CALIFORNIA STATE UNIVERSITY SAN MARCOS

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THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

MASTER OF SCIENCE

IN

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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 spawned a revolution that is opening up new opportunities for agriculture, medicine and industry. This revolution has enabled microbiologists during the past few years to identify over 20 new major evolutionary groups of microbial life - the diversity of which dwarfs Earth's "traditionally-discussed" biological diversity (plants, animals, and fungi). The ability to survey this microbial diversity in situ has shown that less than 0.1% of the organisms present in a given environment have ever been cultured in the laboratory (80). Thus, the 99.9% of microorganisms that remain uncultured provide a vast untapped reservoir of genetic and metabolic diversity, the harvesting of which will have far-reaching positive effects for society in areas such as enhanced food production, medicine (e.g. antibiotic discovery), bioremediation of waste materials, and agriculture. Bioremediation applications include the treatment of waste, the degradation of aromatic compounds, the removal of reduced sulfur compounds in gas streams, and the removal of chlorinated hydrocarbons from waste water (40).

Biotechnology companies are presently exploiting this untapped genetic potential both globally, and here in San Diego County; with research efforts directed toward the discovery of novel proteins for biotechnological applications. As an example, the enzyme, Taq polymerase, isolated from 3 the extremely thermophilic bacterium, Thermus aquaticus (7), is routinely employed in the Polymerase Chain Reaction (PCR) for molecular techniques such as DNA fingerprinting and RFLP analysis, with annual sales surpassing $80 million. Other extremophile products have now been introduced on the market, including an enzyme called cellulase 103, which is being used as a detergent additive that promises to make cotton clothes "look like new" through hundreds of washings. This enzyme is fully active at temperatures over 100°C and a pH of 10. The company producing cellulase, Genencor International, is looking at a potential $600 million market for detergent enzyme additives.

The light-driven process of CO2 fixation and its conversion into a myriad of larger organic compounds is a remarkably successful strategy employed by all plants and several bacteria capable of chemoautotrophic and/or photoautotrophic metabolism. This process has spawned a considerable research initiative by a number of biotechnology companies focusing on microorganisms that can perform unusual metabolisms. In particular, there is considerable interest in the scientific community for isolating anoxygenic photosynthetic bacteria from extreme environments and exploiting their remarkable metabolic versatility and autotrophic CO2 fixation abilities. For example, a photosynthetic bacterium capable of C02 fixation was recently genetically engineered to convert CO2 to ethanol (78). The premise of this investigation relied upon the fact that fully one-third of the biomass (corn starch) used for ethanol production in the u.s. annually is lost as C02 gas due to the inherent inefficiency of biological fermentation (8, 78). The ability to recycle this waste CO2 represents a potential annual savings of over a half billion dollars a year for the ethanol production industry. Because CO2 is a major waste product in many industrial processes, this 4 bacterium, and others like it could also be genetically engineered to convert CO2 to other chemicals or products of interest.

Alkaline detergent enzymes are another example of a commercially viable benefit of alkaliphile research (29, 72). Because of the alkaline nature of soap, only those enzymes that are naturally adapted to an alkaline environment can be used as additives in commercially available detergents. To date, cellulases, proteases, a-amylases, and debranching enzymes have succeeded in large-scale industrial production (38). In addition, the recent discovery of alkaliphilic xylanases may allow for better bleaching of kraft pulp in the pulp and paper industries. To date, the only isolated xylanases are active at acidic to neutral pH, while the paper bleaching process has inherently alkaline conditions (21).

Evolution of Primitive Metabolisms It has now well established that the early Earth atmosphere was anoxic (53). This was also a highly reducing environment consisting of methane

(CH 4), ammonia (NH 3), and water vapor, in addition to other gases such as carbon dioxide (C02), cyanide (HCN) and sulfide (H2S). Chemical reactions between these gases catalyzed by energy from ultraviolet radiation, lightning and geothermal activity produced a variety of organic molecules required for life, including amino acids and sugars. Under these conditions, the first metabolism must have been anaerobic and the source of carbon most likely these organic molecules from the environment. All that may have been required for a primitive energy-yielding metabolism and a primitive form of "life" was a lipid-type membrane that could sequester these chemical reactions, and then a means for transferring this information to another cell (i.e. some hereditary mechanism). The simplest metabolic system thus far proposed which accounts for these conditions is known as 5 the pyrite scenario (82). Briefly stated, the abundance of the reduced (ferrous) iron compounds (FeCD3 and FeS) present early in the Earth's history served as electron donors for the production of pyrite (FeS2). The presence of these ferrous iron compounds allowed the highly electronegative and abundant molecule, H2S (Eo' So IHS- = - 0.27 V) to serve as an electron acceptor for the oxidation of ferrous carbonate and ferrous sulfide (FeCD3 and FeS) to pyrite (FeS2) with a net release of free energy (ilGo' = -67kJ and -41.9 kJ/reaction, respectively). Each of these reactions could have provided more than enough energy for the synthesis of ATP from ADP and inorganic phosphate (Pi) (ilGO' = -31.7kJ). The energy for the condensation of ADP. and Pi could also have come from the separation of protons and electrons from H2 molecules with a primitive hydrogenase, and the subsequent establishment of a proton gradient through a primitive ATPase (6, 15, 82).

Whatever the origin of the first energy-yielding metabolisms, these protobionts soon became the first unicellular prokaryotic organisms. The first fossil evidence of these organisms can be dated at -3.6-3.8 x 109 years ago, less than a half billion years after the formation of the Earth (66). Although these primitive organisms probably survived employing a chemolithotrophic metabolism (inorganic chemical energy and electron source) as described above, they must have relied on the abundance of already synthesized organiC macromolecules for assimilation into cell membranes and primitive proteins. The complexity of even simple fermentations and autotrophic carbon fixation mechanisms required a much longer time period to evolve, with simple fermentation pathways the most likely to have occurred first, followed by the development of complex fermentation pathways. Arguably, the most important step in the evolution of autotrophy and photosynthesis must have been the synthesis of the 6 porphyrin ring (62). This molecule is the key component in the structure of cytochromes, chlorophylls and other tetrapyrroles, and was an absolutely essential step in the evolution of photosynthesis and electron transport. The next significant evolutionary leap was the development of a second photosynthetic reaction center with a redox potential sufficiently high enough to allow H20 to act as an electron donor. This event led to the oxygenation of the atmosphere, and subsequently the evolution of multicellularity (5).

Structure of the Photosynthetic Reaction Center All anoxygenic phototrophic bacteria perform photosynthesis in electron transport complexes consisting of a single photosystem (PS), unlike the two photosystems utilized by cyanobacteria and higher plants (5). Sequence data from the reaction center proteins of plants, cyanobacteria and anoxygenic phototrophs suggest that the photosystems present in higher plants are most likely derived from those of the anoxygenic phototrophs. Namely, PSII is derived from the purple bacteria and PSI from the green bacteria (9, 11, 24). These complexes are embedded in various membrane arrangements which are extensions of the cytoplasmic membrane and are characteristic for each taxonomic group of anoxygenic phototrophs. The photosynthetic reaction center in anoxygenic purple bacteria has been crystallized and its structure and electron transport mechanisms studied in detail. As such, it will be used as the general model in this discussion. The basic structure consists of four pigment-protein complexes: (1) reaction center, (2) light-harvesting I, (3) light-harvesting II, and (4) cytochrome bCl complex. The light-harvesting complexes (antenna complexes) possess two forms of Bchl a (B800-850 = LHI; B870 = LHII) which transfer the absorbed light energy to the reaction center (10, 11, 24, 48). Research data from nonsulfur purple bacteria indicate that the amount of light-harvesting Bchl 7 a per reaction center may be as high as 32:1 (10). The reaction center contains a pair of bacteriochlorophyll a molecules, called the special pair, which upon excitation from absorbed solar energy are transformed from a poor electron donor (Eo' = +0.5 V) to a good electron donor (Eo' = -1.0 V). This change in redox potential allows the Bchl a pair to reduce an acceptor molecule of very low redox potential (e.g. bacteriopheophytin, Eo' = -0.8 V). Reduction of this primary electron acceptor sets up a cascade of electron transport steps sufficient for the generation of a proton gradient and the subsequent synthesis of ATP via a membrane bound ATPase complex. The antennae light-harvesting complexes increase photosynthetic efficiency by increasing the rate of photon energy absorbed and transferring this energy to the reaction center (10, 24). Additional accessory pigment molecules called carotenoids absorb light energy outside the range of bacteriochlorophylls and can also transfer this energy to the reaction center. Their main function, however, is to serve as photoprotective agents that quench singlet oxygen generated from photooxidation reactions (19, 45) and they are in extremely close proximity to the bacteriochlorophyll molecules for this reason (19, 24).

The molecular genetics of anoxygenic photosynthesis is complex and is thought to be one of the most highly conserved genetic and physiological processes in phototrophic bacteria (83). Much of the knowledge gained about gene organization and regulation of anoxygenic photosynthesis comes from the genetically amenable genus Rhodobacter; especially R. sphaeroides and R. capsulatus (18, 45, 46). The photosynthetic gene cluster of both R. sphaeroides and R. capsulatus is located within a 45- to 50-kb region of the chromosome, and the operons are arranged into what is referred to as a superoperon (45, 47). This superoperonal organization allows for the coordinated regulation and expression of the numerous genes 8 required for anoxygenic photosynthesis at the transcriptionaL, transLationaL, and post-transLationaL levels (18, 45, 60). The genes within these operons are regulated by light quality and intensity, oxygen status and the redox state of the cell (4, 14, 16, 45, 46, 60, 83, 86).

Anoxygenic Photosynthesis Anoxygenic phototrophs are Gram negative phototrophic bacteria which inhabit aquatic environments with a high sulfur content; including stagnant water, salt lakes, estuaries and sulfur springs (49, 61). Within these organisms the first division to have evolved photosynthetic capabilities includes members of the genera Chloroflexus, Heliothrix, Herpetosiphon and Thermomicrobium (67). All prokaryotic photosynthesis, excluding that of cyanobacteria, involves the use of a chemical compound other than H20 as an electron donor and in turn liberates some other metabolite as a waste

product. Reducing power for fixation of CO2 in anoxygenic phototrophs must then come from some reduced substance in the environment. The most commonly utilized electron donors include hydrogen suLfide (H25), elemental sulfur (5°), and thiosulfate (520l-), all of which can be oxidized anaerobically in the light to sulfate (50i-) (24, 61). When H20 is not used as an electron donor no oxygen is liberated as a byproduct of the reaction, and thus photosynthesis is termed anoxygenic (33, 49).

Photosynthetic growth in anoxygenic phototrophs requires strict anaerobic conditions and electron donors with a higher redox potential than water to generate the reducing power necessary for CO2 assimilation (33). In addition, photosynthetic growth is most often limited to anoxic environments as the synthesis of bacteriochlorophylls and carotenoids is strongly repressed by 02 in most anoxygenic phototrophs (33, 51, 83). Interesting exceptions include R. centenum which is atypical in that oxygen 9 does not repress bacteriochlorophyll synthesis (83) and deep-sea aerobic anoxygenic phototrophs which utilize geothermal light for photosynthesis (26, 85).

Anoxygenic photosynthetic Bacteria are classified in the Order Rhodospirillaceae which is further divided into two suborders, the Green and Purple Bacteria, based upon both physiological and ecological differences. These anoxygenic photosynthesizers comprise six families (5, 11) and can be easily identified by the characteristic absorbance spectra of their photosynthetic pigments (Table 1):

Table 1: Absorption spectra of various bacteriochlorophyll molecules in vivo (33). Representative group Bchlo absorption maxima (nm) in living cells Purple Bacteria a 375, 590, 800-810, 830-890 Purple Bacteria b 400,605,835-850,1015-1035 Green sulfur Bacteria c 335,460, 745-760 Green Sulfur Bacteria d 325,450, 725-745 Green Nonsulfur Bacteria e 345,450-460,715-725 Heliobacteria g 370,419, 575, 670, 780-790 °bactenochlorophyll 1) Rhodospirillaceae (Nonsulfur Purple Bacteria) which can grow both phototrophically and heterotrophically and are the most nutritionally diverse of the anoxygenic bacteria; 2) Chromatiaceae (Purple Sulfur Bacteria) which utilize reduced sulfur compounds as electron donors for photosynthetic growth and deposit elemental sulfur internally; 3) Ectothiorhodospiraceae (Purple Sulfur Bacteria) which also utilize reduced sulfur compounds as electron donors for photosynthetic growth, but deposit elemental sulfur extracellularly; 4) Chlorobiaceae (Green Sulfur Bacteria) which utilize reduced sulfur compounds as electron donors for photosynthetic growth, are 10 obligately phototrophic and tolerate the highest levels of environmental sulfide of all anoxygenic bacteria; 5) Chloroflexaceae (Green Gliding Bacteria) which grow primarily photoheterotrophically, are morphologically distinct from other green bacteria and are characterized by their gliding motility; 6) Heliobacteriaceae which are unique among anoxygenic phototrophs in that they contain bacteriochlorophyll g while bacteriochlorophyll a is absent. These organisms are metabolically distinct from the other anoxygenic groups described above in that they appear to be incapable of autotrophic growth.

Each of these groups have unique metabolic and genetiC capabilities that can be exploited in preparing selective media for isolation of bacterial species from each group (1, 33, 51, 71, 79).

From an ecological perspective, an inverse relationship exists between the abundance of reduced sulfur compounds (H2S, S°, S20l") present in a given environment and light intensity. Sulfide levels are highest in the lower portions of the water column near the sediment where sulfate-reducing bacteria reduce sulfate (SOl") to H2S. H2S combines abiotically with 02 to form elemental sulfur (S°) and H20 resulting in an anoxic environment (27). These conditions create a vertical stratification (microbial mat) of anoxygenic phototrophic species based upon sulfide tolerance and the photosynthetic pigments possessed by a given organism (20). A three­ millimeter section of such a mat would find Nonsulfur Purple Bacteria occupying the top layer, followed by the Purple Sulfur Bacteria and finally Green Sulfur Bacteria (49, 63). 11 Purple Sulfur Bacteria Purple Sulfur (PS) Bacteria include members of the Ectothiorhodospiraceae and Chromatiaceae families. In PS Bacteria, the photosynthetic pigments are located in an extended system of intracytoplasmic membranes that are continuous with the cytoplasm (23, 24, 33). PS Bacteria are commonly found in salt water, hypersaline, alkaline and many haloalkaliphilic environments, including salt or soda lakes with reddish water (20, 33, 61). Representative species contain either Bacteriochlorophyll (Bchl) a or Bchl b, and the accessory carotenoid pigments of the spirilloxanthin, rhodopinal spheroidene or okenone series (33). The two groups of PS bacteria can be distinguished by the location of their deposited sulfur granules. Ectothiorhodospiraceae deposit sulfur granules externally while Chromatiaceae typically contain internal sulfur granules (33, 61). All members of the PS bacteria utilize the Calvin Cycle for CO2 fixation (52). While Ectothiorhodospiraceae grow photoheterotrophically (61), Chromatiaceae (having a limited ability to uptake organic compounds) grow primarily photoautotrophically (33). Purple Sulfur Bacteria are motile and use flagella to move throughout their aqueous environment. In addition, some species possess gas vesicles that allow vertical movement through the water column (33).

Halophilic and Alkaliphilic Adaptations Members of the Ectothiorhodospiraceae are capable of growth in extreme environments, with representative species commonly found in highly saline, extremely alkaline, and haloalkaline environments (32, 58). Because of the magnitude of molecular and morphological diversity within this family, a recent genus-level division was proposed. The family Ectothiorhodo­ spiraceae now includes the genus HaLorhodospira in addition to the described genus, Ectothiorhodospira (35). 12

In order to inhabit a saline environment, organisms must find a way of combating the unique situation of being in an environment where the solute concentration is higher than that of the cytoplasm. As in any environment, the cell must retain turgor pressure by remaining hyperosmotic to its surroundings. Prokaryotes have adapted to their high solute environment in one of two ways. One, the "salt in" strategy, is used mainly by Archaeans who have adapted all of their intracellular enzymes and components to withstand increased levels of salt. The second, "compatible solute" strategy, is used by Bacteria who maintain osmotic balance by keeping an internal level of compatible solutes balanced to that of the extracellular salt. For the most part, these organic solutes are low molecular weight, uncharged molecules with an extremely high solubility in water. Either of these two scenarios is energetically more expensive than life in a comparable but low-salt environment. However, it is proposed that the "compatible solute" strategy is the more costly of the two, but allows for a more rapid adaptation to the environment because components and enzymes in the internal environment do not have to be adapted to high salt. Because of the increased energy expenditure caused by the high salt environment, the energetically expensive process of dinitrogen fixation is usually not seen in many halophilic organisms (57, 74). It is interesting to note that a number of unique metabolic capabilities including sodium­ dependent solute transport mechanisms have evolved in halophilic organisms; including a sodium-dependent glutamate anti porters in thermophilic Bacillus species (59).

Life in an alkaline environment poses a whole different set of challenges to its inhabitants. Namely, the organism must find a way to maintain an electrical membrane potential (tnI') when the external environment is 13 typically one to two pH units more acidic than the environment. A favorable membrane potential is often accomplished through the activity of various cation transport systems, with uptake and extrusion of cations specific to taxonomic groups (44). One rather popular system to combat the problems associated with the aforementioned lack of a transmembrane pH gradient is to employ a Na+ /W antiporter system (37, 55).

Central to this problem of living in an alkaline environment is the fact that membrane proteins are exposed to an extreme environment. Ito et at., 1997, have described an adaptation of these proteins to include a decrease in the number of aspartate, glutamate and lysine residues with a corresponding increase in the amount of arginine, histidine, asparagine and glutamine residues present in alkaliphilic proteins (38). The substitution of these amino acids is thought to impart a net positive charge to membrane proteins, thus creating an acidic "microenvironment" immediately surrounding the organism. In addition, certain cell wall components increase with an increase in pH. One of these, the cell wall component teichuronopeptide (TUP), increases markedly as the culture pH increases in the alkaliphile Bacillus lentus (3). Another cell wall component, the surface layer protein SlpA, was seen to increase markedly as cells were grown in increasing pH (22). However, there is no evidence that membrane ultrastructure changes with an increase in pH (41). Both of these proteins are thought to support Na+ -dependent pH homeostasis of this facultative alkaliphile. With all of these adaptations to the alkaline environment, it is not surprising that experimental evidence suggests that alkaliphilic organisms are capable of growth rates as high as their neutrophilic counterparts (25). 14 The purpose of the research described herein was to isolate and characterize an anoxygenic phototroph adapted to the pressures of life in a haloalkaline environment. It is anticipated that an isolate from such an environment will provide valuable insight into adaptations of haloalkaliphiles that make these seemingly inhospitable environments a requirement for survival. Gram negative anoxygenic phototrophs are unique in that while they comprise a large portion of the organisms adapted to these environments, very little information regarding their specific adaptations has been completed. In addition, the autotrophic nature of these organisms may uncover unique biochemical pathways that can be used for biotechnological applications (e.g. the fixation of CO2 into useful metabolites and products).

Mono Lake, CA Mono Lake is a saline (ca. 8% NaCl) alkaline (pH 10) lake located in Mono County, California roughly 190 miles east of San Francisco and 300 miles north of Los Angeles. The lake's area became a closed basin roughly 3 million years ago due to tectonic action and the eastern scarp of the Sierra Nevada Mountain Range. The lake itself is approximately one-half million years old, one of the oldest in North America. The salinity of Mono Lake is attributed to the fact that ions dissolved in stream flow emptying into Mono Lake are concentrated when water evaporates in the summer. Because of these conditions, there is a relative paucity of macroscopic organisms in Mono Lake. While Mono Lake is an important waystation for many transient populations of birds and other wildlife, Mono Lake supports resident populations of only the brine fly Ephydra hians and the brine shrimp Artemia monica (12). 15

In the present study, sediment samples from Mono Lake were enriched for the presence of purple sulfur bacteria. Sample sites included: (1) the sandy shore of the lake (sample MLS) and (2) a dried mat adjacent to the main body of water (sample MLM). Mono Lake is a triple water lake, with high levels of sodium chloride, sodium carbonate and sodium sulfate (2, 12, 39). Geochemical data obtained in 1980 determined the major cation of the lake to be: Na+ (96.8%) and the major anion composition consisted primarily of: CO{ and HC03- (46.3%); cr (37.8%); and soi- (15.9%) (12). Based upon these data, and other geophysical parameters (e.g. light and temperature) an enrichment strategy was designed to isolate anoxygenic phototrophic bacteria.

This thesis describes the isolation and characterization of a novel purple sulfur bacterium from Mono Lake. This bacterium is phylogenetically related to members of the genus Ectothiorhodospira, within the recently reorganized family Ectothiorhodospiraceae, and possesses a number of interesting properties not previously described in Ectothiorhodospiraceae. Based upon the data described herein, this new isolate (strain MLS1) is proposed as a new species of the genus Ectothiorhodospira, Ectothiorhodospira monomense, sp. nov. 16 MATERIALS AND METHODS Source of the Organism All strains of bacteria used in this study were isolated from sediment samples collected along the shores of Mono Lake (Mono Lake Basin, Mono County, California). The samples were kindly donated by Or. Betsy Read.

Morphology and Microscopy Microscopic AnaLysis Microscopic observations of pure cultures and cell measurements were performed using a Nikon Alphaphot 2 phase contrast microscopy at 400X and 1000X.

Enrichment and Maintenance Media Primary Enrichment Media: 051(-1 Initial enrichment media was a modification of medium 051C routinely employed for the growth of haloalkaliphilic nonsulfur purple bacteria in soda lakes (50). Medium 051C was modified in this study to reflect the alkaline and saline conditions of Mono Lake (designated as medium 05IC-I), and consisted of (per liter of deionized water): NaCl, BOg; NH4Cl, 0.5g; KH2P04, 0.6g; K2504, 2.5g; Na25203-5H20, 0.1g; vitamin solution (biotin, 10mg; nicotinamide, 35mg; thiamine dichloride, 30mg; pyridoxyl chloride, 10mg; Ca-panthenoate, 10mg; vitamin B12, 5mg; 100ml dH20), 1ml (36); trace elements (EOTA, 5.2g; CoCh-2H20, 190mg; MnCh-4H20, 0.1g; FeCh-4H20, 1.5g; H3B03, 6mg; CuCh-2H20, 17mg; Na2Mo04-2H20, 1BBmg; NiCh-6HzO, 25mg; ZnCh, 70mg; V0504-2H20, 30mg; Na2W04-2HzO, 2mg; NaH5e03, 2mg; 1l dHzO), 1ml (77). The following components were autoclaved separately as solutions: Mg504-7H20, 0.2g; CaCh-2H20, 0.2g; NaHC03, 20g. Following autoclaving and cooling to room temperature, each of these components was individually added, very slowly, with constant 17 stirring. The pH of this media was approximately pH 9.4. For growth on solid media, agar select (Sigma Co., St. Louis, MO) was added to the primary components listed above (1.S%w/v). Organic carbon substrates were added as described in the results; secondary components were added as described above.

Medium OSIC-I was modified slightly by increasing the NaHC03 to 20g per liter, with no additional adjustment of the pH necessary to maintain a pH of -9.4. The increased carbonate caused a slight precipitation to form after the addition of all the secondary components. This precipitate did not alter the pH of the media, nor did it have any apparent effect on the growth of the cultures. However, the presence of precipitate interfered with spectrophotometer readings, and consequently, the media was filter sterilized (O.2f.!m filter) to remove all particulate matter. Filter sterilization did not alter the pH of the media, nor its capacity to support growth of the organisms.

Maintenance Media II: Ectothiorhodospira Maintenance (EM-II) Media This media was designated as medium EM-II and consisted of the following components (per liter of deionized water): NaCl, 70g; Na2S04, 10g; KH2P04, O.8g; NH4Cl, O.8g; SLA trace elements (FeCh.4H20, 1.8g; CoCh.6H20, 2S0mg; NiCh.6H20, 10mg; CuCh.SH20, 10mg; MnCh.4H20, 70mg; ZnCh, 100mg; H3B03, SOOmg; Na2Mo04.2H20; 30mg Na2Se03.SH20; 10mg 1l dH20), 1ml (36). The following components were autoclaved as separate solutions and added slowly with stirring, to the cooled primary components: CaCh.2H20, SOmg; MgCh.6H20, O.1g; NaHC03, 20g. Sodium sulfide (Na2S.9H20), O.8g was dissolved in 10ml dH20, filter sterilized, and added to the media components. Medium EM-II was stored anaerobically to avoid the abiotic oxidation of H2S to elemental sulfur (SO). 18

Growth Conditions and Physiological Characterization Primary Enrichment Conditions Sediment samples (-1ml suspensions) from Mono Lake Shore (MLS) and Mono Lake Mud Pond (MLM) were inoculated into 10ml screw-cap tubes and filled completely with medium OSIC-I, containing malate (30mM) as a carbon source. Enrichment cultures were incubated phototrophically (40W incandescent bulbs, ca 500 lux) at 18°C in temperature controlled circulating water baths.

Following growth and spectrophotometry of enrichment cultures to confirm the presence of anoxygenic phototrophic bacteria, cells were streaked onto agar plates and incubated in anaerobic jars (90: 1% N2: H2) to obtain single colonies. Cells from single colonies were repeatedly streaked onto the same media to obtain a pure culture of the phototrophic organism.

Routine culture maintenance and characterization of strain MLS1 initially employed medium OSIC-II. Based upon subsequent extensive biochemical, physiological and phylogenetic analyses, described in this thesis, medium EM-II was designed and was a better culture medium for strain MLS1. The major difference between medium EM-II and OSIC-II is that the former contains sulfide (-3.2mM) as a reduced sulfur source.

Salinity Strain MLS1 was grown under photo heterotrophic conditions in medium OSIC-II (malate, 30mM) at the following NaCl concentrations: 0.7M, 1.4M, 2.1M, 2.8M, 3.5M and 4.2M. Medium OSIC-II was prepared minus the NaCl, and just prior to autoclaving, the appropriate amount of solid NaCl was added to each experimental solution. Controls were employed in each of these experiments as follows: (1) negative control (no organic carbon, 1.4M 19 NaCl), (2) positive control (30mM malate, 1.4M NaCl) and (3) positive control (30mM malate, added solid 1.4M NaCl). pH Strain MLS1 was grown in medium DSIC-II (30mM malate) at the following pH values: 6, 7, 8, 9, 10 and 11. Medium DSIC-II was made without the addition of the buffering agent (NaHC03)' The secondary components MgS04• 7H20 and CaClz.2H20, were added as described previously. The pH was adjusted with sodium bicarbonate (NaHC03) and sodium carbonate (Na2C03) added to the desired pH value. The control cultures for each experiment were as follows: (1) negative control (no organic carbon, pH 9.4), (2) positive control (30mM malate, pH 9.4).

Bacteriochlorophyll and Protein Analyses Pigment Extraction Absorption spectra of whole-cell and pigment extractions were performed using a Shimadzu UV160U spectrophotometer. Spectra of pigment extractions was performed by treating cell pellets resuspended in 200~l sterile dH20 with 2ml acetone:methanol (7:2) solution for -2.5 hours in darkness at -20°e. Following centrifugation at 3100 x g for 5 minutes, absorption spectra on supernatants were obtained. Bacteriochlorophyll a concentrations were quantified from 5ml methanol extracts collected in the same fashion. These were analyzed for spectrophotometric absorption at 1 1 772nm using the extinction coefficient 46.1.g- .cm- •

Protein concentrations were was determined using a dye-binding colorimetric assay (BioRad Laboratories, Richmond CAl according to manufacturer's instructions. Cell pellets obtained following pigment 20 extractions were first boiled in 3ml 1N NaOH for 20 minutes to lyse the cell pellets. Additionally, the cell pellets from all nutritional/physiological experiments were stored at -20°C and total protein content was determined as above.

N2 Fixation and Nitrogen Metabolism For growth on N2, cultures were grown photosynthetically in 500ml Erlenmeyer flasks containing 100ml medium OSIC-II/N2/malate (30mM). The flasks were placed in an oxoid anaerobic jar (Unipath, Ogdensburg NY) and anaerobic conditions were established with a GasPak (BBL) envelope (HrC02 generator), providing a heads pace of Nr H2 (-99:1%). Cultures were inoculated (0.9% volume) and incubated in an illuminated (-500 lux) glass water bath at 18°C for -28 days and culture density was determined using a Milton Roy Spectronic 20 spectrophotometer.

Cultures were grown on ammonia (NH4Cl/[NH4hS04), glutamine, or glutamate as a nitrogen sources in screw-cap tubes filled completely with medium OSIC-II and incubated as described above until stationary phase.

Isolation of DNA and 16S rONA Sequencing DNA Isolation ONA was isolated from strain MLS1 isolate using standard isolation techniques (81) with slight modifications. Cells were grown to 00600 >1.99 (14 days of growth), from which aliquots (1.5ml) were transferred into Eppendorf tubes and centrifuged at 10,000 RPM for 5 minutes in a tabletop microcentrifuge. Pellets were resuspended in 600)ll TE buffer (10mM Tris.Cl [pH 7.5], 1mM EOTA [pH 8.0]), containing 50S (0.5%) and proteinase K (200)lg). Cells were lysed by incubation at 65°C for 1.5hr, after which NaCl (0.7M) was added. Next, the solution was vortexed and 80)lL of 21 CTAB/NaCl solution (10% CTAB; 0.7M NaCl) was added and mixed by inverting the tubes several times. Cells were then incubated for 1 hour at 65°C. DNA was extracted using an equal volume chloroform/isoamyl alcohol (24: 1) mix. The tubes were inverted several times and then spun at 10,000 RPM for 5 minutes. The lysate was extracted a second time with an equal volume of a phenol/chloroform (1: 1) mix. Following centrifugation, the DNA was precipitated overnight with two volumes of cold 100% ethanol followed by the addition of 0.1 volume 3M NaOAc, and dissolved in 1OO~L TE buffer. DNA concentration and purity were determined spectrophoto­ metrically and by agarose gel electrophoresis (1% agarose gel).

16S rONA Sequencing and Analysis Initial 165 rDNA sequence analysis of strain MLS1 was determined from amplification of a -460bp interior portion of the 165 ribosomal gene from genomic DNA (54). Oligonucleotide primers were designed based on a published protocol by the CSUPERB Microchemical Core Facility at San Diego State University: "Ribosomal A" 5'-CGGCCCAGACTCCTACGGGAGGCAGCA-3'; "Ribosomal B" 5'-GCGTGGACTACCAGGGTATCTAATCC-3'.

Amplification of 165 rDNA from the genomic DNA of strain MLS1 employed the Qiagen HotStarTaqTM Master Mix Kit (Qiagen Co, Valencia CA). Reactions were run using template DNA (100ng or 10ng) added to 25~l Qiagen

HotStarTaq ™ master Mix, 21 ~l sterile dH20, and 2~l of ribosomal A and ribosomal B primers, in a 50~l total volume. PCR reactions were run using a MJ Research Minicycler™ thermocycler with the following cycles: A 15 minute hot start at 95°C served to completely denature the template DNA and activate the Taq polymerase. This was followed by 30 cycles of 95°C denaturation (1 minute), 50°C annealing (30 seconds), and 72°C extension (1 minute). A 10 minute final extension was employed at the end of the cycles 22 and agarose gel electrophoresis was used to confirm the presence of the -460bp PCR product.

The PCR product was purified from the PCR reaction using the Qiagen QIAQuick™ PCR Purification Kit and resuspended in 30fll TE buffer. The purified product was ligated into the pCR®2. 1-TOPO® plasmid and transformed into Escherichia coli using the TOPO TA Cloning® Kit version K (Invitrogen, Carlsbad CA). The recombinant plasmids were purified from the E. coli transformants using a standard plasmid miniprep, and then the purified plasmid was resuspended in 20fll sterile dH20. The presence of insert was confirmed by linearizing the plasmid with Hind III. The purity and concentration of the plasmid DNA was confirmed by spectrophotometric analysis using a Shimadzu UV160U spectrophotometer at 260nm and 280nm and the concentration was adjusted to O. 3flgl fll for sequencing. Sequencing was accomplished using the universal M13 Reverse and M13 Forward primers. Sequencing was completed by the CSUPERB Microchemical Core Facility at San Diego State University. Sequence results were uploaded into BLAST for phylogenetic analysis and isolate identification. A phylogenetic tree was constructed using the online computer program MultAlin (13).

After the initial amplification of an interior -460bp region of the 16S subunit rDNA gene indicated a possible Ectothiorhodospiraceae purple sulfur bacteria, a larger region of the 16S rDNA gene was amplified using primer "CDRDNA1" 5'-GTTTGATCCTGGCTCAG-3' and reverse primer "CDRDNA2" 5'­ TACCTTGTTACGACTT-3' (35). 23 RESULTS Enrichment and Isolation Phototrophic enrichment cultures were established from Mono Lake sediment using a medium selective for the growth of nonsulfur purple bacteria (DSIC media). The sediment samples were collected from a highly saline (8% salinity), highly alkaline (pH 9-10) location (2, 12, 39) and gave rise to red-pigmented suspensions within 2 weeks. Prominent peaks (at 800 and 866nm) from whole cell absorption spectra of the primary enrichment cultures of both Mono Lake Mud (data not shown) and Mono Lake Shore enrichments indicated the presence of phototrophs containing bacteriochlorophyll a (Figure 1).

+2.00A r--c--.....--___ ---t--__t---t---_-_--;

0.500A (A/DIV) 866nmf\ 800Qm

I

+O.OOA '----4---_--I----+--_----!I---__-~ 300 400 500 600 700 800 900 1000 1100 wavelength (nm)

Figure 1: Absorption spectrum of whole cell samples from the Mono Lake Shore primary enrichment. Bacteriochlorophyll a representative peaks are indicated at 800nm and 866nm.

Repeated streaking of individual pigmented colonies onto agar plates failed to yield a pure culture of the anoxygenic phototroph. Indeed, two distinct colony types were present; a burgundy-pigmented colony and an unpigmented beige colony (Figure 2). Absorption spectra of cells from colony suspensions confirmed that the cells from the burgundy-pigmented 24 colony indicated the presence of bacteriochlorophyll Q, as observed in the primary enrichment (Figure 3A). The unpigmented colony was a non photosynthetic contaminant, as no peaks indicative of photosynthetic pigment were present when whole cell extracts were scanned (Figure 3B). When cells of the photosynthetic and non photosynthetic organisms were grown under photoheterotrophic conditions, no growth was observed in the tube inoculated with the unpigmented contaminant. This suggested the requirement of some growth factor or metabolite(s) produced by the phototroph for growth of the heterotrophic contaminant in D51(-1I media.

Figure 2: Agar streak plate of the MLS enrichment showing the presence of the two distinct colony types: (A) a pigmented phototrophic organism and (B) an unpigmented contaminant. +2.00A A B

0.500A (AIDIV)

+O.OOA 300 400 500 600 700 800 900 1000 300 400 500 600 700 800 900 1000 wavelength (nm) wavelength (nm)

Figure 3: Whole cell absorption spectra of the phototrophic bacterium (A) and the unpigmented contaminant (B). Note the presence of peaks corresponding to bacteriochlorophyll a from the anoxygenic phototroph A and the absence of any peaks corresponding to photosynthetic pigments in organism B. 25 Several colonies of the purple ,anoxygenic phototroph were picked and re- streaked to confirm that a pure culture had been obtained. One of these isolates was chosen as the type strain for this study, and designated strain MLS1 (Figure 4).

Figure 4: Growth of strain MLS1 on solid medium OSIC-II (A) and liquid culture (8).

Phylogenetic Analysis PCR amplification of rONA gene from strain MLS1 using primers specific for an internal region within the 16S rONA gene yielded the expected 460bp product (Figure 5). Sequence analysis of the PCR product using BLAST identified strain MLS1 as a purple sulfur bacterium belonging to the Family Ectothiorhodospiraceae. Sequence analysis of strain MLS1 indicated a 98% homology to both Ectothiorhodospira haioaikaliphila (BN 9902) and an unpublished bacteriochlorophyll b-containing Ectothiorhodospira species previously isolated from Mono Lake, designated "Borgoria Red" (28).

Alignment of the partial sequence of strain MLS1 with the complete 16S sequences of other purple sulfur bacteria also indicated strain MLS1 was most closely related to Ectothiorhodospira haioaikaliphila (Table 2), and consequently further sequencing of the 16S rONA gene from strain MLS1 was 26 not warranted. The relationship of strain ML51 to both strains of E. haioaikaliphila is interesting in that although these two strains share a 98.4% homology with each other, strain ML51 shares a 98% homology with organism 11 (BN 9902), and is only 94% homologous with organism 13 (BN 9903). Within the genus Ectothiorhodospira strain ML51 shares greater than 90% homology with the following species: E. shaposhnikovii, E. vacuoiata, E. marismortui and E. marina.

Figure 5: peR amplification of 165 rONA from strain ML51. Primers were constructed to amplify an -460bp fragment (lanes 2-4). Lane 1, 1kb ladder molecular weight marker in 100bp increments (bright band is 1000bp).

The 460bp 165 rONA peR amplification product of strain ML51 was aligned to the species most closely related to strain ML51 from Table 2. However, in order to make a more accurate comparison of these organisms, their complete 165 rONA sequences were trimmed to include only the region comparable to that sequenced from strain ML51 (Figure 6). These data were used to generate a phylogenetic tree based on parsimony analysis in order to show the relationship of strain ML51 to other members of the Ectothiorhodospiraceae (Figure 7). These results are significant in that although only a portion of the 165 rONA sequence was used in the analysis, the results were in agreement with those obtained when the entire 165 27 rDNA gene sequences of these organisms were aligned with each other. The highly conserved nature of this region of the 165 rDNA gene (nucleotides -700-1100) among Ectothiorhodospiraceae can be observed in the alignment of the sequences (Figure 6) (54).

Table 2: The uncorrected percent similarity of 16S rONA sequences of various Purple Sulfur Bacteria, reference species and strain MLS 1.

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 D IH. halochloris 1BN 9850 99.9 98.3 94.8 94.1 94.5 89.7 89.0 88.8 88.9 87.6 87.8 88.1 87.4 89.0 87.0 82.4 83.2 81.5 85 D ~. halochloris 2BN 9851 98.2 94.6 93.9 94.4 89.6 88.8 88.7 88.7 87.3 87.4 87.8 87.2 88.8 86.9 82.3 83.1 81.4 87 ~. abdelmalekiiD 3~N 9840 93.8 93.5 93.9 89.2 89.4 89.4 89.0 87.9 98.0 88.1 86.9 89.2 86.8 82.6 83.5 81.6 88 D ~. halophiia 4BN 9632 98.7 98.0 91.9 89.3 88.9 88.9 88.0 87.8 88.9 88.4 87.9 87.5 82.3 83.0 81.5 84 D IH. halophiia 5BN 9624 97.8 91.4 88.9 88.8 88.6 87.7 87.7 88.6 88.1 87.7 86.8 82.0 82.6 81.0 87 D ~. halophiia 6BN 9630 91.2 89.0 88.9 88.8 87.7 87.8 88.6 88.0 87.4 86.9 81.9 82.8 81.1 87 IA. aquaeoleiD 7IA Tee 49307 89.0 88.6 88.9 88.6 88.3 89.1 88.4 87.8 88.7 83.6 83.3 81.8 87 c IF. shaposhnikovii 8BN 9711 99.2 98.2 96.0 96.3 96.6 95.3 95.2 90.3 84.5 83.4 82.2 93 c 1<". shaposhnikovii 9iBN 9912 93.8 95.7 95.9 96.2 95.0 94.9 89.9 84.1 83.2 82.0 95 C I.... vacuolata 1C BN 9512 95.3 95.4 96.3 95.5 95.2 89.9 89.3 83.1 82.6 93 c IF. haloalkaliphila 11 BN 9902 98.1 98.4 95.7 94.7 89.4 83.5 82.2 81.3 98 1<". marina C 12 BN 9914 97.9 95.5 94.9 88.9 83.3 91.9 81.1 97 c . haloalkaliphila 13 BN 9903 96.5 95.3 89.7 84.1 82.4 81.7 94

I.... marismortui' 14BN 9410 95.1 89.4 84.2 82.3 81.4 91 F. mobilis' 15 DSM 237 89.5 83.4 82.7 81.1 90 C. vinosumD 16IATee 17899 85.3 83.2 80.9 87

17Fscherichia coli 80.8 78.6 86 jR. rubruml? 18IATCC 11170 86.3 80 191R. palustris' 81 ctothiorhodospira 20 train MLS 1 QGenus Ha/orhodosplra bGenus Arhodomonas cGenus Ectothiorhodospira dGenus Chromatium "Genus Rhodospirillum 'Genus Rhodopseudomonas 28

.1 .1" £:IJ ,jU qU ::)0 bU 10 uu ::110 J.uu HU .L'" .L,jU 1------+------+------+------.. -+--'------+-----.. --+------+------+--... ----+------+--... ----+------+-...... ---1 "LS1 TGTTTGCECCC-RCGCTTT cGCACCTNRGTGT lOOT ITTHGTCf ilbllRGl CGCCTTCGC' RCTGGTGTTCCTTCCGRTR-TCTRCGCRTTTCRCCGCTRCRCCGGHRRTTCCRCr: TCCTCTRCCll 'R BN9902 TGTTTGCTCCCCCRC-CTTT I. ',CACCTCRGTGT .RGT TTTRGTCI, nl,HIRG 'CGCCTTCGC RCTGGTGTTCCTTCCGRTR-TCTRCGCRTTTCACCGCTRCRCCGGHRRTTCCRCfJ iCCTCTRCCrll'R 8orgoria TGTTTGCT~CCC-RCGCTTI fllCACCTCRGTGT 'RGT 'TTRGTC' [I II CRG TCGCCTTCGC· 'RCTGGTGTTCCTTCCGRTR-TCTRCGCRTTTCRCCGCTRCRCCGGRRRTTCCRCF 'CCTCTRCC!' 'R OS"2111 TGTTTGCT,TCCC-RCGCTTi [CCACCTCRGTGT' RGT ERGIe'." 'RG iCGCCTTCGC RCTGGTGTTCCTTCCGRTR-TCTRCGCRTTTCRCCGCTRCRCCGGHRRTTCCRCT' ICCTCTRCC: TR O5H241 TGTTTGCTCCCC-RCllCTT r TTCflCCTCRGTGT lRGT fTCRGTCC,,[,l rRG iCGCCTTCGC RCTGGTGTTCCTTCCGRTR-TCTRCGCRTTTCRCCGCTRCRCCGGRRRTTCCRCT 'CCTCTRCC." R RTCC51935 TGTTTGCT':CCC-RCClTT II! :C!lCCTCRGTGEOOT ITTRGTCCiltiC,iRG rCGCCTTCGC 'RCTGGTGTTCCTTCCGRTR-TCTRCGCRTTTCRCCGCTRCRCCGGRRRTTCCRCF ;CCTCTRCCTTR OSH243 TGTTTGCTCCCC-RCGI TTNH.iCHCCTCRGTGTIRGTTTNRGTcr:IGfl'iRG! CGCCTTCGC.RCTGGTGTTCCTTCCGRTR-TCTRCGCRTTTNRCCGCTRCRCCGGRRRTTCCRCTI! :CCTCTRCCII"R OSH239 TTGCTCCCC-RCC-TT: '. ICI ICCTCRGTGTCRGTTTCRGTCiCGI'T,RG' CGCCTTCGCIRCTGGTGTGCCTTCCGRTR-TCTRCGCRTTTCRCCGCTRCRCCGGRRRTTCCRCT:' [CCTCTRCCTIIR OSH418O TGTTTGCTCCCC-RCfl TT I,CCrlCCTCRGTGTCRGT !TTCGTCCHI; 11 ,RGCCGCCTTCGC RCTGGTGTTCCT '[CGRTR-TCTRCGCRTTTCRCCGCTRCRCCGGCRRTTCCRCTI. !CCTCTRCC[,. iR O5H237 TGTTTGCTCCCC-RCl ,TTTNHNCHCCTCRG,.GT .RGT rn GGTCCYYllCRGeCGCCTTCGC-RCTGGTGTTCCT. CCGRTR-TCTRCGCRTTTCRCCGCTRCRCCGGGARTTCCRCTI rCCTCTRCC! 'R 05"244 TGTTTGCTRCCC-RC:' 'WH· CGCCTCRGLGTCRGT lNCGGTC,. lIGGeRG! CGCCTTCGC.ACTGGTGTTCCT CCGRTR-TCTRCGCRTTTCRCCGeTRCRCCGG!;RRTTCCRCTRCCCTCTRCCGHA 8119630 TGTTTGCTRCCC-RWrTT ;'.' .CGCCTCAGI,GFRGT rTf "GTU ACGeRG! CGCCTTCGC.RCTGGTGTTCCT CCGRTRATCTRCG6RTTTCRCCGCTRCRCCGGCRRTTCCRCTRCCCTCTACCG, R BN9624 TGTTTGCTRCCC--CRCTT' ·l'TCGCCTCAG· GT lRGT: TC',GTCI i!I!GeRG rCGC- TTCGC RCTGGTGTTCCT' CCGATH-TCTRCGCRTTTCACCGCTRCRCCGGIiRRTTCCACTRCCCTCTRCCG,R OSHl059 TGTTTGCECCC-RCII!!TTTNNC6CCTCRG'!GTNRGTCTlrl,GTCNII11GCRG rCGCCTTCGC ACTGGTGTTCCT CCGRTH-TCTRCGCRTTTCACCGCTRCRCCGGGAATTCCACTRCCCTCTRCCGGR Bll9851 TGTTTGCTCCCC-RCCm ,N"CGCCTCR&; GTRRGTCT1r'lGTCNflG6CAG 'CGCCTTCGC' RCTGGTGTTCCT CCGRTH-TCTRCGCRTTTCACCGCTRCRCCGGiARTTCCRCTRCCCTCTRCCGGA O5H2110 TGTTTGCTCCCC-RC--TT II CCGCCTCAG'GFRGTCTCiiGTC('i!liGCRGTCGCCTTCGCcRCTGGTGTTCCTi CCGRTH-TCTACGCRTTTCRCCGCTRCRCCGGfiRRTTCCRCTRCCCTCTRCC R Consensus TGTTTGCLCCC ACdI' '0 iC lCCTCAG, GT lRGT' T,lSGTC 'IRG. CGCCTTCGC· ACTG6TGTTCCT CCGRTR TCTRCGCRTTTCRCCGCTRCRCCGG',AATTCCRCT I CCTCTRCC ·R

ill M ~ ~ m ~ ~ ~ m ~ ~ ~ ~ ~ 1------+------+------+------+------+.. ------+------+-_.. _-----+------+------+------+------+------1 "LS1 CT -CTII-GCC cfGCAGTRTCRGATGCRi1TTCC IOOGTTRRGCCCfiGGG-TTTCRCA ICTGACTGRCrrr ' I.RCCTRCGTGC CTTTRCG-CCRGT'RTTCCGA ITAhCGC· C'C-AC' 'ICCGTRTTR BH9902 CT-Cle--GC' . 'GCAGTRTCRGATGCRllTTCLRGGTTRRGCCCIGGGCTTTCRCR ICTGRCTGRCRT,I", "RCCTRCGTGC .cTTTACGICCRGT '.RTTCCGA' TA"CGC 'C IC-ACl' ICCGTRTTA Borgoria CT -CTA-GCl H'GCRGTRTCRCRTGCRATTCCLRGGTTRRGCCC lGGGCTTTCRCR ICTGACTGRCIlF :1. RCCTACGTGCi£TTTACGICCAGlIRTTCCGR ITRI' CGC :CilC-ACi. I lCCGTRTTR OSH2111 CT -CT!1--GCI rl !GCAGTATCR.IRTGCRflTTCCCRGGTTRRGCCCGGGCTTTCRCA ICTGACTGRC,ITl'I' RCCTRCG6GC.cTTTACG6CCRGT"RTTCCGR TRI'CGC'CiiCCAW ,CCGTRTTR O5H241 CT-CTR--GCI'II IGCRGTRTCRLRTGCRf!TTCC"RGGTTRAGCCC.IGGGCTTTCRCR1CTGRCTGRCIITc·11' .IRCCTRCG6GC'XTTTRCG6CCOOT. RTTCCGR. TR· ,CGC'C :CCRCC' rCCGTATTA RTCC51935 CT -CTH--GC,.! IGCRGTRTCR iRTGCRIITTCCCRGGTTRAGCCCIGGGCTTTCACR I TlCGRCT TRC'T, " RCCTRCGYGCSCTTTRCG'.CCAGTRRTTCCGR TRI.CGC;' "C-RC;I TCCGTRTTA O5H243 CT -CTH--GCCII fGCRGTRTNRGRTGCOOTTCCCRGGTTGRGCCCRGGGCTTTCRCR . CTGRCTGRC.IT,· '. 'RCCTRCGTGChCTTTRCG' CCRGTIIRTTCC6R i TRflCGCTNCC-ACNIICCGTATTA O5H239 CT -CTR--GCC iITGCRGTRTCRIiRTGCOOTTCC'RGGTTGRGCCCRGGGCTTTCRCR: CTGRCTGRC.r' I RCCTRCGTGcr,cTTTRCG CCAGH1RTTCCGR 'TRRCGC 1'liC-RCI CCGTRTTR OSH4180 CTTCT?CTGC---GCRGTRTC·II,RTGCRI'TTCCIRGGTTGRGCCCiiGGGCTTTCACR ,C, GACT 'RCT. ---liCCTRCGTGCI .cTTTACG, 'CCRGTHRTTCCGATRflCGC IC[C-nC [CCGTATTR O5H237 CT-Cn--GCl9'GCRGTRTC"uRTGCAilTTCC-RGGTTfiAGCCCGGGGCTTTCRCR 'C 'GRCT 'RCI Tel . 'rRCCTRCGTGC'ICTTTRCG' CCAGT..1RTTCCGRATACCGCGC-CTTHNHNNCGTRTTR' O5H244 CT -CTN--GCt nAGCRGTRTCC[;RTGCRRTTCCTRGGTTllRGCCCRGGGCTTTCACRCC.IGACT . RCCT6G' ICCTRCGeGCCCTTTRCG ,CCRGTGRTTCCGR ,'TAACGCT 11ICGe-C' : TCGTRTTR 8119630 CT-CTr:--GTCRRGCRGTRTCGliRTGCA:lTTCCTRGGTTGAGCCCRGGGCTTTCRCRCCI'GRCT iRCCTGll' ;CCTRCGeGCCCTTTRCGCCCAGTGRTTCCGRITARCGC' CGe- TCGTRTTR 8119624 CT -CT·j--GCCHRGCRGTRTCCGRTGCRIlTTCCTRGGTTGAGCCCRGGGCTTTCRCRCCCGRCT 'RCCTGGl, l,cCTRCG6GCCCTTTRCG6CCRGTGRTTCCGR fTARCG6 : IrrCGe-' ITCGTRTTR OSH1059 CT -CTN--GCTRCGCRGTRTCIIftRTGCRflTHCCIRGGTTGRGCCCflGGGCTTTCRCAIC',6ACT JRC'T "G,," .cCTRCGeGCCCTTTRCGC CCRGTGRTTCCGA 'TRflCGC I I,CGe- TCGTRTTR 8119851 CT-CTn--GCTHCGCRGTRTC·IRATGCR."TTCC:RGGTTIIAGCW,GGGCTTTCRCR ;C[GRCT RC'ITi'GI I '[CCTRCGeGC"CTTTRCGCCCAGTGRTTCCGR! TRPCGC' CGe-, TCGTRTTR OSH2110 CT -CT.I--GCYRTGCAGTRTCHGRTGCOOTTCC,IRGGTTfiRGCCCRGGGCTTTCRCA' CTGRCTGACIIT'lfll ICCTRCGeGC,HTTACGrCCAGF,RTTCCGAl TR,ICGC' . CGeN,', lTCGTRTTR Consensus CT.CT a•• GC·' ,1 GCRGTRTC,.;ATGCR" TTCCcRGGTT ,RGCCC,GGGCTTTCRCR'.C.1GRCT, RC ,T ,. ",CCTRCG.GC,CTTTACG, CCAGT·,RTTCCGW TA .CGCI ,:C ••• u.CGTRTTA

~ m ~ ~ ~ m ~ ~ ~ ~ ~ m ~ ~ 1------+------+------+------+------+------.------+------+------+------+------+------+------I "LS1 cm GGCTGNTGGCRCCGIIRGTT HTGCCGGG iGCTTCTTCFl GGG-TGRTGTCRRc '.C RGCI' C---' TRTT.: ',C',I 'C---' 'TTTCTTCCCCHCTGARRGTGCTTTRCRRCCCGCR· .-CCTTCTT 8119902 CCG', GGCTGCTGGCRC-Gi,RGTTIi-GCCGG- 'GCTTCTTCTi, !GGG- TGRTGTCRR,~ 'iC RGCHC---liTRTTi .... iC· , CC--- .' . TTTCTTCCWrf'TGAARGTGCTTTRCRACCCGCRI,GCCTTCTT Borgoria CCGi,GGCTGCTGGCRC-GGRGTT:I-GCCGG- 'GCTTCTTtn IGG6- TGRTGTCAA['C.RGC, IC---,TRTT!",r;c" TC--- '. TTTCTTCCC- ·1 TGAAAGTGCTTTRCRRCCCGCRbGCCTTCTT 05"2111 CCGI GGCTGCTGGCRC-GGRGTFI-GCCGG-. GCTTCTTCTI. 'GGG-TGRTGTCRRli:C?AGCI'C---iiTRTTi" .iC IIC---.. TTTCTTCCCC " TGRARGTGCTTTRCRACCCGCAhGCCTTCTT 05"241 CCG! GGCTGeTGGCRC-GGRGTT2-GCCGG- TGCTTCTTCr. . GGG-TGRTGTCAA! :CI·RGCi'C---fTRTT 'C.' 'C---, II TTTCTTCCCC:ITGAAAGTGCTTTRCRRCCCGCR'CGCCTTCTT RTCC51935 CCGCGG-TGCTGGCRC-GGRGTT. '-GCCGG- TGCTTCTTCT' iGGGRNGRTGTCRR','C:6GC··C---G TRTT .CR, 'C--C " TTTCTTCCCC 'CTGRRRGTGCTTTRCRACCCGCAGCCRTCTT OSH243 CCGNGGCTGCTGGCRC-G ·RGTT.!-GCCGG- !GCTTCTTCT .. !GG6-T!·R,,6TCAW,TC'RGC'C---·,TATT. G.C. C--- i' TTTC TCCCCiiCTGAAAGTGCTTTRCRACCCGCA,GCCTTCTT OSH239 CCG'GGCTGCTGGCRC-G:RGTTrl-GCCGG- 'GCTTCTTCF.I GGG- H,Ri GTCRR, TCiHGC·.C---GTRTTG'iC ". i C--- .. TTTC. TCCCCiITGAAAGTGCTTTRCRRCCCGCA.,GCCTTCTT 05"4180 CCG·:GGeTGCTGGCRC-GGRGTTI!-GCCGG- 'GCTTCTTCT', 'GGG-TGRTGTCRA'.·(,GGCGC---TTRTTCGI ,CRI.I.C--- . TTTCTTCCCCi ,. TGRAAGTGCTTTRCRACCCGCA! IGCCTTCTT OSH237 CCGC GGCTGCTGGCRC-G'iRGTT8-GCCGG- [GCTTCTTCr', . GGG-T!.A'.GTCRRI, TC·IGGC'C---'JTRTT.IGuCRr .1.[---. 'TTTC' .TCCCC;I .TGARRGTGCTTTRCRRCCCGCRI:GCCTTCTT 05"244 CCGNGGCTGCTGGCRC-GRRGTT -TGCCGGC-GCTTCTTCTTNGGG-TiR' GTCNR" 'C--GC-CRGGI, TRTTi'I,CC---CGRC . TTTCITCCCCGRTGRARGTGCTTTRCRRCCCGNAHGCCTTCTT BN9630 CCGI' GGCTGCTGGCRC-GRRGTT-TGCCGGC-GCTTCTTCTTCGGG-TflRCGTCRA 'C--GC-CRGGcTRTT. 'fCC--CRRC,·, : TTTCr!TCCCCGRTGAAAGTGCTTTRCRRCCCGCAGGCCTTCTT BN9624 CCG'iGGCTGCTGGCAC-GRR6TT-TGCCGGC-GCTTCTTCTTCGGG-T,'RI'GTCAAI ,'.C--GC-CRG6ll TRTTI,I'CC--CGRC I TTTC .. TCCCCGRTGRRRGTGCTTTRCAACCCGCR-GCCTTCTT OSHl059 CCG'~GGCTGCTGGCRC-GHRGTT-TGCCGGC-GCTTCTTCTT'GGG-T"R"GTCRRCTC--GCI,CRGC-TRTTfiICC·, TGCRRC--TTTCCTCCCCNRTGRRR6TGCTTTRCRRCCCGCRCGCCTTCTT Bll9851 CCGCGGCTGCTGGCRC-GRR6TT -TGCCGGC-GCTTCTTCm GGG-T R GTCRACTC--GCHCRGe-TATTi'CC ,TGCRRC---TTTCI, TCCCC· RTGRRRGTGCTTTACRRCCCGCRCGCCTTCTT OS"2110 CCG·:GGCTGCTGGCAC-GRRGTT -TGCCGGC-GCTTCTTCT. GGG-rR, GTCRACTC--GC·CRGC-TRTTI" ICC, TGCR-C---TTTCL TCCCC ·RTGRRRGTGCTTTACAACCCGCR-GCCTRCTT Consensus CCG"GGCTGCTGGCRC GiAGTT i.GCCGG.,GCTTCTTCL. GGG T .R, GTCRA ,C.I.GC ,C ••• JRTT· C C... I TTTC' TCCCC."TGARRGTGCTTTRCRACCCGCA GCCTTCTT 391 400 410 420 430 440 447 1------+-----_... _+------+------+------+-----1 "LS1 NRCRCRCGCGGCRTTGCTGGRTGAGICGTT"CCCCCRTTGTCCRRTRTTCCCCRCTGC Bll9902 CRCACACGCGGCRTTGCTGGRTi.RGE-TT .. CCCCCRTTGTCCRRTRTTCCCCRCTGC Borgoria CRCRCACGCGGCRTTGCTGGRTI.RGC-TTfCCCCCRTTGTCCRRTRTTCCCCRCTGC OSH2111 CACRCACGCGGCRTTGCTGGRTRGCGTTI'CLCCCRTTGTCCARTRTTCCCCRCTGC DSH241 CRCRCACGCGGCATTGCTGGRT. RG6TT,C"CCCRTTGTCCRRTRTTCCCCRCTGC RTCC51935 CRCACRCGCGGCRTTGCTGGRT"RG·TGTT 'C'ICCCRTTGTCCARTRTTCCCCRCTGC 05"243 CRCRCRCGCGGCATTGCTGGRTNAG,:'TT 'CCCCATTGTCCRRTRTTCCCCRCTGC 05"239 CRCRCRCGCGGCRTTGCTGGRTCAGII.ITT·CC·XCCATTGTCCRRTATTCCCCRCTGC OSIt4180 CRCRCRCGCGGCRTTGCTGGRTCRG-ITTTTC' CCCRTTGTCCRRTRTTCCCCRCTGC O5H237 CRCRCRCGCGGCATTGCTGGRTtRGh6H C' CCCRTTGTCCRRTRTTCCCCRCTGC O5H244 CACACACGCGGCRTNGCTGGRTNNGl,LTTTC"CCCRTTGTCCAATRTTCCCCACTGC Bll9630 CRCRCACGCGGCRTTGCTGGRT ,RG ,CTTTC"CCCRTTGTCCARTRTTCCCCRCTGC 8119624 CACRCRCGCGGCRTTGCTGGAF.RG,i TTYCiCCCATTGTCCARTRTTCCCCACTGC OSH1059 CRCRCRCGCGGCRTTGCTGGRL RGf.'CTTCCI!CCCRTTGTCCRRTRTTCCCCRCTGC Bll9851 CRCRCRCGCGGCRTTGCTGGRT·.RGili TT ,C',CCCATTGTCCAATRTTCCCCACTGC OSH2110 CRCRCRCGSGGCRTTGCTGGRTCRGG-TTl CiCCCATTGTCCARTRTTCCCCRCTGC Consensus CRCRCRCGCGGCRTTGCTGGRTcRG," HiC ;CCCATTGTCCARTRTTCCCCRCTGC

Figure 6: Alignment of strain MLS 1 partial 16S rONA with comparable regions from related organisms shown in Table 2. Alignment is based on consensus of the sequence at high, 90% (blue), moderate, 50% (purple), and low consensus,<50% (black) of the organisms. An overall consensus for each nucleotide position is included in the bottom row. Consensus letters in upper case indicate those with high (~90%) consensus across species, lower case letters represent moderate consensus (~50%) between the organisms. 29

...--- H. abdeLmalekii H. haLochLoris r----L~~ H. halochloris H. halophiLa .....__ ...... ---- H. halophila H. halophila ,....------E. mobilis 11..... -- E. marismortui --t.-=E.:...~sh.:.:a::poShnikovii r E. shaposhnikovii a..---t .--___ E. haloaLkaliphila BN 9903 E. marina E. vacuoLata Ectothiorhodospira strain MLS1 E. Borgoria Red E. haLoalkaUphiLa BN 9902 ---10 PAM

Figure 7: Phylogenetic tree of strain MLS1 from Mono Lake to other members of the genera Ectothiorhodospira and Halorhodospira. PAM is the accepted point mutations per 100 amino acids.

Morphology Individual cells of Ectothiorhodospira strain MLS1 were motile, rod shaped to slightly-curved, and stained Gram negative. The average

Ectothiorhodospira strain MLS1 cell was 1 X 3~m (Figure 8).

Figure 8: Phase contrast micrograph of Ectothiorhodospira strain MLS1. Magnification, 1000X. Photosynthetic Pigments Densely grown cultures of Ectothiorhodospira strain MLS1 were intensely burgundy in color. Absorption spectra of intact cells showed maxima at 30 800nm and 866nm corresponding to bacteriochlorophyll a. A peak at 560nm in the extracted spectra was indicative of the major carotenoid spirilloxanthin (Figure 9). A second peak at 771 nm in the extract confirmed the presence of bacteriochlorophyll a. Furthermore, based on the absence of a distinct peak at 794nm in the extract, there was no evidence of any significant amount of bacteriochlorophyll b, which is the major bacteriochlorophyll in many Ectothiorhodospira species, including the closely related Mono Lake isolate "Borgoria Red" (28).

The average specific bacteriochlorophyll a content of Ectothiorhodospira strain MLS1 under optimal conditions was 24.4~g Bchl a/ml, with a pigment to protein ratio of 17.6~g Bchl a/mg total cellular protein.

+2.00A • 771nhi -- A B

O.SOOA . Brc \ 5600m (A/DIV) f\ '\~:A '\. vr\.\ /\ ' . J l\~. \ \ > ~! •

+O.OOA 300 400 500 600 700 BOO 900 1000 300 400 500 600 700 BOO 900 1000 wavelength (nm) wavelength (nm)

Figure 9: Absorption spectra of intact cells (A), and methanol:acetone extracts (8) of Ectothiorhodospira strain MLS1.

Growth and Nutritional Properties Ectothiorhodospira strain MLS1 was screened for its ability to photoassimilate various carbon sources. Growth of Ectothiorhodospira strain MLS1 was optimal under photoheterotrophic conditions in minimal media supplemented with either malate or sodium acetate (Table 3). 31 Interestingly, growth on acetate required that it be present as a sodium salt at very high molar concentrations (100 mM and 200 mM). Substitution of ammonium acetate at these concentrations failed to support the growth of strain MLS1. Decreasing the sodium acetate and ammonium acetate to 30 mM resulted in similar, albeit low, growth yields of Ectothiorhodospira strain MLS1. Other carbon sources found to support the growth of Ectothiorhodospira strain MLS1 were fumarate, pyruvate and succinate (Table 3).

Table 3: Cell yields of Ectothiorhodospira strain MLS1 with organic components photoassimilated as carbon source and electron donora. Cell Yieldc per Carbon Source (mM) Substrateb 200 100 30 10 2 Ammonium Acetate - - ++ + + Butyrate nd nd - - - ... aproate - - - - - Dextrose (D-glucose) - - - - - Fructose (D-levulose) + - - - - Fumarate - - ++ ++ + ~lycerol - - - - - Lactate - - - - + ifJ-malate +++ +++ +++ ++ + D-mannitol - - - - - Propionate - - - - - fyruvate ++ ++ + - - Sodium Acetate +++ +++ ++ ++ + !Sodium Citrate - - - - - ~odium Tartrate - - - - - ~orbitol - - - - - Succinate - - ++ ++ + aData obtamed followmg a secondary transfer mto the same carbon source. bMinimal salts medium (DSIC-II) with 1.4M NaCI at pH 9.4 containing the listed carbon sources. cAbundant growth (+++; OD600nm >1.999), moderate growth (++; 0.951-1.999), poor growth (+; 0.300-0.950) and no growth (-; <0.299), nd; not determined.

The inability of Ectothiorhodospira strain MLS1 to photoassimilate propionate at any of the tested molar concentrations is particularly interesting in that most members of the Genus Ectothiorhodospira are able 32 to utilize propionate as a sole carbon source under phototrophic conditions (31). Of particular note is that Ectothiorhodospira strain ML51 was unable to grow photoautotrophically utilizing H25 as an electron donor under anaerobic conditions, in contrast to other Ectothiorhodospira species. Growth under photoautotrophic conditions on solid media with H2 gas as electron donor was also negative. In addition, Ectothiorhodospira strain ML51 was unable to use sodium thiosulfate for photoautotrophic growth.

Salinity (Nael) Optimum Growth of Ectothiorhodospira strain ML51 under varying NaCl concentrations indicated an optimal growth rate (g = 84.5h) and maximum cell density at 1.4 M (8%) NaCl. Moderate growth was also noted at 0.7 M (4%) and 2.1 M (12%) NaCl, with culture densities approximately 72% and 54% of maximum, respectively (Figure 10) .

.c 0.9+---­ ~ 0.8+----­ ~ 0.7 ~ 0.6 .~ 0.5 0.4 ~o t: 0.3 ~ 0.2 Q) a.. 0.1 o U III e.., e.., Z c oc o ~ u u 00 N +

Figure 10A: Effect of NaCI concentration on the growth of Ectoth;orhodospira strain MLS1. Each point represents the mean of two separate experiments. Positive and negative controls were grown in 1.4M NaCI with and without malate (30mM), respectively. 33

Figure 10B: Photo showing the color and relative densities of Ectothiorhodospira strain MLS1 cultures from the salinity optimization experiments shown in Figure 10A. Pictured are the (1) negative control (minus malate, 104M NaCI), (2) positive control (30mM malate, 104M NaCI) and the experimental tubes (each with 30mM malate); (3) 0.7M NaCI, (4) 2.1M NaCI, (5) 2.8M NaCI, (6) 3.5M NaCI and (7) 4.2M NaCI.

The salinity growth data indicate that Ectothiorhodospira strain MLS1 is a moderate halophile, with an absolute requirement for NaCl in the culture medium. To confirm that variation in NaCl concentrations did not affect the media pH, the pH of each culture was determined after the completion of the experiments and no significant difference in the pH was observed at any NaCl concentration (average -9.4). pH Optimum The pH optimum for growth of Ectothiorhodospira strain MLS1 was determined to be that of the positive control, 9.4 (Figure 11). Moderate growth was also noted at pH 7 (20% maximum), pH 8 (80% maximum), pH 10 (80% maximum) and pH 11 (30% maximum). No growth was observed at pH 6.

Cell yields (OD6oonm ) of Ectothiorhodospira strain MLS1 indicated that the isolate is equally able to grow well at pH 8 and pH 10, with growth of both approximately 81% of that obtained at optimum pH (9.4). However, analysis 34 of total cellular protein indicated that Ecto.thiorhodospira strain MLS1 at pH 10 was 98% maximum obtained at pH 9.4, whereas the protein content of cells at pH 8 was only 90% maximum. This may reflect differences in the amount of cell membrane proteins, like those mentioned in the results, which infer survivability in an alkaline environment. 1---- I 1 ~------

I .s:: 0.9 I ~ 0.8 f------I ~ 0.7 1------i ~ 0.6 +------­ .~ 0.5 +------­ :E '0 0.4 +------

~ 0.3 -j------u t 0.2 +------

...... co 0 ~ ::c ::c ~ ~ e e I a. a. ::c ::c ...c ...c a. a. 0 0 I ___i __ u u [_O·~ + I ! ------.! Figure 11 A: Effect of pH on the growth of Ectothiorhodospira strain MLS 1. Each point represents the mean of two separate experiments. Positive and negative controls were grown at pH 9.4 with and without malate, respectively.

Figure 11 B: Photo showing the color and relative densities of Ectothiorhodospira strain MLS 1 cultures from the pH optimization experiments shown in Figure 11 A. Pictured are the (1) negative control (minus malate, pH 9.4), (2) positive control (30mM malate, pH 9.4) and the experimental tubes (each with 30mM malate); (3) pH 7, (4) pH 8, (5) pH 10, and (6) pH 11. No growth was noted at pH 6 (not pictured). 35

Cell Yield Analyses of Strain MLS1: Optical Density versus Total Cellular Protein In order to confirm whether culture turbidity (OD600) was an accurate reflection of true cell number increase, Ectothiorhodospira strain MLS1 was grown photoheterotrophically in medium DSIC-II with 30mM malate as a carbon source. The protein content of these cells was determined using the Bradford Microassay procedure (see Methods) and compared to the corresponding culture density (OD600nm). The results clearly indicated that increases in optical density correlated strongly with total cellular protein (Figure 12).

2 1.8 ./'"- .., 1.6 1.4 ./'" ./'" 0 1.2 0 c'" 1 ./'" 0 0.8 ~ ./" 0.6 ./'" 0.4 ./+ 0.2 r 0 10 20 30 40 50 60 Total Cellular Protein (ug/ml)

Figure 12: Relationship between total cellular protein (J..1g/ml) and absorbency of cultures at 600nm. Dots represent the actual data points, solid line is the best-fit line through the data (r2 =0.9794).

Data on the physiology of Ectothiorhodospira strain MLS1 described above indicate that growth was optimal under photoheterotrophic conditions (30mM malate; at pH 9.4 and 1.4M NaCl). These results, based on spectrophotometric data, are also supported when one compares total 36 protein content from stationary phase cells following growth under various experimental conditions. Since the data above confirmed that cellular protein increased proportionally with the growth of Ectothiorhodospira strain MLS1, the total protein content of Ectothiorhodospira strain MLS1 cells then should accurately reflect total cell yield under each experimental condition.

1 0.98 0.96 0.94 .~ ~ 0.92 E 0.9 b.... ::J c... E 0.88 :::: 'x ~ ~ 0.86 ttl * 0.84 ~ ~ 0.82 0.8 0.78 0.76 0.74

"- 00 0 ~ OJ OJ OJ OJ OJ -0.... U U ~ ~ ...... ttl ttl ::c ::c ttl ttl ttl ttl ttl <:: Z Z a. a. ::c ::c ...... > .... <:: 0 a. a. OJ ttl ::J OJ 'u U ::E ::E u .... u ttl E ttl U "- ~ ::J >- ::J + '- a. n, 0 N ::c..r z '" z

Figure 13: Comparison of total cell protein content of Ectoth;orhodosp;ra strain MLS1 under various culture conditions. Total protein content of cells under each experimental condition was normalized as percent maximum of the control culture (see text).

The total protein content of Ectothiorhodospira strain MLS1 cells grown at pH 10 was only ca. 3% lower than control cells (Figure 13), whereas spectrophotometric data from these same cells indicated culture densities to be about 20% lower than control conditions. This lends support to the hypothesis that higher levels of cell membrane proteins are typically present in alkaliphilic bacteria at increased pH levels, as mentioned above. However, in general, changes in optical density were proportional to total cellular protein data. 37

Nitrogen Assimilation Ammonium chloride (NH4(l) served as the nitrogen source for routine culture of Ectothiorhodospira strain ML51. Ammonium sulfate ([NH4h504) and glutamine were also utHlzed as sole sources of fixed nitrogen. No difference was seen in the growth rate of Ectothiorhodospira strain ML51 with either NH4Cl or (NH4h504 at concentrations of 9 and 18mM in medium 051(-11. However, when glutamine was used as the source of fixed nitrogen in medium 051(-11, the generation time was reduced by 65%. Glutamate was unable to support the growth of Ectothiorhodospira strain ML51 as a sole source of nitrogen. However, Ectothiorhodospira strain ML51 was able to grow on atmospheric dinitrogen (N2), suggesting that this organism synthesized a functional nitrogenase system. As expected from the bioenergetics of nitrogenase systems from other organisms, growth on N2 was much slower than growth with a source of fixed nitrogen.

Optimization of Culture Media for Ectothiorhodospira strain MLS1 Phylogenetic analysis of Ectothiorhodospira strain ML51 indicating that it was a purple sulfur bacterium led to the adjustment of the original enrichment media (051(-11), the most significant change of which was increasing the amount of reduced sulfur available as an electron donor. The new medium was adjusted to reflect the haloalkaliphilic conditions of Mono Lake and designated medium EM-II. Growth in medium EM-II resulted in a 25% reduced generation time (g = 62.9h) from that obtained in 051(-11 medium (g = 84.5h). Although the growth rate was increased dramatically, final cell yields (as determined spectrophotometrically) in medium 051(-11 and medium EM-II were identical. 38 The generation time of Ectothiorhodospira strain ML51 with glutamine (14mM) as a nitrogen source decreased to 29.75 hours from 84.5 hours. This represented a decrease of 65% from similar medium D5IC-1I culture conditions with NH4Cl (9mM) and a reduction of 53% from medium EM-II culture supplemented with NH4Cl (9mM) (Figure 14).

1.92

1.32 +------#------I"~-~~-----__l

E c: 8 ::£. o o

24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 408 time (hours)

Figure 14: Growth curve of Ectothiorhodospira strain MLS 1 using medium OSIC-II with 9mM NH 4CI (+) and medium EM-II with 9mM NH4CI (WI), and medium OSIC-II with 14mM glutamine (e). 39 DISCUSSION The phylogeny of purple sulfur bacteria has been a source of debate since the inception of modern microbiology. In 1907, all purple anoxygenic phototrophs were grouped together within the Order Rhodobacteria. The members of this order were further subdivided based on the ability (Thiorhodaceae) and lack thereof (Athiorhodaceae) to store elemental sulfur internally. In 1974, members of the Athiorhodaceae that were able to store elemental sulfur externally were reassigned to the genus Ectothiorhodospira and placed within the newly characterized Family Chromatiaceae (31). The Chromatiaceae included all those purple anoxygenic phototrophs possessing the ability to store elemental sulfur intra- or extracellularly. The Chromatiaceae was further defined to include only those species depositing elemental sulfur within the cell, while the Family Ectothiorhodospiraceae was proposed to include only species which has the capability to store elemental sulfur extracellularly, and these organisms were placed into the genus Ectothiorhodospira.

The family Ectothiorhodospiraceae is relatively new, having been recognized in modern-era microbiology within the past twenty years. The most recent edition of Bergey's Manual of Systematic Bacteriology recognizes six species of Ectothiorhodospira: E. mobilis, E. shaposhnikovii, E. vacuoLata, E. haLophila, E. haLochLoris and E. abdeLmaLekii (31). However, recent extensive 165 rDNA analysis has reevaluated and resolved the phylogenetic relationships among members of the Ectothiorhodospiraceae, establishing two separate genera: Ectothiorhodospira and HaLorhodospira. These genera were established based upon both molecular (165 rDNA) and physiological properties including bacteriochlorophyll and carotenoid content, fatty acid composition and metabolic requirements (35). Interestingly, the new 40 taxonomic scheme has aligned all of the bacteriochlorophyll b-containing members of the Ectothiorhodospiraceae into the genus, Halorhodosp;ra, whereas the genus Ectoth;orhodosp;ra contains exclusively bacteriochlorophyll a-containing species. This reevaluation also included the reclassification of various strains of E. mobWs, with three of the sequenced strains moved to the already established species E. shaposhn;kovU. In addition, this reorganization resulted in the creation of two previously undescribed species, E. haloalkaHphHa (BN 9902 and BN 9903) and E. marina (BN 9914) (35). Soon after the publication of the 1996 Imhoff and SUling paper detailing this reevaluation, independent reviews confirmed the newly characterized E. haloalkaHphHa as a bacterium with a distinct lineage within the y subdivision of the proteobacteria (67, 73, 75).

The intraspecies rearrangement within E. mobWs is of particular interest in this study because Ectoth;orhodosp;ra strain MLS1 showed a maximum similarity of 98% to Ectoth;orhodosp;ra haloalkaHphHa (BN 9902) which was previously classified as an E. mobWs species. Ectoth;orhodosp;ra strain MLS1 had only a 90% similarity to the E. mobWs type strain (BN 9911). In fact, of those Ectothiorhodosp;ra species described in Bergey's Manual of Systematic Bacteriology (31), the closest relationship of Ectothiorhodosp;ra strain MLS1 is to E. shaposhn;kovU and E. vacuolata at 93% (Table 4). The 16S rDNA analysis of Ectothiorhodosp;ra strain MLS 1 conclusively identifies this bacterium as a member of the genus Ectoth;orhodosp;ra, most closely related to E. haloalkaHphHa (BN 9902). However, as will be discussed below, there are a number of significant biochemical and physiological differences between Ectoth;orhodosp;ra strain MLS1 and other characterized members of the genus. The recently isolated bacteriochlorophyll b-containing Ectoth;orhodosp;ra species, E. Borgoria Red (28), is 98% similar to Ectothiorhodosp;ra strain MLS1, however other 41 than a brief study as part of a master's thesis, characterization of this organism has not been published.

Table 4: Comparison of morphological and physiological characteristics of Ectothiorhodospira strain MLS1 to Ectothiorhodospiraceae type strains described in Bergey's Manual of Systematic Bacteriology (31), as well as E. haloalkaliphila (BN 9903) (35).

cell size cell major major Salinity 16S c (length) flagellation suspension carotenoida pigmentb tolerance rDNA Ectothiorhodospira strain MLS1 3.01Jm bipolard red sp Bchla 4-12% E. haloalkaliphila BN 9903 nd nd nd nd Bchl Q 5%e 94%

E. mobilis 0.7-1.0IJm polar tuft red sp Bchl a 1-7% 90%

E. shaposhnikovii 0.8-0.9IJm polar tuft red sp Bchl Q 1-7% 93%

E. vacuolata 1.51Jm bipolar red sp Bchl a 1-6% 93%

H. halophila 0.6-0.9IJm bipolar red rh Bchl b 11-32% 84%

H. halochloris 0.5-0.6IJm bipolar green rh Bchl b 14-27% 85%

H. abdelmalekii 0.9-1.2IJm bipolar green rh Bchl b 12-18% 88% asp, spmlloxanthm;.. rh, rhodopm bbacteriochlorophyll a or b Cinterior 460bp region of 16S small subunit rONA (E. coli positions -700-1100bp) dbased on motility observed using phase contrast microscopy eonly NaCI optimum is available for this organism

In addition to the rONA data, several major properties described in this thesis demonstrate that Ectothiorhodospira strain MLS1 belongs within the genus Ectothiorhodospira within the family Ectothiorhodospiraceae; Its rod­ shaped morphology, the presence of bacteriochlorophyll a, its obligately anaerobic and alkaliphilic metabolism, and anoxygenic phototrophy are typical characteristics of members of the purple sulfur branch of the Domain Bacteria. However, there are significant differences between Ectothiorhodospira strain MLS1 and other members of this newly reorganized family that warrant further analysis and discussion and argue strongly for its classification as a new species in the genus Ectothiorhodospira . 42

Perhaps most novel with respect to other members of its genus is the salinity optimum and tolerance of Ectothiorhodospira strain MLS1, with optimal growth at 1.4M (8%) NaCl, the absencelinhibition of growth at NaCl concentrations below 0.7M (4%), and growth up to 2.1 M (12%) NaCl. The salinity requirement of Ectothiorhodospira strain MLS1 contrasts sharply with that of other members of the newly reorganized genus, all of whom grow optimally at ca. 5% NaCl, with tolerances between 1-7% (Table 4) (31, 34, 35).

Initial enrichments were aimed at identifying the in vitro conditions that would enrich for the growth of purple anoxygenic phototrophs while reflecting the in situ conditions of Mono Lake. An enrichment media (DSIC­ I) was formulated to isolate nonsulfur purple bacteria, containing sodium bicarbonate (NaHC03, 0.5%w/v) and NaCl (8% w/v). Mono Lake is a triple water lake, with high levels of sodium chloride, sodium carbonate and sodium sulfate (2, 12, 39). Geochemical data obtained in 1980 determined that the lake consisted of Na+ (96.8%) as the major cation, and several anion species: cot and HC03-(46.3%); cr (37.8%); and soi- (15.9%) (12). Consequently, organisms isolated from this environment would be expected to be adapted to these levels of inorganic compounds. The ecological/physiological role of sulfate in Ectothiorhodospira strain MLS1 was not examined in this thesis, however medium DSIC-I contained a much lower amount of soi- (0.04% w/v) than that described for Mono Lake. However, increasing the sulfate level in the media would only serve to decrease pH and the maintenance of enrichment pH (pH 9.4) would require too high a level of HC03-/C03- for adequate buffering of the media. 43 Following initial enrichments and isolation of Ectothiorhodospira strain ML51 , medium D5IC-1 was buffered almost exclusively with sodium bicarbonate, with an increased concentration of 200 mM. During experiments aimed at determining the pH optima of Ectothiorhodospira strain ML51, 200 mM carbonate was also used as a buffering agent, with a change only in the ratio of bicarbonate to carbonate in each of the experimental conditions. For experimental conditions at pH 6, 200 mM NaHC03 was added to the media with subsequent pH adjustment using HCl. Phototrophic growth of Ectothiorhodospira strain ML51 was completely inhibited at pH 6, poor at pH 7.0 (20% maximum growth) and greatly enhanced at pH 8 and 10 (80% maximum). These results most likely reflect dramatic changes in membrane potential, affecting both the ability of Ectothiorhodospira strain ML51 to transport nutrients and to synthesize ATP.

Membranes are energized by proton electrochemical gradients and protons extruded from the cell must return via specialized transmembrane proteins, some of which include solute transporters and ATPases. Thus, changes in membrane potential of the cell would be reflected in the inability to exchange nutrients and to generate sufficient ATP for biosynthesis. Under anaerobic conditions, an ATPase couples proton flow to ATP hydrolyses (an endothermic reaction derived from substrate level phosphorylation) to maintain the electrical potential and proton gradient across the membrane. Fortunately for anoxygenic phototrophic bacteria living in these anaerobic environments, the energy source is inexpensive (light) and so translocation of protons and synthesis of ATP is not a problem. However, alkaliphilic bacteria exist in an environment where the external pH is at least 1.5 to 2 pH units more alkaline than the internal pH, requiring them to constantly bring protons into the cell to keep the cytoplasmic pH relatively acidic (pH 8.5-9.0). Recent research has suggested that acidification of the cytoplasm 44 of alkalinophiles is accomplished with Na+ /W antiporters which in turn are driven by the electrical membrane potential (L1 '1') and the proton gradient (L1 pH) (37, 74, 76). Thus limitations on the growth of Ectothiorhodospira strain MLS1 below pH 8 and above pH 10 most likely reflects changes in the membrane bioenergetics of the cell affecting solute transport, ATP production and internal homeostasis.

Following the completion of preliminary phylogenetic analyses, Ectothiorhodospira strain MLS1 was grown in media used for the routine culture of Ectothiorhodospira species (31) (designated medium EM-II) which contained sulfide (3.3mM) as electron donor. Medium DSIC-II used in most of the studies described herein had a low level of sodium thiosulfate (O.4mM) as the reduced sulfur source and malate (30mM) as a carbon source, providing metabolic conditions more selective for the isolation of purple nonsulfur bacteria over a purple sulfur or green sulfur anoxygenic phototroph. The fact that these enrichment conditions yielded a purple sulfur bacterium indicates that these bacteria may predominate in this saline alkaline lake. By providing a more reduced and higher level of sulfur (H2S), growth of Ectothiorhodospira strain MLS1 was greatly enhanced. This contributed dramatically to the reduction of the generation time of Ectothiorhodospira strain MLS 1 from 89.5 hours to 62.9 hours and suggests that this organism is able to use sulfide as a photosynthetic electron donor. Sulfide oxidation is common in PS bacteria and under alkaline conditions often results in the formation of polysulfides as an intermediate during oxidation of sulfide to sulfur (from H2S to S°). Members of the family Ectothiorhodospiraceae differ in their abilities to utilize reduced sulfur compounds and thus further studies on sulfur metabolism in Ectothiorhodospira strain MLS1 will be required to determine what pathways are utilized during photoheterotrophic growth and whether sulfide 45 is completely oxidized to sulfate or to some other intermediate(s) (e.g. S°, S20{,S40{).

Purple photosynthetic bacteria exhibit tremendous versatility with respect to carbon metabolism, with nonsulfur purple bacteria being more metabolically diverse than purple sulfur bacteria. Ectothiorhodospiraceae species have been shown to grow photoautotrophically, photoheterotrophically, chemoorganotrophically, aerobically and microaerobically. No species to date has been shown to be capable of growth chemolithotrophically (31). Carbon utilization experiments performed on Ectothiorhodospira strain MLS1 indicated that of the substrates tested, photoheterotrophic growth was obtained with only acetate, succinate, fumarate, pyruvate, malate, fructose and lactate. In addition, attempts to grow Ectothiorhodospira strain MLS1 photoautotrophically, either with sulfide or thiosulfate as electron donors were negative. In contrast, E. haloalkaliphila BN 9903 is capable of photoautotrophic growth, suggesting significant physiological differences between these two organisms (Table 5).

In order to further test the photoautotrophic capabilities of Ectothiorhodospira strain MLS1, it is first necessary to study the enzymes responsible for C02 fixation in this organism in order to determine whether they are functional. All purple sulfur bacteria tested have been found able to utilize the Calvin Cycle for C02 fixation. Expression of Rubisco (ribulose- 1,5-bisphosphate carboxylase/oxygenase), the key enzyme of this cycle, is strongly repressed in the presence of organic carbon. Consequently, before pursuing further experiments on photoautotrophy in Ectothiorhodospira strain MLS1, Southern hybridization with heterologous Rubisco probes will 46 be employed to determine if this gene is present in the genome of this organism (34, 35).

Table 5: Photosynthetic electron donors and carbon sources used by species of the family Ectothiorhodospiraceae (31, 34).

(I) (I) ...... (I) ...... (I) I1l (I) (I) I1l (I) (I) ...... c: ...... (I) I1l ...... 0 "- VI (I) I1l (I) ...... 0 I1l I1l (I) VI I1l ...... c: .... "'5 0 ...... 5. > I1l .... (I) "0 VI ...... 0 I1l I1l ...... ::l ·u >. u 0 u u ...... (I) 0 .... u ...... 5 ::l ::l E U U .... >. ro ::l ::l Z. ::l :E .... ::l donor/source I1l a. a. E VI ..c 011 VI ...... "- on "- ~ Ectothiorhodospira + - + + + - - - - + - + + strain MLS1 E. haloalkaliphila + - + + + - - + + - - + - BN 9903 + + + + + - + + + + E. mobilis - - - + + + + + + - + + + + + + E. shaposhnikovii + + + + + + + + + + + + E. vacuolata - - - - + + + + + + + - + + H. halophila - - - - + + + + + - - + - - - + H. halochloris - + + + + + - 0 + - - - + - H. abdelmalekii.. +posltlVe m most strams -negative in most strains !:positive in some strains but negative in others 0 not tested

A significant feature distinguishing Ectothiorhodospira strain MLS1 from other members of its genus is its inability to grow photoheterotrophically utilizing propionate as a carbon source (31). In most purple bacterium, growth on propionate is dependent upon C02 being present in the medium, and this carbon source is present at high levels in Ectothiorhodospira strain MLS1 media. Propionate metabolism has been extensively studied in Rhodospirillum rubrum (43) where it was found that propionate was photoassimilated to succinate via the enzyme propionyl CoA carboxylase. The inability of Ectothiorhodospira strain MLS1 to utilize propionate is particularly interesting since it grows well on succinate which in some phototrophic organisms is fermented to propionate via the enzyme 47 propionyl CoA carboxylase (70). This enzyme is negatively regulated (transcriptionally and post-translationally) in response to malate, the preferred carbon source for phototrophic growth in R. rubrum. Thus it may be possible that this alkaliphilic phototroph possesses a propionyl CoA carboxylase activity only in the succinate to propionate (oxidative) direction. Future studies will investigate the apparent inability of Ectoth;orhodosp;ra strain MLS1 to utilize propionate by measuring propionyl CoA carboxylase activity under a range of substrate concentrations and physiological conditions.

The ability of Ectoth;orhodosp;ra strain MLS1 to grow in medium with N2 as the sole source of cellular nitrogen indicates that it expresses a functional nitrogenase system. Among purple sulfur bacteria (Chromatiaceae and Ectothiorhodospiraceae) dinitrogen fixation has been recorded for only about seven species of Chromat;um, four species of Ectoth;orhodosp;ra and only a few species in the genera Th;ocapsa, Amoebobacter, Thiocystis and Lamprobacter (73, 84). The data described in this study supporting diazotrophy in Ectoth;orhodosp;ra strain MLS1 comes exclusively from growth studies, and actual ;n vivo nitrogenase assays of suspensions of intact cells (i.e. acetylene reduction assays) will be required to unequivocally confirm these growth data. Although the generation time of Ectoth;orhodosp;ra strain MLS1 under diazotrophic conditions was not determined, maximum culture density was obtained in roughly twice the incubation time as control cultures grown with ammonium chloride (9mM).

As observed in many other diazotrophic anoxygenic phototrophs, glutamine could serve as a nitrogen source Ectoth;orhodosp;ra strain MLS1. In fact, this compound was the preferred source of fixed nitrogen in Ectothiorhodosp;ra strain MLS1, reducing the generation time from 62.9 48 hours to 29.75 hours. Not surprisingly, Ectothiorhodospira strain MLS1 was unable to utilize glutamate as a source of fixed nitrogen. In most diazotrophic anoxygenic phototrophs, N2 fixation is regulated by a number of transcriptional and posttranslational mechanisms. One such molecular switch involves the cell's sensing of the ratio of a-ketoglutarate to glutamate. Glutamine can be deaminated to glutamate, the deamination of which yields a-ketoglutarate. Thus, increasing levels of glutamate serves as a signal for a reduction in fixed nitrogen and thus diazotrophic growth is upregulated in the presence of glutamate. Gest and Kamen first noted this when cells grown with glutamate were seen to liberate large quantities of hydrogen gas, a by-product of the first reaction in nitrogen fixation (17). Subsequent experiments indicated that labeled 15N2 was incorporated under these conditions (64). Future studies on nitrogen metabolism and N2 fixation in Ectothiorhodospira strain MLS1 will involve enzyme assays of nitrogenase, glutamine synthetase and glutamate dehydrogenase in order to analyze the regulatory aspects of these processes.

Ectothiorhodospira strain MLS1 is clearly a member of the genus Ectothiorhodospira, based on the morphological, biochemical and molecular data presented in this thesis. Although further metabolic, biochemical and genetic analysis will provide more detailed information about Ectothiorhodospira strain MLS1, the distinct properties discussed herein may warrant the classification of Ectothiorhodospira strain MLS1 as a novel species of the genus Ectothiorhodospira. Thus, the newly isolated purple sulfur bacterium, Ectothiorhodospira strain MLS1, described herein is proposed as a new species of the Ectothiorhodospiraceae, Ectothiorhodospira monomense, sp. nov. (mense N.L. adj. pertaining to Mono Lake). 49 LITERATURE CITED

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CURRICULUM VITA OF CRISTA D. DiBERNARDO

June 2001

PERSONAL

Home Address: Post Office Box 1856 San Marcos, CA 92079

E-mail address: [email protected]

EDUCATION

M.S. 2001. California State University, San Marcos, CA. Thesis title: The Isolation and Characterization of a Novel Haloalkaliphilic Purple Sulfur Bacterium, Ectothiorhodospira monomense, sp. nov., from Mono Lake. Advisor: Dr. Thomas M. Wahlund, Professor of Biological Sciences

B.S. 1997. California State University, San Marcos, CA. Major: Biological Sciences

PUBLICATIONS

DiBernardo, Crista, D. Garcia and R. Bray. 1999. Laboratory Exercises for GES 102, General Education Science, Biology. Aztec Shops, Ltd.

PROFESSIONAL EXPERIENCE AND CURRENT POSITION

Current Position: Adjunct Faculty, Biological Sciences, San Diego Community College District, Fall 2000 to present