IDENTIFICATION AND STRUCTURAL CHARACTERIZATION OF SIDEROPHORES PRODUCED BY HALOPHILIC AND ALKALIPHILIC BACTERIA By Abigail Marie Richards A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Department of Chemical Engineering AUGUST 2007 To the Faculty of Washington State University The members of the Committee appointed to examine the dissertation of ABIGAIL MARIE RICHARDS find it satisfactory and recommend that it be accepted. _________________________________________ Chair _________________________________________ _________________________________________ ii ACKNOWLEDGEMENT I would like to especially thank my committee members Dr. Brent Peyton, Dr. William Apel, Dr. James Petersen, and Dr. David Yonge and especially Dr. Richard Zollars who was willing to fill in at the last minute. It was through their encouragement that I decided to embark on my Ph. D. and I would like to commend them for their efforts in providing such an excellent, well balanced education. They have each been excellent mentors to me and I would like to thank them for all of their advice and input. I would also like to thank the Chemical Engineering Department at WSU for all of the support throughout both my undergraduate and graduate education. Special thanks to Jo Ann McCabe for helping me to remotely order supplies while I was at the CBE. I was able to perform the work for this project at a number of locations and would like to thank my gracious hosts at each of those sites: Special thanks to Dr. Antonio Ventosa for allowing me to work in his laboratory in Sevilla, Spain and to all of his students who were fantastic hosts and gave me my first taste of molecular biology. Thank you to those at the INL, in particular, Dr. William Apel, Dr. Vicki Thompson, Dr. Gary Groenewold and Dr. Garold Gresham for generously providing me with time on their mass spec. instrumentation at the Idaho National Laboratory throughout my Ph. D. work and thoughtful discussions about my results. I’d like to thank Dr. Anne Camper, my host at the Center for Biofilm Engineering and all of the people in her lab and throughout the CBE who made my experience at Montana State University so enjoyable including Mark Shirtliff, Mark Burr, Stewart Clark, Ben Klayman, Jennifer Faulwetter and Erin Field. Everyone at the CBE there immediately made me feel at home and I iii feel privileged to have had this experience. Dr. Robin Gerlach was infinitely helpful with the identification of all of my siderophores by allowing me to use his LC-MS system and the time that he spent helping me developing my LC-MS methods. John Newman, also at the Center for Biofilm Engineering, was also instrumental in methods development, particularly with HPLC. I’d like to thank my family for their support throughout all of my schooling, and my husband Lee for helping with the editing of this document and encouragement. This work was supported almost entirely by the Inland Northwest Research Alliance through a three year research grant as well as a two year individual fellowship which provided my support for the past two years. Through the INRA program I was able to continue my interdisciplinary education and this work could not have been accomplished without their generous financial support. The LC-MS instrument used for siderophore identification was provided by the Defense University Research Instrumentation Program (DURIP) Contract Number: W911NF0510255. iv IDENTIFICATION AND STRUCTURAL CHARACTERIZATION OF SIDEROPHORES PRODUCED BY HALOPHILIC AND ALKALIPHILIC BACTERIA Abstract By Abigail Marie Richards, Ph. D. Washington State University August 2007 Chair: Brent M. Peyton The first two chapters of the present dissertation focus on a description of two main topics. The first addresses siderophore production by plants and microbes as a means of acquiring ferric iron. Also described is the ability of siderophores to coordinate metals other than ferric iron, such as heavy metals and radionuclides, which potentially alters their speciation and mobility. The second chapter give an overview of the biology of halophilic and alkaliphilic microorganisms. The third part of this dissertation involves the identification and characterization of siderophores produced by the halophilic and alkaliphilic bacterium Halomonas campisalis. Several desferrioxamine siderophores including desferrioxamines G1, G1t, X3, X7, D2, and E were isolated from low-iron, culture supernatant and structurally characterized by ESI-MS and ESI- MS/MS. This work represents the first documentation of ferrioxamine production by a halo- alkaliphilic bacterium. v The fourth part of this dissertation is an assessment of siderophore production in a naturally saline and alkaline environment, the soda lake Soap Lake, located in eastern Washington State, USA. Eight siderophore producing halo-alkaliphiles were isolated from Soap Lake. Of these isolates, several were found to belong to the genus Halomonas. The isolate SL28, most closely related to Halomonas pantelleriense, was found to produce a new family of six of amphiphilic siderophores, named the sodachelins. The sodachelin siderophores are of particular interest because, when exposed to UV light, they facilitate a photolytic reduction of Fe(III) to Fe(II) along with a cleavage of the ligand located at the b-hydroxyaspartate residue. To my knowledge, this is the first characterization of amphiphilic siderophores produced by a bacterium from a soda lake environment that is capable of reducing Fe(III). The final portion of this dissertation contains suggestions for future work. Much of this work focuses on the identification of the siderophores produced by other halophilic and alkaliphilic isolates obtained in an earlier portion of this work. Siderophore production in halo- alkaliphiles (and extremophiles in general) is poorly characterized and some of the isolates appear to produce siderophores that may constitute new compounds. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS…………………………………………………………………… iii ABSTRACT………………………………………………………………………………….... iv LIST OF TABLES…………………………………………………………………………….. xii LIST OF FIGURES…………………………………………………………………………… xiii CHAPTER ONE: A BRIEF OVERVIEW OF MICROBIAL IRON TRANSPORT AND SIDEROPHORE PRODUCTION………………...……………………. 1 1.0 Introduction…………...………………………………………………………………….... 1 1.1 Microbial siderophore production ………………………………………………………… 4 1.2 Siderophore based iron acquisition………………………………………………………... 5 1.3 Iron, siderophores and disease causing microorganisms………………………………….. 8 1.4 General siderophore structural traits……………………………………………………... 10 1.5 Siderophore production by marine microorganisms………………………………………. 13 1.6 Siderophore affinity for divalent heavy metal cations and radionuclides…………………. 14 1.7 Concluding remarks………………………………………………………………………... 15 1.8 References………………………………………………………………………………….. 15 CHAPTER TWO: HALOPHILIC AND ALKALIPHILIC MICROORGANISMS………………………………………………………………………… 30 2.0 Extremophiles……………………………………………………………………………… 30 2.1.0 Halophiles……………………………………………………………………………….. 30 vii 2.1.1 Mechanisms of halotolerance……………………………………………………............ 32 2.1.2 Saline and hypersaline environments……………………………………………………. 32 2.2.0 Alkaliphiles………………………………………………………………………………. 34 2.2.1 Specific mechanisms of alkaline tolerance………………………………………………. 34 2.3.0 Soda lakes.……………………………………………………………………………….. 35 2.3.1 Soap Lake………………………………………………………………………………... 36 2.5 Concluding remarks………………………………………………………………………... 38 2.4 References………………………………………………………………………………….. 39 CHAPTER THREE: IDENTIFICATION AND CHARACTERIZATION OF A SUITE OF NATURAL FERRIOXAMINE SIDEROPHORES PRODUCED BY A HALO-ALKALIPHILC BACTERIUM…………………………………. 42 3.0 ABSTRACT………………………………………………………………………………... 43 3.1 INTRODUCTION………………………………………………………………………….. 44 3.2 MATERIALS AND METHODS…………………………………………………………….50 3.2.1 Growth conditions………………………………………………………………………… 50 3.2.2 Siderophore detection………………………………………………………………........ 50 3.2.3 Siderophore isolation……………………………………………………………………. 51 3.2.4 Siderophore characterization…………………………………………………………….. 52 3.3 RESULTS………………………………………………………………………………….. 52 3.4 DISCUSSION……………………………………………………………………………… 53 3.5 CONCLUSION……………………………………………………………………………. 58 3.6 ACKNOWLEDGEMENTS……………………………………………………………….. 58 3.7 LITERATURE CITED……………………………………………………………………. 59 viii CHAPTER FOUR: NOVEL AMPHIPHILIC SIDEROPHORES PRODUCED BY A BACTERIUM ISOLATED FROM A SODA LAKE………………………………….. 79 4.0 ABSTRACT………………………………………………………………………………… 80 4.1 INTRODUCTION………………………………………………………………………….. 81 4.2 MATERIALS AND METHODS………………………………………………………….. 85 4.2.1 Sample collection…………………………………………………………………………. 85 4.2.2 16S rRNA sequencing…………………………………………………………………..... 86 4.2.3 Growth medium……………………………………………………………………………86 4.2.3.1 Initial enrichment and growth medium…………………………………………………. 86 4.2.3.2 Growth medium for Halomonas strains………………………………………………… 87 4.2.3.3 Iron removal from complex media components………………………………………... 88 4.2.3.4 Iron limit medium for Halomonas strain SL28…………………………………………. 88 4.2.4 Siderophore detection and characterization………………………………………………. 88 4.2.5 Siderophore isolation………………………………………………………………………89 4.2.6 Structure determination…………………………………………………………………… 90 4.2.7 Photochemical experiments………………………………………………………………. 90 4.2.8 Fatty acid analysis………………………………………………………………………… 92 4.3 RESULTS……………………………………………………………………………………92 4.3.1 Isolate identification………………………………………………………………………. 92 4.3.2 Siderophore isolation………………………………………………………………………93 4.3.3 Structure