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Understanding the Impacts of Viruses on Microbial Methanol Utilisation in Seawater

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Understanding the Impacts of Viruses on Microbial Methanol Utilisation in Seawater Understanding the impacts of viruses on microbial methanol utilisation in seawater Kevin Jamie Purves Doctor of Philosophy University of East Anglia, Norwich, UK School of Environmental Sciences In collaboration with Plymouth Marine Laboratory September 2019 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Abstract Methylotrophs are bacteria which utilise methanol and other one-carbon compounds for assimilative (growth) and dissimilative (energy) metabolism. Methylotrophy has been demonstrated by a large proportion of marine bacteria, influencing carbon cycling and is a significant sink of methanol in the marine environment. Viruses are understood to considerably influence biogeochemical cycles in the oceans, however the extent to which they influence methylotrophs and methanol cycling has remained unclear. Virus-like particles associated with a methanol-utilising methylotroph were isolated to this end and characterised using electron microscopy, indicating morphologies resembling enveloped viruses. A comprehensive seasonal survey in the Western Channel Observatory (WCO) combined virus abundance data with a taxonomic investigation of the microbial community; environmental variables; plankton abundance; bacterial production data and methanol uptake rates. The latter ranged between 0.1 – 10.6 nmol L-1 h-1 throughout the water column in the WCO with little depth variation. Seasonal trends were also consistent throughout the water column, with the highest uptake rates occurring during winter months. For the first time, seasonal virus abundance data was also determined with methanol uptake rates and bacterial production and showed a significant negative correlation with rates of methanol dissimilation. This could suggest that under high viral load bacteria are unable to utilise methanol as readily to meet their energy requirements. However, this reflects total virus abundance and not those that specifically infect methylotrophic bacteria. Furthermore, the diversity and distribution of a recently discovered alternate methanol dehydrogenase utilised by a portion of the methylotrophic community was explored. Sequencing of the xoxF5 gene was revealed to be highly conserved within the Rhodobacteraceae throughout the year in the WCO and was also the dominant associated bacterial group along a transect of the Atlantic. Future studies should focus on viruses specific to known methanol utilisers to understand their role in controlling marine methanol concentrations. 2 Access Condition and Agreement Each deposit in UEA Digital Repository is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of the Data Collections is not permitted, except that material may be duplicated by you for your research use or for educational purposes in electronic or print form. You must obtain permission from the copyright holder, usually the author, for any other use. Exceptions only apply where a deposit may be explicitly provided under a stated licence, such as a Creative Commons licence or Open Government licence. Electronic or print copies may not be offered, whether for sale or otherwise to anyone, unless explicitly stated under a Creative Commons or Open Government license. Unauthorised reproduction, editing or reformatting for resale purposes is explicitly prohibited (except where approved by the copyright holder themselves) and UEA reserves the right to take immediate ‘take down’ action on behalf of the copyright and/or rights holder if this Access condition of the UEA Digital Repository is breached. Any material in this database has been supplied on the understanding that it is copyright material and that no quotation from the material may be published without proper acknowledgement. Contents Abstract ............................................................................................................. 2 List of figures .................................................................................................... 8 List of tables .................................................................................................... 12 Acknowledgements ........................................................................................ 13 1. Introduction .............................................................................................. 15 1.1. Methanol in the environment .................................................................... 15 The global methanol budget .............................................................. 15 Methanol in the oceans ..................................................................... 16 Methanol production in the marine environment ............................... 19 1.2. Methylotrophy in the environment ............................................................ 19 Methylotrophic bacteria ..................................................................... 19 Metabolism pathways of aerobic methylotrophs ................................ 21 Methanol dehydrogenase .................................................................. 22 A rare earth element-dependent methanol dehydrogenase .............. 24 Marine methylotrophy ........................................................................ 27 1.3. Viruses in the environment ...................................................................... 31 An introduction to viruses .................................................................. 31 Phage in the marine environment...................................................... 34 Marine biogeochemical influences .................................................... 38 Phage of methylotrophic bacteria ...................................................... 41 1.4. Project aims and objectives ..................................................................... 43 2. Materials and methods............................................................................. 46 2.1. Bacterial strains ....................................................................................... 46 2.2. Cultivation and maintenance of strains .................................................... 48 Methylophaga spp. ............................................................................ 48 Marinibacterium anthonyi La6 ........................................................... 48 2.3. Timeseries sampling and process ........................................................... 49 3 Supplemental timeseries and cruise data .......................................... 50 2.4. Isolation of methylotrophic bacteria ......................................................... 52 2.5. Analytical Flow Cytometry (AFC) ............................................................. 53 Sample preparation and storage ....................................................... 53 Bacterial numbers analysis by flow cytometry ................................... 53 Viral-like particle analysis by flow cytometry ..................................... 54 2.6. Virus Techniques ..................................................................................... 56 Filtrate ............................................................................................... 56 Incubation variables .......................................................................... 57 Growth of methanol-utilising methylotrophic bacteria in agar ............ 57 Plaque assay ..................................................................................... 57 Spot testing ....................................................................................... 58 Phage enrichment in liquid culture .................................................... 59 Prophage induction by ultraviolet, antibiotic and thermal stress ........ 61 Tangential Flow Filtration (TFF) ........................................................ 62 PEG Precipitation and CsCl gradient purification .............................. 62 2.7. Virus dilution and reduction experiments ................................................. 63 Virus reduction .................................................................................. 63 Virus dilution ...................................................................................... 63 2.8. Transmission Electron Microscopy (TEM) ............................................... 64 2.9. Extraction of nucleic acids ....................................................................... 64 Environmental DNA extraction .......................................................... 64 Genomic DNA extraction ................................................................... 65 Pulsed Field Gel Electrophoresis (PFGE) ......................................... 66 2.10. Nucleic acid manipulation techniques ................................................... 66 Quantification of DNA .................................................................... 66 Standard polymerase chain reaction (PCR) ................................... 67 Agarose gel electrophoresis ........................................................... 69 DNA purification ............................................................................. 69 4 Sanger sequencing of PCR amplicons and analysis ...................... 69 Primers used for Illumina sequencing
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