Copyright by Nicola Mary Lisa Davies 2006 The Dissertation Committee for Nicola Mary Lisa Davies Certifies that this is the approved version of the following dissertation: Iron Acquisition by Shigella dysenteriae and Shigella flexneri Committee: Shelley M. Payne, Supervisor Charles F. Earhart Richard J. Meyer Ian J. Molineux Martin Poenie Iron Acquisition by Shigella dysenteriae and Shigella flexneri by Nicola Mary Lisa Davies, B.Sc.H. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August 2006 Dedication To Dad and Mark Acknowledgements I would like to thank Shelley Payne for her patience, support and guidance over the last five years. I would also like to acknowledge the support and assistance of all of the members of the Payne lab. I am especially grateful to Erin Murphy and Alexandra Mey for many scientific discussions and excellent suggestions, and to Keren Hilgendorf for technical assistance. v Iron Acquisition by Shigella dysenteriae and Shigella flexneri Publication No._____________ Nicola Mary Lisa Davies, PhD The University of Texas at Austin, 2006 Supervisor: Shelley M. Payne Shigella dysenteriae and Shigella flexneri are Gram-negative facultative intracellular pathogens that cause bacillary dysentery. Iron is an essential micronutrient for these pathogens and they have evolved several systems to obtain iron. The Feo and Sit systems are involved in acquisition of ferrous iron and the TonB-dependant systems transport ferric iron and heme. In S. flexneri, a strain with a defect in the Sit system exhibited decreased fitness in the intracellular environment compared to the wild type. Although the TonB, Sit and Feo iron transport systems are present in both S. dysenteriae and S. flexneri, S. dysenteriae strains with defects in iron transport exhibited different phenotypes than similar mutants in S. flexneri. In S. dysenteriae, the TonB-dependent iron transport systems were important for growth in low-iron environments, while the Feo system was important for growth when iron was abundant. The Feo system was the only system found to be important for competition with the wild type strain in vitro or in the intracellular environment. An S. dysenteriae strain with a mutation in feoB formed vi smaller colonies than the wild type and overproduced enterobactin when grown in media containing supplemental iron, but exhibited no defects when grown in low-iron conditions. Despite these phenotypes, when grown in media containing abundant iron the feoB mutant did not appear to be starving for iron, indicating that the overproduction of enterobactin is not in response to iron starvation and suggesting that the Feo system plays a role in the regulation of enterobactin biosynthesis in S. dysenteriae. An S. dysenteriae mutant, defective in all three of the known iron transport systems, was able to grow well in media supplemented with iron, indicating the existence of an additional iron transport system. This system appears to transport free iron and is able to support growth to wild type levels in medium containing only 1 µM FeSO4, suggesting that the system has a high affinity for iron. Taken together, the results of this study indicate that several redundant iron transport systems are employed by S. dysenteriae, highlighting the importance of iron acquisition to the survival and pathogenesis of this organism. vii Table of Contents List of Tables .................................................................................................................... xii List of Figures.................................................................................................................. xiii I. INTRODUCTION................................................................................................................1 A. Pathogenesis of Shigella species..............................................................................1 B. Differences Between S. flexneri and S. dysenteriae ................................................5 C. Iron Acquisition by S. dysenteriae and S. flexneri...................................................7 1. TonB-Dependent Iron Uptake............................................................................9 2. The Feo Iron Transport System .......................................................................16 3. The Sit Iron and Manganese Transport System...............................................18 D. Regulation of Iron Acquisition by S. dysenteriae and S. flexneri..........................20 1. Regulation By Fur............................................................................................20 2. Positive Regulation in the Presence of Abundant Iron: The Role of the Small RNA RyhB ............................................................................................21 E. Purpose of This Study............................................................................................23 II. MATERIALS AND METHODS.........................................................................................24 A. Bacterial Strains and Plasmids...............................................................................24 B. Media and Growth Conditions...............................................................................24 C. Determination of Iron Concentration in Media .....................................................30 D. Recombinant DNA Methods..................................................................................31 E. Polymerase Chain Reaction ...................................................................................31 F. Oligonucleotides ....................................................................................................32 G. DNA Sequencing...................................................................................................32 H. Transformation of Bacterial Strains.......................................................................35 1. Transformation of CaCl2-Competent E. coli ...................................................35 viii 2. Electroporation of Shigella ..............................................................................36 3. Conjugation......................................................................................................36 I. Construction of Recombinant Plasmids.................................................................37 J. Construction of Mutant Strains..............................................................................38 1. NDS100............................................................................................................38 2. NDS115............................................................................................................39 3. NDS116 and NDS117......................................................................................40 4. NDS118, NDS119, NDS120 and NDS126......................................................40 5. NDS121............................................................................................................41 6. NDS122............................................................................................................41 7. NDS129............................................................................................................42 K. Tissue Culture........................................................................................................43 1. Invasion Assays...............................................................................................43 2. Plaque Assays..................................................................................................44 L. Siderophore Assays................................................................................................44 1. Arnow Assays..................................................................................................44 2. Hydroxamate Assays.......................................................................................45 M. Utilization of Iron, Siderophores and Heme: Bioassays.......................................45 N. Sensitivity to Metal Salts .......................................................................................46 O. β-Galactosidase Assays..........................................................................................46 P. Colony Size Assays................................................................................................47 Q. Southern Blot Analysis ..........................................................................................48 R. Frequency of Loss of the T7 Promoters and sit Locus ..........................................48 S. Viability in the Presence of T7 RNA Polymerase Expression ..............................49 T. Competition Assays...............................................................................................49 1. In Vitro Competition........................................................................................50 2. Intracellular Competition.................................................................................50 ix U. Microarray Analysis...............................................................................................51 III. THE SIT SYSTEM AND THE SHI-4 ISLAND IN S. FLEXNERI...........................................53 A. Introduction.........................................................................................................53