SITE AREAS:
VIRUSES
Exploiting Marine and Freshwater Microbial Biodiversity Research for Biotechnology |
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SITE AREAS: |
VIRUSES |
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4th veg workshop 16 - 17 February 2005
AbstractsVIRAL DIVERSITY IN THE MARINE ENVIRONMENT. EFFECT ON THE SPECIES RICHNESS OF PLANKTONIC ORGANISMS Sandaa, R.-A., Virioplankton are the most commonly found biological agents in the marine environment. Knowledge about the diversity and function of these plankton organisms in the aquatic food web remains a relatively unexplored field of study. In this talk we will focus on the ecological importance of viruses in the marine ecosystem demonstrated by different studies carried out on seasonal samples from coastal water and samples from different mesocosms. In these experiments we employed flow cytometry (FCM) to monitor the succession and numbers of dominating algal, bacterial and viral populations, while the relationship and changes in the bacterial and viral community structure were investigated by denaturant gradient gel electrophoresis (DGGE) and pulse field gel electrophoresis (PFGE). Some dominating PFGE and DGGE bands are also further characterised by PCR and sequencing. These experiments demonstrates how changes in the viral community correlates with changes in the bacterial and phytoplankton community structure, and the experimental results will be discussed in light of theories for biodiversity control.
Giant Viruses of the Marine Brown Algae Bellas, C.M., Weynberg , K.D., The Ectocarpales are a group of marine brown algae that are important components of near-shore communities. They are frequently subjected to a persistent virus infection which often remains dormant for long periods of time. When symptoms occur, they do so only in the hosts spore or gamete producing cells. Molecular probes have been developed to detect several species of dormant viruses within this group. The design of the probes, the virus-host relationship and the relevant ecological implications are discussed.
CORAL BLEACHING: ARE VIRUSES INVOLVED?Lohr J. E.1,2, Wilson W. H. 2,3 1Department of Biological Sciences, Over the past few years a sequence of major bleaching events has threatened the world’s coral reefs. Coral bleaching manifests itself as a loss of zooxanthellae and/or chlorophyll from cnidarian hosts. The underlying cause of bleaching and the mechanisms involved are largely unknown. Here we investigate the hypothesis for bleaching that zooxanthellae harbour a latent virus. If zooxanthellae harbour latent viruses, environmental triggers, such as increased temperatures and UV light, will induce a lytic cycle which may lead to the deterioration and expulsion of the symbionts by their host and hence cause bleaching of the host animal. Evidence from transmission electron microscopy (TEM) and flow cytometry (FC) suggests that viruses are induced from the zooxanthellae. It is therefore possible that environmentally-regulated viral induction mechanisms contribute to bleaching events in coral reef environments. Ultimately, we plan to lay the foundations for the development of molecular probes to detect viruses in field samples through the isolation, characterisation and genomic sequencing of viruses present in the symbiotic zooxanthellae of corals.
Production of DMS and related compounds during viral infection of algae and their antiviral propertiesClaire Evans1,2,3, Gill Malin2, Peter Liss2, Willie Wilson1,3 1
Dimethyl sulphide constitutes the major source of biogenic sulphur to the atmosphere over remote ocean regions and can influence climate via aerosol formation and the subsequent production of cloud-condensation nuclei. The primary source of DMS is dimethylsulphoniopropionate (DMSP) a compatible solute found in certain marine phytoplankton. Equimolar amounts of DMS and acrylic acid are formed when DMSP is cleaved by phytoplankton DMSP lyase isozymes. It has been suggested that DMSP is also the base of an antioxidant cascade involving DMS, acrylic acid and their downstream products, including dimethylsulphoxide (DMSO). DMSO may also be biosynthesised by phytoplankton cells. In studies on axenic cultures of the phytoplankton Emiliania huxleyi we followed the production and fate of these compounds during viral infection. DMSO concentrations increased rapidly after viral addition. DMS and acrylic acid production occurred simultaneously during the later stages of the culture crash, but DMS concentrations were lower than those of acrylic acid suggesting that some of this pool may have been converted to another compound. Further culture studies revealed that during infection cells produce hydrogen peroxide and exhibit elevated levels of reactive oxygen species which supports the idea that some of the DMS produced during viral infection could act as an antioxidant. DMS production during viral infection of E. huxleyi was also observed during a mesocosm study in
HOST AND VIRUS GENOTYPIC DIVERSITY DURING BLOOMS OF THE MARINE COCCOLITHOPHORID Emiliania huxleyiJoaquin Martinez Martinez*1, Declan Schroeder2, Gunnar Bratbak3 & William Wilson1 1Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, UK; 2Marine Biological Association, Citadel Hill, Plymouth, PL1 2PB, UK; 3Dept. Biology, Microbiology, University of Bergen, N-5020 Bergen, Norway Viruses play an important role in the life of plankton populations. Plankton are the producers and recyclers of most of the energy that flows through the oceanic ecosystem. Consequently, the viruses that infect them are likely to significantly affect the structure, function and biogeochemical cycling of marine food webs. Emiliania huxleyi, a marine microalga that forms dense blooms in coastal and mid-ocean waters, is an important species with respect to past and present marine primary productivity, sediment formation and climate. We set out to determine whether the gene encoding a protein with calcium-binding motifs (GPA) from E. huxleyi displayed any significant differences that could be attributed to the A and B morphotypes. Primers design for the GPA cDNA of E. huxleyi strain L were used to amplify the DNA extracted from 15 E. huxleyi isolates. DGGE and sequencing analysis were used to assess the natural genetic diversity of host and viruses based on variations in the GPA gene from E. huxleyi and the major capsid protein gene from E. huxleyi-specific viruses. In the current study we have monitored the progression of three different blooms of E. huxleyi; a natural bloom in the North Sea in June 1999 and two induced blooms in seawater enclosures off western Norway in June 2000 and 2003. - We have discovered a genetic marker that correlates significantly with the separation of the most widely recognised E. huxleyi A and B morphotypes. - The E. huxleyi A-morphotype dominated during the Bergen mesocosm studies however we discovered it can be further separated into different genotypes. Both morphotypes were present during the bloom in the North Sea. - The virus population during the E. huxleyi crash was dominated by 2 genotypes. - Host and viral communities were more diverse and dynamic in the North Sea than in the induced experiments, and they showed temporal and depth variations.
Cyanophages and pseudolysogenyA.Millard & N.H.Mann. University of warwick, Coventry CV4 7AL. Marine Synechococcus is found in the oligotrophic regions of the world`s oceans. In these regions phosphate and nitrogen are thought to be important factors in limiting the growth of cyanobacteria; consequently they are likely to be important in influencing phage infections. It is already known that phosphate limitation of Synechococcus WH7803 leads to a pseudolysogenic state when infected with cyanophage S-PM2 (Wilson et al., 1996). This pseudolysogenic state is to be further investigated by the use of proteomics and transcriptomics. The approach and progress made to be date will be discussed.
Picocyanobacteria – The sweetcorn of the microbial world?Mandy Dillon, Jill Thurman, Janice Drinkall and Jackie Parry Dept Biological Sciences, Lancaster University, Lancaster LA1 4YQ Protozoa have long been considered the major predators of bacteria, playing an important role in the transfer of bacterial carbon along the aquatic food-web. There is no doubt that many bacterial strains are adequately digested by these predators, however, it has often been assumed that the level of prey ingestion is proportional to the level of prey digestion. Our work has shown this not to be true, particularly for the picocyanobacteria. Although many protozoa can ingested vast numbers of these bacterial cells, many are unable to digest them and the picocyanobacterial cells are excreted in a viable form. Thus, many protozoa do not appear to effectively transfer picocyanobacterial carbon along the food-web, they just repackage it. Attention has now turned to the potential role of viruses in controlling the mortality of this group and freshwater cyanophage are currently being isolated and characterised.
The Development of a Molecular Probe to Detect Freshwater CyanophagesAndrea Baker1&2, Dr Victoria Goddard1, Dr Dave G Adams2 & Dr Willie Wilson1 1 The Marine Biological Association of the United Kingdom, Citadel Hill, Plymouth, PL1 2PB, 2The Department of Biochemistry and Microbiology, The University of Leeds, Leeds, LS2 9JT
Cyanophages are a group of viruses that infect the globally important primary producers, the cyanobacteria. They are considered to play major roles in influencing host community diversity, terminating toxic cyanobacterial water blooms and influencing biogeochemical cycles. A considerable amount of research has been directed towards the study of marine cyanophage communities, however there is still a great deal to discover regarding their freshwater counterparts. PCR-based probes have been designed based on the major capsid protein gene that specifically amplify freshwater cyanomyoviruses. They have proved successful for use on environmental samples and in combination with sequencing analysis, they can provide information concerning cyanophage community diversity. It is proposed that these probes are a more suitable marker for the amplification of freshwater cyanophages compared to those presently being used.
Effect of light on the interaction between cyanophage S-PM2 and Synechococcus sp. WH7803YING JIA and NICHOLAS H. MANN Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK S-PM2 is a bacteriophage, with a double-stranded, circular genome, which infects the ecologically important marine cyanobacterium Synechococcus sp. WH7803. We are currently investigating fundamental features of phage-host interactions. An initial investigation of the role of light on phage attachment has indicated striking light-dependence. In the darkness, phages were not capable of adsorbing to cells, but adsorption resumed as soon as the light switched on. A collection of marine cyanophages is being screened in order to see if this is a common phenomenon. This will offer a significant insight into one of the factors affecting cyanophage and host dynamics under natural environmental conditions. Because of the time-consuming and labour-intensive nature of the plating method, we are developing an alternative technique that allows us to count phages stained with SYBR Green I by flow cytometry. This approach has been verified on CsCl-purified phage, and we are attempting to apply it to adsorption experiments.
The use of real-time PCR to determine phage gene expression throughout infectionMartha R.J. Clokie, Jinyu Shan, Yin Jia, Steve West and Nicholas H. Mann Department of Biological Sciences, University of Warwick, Coventry CV4 7AL The E. coli phage T4 takes 30 minutes from infection to lysis, whereas phage S-PM2, which infects the cyanobacterium Synechococcus, takes around 9 hours. The expression of T4 genes can be divided into immediate early, delayed early, middle and late. S-PM2 has homologues of T4 genes of each of these expression categories. Representative genes were selected and probes and primes were designed for an S-PM2 analysis using quantitative real-time PCR. Expression profiles were similar to those from T4, but with some notable differences. These data are consistent with promoter analysis of the S-PM2 genome. Other S-PM2 genes were also investigated. These include the phage encoded photosynthesis gene in the genome psbA. This could be distinguished from the host psbA gene. These data can then be interpreted in the framework of general phage expression.
The effect of cyanophage S-PM2 infection on phycoerythrin gene expression, phycoerythrin synthesis and breakdown in Synechococcus sp. WH7803JINYU SHAN, MARTHA R. CLOKIE and NICHOLAS H. MANN Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK Marine members of the genus Synechococcus are photosynthetic cyanobacteria, which are of great ecological significance. Unlike higher plants, light harvesting for photosynthesis depends on a specific structure, the phycobilisome, composed mainly of phycobiliproteins together with non-pigmented linker polypeptides. Due to the abundance of marine cyanophages these cyanobacteria are often subject to infection. The phage S-PM2 has been extensively characterized in this laboratory. Spectrophotometry has revealed that phycoerythrin, the dominant phycobiliprotein in Synechococcus, is progressively degraded during the process of S-PM2 infection, presumably to serve as a source of amino acids for the synthesis of virion proteins. We have identified an S-PM2 gene encoding a homologue of the cyanobacterial CpeT protein which may have a role in regulating the expression of the phycoerythrin genes. Using quantitative real-time PCR, we found that phage cpeT gene expression was greatest at the early stage of infection. We are currently investigating how phage S-PM2 controls the synthesis of phycoerythrin and the degradation of the phycobilisome.
'A sweet attraction; gycoprotein biosyntheiss in Streptomyces discovered through studies on a phage receptor.' Debbie Cowlishaw, Silvia Wehmeier, Anpu Varughese and Maggie Smith. Bacteriophages use their host’s surface macromolecules as receptors to trigger the initial stages of phage infection. Studies of these receptors frequently provide insights on the nature of the host cell wall. Here we present the identification of two genes required for the synthesis of the phiC31 receptor. One of the gene products SCO3154 (renamed pmt) is similar to dolichol phosphate-D-mannose:protein O-D-mannosyltransferases (Pmt proteins) found in eukaryotic cells from yeasts to humans. The Pmts transfer mannose from the donor, dolichol phosphate mannose, to serine or threonine residues in the first step in O-glycosylation. Homologues of pmt are detected by database searches in other actinomycetes including mycobacteria and corynebateria. We propose that the actinomycetes use these pmt homologues to glycosylate proteins using a polyprenol-phosphate-mannose as a mannose donor. S. coelicolor strains that lack either pmt or SCO1423 (renamed ppm1) that encodes a putative polyprenol-phosphate-mannose synthase are retarded in growth. We are using proteomics to characterise the putative ‘glycoproteome’ of Streptomyces coelicolor. We are also undertaking a phenotypic analysis of the pmt and ppm1 defective mutants to try to reveal the biological role of glycosylation. Investigations on the co-evolution of wild cabbage (Brassica oleracea subsp. oleracea) populations and Turnip mosaic virus isolates in the U.K.C. OBERMEIER*, P. J. HUNTER*, K. OHSHIMA† and J. A. WALSH* *Warwick HRI, Wellesbourne, Warwick, CV35 9EF, UK †Laboratory of Plant Virology, Saga University 840-8502, Japan Turnip mosaic virus (TuMV) isolated from cabbage and other plants from the UK and worldwide shows a high degree of biological and molecular diversity. The extent of plant-virus adaptation and co-evolution in wild forms of the perennial Brassica oleracea subsp. oleracea is being studied. Plants grown from seed collected from two TuMV-infected wild B. oleracea plants one in Wales and the other in Dorset and the two genetically diverse TuMV isolates (GBR 83 and GBR 98) recovered from these original plants were used to study genetic co-adaptation of TuMV isolates and their native hosts. Competition experiments involving mechanical co-inoculation of native and non-native TuMV isolates to wild B. oleracea plants from Wales and Dorset were performed. Up to 5 rounds of consecutive transmission were carried out to test the relative fitness (adaptation) of virus isolates in terms of their ability to replicate and move efficiently within different host plants. Molecular and biological assays revealed that in wild B. oleracea plants from Dorset, the native isolate GBR 98 predominated after 2 rounds of transmission, whereas in wild B. oleracea plants from Wales and susceptible B. napus control plants, both virus isolates were detected in mixed infection at the end of 5 rounds of transmission. This suggests that there is some adaptation of virus isolates and their native hosts within the Brassica population in Dorset. The adaptation of these TuMV isolates and their wild B. oleracea hosts will be further investigated in hybridisation experiments comparing plant RNA expression profiles following challenge with their native or non-native TuMV genotypes. This will identify plant genes differentially up-, or down-regulated. For this purpose oligonucleotides derived from 15,000 expressed sequence tags (ESTs) from the B. napus (a natural hybrid of B. rapa and B. oleracea) genome have been produced and used to generate microarrays for hybridisation analysis.
The role of viral pathogens in lepidopteran host populations: the winter moth and its natural enemiesRobert Graham NERC Centre for Ecology and Hydrology, Mansfield Road, Oxford, OX1 3SR A lepidopteran system is reported, in which viral pathogens are both abundant and genotypically variable. Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles to investigate the prevalence of the pathogens, Operophtera brumata Nucleopolyhedrovirus (OpbuNPV) and O. brumata Reoviruses, within the natural system. OpbuNPV was recorded in eleven of the thirteen winter moth populations sampled, with two populations suffering viral mortality at rates of over 50%. Restriction Endonuclease and sequence analysis has allowed the discovery of 41 genotypically-variant strains of OpbuNPV. Three species of reovirus have been discovered, all of which are believed to be novel. Electron microscopy and sequence data places two of the viruses within the Cypoviridae genus. The polyhedrin gene of these two viruses was sequenced, allowing their relationship with other cypoviruses to be portrayed as a cladogram. The third reovirus was also isolated from a hymenopteran parasitoid wasp (Phobocampe neglecta) which may have a commensal or mutualistic relationship with the virus, acting as a vector. Sequence data classifies this virus as an unspecified reovirus.
Investigating the genetic parameters that affect horizontal transmission parametersFiona King School of Biological and Molecular Sciences, Oxford Brookes University Transmission of pathogens is fundamentally important for their survival in the envionment. There is limited knowledge on how transmission is efficently maintained particularly with viruses. It is acknowlegded that conditions like heat, light and dessication can have an effect on the sustainibility of virus infectivity, especially when the virus is outside its normal host. Baculovirus pathogenesis has been described in great detail for the members of the genus nucleopolyhedrovirus (NPVs) where the final stages of baculovirus infection is characterised by terminal liquefaction of host. Terminal liquefaction ensures efficient horizontal transmission of the virus. Two virally encoded proteins, chitinase and cathepsin, are required for terminal liquefaction to occur. Extensive studies on Autographa californica (Ac) multiple NPV has suggested that they work in synergy to breakdown the chitin-rich tissues, resulting in liquefaction (Hawtin et al., 1997). The focus of this research is to further this work through the analysis of recombinant AcMNPV mutants. Previously mutants have lacked either chitinase or cathepsin activity through insertional inactivation by the lacZ casette. These mutants are not full gene deletions and recent evidence has shown that the lacZ casette can interfere in larval bioassays. Currently a chitinase and cathepsin double knockout AcMNPV mutant (AcDchiADvcath) is being produced. In the mean time a virus that lacks the promoters for both chitinase and cathepsin, thus making the genes redundant, is being used (Acchit-cath-). To date AcDchiA (full gene deleion of chitinase) has been produced. Studies on the effect a partial p10 deletion has on transmission is also being examined. An area of this research aims to determine the effect these deletions may play in transmission dynamics e.g. dose required to induce a lethal response, time to death, modes and rates of transmission. All results have been compared to AcMNPV infection (the prototype for NPV infection). Comparison of the LD50 (lethal dose required to kill half the population) for each of the viruses illustrates the significant impact each gene deletion has on virus lethality. Transmission analysis shows that a virus that liquefies it host (AcMNPV) is more rapid in its speed of transmission to a second population compared to one that does not liquefy its host (Acchit-cath-). The role of frass (larval excrement) has also been examined in its role of horizontal transmission. Results have been compared with those obtained for each of the recombinants. Frass from all recombinants and AcMNPV infected larvae is capable of inducing a lethal response in a second generation of non-infected larva. AcchiA and Acchit-cath- infected larvae appear to release infective frass earlier on in infection compared to the other recombinants and AcMNPV. Frass from a AcP10 infected larva does not appear to be as infective. Supervisors: Prof. L King; Prof. R. Possee; Dr. R. Hails Funding: National Environmental Research Council (NERC): CASE studentship with Centre for Ecology and Hydrology (CEH), Oxford
What would it take to sequence an entire virome in a single experiment?Simon T. Bennett simon.bennett@solexa.com Solexa Limited, Chesterford Research Park, Little Chesterford, Saffron Walden, Essex CB10 1XL Direct analysis of large numbers of informative DNA markers without the need for time-consuming sample preparation, even across entire genome entities, would be highly desirable. Revolutionary new technologies capable of sequencing DNA directly, a base at a time and at the single molecule level are beginning to transform the economics of DNA sequencing, which, in turn, will lead to rapid, low-cost DNA sequencing. With densities of up to 100 million molecules per square centimetre on an array, Solexa’s revolutionary Sequencing by Synthesis (SBS) approach leads the new wave of single-molecule based sequencing technologies. SBS is expected ultimately to offer up to five orders of magnitude greater efficiency, in terms of both costs and throughput, compared with conventional DNA sequencing methods. Ultra-high density arrays of this nature will enable analysis of large genome entities, including individual whole genomes, and, for small genomes, could be applied to meta-genome analysis such as, for example, of entire viral communities or “virome”. With further refinement and miniaturization, later generations of this technology could be adapted for use directly in the field. |
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