INDUCIBLE TOLERANCE AND SENSITIVITY TO STRESS RESPONSES IN Escherichia colt w i t h p a r t i c u l a r r e f e r e n c e t o c o p p e r AND pH This Thesis Is Submitted In Fulfilment Of The Requirement For The Degree Of Doctor Of Philosophy From The University Of London NOOR HANA HUSSAIN 1996 UCL Department of Biology DARWIN BUILDING UNIVERSITY c o l l e g e LONDON -1- ProQuest Number: 10106502 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10106502 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 -2- ACKNOWLEDGEMENTS I would like to thank my supervisor, Professor R.J Rowbury, for his guidance and help throughout the duration of my PhD studentship. I would also like to thank Margaret Goodson for her help and friei^hip. I also wish to thank the following individuals; Carol Allman, Zoulikha Lazim, Steve Athwal and Dominic Chaloner for their help and friendship; Chris Prodromou for letting me invade his desk while writing my thesis, John Hinks (who grins like the devil) for relieving my toner-induced stress, Bernard O’Hara, Tony Headley, Carlos Aguillar, Renos Savva and Malcolm Mac Arthur for putting up with my presence and for their help; Peh Eng Kok and Sushela Somanath for their constant concern and encouragement. A special thank you to Shradha Singh (’Beti’) for her tremendous help and for being such a wonderful friend. I am very grateful to my family; Mak, Yong, Yong ’A’, Abang Chik, Kak Nga, Abang G, Kak Nik, Abang Lee, Rahim and Muni for their love, support and encouraient throughout my studies. Lastly a very special thank you to Chanakya for his friendship, kindness, concern and for putting up /\j with my stress-induced phenotypic changes! Finally I am grateful to (j2 n t^ ^ sponsors, the Public Services Department of Malaysia and MARA Institute of Technology Malaysia for awarding me this PhD studentship. -3- ABSTRACT Stress responses to copper and alkali were studied in Escherichia coli. E. coli 1829 and its derivatives, were able to tolerate lethal doses of CuSO^ (58.92 pg/ml and 117.84 pg/ml) after pre-exposure to sublethal doses of CuSO^ (14.73 pg/ml and 29.46 pg/ml). The observed copper tolerance was due to a phenotypic change induced during the pre­ exposure period which depends on de novo protein synthesis. Cytoplasmic membrane proteins of molecular weights 26 and 24.5 kDas and outer membrane proteins of molecular weights 16.5, 18, 31.5 and 65 kDas were overexpressed in the copper-induced cells. The DNA from the copper-induced cells was also less damaged than that from the uninduced cells. Pre-exposure to 14.73 pg/ml CuSO^ also confers cross-protection to heat, acid, alkali and cadmium sulphate but not to hydrogen peroxide . Pre-exposure to mildly acidic pH which would normally induce acid tolerance was shown to also induce alkali sensitivity. When E.coli 1829 cells were transferred from pH^ 7.0 to pHq 5.5 for one hour they became alkali sensitive upon challenge with pHs 9.5 and 9.75 for 30 minutes. Substantial induction also occurs at pH 6.0 but there was less at pH^ 5.0 and practically none at pH^ 6.5. The response was triggered by cytoplasmic acidification by protons entering the cells possibly via OmpC, LamB, PhoE, NhaA and NhaB. The induction of alkali sensitivity also depends on de novo protein synthesis of components involved in alkali sensitization. Cytoplasmic membrane proteins of molecular weights 14 and 18 kDas were overexpressed in the pH 5.5 induced cells. The induction of the alkali sensitization components is not subject to catabolite repression nor affected by deletion in rpoS but appeared to be under the control of Fur, RelA, CysB and Lrp. Mutants with a deletion in tonB showed derepressed alkali sensitivity; the response being observed in pH 7.0 induced cells instead. The expression of the alkali sensitization components also appeared to be affected by changes in the DNA supercoiling and is influenced by HimA, HimD and H-NS. -4- CONTENTS PAGE DEDICATION 2 ACKNOWLEDGEMENTS 3 ABSTRACT 4 CONTENTS 5 LIST OF TABLES 15 LIST OF FIGURES 20 LIST OF ABBREVIATIONS 23 CHAPTER 1 INTRODUCTION 26 1.1 Prologue 26 1.2 Escherichia coli-'xis importance in public health 27 1.2.1 Intestinal infections and diseases in man and animalsdue to 27 Escherichia coli 1.2.2 Extraintestinal infections and diseases in man andanimals 28 due to Escherichia coli 1.3 The Cell Envelope Of Escherichia co/i-structures and functions 29 1.3.1 Outer membrane 29 1.3.1.1 Lipopolysaccharide 32 1.3.1.2 Phospholipids 34 1.3.1.3 Proteins 34 1.3.1.3.1 Lipoprotein 35 1.3.1.3.2 OmpA protein 35 1.3.1.3.3 Porins 36 1.3.1.3.4 Specific diffusion channels 39 1.3.1.3.5 High-affinity receptors-TonB-dependent transport 40 proteins 1.3.2 Peptidoglycan 44 1.3.3 Cytoplasmic membrane 44 1.3.3.1 Functions of the cytoplasmic membrane 47 1.3.4 Zones of adhesions 54 1.3.5 Periplasmic space 55 -5- 1.4 Biological Role of Copper in Escherichia coli 56 1.5 Toxicity of Copper in Escherichia coli 56 1.6 Sources Of Copper Stress Exposure 57 1.7 Metabolism of copper in Escherichia coli 57 1.8 Copper Resistance in Gram-Negative Bacteria 59 1.8.1 Mechanism of copper resistance in Escherichia coli 61 1.8.2 Mechanism of copper resistance in other Gram- negative 62 bacteria 1.9 pH Exposures 63 1.10 pH Homeostasis In Escherichia coZf-mechanism of pH stress 63 management 1.10.1 Components of pH homeostasis 64 1.10.1.1 Electrogenic KVATPase uptake system 65 1.10.1.2 Potassium/proton antiporters 65 1.10.1.3 Calcium/proton antiporters 6 6 1.10.1.4 Sodium/proton antiporters 6 6 1.10.1.4.1 Other physiological roles of sodium/proton 6 8 antiporters in Escherichia coli 1.10.1.4.2 Regulation of transcription of sodium/proton 6 8 antiporters 1.11 Inducible Stress Tolerance in Escherichia coli 69 1.11.1 Inducible Thermotolerance 69 1.11.2 Inducible toleranceto alkylating agents 70 1.11.3 Inducible toleranceto oxidativestress agents 70 1.11.4 Inducible toleranceto low pH 71 1.11.5 Inducible tolerance to high pH 72 1.12 Inducible Cross-Tolerance to Stress in Escherichia coli 72 1.13 Inducible Cross-Sensitivity to Stress in Escherichia coli 74 1.14 Stress Response Systems in Escherichia coli 74 1.14.1 Heat-shock response 77 1.14.1.1 Functions of heat-shock proteins 81 1.14.1.2 Inducers, sensors and signals for heat-shock regulon 84 -6- 1.14.2 Oxidative stress-response 8 8 1.14.2.1 Peroxide stress response-OxyR regulon 8 8 1.14.3 The stringent response 89 1.14.3.1 Other inducers of stringent response 91 1.15 Transmembrane Signal Transduction 91 1.15.1 Regulation of ompF and ompC expressions by EnvZ/OmpR 93 1.16 Global Regulatory Proteins 97 1.16.1 The sigma factor S (RpoS) 97 1.16.2 Ferric uptake regulatory (Fur) protein 101 1.16.2.1 Regulation of internal iron by fur 102 1.16.2.2 Involvement of fur in pH regulated genes 102 1.16.3 Leucine-responsive regulatory protein. 103 1.17 pH-Induced Stimulons 106 1.18 The Role of Antisense RNA in Posttranscriptional Regulation of 109 Gene Expression 1.18.1 Involvement of MicF antisense RNA in posttranscriptional 109 repression of ompF 1.19 The effects of glucose on gene expression in Escherichia coli 112 1.20 The Influence Of DNA Topology On Gene Expression. 113 1.20.1 Influence of supercoiling on gene expression in Escherichia coli 115 1.20.1.1 Influence of environmental factors on supercoiling 115 1.20.2 Influence of histone-like proteins on gene expression in 117 Escherichia coli 1.20.2.1 H-NS 118 1.20.2.1 Integration host factor (IHF) 121 1.21 Aims of Study 125 CHAPTER 2 MATERIALS AND METHODS 127 2.1 Bacterial Strains and Phages 127 2.2 Maintenance Of Bacterial Strains 127 2.3 Growth Media 127 2.4 Antibiotics 132 -7- 2.5 Chemicals 132 2.6 Growth Conditions 132 2.7 Measurement Op Optical Density 132 2.8 Measurement Of Viable Cells 133 2.9 External And Internal pH 133 2.10 Sensitivity To Copper(II) Sulphate 133 2.11 Recovery Of Copper-Treated Cells 134 2.12 Sensitivity To Copper(II) Sulphate 134 2.13 Sensitivity To Potassium Sulphate 134 2.14 Induction Of Tolerance To Copper(II) Sulphate 134 2.14.1 Pre-exposure (induction period) 134 2.14.2 Test of copper tolerance 135 2.15 Tests Of Copper Tolerance In Copper (Il)Chloride And Potassium 135 Sulphate Pre-Bxposed Cells 2.16 Determination Of Whether Copper-Resistant Mutants Arose During 135 Induction 2.17 Kinetics Of Induction Of Copper Tolerance 136 2.18 Kinetics Of Tolerance Induced In Copper-Habituated Cells 136 2.19 Induction To Copper Tolerance A t 30°C 136 2.20 Measurement Of Protein Synthesis Using Radiolabelled *"^C Uracil and 136 ^"^C phelnyalanine 2.20.1 Incorporation of isotopes into cells 136 2.20.2 Preparation of samples for radioassay 137 2.21 Induction Of Tolerance To Copper In The Presence Of Chloramphenicol 137 2.22 Cross-Tolerance Responses In Copper-Induced Cells 137 2.22.1 Heat 138 2.22.2 Hydrogen peroxide 138 2.22.3 Cadmium sulphate 138 2.22.4 Acid 138 2.22.5 Alkali 138 2.23 Induction Of p-Galactosidase In Copper-Habituated And Non-Habituated 139 Cells 2.24 Assay For p-Galactosidase Production 139 2.25 Plasmid DNA Isolation 140 2.25.1