Fluorescence mediated particle analysis of large populations of prokaryotic cells and viruses

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We request funds to support the purchase of an A40-MiniFCM flow cytometer (Apogee Flow Systems), suitable for the analysis of small prokaryotes and large viruses. The application arises from the recognition that single-cell approaches that have been made possible by recent advances in microscopy, the use of fluorescent proteins and improved fluorescent dyes, have transformed our understanding of the biology of microorganisms and viruses. We describe four diverse projects that will utilise state-of-the-art particle analysis techniques to address biological questions that have relevance to both basic and applied science. Project 1 addresses basic questions concerning DNA replication and cell proliferation, features common to all forms of life. Using an archaeal system with high homology to eukaryotic systems as a sample, genetically tractable model, the goal is to identify novel genes essential for these pathways. Project 2 addresses basic questions on the relationship between DNA replication and the maintenance of DNA methylation patterns that are essential to epigenetic regulation using bacteria as a model system. In project 3, method development for rapid and economical virus titering is described. The final project concerns research on bacteria with highly reduced genomes, specifically to understand the biological significance of variable genomic content in the bacterium Buchnera, which forms beneficial infections in insect cells. The A40-MiniFCM possesses a number of attributes that are essential for the success of our investigations: (1) Exceptional detection capabilities. The A40-MiniFCM uses a high numerical aperture lens to provide an excellent signal to noise ratio. This allows the differentiation of small differences associated with genome copy number and proliferation (required for Projects 1, 2 and 4). In addition, we show here for the first time that this arrangement allows the detection and quantification of unlabelled Baculovirus particles. This ability will be refined and exploited in the development of novel methods for viral titering as described in Project 3. (2) 405nm laser for enhanced DNA analysis. With the use of specially selected fluorescent DNA stains this allows the accurate detection and quantification of the low fluorescence signal associated with very small genomes of a number of prokaryotes, and allows discrimination of the small variations associated with proliferation of these organisms. This feature is key for the study of proliferation control in the archaea, epigenetic regulation in bacteria, and understanding the mechanisms of transmission and variation of obligate intracellular bacteria in aphids (projects 1, 2 and 4). (3) Accurate volumetric sample injection. The A40 has a true volumetric injection system using a calibrated syringe. Accurate control of the injection volume allows precise determination of the number of particles in an exact volume. This will allow the development of rapid virus titering protocols as outlined in project 3. (4) analysis of limited material. Samples as small as 100 microlitres can be loaded and injected on the system. The bacteria to be analysed in project 4 are obligate symbionts, and unculturable outside of their host aphids. This system allows the analysis of samples available in only very limited quantities to be carried out. There is currently no Apogee A40-MiniFCM in the UK, and Apogee will use the University of York as their UK reference site. An additional benefit of this relationship with Apogee will be the customisation of this system to further enhance its capabilities. To this end, Apogee will install higher-powered 405 nm and 488 nm lasers that those currently offered. This enhancement will increase the sensitivity of the instrument to particle size (as determined by light scatter). Furthermore, the University of York will act as the primary beta-test site for the development of a new optical bench design that will be developed by Apogee. These mo