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Francisella Tularensis Insertion Sequence Elements Contribute to Differential Gene Expression University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-5-2018 Francisella tularensis Insertion Sequence Elements Contribute to Differential Gene Expression Amanda M. Bartling University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Pathogenic Microbiology Commons Recommended Citation Bartling, Amanda M., "Francisella tularensis Insertion Sequence Elements Contribute to Differential Gene Expression" (2018). Theses & Dissertations. 285. https://digitalcommons.unmc.edu/etd/285 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. 1 FRANCISELLA TULARENSIS INSERTION SEQUENCE ELEMENTS CONTRIBUTE TO DIFFERENTIAL GENE EXPRESSION. by Amanda Marie Bartling A THESIS Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of Requirements for the Degree of Master of Science Pathology and Microbiology Graduate Program Under the Supervision of Professor Marilynn A. Larson University of Nebraska Medical Center Omaha, Nebraska December 2017 Advisory Committee: Marilynn A. Larson, PhD Steven H. Hinrichs, MD Paul D. Fey, PhD ii FRANCISELLA TULARENSIS INSERTION SEQUENCE ELEMENTS CONTRIBUTE TO DIFFERENTIAL GENE EXPRESSION. Amanda M. Bartling, M.S. University of Nebraska Medical Center, 2018 Advisor: Marilynn A. Larson, PhD Francisella tularensis is the causative agent of the disease tularemia and a select agent. The genome of this pathogen contains many insertion sequence (IS) elements with ISFtu1 being the most abundant. ISFtu1 expression and the contribution of this IS element to differential gene expression in the F. tularensis subpopulations was evaluated. Full-length and truncated ISFtu1 sense and antisense transcriptional expression was detected. The prototype A.I strain (Schu S4) had considerably higher expression levels of ISFtu1 and the adjacent genes than the wild type A.I strain (NE061598). The A.II strains (WY96-3418 and WY-00W4114) had similar expression levels for ISFtu1 and the adjacent genes. In the highly virulent A.I strains, a bicistronic transcript encoding the universal stress protein (Usp) and a downstream ISFtu1 was highly expressed during early and mid log growth phase when provisions were plentiful, and was induced 2-fold by nitric oxide and a polyamine. During late log growth and stationary phase, only monocistronic transcripts for usp and ISFtu1 were being moderately expressed. Intrinsic transcription termination sequences were not apparent between usp and ISFtu1. Secondary RNA structure models indicated that the bicistronic transcript will form more readily than the monocistronic usp transcript. The co-expression of ISFtu1 and the adjacent gene(s) may provide a fitness advantage by regulating expression and/or transcript stability of the co-transcribed gene(s). Additional study will provide a better understanding of the contribution of IS elements to the differential gene expression and virulence observed for the various F. tularensis clades. ii TABLE OF CONTENTS ABSTRACT. .................................................................................................................... ii TABLE OF CONTENTS .................................................................................................. ii LIST OF FIGURES ......................................................................................................... iv LIST OF TABLES ........................................................................................................... vi LIST OF ABBREVIATIONS ........................................................................................... vii INTRODUCTION ............................................................................................................ 1 Mobile Genetic Elements in Bacteria........................................................................... 1 Insertion Sequence Elements ..................................................................................... 6 Regulation of IS Elements in Bacteria ......................................................................... 7 Mobile Genetic Elements Influence on Virulence .......................................................10 Francisella tularensis Classification and Historical Perspective ..................................11 Francisella Pathogenicity ...........................................................................................15 Francisella and Insertion Sequence Elements ...........................................................20 MATERIALS AND METHODS .......................................................................................25 Bacterial Strains and Culturing ...................................................................................25 RNA Isolation .............................................................................................................27 Nucleic Acid Quantitation ...........................................................................................28 DIG Labeled Probe Synthesis ....................................................................................28 Northern Blot Analysis ...............................................................................................29 DNase Treatment of RNA ..........................................................................................32 Ribosomal RNA Depletion of RNA .............................................................................32 RNA-Seq Library Synthesis .......................................................................................33 Purification of cDNA for RNA-Seq ..............................................................................35 Reverse transcription-PCR ........................................................................................36 5’ Rapid Amplification of cDNA Ends (RACE) ............................................................38 In silico Analysis ........................................................................................................39 RESULTS ......................................................................................................................43 F. tularensis Genomic Content ...................................................................................43 F. tularensis Growth Curves .......................................................................................45 ISFtu1 Open Reading Frame Analysis .......................................................................49 Reverse transcription PCR (RT-PCR) of ISFtu1 transcripts .......................................54 Northern Blot Analysis of ISFtu1 ................................................................................59 iii Differential Expression and Co-Expression of ISFtu1 with Adjacent Genes ...............62 Genes Co-transcribed with ISFtu1 ........................................................................... 102 Genetic Analysis of Co-transcribed Universal Stress Protein and ISFtu1 ................. 111 DISCUSSION .............................................................................................................. 124 F. tularensis Genotypic and Phenotypic Comparisons ............................................. 124 F. tularensis Type A.I Response to Nitric Oxide ....................................................... 125 F. tularensis Type A.I Response to Polyamine ......................................................... 126 F. tularensis Insertion Sequence Elements .............................................................. 129 ISFtu1 Transcript Analysis ....................................................................................... 130 Co-expression of ISFtu1 and Adjacent Genes ......................................................... 131 Transcriptional Expression of Bicistronic ISFtu1 and the Universal Stress Protein ... 134 REFERENCES ............................................................................................................ 136 iv LIST OF FIGURES Figure 1: Recombination between two insertion sequence elements ...................... 8 Figure 2: Growth curves of various F. tularensis strains .........................................47 Figure 3: Growth of wild type A.I F. tularensis NE061598 in the absence or presence of nitric oxide producing SNAP or the polyamine spermine ...................48 Figure 4: F. tularensis ISFtu1 open read frame, including nucleotide and protein sequence ......................................................................................................................51 Figure 5: Alignment of representative open reading frames for the ISFtu1 protein in F. tularensis type A.I Schu S4, A.II strain WY96, and attenuated type B LVS .....52 Figure 6: Representative F. tularensis ISFtu1 gene showing direct and inverted repeats .........................................................................................................................53 Figure 7: RT-PCR detection of 5', middle, and 3' region of expressed ISFtu1 RNA in F. tularensis A.I and A.II strains. ................................................................................55 Figure 8: RT-PCR detection of full length and truncated ISFtu1 transcripts in F. tularensis type A.I strains Schu S4
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