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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 CHARACTERIZATION OF THE PSEUDOMONAS AERUGINOSA PILT GENE WHICH IS REQUIRED FOR TWITCHING MOTILITY AND PILUS FUNCTION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Patricia Lynn Antalis, B. S. * * * * * The Ohio State University 1996 Dissertation Committee: Approved by Kathleen E. Kendrick, Adviser Aldis Darzins Neil R. Baker Adviser Charles J. Daniels Department of Microbiology UMI Number: 9630843 UMI Microform 9630843 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 DEDICATION To my husband, and best friend, George ACKNOWLEDGMENTS I would like to express my appreciation to each of my committee members for their guidance, especially my advisers Dr. Aldis Darzins and Dr. Kathleen Kendrick. Thanks also go to Lynn O'Donnell and Mary Russell for their camaraderie, advice and wit. I am indebted to family for their continual support, especially my sister and brother-in-law for their generosity and humor during this last year. I am most grateful to my husband for his patience and for his reminding me of what is truly important. VITA November 12,1966 ......................Bom - Wheeling, West Virginia 1989............................................. B. S. in Biology, The Ohio State University, Columbus, Ohio 1987 ............................................... Research Assistant, Department of Zoology, The Ohio State University 1987 -1989...................................Research Assistant, Department of Entomology, The Ohio State University 1989 - present...............................Graduate Teaching Associate, Department of Microbiology, The Ohio State University FIELDS OF STUDY Major Field: Microbiology Studies in Bacterial Gene Regulation Dr. K. E. Kendrick and Dr. A. Darzins, Advisers TABLE OF CONTENTS DEDICATION.................................................................. ACKNOWLEDGMENTS................................................. VITA................................................................................ TABLE OF CONTENTS................................................. LIST OF FIGURES........................................................... LIST OF TABLES............................................................ LIST OF ABBREVIATIONS............................................ CHAPTER I: Introduction ........................................... Role of Pseudomonas aeruginosa pili in adherence Structure of P. aeruginosa pilin............................. P. aeruginosa pilus biogenesis .............................. Occurrence of type IV pili in other bacteria .......... Pilus retraction ..................................................... Twitching motility Goals of this project................................................................................. 39 CHAPTER II: Materials and Methods........................................................... 40 Media and culture conditions .................................................................. 40 Bacterial strains, plasmids, and bacteriophages......................................41 Phage sensitivity and twitching motility assays................................... 46 Electron microscopy techniques ....................................................... 47 Mapping of the HOD1 phage-resistance m utation................................. 48 LDgo studies ................................................................................................ 48 Standard DNA isolation and manipulation techniques ........................... 49 Transfer of plasmid DNA intoPseudomonas ......................................... 52 Generation of nested deletions for DNA sequence analysis .................... 54 DNA hybridization methods .................................................................... 55 Standard methods for protein analysis..................................................... 58 Overexpression ofpilT in E.coli and Pseudomonas using T7 promoter-based systems.............................................................60 RNA isolation and manipulation ............................................................. 61 PCR conditions for generation of SI nuclease probes ..............................66 Enzyme assays ......................................................................................... 67 Generation of random mutations in p ilT.................................................. 69 CHAPTER III: pilT is Required for Phage Sensitivity and Twitching Motility in P. aeruginosa P A O l................................................. 71 Introduction ............................................................................................ 71 vi R esults ........................................................................................................73 Isolation of phage-resistant mutants of PAOl defective in pilus biogenesis ................................................................73 Complementation of the hyperpiliated m utants......................76 Conjugations! mapping of the HOD1 phage-resistance locus ..................................................................................... 80 Overexpression ofpilT in E.coli and Pseudomonas using T7 promoter-based system s ...............................................82 Nucleotide sequence of the pilT open reading frame............... 90 Disruption of pilT in PA O l......................................................... 90 Characterization of the mutated pilT allele from HOD1 95 Generation of pilT mutants by using random mutagenesis . 99 Virulence of HOD1 in a mouse infection model ...................... 104 Discussion ............................................................................................... 107 CHAPTER IV: Construction and Analysis of pilU and pilT pilU Mutants in P. aeruginosa P A O l ........................................................... 130 Introduction ................................................................................................130 R esults ..................................................................................................... 132 Introduction of pilU into the pilT mutant HOD1 ................ 132 Construction and complementation analysis of a pilU mutant ........................................................................ 133 Construction and complementation analysis of a pUTpilU double m u ta n t ................................................... 140 Discussion................................................................................................ 145 CHAPTER V: Expression ofpilT is Differentially Regulated at the Transcriptional Level...............................................................147 Introduction ................................................................................................147 Results .......................................................................................................149 Northern analysis of the pilT transcript................................. 149 pilT expression does not require the alternative sigma factor RpoN.............................................................152 Identification of promoter activity in thepilT pilU intergenic region................................................................ 152 Identification of the region required for pilT promoter activity............................................................................... 155 Quantitative analysis of pilT expression on solid and liquid m edia ................................................................ 162 Mapping of the transcription start sites ofpilT and analysis of transcript populations.....................................168 Identification of potential regulatoiy regions upstream ofp ilT................................................................................. 182 Discussion ................................................................................................
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