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Conserved and Specific Proteome Responses To CONSERVED AND SPECIFIC PROTEOME RESPONSES TO GROWTH ON FERRIC CITRATE VERSUS FUMARATE IN VARIOUS GRAM- NEGATIVE DISSIMILATORY IRON REDUCING BACTERIA: Anaeromyxobacter dehalogenans 2CP-1, Shewanella oneidensis MR-1, Geobacter sulfurreducens PCA AND Geobacter bemidjiensis Bem A Thesis Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Master of Science by Qiaochu Wang August 2015 © 2015 Qiaochu Wang ABSTRACT A growing list of Gram-negative iron reducing bacteria are known to be also capable of reducing soluble U(VI) to insoluble U(IV) biologically, providing potential uranium bioremediation strategies. Though model organisms have been studied for years, our knowledge of them is still limited and iron reductases of many other poorly characterized bacteria remain unknown. In this thesis, bottom-up proteomics using iTRAQ labeling was employed to analyze differential protein expression in Anaeromyxobacter dehalogenans 2CP-1, Shewanella oneidensis MR-1, Geobacter sulfurreducens PCA and Geobacter bemidjiensis Bem grown on ferric citrate versus fumarate. In all organisms, there were modest increases in enzymes associated with overall activity (oxidative phosphorylation, TCA cycle, RNA polymerase) – consistent with iron being a more energetic electron acceptor than fumarate. For A. dehalogenans, our analyses suggested which cytochromes are iron reductases (A2cp1_0127 and A2cp1_1731). Additional studies such as heterologous expression or knock out mutations will be needed to prove this function. BIOGRAPHICAL SKETCH Qiaochu had obtained a Bachelor of Engineering degree from Southwest Jiaotong University in China before he came to the U.S. for his graduate study at Cornell University in 2013. He knew that he would probably put his whole career in environmental engineering when he gave up the chance to switch his major from environmental engineering to electrical engineering in his sophomore year. During his undergraduate study, his main concentration is technologies can be applied in wastewater treatment plants and his graduation project for bachelor’s degree is the technological design of a hospital wastewater treatment plant. He also did research in the application of enhanced coagulation technique in the removal of heavy metals from source water. After the first academic year of his M.S. program at Cornell University, he became interested in environmental proteomics, which made his way finishing this thesis research. Although his undergraduate study was more focusing on physical and chemical processes, which made him a novice in environmental microbiology, he made efforts to learn knowledge of microbiology, biochemistry, etc. to strengthen his background, enabling him to continue his proteomics research. Though it may not be a field to make money, he decided to stay in Environmental Engineering field and will try to find a chance to become a PhD student in this field. iii To my beloved Mum and Dad &Xiaowei iv ACKNOWLEDGEMENTS Firstly, I would like to give my appreciation to Prof. Ruth Richardson and Prof. James Gossett, for their unstoppable wisdoms and great patience in guiding me through the two- year academic journey and finally finished my thesis successfully. Their ways of thinking about problems are the most valuable things I have learned at Cornell University. Secondly, I would like to express my love and appreciation to my parents who have always been giving their endless love to me, supporting my education financially and spiritually. All these can never be paid back but I would try my best to guarantee their health and happiness. I would also like to thank my girlfriend for her love and accompanying, as well as her support and understanding when I face difficulties. I will try my best to let her feel the same. Thirdly, I want to give my special thanks to my colleagues Annie Otwell and Cristina Fernandez-Baca for their friendliness and patience in helping me to learn many laboratory techniques during my research, which truly benefit me a lot. Then I would like to thank all other faculty members of Environmental Processes: Prof. Leonard Lion, Prof. James Bisogni, Prof. Damian Helbling and Dr. Monroe Weber-Shirk for their selflessly spreading knowledge both inside the class and outside the class. v Last but not least, I want to give my thanks to all the people that have helped me, laughed with me and been my friend. This includes the entire Richardson Lab Group, all advisors and friends I met at Cornell University. vi TABLE OF CONTENTS ABSTRACT ................................................................................................................................................................. III BIOGRAPHICAL SKETCH ..................................................................................................................................... III ACKNOWLEDGEMENTS ......................................................................................................................................... V TABLE OF CONTENTS ......................................................................................................................................... VII LIST OF TABLES ........................................................................................................................................................ X LIST OF FIGURES ................................................................................................................................................... XII LIST OF ABBREVIATIONS .................................................................................................................................. XV CHAPTER 1. INTRODUCTION ............................................................................................................................. 1 1.1 URANIUM CONTAMINATION ....................................................................................................................... 1 1.2 BIOREMEDIATION OF URANIUM ................................................................................................................. 2 CHAPTER 2. BACKGROUND ................................................................................................................................. 4 2.1 DISSIMILATORY IRON REDUCTION............................................................................................................. 4 2.1.1 Concept ................................................................................................................................................... 4 2.1.2 Access to Iron ....................................................................................................................................... 4 2.1.3 Dissimilatory Iron Reducing Bacteria (DIRB) ....................................................................... 5 2.1.4 Uranium Reduction ........................................................................................................................... 6 2.2 DIRB IN BIOREMEDIATION AND BIOENERGY .......................................................................................... 7 2.2.1 Heavy Metal and Uranium Contamination .............................................................................. 7 2.2.2 Chlorinated Solvents Dechlorination ..................................................................................... 10 2.2.3 Microbial Fuel Cells ........................................................................................................................ 11 2.3 DIRBS IN THE ENVIRONMENT ................................................................................................................ 12 2.3.1 As Members of Microbial Communities ................................................................................ 12 2.3.2 Environmental Significance ........................................................................................................ 13 2.4 SELECTED DIRBS STUDIED IN THIS THESIS ......................................................................................... 15 2.4.1 Overview ............................................................................................................................................. 15 vii 2.4.2 Anaeromyxobacter dehalogenans 2CP-1 .............................................................................. 16 2.4.3 Shewanella oneidensis MR-1 ..................................................................................................... 20 2.4.4 Geobacter sulfurreducens PCA .................................................................................................. 23 2.4.5 Geobacter bemidjiensis Bem ...................................................................................................... 26 2.5 PROTEOMICS METHODS FOR STUDYING MICROORGANISMS............................................................... 28 2.5.1 Overview ............................................................................................................................................. 28 2.5.2 Comparison to Genomics Study ................................................................................................ 28 2.5.3 LC-MS/MS Based Proteomics .................................................................................................... 29 2.5.4 Proteomics Methods ...................................................................................................................... 30 CHAPTER 3. OBJECTIVES ..................................................................................................................................
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