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MIAMI UNIVERSITY the Graduate School MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Jenna Dolhi Candidate for the Degree: Doctor of Philosophy Dr. Rachael Morgan-Kiss, Director Dr. Annette Bollmann, Reader Dr. Gary Janssen, Reader Dr. D.J. Ferguson Dr. Melany Fisk, Graduate School Representative ABSTRACT ENVIRONMENTAL IMPACTS ON RUBISCO: FROM GREEN ALGAL LABORATORY ISOLATES TO ANTARCTIC LAKE COMMUNITIES by Jenna M. Dolhi Ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO) is found in a variety of autotrophic microorganisms ranging from green algae, cyanobacteria, and chemoautotrophic bacteria. As this enzyme has the potential to catalyze carboxylation (carbon fixation) or oxygenation (photorespiration) reactions, it is regulated in response to environmental variables at the levels of transcription, translation, and post-translation by the enzyme, RubisCO activase. A combination of laboratory experiments on green algal isolates and field experiments were utilized to gain insight on carbon fixation in permanently ice-covered Antarctic lakes. RubisCO was investigated as a potential target for cold adaptation of carbon fixation in the psychrophilic green alga, Chlamydomonas raudensis UWO241 (UWO241), isolated from Lake Bonney, Antarctica. RubisCO activity, stability, and whole cell carbon fixation were measured for the psychrophile and compared to a closely related mesophilic alga, C. raudensis SAG49.72 (SAG49.72). The effect of environmental factors including light and temperature on UWO241 and SAG49.72 RubisCO activation state, an indirect measurement of RubisCO activase activity, and abundance was investigated using a modified RubisCO carboxylase assay and immunoblotting, respectively. Lastly, maximum potential RubisCO carboxylase activity was determined using a modified activity assay in multiple ice covered Antarctic lakes including Lake Bonney. This data was complemented with lake depth profiles of enzyme abundance determined by quantitative real- time PCR and RubisCO-harboring organism diversity. While purified RubisCO of the psychrophilic green alga did not function optimally at low temperature, whole cell carbon fixation was greater under such conditions, suggesting that the overall process of carbon fixation is modified to function in UWO241. Increased RubisCO abundance at low temperature may contribute to this phenomenon. Low light levels may be important in regulation of RubisCO via RubisCO activase and should be further investigated. Based on community level RubsiCO activity and enzyme abundance, light and RubisCO harboring organisms including eukaryotic algae and cyanobacteria were positively correlated, but this was variable between lakes. Dark carbon fixation was potentially important in lakes west lobe Bonney and Fryxell and this community was negatively correlated with light. Results of targeted physiology and community level experiments led to development of a carbon fixation model for Lake Bonney. ENVIRONMENTAL IMPACTS ON RUBISCO: FROM GREEN ALGAL LABORATORY ISOLATES TO ANTARCTIC LAKE COMMUNITIES A DISSERTATION Submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Jenna M. Dolhi Miami University Oxford, OH 2014 Advisor: Dr. Rachael Morgan-Kiss Reader: Dr. Annette Bollmann Reader: Dr. Gary Janssen TABLE OF CONTENTS Page Chapter I: Introduction 1 1.1. Natural habitat and identity of UWO241 2 1.2. Photosynthetic electron transport in UWO241 3 1.2.1. Photosynthetic electron transport 3 1.2.2. Adaptations and acclimations of photosynthetic electron transport to irradiance 4 1.2.3. Adaptations of energetics to low temperature 6 1.3. Photosynthetic carbon fixation reactions in UWO241 7 1.3.1. Photosynthetic carbon fixation reactions 7 1.3.2. Adaptations and acclimations of enzymes to low temperature 7 1.3.3. RubisCO: the rate limiting enzyme of the CBB cycle 8 1.3.4. Adaptation of RubiscO to atmospheric CO2 and O2 concentrations 9 1.3.5. Regulation of carbon fixation: RubisCO 10 1.4. Photosynthetic microbial eukaryote diversity and the impacts of climate change 12 in the dry valley lakes 1.5. Dissertation objectives 13 Chapter II: Functional characterization of RubisCO from psychrophilic and 20 mesophilic green algal isolates 2.1. INTRODUCTION 21 2.2. METHODS 24 2.2.1. Growth conditions 24 2.2.2. rbcL and rbcS sequencing 24 2.2.3. RubisCO enzyme extraction and purification 25 2.2.4. RubisCO carboxylase activity assays 26 2.2.5. RubisCO carboxylase activity assay of crude lysates 27 2.2.6. Carbon fixation by whole cells 27 2.2.7. Chlorophyll fluorescence measurements 28 2.3. RESULTS 29 2.3.1. RubisCO subunit sequences 29 2.3.2. RubisCO purification 29 2.3.3. Temperature response of partially purified RubisCO carboxylase activity 29 2.3.4. Temperature response of RubisCO carboxylase activity of crude lysates 30 2.3.5. Temperature response of whole cell carbon fixation and photosynthesis 31 2.4. DISCUSSION 32 Chapter III: The effect of environmental factors (growth irradiance and temperature) 45 on modulation of RubisCO activity and abundance in psychrophilic and mesophilic green algal isolates 3.1. INTRODUCTION 46 3.2. METHODS 48 3.2.1. Strains and growth conditions 48 3.2.2. Chlorophyll fluorescence measurements 49 3.2.3. Lysate extraction and protein determination 49 3.2.4. RubisCO carbamylation assay 50 ii 3.2.5. SDS-PAGE and Western blotting 50 3.2.6. RubisCO activase gene sequencing 51 3.3. RESULTS 52 3.3.1. Effect of growth irradiance and temperature on growth rates 52 3.3.2. Effect of growth irradiance and temperature on photochemical function 53 3.3.3. Effect of growth irradiance and temperature on RubisCO activity and 54 carbamylation state 3.3.4. Effect of growth irradiance and temperature on RubisCO abundance 55 3.3.5. Determination of the RubisCO activase sequence for C. raudensis UWO241 55 3.4. DISCUSSION 56 Chapter IV: Diversity and distribution of carbon fixation genes in ice-covered lakes 67 of the McMurdo Dry Valleys, Antarctica 4.1. INTRODUCTION 68 4.2. METHODS 71 4.2.1. Site description 71 4.2.2. Sample collection 72 4.2.3. Limnological parameters 72 4.2.4. DNA extraction 73 4.2.5. Quantitative PCR 73 4.2.6. PCR, cloning, and sequencing 74 4.2.7. Phylogenetic analysis 74 4.2.8. Statistical analysis 75 4.3. RESULTS 75 4.3.1. Lake chemistry and biology 75 4.3.2. Spatial distribution of rDNA genes 76 4.3.3. Spatial distribution of major autotrophic genes 76 4.3.4. Functional gene diversity 77 4.3.5. Structure of autotrophic communities in relation to abiotic variables 79 4.4. DISCUSSION 79 4.5. ACKNOWLEDGEMENTS 84 Chapter V: Functional characterization of autotrophic and protist-specific 104 heterotrophic activity in permanently ice-covered lakes of the McMurdo Dry Valleys, Antarctica 5.1. INTRODUCTION 105 5.2. METHODS 108 5.2.1. Field sampling 108 5.2.2. Limnological parameters 108 5.2.3. Lysate extraction and RubisCO carboxylase activity assay 109 5.2.4. Lysate extraction and βGAM activity assay 109 5.2.5. Protein concentration determination 110 5.2.6. Bacterial enumeration 110 5.2.7. Statistical analyses 110 5.3. RESULTS 110 5.3.1. Limnological parameters 110 iii 5.3.2. MDV lake community RubisCO carboxylase activity 111 5.3.3. MDV lake community βGAM activity 112 5.3.4. Correlations of autotrophic and heterotrophic enzyme activity with 112 physicochemical lake parameters 5.4. DISCUSSION 113 Chapter VI: Conclusions 125 References 135 iv LIST OF TABLES Page 1. Impact of low temperatures on photostasis in Chlorella vulgaris and C. 15 raudensis UWO241 2. Anion exchange column chromatography purification of RubisCO from C. 36 raudensis UWO241 and SAG49.72 3. General physical and chemical characteristics for study sites 85 4. Primer set sequences with annealing temperatures for qPCR and/or clone library 86 construction of functional carbon fixation genes 5. Checklist for MiQE guidelines 87 6. Autotrophic gene clone libraries 89 7. Pearson’s correlation coefficients for the relationship between lake biological 90 and environmental parameters and abundance of functional genes in MDV lakes 8. Pearson correlation coefficient values for average RubisCO and βGAM activity 118 with lake physical, chemical, and biological parameters for all lakes combined 9. Pearson correlation coefficient values for average RubisCO and βGAM activity 119 with lake physical, chemical, and biological parameters for East and West lobe Bonney, Fryxell, and Vanda 10. UWO241 cDNA sequence library contigs coding for homologs of β- 133 carboxylation and TCA cycle enzymes v LIST OF FIGURES Page 1. General physicochemical characteristics of Lake Bonney in Taylor Valley, 16 Antarctica 2. Photosynthetic electron transport chain in green algae and plants 17 3. Calvin Benson Bassham cycle showing carboxylation and oxygenation reactions 18 catalyzed by the RubisCO enzyme 4. Predicted climate effects in Antarctic lakes 19 5. Neighbor-joining phylogenetic trees based on translated DNA sequences of C. 37 raudensis UWO241 and SAG49.72 RbcL and RbcS 6. SDS-PAGE of purified protein fractions from exponentially growing C. 38 raudensis UWO241 and SAG49.72 7. Activity of purified RubisCO from the psychrophilic and mesophilic C. raudensis 39 measured at various assay temperatures 8. Thermolability of purified RubisCO from C. raudensis UWO241 and SAG49.72 40 incubated at 50 °C 9. Thermolability of purified RubisCO from C. raudensis UWO241 and SAG49.72 41 incubated at various temperatures 10. RubisCO activity in crude lysate of psychrophilic (Chlamydomonas sp. CCMP 42 681, Chlamydomonas sp. ARC, Chlorella sp. BI, C. raudensis UWO241) and mesophilic green algae (C. raudensis SAG49.72) 11. Inorganic carbon uptake at various temperatures and 50 µmol photons m-2 s-1 for 43 C. raudensis UWO241 and C. raudensis SAG49.72 12. Effect of temperature on electron transport efficiency and energy distribution in 44 C. raudensis UWO241 and SAG49.72 13. Effects of irradiance and temperature on growth rates of psychrophilic C. 60 raudensis UWO241 and mesophilic C.
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