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Studies of Phou in Escherichia Coli: Metal Binding, Dimerization,Protein/Protein Interactions, and a Signaling Complex Model

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Studies of Phou in Escherichia Coli: Metal Binding, Dimerization,Protein/Protein Interactions, and a Signaling Complex Model Brigham Young University BYU ScholarsArchive Theses and Dissertations 2014-12-01 Studies of PhoU in Escherichia coli: Metal Binding, Dimerization,Protein/Protein Interactions, and a Signaling Complex Model Stewart G. Gardner Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd BYU ScholarsArchive Citation Gardner, Stewart G., "Studies of PhoU in Escherichia coli: Metal Binding, Dimerization,Protein/Protein Interactions, and a Signaling Complex Model" (2014). Theses and Dissertations. 5685. https://scholarsarchive.byu.edu/etd/5685 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Studies of PhoU in Escherichia coli: Metal Binding, Dimerization, Protein/Protein Interactions, and a Signaling Complex Model Stewart G. Gardner A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy William R. McCleary, Chair Joel Shelby Griffitts Brent Lynn Nielsen Julianne House Grose Zachary Thomas Aanderud Department of Microbiology and Molecular Biology Brigham Young University December 2014 Copyright ©2014 Stewart G. Gardner All Rights Reserved ABSTRACT Studies of PhoU in Escherichia coli: Metal Binding, Dimerization, Protein/Protein Interactions and a Signaling Complex Model Stewart G. Gardner Department of Microbiology and Molecular Biology, BYU Doctor of Philosophy Phosphate is an essential nutrient for all forms of life. Escherichia coli has a PhoR/PhoB two component regulatory system that controls the expression of various genes whose products allow the cell to thrive in low phosphate environments. The signaling mechanism of the PhoR/PhoB system has been studied and the phosphorylation cascade that controls gene expression is well understood. What is still unknown is how PhoR senses the phosphate level of the environment. The PstS, PstC, PstA, PstB, and PhoU proteins play a role in this signal sensing. This work confirms the hypothesis that the PstSCAB complex senses the environmental phosphate and that phosphate signal is passed through PhoU to PhoR. Further, this work characterizes residues important for interaction on PhoU and PhoR and identifies a structural model for interaction. This model points to a potential mechanism for PhoU mediated signaling to PhoR. We tested this model with direct coupling analysis and obtained further confirmation. Further use of these techniques may elucidate more of the interactions necessary for proper phosphate signaling. Keywords: PhoR, PhoU, PstSCAB, Pho regulon, two component signal transduction, PAS domain, direct coupling analysis, protein structure modeling ACKNOWLEDGEMENTS I would like to thank Dr. William R. McCleary for his guidance, encouragement, and mentoring throughout my academic career. I would also like to thank my advisory committee: Drs. Joel Griffitts, Brent Nielsen, Julianne Grose, and Zachary Aanderud. Thanks in addition to Dr. Allen R. Buskirk for serving on my committee. I would also like to thank Dr. Perry Ridge and Dr. Byron Adams for help with bioinformatic and phylogenetic analyses. Thanks to Casey Callison, Kristi Johns, and all the other students who have worked with me in Dr. McCleary’s laboratory. Most importantly, thanks to my wife Jamie Gardner, for her support, love, and confidence. For the research presented in Chapter 2 “The PhoU protein from Escherichia coli interacts with PhoR, PstB and metals to form a phosphate-signaling complex at the membrane” (1). I thank my co-authors: Kristine D. Johns, Rebecca Tanner, and William R. McCleary Additionally, I thank Dr. John Bell, Jen Nelson, and Liz Gibbons for their assistance with the fluorescence assays and Lance Moses for help with the ICP-MS analysis. I thank several undergraduate students; Geoffrey Johnston, Rachel Winn, Austin Callison, and Annaliese Gabrielle for help in the initial characterization of the mutant strains and Casey Callison, Kirk Richardson, Michael Barrus, and Gregory Bowden for excellent technical assistance. That work was supported by Public Health Service grant R15GM96222 from the National Institute of General Medical Sciences. R. T. was partially supported by an Undergraduate Mentoring Environment Grant from Brigham Young University. For the research in Chapter 3 “Genetic analysis, structural modeling, and direct coupling analysis suggest a mechanism for phosphate signaling in Escherichia coli”, I thank my co- authors: Justin B. Miller, Tanner Dean, Tanner Robinson , McCall Erickson, Perry Ridge, and William R. McCleary. Also, I thank Kathryn Hanks, Alex Cummock, Evan Christensen, Bethany Evans, Gregory Bowden, and Michael Barrus for help with sequence collection and mutant characterization. This work was supported by Public Health Service grant R15GM96222 from the National Institute of General Medical Sciences. Table of Contents Title Page ......................................................................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii Table of Contents ............................................................................................................................ v List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x Chapter 1 The Pho Regulon in Escherichia coli ............................................................................. 1 1.1 Introduction ........................................................................................................................... 1 1.1.1 Histidine Kinases ........................................................................................................... 2 1.1.2 Response Regulators ...................................................................................................... 7 1.1.3 PhoB and PhoR .............................................................................................................. 9 1.1.4 PstSCAB PhoU ............................................................................................................ 14 1.2 Regulation of the Pho Regulon ........................................................................................... 21 1.2.1 Crosstalk ...................................................................................................................... 22 1.2.2 Stochastic activation .................................................................................................... 22 1.2.3 Other Pho Regulon Genes ............................................................................................ 23 1.3 Pho Regulon and Pathogenesis ........................................................................................... 25 1.3.1 PhoU and Pathogenesis ................................................................................................ 26 1.4 Conclusions ......................................................................................................................... 27 v Chapter 2 The PhoU protein from Escherichia coli interacts with PhoR, PstB and metals to form a phosphate-signaling complex at the membrane ......................................................................... 29 2.1 Abstract ............................................................................................................................... 29 2.2 Introduction ......................................................................................................................... 30 2.3 Materials and methods ........................................................................................................ 33 2.3.1 Bacterial stains, growth media, and growth conditions. .............................................. 33 2.3.1.1 E. coli PhoU structure prediction .............................................................................. 36 2.3.1.2 Bacterial adenylate cyclase two-hybrid analysis ...................................................... 36 2.3.2 β-galactosidase activity assays. .................................................................................... 36 2.3.3 PstB/PhoU co-elution experiments .............................................................................. 37 2.3.4 PhoR/PhoU co-elution experiments ............................................................................. 38 2.3.5 Alkaline phosphatase (AP) and Western immunoblot assays...................................... 39 2.3.6 PhoU purification and gel filtration. ............................................................................ 39 2.3.7 Inductively coupled plasma mass spectrometry (ICP-MS) ......................................... 40 2.3.8 Fluorescence assays for metal binding. ....................................................................... 40 2.3.9 Integrated strain construction. .....................................................................................
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