University of Plymouth PEARL https://pearl.plymouth.ac.uk 04 University of Plymouth Research Theses 01 Research Theses Main Collection 2008 CHARACTERISATION OF LATENT INFECTIONS lN AQUATIC CYANOBACTERIA AND MICROALGAE LOHR, JAYME ELIZABETH http://hdl.handle.net/10026.1/2443 University of Plymouth All content in PEARL is protected by copyright law. Author manuscripts are made available in accordance with publisher policies. Please cite only the published version using the details provided on the item record or document. In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of content should be sought from the publisher or author. CHARACTERISATION OF LATENT INFECTIONS lN AQUATIC CY ANOBACTERIA AND MICROALGAE by JA YME ELIZABETH LOHR A thesis submitted to the University of Plymouth in partial fulfilment for the degree of DOCTOR OF PHILOSOPHY School of Biological Sciences Faculty of Science April2008 ABSTRACT Characterisation of latent infections in aquatic cyanobacteria and microalgae Jayme Elizabeth Lohr Aquatic photosynthetic microorganisms were surveyed (algae and cyanobacteria) for novel lysogenic/latent viruses and new methodology was established, using a variety of techniques such as AFC, electron microscopy, and molecular tools. The first study assessed Symbiodinium sp. cultures as a model system to in vestigate the induction of potential latent vi ruses. The study of Symbiodinium sp. showed that ea. 37% of the strains tested had a group of filamentous VLPs that is inducible by UV -C treatment. Extrapolation of this virus-host interaction and its effects on zooxanthellae viability provides a novel link to the impact of latent infection on symbiotic dtnotlagellates of cnidarians and the subsequent disruption of the reef ecosystems. The second study examined the interaction of a freshwater cyanobacterium and its inducible VLPs. The work carried out on Pseudanabaena, strain PPt I 0905 suggests that this freshwater cyanobacterium harbours a prophage. An unusua l interaction was observed in this freshwater cyanobacteriu m, where the abundance of carboxysome-like particles tncreased I 0 times in heat-treated cultures. The cyanobacterium PPt I 0905, its inducible VLPs and the eo-occurring increase in carboxysomes could be a new mechanism in which lysogeny benefits freshwater cyanobacteria, possibly increasing the host's photosynthetic efficiency. Finally, the thi rd study investigated the presence oflatent viruses in the Plymouth culture collection of marine algae. The characterisation and isolation of inducible viruses from this alga l culture co llection has revealed much novel information on the prevalence of latent viruses in algae. From the 30 algal species examined in this study, over 35% appear to contain an inducible infectious agent. AFC and TEM images have confim1ed the presence of VLPs, and thin sections of UV­ induced cultures fu tther supported the presence of VL Ps in the UV -induced cultures. This work's contribution increases the knowledge of latent and temperate viruses of aquatic microbes, which are unden·epresented in previous studies. Additional ly, this research estab lished novel tec hniques for the study of unique biological interactions between aquatic viruses and their hosts that will fac ili tate and improve subsequent investigation of simi tar systems. TABLE OF CONTENTS Table of Contents 11 List of Tables v List of Figures v1 Acknowledgements v111 Author's Declaration IX Chapter I Introduction I 1.1 The global importance of aq uatic viruses 1 1.1.1 Development of aquatic virus research I 1.1.2 Viral dynamics 3 1.2 Capsid structure and symmetry 4 1.2. 1 lcosahedral symmetry 4 1.2.2 Helical symmetry 5 1.2.3 Enveloped viruses 6 1.2.4 Complex viruses 7 1.3 Replication strategies 8 1.4 Taxonomy I 0 1.4.1 Bacteriophages I 0 1.4.2 Algal viruses 13 1.5 Life cycles 15 1.5. 1 Lytic 15 1. 5.2 Lysogeny/latency 16 1. 5.3 Chronic infections 18 1.5.4 Gene transfer 20 1.6 Nature of known aquatic viruses 20 1.6. 1 Freshwater viruses 2 1 1.6.2 Marine viruses 22 1.7 Aims and objectives 23 1.7.1 Do zooxanthellae harbour latent viruses? 23 1.7.2 What are the potential interactions of inducible VLPs from a fres hwater cyanobacterium? 24 I. 7.3 Are latent viruses prevalent in marine algae? 25 Chapter 2 Methods and Materials 26 2. 1 Cultures 26 2.1.1 Zooxanthellae 26 2.1.2 Cyanobacterium 30 2.1.3 Plymouth Culture Collection (PCC) of Marine Algae 31 2.2 Induction conditions 34 2.2.1 Zooxanthellae induction 34 2.2.2 Cyanobacterium induction 34 2.2.3 PCC phytoplankton induction 35 2.3 Enumeration of hosts, VLPs and bacteria 36 2.4 Transmission Electron Microscopy (TEM) 37 2.5 Concentration ofYLPs 38 2.5.1 Tangential flow fi ltration 38 2.5.2 QuikStand filtration system 39 2.5.3 Polyethylene glycol (PEG) precipitation 40 2.5.4 Ultracentrifugation 41 2.5.5 CsCl gradi ents 41 11 2.6 Isolation ofVLPs 4 1 2.6.1 Isolation of VLPs from zooxanthellae 4 1 2.6.2 1solation ofVLPs from cyanobacteri a and PCC phytoplankton 42 2. 7 Extraction of nucleic acid 42 2.7.1 Algal DNA extractions 42 2.7.2 Vi ral RNA extractions 43 2.7.3 Viral DNA extractions 43 2.7.4 RNase and DNase treatments 43 2.8 Pulsed fi eld gel electrophoresis (PFGE) 44 2.9 Polymerase chain reaction (PCR) 45 2.9. 1 Electrophoresis 46 2.9.2 Gel Purification of DNA 46 2.9.3 Sequencing reactions 46 2. 10 Cloning 4 7 2.10. 1 Preparati on of DNA for cloning 47 2. 10 .2 Cloning 48 2. 10 .3 Screening and sequencin g 49 2. 10 .4 Sequence analysis 49 Chapter 3 Latent viruses of zooxanthell ae 5 1 3. 1 Introduction 5 1 3.2 Results 57 3.2.1 Evaluati on of induction methods 57 3.2.2 Enumeration ofzooxanthellae 62 3.2.3 Enumeration ofVLPs 64 3.2.4 Inducible VLPs 66 3.2.5 Effect of UV treatment on in duction of VLPs over time 7 1 3.2.6 Isolation and concentration ofVLPs 75 3.2.7 Visualization ofVLPs 76 3.2.8 Thin sections 79 3.2.9 Molecular characterisation of the filamentous VLPs 82 3.2.9. 1 Preparation of nucleic acid 82 3.2.9.2 PFGE 83 3.2.9.3 Clone Libraries 84 3.2.9.4 RNA methodology 86 3.2.9.5 Contigs and sequencing 87 3.3 Discussion 92 3.3. 1 Evaluation of induction methods 92 3.3 .2 Enumeration of zooxanthell ae 94 3.3.3 Identi fi cation ofVLPs by AFC 95 3.3 .4 Effect of VV treatment on induction of VLPs over time 96 3.3.5 Isolation and concentration ofVLPs 97 3.3.6 Visualization ofVLPs 98 3.3.7 Molecular work 99 3.4 Implications for coral bleaching I 02 3.5 Conclusions I 03 Chapter 4 Inducible VLPs of a freshwater cyanobacterium I 04 4. 1 Introduction 104 4.2 Results 110 4.2. 1 Growth curves 110 4.2.2 Enumerati on of VLPs Ill Ill 4.2.3 Inducti on curves 11 3 4.2.4 Visualization of VLPs 115 4.2.5 Molecular studies 11 8 4.2.5.1 Preparation of nucleic acid 118 4.2.5.2 PFGE 11 9 4.2.5.3 Clone libraries 122 4.2.5.4 Contigs and sequenci ng 123 4.3 Discussion 127 4.3.1 Growth curves 127 4.3.2 Identification and enumeration of VLPs by AFC 128 4.3.3 Visualization of VLPs 129 4.3.4 Molecular stud ies 130 4.3.5 Contigs and sequencing 131 4.4 Conclusions 133 Chapter 5 Latency in algal culture collections 134 5. 1 Introduction 134 5.2 Results 140 5.2.1 Amplification of algal culture with virus specific primers 140 5.2.2 inducible VLPs 14 1 5.2.3 Effect of UV treatment on induction ofVLPs over time 148 5.2.4 Visualization of VLPs 158 5.2.5 Thin sections 16 1 5.2.6 Molecular studies 168 5.2.6. 1 Preparation of nucleic acid 168 5.2.6.2 PFGE 169 5.2.6.3 Amplification of DNA polymerase gene 171 5.2.6.4 Clone libraries 173 5.2.6.5 Contigs and sequencing 173 5.3 Discussion 179 5.3.1 Amp li fication wi th algal virus-specific primers 179 5.3.2 Identi fication of inducible VLPs by AFC 180 5.3 .3 Effect of UV treatment on induction of VLPs over time 182 5.3.4 Visualization of VLPs 182 5.3 .5 Molecular analysis 185 5.4 Conclusions 188 Chapter 6 Conclusions and Suggestions for Future Work 190 6.1 General overview 190 6.2 Latent viruses of Symbiodinium sp. 190 6.3 Freshwater temperate cyanophages 192 6.4 Latent vi ruses in algal culture collections 194 6.5 Future directions 195 Appendix I 198 Supplemental Material CD ROM References 2 17 Publication 236 IV LIST OF TABLES Table 1.1 Replication strategies 9 Table 1.2 Examples of algal vimses 14 Table 2.1 Zooxanthellae strains 27 Table 2.2 ASP-8A medium 28 Table 2.3 BG-1 I medium 30 Table 2.4 Plymouth culture collection of algal strains 32 Table 2.5 F/2 medium 33 Table 2.6 List of primers 45 Table 2.7 Restri ction enzymes 48 Table 2.8 Composition of cloning media 49 Table 3.1 Named Symbiodinium species 53 Table 3.2 Presence ofVLP clusters 70 Table 4.1 BLAST results 125 Table 5.1 List of the main algal culture collections 137 Table 5.2 Summary of changes in UV induced algal cultures 146 Table 5.3 BLAST results 176 V LIST OF FIGURES Figure 1.1 Microbial loop wi th vims shunt 3 Figure 1.2 lcosahedral symmetry 5 Figure 1.3 Helical symmetry 6 Figure 1.4 Enveloped virus 7 Figure 1.5 Complex virus 8 Fi gure 1.6 Bacteriophage morphotypes 12 Figure 1.