University of Bath PHD The structural and biological properties of Photorhabdus Mns, and CYP6G1-mediated insecticide resistance in Drosophila melanogaster Jones, Robert Award date: 2007 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 05. Oct. 2021 The Structural and Biological Properties of Photorhabdus Mns, and CYP6G1-Mediated Insecticide Resistance in Drosophila melanogaster Robert Jones A thesis submitted for the degree of Doctor of Philosophy Department of Biology and Biochemistry University of Bath September 2007 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with its author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and they must not copy it or use material from it except as permitted by law or with the consent of the author. 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ABSTRACT Photorhabdus colonise the gut of insect-pathogenic nematodes, and are themselves insect pathogens that produce variety of toxicity factors. One protein, Mns, is highly conserved within the genus and constitutes over 30% of the total protein secretion from Photorhabdus luminescens, but is shown in this study not to be an insect toxin. Mns associates with extracellular material when Photorhabdus grows in colony biofilms, and modifies the attachment of cells to surfaces. The protein is also detected in aggregations of Caenorhabditis elegans nematodes induced by Photorhabdus asymbiotica supernatant, but appears not to interact with its symbiotic host nematodes, Heterorhabditis. Here, we use circular dichroism, dynamic light scattering and differential scanning calorimetry to characterise Mns, and propose that the high propensity of the protein to aggregatein vitro may relate to its function in vivo. In this report, cytochrome P450-mediated insecticide resistance in Drosophila melanogaster has also been investigated. In Hikone-R flies, resistance to DDT and other insecticides is conferred by overexpression of a single cytochrome P450 gene, Cyp6gl. Here, CYP6G1 is purified from Escherichia coli as a recombinant protein, and used to prepare anti-CYP6Gl antibodies to show the expression pattern of the protein in insecticide-resistant and -susceptible flies. CYP6G1 is expressed in Hikone-R fly sperm, indicating that the protein has a role in reproduction. Furthermore, a homology model of CYP6G1 is presented, and shows not only how a variety of insecticides can be accommodated by the active site cavity, but also that the enzyme may contribute to hormone titres in the fly. How this relates to life history traits previously identified in Hikone-R flies is discussed. ACKNOWLEDGEMENTS I am very grateful for the help I’ve received. My biggest thanks go to Nick Waterfield, Stefan Bagby, Jean van den Elsen and Richard ffrench-Constant, whose ideas, expertise and time have been invaluable and are much appreciated. Many people have provided practical assistance and materials during the data collection stages of this report. For this I mostly thank Michelle Hares and Andrea Dowling, Isabella Vlisidou, Guowei Yang and Abhishek Upadhyay, to each of whom I’m very grateful. I have also benefited from the knowledge and generosity of Stuart Reynolds and Ioannis Eleftherianos, Sam Boundy, Tim Karr, Ben Heath and Kim Steeds. Toby Jenkins and Xavi Munez, Rene Feyereisen, Alan Cooper and Sharon Kelly, Todd Ciche, Phillip Dabom, Ursula Potter, Kevin Balbi and Kay Uppington have all been valuable collaborators and must be thanked. Sandra Barnes, Paul Wilkinson, Rob Jackson, David Clarke and his group, Tim Bearder, Sophie Ainsworth, Matt Amos and Maria Sanchez-Contreras have contributed to this work. Saskia Bakker did the majority of the P450 homology modelling and has been a very helpful colleague. I also have to thank Sally Shuttleworth and Kenneth Holboum for help in this area. It was a pleasure to work with Caroline McCart, who helped with all of my Drosophila- related work and has patiently continued to answer all of my questions. Lastly I thank my parents and Stewart, Matthew and Zoe, and those who may not have contributed directly, but like many of those listed above, have enriched my time at the University of Bath. Here I include all members past and present of our lab, Ronald Jenner, Peter Millichap, Samantha McCavera, Victoria Eastham, Joanna Clark and Robin Francis, Tamsin Greenway, TeamBath, CrewBath, and the Sports Training Village. ABBREVIATIONS AChE Acetylcholinesterase amp Ampicillin BSA Bovine serum albumin CD Circular dichroism CFU Colony forming units Cif Cycle inhibiting factor CNF Cytotoxic necrotising factor COSY Correlation spectroscopy CR Congo red CV Crystal violet DDT Dichloro-diphenyl-trichloroethane 8-ALA 5-aminolevulinic acid DLS Dynamic light scattering DNA Deoxyribonucleic acid DSC Differential scanning calorimetry DTT Dithiothreitol EDTA Ethylenediamine tetraacetic acid GABAR y-aminobutyric acid receptor GASP Growth-advantage-in-stationary-phase GFP Green fluorescent protein GST Glutathione-S-transferase Heal Calorimetric enthalpy H vh van’t Hoff enthalpy HEPES 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid Hms Hemin storage HSQC Heteronuclear single quantum correlation HTV High tension voltage HWI Hauptman Woodward Institute IJ Infective juvenile IPTG Isopropyl-|3-D-thiogalactopyranoside IS Insertion sequence JH Juvenile hormone kan Kanamycin KDR Knockdown resistance LB Luria Bertani Lysogeny Broth MBP Maltose binding protein Mcf Makes caterpillars floppy Mns Makes nematodes sticky MOE Molecular Operating Environment MROD Methoxy-resorufm O-Demethylation Mrf Mannose-resistant fimbriae MWCO Molecular weight cut-off NMR Nuclear Magnetic Resonance NOE Nuclear Overhauser effect NOESY Nuclear Overhauser effect spectroscopy NRMSD Normalised root mean standard deviation ODM Octadecylmercaptan P450 Cytochrome P450 PBS Phosphate buffered saline PCR Polymerase chain reaction PDB Protein databank PEG Polyethylene glycol Pi Isoelectric point Pnf Photorhabdus necrotising factor PVC Photorhabdus virulence cassette Rdl Resistant to dieldrin rDNA Ribosomal DNA, genes coding for ribosomal RNA RFLP Restriction fragment length polymorphism rif Rifampicin RT Room temperature SAM Self-assembled monolayer SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM Scanning Electron Microscopy SPR Surface plasmon resonance SRS Substrate recognition site SVD Singular value decomposition Tm Transition midpoint TB Terrific Broth Tc Toxin complex TCA Trichloroacetic acid TEM Transmission electron microscopy tet Tetracyclin TEV Tobacco etch virus TOCSY Total correlated spectroscopy TPBS Tween, milk powder PBS Tris T ris(hydroxymethyl)aminomethane TROSY Transverse relaxation optimised spectroscopy TT01 Photorhabdus luminescens strain TT01 TTSS Type III secretion system u v Ultraviolet 20E 20-hydroxyecdysone AMINO ACIDS Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutamine Gin Q Serine Ser S Glutamic acid Glu E Threonine Thr T Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine lie I Valine Val V vi CONTENTS Title page i Abstract ii Acknowledgements iii Abbreviations iv Contents vii List of Figures and Tables xiii CHAPTER 1 Introduction to Photorhabdus 1 1 INTRODUCTION 2 1.1 Biochemical and Physiological Characterisation 2 1.2 Taxonomy and Phylogeny 2 1.3 Photorhabdus Lifecycle 4 1.4 Phase Variation 6 1.4.1 Photorhabdus Phase Variants 8 1.4.2 Causes of Phenotypic Variation 9 1.4.3 Proteomics of Phenotypic Variation 10 1.5 Virulence Mechanisms of Photorhabdus 10 1.5.1 Toxin Complexes 10 1.5.2 Makes