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VIRUSES
Exploiting Marine and Freshwater Microbial Biodiversity Research for Biotechnology |
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1st VEG Workshop 14-15 February 2002
AbstractsThursday 14th February 2002Marine Cyanophages13:40 Marine cyanophage evolutionNicholas H. Mann and Emma Hambly Department of Biological Sciences, University of Warwick, Coventry CV4 7AL The large majority of phages isolated from the marine environment by virtue of their ability to form plaques on marine Synechococcus strains are myoviruses with icosahedral heads, contractile tails and baseplates. One particular cyanomyovirus, S-PM2, has been studied in considerable detail. Initial sequence analysis of a 10 kb region of its genome revealed that S-PM2 and the archetypal myovirus T4, despite the considerable phylogenetic and ecological separation of their respective hosts, both contain an evolutionarily conserved module that determines the structural components of the phage head and contractile tail. This suggests that the enormous diversity of phages in the sea could be a result of genetic shuffling between disparate phages mediated by such commonly shared modules. These conserved sequences could facilitate genetic exchange by providing sequence-related substrates for recombination between otherwise divergent phage genomes. Such a mechanism would thus expand the pool of phage genes accessible by recombination to all those phages that share common modules. The genome of S-PM2 has now been fully sequenced and preliminary analysis of the complete sequence throws further light on the modular nature of this phage. 14:10 Use of a SMART technique to detect marine cyanophagesHall, M.J.1, Wharam, S.D.2, Weston, A.2, Cardy, D.L.N.2 and Wilson, W.H.1 1Marine Biological Association of the UK, The Laboratory, Citadel Hill, Plymouth. PL1 2PB. UK. 2Cytocell Ltd, Banbury Business Park, Adderbury, Banbury, Oxfordshire. OX17 3SN. UK. Ecological studies of algal viruses require reliable, specific techniques for detection, quantification and analysis of diversity. These would allow some understanding of the effect of viruses on bloom dynamics. However, many of the available techniques lack the specificity which ecological studies of algal viruses require. Molecular techniques have been instrumental in changing our understanding of the composition of virus communities. A novel isothermal nucleic acid amplification assay has been developed to detect and discriminate specific DNA or RNA targets. The amplification process, which does not require thermal cycling or involve copying the target sequence, uses two target-specific probes to generate large amounts of an RNA signal. A colorimetric system then detects and quantifies the signal. The assay; SMART (Signal Mediated Amplification of RNA Technology), is simple to perform and when fully developed will be suitable for high throughput screening. SMART probes designed for specific cyanophages were tested on synthetic targets, purified cyanophage genomic DNA and crude cyanophage lysates. The assay was shown to be highly strain specific in these systems. By using the more conventional techniques of DGGE (Denaturing Gradient Gel Electrophoresis) and sequencing, a new set of SMART probes was developed to detect a dominant strain that was identified during a mesocosm experiment in Bergen, Norway. These, along with the existing probes, have been used to rapidly screen samples from the different bags throughout the experiment. We are currently optimising the assay for the detection of cyanophage RNA within infected host cells. 14:30 Isolation and characterisation of cyanophages from the Red Sea.Andrew Millard and Nicholas H. Mann Department of Biological Sciences, University of Warwick, Coventry CV4 7AL Cyanophages have been shown to be important in the marine environment. Water samples collected off the coast of Eilat over a 12-month period have been used to isolate cyanophages. Isolation has been done through a plaque assay procedure, using three different Synechococcus sp. strains as hosts .The data reveal that cyanophage numbers rise and fall with that of Synechococcus sp concentrations. 14:50 Marine viruses and cyanobacterial community structureMartin Mühling1,3, Nick J. Fuller1, Andrew Millard1, Dave J. Scanlan1, William H. Wilson3, Anton F. Post2 and Nick H. Mann1 1Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK 2Interuniversity Institute for Marine Science, Eilat, Israel 3Marine Biological Association, Citadel Hill, Plymouth, PL1 2PB, UK Viruses infecting marine Synechococcus strains, cyanophages, are extremely numerous in surface seawater and they are genetically diverse. The large majority of infective cyanophages which have been isolated appear to belong to the family cyanomyoviridae, possessing a dsDNA genome, icosahedral head, contractile tail and base plate. There is conflicting evidence regarding the impact of viruses on natural assemblages of Synechococcus strains. This project is aimed at investigating the impact of cyanophages on the population structure of marine Synechococcus strains from the Red Sea during an annual cycle. PCR primers specifically amplifying rpoC1 fragments from Synechococcus spp. were designed and used to prepare 20 clone libraries covering an annual cycle and three sampling depths. RFLP analysis of ca. 36 clones from each of the libraries suggested that during the summer the Synechococcus population was dominated by only a few genetically different clones, while there was great genetic diversity during the winter. This general trend was found to be independent of the depth from which the samples were taken. Plaque assays showed that viruses able to infect Synechococcus sp. strain WH7803 were abundant during the summer months. Fluctuations in the overall abundance of Synechococcus coincided with increases in cyanophage abundance. The genetic diversity of the cyanophage population during this annual cycle was also investigated. Cyanophage specific DGGE primers amplifying a fragment of the g20 gene, which encodes a capsid assembly protein, were used to prepare viral clone libraries. 36 of the clones from each of the libraries were screened by DGGE to elucidate the genetic diversity of the cyanophage population, and those shown to be different were sequenced. The comparison of these data to those on the genetic diversity of the Synechococcus population will be discussed. Marine Viruses15:50 Are viruses involved in coral bleaching.William H. Wilson*, Isobel Francis†, Keith Ryan*, Simon K. Davy† *Marine Biological Association, Citadel Hill, Plymouth, PL1 2PB, UK. †Institute of Marine Studies, University of Plymouth, Plymouth, PL4 8AA, UK. Corresponding author. E-mail: whw@mba.ac.uk Bleaching manifests itself as a loss of symbiotic dinoflagellates (zooxanthellae) and/or chlorophyll from a variety of symbiotic hosts, including corals and sea anemones. Bleaching is known to result from a range of environmental stresses, the most significant of which is elevated temperature, and how these stresses elicit a bleaching response is currently the focus of intense research. One consequence of environmental stress that has yet to be considered is viral attack. Here we have isolated a transferable infectious agent believed to be a virus, from zooxanthellae of the temperate sea anemone Anemonia viridis. The infectious agent is induced by elevated temperature. Once induced, the filterable agent can be further propagated without heat induction, thus fulfilling Koch’s postulates. We propose that zooxanthellae harbor a latent viral infection that is induced by exposure to elevated temperatures. If such a mechanism also operates in the zooxanthellae harbored by reef corals, and these viruses kill the symbionts, then this could contribute to temperature-induced bleaching. 16:10 Coccolithovirus: characterisation of a new large dsDNA virus that infects Emiliania huxleyi. Declan C. Schroeder1, Joanne Oke1, Gillian Malin2 and William H. Wilson1 Emiliania huxleyi-specific viruses (EhV) were isolated from E. huxleyi blooms off the coast of Plymouth, UK in July 1999 and July/August 2001, and from an E. huxleyi bloom induced during a mesocosm experiment in a fjord near Bergen, Norway during June 2000. Transmission electron microscopy revealed that all 10 virus isolates are 150 -200 nm in diameter with an icosahedral symmetry. Their density is approximately 1.2 in CsCl gradients and they have large double stranded DNA genomes approximately 410 kb in size. Phylogenetic analysis of DNA polymerase gene fragments of these viruses suggests that EhVs belong to a new genus within the family of algal viruses, Phycodnaviridae. We propose to name this new virus genus Coccolithovirus. Differences within members of the Coccolithovirus were elucidated by host range analysis of the virus isolates and sequence analysis of a gene fragment encoding part of their putative major capsid protein. All 10 virus isolates within this new genus only infected E. huxleyi strains that have previously been shown to exhibit low dimethylsulphoniopropionate lyase (DMSP-lyase) activity (CCMP1516, CCMP374 and L), while E. huxleyi strains with high DMSP-lyase activity (CCMP373 and CCMP379) were resistant to infection 16:30 Biogeochemical effects of algal viruses; The case for sulphur Claire Evans1, William H. Wilson2 and Gillian Malin1 Dimethyl sulphide (DMS) constitutes the most important source of biogenic sulphur to the atmosphere over the open oceans and has been implicated in global climate. Most DMS originates from dimethylsulphioniopropionate (DMSP) a compatible solute found in certain marine phytoplankton. Enzymatic cleavage of DMS to DMSP occurs via a group of isozymes termed DMSP lyases present in many marine microbes. Two previous studies have shown that viral lysis of DMSP containing phytoplankton cells results in the release of DMSP, however it is still unknown as to whether DMS production during infection is as a result of algal DMSP lyase. This project focuses on the Emiliania huxleyi host virus system. Different E. huxleyi strains have been shown to exhibit different levels of DMSP lyase activity and so far only viruses infectious to the ‘low lyase’ activity strains have been isolated. Axenic culture studies of the ‘low lyase’ E. huxleyi strain 1516 have revealed that only small amounts of DMS are produced during the viral infection cycle. Results of ongoing experiments will be presented showing the effect of viral infection on the activity of DMSP lyase. 16:50 Viruses vs. grazing as controls of phytoplankton populations.Stephen Archer. Plymouth Marine Laboratories, Prospect Place, The Hoe, Plymouth, PL1 3DH 17:10 The Ecology of Virus Infections in the Marine Filamentous Brown AlgaeLeadbeater BSC, Dixon NE, Wood, KR School of Biosciences, University of Birmingham Virus infections are common amongst marine filamentous brown algae. Members of the Ectocarpales (Ectocarpus, Hincksia, Pylaiella) on a global basis display symptoms of virus infection. These are swollen, hyaline sporangia, which instead of containing flagellated spores produce millions of icosahedral virus particles. The viral genome is present in all cells of an infected alga but the symptoms are only expressed in some of the reproductive structures. Virus infection of Ectocarpus fasciculatus has been studied in detail throughout a period of three years on a shore in Dorset, S. England. Occurrence of the symptoms of infection was more frequent in winter months than in summer months and experiments on cultured material confirm that lower temperatures result in a greater number of sporangia showing overt symptoms. The occurrence and distribution of the virus in this natural population will be detailed and the possible means of its transmission from one plant to another discussed. Friday 15th February 2002Baculoviruses09:00 Survival of baculovirus genotypes in natureIan Smith Central Science Laboratory, Sand Hutton, York YO41 1LZ The Baculoviridae is a large family of invertebrate-specific DNA viruses, many of whose 600-odd members are pathogenic for serious insect pests of agriculture, horticulture, and forestry. In this talk, I will give a broad introduction to the baculoviruses in terms of their taxonomy, pathology, and contribution to plant protection. Because many of the host species of these viruses, primarily belonging to the Lepidoptera, undergo only one generation each year, virus from an infected cadaver must be able to survive in the environment for prolonged periods. We do not understand very clearly how this happens. During infection, baculovirus particles become embedded within a protein matrix, the occlusion body, and occluded virus can survive externally for the requisite period between host generations but only if it is shielded from sunlight. I will review what is known about this aspect of survival outside the host; in the following talk, John Burden will describe an alternative strategy for trans-generational survival, in which baculoviruses may persist in a 'latent' state within the host. At the onset of a lethal infection, baculoviruses are ingested by a host larva during feeding and virus particles are liberated as the occlusion body matrix dissolves in the gut lumen. The transition from this exposed extracellular state to penetration of gut epithelial cells, another critical phase of baculovirus survival in which individual genotypes may either flourish or be eliminated, will also be discussed. 09:30 Persistent baculovirus infections – Virus survival between epizootics.
Centre for Ecology and Hydrology, Mansfield Road, Oxford, OX1 3SR 1Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP 2University of Liverpool, Brownlow St, Liverpool, L69 3GS Phage-mediated gene transfer09:50 Bacteriophage Survival and Transfer of Verocytotoxin Genes amongst Diverse Enteric Bacterial Hosts and in Soil Microcosms.James, Chloe, E.,1* Allison, Heather, E.,1 McCarthy, Alan, J.,1 Sharp, Richard, J.,2 and Saunders, Jon, R.1. 1University of Liverpool, UK, 2The Centre for Applied Microbiological Research (CAMR), Porton Down, Salisbury, UK. Verotoxin (VT) genes, carried by temperate bacteriophages (VT phages), are major virulence determinants of verotoxigenic Escherichia coli (VTEC) and occur in a wide range of other enteric bacterial species. It has been hypothesised that VT phages have a broad host range that could potentiate transfer of VT genes to less virulent pathogens or enteric commensals. It is possible that phages could enable VT genes to persist in the environment following shedding in cattle faeces. Consequently, such phages may represent an underestimated mechanism for survival and transfer of the VT genes themselves. Although the survival of VTEC has been extensively studied, similar investigations on VT phages have not been reported. A VT2 phage isolated from an E. coli O157 strain and tagged with reporter genes was used as a tool to study the host range and survival of VT phages. This phage was found to vary in its ability to infect via lytic and lysogenic pathways in a range of laboratory and clinical enteric bacterial strains. 7/24 clinical E. coli and 4/4 clinical Shigella isolates were susceptible to VT-phage infection. Many susceptible strains were smooth. Soil microcosm models have been developed and used to examine the survival and transfer of VT phages with emphasis on its relevance to the cattle farm environment. Novel use of a RecA441 mutant indicator host in plaque assays enabled efficient detection of all phage particles present due to inhibition of the lysogenic pathway. The phage was found to survive in soil for long periods (> 1 month) and was able to infect indigenous bacterial hosts in the microcosm environment. The potential clinical and agricultural significance of this VT phage emphasises the need to determine the nature of VT phage /host relationships in relation to toxin gene survival and dissemination in the environment. 10:10 Cyanophage-mediated gene transfer in the marine environment.1Martha Clokie, 2William Wilson & 1Nick Mann 1Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL. 2Marine Biological Association, Citadel Hill, Plymouth, PL1 2PB. Horizontal gene transfer has been shown to occur between bacteria in both terrestrial and aquatic environments. However, it has not been demonstrated for marine components of the picoplankton. A mutant strain of Syenchococcus containing a chromosomal marker for kanamycin resistance was infected with a range of phages. Uptake of the kanamycin marker from Synechococcus into phage heads was established and quantified using real-time PCR. Low freqency transduction of the kanamycin marker to a wild type strain of Synechococcus was demonstrated. Results from these and other studies will be presented. Soil environment10:30 Serratia phages from sugar-beet rhizosphere Ashelford, K.E., Day, M.J., Bailey, M.J., Lilley, A.K. & Fry, J.C. (1999). In situ population dynamics of bacterial viruses in a terrestrial environment. Applied and Environmental Microbiology 65, 169-174. Ashelford, K.E., Fry, J.C., Bailey, M.J., Jeffries, A.R. & Day, M.J. (1999). Characterisation of six bacteriophages of Serratia liquefasciens CP6 isolated from the sugar beet phytosphere. Applied and Environmental Microbiology 65, 1959-1965. Ashelford K.E., Norris S.J., Fry J.C., Bailey M.J., Day M.J. (2000). Seasonal population dynamics and interactions of competeing bacteriophages and their host in the rhizosphere. Applied and Environmental Microbiology 66, 4193-4199. Ashelford, K.E., Fry, J.C., Bailey, M.J. & Day, M.J. (Submitted). Taxonomic and ecological implications of the characterisation of Serratia isolates from soils, their interactions with each other and their predatory bacteriophages. International Journal of Systematic and Evolutionary Microbiology.
Freshwater ecosystems11:30 Cyanophage-host interactions in the AN-15:Anabaena PCC 7120 model systemDave G. Adams Division of Microbiology, University of Leeds, Leeds, LS2 9JT Cyanophage AN-15 was originally isolated from a sewage settling pond and is a cyanomyovirus with an isometric head, a central tail core and a contractile sheath. AN-15 infects cyanobacteria of the genera Anabaena and Nostoc and for this study Anabaena sp. strain PCC 7120 was chosen as host. Infection of Anabaena 7210 required 1 mM Ca2+, whereas stability of the cyanophage could be maintained with either 1 mM Ca2+ or 1 mM Mg2+. Following lysis of the host, either in liquid cultures or on agar plates, regrowth occurred as a result of the selection of phage-resistant host strains, which arose at a frequency of approximately 10-5. This resistance was stably maintained and was due to mutation, not to lysogeny. The resistant Anabaena 7210 strains fell into two categories, those that adsorbed the AN-15 to a much lower degree than the wild-type and those that adsorbed the cyanophage as effectively as the wild-type, but in which phage multiplication was defective. Mutant strains of AN-15, capable of lysing the cyanophage-resistant Anabaena 7120 strains, could be isolated by increasing the multiplicity of infection. These mutant cyanophages arose at a frequency of approximately 10-7 and retained the ability to lyse wild-type Anabaena 7120. 11:50 Antarctic Virus ecologyDavid Pearce British Antarctic Survey, Cambridge This talk will begin with a brief review of some of the more interesting aspects of Antarctic virus ecology to date, and then describe some of the work currently underway at the British Antarctic Survey, to identify and understand the role of viruses in Antarctic microbial ecology. Modelling virus interactions12:10 A mechanism allowing viruses to co-exist with phytoplankton blooms.Sandy Murray University of Aberdeen A model has been developed that provides a mechanistic explanation for the co-existence of viruses and phytoplankton blooms. Because viruses seem ubiquitous have the potential to reproduce rapidly at high host density, it is difficult with standard reproduction models to explain how mono-specific blooms could develop and persist. The modelling presented consists of two parts. Firstly we demonstrate that at high host densities optimal viral reproduction strategy switches from lytic to lysogenic as an increasing proportion of the hosts become infected. Secondly, we suggest a mechanism that we demonstrate can produce a response close to this optimal strategy: lysogeny in response to superinfection. The result is a model in which viral mortality is reduced at high host density, however as almost all the hosts are lysogens the bloom can be rapidly lysed if subjected to stress. In the absence of such stress, viruses serve to protect the bloom, because rival phytoplankton species with moderate populations are subjected to relatively greater viral mortality. 12:30 The Potential Significance of Marine Viruses as a Control on the Cycling of Carbon in Extreme Oligotrophic SystemsJ. I. Allen1*, G. Triantafyllou2 and G. Petihakis2 1. Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth, PL1 3DH. 2. IMBC, Institute of Marine Biology of Crete, Heraklion P.O. Box 2214, Crete, Greece, GR 71003 *Corresponding author email jia@pml.ac.uk A theoretical model has been developed which considers the host parasite relationship between free living heterotrophic bacteria and a lytic virus and its effect on the biogeochemical cycling of carbon and nutrients in marine ecosystems. This has been embedded into the European Regional Seas Ecosystem Model (ERSEM) and used to simulate the seasonal cycle of the highly oligotrophic Cretan Sea. Our simulations suggest that host parasite interactions between bacteria and marine viruses may have a significant influence on the cycling of carbon and nutrients in oligotrophic waters. In situations such as those observed in the eastern Mediterranean sea where bacterial activity is presumed to be nutrient rather than DOC limited, the virus may act against the microbial loop and allow primary production to compete, leading to a shift away from a microbial loop ecosystem functionality and a consequent increase in carbon fixation. Phage therapy14:00 Bacteriophage mediated control of Clostridium perfringens in poultry.Richard Sharp & Helen Rowe, Centre for Applied Microbiology, Porton Down, Salisbury, SP4 OJG While the aetiology of necrotic enteritis in poultry remains uncertain it is generally accepted that the ultimate causative organism is C. perfringens. The events that lead up to infection, however, remain uncertain. Other factors and organisms appear to play a role in establishing conditions that predispose the birds to the development of necrotic enteritis. For example, infection with Eimeria spp, (coccidiosis) predisposes poultry to the development of necrotic enteritis following changes in the pattern of infection of the gut with C. perfringens. We are working towards the development and delivery of bacteriophage to lyse C. perfringens at the site of necrotic enteritis through either the feed or preferably drinking water. Unlike antibiotics, bacteriophage are able to target a single group of organisms without disruption to the rest of microflora. Successful phage therapy strategies will improve the health of birds in the poultry industry without recourse to antibiotics (of particular concern being those with analogues used in human medicine). Feeding antibiotics to poultry has been demonstrated to improve performance, reduce ileal weight, and decrease the population of growth inhibiting bacteria such as Clostridium perfringens. The UK and the EU are now moving away from the inclusion of antibiotics as feed additives. Bacteriophage therapy offers a number of advantages over antibiotic therapy including, activity against drug resistant organisms. It can also be used prophylacticly to combat the spread of infection. Bacteriophages offer high specificity towards the target organism and have no affect on non-target organisms. They are self-replicating and self-limiting; when the target organism is present, phage replicate until the target bacteria are infected and lysed. Bacteriophage naturally mutate to combat host resistance mutations or they can be mutated deliberately in the laboratory to modify their host range. We are currently isolating a range of bacteriophages that are active against poultry strains of C. perfringens. These will be applied in in vivo studies in necrotic enteritis poultry models later this year. 14:30 Pseudomonas phage and application for the control of infection in CF patients.Gavin Hughes & Richard Sharp Centre for Applied Microbiology, Porton Down, Salisbury, SP4 OJG Cystic fibrosis (CF) is an inheritable disease that develops in individuals with mutations in the cystic fibrosis transmembrane regulator protein. The defective gene affects the exocrine glands of the body causing thick mucus to form in the bronchial tree. This predisposes the patient to recurrent chronic Pseudomonas aeruginosa respiratory infections that grow as an endo-bronchial biofilm. During prolonged infection the bacteria differentiate into a mucoid phenotype that over produces alginate exopolysaccharide which acts as a significant barrier to the penetration of antibiotics and effective host immune response. Treatment of these pulmonary infections is through early aggressive antibiotic regimes, however once established infections remain refractory to all treatments and leads to a gradual decline in lung function. With the emergence of antibiotic resistance alternatives must be found to combat these infections, and we are therefore investigating the use of bacteriophage as antimicrobial agents. Bacteriophages infecting Ps. aeruginosa have been detected in sputum samples from CF patients and phage have also been identified which are able to lyse cells within environmental biofilms. We have isolated a range of bacteriophages from clinical and environmental sources that are able to infect Ps. aeruginosa and which mediate the production of alginate lyase. Biofilms of Ps. aeruginosa developed using an in vitro biofilm model were degraded by administration of alginate lyase phage, reducing the overall biofilm population by three logs following four exposures to phage over 24 hours. Biofilms treated with x10 the MIC of Tobramycin had no effect on the Pseudomonas sessile community. We are currently working towards the development of an in vivo model to assess the efficacy of alginate lyase bacteriophage to eradicate pulmonary infections in CF mice. |
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