LIFE AT THE LIMITS: DIVERSITY, PHYSIOLOGY AND BIOENERGETICS OF HALOALKALITHERMOPHILES by KAREN JEAN BOWERS (Under the Direction of Juergen Wiegel) ABSTRACT Haloalkalithermophiles are poly-extremophiles adapted to grow at high salt concentrations, alkaline pH values and temperatures greater than 50ºC. Halophilic alkalithermophiles are of interest from physiological perspectives as they combine unique adaptive mechanisms and cellular features that enable them to grow under extreme conditions. Water and sediment samples from the lakes of the Wadi An Natrun, Egypt and from Lake Magadi, Kenya were investigated for the presence of novel haloalkalithermophiles. These athalassohaline lakes are noted for their high temperature, alkaline pH and high Na+ concentrations. Two novel bacterial species, Natranaerobius jonesii and Natranaerobius grantii, and one novel archaeal species, Natronolimnobius aegyptiacus, were isolated and characterized. Furthermore, the adaptive mechanisms and bioenergetic properties of the species belonging to the order Natranaerobiales were investigated. Collectively, these microorganisms display a ΔpH homeostasis, rather than an intracellular pH homeostasis; a growth requirement for Cl-; and are moderately UV resistant. INDEX WORDS: Halophile, Alkaliphile, Thermophile, Extremophile, Alkalithermophile, Haloalkalithermophile, Natranaerobiales, Natranaerobius jonesii, Natranaerobius grantii, Natronolimnobius aegyptiacus, Wadi An Natrun, Kenya Rift Valley, Adaptive mechanisms, Intracellular pH, Proton motive force, Membrane potential LIFE AT THE LIMITS: DIVERSITY, PHYSIOLOGY AND BIOENERGETICS OF HALOALKALITHERMOPHILES by KAREN J. BOWERS B.A., University of Georgia, 2003 M.Ed., Troy University, 2008 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2010 © 2010 Karen Jean Bowers All Rights Reserved LIFE AT THE LIMITS: DIVERSITY, PHYSIOLOGY AND BIOENERGETICS OF HALOALKALITHERMOPHILES by KAREN J. BOWERS Major Professor: Juergen Wiegel Committee: Lawrence J. Shimkets William B. Whitman Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2010 DEDICATION To my mother, who always said I could. iv ACKNOWLEDGEMENTS There are numerous people who deserve thanks for making this thesis possible, whether by providing academic advice and support, personal encouragement, a willing ear or a swift kick, when needed. First and foremost, I need to thank my advisor, Juergen Wiegel, for all of his support and guidance. I would have been hard-pressed to find a better mentor. The assistance of my committee members, Larry Shimkets and Barny Whitman has also been extremely helpful and is most appreciated. I also owe an enormous debt of gratitude to my former labmate, Noha Mesbah, who helped me in more ways than I can ever express. My labmates Isaac Wagner and Elizabeth Burgess, along with undergraduate lab members Jacob Gilleland and Litty Varghese also deserve thanks for assistance with keeping my perspective, sense of humor, with lab work and for a fresh eye when needed. Other friends at the University of Georgia, Dana Cook, Noreen Lyell, Chandra Carpenter, Dawn Adin and John Buchner, Lyla Lipscomb, and Dave Samuels have been great sounding boards and, at times, comic relief. I also cannot neglect to thank Wendy Dustman, who seems to be everything to everyone and has helped me in innumerous ways, large and small. My ―outside‖ friends have been as much a part of this effort as my academic colleagues. I appreciate all of the reminders that a real-world exists outside the lab and not being told I am crazy more often than was absolutely necessary. Finally, I have to thank my family, particularly my parents and brother, for all of their support and encouragement. Without them, this pursuit would have been much more difficult. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW .....................................................1 Biodiversity of poly-extremophilic Bacteria: Does combining the extremes of high salt, alkaline pH and elevated temperature approach a physico- chemical boundary for life? .................................................................................1 Temperature and pH optima of extremely halophilic Archaea: a mini-review ......13 2 NATRONOLIMNOBIUS AEGYPTIACUS SP. NOV., AN AEROBIC EXTREMELY HALOPHILIC ALKALITHERMOPHILIC ARCHAEON ISOLATED FROM THE ATHALASSOHALINE WADI AN NATRUN, EGYPT ....................................................................................................................42 3 NATRANAEROBIUS JONESII SP. NOV. AND NATRANAEROBIUS GRANTII SP. NOV., TWO ANAEROBIC HALOPHILIC ALKALITHERMOPHILES ISOLATED FROM THE KENYAN-TANZANIAN RIFT ....................................54 4 ADAPTIVE MECHANISMS AND INTRACELLULAR PH REGULATION IN NATRANAEROBIUS SPECIES, ANAEROBIC, HALOALKALITHERMOPHILIC BACTERIA ......................................................62 5 CONCLUSIONS..........................................................................................................96 vi APPENDIX A CALCULATION OF BIOENERGETIC PARAMETERS .........................................98 vii LIST OF TABLES Page Table 1.1: Growth characteristics of extremophiles ......................................................................34 Table 1.2: [Na+], pH and temperature optima and ranges for extremely halophilic Bacteria .......35 Table 1.3: [Na+], pH and temperature optima and ranges for extremely halophilic Archaea .......38 Table 2.1: Differential characteristics of strain JW/NM-HA 15T and closely related strains........53 Table 3.1: Substrate utilization profile of strains JW/NM-KB 43T and JW/NM-KB 411T ...........60 Table 3.2: Differential characteristics of strain JW/NM-KB 43T, JW/NM-KB 411T and closely related strains ....................................................................................................61 Table 4.1: Changes in intracellular [K+] and [Na+] under varying conditions ..............................94 Table 4.2: Survival of Natranerobius thermophilus and Natronovirga wadinatrunensis after UV radiation exposure...................................................................................................95 viii LIST OF FIGURES Page Figure 1.1: Correlation of [Na+] optimum and pH or temperature optima of extreme halophiles.......................................................................................................................28 Figure 1.2: Clustering of poly-extremophiles relative to other extreme halophiles ......................30 Figure 1.3: Correlation of [Na+] optimum and pH or temperature optima of extremely halophilic Archaea ........................................................................................................31 Figure 1.4: Correlation of [Na+] optimum and pH or temperature optima of extremely halophilic Archaea ........................................................................................................33 Figure 2.1: Light microscopic image of Strain JW/NM-HA 15T ..................................................51 Figure 2.2: Phylogenetic relationship of strains to closely related microorganisms .....................52 Figure 3.1: Light microscopic images of strains JW/NM-KB 43T and JW/NM-KB 411T ............68 Figure 3.2: Phylogenetic relationship of strains JW/NM-KB 43T and JW/NM-KB 411T to closely related microorganisms .....................................................................................69 Figure 4.1: Bioenergetic properties of N. jonesii and N. grantii ....................................................91 Figure 4.2: Effect of extracellular pH on membrane potential and ΔpH .......................................92 Figure 4.3: γ-radiation resistance of Natranaerobius thermophilus ..............................................93 ix CHAPTER ONE INTRODUCTION AND REVIEW OF THE LITERATURE This introduction describes the diversity of extremely halophilic Bacteria and Archaea, according to pH and temperature optima and specifically discusses the physiological properties of poly-extremophiles, e.g., haloalkalithermophiles. Figures 1.1a and b and 1.3a and b illustrate the frequency of pH and temperature optima amongst extremely halophilic Bacteria and Archaea, and show that most extremely halophilic microorganisms cluster at near-neutral pH and mesophilic temperature optima. Figures 1.2 and 1.4 show the relationship of poly-extremophilic Bacteria and Archaea, respectively, with relation to other extremely halophilic microorganisms. Throughout this discussion, the uniqueness of these poly-extremophiles is highlighted and sets the stage for the work discussed in Chapters 2-4. Finally, the main objectives of this work are outlined in the final section. ______________ Adapted from: Bowers, K.J., Mesbah, N.M, & Wiegel, J. (2009). Biodiversity of poly-extremophilic Bacteria: Does combining the extremes of high salt, alkaline pH and elevated temperature approach a physic-chemical boundary for life? Saline Systems. 5, 9 (23 November 2009). Bowers,