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VIRUSES
Exploiting Marine and Freshwater Microbial Biodiversity Research for Biotechnology |
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2nd VEG Workshop 20-21 February 2003
AbstractsThursday 20th FebruaryThe Diversity and Evolution of the T4-type BacteriophagesCarine Desplats, Claire Bertrand, Françoise Tétart and H. M. Krisch: Laboratoire de Microbiologie et Génétique Moléculaire du CNRS, UMR 5100,118 Route de Narbonne, 31062 Toulouse Cedex, France Recent studies suggest that viruses are the most numerous entities in the biosphere; bacteriophages, the viruses that infect Eubacteria and Archaea, constitute a substantial fraction of this population. In spite of their ubiquity, the vast majority of phages in the environment have never been studied and nothing is known about them. For the last 10 years our research has focused on an extremely widespread group of phages, the T4-type. It has now become evident that phage T4 has a myriad of relatives in nature that differ significantly in their host range. The genomes of all these phage have homology to the T4 genes that determine virion morphology. Although phylogenetically related, these T4-type phages can be subdivided into four groups that are increasingly distant from T4: the T-evens, the pseudo T-evens, the schizo T-evens and the exo T-evens. Genomic comparisons between the various T4-type phages and T4 indicate that these genomes share homology not only for virion structural components but also for most of the essential genes involved in the T4 life cycle. This suggests that horizontal transmission of the genetic information may have played a less general role in the evolution of these phages than has been supposed. Nevertheless, we have identified several regions of the T4-type genome, such as the segment containing the tail fiber genes that exhibit evidence of extensive modular shuffling during evolution. The T4-type genomes appear to be a mosaic containing a large and fixed group of essential genes as well as highly variable set of non-essential genes. These non-essential genes are probably important for the adaptation of these phages to their particular life-style. Furthermore, swapping autonomous domains within the essential proteins may slightly modify their function(s) and contribute to the adaptive ability of the T4-type phage family. Regulatory sequences also display considerable evolutionary plasticity and this too may facilitate the adaptation of phage gene expression to new environments and stresses. Developing a universal molecular probe to detect freshwater cyanomyovirusesAndrea Baker1&2, Vic Goddard2, Jo Oke2, Dave Adams1, Willie Wilson2 1University of Leeds 2The Marine Biological Association Cyanophages are significant components of aquatic microbial communities, playing important roles in ecosystem dynamics. In recent years the development of molecular probes has allowed a greater understanding of marine cyanophage communities, however little work has been carried out on freshwater cyanophages. This project focuses on the development of a PCR-based probe to detect freshwater cyanomyoviruses. The major capsid protein (MCP) was purified from the freshwater cyanomyovirus, AN-15, which infects cyanobacteria of the genera Anabaena and Nostoc. Database searches revealed that the AN15 N-terminal polypeptide sequence of the MCP showed homology to g23, the MCP from a range of other myoviruses. We used this sequence to design an oligonucleotide forward primer then amplified a putative g23 fragment from AN15 also using a g23 conserved region reverse primer. Future work, after sequence analysis of AN15 and other cyanomyoviruses, will involve further developing the g23 target region as a universal freshwater cyanomyovirus probe for temporal and spatial analysis of cyanomyoviruses in freshwater ecosystems. Virus community dynamics in a fresh water ecosystem.Victoria Goddard1, Andrea Baker1 Jo Oke1, Dave Adams2 and Willie Wilson1. 1Marine Biological Association UK 2University of Leeds Marine virus communities have been relatively well characterised in recent years, but their fresh water counterparts remain poorly understood. The aim of this project is to investigate the virus community in Priest Pot, a small fresh water lake in Cumbria. Accumulated data will then be compared with that already obtained from marine studies. Samples have been collected over an annual cycle from Priest Pot. The viral fraction has been concentrated and community profiles obtained by PFGE. Virus counts obtained by flow cytometry have also be used to determine fluctuations in diversity and abundance of different virus groups over time and space. TEM studies will also be used to characterise viruses that have not been isolated. Preliminary results show that viruses are present in the water column, at all times. Total virus counts do fluctuate over the annual cycle however, apparently reflecting the mixed or stratified state of the lake. A large diversity of virus morphologies has been observed by TEM, and PFGE has also shown there to be a variety of virus genome sizes present, ranging in size from approximately 20 to 500 kb. Friday 21st FebruaryAnalysis of an Emiliania huxleyi virus genome.1,2,Wilson, W.H., 1Schroeder, D.C., 1Oke, J. and 3Parkhill, J. 1Marine Biological Association and 2Plymouth Marine Laboratory. Current address: 1Marine Biological Association, Citadel Hill, Plymouth, PL1 2PB, UK. 2The Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK Emiliania huxleyi (Haptophyta) is a unicellular alga found throughout the world’s oceans. It is best known for its immense coastal and open ocean blooms at temperate latitudes that can cover 10,000 km2 or more. The size and intensity of these blooms makes E. huxleyi important for nutrient and CO2 cycling and biogenic sulphur production (in the form of dimethyl sulphide) in the marine environment. Consequently it is a key species for current studies on global biogeochemical cycles and climate modelling. We have isolated several strains of viruses that infect E. huxleyi. They are large double stranded DNA viruses and all strains isolated to date have a genome of approximately 410 kbp. We have sequenced the genome of one of these viruses (EhV86) and at the time of writing the shotgun sequencing is complete and we are in the process of finishing. Analysis of the EhV86 genome will be discussed at the meeting. The Baculovirus Updated Genome site (BUGs), a prototype pipeline for small genome annotation and comparisons.Sarah L Turner & Dawn Field, CEH Oxford Whole genome sequence comparisons of closely related organisms that occupy distinct niches enables species specific genes, which might confer adaptations to the niche, to be identified for future study and possible exploitation. Whole genome sequences are being published with increasing frequency and a major challenge facing research scientists is keeping pace with the additional information that each new genome provides. We are developing a prototype system that identifies, collects and maintains a local database of genomes of interest, in this instance, those of baculoviruses. These are large DNA viruses (>100kbp) that infect and kill the larvae of many lepidopteran pests of agricultural importance. Baculoviruses have restricted host ranges, i.e. each virus infects only a limited number of lepidopteran species, and have been used as species-specific, environmentally friendly bio-control agents. The prototype pipeline takes the genome data and automatically undertakes a suite of analyses tailored to provide easy access to up-to-date genome comparisons and facilitate genome annotation. These can be used to better understand baculovirus ecology and evolution. Lambda integrase gene family: markers for phage diversity, environmental regulation, and gene transfer in freshwater bacteria.S.A Bromley1, Prof. R.W. Pickup2 and Prof. J.R Saunders1 1 The University of Liverpool. 2 CEH Windermere The project will utilise lambda DNA integrase family genes as markers for defining the distribution and diversity of temperate (lysogenic) phages in freshwater bacteria. Studies on virus interaction with bacterial populations have concentrated on lytic phages and their effects on population dynamics. However temperate phages, with lethal functions repressed, may integrate into/remodel bacterial genomes and can transmit non-viral genes by transduction/phage conversion and probably play an even greater role in the evolution and adaptation of bacterial populations. The project will ask:
Priest pot will be used as the experimental site, since we already have extensive data, stored cultures and DNA samples from it and are developing, under NERC-funded work, primers for DNA integrases of P4 phage and lambda subfamilies that are relatively selective for temperate bacteriophages present in many freshwater bacteria. We will analyse phage diversity in the total virus (from induced lysogens in recovered bacteria and in cell filtrates) and viral DNA fraction and determine whether newly identified phage sequences match with those in dominant freshwater phages.
Integrase Profiling – A Means of Identifying Prophage and Pathogenicity Island Carriage by Staphylococcus aureus.F.A. BURDEN, M.E. RAPSON & N.H.MANN University of Warwick, Biological Sciences Dept., UK. Almost all Staphylococcus aureus isolates have been shown to contain at least one temperate bacteriophage integrated into their chromosome. In S. aureus phages, integration is facilitated by the enzyme integrase. Integrase genes are also found in pathogenicity islands within S. aureus genomes. By developing PCR probes towards the integrase gene it is possible to identify patterns of prophage and pathogenicity island carriage within the S. aureus chromosome. Published integrase gene sequences were aligned and phylogenetic analysis was carried out. Eight distinct integrase gene ‘families’ were defined. Family specific PCR primers were designed which permitted identification of the integrase genes within S. aureus genomes. This identification allows a database of prophage and pathogenicity island carriage within isolates to be developed. A number of MRSA and MSSA strains were subjected to the family-specific PCR probes allowing characterization of the integrase profiles of these isolates. The results have shown that every S. aureus isolate screened contains at least one integrase type, with many containing two or even three members of different integrase families. The family of integrase genes known as Family 5 is of particular interest as carriage of members of this integrase family in S. aureus isolates tested is extremely common. In conclusion integrase carriage and hence prophage and pathogenicity island carriage by S. aureus isolates appears to be ubiquitous. Carriage of multiple members of different integrase families is common with some isolates carrying up to four members of different integrase families. Integrase profiles of S. aureus isolates appear to change over time with some members of different families only having appeared within S. aureus isolates recently and others not present in later isolates.
Factors affecting viral load in a population of insects harbouring a persistent baculovirus infection.C. Nixon1, J. Burden2, A. Hodgkinson2, L. King1, R. Possee2, and R. Hails2. 1 Oxford Brookes University, Gipsy Lane campus, Headington, Oxford OX3 0BP. 2 NERC Centre for Ecology and Hydrology, Mansfield Road, Oxford, OX1 3SR. The cabbage moth, Mamestra brassicae, is a serious pest of brassica and other crops in the UK and Europe and can be controlled by lethal baculovirus infections. We have previously described methods for the detection and prevalence of a persistent, non-lethal baculovirus infection within laboratory and field populations of M. brassicae. Current work has been to develop sensitive molecular techniques to monitor relative viral load in individual insects. This has allowed comparisons between different persistently-infected populations and has also been used to study the effects of various host pressures, such as diet quality, on the state of the viral infection in these hosts. The consequences of a persistent virus and its activation are described in relation to the host.
Characterisation and exploitation of a pool of Plutella xylostella granulovirus variants in KenyaDavid Woodward A granulovirus shows significant potential as biocontrol agents for Plutella xylostella, an important pest of cruciferous crops world-wide. A large number of genetically distinct PlxyGV isolates were discovered in 1998 in a survey of smallholdings around Nairobi. Five of the most distinct and homogeneous isolates were compared to 3 previously characterised Asian isolates. These isolates are being studied to; assess the efficacy of the isolates; determine genetic differences in specific regions of the viral genomes; to correlate these changes with differences in biological activity; and finally to improve in vivo production of the virus. Tools have been developed to study the interaction of virus isolates, virus persistence and the maintenance a heterogeneous gene pool.
Elevated Abundance of Bacteriophage Infecting Bacteria in SoilKevin E. Ashelford, Martin J. Day, and John C. Fry Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3TL, United Kingdom Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean 1.5 x 107 g-1), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 108 g-1, which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.
Isolation of a putative temperate cyanophage from marine Synechococcus sppA. Millard & N.Mann University of Warwick It has only recently been shown that lysogeny does occur in marine Synechococcus spp (McDaniel L, Houchin LA, Williamson SJ, et al 2002). All reported cases of lysogeny within marine Synechococcus are based on the use of mitomyocin C to induce the phage from its host. To date there have been no reports of any of these induced phage being isolated. A new technique of co-culturing has been used to isolate a putative temperate cyanophage from its lysogenic host. The basis of this method is discussed, along with the problems in the characterisation of this putative temperate phage.
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