Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2017 Pyoverdine production and its social effects in natural communities of Pseudomonas bacteria Butaitė, Elena Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-147572 Dissertation Published Version Originally published at: Butaitė, Elena. Pyoverdine production and its social effects in natural communities of Pseudomonas bacteria. 2017, University of Zurich, Faculty of Science. Pyoverdine Production and its Social Effects in Natural Communities of Pseudomonas Bacteria Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Elena Butaitė aus Litauen Promotionskommission Prof. Dr. Rolf Kümmerli (Vorsitz) Prof. Dr. Jakob Pernthaler Prof. Dr. Gregory J. Velicer Zürich, 2017 Pyoverdine Production and its Social Effects in Natural Communities of Pseudomonas Bacteria Dissertation for Obtainment of a Scientific Doctorate (Dr. sc. nat.) submitted to the Faculty of Mathematics and Natural Sciences of the University of Zurich by Elena Butaitė from Lithuania Promotion Committee Prof. Dr. Rolf Kümmerli (Chairman) Prof. Dr. Jakob Pernthaler Prof. Dr. Gregory J. Velicer Zurich, 2017 Dedicated to my parents, especially to the memory of my wonderful mother, Olga Butas, who always believed in me, immensely loved and supported me, and whose selfless kindness and love to others will always inspire me. ‘Somewhere, something incredible is waiting to be known.’ ~ Sharon Begley (1977) Contents Summary ................................................................................................................................................. 1 Zusammenfassung ................................................................................................................................... 3 Chapter 1. Introduction............................................................................................................................ 5 1.1 The struggle for iron ...................................................................................................................... 6 1.1.1 Iron limitation ......................................................................................................................... 6 1.1.2 Siderophores ........................................................................................................................... 6 1.1.3 Siderophore-independent iron-acquisition systems ................................................................ 7 1.2 Cooperation and cheating in microbes .......................................................................................... 8 1.3 Siderophore non-producers in nature .......................................................................................... 10 1.4 Bacteria and siderophores studied in this thesis .......................................................................... 10 1.4.1 Fluorescent pseudomonads and pyoverdine ......................................................................... 10 1.4.2 Pyoverdine production and diversity .................................................................................... 11 1.5 Aims of the thesis ........................................................................................................................ 12 Chapter 2. Project 1 ............................................................................................................................... 14 2.1 Siderophore cheating and cheating resistance shape competition for iron in soil and freshwater Pseudomonas communities ............................................................................................................... 15 2.2 Supporting material ..................................................................................................................... 28 Chapter 3. Project 2 ............................................................................................................................... 40 3.1 Environmental determinants of pyoverdine production, exploitation and competition in natural Pseudomonas communities ............................................................................................................... 41 3.2 Supporting material ..................................................................................................................... 66 Chapter 4. Concluding discussion ......................................................................................................... 77 4.1 Overview ..................................................................................................................................... 78 4.2 What does the ability or inability to use heterologous siderophores mean?................................ 80 4.2.1 Why does siderophore exploitation not necessarily lead to cheating? ................................. 80 4.2.2 The different roles of heterologous siderophore receptors ................................................... 80 4.2.3 Changing pyoverdine structure as a mechanism to resist cheating ...................................... 81 4.3. Diversity of microbial communities ........................................................................................... 83 4.3.1 Factors promoting and stabilizing diversification of bacterial communities ........................ 83 4.3.2 ‘Key-lock’ diversity ............................................................................................................. 84 4.3.3 Multiplicity of public-good interactions in natural communities ......................................... 85 4.4. How can siderophore producers and our knowledge on social evolution of siderophore production be applied? ...................................................................................................................... 85 4.4.1 Fighting plant pathogens ...................................................................................................... 86 4.4.2 Heavy-metal detoxification .................................................................................................. 87 4.4.3 Fighting human pathogens ................................................................................................... 88 References ............................................................................................................................................. 90 Acknowledgments ............................................................................................................................... 100 Summary Iron is a key growth-limiting factor for most bacteria. It is required for various essential cellular processes, like DNA biosynthesis and respiration. However, in nature iron is generally limited. To overcome iron limitation, many bacteria secrete siderophores, iron-scavenging molecules that have a high affinity for ferric iron. These molecules bind iron from the surrounding environment and are taken up by cells via specific membrane-embedded receptors. Siderophores are not only needed to acquire iron but also serve in competition with others: the secreted siderophores can lock iron away from competitors with incompatible receptors. Moreover, siderophores can be shared among individuals with compatible receptors. While cooperative siderophore sharing might be a beneficial strategy, it can also select for siderophore-negative strains with one or many receptors. Such non- producers can act as cheaters exploiting siderophores of others, which benefits cheaters but reduces the fitness of producers. Siderophore production and siderophore-mediated interactions have been extensively studied in laboratory conditions. However, we know only little about the determinants of siderophore production and their sociality in the complex bacterial communities in nature. The aim of my thesis was to bridge this gap in knowledge. I carried out two main projects: (i) project 1 mainly focuses on siderophore cooperation and cheating, and (ii) project 2 focuses on the environmental determinants of siderophore production and siderophore-mediated interactions. For this, I first isolated 930 Pseudomonas strains from 48 different soil and pond communities. I was interested in their main, high-affinity siderophore pyoverdine, whose structure can vary between different pseudomonads. Pyoverdine production has been extensively studied in laboratory conditions (especially in the opportunistic human pathogen P. aeruginosa) and has become a model example of bacterial cooperation. In the first project, using 315 isolates, I focused on pyoverdine production to study its exploitability among community members, the fitness consequences of the ability or inability to exploit heterologous pyoverdines, and the genetic basis of the observed pyoverdine-mediated interactions. I found that pyoverdine non-producers occur in many soil and pond communities. Some non-producers could act as cheaters on the producers secreting a compatible pyoverdine. Conversely, non-producers could also be inhibited by structurally different pyoverdines from other community members. The results of this project suggest that in nature there is both selection for cheating and resistance against cheating, which could drive antagonistic co-evolution and diversification in bacterial communities. In the second project, using all 930 isolates, I studied