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Genetic and Biochemical Analysis of a Conserved, Multi-Gene System Regulation Spore-Associated Proteins in Streptomyces Coelicolor

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Genetic and Biochemical Analysis of a Conserved, Multi-Gene System Regulation Spore-Associated Proteins in Streptomyces Coelicolor Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Fall 12-20-2019 Genetic and Biochemical Analysis of a Conserved, Multi-Gene System Regulation Spore-Associated Proteins in Streptomyces Coelicolor Joseph Sallmen Follow this and additional works at: https://dsc.duq.edu/etd Part of the Bacteriology Commons, Genetics Commons, and the Molecular Genetics Commons Recommended Citation Sallmen, J. (2019). Genetic and Biochemical Analysis of a Conserved, Multi-Gene System Regulation Spore-Associated Proteins in Streptomyces Coelicolor (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/1830 This One-year Embargo is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. GENETIC AND BIOCHEMICAL ANALYSIS OF A CONSERVED, MULTI-GENE SYSTEM REGULATING SPORE-ASSOCIATED PROTEINS IN STREPTOMYCES COELICOLOR A Dissertation Submitted to the Bayer School of Natural and Environmental Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctor of Philosophy By Joseph Wade Sallmen II December 2019 Copyright by Joseph Wade Sallmen II 2019 GENETIC AND BIOCHEMICAL ANALYSIS OF A CONSERVED, MULTI-GENE SYSTEM REGULATING SPORE-ASSOCIATED PROTEINS IN STREPTOMYCES COELICOLOR By Joseph Wade Sallmen II Approved October 1, 2019 ________________________________ ________________________________ Joseph R. McCormick, Ph.D. Jana Patton-Vogt, Ph.D. Professor of Biological Sciences Professor of Biological Sciences (Committee Chair) (Committee Member) ________________________________ ________________________________ Nancy Trun, Ph.D. Michael Cascio, Ph.D. Associate Professor of Biological Associate Professor of Chemistry and Sciences Biochemistry (Committee Member) (Committee Member) ________________________________ ________________________________ Phillip Reeder, Ph.D. Joseph R. McCormick, Ph.D. Dean, Bayer School of Natural and Professor and Chair of Biological Environmental Sciences Sciences Professor of Biological Sciences iii ABSTRACT GENETIC AND BIOCHEMICAL ANALYSIS OF A CONSERVED, MULTI-GENE SYSTEM REGULATING SPORE-ASSOCIATED PROTEINS IN STREPTOMYCES COELICOLOR By Joseph Wade Sallmen II December 2019 Dissertation supervised by Joseph R. McCormick, Ph.D. Streptomyces coelicolor is a Gram positive, filamentous soil dwelling bacterium that exhibits a complex life cycle including the transition from a vegetative mycelium to a sporulating, aerial mycelium. Early genetic studies identified two classes of genes that resulted in developmental blocks, bald and white. The bald (bld) phenotype occurs when colonies cannot produce aerial hyphae. White (whi) colonies exhibit incomplete sporulation and/or loss of production of the concurrently produced grey pigment. While some of the original mutants have been explored, the developmental functions of many of the identified genes are not well understood. Of particular interest is a multi-gene regulatory system, with multiple paralogs of each gene present in S. coelicolor chromosome, which are well conserved among other streptomycetes and morphologically complex actinomycetes. The three genes encode 1) a predicted helix-turn-helix protein iv (WhiJR-like proteins), 2) a small, acidic protein of unknown function (WhiJS-like proteins), and 3) an anti-sigma factor-like protein (WhiJA-like proteins). In order to explore the roles of these genes in development, one such gene system involved in the regulation of the spore-associated protein (sap) operon, sapCED, was analyzed. sapR (whiJR-like), sapS (whiJS-like) comprise a divergently transcribed operon from sasA (whiJA-like), which is the first gene in the sapCED operon. In order to investigate the potential roles of this gene system in the developmental regulation of this spore- associated protein operon, null mutants were isolated by recombineering. Single and double null mutants were isolated and the effects on expression were assayed by the extraction of spore-associated proteins and a transcriptional fusion of the sapCEDp to luciferase. These assays indicate that in the absence of sapR and sapS, (either individually or together) there was an increase in expression of the sapCED operon. However, a single deletion of sasA has no observed phenotype. To determine protein- protein interactions, sapR, sapS, and sasA were cloned into a bacterial adenylate cyclase two-hybrid (BACTH) system. Analysis suggests self-interactions of SapR, SapS, and SasA. SapR was shown to interact with SapS; and, SapS was shown to interact with SasA. These interactions, save for SapS interacting with SasA, were recapitulated in a homologous multi-gene system (SCO3421, SCO3423, SCO3424). Using an electrophoretic mobility shift assay, SapR has been shown to interact with the promoter region of sapCED. These results suggest a model for SapR and SapS in repressing the development-associated sapCED operon, while SasA may function as a feedback mechanism through its interactions with SapS. Additionally, nine spore associated- v proteins were identified in common laboratory strains of S. coelicolor that may play important roles for nutrient harvesting during germination. vi DEDICATION This dissertation is dedicated to the memory of Mr. John Thompson. Mr. Thompson, or Mr. T as he was often called, was my Biology teacher in Neshannock High School. He inspired me, and many others, with his joyfulness and fervent passion for biology and education. Almost every morning and many an afternoon, we would spend in his classroom/laboratory studying, thinking, and chatting, all the while he would be setting up demonstrations or explaining difficult concepts. He always made time for us and always tried to give us his full and undivided attention when we were in need of it. His dedication to his craft and his unwavering commitment to excellence imparted upon me a sincere life-long interest in the biological sciences and a robust desire to educate people. If it were not for his palpable love for biology and education, I genuinely do not believe that I would have pursued an education in biology, notwithstanding a Ph.D. Thus, in honor of his memory and his role in my life, I dedicate this dissertation to him. vii ACKNOWLEDGEMENT There is no shortage of people that played a role in my graduate school career and certainly more than I could ever recount here, but despite this, I will do my best to acknowledge those that have made an impact on me and shaped my career at Duquesne and my life going forward. I will begin by thanking and acknowledging my mentor, Dr. Joseph R. McCormick. He was instrumental in shaping my research outlook and style, in how I think about scientific problems and how I execute the solutions to those problems. His mentorship has invariably left a mark on my career and pathos as a scientist and certainly will continue to shape my viewpoints, long after I have left Duquesne. Next, I must acknowledge the thoughtful role that my committee has played throughout my graduate career. Drs. Michael Cascio, Jana Patton-Vogt, and Nancy Trun have always provided thoughtful and constructive criticism to my work, in addition to meaningful discussions that have helped shape my perspective on my own science and how that relates to others. In addition, each of them have been incredibly supportive and helpful, whether by writing letters of recommendation, providing resources, keeping me informed on outside opportunities, and just in general, a wealth of inspiration. I should also include, all the faculty in the Department of Biological Sciences, especially Drs. Benedict Kolber, Michael Jensen-Seaman, Kyle Selcer, David Lampe, John Stolz, Dr. Allyson O’Donnell all who provided not only their time, but their resources, in helping me through my time in graduate school. In addition to the faculty, many people from across the Bayer School and Duquesne have been incredibly important to my progress and education as a graduate viii student. Dean Phillip Reeder and before him, Dean David Seybert, were always supportive of my place at Duquesne and through the Assistant Dean, Phillip Palmer, supportive of my initiatives, such as creating the BSNES Association of Graduate Students (BAGS). Through the Center for Teaching Excellence, Drs. Steven Hansen and Erin Rentschler, along with Christina Frasher, helped to instill the importance of University level teaching through my completion of the Certificate and Advanced Certificate of University Teaching. They provided thoughtful help and criticism of my teaching portfolio and work and for that I am very grateful. Finally, I must thank all of the support personnel in the Bayer School from the machine shop (Dan Bodner, Chris Lawson, and Lance Crosby) who never let me down when it came to fixing broken lab equipment to all of the paperwork and advising people in the Dean’s Office (Heather Costello, Denise Veselic, and Amie Walters) or the computer support of John Astleford to all of the day to day support from the office in the Department of Biological Sciences (Pamela Ferchak, Terri Widenhofer, Judy Quinque, and all the student aides). A special thanks to Drs. Jason Heming and Lisa Ludvico, and Mrs. Lalitha Rajakumar who have all been incredible mentors in teaching. Additional thanks to Dr. Heming for his constant support as a friend and always had whatever supplies I could ever need. I was just a small part in a much larger group
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