Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2015 Iron transport strategies of the genus Burkholderia Mathew, Anugraha Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-113412 Dissertation Published Version Originally published at: Mathew, Anugraha. Iron transport strategies of the genus Burkholderia. 2015, University of Zurich, Faculty of Science. Iron transport strategies of the genus Burkholderia Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Anugraha Mathew aus Indien Promotionskomitee Prof. Dr. Leo Eberl (Vorsitz) Prof. Dr. Jakob Pernthaler Dr. Aurelien carlier Zürich, 2015 2 Table of Contents Summary .............................................................................................................. 7 Zusammenfassung ................................................................................................ 9 Abbreviations ..................................................................................................... 11 Chapter 1: Introduction ....................................................................................... 14 1.1.Role and properties of iron in bacteria ...................................................................... 14 1.2.Iron transport mechanisms in bacteria ...................................................................... 14 1.2.1 Iron chelation by siderophores ........................................................................... 15 1.2.2 Ferric reduction and transport of ferrous iron..................................................... 16 1.2.3 ATP-binding Cassette (ABC) type metal transporters .......................................... 17 1.3 Iron transport systems in different environments ...................................................... 17 1.3.1 Iron acquisition by pathogens ............................................................................ 17 1.3.2 Iron acquisition by plant-associated as well as soil microbes ............................... 19 1.4 The genus Burkholderia ............................................................................................ 19 1.4.1 The Bcc as human opportunistic pathogens ........................................................ 22 1.4.2 Iron transport systems in Burkholderia............................................................... 25 1.5 Preliminary results leading to this study .................................................................... 26 Chapter 2. Objective of the study ........................................................................ 36 Chapter 3: Results I. A novel siderophore-independent strategy of iron uptake in the genus Burkholderia ............................................................................................... 38 3.1 Abstract ................................................................................................................... 39 3.2 Introduction ............................................................................................................. 39 3.3 Results ..................................................................................................................... 41 3.3.1 Both pyochelin and ornibactin are dispensible for the growth of B. cenocepacia H111 .......................................................................................................................... 41 3.3.2 A novel iron uptake system in Burkholderia ....................................................... 43 3.3.3 A siderophore null mutant of H111 depends on the FtrBccABCD system to grow in iron limited conditions ............................................................................................... 44 3.3.4 FtrBccABCD is not required for uptake of ferrous iron in Bcc H111 ........................ 45 3.3.5 FtrBccABCD is not involved in the uptake of complex iron substrates such as, hemin and lactoferrin ........................................................................................................... 46 3.3.6 FtrBccABCD is a high affinity iron transporter ....................................................... 48 3.3.7 Heterologous expression of ftrBccABCD rescues the growth of an E. coli strain lacking all known high affinity iron uptake systems ..................................................... 49 3.3.8 All four components of FtrBccABCD are essential for iron uptake.......................... 50 3.3.9 Transcription of ftrBccABCD is regulated by iron limitation, not by pH .................. 52 3.3.10 The role of FtrBccABCD for virulence in Galleria mellonella infection model ........ 53 3.3.11 Distribution of FtrBccABCD in Burkholderia species ............................................ 53 3.4 Discussion ................................................................................................................ 56 3.5 Materials and Methods ............................................................................................ 60 3.6 Unpublished results .................................................................................................. 85 3 Chapter 4. Results II: Ornibactin production increases the pathogenic potential of the environmental isolate, Burkholderia sacchari ................................................ 96 4.1 Introduction ............................................................................................................. 96 4.2 Results ..................................................................................................................... 98 4.2.1 Construction of transgenic B. tuberum and B. sacchari strains that produce ornibactin .................................................................................................................. 98 4.2.2 Production of siderophores renders B. sacchari pathogenic in a G. mellonella infection model........................................................................................................ 104 4.2.3 B. sacchari producing ornibactin can grow inside G. mellonella ......................... 106 4.2.4 Ornibactin does not contribute towards pathogenicity in C. elegans ................. 107 4.3 Discussion .............................................................................................................. 108 4.4 Materials and Methods .......................................................................................... 111 Chapter 5: Results III. Role of the Ftr system and siderophores in different habitats of Burkholderia .................................................................................................. 122 5.1 Introduction ........................................................................................................... 122 5.2 Results ................................................................................................................... 124 5.2.1 The Ftr system is unnecessary for nodulation and symbiosis of Burkholderia with legumes ................................................................................................................... 124 5.2.2 Ornibactin production does not increase the efficiency of nodulation of legumes by B. tuberum .......................................................................................................... 126 5.2.3 Ornibactin provides a competitive advantage for nodule occupancy ................. 127 5.2.4 The Ftr system is important for growth of Burkholderia in a soil microcosm ...... 128 5.3 Discussion .............................................................................................................. 130 5.4 Materials and methods ........................................................................................... 133 Chapter 6. Results IV: The role of Burkholderia siderophores in metal homeostasis .......................................................................................................................... 141 6.1 Introduction ........................................................................................................... 141 6.2 Results ................................................................................................................... 143 6.2.1 H111 producing siderophores can tolerate toxic metals better than non-producers. ................................................................................................................................ 143 6.2.2 The Toxic effect of metals can be relieved by the exogenous addition of ornibactin in Burkholderia strains lacking siderophores ............................................................. 146 6.2.3 Copper and aluminium induce siderophore production .................................... 148 6.3 Discussion .............................................................................................................. 150 6.4 Material and methods ............................................................................................ 153 Chapter 7. Discussion and outlook ..................................................................... 157 7. 1 Discussion ............................................................................................................. 157 7.2 Outlook .................................................................................................................. 158 Chapter 8. Material and methods