Molecular Ecology of Marine Isoprene Degradation Antonia Johnston University of East Anglia 2014 A thesis submitted to the School of Environmental Sciences in fulfilment of the requirements for the degree of Doctor of Philosophy University of East Anglia, Norwich, UK September 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 derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Declaration I declare that the content of this thesis entitled “Molecular Ecology of Marine Isoprene Degradation” was undertaken and completed by myself under the supervision of Professor J.C.Murrell, unless otherwise acknowledged and has not been submitted in support of an application for another degree or qualification in this or any other university or institution Antonia Johnston Acknowledgements I would like to thank my primary supervisor, Professor J.C.Murrell, for his help and guidance throughout my PhD. I would also like to thank past and present members of the Murrell lab at the University of Warwick and the University of East Anglia, especially Myriam El Khawand and Dr. Andrew Crombie for their help and collaboration on this project. I thank my secondary supervisor Dr. Jonathon Todd for his advice. I would especially like to thank our collaborators on this project, including Dr. Terry McGenity at the University of Essex, Professor Dan Arp at Oregon State University, Capt. Collett at the 47th Regiment Royal Horse Artillery for assistance with sampling, and Sue Slade at the University of Warwick proteomics facility. Special thanks go to my family for their continued support. Table of Contents List of Abbreviations 1 Abstract 3 Chapter 1: Introduction 4 1.1 The structure of isoprene 5 1.2 Industrial uses of isoprene 5 1.3 The effect of isoprene on the atmosphere and global climate 6 1.4 Isoprene cycling and the global isoprene budget 8 1.4.1 Isoprene sources and production 8 1.4.1.1 Isoprene production by humans and animals 8 1.4.1.2 Production of isoprene by terrestrial vegetation 9 1.4.1.3 Pathways of isoprene production 12 1.4.1.4 Algal isoprene production in the marine environment 14 1.4.1.5 Isoprene production in bacteria 16 1.4.2 Isoprene sinks and degradation 17 1.4.2.1 Microbial consumption of isoprene in the terrestrial environment 17 1.4.2.2 Isoprene degradation in the marine environment 17 1.5 Isoprene monooxygenase in Rhodococcus AD45 19 1.6 The soluble diiron centre monooxygenases 22 1.6.1 SDIMOS in the Actinobacteria 25 1.6.2 Alkene monooxygenase of Xanthobacter Py2 25 1.7 Use of soluble diiron centre monooxygenases in bioremediation 26 1.8 Microbial biodegradation of natural rubber 26 1.9 Biology of the genus Gordonia 27 1.10 Stable isotope probing (SIP) 27 1.10.1 Phospholipid fatty acid (PLFA) and RNA SIP 28 1.10.2 DNA Stable Isotope Probing 28 1.11 Project hypotheses and aims 30 Chapter 2: Materials and Methods 31 2.1. Chemicals and reagents 32 2.2. Bacterial strains 33 2.3 Culture media and growth of organisms 34 2.4 Acetylene inhibition assays 38 2.5 DNA extraction, processing and storage 39 2.6 Genomic DNA extraction for genome sequencing 39 2.7 Genome sequencing and analysis 39 2.8 Agarose gel electrophoresis 39 2.9 Polymerase chain reaction (PCR) 40 2.10 Cloning of PCR products 42 2.11 RFLP analysis of cloned PCR products from enrichment samples 42 2.12 DNA sequencing and phylogenetic analysis 42 2.13 Extraction of protein and protein purification 43 2.14 SDS polyacrylamide gel electrophoresis (SDS-PAGE) 43 2.15 RNA extraction and storage 45 2.16 Quantitative reverse transciptase polymerase chain reaction (qRT- PCR) 45 2.17 Oxygen electrode assays 47 2.18 Environmental sampling 47 2.19 Enrichment and isolation of isoprene degrading microorganisms 48 2.20 DNA Stable isotope probing (DNA-SIP) of estuarine samples 48 2.21 Denaturing gradient gel electrophoresis (DGGE) 49 2.22 Illumina sequencing of 16S rRNA and isoA amplicons from DNA-SIP fractions 50 Chapter 3: Isolation and characterisation of marine isoprene degrading bacteria 52 3.1 Introduction 51 3.2 Enrichment and isolation of isoprene degrading bacteria from marine, coastal and estuarine environments 53 3.3 SDS-PAGE of isolates grown on glucose vs isoprene 79 3.4 Discussion 93 Chapter 4: Physiology and regulation of isoprene and propane metabolism in Gordonia polyisoprenivorans i37 and Mycobacterium hodleri i29a2* 95 4.1 Growth of Gordonia polyisoprenivorans i37 and Mycobacterium hodleri i29a2* on short chain hydrocarbons 95 4.2 SDS-PAGE and polypeptide analysis of Gordonia polyisoprenivorans i37 and Mycobacterium i29a2* 98 4.3 Oxygen electrode assays in Gordonia polyisoprenivorans i37 and Mycobacterium hodleri i29a2* 106 4.4 qRT-PCR analysis of Gordonia polyisoprenivorans i37 111 4.5 Acetylene inhibition assays of isoprene monooxygenase in Gordonia polyisoprenivorans i37 and Mycobacterium hodleri i29a2* 116 4.6 Antibiotic resistance in Gordonia polyisoprenivorans i37 and Mycobacterium hodleri i29a2* 117 4.7 Discussion 119 Chapter 5: Genome mining in Gordonia polyisoprenivorans i37 and Mycobacterium hodleri i29a2* 120 5.1 Introduction 121 5.2 The genome of Gordonia polyisoprenivorans i37 121 5.2.1 Analysis of histidine biosynthesis pathways and aminoacyl tRNA biosynthesis in Gordonia polyisoprenivorans 122 5.2.2 Analysis of Gordonia polyisoprenivorans isoprene gene cluster sequences 123 5.2.3 Analysis of propane monooxygenase gene sequences in Gordonia polyisoprenivorans 125 5.2.4 Analysis of polyisoprene degradation-related gene sequences in Gordonia polyisoprenivorans i37 128 5.2.5 Other monooxygenases present in Gordonia polyisoprenivorans i37 genome 131 5.2.6 Kegg metabolic pathway analysis for Gordonia polyisoprenivorans 132 5.3 The genome of Mycobacterium hodleri i29a2* 143 5.3.1 Analysis of isoprene gene cluster sequences in Mycobacterium hodleri 144 5.3.2 Analysis of propane monooxygenase gene sequences in Mycobacterium hodleri 145 5.3.4 Analysis of monooxygenase enzyme systems in Mycobacterium hodleri 146 5.3.4 Kegg metabolic pathway analysis for Mycobacterium hodleri i29a2* 149 5. 4 Discussion 155 Chapter 6: Design and testing of PCR primer set based on isoprene monooxygenase alpha subunit 158 6.1 Introduction 159 6.2 Design and testing of isoA primers based on Rhodococcus AD45 gene sequence 159 6.3 Design of isoA primers based on isoA sequences from the genomes of Gordonia, Mycobacterium and terrestrial Rhodococcus isolates 161 6.3.1 Testing of isoA primers in marine and estuarine isolates 162 6.3.2 Testing of isoA primers with DNA from marine, coastal and estuarine environments and assessment of isoA diversity in environmental samples 164 6.3.3 Testing of isoA primers with negative controls 170 6.4 Discussion 171 Chapter 7: Using DNA Stable Isotope Probing to identify active isoprene degraders in the Colne Estuary, Essex 173 7.1 Introduction 174 7.2 Experimental Design 175 7.3 Results – Hythe incubations 176 7.4 Results – Wivenhoe incubations 189 7.5 Discussion 207 Chapter 8: Discussion 209 8.1 Isoprene degradation occurs in marine samples from several different environments 210 8.2 The Gordonia genome contains a gene cluster encoding a soluble diiron centre isoprene monooxygenase. In Gordonia polyisoprenivorans, polypeptides associated with isoprene degradation are induced during growth on isoprene 212 8.3 A PCR primer set has been designed that can amplify the isoA gene in environmental samples – a future gene probe for isoprene degraders 214 8.4 DNA-SIP experiments indicated that Rhodococcus sp., Mycobacterium sp. and other Actinobacteria are active isoprene degraders in the Colne estuary 215 References 217 List of Figures and Tables Figure 1.1: The structure of isoprene 5 Figure 1.2: An example of isoprene interaction with reactive oxygen species to form ozone 7 Figure 1.3: The isoprene cycle as based on current literature 8 Figure 1.4: The intercalation of isoprene in the cellular membrane lipid bilayer in increasing temperatures 11 Figure 1.5: The Mevalonate pathway of isoprene production 13 Figure 1.6: The Non mevalonate pathway for isoprene production 14 Figure 1.7: Gene cluster encoding isoprene degradation enzymes in Rhodococcus AD45 21 Figure 1.8: Putative pathway for isoprene degradation in Rhodococcus AD45 24 Figure 1.9: Alignment of α-subunit of SDIMO hydoxylases 29 Figure 1.10: DNA based stable isotope probing (DNA-SIP) 30 Figure 1.11: A schematic of a 13C-labelled isoprene incubation, DNA extraction and downstream analysis 31 Figure 3.1: Map of United Kingdom showing environmental sampling sites 53 Figure 3.2: GC measurements of enriched environmental samples from a variety of marine, coastal and estuarine sites 54 Figure 3.3: 16S rRNA gene phylogeny of isolates 59 Figure 3.4: Microscopy of isolates 62 Figure 3.5: Growth curves of isolates on isoprene in liquid culture 65 Figure 3.6.1 Comparative SDS-PAGE of Rhodococcus wratislaviensis i48 and Rhodococcus erythropolis i24 strains grown on glucose and isoprene as sole carbon source 80 Figure 3.6.2: Comparative SDS-PAGE of cell free extracts from Rhodococcus globerulus i8a2 grown on glucose and isoprene as sole carbon source 87 Figure 3.6.3: Comparative SDS-PAGE of Micrococcus luteus i61b grown on glucose and isoprene as sole carbon source 89 Figure 3.6.4: Comparative SDS-PAGE of cell extracts of Loktanella i8b1 grown on glucose and isoprene as sole carbon source