ECOLOGICAL ASPECTS OF VIRUSES IN SOILS by Kurt Elliott Williamson A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant and Soil Sciences Fall 2006 Copyright 2006 Kurt E. Williamson All Rights Reserved UMI Number: 3247690 3247690 2007 Copyright 2006 by Williamson, Kurt Elliott UMI Microform Copyright All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 All rights reserved. by ProQuest Information and Learning Company. ECOLOGICAL ASPECTS OF VIRUSES IN SOILS by Kurt Elliott Williamson Approved: __________________________________________________________ Donald L. Sparks, Ph.D. Chair of the Department of Plant and Soil Sciences Approved: __________________________________________________________ Robin Morgan, Ph.D. Dean of the College of Agriculture and Natural Resources Approved: __________________________________________________________ Daniel Rich, Ph.D. Provost I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ K. Eric Wommack, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Mark Radosevich, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Yan Jin, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ David W. Smith, Ph.D. Member of dissertation committee iv ACKNOWLEDGMENTS I would like to thank Mark Radosevich, Ph.D. for his support, enthusiasm, and his faith in my abilities as a scientist. While other graduate programs had their doubts about my potential as a scholar and scientist, Mark took his chances, and it has made all the difference in the world. Thanks are also due to Kirk Czymmek and Debbie Powell in the DBI BioImaging suite, for keeping the TEM running and helping me capture the beautiful phage images contained within these pages. To my research group: Shellie Bench, Rebekah Helton, Matthew Simon, Sharath Srinivasiah, Danielle Winget, and Shannon Williamson, you are not only excellent colleagues, but also worthy friends. Thanks for sharing of your ideas, your time, and your humor during our brief overlap. I am hopeful that our paths will continue to cross for many years to come. Finally, and most importantly, I owe an incredible debt of gratitude to my wife and best friend, Beth, and to my parents, Kenneth and Sylvia Williamson. Without their unfaltering love and support, this achievement would not be possible. This manuscript is dedicated to the memory of my grandparents: Frederick and Audrey Williamson, and Mervin and Edith Dale. v TABLE OF CONTENTS LIST OF TABLES vii LIST OF FIGURES viii ABSTRACT x Chapter 1 GENERAL INTRODUCTION 1 Introduction 1 Virus life cycle 2 Impacts of viral lysis 3 Genetic interactions between virus and host 5 Lysogenic conversion 5 Transduction 6 Viruses in soils 8 Historical overview 8 Dynamics of phage-host systems in soils 9 Transduction in soils 12 A prevalence of lysogeny in soil? 13 Progress in soil viral ecology 15 Methods 16 Extraction and enumeration of autochthonous viruses in soil 16 Extraction 16 Enumeration 17 Assessing viral diversity in soils 19 Assessing lysogenic interactions in soils 20 2 EXTRACTING NATURAL VIRAL COMMUNITIES FROM SOILS 22 Introduction 22 Materials and Methods 23 Results 28 Discussion 30 3 ABUNDANCE AND DIVERISTY OF VIRUSES IN SOILS 41 Introduction 41 Materials and Methods 42 vi Results 49 Discussion 54 4 CULTIVATION-DEPENDENT ASSESSMENT OF LYSOGENIC INTERACTIONS IN SOILS 72 Introduction 72 Materials and Methods 74 Results 78 Discussion 83 5 CULTIVATION-INDEPENDENT ASSESSMENT OF LYSOGENIC INTERACTIONS IN SOILS 97 Introduction 97 Materials and Methods 98 Results 103 Discussion 107 6 GENERAL DISCUSSION 126 Viral Abundance, Bacterial Abundance, and the VBR 127 Viral Diversity 134 Selection for Viral Replication Strategy 136 Isolate Inductions 136 Induction of Soil Bacterial Assemblages 138 Questions Remaining 140 BIBLIOGRAPHY 143 vii LIST OF TABLES Table 2.1. Physical and chemical properties of soils 37 Table 2.2. Properties of bacteriophages and their recovery from soil 37 Table 2.3. Effect of eluant on recovery of viable phages and virus-like particles (VLP) from soil 38 Table 2.4. Effect of Soil X Eluant of EFM Direct counts of VLPs 39 Table 3.1. Global properties of soils 62 Table 3.2. Characteristics of Phages 63 Table 3.3. Two-tailed Pearson correlations between bacteriophage abundance, diversity, and soil physical factors 63 Table 4.1. Physical properties of soils 92 Table 4.2 Identification of soil isolates based on 16S rRNA gene sequence 93 Table 5.1. Soil physical properties 118 Table 5.2a. Two-tailed Pearson Correlations for Antarctic soils 123 Table 5.2b. Two-tailed Pearson Correlations for Delaware soils 123 Table 6.1. Comparisons of virus-to-bacterium ratios across ecosystems 129 viii LIST OF FIGURES Figure 2.1. DNase treatment of soil virus extracts 39 Figure 2.2. Transmission electron micrograph of phage from Matapeake soil extracted with sodium pyrophosphate 40 Figure 3.1. Abundance of virus-like particles in six Delaware soils based on single extractions with 1% potassium citrate 64 Figure 3.2. Viral abundance in sequential soil extractions 65 Figure 3.3. Pulsed-field electropherogram of virus genomes extracted from two soils 66 Figure 3.4. Pulsed-field electropherogram of soil virus concentrates from five Delaware soils 67 Figure 3.5. RAPD-PCR of soil viral concentrates 68 Figure 3.6. Frequency distribution of viral morphologies from six Delaware soils and known phage isolates 69 Figure 3.7. Example transmission electron micrograph demonstrating degrees of tail breakage incurred to phage T4 during extraction from Evesboro soil 70 Figure 3.8. Incidence of phage tail breakage by morphology 71 Figure 4.1. Comparison of induction responses across isolates 94 Figure 4.2. Pulsed-field gel electropherogram of induced viruses 95 Figure 4.3. Examples of phage particles induced from each lysogen 96 Figure 5.1. Optimizing extraction of bacteria from soil 119 Figure 5.2. Effect of bacterial extraction procedures on induction response 120 Figure 5.3. Comparison of induction approaches 121 ix Figure 5.4. Reproducibility of estimates of burst size and inducible fraction by the Extract-Induce-18h method 122 Figure 5.5. Biological parameters of soils 125 [...]... Colwell, 2000b)] In contrast to growing knowledge of the role of viruses in marine microbial communities, however, the ecological role of viruses in soil microbial communities remains relatively unexplored The first step in any analysis of soil viruses is the effective extraction of viruses from soil As outlined in the General Introduction, several studies have detailed the mechanics and kinetics of virus... community fingerprinting was attempted using PFGE and RAPDPCR, but results were inconsistent Assessing Lysogenic Interactions in Soils Generally, investigations of lysogeny depend upon induction assays using UV light or mitomycin C (MC) (Roberts, 1983) In aquatic samples, the inducing agent can freely diffuse (MC), or penetrate when samples are spread in thin layers (UV), to most of the cells within the... 2 of this text compares several extraction-enumeration approaches aimed at determining viral abundance in soils, and includes a comparison of EFM and TEM direct counting of extracted viruses The results of these studies are applied to the extraction and enumeration of viruses from a larger set of soils in Chapter 3 Assessing Phage diversity in soils Current methods for assessing genetic diversity of. .. appropriate methods specific to the study of viruses in soils Consequently, any record of progress in soil viral ecology will initially be one of methods development METHODS Extraction and Enumeration of autochthonous viruses in soil Extraction At the heart of any attempt to enumerate viruses in soils is the extraction of viruses from soil Consequently, extraction efficiency is of critical importance to any method... important for phages inhabiting Saharan sands, they may be more indicative of the restrictive nature of desert soils (i.e., lack of free water or organic substrates for host growth) Hence, extrapolating these results to other, more productive soils is not justified Furthermore, despite the convincing arguments of Stewart and Levin (Stewart and Levin, 1984), a preponderance of lysogeny in soils need not be... (Craun, 1991), hence the high level of interest in pathogenic viruses in soils Many studies geared toward assessing potential hazards associated with land application of wastewater have yielded insights into virus-soil interactions [for a review, see (Gerba, 1984)] Initially, the prevailing view of virus-soil interactions was that adsorption to soil surfaces resulted in inactivation: the mechanism through... extracted viruses from rhizosphere soil by bead-beating samples in water The resulting soil extracts were used in conjunction with transmission electron microscope direct counting (described below) Analyses of viruses in other porous media, namely sewage sludge and marine sediments, have relied on a variety of extraction techniques, many aimed at direct counting of viruses Shaking in glycine buffer... 1995) which indicated that transduction frequency in aquatic systems was enhanced as much as 100 fold in the presence of suspended particulate matter Apparently, such particulates increased aggregation of phages and bacteria at particle surfaces, enhancing interactions between phage and host 7 VIRUSES IN SOILS Historical overview In contrast to growing knowledge regarding the viral ecology of marine ecosystems,... regarding the viral ecology of marine ecosystems, the ecological role of viruses in soil microbial communities remains relatively unexplored Historically, a large portion of research concerning viruses in soils has been from a public health perspective, dealing with the fate, transport, and detection of pathogenic viruses exogenous to soils In terms of waste management, soil has been viewed as a biological... The importance of viruses in aquatic ecosystems has been established over the past decade By contrast, the abundance and distribution of viruses in soils is almost completely unknown An essential first step in any investigation of viruses in soil is evaluation of viral recovery methods suitable for subsequent cultivationindependent analyses A comparison of four common elution buffers indicated that . Transduction 6 Viruses in soils 8 Historical overview 8 Dynamics of phage-host systems in soils 9 Transduction in soils 12 A prevalence of lysogeny in soil? 13 Progress in soil viral. enumeration of autochthonous viruses in soil 16 Extraction 16 Enumeration 17 Assessing viral diversity in soils 19 Assessing lysogenic interactions in soils 20 2 EXTRACTING NATURAL. sparked initial curiosity about viruses in the environment. Since then, the literature has reflected an explosion of interest in the prevalence of viruses in various environments and the effects of