provided by the Gordon and Betty Moore Foundation. This document is based on work partially supported by the U.S. National Science Foundation (NSF) to the Scientific Committee for Oceanographic Research under Grant OCE-0608600. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF. ISBN 978-0-9845591-0-7, DOI 10.4319/mave.2010. 978-0-9845591-0-7.9 Suggested citation format: Sandaa, R. -A. , S. M. Short, and D. C. Schroeder. 2010. Fingerprinting aquatic virus communities, p. 9–18. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. MAVE Chapter 2, 2010, 9–18 © 2010, by the American Society of Limnology and Oceanography, Inc. MANUAL of AQUATIC VIRAL ECOLOGY method was used in conjunction with DGGE to investigate the relationship between bacteriophages and their hosts in nutrient-manipulated mesocosms and the effect of both topdown and bottom-up control of the bacterial community (Øvreås et al. 2003). Among the first reports of applying DGGE as a tool for aquatic virus research were studies of marine cyanophage diversity. Wilson et al. (2000) initiated this work by examining the diversity of a viral capsid protein (g20) in marine environments. Later, Frederickson et al. (2003) adopted this DGGE-based technique and demonstrated that patterns in cyanophage communities were related to the physical structure of the water column in marine environments. Other studies that were also based on this DGGE approach revealed that cyanophage diversity was temporally variable in Lake Bourget, France (Dorigo et al. 2004), and that highly similar phage sequences could be obtained from a variety of marine and freshwater samples (Short and Suttle 2005). More recently, DGGE was used to study the diversity of g20 in Norwegian coastal waters, revealing surprising genetic diversity and seasonal shifts in cyanophage communities (Sandaa and Larsen 2006). Around the same time that the first reports of DGGE analysis of cyanophage genes appeared in the literature, other investigators were using DGGE to examine the diversity of DNA polymerase genes belonging to viruses that infect eukaryotic algae, i.e., the phycodnaviruses (Short and Suttle 1999). Eventually, these researchers used DGGE-based methods to show that some phycodnaviruses were widely distributed in nature (Short and Suttle 2002), and some persisted in British Columbia coastal waters throughout most of a year-long study (Short and Suttle 2003). Similarly, to study the population dynamics of viruses that infect the bloom-forming alga E. huxleyi, Schroeder et al. (2003) developed a DGGE method to examine the diversity of the major capsid protein of E. huxleyi–specific viruses and demonstrated that only a few members of the E. huxleyi virus community actually caused the demise of a bloom observed in a mesocosm experiment. This observation was made again 3 years later at the same study site (Martinez et al. 2007). Although the use of DGGE/PFGE to directly study aquatic viruses is limited to a relatively small number of reports, several studies have used DGGE to link changes in host community composition to changes in virus abundance and/or virus diversity as inferred from PFGE analysis of virus genome sizes (e.g., Castberg et al. 2001, Goddard et al. 2005, Larsen et al. 2001, Riemann and Middelboe 2002, Simek et al. 2001, Van Hannen et al. 1999b, Weinbauer et al. 2007, Zhang et al. 2007). Together, the body of literature describing DGGE/PFGE-based investigations of aquatic virus communities demonstrates the importance of these methods for aquatic virologists and convincingly demonstrates that fingerprintbased investigations have had a profound impact on current understanding of virus community ecology. Materials and procedures An overview of the methods involved in fingerprinted aquatic virus communities is shown in Fig. 1. Because the two methods considered in this chapter (PFGE and DGGE) are very different, each will be considered independently. Because the analysis of the fingerprint patterns generated by each method does not differ, this section will conclude with a brief comment on fingerprint analysis. Furthermore, in this section we have provided the formulation for many of the reagents used for PFGE and DGGE; nevertheless, more detailed recipes for many of them (e.g., electrophoresis buffers) can be found in most molecular biology laboratory manuals (e.g., Ausubel et al. 2002, Sambrook et al. 1989). PFGE—The viral concentrate used for PFGE analysis must be molded into plugs, followed by lysis of the virus particles to release their DNA. It is possible to run solution-based preparation of viral DNA for PFGE (Steward 2001); however, large DNA molecules (>100 kb) are extremely sensitive to mechanical shearing in aqueous solution (Bouchez and Camilleri 1997). The consensus is that lysis inside viral plugs prevents mechanical shearing of the DNA, resulting in more discrete PFGE bands. Intact viral genomes are then separated by size by PFGE. After separation, the banding pattern is visualized by staining with a fluorescent DNA stain. This banding pattern provides a visual record of the genome size distribution that can be used for qualitative and quantitative comparisons between samples. Equipment and reagents: • Pulsed field gel electrophoresis system. We recommend the CHEF-DR II (Bio-Rad); • Casting stand, comes in 14 × 13 cm (small gel, 10 wells) or 21 × 14 cm (large gel, 15 wells) frame and platform; • Combination comb holder; • Combs, 1.5 mm thick; • Plug molds (Bio-Rad), each well holds 80 μL; • Screened caps (Bio-Rad); • DNA molecular weight standards, e.g., lambda ladder (available in blocks that have to be sliced into smaller pieces before use) and 5-kb ladder (e.g., Bio-Rad); • Dilution or storage buffer: SM buffer (0.1 M NaCl, 8 mM MgSO 4 7H 2 O, 50 mM Tris-HCl, 0.005% (wt/vol) glycerin); • Lysis buffer: must be freshly made (250 mM EDTA, pH 8.0, 1% SDS, 1 mg/mL Proteinase K); 5 mL lysis buffer is needed per sample; • 1.5% PFGE-grade agarose (e.g., SeaKem GTG agarose; Cambrex) for preparing plugs dissolved in TE 10:1 (10 mM Tris, 1 mM EDTA, pH 8.0); the agarose can be stored at 4°C in between uses; • Washing buffer: TE 10:1 (10 mM Tris, 1 mM EDTA, pH 8.0), 150 mL per sample; • Storage buffer: TE 20:50 (20 mM Tris, 50 mM EDTA, pH 8.0), 20 mL per sample; Sandaa et al. PFGE and DGGE for describing aquatic virus communities