CALIFORNIA STATE UNIVERSITY SAN MARCOS THESIS SIGNATURE PAGE THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE MASTER OF SCIENCE IN BIOLOGICAL SCIENCES THESIS TITLE: The Isolation and Characterization ofa Novel Haloalkaliphilic Purple Sulfur Bacterium, Ectothiorhodospira monomense, sp. nov., from Mono Lake AUTHOR: Crista DiBernardo DATE OF SUCCESSFUL DEFENSE: May 25, 2001 THE THESIS HAS BEEN ACCEPTED BY THE THESIS COMMITTEE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOLOGICAL SCIENCES. Thomas Wahlund, Ph.D. ~idJ-fJ{.Wt<-~aY25'OI THESIS COMMITTEE CHAIR (TYPED) SIGNATURE DATE Betsy Read, Ed.D. ~a R~c~ May25,01 THESIS COMMITTEE MEMBER (TYPED) SI URE DATE Victoria Fabry, Ph.D. THESIS COMMITTEE MEMBER (TYPED) ii The Isolation and Characterization of a Novel Haloalkaliphilic Purple Sulfur Bacterium, Ectothiorhodospira monomense, sp. nov., from Mono Lake by Crista Danielle DiBernardo Bachelor of Science, Biological Sciences A thesis written in partial fulfillment of the requirements for Master of Science in Biological Sciences Department of Graduate Studies California State University San Marcos June 2001 iii ACKNOWLEDGMENTS Getting a Master's Degree has been an experience that I never would have made it through without the support and encouragement of family and friends. It is because of you that this thesis is finally published. Thank you, from the bottom of my heart! Dr. Tom Wahlund, thanks for taking on a graduate student, wholly uneducated in microbiology, and teaching me to love germs! Your support, feedback and high expectations have helped me to look back on the past few years with pride and accomplishment. Dr. Betsy Read, your encouragement and support from the moment I stepped foot on CSU San Marcos soil have meant more than you will ever know. Thank you for always being there with advice, moral support and pom-poms when needed. Dr. Vicki Fabry, you've always made me feel accomplished and worthy of any challenge, both scholastic and personal. Thank you for always believing in me. To Shannon and Alicia, who have easily spent hours in complete silence listening to my complaints, and commiserating with me even when I was at my most irrational. To Jason and Dande, for making life in Science Hall fun. To Jose, Mike and Kevin whose friendships remind me that there's more to life than biology. Thanks! Huge thanks to Jenni & Mario, for believing in me and giving me a place to call home. To my little brother Mario, thank you for always thinking I'm the smartest person in the world, true or not. iv Above all, I thank my parents for their support, love and belief that I could do anything. To my Dad; whose childhood dreams of being a scientist inspired my reality. And to my Mom; whose hugs & encouragement still make everything all right, even when you're 27. v ABSTRACT Life exists in a wide range of environments with prokaryotic organisms inhabiting those considered most hostile, including extremes of temperature, salinity, water activity, and pH. The microorganisms that inhabit these extreme environments, and in particular anoxygenic phototrophs that inhabit salt and soda lakes, are the focus of this thesis. These environments are typified by (1) high salinity, and the associated problem of a hypoosmotic external environment, and (2) an alkaline environment, where the cell's membrane potential is disrupted. In the present study, a novel purple sulfur bacterium of the genus Ectothiorhodospira was isolated from the highly saline (8%) highly alkaline (pH 8-10) Mono Lake in Mono County, CA. This isolate exhibited optimal growth under photoheterotrophic conditions at 1.4 M NaCl and pH 9.4, and possessed many metabolic properties unique among related organisms. The classification of the organism isolated in this study, Ectothiorhodospira strain MLS1, as a novel species, Ectothiorhodospira monomense (N.L. adj. pertaining to Mono Lake), is thus proposed. This thesis describes the isolation and characterization of this organism, and lays the foundation for future studies of its metabolism and genetics, including aspects of nitrogen and carbon fixation; as well as the synthesis, regulation and stability of cell wall and cell membrane components under highly saline and alkaline conditions. vi TABLE OF CONTENTS Thesis Signature Page Title Page ii Acknowledgements iii Abstract v Table of Contents vi List of Tables vii List of Figures viii Introduction 1 Materials and Methods 16 Results 23 Discussion 39 Literature Cited 49 Curriculum Vita 58 vii LIST OF TABLES Table 1: Absorption spectra of various bacteriochlorophyll 9 molecules in vivo Table 2: The uncorrected percent similarity of 16S rDNA sequences 27 of various Purple Sulfur Bacteria, reference species and strain MLS1 Table 3: Cell yields of Ectothiorhodospira strain MLS1 with organic 31 components photoassimilated as carbon source and electron donor Table 4: Comparison of morphological and physiological 41 characteristics of Ectothiorhodospira strain MLS1 to Ectothiorhodospiraceae type strains described in Bergey's Manual of Systematic Bacteriology as well as E. haioaikaliphila (BN 9903) Table 5: Photosynthetic electron donors and carbon sources used by 46 species of the family Ectothiorhodospiraceae viii LIST OF FIGURES Figure 1: Absorption spectrum of whole cell samples from the Mono 23 Lake Shore primary enrichment Figure 2: Agar streak plate of the MLS enrichment showing the 24 presence of the two distinct colony types Figure 3: Whole cell absorption spectra of the phototrophic 24 bacterium and the unpigmented contaminant Figure 4: Growth of strain MLS1 in DSIC-II media agar plate and 25 liquid culture Figure 5: PCR amplification of 16S rDNA from strain MLS1 26 Figure 6: Alignment of strain MLS1 partial 16S rDNA with 28 comparable regions from related organisms shown in Table 2 Figure 7: Phylogenetic tree of strain MLS1 from Mono Lake to other 29 members of the genera Ectothiorhodospira and Haiorhodospira Figure 8: Phase contrast micrograph of Ectothiorhodospira strain 29 MLS1 Figure 9: Absorption spectra of intact cells and methanol:acetone 30 extracts of Ectothiorhodospira strain MLS1 ix Figure 10A: Effect of NaCl concentration on the growth of 32 Ectothiorhodospira strain MLS1 Figure 10B: Photo showing the color and relative densities of 33 Ectothiorhodospira strain MLS1 cultures from the salinity optimization experiments shown in Figure 10A Figure 11A: Effect of pH on the growth of Ectothiorhodospira strain 34 MLS1 Figure 11 B: Photo showing the color and relative densities of 34 Ectothiorhodospira strain MLS1 cultures from the pH optimization experiments shown in Figure 11A Figure 12: Relationship between total cellular protein (f,lg/ml) and 35 absorbency of cultures at 600nm Figure 13: Comparison of total cell protein content of 36 Ectothiorhodospira strain MLS1 under various culture conditions Figure 14: Growth curve of Ectothiorhodospira strain MLS1 using 38 medium DSIC-II (9mM NH4Cl) and medium EM-II (9mM NH4Cl), and medium DSIC-II with glutamine (14mM) 1 INTRODUCTION Life in Extreme Environments: An Overview Life flourishes on Earth in an incredibly wide range of environments, from high-salt deserts to volcanoes to polar ice. Yet it was only about 30 years ago when scientists began to discover that microbial life was not restricted to a limited range of environments, defined by a relatively narrow range of environmental factors (temperature, pH, water availability, and nutrient source). It has clear that there is no environment on the planet that contains eukaryotic organisms but is devoid of bacteria. However, there are numerous bacterial habitats that are devoid of eukaryotic organisms. From a eukaryotic (human) perspective, these latter environments are classified as extreme. The noted microbiologist Thomas Brock defined an extreme environment as one in which the physical or nutritional conditions present deviate radically from those same conditions normally associated with the taxonomic group in question (7). This definition is unique in characterizing an extremophile, as it is dependent not solely upon the extreme nature of the environment. More specifically, it also allows for defining extreme as deviating from the normal conditions within which the relatives of an organism are normally found. These habitats include extremes of 0 temperature (e.g. > 80 ( to < O°C), pH (> 10 to < 0), salinity (3 - 15% NaCl), high pressure (400-1110 atm), low water activity (aw < 0.800) and/or combinations of these parameters (30, 42, 56, 59, 65, 73). Although these conditions are extreme from the perspective of most higher animals, they are usually absolutely essential for the survival of a given organism. For example, the hyperthermophilic Archaean Pyrodictium has an optimal growth temperature of 105°(, with a growth range between 82° 0 and 110 ( (68, 69). At temperatures lower than 70°(, metabolic activity ceases and the cells die due to the denaturation of critical metabolic 2 enzymes and the instability of the plasma membrane. A seemingly hospitable environment is actually extreme for Pyrodictium. The study of microbial life forms and the extreme environments in which they exist is providing new insights into how organisms have evolved and adapted to diverse environments. This knowledge will provide the basis to understand not only how life originated and evolved on Earth, but also how life may thrive on other planets. In addition, the development of new technologies and experimental approaches in microbiology has