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Download (7Mb) University of Warwick institutional repository: http://go.warwick.ac.uk/wrap A Thesis Submitted for the Degree of PhD at the University of Warwick http://go.warwick.ac.uk/wrap/49461 This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. Gene regulation in methanotrophs: Evidence from Methylococcus capsulatus (Bath) and Methylosinus trichosporium (OB3b) Ashraf Khalifa A thesis submitted to the School of Life Sciences in fulfilment of the requirements for the degree of Doctor of Philosophy February 2012 University of Warwick Coventry, UK Contents List of figures ............................................................................................................. vi List of tables ............................................................................................................... ix Acknowledgements .................................................................................................. xi Declaration ............................................................................................................... xii Abbreviations........................................................................................................... xiii Chapter 1 Introduction ........................................................................................ 1 1.1 Introduction ............................................................................................................2 1.2 A brief history of the taxonomy of methanotrophs ...................................................3 1.3 Phylogeny of methanotrophs ....................................................................................7 1.4 Distribution of methanotrophs ..................................................................................9 1.5 Ecological significance of methanotrophs .............................................................10 1.6 Biotechnological application of methanotrophs ....................................................11 1.6.1 Pollutant degradation ................................................................................11 1.6.2 Production of fuels .....................................................................................11 1.6.3 The pharmaceutical industry.......................................................................12 1.6.4 Single cell protein (SCP) ............................................................................13 1.7 The methane oxidation pathway..............................................................................13 1.8 Particulate methane monooxygenase (pMMO) .....................................................15 1.8.1 Biochemistry of pMMO ...........................................................................16 1.8.2 Genetics of pMMO...................................................................................18 1.9 Methanobactin (Mb) ............................................................................................20 1.9.1 Polyketide synthases (PKS) ........................................................................22 1.9.2 Non-ribosomal peptide synthetases (NRPS) .................................................23 1.10 Soluble methane monooxygenase (sMMO) .........................................................24 1.10.1 Biochemistry of sMMO ............................................................................24 1.10.2 Genetics of sMMO ...................................................................................27 1.11 Genetic systems for methanotrophs.........................................................................30 1.12 The Copper Switch ................................................................................................32 1.13 Copper uptake in methanotrophs .............................................................................35 1.13.1 P-type ATPases .........................................................................................37 1.14 DNA microarrays ..................................................................................................39 1.15 Project aims..........................................................................................................40 Chapter 2 Materials and Methods .................................................................... 41 2.1 Chemicals .............................................................................................................42 2.2 Antibiotics.............................................................................................................42 2.3 Bacterial growth media ..........................................................................................42 2.3.1 Nitrate Mineral Salts medium (NMS) .......................................................42 2.3.2 Luria–Bertani (LB) medium .....................................................................45 2.4 Bacterial strains ....................................................................................................45 2.4.1 Bacterial strains and examination for purity ..............................................45 ii 2.4.2 Maintenance of bacterial strains................................................................46 2.4.3 Calculation of specific growth rate ..............................................................46 2.5 Nucleic acid extraction from methanotrophs .........................................................48 2.5.1 Extraction of genomic DNA from Mc. capsulatus and Ms. trichosporium ...48 2.5.2 Quantification and storage of DNA...........................................................49 2.5.3 Total RNA extraction from Mc. capsulatus...............................................49 2.5.4 Estimation of RNA quality using Bioanalyzer..............................................50 2.5.5 Plasmid extraction from E. coli (mini-prep) ..............................................51 2.6 DNA manipulation techniques ..............................................................................51 2.6.1 DNA restriction digests ............................................................................51 2.6.2 DNA purification from agarose gels .........................................................51 2.6.3 Dephosphorylation ...................................................................................51 2.6.4 DNA ligations ..........................................................................................52 2.6.5 Cloning PCR products ..............................................................................52 2.6.6 Agarose gel electrophoresis ......................................................................52 2.7 Bacterial genetics .................................................................................................52 2.7.1 Transfer of plasmids into methanotrophs by conjugation ..........................52 2.7.2 Preparation and transformation of chemically-competent Escherichi coli.......54 2.7.3 Transformation of chemically-competent E. coli.......................................54 2.7.4 Preparation of electrocompetent E. coli ....................................................54 2.7.5 Transformation of electrocompetent E. coli by electroporation .................55 2.8 Polymerase chain reaction (PCR) .........................................................................55 2.9 Reverse transcriptase PCR (RT–PCR) ..................................................................56 2.10 DNA sequencing and analysis ..............................................................................57 2.11 Biochemical analysis ............................................................................................57 2.11.1 Cell–free extract preparation.....................................................................57 2.11.2 Estimation of protein concentration............................................................58 2.11.3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis ............58 2.11.4 Analysis of polypeptides using MS/MS .......................................................60 2.12 Enzyme Assays ....................................................................................................60 2.12.1 Naphthalene oxidation assay for sMMO activity.......................................60 2.12.2 Determination of whole-cell cytochrome oxidase activity .............................61 2.13 Methanobactin production assay ...........................................................................61 2.13.1 Preparation of chrome azurol S – copper (Cu–CAS) agar ..........................62 2.14 Estimation of oxygen consumption rate using the oxygen electrode...........................62 2.15 Statistical analysis..................................................................................................63 Chapter 3 Investigating potential copper transport systems by targeted mutagenesis in Mc. capsulatus (Bath) ..................................................................... 64 3.1 Introduction...........................................................................................................65 3.2 Features of CopA of Mc. capsulatus........................................................................67 3.3 Inactivation of the copA2 and copA3 genes ..............................................................73
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