UNIVERSITY OF READING SCHOOL OF ANIMAL AND MICROBIAL SCIENCES ADAPTATION OF RHIZOBIUM TO ENVIRONMENTAL STRESS by Marc A. Fox Submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy September 2005 i I declare that this is my own account of my research and that this work has not been submitted for a degree at any other university. However, I would like to acknowledge that certain vectors and strains were constructed by members of the laboratory, as described in the text. I also acknowledge the help I received from the undergraduate project students Claire Vernazza, Lara Clewes-Garner and David Stead in long and arduous task of screening the LB3 library, under my joint supervision with Professor Philip Poole. Marc Fox ii ACKNOWLEDGEMENTS Firstly, I would like to thank Philip Poole for supervising me throughout this project. His guidance over the years has proved fundamental to this research. Thanks too should go to my colleagues in Lab 160. James White for assistance with radioactive assays, Tim Mauchline for development of colony PCR, Karunakaran for the creation of vectors, Alexandré Bourdes for aid given with plants, Laura Fooks, Alex Pudney and Elham Moslehi-Mohebi for help with protein work and Arthur Hosie, Mary Leonard, Michelle Barr, Alison East, Jon Seaman and Emma Lodwig for their assistance in general. Thanks are also due to the staff of Central Science Services, AMS, especially Jane Clarke for the thousands of plates she prepared for me. I would also like to thank the University of Reading and the BBSRC for awarding me the funding to carry out this research. Thanks to the other labs I visited whilst carrying out this project, whose staff were both welcoming and helpful. Thanks to Alan Williams, Martin Krehenbrink and Allan Downie from the John Innes Centre, Norwich, for allowing me use of their Tn5 library; and to Laurence Dupont, Geneviève Alloing and Daniel LeRudulier from INRA-CNRS, Université de Nice Sophia-Antipolis, for allowing me to carry out proline betaine transport assays with them. Finally, a big thank-you to my parents and my sister Sarah, for their continued support throughout all my academic studies and also to John, Emma, Jenny and Rach for helping me get through this. iii ABSTRACT A previously created promoter probe library of Rhizobium leguminosarum 3841, LB3, was investigated to identify genes that are induced under stressful conditions. Each bacterium in the library contains a plasmid with a random chromosomal insert, upstream of a promoterless gfpUV reporter. If the insert contains a promoter that responds to a stress it will activate production of green fluorescent protein (GFP) and colonies will fluoresce bright green when examined under UV light. Over 30,000 colonies were screened on various media designed to reproduce hyper- osmotic stress, acidic stress and metal toxicity and 32 were induced. The release of the preliminary genome of 3841 allowed the genes, or operons, associated with each of the isolated stress-induced fusions from LB3 to be identified. Mutations were made in ten of the genes selected from LB3 that are upregulated by hyper-osmosis. The mutants were then tested to see how they would grow in standard and stressed conditions, and if the way which they interacted with pea plants was altered. This led to the discovery of a two-component response regulator system (RL1156 and RL1157) responsible for controlling the transcription of RL1155 in response to low pH and hyper- osmosis. One of the genes isolated from LB3 was upregulated by hyper-osmosis and is part of an operon for an ABC transporter that shares sequence identity to the well characterised glycine betaine transporter (ProU). This led to the identification of five other ABC systems that shared a significantly similar sequence identity to this transporter. One of these transporters (termed QAT1 in this work) appears to be the homologue of the Cho system in S. meliloti as it is induced by choline and is responsible for its uptake. Studies also demonstrated that hyper-osmosis temporarily inactivates solute uptake via ABC transporters (but not secondary permeases). iv LIST OF ABBREVIATIONS USED aa Amino acids ABC ATP-binding cassette AIB 2-Amino-isobutyric acid ALA δ-Aminolevulinic acid AlCl3 Aluminium chloride AMA Acid minimal agar Amp Ampicillin AMS Acid minimal salts ASP Acid shock protein ATP Adenosine triphosphate BAP Bacterial alkaline phosphatase bp Base pair cfu Colony forming units CSP Cold shock protein CuCl2 Copper chloride DFI Differential fluorescence induction DNA Deoxyribonucleic acid dNTP 2’-deoxynucleoside 5’-triphosphate EDTA Ethylenediaminetetraacetic acid EPS Exopolysaccharide et al. et alii Fix Fixation GABA γ-amino-n-butryic Acid Gen Gentamycin GFP Green fluorescent protein glc Glucose GDW Glass distilled water H2O2 Hydrogen peroxide HSP Heat shock protein IMP Integral membrane permease IPTG Isopropyl β-D-thiogalactoside IS50 Insertion sequence 50 Kan Kanamycin v Kb(p) Kilobase (pairs) KCl Potassium chloride LA Luria-Bertani agar LB Luria-Bertani broth LPS Lipopolysaccharide MES 2-Morpholinoethanesulfonic acid MFS Major facilitator superfamily MgCl2 Magnesium chloride MgSO4 Magnesium sulphate MGT Mean generation time MIC Minimal induction concentration MOPS 3-[N-morpholino]propanesulfonic acid N-free Nitrogen free NaCl Sodium chloride Nal Naladixic acid Neo Neomycin nH2O Nanopure water NH4 Ammonium Nod Nodulation Nys Nystatin OD Optical density OEP Outer membrane efflux protein ORF Open reading frame PCR Polymerase chain reaction PEG Polyethylene-glycerol PHB Polyhydroxybutyrate QAC Quaternary amine compound QAT Quaternary amine transporter r Resistant RBS Ribosome binding site RMS Rhizobium minimal salts rpm Revolutions per minute s Sensitive SBP Solute binding protein Spc Spectinomycin vi Str Streptomycin TAE Tris acetate EDTA TCA Tricarboxylic acid Tet Tetracyclin Tn5 Kanamycin/Neomycin resistant transposon TY Tryptone-Yeast media UV Ultraviolet VS Vincent’s sucrose wt Wild-type X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactoside X-Glc-A 5-bromo-4-chloro-3-indolyl-β-D-glucuronide ZnCl2 Zinc chloride vii CONTENTS 1. INTRODUCTION 1 1.1. Rhizobium 2 1.1.1. Taxonomy 2 1.1.2. Symbiosis 5 1.1.2.1. The nod Genes 5 1.1.2.2. Nodule Formation 6 1.1.2.3. Nitrogen Fixation 7 1.2. Stress Response 9 1.2.1. What is a Stress Response? 9 1.2.2. Examples of Stress Response in Rhizobium 10 1.2.2.1. Osmotic Stress 10 1.2.2.2. pH Stress 17 1.2.2.3. Oxygen/Oxidative Stress 20 1.2.2.4. Metal Stress 21 1.2.2.5. Temperature Stress 22 1.2.2.6. Starvation Stress 24 1.3. Research Objectives 26 2. MATERIALS & METHODS 27 2.1. List of Strains 28 2.2. List of Plasmids/Cosmids 32 2.3. Primers Used 36 2.4. Media & Growth Conditions Used 43 2.5. Antibiotics Used 44 2.6. Molecular Techniques 44 2.6.1. DNA Isolation 44 2.6.2. Agarose Gel Electrophoresis, Staining and Extraction 44 2.6.3. DNA Digests 45 2.6.4. Ligation 45 2.6.5. Transformation 45 2.6.6. Polymerase Chain Reaction (PCR) 45 2.6.7. Enzyme/Nucleotide Removal 46 2.6.8. DNA Purification 46 viii 2.6.9. DNA Sequencing 46 2.7. Conjugation 47 2.8. Mutagenesis 48 2.8.1. Tn5 Mutagenesis 48 2.8.2. pK19mob Mutagenesis 48 2.9. Transduction 48 2.9.1. Phage Propagation 48 2.9.2. Non-UV Transduction 49 2.10. GFP-UV Quantification 49 2.11. Plant Experiments 50 2.12. Transport Assays 50 2.13. Protein Assays 51 2.13.1. Periplasmic Fraction Isolation 51 2.13.2. SDS-PAGE 51 3. IDENTIFICATION OF KEY STRESS CONDITIONS & STRESS INDUCED FUSIONS 53 3.1. Introduction 54 3.2. Results 57 3.2.1. Minimum Induction Concentrations (MICs) 57 3.2.2. Mass Screenings 58 3.2.3. Cross Induction of Stress-Induced Fusions in R. leguminosarum 60 3.2.4. Further Cross Induction 61 3.3. Discussion 66 3.3.1. Initial Screens 66 3.3.2. Cross Induction Screens 67 4. CHARACTERISATION OF STRESS INDUCED FUSIONS 69 4.1. Introduction 70 4.2. Results 71 4.2.1. Sequencing Fusions 71 4.2.2. Analysing Sequence Data 72 4.2.3. Quantifying GFP Induction in AMS Cultures 74 4.2.4. Overall Results 75 4.2.4.1. pRU843/RU1507 76 4.2.4.2. pRU844/RU1508 79 ix 4.2.4.3. pRU845/RU1509 82 4.2.4.4. pRU846/RU1510 87 4.2.4.5. pRU848/RU1512 89 4.2.4.6. pRU849/RU1513 92 4.2.4.7. pRU850/RU1514 95 4.2.4.8. pRU853/RU1517 98 4.2.4.9. pRU854/RU1518 102 4.2.4.10. pRU855/RU1519 104 4.2.4.11. pRU857/RU1521 108 4.2.4.12. pRU858/RU1522 112 4.2.4.13. pRU859/RU1506 113 4.2.4.14. pRU861/RU1523 116 4.2.4.15. pRU862/RU1524 119 4.2.4.16. pRU863/RU1525 121 4.2.4.17. pRU865/RU1527 125 4.2.4.18. pRU866/RU1528 128 4.2.4.19. pRU867/RU1529 131 4.2.4.20. pRU868/RU1530 134 4.2.4.21. pRU869/RU1531 137 4.2.4.22. pRU870/RU1532 140 4.2.4.23. pRU871/RU1533 143 4.2.4.24. pRU872/RU1534 146 4.2.4.25. Summary 148 4.3. Discussion 150 5. ISOLATION AND CHARACTERISATION OF MUTATIONS IN STRESS-INDUCED GENES 154 5.1. Introduction 155 5.2. Results 156 5.2.1. Identification of Stress Regulation Pathways 156 5.2.2. Generation of Specific Mutants 156 5.2.3. Hyper-Osmotic MICs 163 5.2.4. Mutant Growth Rates and in Planta Phenotypes 166 5.2.5.