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Post-Transcriptional Regulation of Rsma in Pseudomonas Aeruginosa Ian Miller East Tennessee State University

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Post-Transcriptional Regulation of Rsma in Pseudomonas Aeruginosa Ian Miller East Tennessee State University East Tennessee State University Digital Commons @ East Tennessee State University Electronic Theses and Dissertations Student Works 8-2018 Post-transcriptional Regulation of RsmA In Pseudomonas aeruginosa Ian Miller East Tennessee State University Follow this and additional works at: https://dc.etsu.edu/etd Part of the Biology Commons, and the Microbiology Commons Recommended Citation Miller, Ian, "Post-transcriptional Regulation of RsmA In Pseudomonas aeruginosa" (2018). Electronic Theses and Dissertations. Paper 3481. https://dc.etsu.edu/etd/3481 This Thesis - Open Access is brought to you for free and open access by the Student Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact [email protected]. Post-transcriptional Regulation of RsmA In Pseudomonas aeruginosa _______________ A thesis presented to the faculty of the Department of Biology East Tennessee State University In partial fulfillment of the requirements for the degree Master of Science in Biology _______________ by Ian Miller August 2018 _______________ Dr. Christopher Pritchett, Chair Dr. Bert Lampson Dr. Michael Kruppa Keywords: Pseudomonas aeruginosa, RsmA, mRNA stem-loop ABSTRACT Post-transcriptional Regulation of RsmA In Pseudomonas aeruginosa by Ian Miller Pseudomonas aeruginosa is an infectious Gram-negative bacillus that is found in a variety of environments. Gene regulatory mechanisms control the expression of virulence factors that allow P. aeruginosa to initiate acute infections and persist as a chronic infection of its host. Two- Component Systems (TCS), transcriptional and post-transcriptional regulators, small non-coding RNAs (sRNA), mRNA secondary structure, and others are involved in the coordination of specific virulence genes. A significant post-transcriptional regulator involved in this regulatory network is the Regulator of Secondary Metabolites (RsmA). In this study, we investigated the contribution of a putative stem-loop has on expression of RsmA using bioinformatics and molecular techniques. We constructed rsmA leader fusions to measure translation with and without the secondary structure present. Secondly, we introduced point mutations in the stem of the stem-loop of the leader fusions to disrupt the formation of the stem-loop. Finally, we performed Site-Directed Mutagenesis on the rsmA leader to examine protein levels in vivo by western blot analysis using an HA-tagged rsmA. Our data suggests that the segment of RNA that contains the putative stem-loop structure serves some function in post-transcriptional regulation of RsmA. 2 ACKNOWLEDGMENTS I would like to thank my committee for being patient with me as I am a procrastinator. Thank you Dr. Kruppa for providing teaching moments during my seminars and committee meetings. Dr. Lampson, for initiating my first experience with molecular biology research in Dr. Pritchett’s lab during my undergraduate career and thank you for serving on my committee. A special thanks to Dr. Pritchett for putting up with me for a brief time in undergrad and allowing me to work on this novel project as a graduate student. Thank you to all of my educators for sharing your knowledge with me. A big thanks to Dr. Sean Stacey and Danielle Williams for everything you all have done for me. You all are excellent lab mates and more importantly, great friends. I will miss Friday afternoon half-priced margaritas at Amigo’s with you all. Dr. Fox, for providing food at our meetings and endless conversation about running. Thank you, Robin Grindstaff, for maintaining our teaching labs and media. Thank you to Brenda Watson and Dolores Britt for always being kind. I owe a big thanks to Eva Psimer for teaching me how to relax and breathe during the tough times. Thanks to Caity Baker, Walter Witt, Michael Hawkins, and Jessie Lewis for your hospitality. Thanks to the ETSU Molecular Biology Core for sequencing our plasmids and ordering our primers. Funding for this research was provided by Major and Minor RDC Grants. 3 TABLE OF CONTENTS Page ABSTRACT .....................................................................................................................................2 ACKNOWLEDGMENTS ...............................................................................................................3 LIST OF TABLE .............................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 Chapter 1. INTRODUCTION .......................................................................................................................9 Pseudomonas aeruginosa ....................................................................................................9 Pseudomonas aeruginosa Virulence..................................................................................10 RsmA and Post-Transcriptional Regulation ......................................................................13 Gene Regulation by RNA & RNA Secondary Structures .................................................17 Regulation of RsmA ..........................................................................................................21 2. MATERIALS AND METHODS ...............................................................................................25 Bacterial Strains and Growth Conditions ..........................................................................25 Colony PCR .......................................................................................................................25 Genomic DNA Isolation ....................................................................................................26 Plasmid DNA Isolation ......................................................................................................26 Site-Directed Mutagenesis .................................................................................................28 For Deletion ...........................................................................................................28 For Substitution ......................................................................................................30 Vector Digest and Ligation ................................................................................................30 Transformation ...................................................................................................................31 4 Triparental Mating .............................................................................................................31 Insertion of Translational Leader Fusion ...........................................................................32 Insertion of Deletion Mutation...........................................................................................33 β-Galactosidase Assay .......................................................................................................34 Western Blot Analysis .......................................................................................................35 Statistical Analysis .............................................................................................................37 3. RESULTS ..................................................................................................................................38 The rsmA mRNA Transcript is Under Post-Transcriptional Regulation ...........................38 The 5’ Untranslated Region of the RsmA Transcript Contains Secondary Structures ......40 The Putative Distal Stem-loop Contributes to the Post-transcriptional Regulation of rsmA ............................................................................................................................................42 Translation of the RsmA Transcript is Not Attributed to the GacS/GacA TCS or RsmA 43 The Putative Stem-loop is Not a RNA Thermometer ........................................................44 Western Blot Analysis Provides Insight into RsmA Levels Among 5’ UTR Mutants ......45 4. DISCUSSION ............................................................................................................................48 Leader Fusion Analysis and Bioinformatics Provide Evidence of Post-transcriptional Regulation ..........................................................................................................................49 Western Blot Reveals that Regulation of RsmA is Multi-tiered ........................................51 REFERENCES ..............................................................................................................................54 APPENDICES ...............................................................................................................................59 Appendix A: Media............................................................................................................59 Appendix B: Buffers ..........................................................................................................61 5 Appendix C: Supplemental Material .................................................................................64 Stem-loop Structures ..............................................................................................64 Table
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