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Detection of Microbial Taxa in Complex Communities: impacts of relative abundance, gene transfer and persistence of target DNA David William Cleary BSc A Thesis submitted for the Degree of Doctor of Philosophy University of Warwick, the School of Life Sciences April 2012 Table of Contents Table of Contents .................................................................................................. i List of Figures ..................................................................................................... vii List of Tables .................................................................................................... xxii Acknowledgements .......................................................................................... xxvi Declaration ...................................................................................................... xxvii Summary ........................................................................................................xxviii List of Abbreviations ...................................................................................... xxix Introduction .......................................................................................................... 1 1.1 Introduction ...................................................................................................... 2 1.2 The Burkholderiaceae ...................................................................................... 3 1.2.1 Burkholderia pseudomallei, B. mallei and B. thailandensis ......................... 4 1.2.2 Burkholderia cepacia Complex .................................................................. 10 1.3 Natural Genetic Transformation of Burkholderia sp. .................................... 14 1.4 DNA Persistence ............................................................................................ 16 1.5 Metagenomics ................................................................................................ 17 1.5.1 Pyrosequencing ........................................................................................... 20 1.5.2 Phylogenetic Markers and Application of High-throughput Sequencing for Analyses of Microbial Community Structure ...................................................... 21 1.5.3 Application of Whole Genome Sequencing to Pathogen Detection ........... 25 1.5.4 Emerging Approaches for Metagenomic Studies - Metatranscriptomics ... 26 1.6 Project Aims ................................................................................................... 29 Materials and Methods ...................................................................................... 30 2.1 Clay Minerals ................................................................................................. 31 2.1.1 Generating Homo-ionic Minerals ............................................................... 31 2.1.2 DNA Adsorption to Homo-ionic Minerals ................................................. 31 2.2 Media and Reagents ....................................................................................... 32 2.2.1 Microbiological Media ................................................................................ 32 2.2.2 Antibiotics ................................................................................................... 33 2.2.3 Buffers and Solutions .................................................................................. 34 2.3 Bacterial Strains ............................................................................................. 34 2.3.1 Bacillus anthracis Ames Spores ................................................................. 34 2.3.2 Bacillus globigii NCTC 10073 ................................................................... 35 2.3.3 Burkholderia cepacia Complex .................................................................. 35 2.3.4 Burkholderia pseudomallei K96243 ........................................................... 36 2.3.5 Determination of Antibiotic Minimum Inhibitory Concentration .............. 37 i 2.3.6 Storage of Strains ........................................................................................ 37 2.4 Nucleic Acid Extractions and Manipulations ................................................ 38 2.4.1 Genomic DNA Extraction ........................................................................... 38 2.4.2 DNA Extraction from Soil .......................................................................... 38 2.4.2.1 MoBio UltraClean® Soil DNA Isolation Kit ............................. 38 2.4.2.2 MoBio PowerSoil® Soil DNA Isolation Kit .............................. 39 2.4.2.3 Modified UltraClean® Soil DNA Isolation Kit (Andrews et al., 2004) 40 2.4.2.4 Phenol / Chloroform (Krsek and Wellington, 1999) .................. 40 2.4.2.5 Epicentre SoilMaster™ DNA Extraction Kit ............................. 41 2.4.2.6 Phenol / Chloroform-based Extraction (Yeates et al., 1998) ..... 42 2.4.2.7 MoBio PowerMax® Soil DNA Isolation Kit ............................. 42 2.4.3 DNA Extraction from Collected-Aerosol Samples ..................................... 43 2.4.4 Plasmid DNA Extraction............................................................................. 44 2.4.5 Agarose Gel Electrophoresis ....................................................................... 44 2.4.6 Nucleic Acid Extraction from Agarose Gels .............................................. 45 2.4.7 Microcon® Purification .............................................................................. 45 2.4.8 Ethanol Precipitation ................................................................................... 45 2.4.9 Quantification of Nucleic Acids.................................................................. 46 2.4.9.1 UV Densitometry ....................................................................... 46 2.4.9.2 Picogreen/Ribogreen Quantification of DNA ............................ 46 2.4.9.3 Agilent 2100 Bioanalyser DNA Analysis .................................. 46 2.4.10 Whole Genome Amplification .................................................................. 47 2.4.11 Removal of Multiple Displacement Amplification-generated, Hyperbranched DNA Structures .......................................................................... 47 2.4.12 Nucleic Acid Manipulations ..................................................................... 47 2.4.12.1 Restriction Endonuclease Digestion of DNA ............................. 47 2.4.12.2 Klenow Treatment ...................................................................... 48 2.4.12.3 Alkaline Phosphatase Treatment ................................................ 48 2.4.13 Plasmids .................................................................................................... 48 2.4.14 Ligations with T4 DNA Ligase ................................................................. 49 2.4.15 Transformation of Competent HB101 E. coli (Promega Ltd, USA)........ 49 2.4.16 One Shot® TOP10 E. coli ......................................................................... 50 2.4.17 Filter Transformations ............................................................................... 50 ii 2.4.18 Polymerase Chain Reaction (PCR) ........................................................... 50 2.4.19 PCR Optimisation ..................................................................................... 51 2.4.19.1 Primers ........................................................................................ 51 2.4.19.2 Magnesium and dNTP Concentration ........................................ 52 2.4.19.3 TaqMan® Real-time PCR Probe ................................................ 52 2.4.19.4 Taq DNA Polymerase ................................................................ 53 2.5 454™ Sequencing .......................................................................................... 57 DNA Persistence in Soil ..................................................................................... 59 3.1 Introduction .................................................................................................... 60 3.1.1 Release and Persistence of Bacterium-derived DNA.................................. 62 3.1.2 Extraction of DNA from Soil ...................................................................... 65 3.2 Aims ............................................................................................................... 67 3.3 Impact of Mineral Concentration on Adsorption of DNA ............................. 68 3.3.1 Comparison of Minerals .............................................................................. 68 3.3.2 Effect of Cation on DNA Adsorption ......................................................... 71 3.3.3 Effect of pH on DNA Adsorption ............................................................... 73 3.3.4 Release of Adsorbed DNA .......................................................................... 74 3.3.5 Protection of Mineral-adsorbed DNA from DNaseI Mediated Degradation