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Characterisation of Methylotrophs in the Rhizosphere

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Characterisation of Methylotrophs in the Rhizosphere Characterisation of methylotrophs in the rhizosphere Michael C. Macey Doctor of Philosophy University of East Anglia, Norwich, UK School of Environmental Sciences September 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. 1 Acknowledgements I would like to thank my supervisory team, Colin Murrell, Giles Oldroyd and Phil Poole. I would like to give special thanks to Colin Murrell for giving me the opportunity to complete this PhD at the UEA and for all of his advice and guidance over the course of four years. I would like to thank the Norwich Research Park and the BBSRC doctoral training program for their funding of my PhD. I would also like to thank the other members of the Murrell lab, both past and present, especially Dr. Andrew Crombie and Dr. Jennifer Pratscher, for their invaluable discussion and input into my research. I would like to thank Dr. Stephen Dye, Dr. Marta Soffker and the staff of Cefas for the opportunity to complete my internship at Cefas Lowestoft. I would like to thank everyone from the UEA I have worked and interacted with over my time here. Finally, I want to thank my wife and my family for their continued support. 2 Abstract Methanol is the second most abundant volatile organic compound in the atmosphere, with the majority of this methanol being produced as a waste metabolic by-product of the growth and decay of plants. There is a large disparity between the amount of methanol estimated as being produced and that which enters the atmosphere. This disparity is believed to be due to the utilisation of methanol by plant associated methylotrophs. The diversity and activity of methylotrophs associated with the root and rhizosphere of pea and wheat plants was assessed through a range of cultivation independent and dependent approaches. Enrichments performed with a range of environmental samples supplemented with methanol resulted in the isolation of several strains of methylotrophic bacteria, including two novel species of methylotroph belonging to the family Methylophilaceae, whose genomes were sequenced and their physiological capabilities assessed. The diversity and abundance of methanol dehydrogenase encoding genes in bulk soil and the pea and wheat rhizosphere was assessed through 454 sequencing and qPCR respectively. Sequencing showed high levels of diversity of methylotrophic bacteria within the bulk soil and also showed a shift in this diversity between the bulk soil and the plant associated soils, in spite of no shift in the abundance of these genes occurring. Active methylotrophs present in the bulk and plant associated soils were identified by DNA stable isotope probing using 13C labelled methanol. Next generation sequencing of the 16S rRNA genes and construction of metagenomes from the 13C labelled DNA revealed members of the Methylophilaceae as highly abundant in all of the soils. A greater diversity of the Methylophilaceae and the genus Methylobacterium were identified as active in the plant associated soils relative to the bulk soil. 13 A CO2 stable isotope probing experiment identified methylotrophs as utilising plant exudates in the pea and wheat root and rhizosphere communities. Several methylotrophic genera were identified as exudate utilising, in addition to heterotrophic genera and Actinomycetes. The specific 13C labelled genera were shown to vary between both the wheat and pea plants and between the rhizosphere and root communities. 3 Abstract .......................................................................................................................... 3 Chapter 1: Introduction ........................................................................................... 16 1.1 Methylotrophic bacteria ....................................................................................... 16 1.1.1 Basic characteristics ....................................................................................... 17 1.1.2 Methanotrophic methylotrophs ................................................................... 19 1.2 Methanol utilising methylotrophs and methanol dehydrogenases ................... 20 1.2.1 The classic methanol dehydrogenase (MxaFI) .............................................. 20 1.2.2 The alternate methanol dehydrogenase (XoxF) ........................................... 22 1.2.2.1 xoxF1 ........................................................................................................ 27 1.2.2.2 xoxF2 ........................................................................................................ 27 1.2.2.3 xoxF3 ........................................................................................................ 27 1.2.2.4 xoxF4 ........................................................................................................ 27 1.2.2.5 xoxF5 ........................................................................................................ 27 1.2.2.6 xoxF outgroups ........................................................................................ 27 1.2.3 The other alternate methanol dehydrogenase (Mdh2) ............................... 27 1.2.4 NAD(P)+ methanol dehydrogenase ............................................................... 28 1.2.5 N,N9-dimethyl-4-nitrosoaniline (DMNA)-dependent nicotinoprotein methanol:DMNA oxidoreductase ........................................................................... 28 1.2.6 Eukaryotic methanol dehydrogenase ........................................................... 29 1.2.7 Methylotrophic and methyl reducing Archaea ............................................. 29 1.3 The Global Methanol Budget ............................................................................... 30 1.3.1 Production of methanol in the marine environment ................................... 32 1.3.2 Production of methanol in the terrestrial environment .............................. 32 1.3.2.1 Plants growing and decaying .................................................................. 32 1.3.2.2 Anthropogenic activity ............................................................................ 33 1.3.3 Disparity in the methanol budget ................................................................. 33 1.4 Methylotrophs in the soil environment ............................................................... 34 1.4.1 Hyphomicrobium ............................................................................................ 34 1.4.2 Methylophilaceae .......................................................................................... 34 1.4.3 Methylobacterium ......................................................................................... 35 1.4.3.1 Plant growth promoting traits confirmed in species of Methylobacterium ............................................................................................... 35 1.5 Methods to study the microbial communities .................................................... 36 1.5.1 Cultivation dependent approaches ............................................................... 36 4 1.5.2 Low resolution approaches ........................................................................... 37 1.5.3 Next generation sequencing .......................................................................... 37 1.5.4 Omics-based approaches ............................................................................... 38 1.5.5 Stable Isotope Probing ................................................................................... 38 1.5.5.1 Identification of exudate utilising bacteria in the rhizosphere through 13 the supply of CO2 ............................................................................................. 40 1.5.5.2 Flaws in the designs of SIP experiments ................................................ 45 1.6 The Plant microbiome .......................................................................................... 47 1.6.1 The phyllosphere ............................................................................................ 47 1.6.2 The rhizosphere .............................................................................................. 47 1.6.2.1 Root and rhizoplane colonising bacteria ................................................ 48 1.6.2.2 Impacts of the rhizosphere community on the plant ............................ 48 1.6.3 Methylotrophs in the rhizosphere ................................................................ 49 1.7 Project aims........................................................................................................... 52 Chapter 2 Materials and Methods........................................................................ 53 2.1 Chemicals and reagents ........................................................................................ 53 2.2 Growth of bacterial strains ................................................................................... 54 2.2.1 Bacterial strains .............................................................................................. 54 2.2.2 Growth media and culturing of organisms ................................................... 55 2.2.2.1 Nitrate reduction ..................................................................................... 57 2.2.2.2 Siderophore
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