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Acinetobacter Baumannii</Em> University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 4-5-2019 Investigating the Proteinaceous Regulome of the Acinetobacter baumannii Leila G. Casella University of South Florida, [email protected] Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the Microbiology Commons Scholar Commons Citation Casella, Leila G., "Investigating the Proteinaceous Regulome of the Acinetobacter baumannii" (2019). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/8345 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Investigating the Proteinaceous Regulome of the Acinetobacter baumannii by Leila G. Casella A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cell Biology, Microbiology, and Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Lindsey N. Shaw, PhD Prahathees Eswara, PhD James Riordan, PhD Burt Anderson PhD Date of Approval April 29, 2019 Keywords: Gene Regulation, Energy, Metabolism, Iron Copyright @ 2019, Leila G. Casella ACKNOWLEDGEMENTS First and foremost, to my mentor Dr. Lindsey Shaw, I want to thank you for having me in your lab and support me not only academically but in times that I was in much need of a hand. I wanted to thank you for those times that you have pushed me to develop my scientific thinking and become a better scientist. Dr Prahathees Eswara,Dr. Burt Anderson, and Dr. James Riordan thank you for your support and the insights provided that raised ideas and guided my projects. I would like to thank everyone in the Shaw lab including former members that have left to continue their journeys for your support and for those times of laughs that made easier the long days in the lab. To my mom, thank you for understanding and let me be away from you to pursue my goals without making me feel guilty, you know it was not an easy decision. TABLE OF CONTENTS List of Tables ................................................................................................................... iv List of Figures .................................................................................................................. v Abstract .......................................................................................................................... vii Chapter I: Introduction .....................................................................................................1 The Genus Acinetobacter ......................................................................................1 Acinetobacter baumannii History and Identifying Features ...................................2 The infections of A. baumannii ..............................................................................4 The Drug Resistance of A. baumannii ...................................................................7 The Mechanisms of Drug Resistance in A. baumannii ..........................................8 A. baumannii as a Public Health Treat ................................................................10 The Virulence Factors of A. baumannii ...............................................................11 Genome Plasticity of A. baumannii......................................................................15 Regulation in A. baumannii .................................................................................16 A. baumannii Metabolism ....................................................................................19 Metal Acquisition in A. baumannii........................................................................22 Project aim ..........................................................................................................24 Chapter II: Towards The Complete Proteinaceous Regulome of Acinetobacter baumannii…………………. ......................................................................................26 Note to Reader ....................................................................................................26 Chapter III: Identification of a Novel Two Component System Governing Membrane Energetics in Acinetobacter baumannii.....................................................................27 Abstract ...............................................................................................................27 Introduction .........................................................................................................28 Materials and Methods. .......................................................................................31 Bioinformatic analysis. ..............................................................................31 Strains and growth conditions...................................................................32 Construction of an arcAB-lacZ transcriptional fusion strain. .....................33 Construction of arcA and arcB complementation strains. .........................33 Transcriptional profiling assays using b-galactosidase. ............................34 Intracellular pH measurement...................................................................35 Membrane Depolarization Assay. .............................................................35 RNA Sequencing and Bioinformatic Analysis. ..........................................36 Quantitative real time PCR. ......................................................................37 Intracellular metabolite quantification assays. ..........................................38 i Antimicrobial susceptibility assays. ...........................................................39 Assaying ATP levels within cells. ..............................................................39 Determination of cytochrome oxidase activity...........................................40 Results. ...............................................................................................................40 Identification of ArcAB homologs in the genome of Acinetobacter baumannii. ............................................................................................... .40 Expression of the ArcAB TCS is induced upon disruption of the proton gradient in A. baumannii ...........................................................................45 Disruption of ArcAB impairs the PMF in A. baumannii. .............................47 Transcriptomic profiling reveals a shift in expression of genes required for glucose utilization upon arcA disruption. ..................................................49 Increased glycolytic influx in the arcA mutant favors activation of the Pta- AckA pathway generating acetate from pyruvate. ....................................53 arcA mutants of A. baumannii use an incomplete TCA Cycle, instead favoring the glyoxylate shunt. ...................................................................54 NADH levels are increased in the arcA mutant as a result of its hyperactive glyoxylate shunt.....................................................................57 The altered metabolic state of arcAB mutants favors ATP production via substrate phosphorylation.........................................................................58 Cytochrome oxidase activity is reduced as a result of arcAB disruption. ..60 The accumulation of oxaloacetate in arcA mutant cells leads to aminoglycoside hyper-susceptibility. ........................................................62 Discussion. ..........................................................................................................63 Chapter IV: Characterization of a novel fec-operon responsive to hemin in Acinetobacter baumannii ..........................................................................................69 Abstract ..............................................................................................................69 Introduction. ........................................................................................................70 Materials and Methods. ..................................................................................................73 Bioinformatic analysis .............................................................................. .73 Strains and growth conditions...................................................................74 Construction of a fecA-lacZ transcriptional fusion strain. ..........................74 Construction of fecR and hemO complementation strains. .......................75 Growth assays. .........................................................................................76 Transcriptional profiling assays using b-galactosidase. ............................76 RNA-seq analsyis. ....................................................................................77 qRT-PCR ................................................................................................. .78 Hemin measurements. .............................................................................79 Human serum survival assay....................................................................80 Results . ..............................................................................................................80 The FecI operon of A. baumannii has a distinct set of genes compared to homologs
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