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Bacterial Degradation of Isoprene in the Terrestrial Environment Myriam

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Bacterial Degradation of Isoprene in the Terrestrial Environment Myriam Bacterial Degradation of Isoprene in the Terrestrial Environment Myriam El Khawand A thesis submitted to the University of East Anglia in fulfillment of the requirements for the degree of Doctor of Philosophy School of Environmental Sciences November 2014 ©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 derive there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Abstract Isoprene is a climate active gas emitted from natural and anthropogenic sources in quantities equivalent to the global methane flux to the atmosphere. 90 % of the emitted isoprene is produced enzymatically in the chloroplast of terrestrial plants from dimethylallyl pyrophosphate via the methylerythritol pathway. The main role of isoprene emission by plants is to reduce the damage caused by heat stress through stabilizing cellular membranes. Isoprene emission from microbes, animals, and humans has also been reported, albeit less understood than isoprene emission from plants. Despite large emissions, isoprene is present at low concentrations in the atmosphere due to its rapid reactions with other atmospheric components, such as hydroxyl radicals. Isoprene can extend the lifetime of potent greenhouse gases, influence the tropospheric concentrations of ozone, and induce the formation of secondary organic aerosols. While substantial knowledge exists about isoprene production and atmospheric chemistry, our knowledge of isoprene sinks is limited. Soils consume isoprene at a high rate and contain numerous isoprene-utilizing bacteria. However, Rhodococcus sp. AD45 is the only terrestrial isoprene-degrading bacterium characterized in any detail. A pathway for isoprene degradation involving a putative soluble monooxygenase has been proposed. In this study, we report the isolation of two novel isoprene-degrading bacteria and characterization of the isoprene gene clusters in their draft genomes. Using marker exchange mutagenesis, transcription assays and proteomics analyses, we provide conclusive evidence that isoprene is metabolized in Rhodococcus sp. AD45 through the induced activity of soluble isoprene monooxygenase, a close relative to well known soluble diiron center monooxygenase enzymes. Metabolic gene PCR assays based on a key component of isoprene monooxygenase were also developed to detect isoprene degraders in the environment. The diversity of active isoprene degraders in the terrestrial environment was investigated using DNA-stable isotope probing experiments combined with 454 pyrosequencing. 2 Dedicated to my loving parents 3 Table of contents Abstract ....................................................................................................................... 2 List of tables ................................................................................................................ 9 List of figures ............................................................................................................ 11 List of abbreviations ................................................................................................ 15 Acknowledgements ................................................................................................... 16 Chapter 1 .................................................................................................................. 17 Introduction .............................................................................................................. 17 1.1 Isoprene ............................................................................................................ 18 1.2 Isoprene and atmospheric chemistry ................................................................ 18 1.3 Isoprene production .......................................................................................... 22 1.3.1 Isoprene biosynthesis by plants ................................................................. 24 1.3.1.1 Environmental factors influencing isoprene emission from leaves .... 28 1.3.1.2 The role of isoprene in plant protection .............................................. 32 1.3.2 Isoprene production by microbes ............................................................... 35 1.3.3 Isoprene production by animals and humans ............................................. 36 1.3.4 Isoprene production in the marine environment ........................................ 38 1.4 Isoprene and climate change ............................................................................ 40 1.5 Bacterial degradation of isoprene ..................................................................... 41 1.6 Project aims ...................................................................................................... 46 Chapter 2 .................................................................................................................. 48 Materials and Methods ............................................................................................ 48 2.1 General purpose buffer and solutions ............................................................... 52 2.2 Cultivation of Rhodococcus sp. strains AD45, LB1 and SC4 .......................... 53 2.3 Purity checks and maintenance of bacterial strains .......................................... 54 2.4 Quantification of headspace concentration of isoprene.................................... 55 4 2.5 Extraction of nucleic acids ............................................................................... 55 2.5.1 Extraction of genomic DNA ...................................................................... 55 2.5.2 Small-scale plasmid extraction .................................................................. 57 2.5.3 RNA extraction .......................................................................................... 57 2.6 Nucleic acid manipulation methods ................................................................. 58 2.6.1 Quantification of DNA and RNA .............................................................. 58 2.6.2 Nucleic acid purification ............................................................................ 58 2.6.3 DNA restriction digests ............................................................................. 58 2.6.4 Polymerase chain reaction (PCR) .............................................................. 58 2.6.5 Cloning of PCR products ........................................................................... 59 2.6.6 Clone library construction and Restriction Fragment Length Polymorphism (RFLP) assays ............................................................................ 59 2.6.7 DNA ligations ............................................................................................ 59 2.6.8 Agarose gel electrophoresis ....................................................................... 59 2.6.9 Reverse transcription PCR (RT-PCR) ....................................................... 60 2.6.10 Quantitative Reverse Transcription - PCR (qRT-PCR) ........................... 60 2.6.11 DNA sequencing ...................................................................................... 61 2.7 Antibiotics ........................................................................................................ 61 2.8 Preparation of SOC medium ........................................................................... 62 2.9 Transformation of chemically competent E. coli ............................................. 62 2.10 Preparation and transformation of electrocompetent E.coli ........................... 62 2.11 Conjugual transfer of pMEK plasmid from E. coli to Rhodococcus AD45 ... 63 2.12 Preparation and transformation of electrocompetent Rhodococcus sp. AD45 ................................................................................................................................ 64 2.13 Sucrose selection for double cross-over mutants ........................................... 64 2.14 Analysis of proteins ........................................................................................ 65 2.14.1 Harvesting of cells and preparation of cell-free extracts ......................... 65 2.14.2 Protein quantification ............................................................................... 65 5 2.14.3 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS- PAGE) ................................................................................................................. 65 2.14.4 Mass spectrometry analysis of polypeptides ........................................... 66 2.15 DNA- Stable Isotope Probing (DNA- SIP) .................................................... 67 2.16 Denaturing gradient gel electrophoresis (DGGE) .......................................... 67 2.17 Bacterial community analysis by 454 pyrosequencing .................................. 68 Chapter 3 .................................................................................................................. 69 Isolation of novel terrestrial isoprene degrading bacteria and preliminary analyses of the genome sequences of Rhodococcus AD45, Rhodococcus SC4 and Rhodococcus LB1 ..................................................................................................... 69 3.1 Introduction .....................................................................................................
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