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Characterisation of Novel Methylotrophs and the Role of Xoxf in Coastal Marine Environments

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Characterisation of Novel Methylotrophs and the Role of Xoxf in Coastal Marine Environments Characterisation of novel methylotrophs and the role of xoxF in coastal marine environments Alexandra M. Howat Doctor of Philosophy University of East Anglia, Norwich, UK School of Environmental Sciences February 2017 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 Methanol is one of the most abundant volatile organic gases in the atmosphere, and whilst much is known about the sources of methanol, much less is known about the sinks. Methylotrophs are able to use one carbon compounds, such as methanol, as their sole source of carbon and energy. Seawater enrichments with methanol gave rise to the isolation of a novel species of the methylotroph Methylophaga. Some methylotrophs require a rare earth element (REE) when using the alternative methanol dehydrogenase (MDH) XoxF for growth on methanol. Addition of REEs to methanol seawater enrichments, using coastal waters from the south coast of the United Kingdom, showed REE stimulated methanol oxidation, whilst amplicon sequencing of the xoxF5 gene revealed relative increases in unknown sequences. Isolation from enrichments containing lanthanum allowed the cultivation of a new member of the Roseobacter clade, strain La 6. A mutant in the only MDH gene in the genome and complementation and enzyme assays of this strain revealed the essential nature of xoxF during growth on methanol and ethanol. Genome sequencing revealed that stain La 6 has the largest genome of all Roseobacters, at 6.79 Mbp. This facultative methylotroph is metabolically very versatile, growing on some alkanes and aromatic compounds but it was also able to degrade and synthesise DMSP. Multilocus sequence analysis suggests that whilst it shares the core genes with subgroup 1 of the Roseobacters, it shares very little of its pangenome, suggesting unique genetic adaptations. Given this data, the new strain is proposed to be a new genus in the Roseobacter clade. Attempts to express different xoxF sequences in the xoxF mutant of La 6 revealed no phenotype, suggesting there may be as yet unidentified regulatory or accessory mechanisms involved during growth on methanol in this bacterium. 2 Contents Abstract………………………………………………………………..…………........2 List of figures…...…………………………………………………………………....9 List of tables……………………………………………………………...12 Acknowledgements………………………………………………………13 1 Introduction ..................................................................................... 15 1.1 The importance of methylotrophy .................................................................... 15 1.2 Aerobic methylotrophy ..................................................................................... 16 1.2.1 An overview of the history and phylogeny of methylotrophs................... 16 1.2.2 Metabolism of aerobic methylotrophs ...................................................... 18 1.3 The oxidation of methanol to formaldehyde .................................................... 19 1.3.1 Methanol dehydrogenases of methylotrophs ............................................ 19 1.3.2 The canonical methanol dehydrogenase, MxaFI ...................................... 21 1.3.3 XoxF as a rare earth element-dependent methanol dehydrogenase .......... 22 1.4 Methanol in the environment............................................................................ 28 1.4.1 The global methanol budget ...................................................................... 28 1.4.2 Methanol in the marine environment ........................................................ 30 1.4.3 Methanol production by microbes ............................................................ 31 1.5 Methylotrophy in the marine environment ....................................................... 31 1.5.1 Methylophaga spp. .................................................................................... 32 1.5.2 SAR11 and OM43 clade ........................................................................... 32 1.5.3 Marine Roseobacter clade ......................................................................... 33 1.6 Aims and objectives ......................................................................................... 36 2 Materials and Methods .................................................................... 38 2.1 Chemicals and reagents .................................................................................... 38 2.2 Bacterial strains, plasmids and primers ............................................................ 39 2.3 Cultivation and maintenance of strains ............................................................ 41 2.3.1 Antibiotics ................................................................................................. 42 3 2.3.2 Escherichia coli ......................................................................................... 42 2.3.3 Preparation and transformation of chemically competent E.coli .............. 42 2.3.4 Methylophaga spp ..................................................................................... 43 2.3.5 Oceanicola strain La 6 .............................................................................. 44 2.3.6 Conjugation of strain La 6......................................................................... 45 2.4 Bacterial purity checks and microscopy ........................................................... 45 2.5 Extraction of nucleic acids ............................................................................... 45 2.5.1 Environmental DNA extraction ................................................................ 45 2.5.2 Genomic DNA extraction ......................................................................... 46 2.5.3 Small scale plasmid extraction .................................................................. 47 2.6 Nucleic acid manipulation techniques .............................................................. 47 2.6.1 Quantification of DNA .............................................................................. 47 2.6.2 Polymerase Chain Reaction (PCR) ........................................................... 47 2.6.3 Restriction digests ..................................................................................... 47 2.6.4 DNA purification ...................................................................................... 48 2.6.5 Agarose gel electrophoresis ...................................................................... 48 2.6.6 DNA ligations and cloning of PCR products ............................................ 48 2.6.7 DNA sequencing and assembly ................................................................ 48 2.6.8 Multi-Locus Sequence Analysis ............................................................... 49 2.6.9 Gene content analysis ................................................................................ 50 2.6.10 Denaturing gradient gel electrophoresis.................................................... 50 2.7 Harvesting cells and preparation of cell free extracts ...................................... 51 2.8 Protein analysis ................................................................................................. 51 2.8.1 Quantification ............................................................................................ 51 2.8.2 SDS-PAGE ................................................................................................ 51 2.8.3 Peptide Mass Fingerprinting ..................................................................... 52 2.9 NAD(P)-independent alcohol dehydrogenase assay ........................................ 53 2.10 Measurements of substrates .............................................................................. 53 2.10.1 Headspace methanol.................................................................................. 53 2.10.2 Methanol in enrichment medium .............................................................. 54 2.10.3 Headspace dimethylsulfide ....................................................................... 54 2.10.4 Total dimethylsulfonioproprionate............................................................ 55 2.10.5 Indole acetic acid in culture media............................................................ 55 2.10.6 Thiosulfate in culture media...................................................................... 55 4 2.11 Physiological characterisation .......................................................................... 56 2.11.1 Gram stain, catalase and oxidase test ........................................................ 56 2.11.2 Carbon source utilisation........................................................................... 56 2.11.3 Temperature, pH and salinity ranges ........................................................ 56 2.11.4 Sensitivity to antibiotics ............................................................................ 57 2.11.5 Dimethylsulfide production ...................................................................... 57 2.11.6 Dimethylsulfoniopropionate degradation.................................................. 57 2.11.7 Dimethylsulfoniopropionate production ................................................... 58 2.11.8 Carbohydrate oxidation and fermentation ................................................
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