P1 BACTERIOPHAGE AND TOL SYSTEM MUTANTS Cari L. Smerk A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2007 Committee: Ray A. Larsen, Advisor Tami C. Steveson Paul A. Moore Lee A. Meserve ii ABSTRACT Dr. Ray A. Larsen, Advisor The integrity of the outer membrane of Gram negative bacteria is dependent upon proteins of the Tol system, which transduce cytoplasmic-membrane derived energy to as yet unidentified outer membrane targets (Vianney et al., 1996). Mutations affecting the Tol system of Escherichia coli render the cells resistant to a bacteriophage called P1 by blocking the phage maturation process in some way. This does not involve outer membrane interactions, as a mutant in the energy transucer (TolA) retained wild type levels of phage sensitivity. Conversely, mutations affecting the energy harvesting complex component, TolQ, were resistant to lysis by bacteriophage P1. Further characterization of specific Tol system mutants suggested that phage maturation was not coupled to energy transduction, nor to infection of the cells by the phage. Quantification of the number of phage produced by strains lacking this protein also suggests that the maturation of P1 phage requires conditions influenced by TolQ. This study aims to identify the role that the TolQ protein plays in the phage maturation process. Strains of cells were inoculated with bacteriophage P1 and the resulting production by the phage of viable progeny were determined using one step growth curves (Ellis and Delbruck, 1938). Strains that were lacking the TolQ protein rendered P1 unable to produce the characteristic burst of progeny phage after a single generation of phage. E. coli strains containing the paralogous ExbB were also unable to produce viable phage progeny in the absence of TolQ, suggesting that this role in phage maturation is unique to the TolQ protein. This role is also independent of the energy harvesting function of TolQ, as a strain containing an energetically inactive TolQ protein with a iii point mutation are able to produce enough viable progeny in one generation of phage to constitute a burst. This data suggests that there is some unique, undetermined function of TolQ that is parasitized by the P1 bacteriophage in order to mature and produce viable, infectious progeny. iv ACKNOWLEDGEMENTS The fulfillment of the requirements for my Masters degree could not have been made without the love, help, and contributions of many people along the way. I would like to take this opportunity to thank those people who have supported me throughout this process. I would first like to thank my advisor, Dr. Ray Larsen. Without your continuous guidance throughout this entire project, from start to finish, the significance of this research may never have been recognized. I would also like to thank you for your help writing this document. Without your seemingly endless knowledge of research that others have done, we may never have seen the culmination of this project either. I would also like to thank my entire committee, Dr. Tami Steveson, Dr. Lee Meserve, and Dr. Paul Moore for their support and collective guidance over the past two years (some of you much longer than that). The time and knowledge you have all provided has given me a great deal of help along the way, and inspired me to always look for the bigger picture. I would also like to thank Dr. George Bullerjahn for his support and assistance throughout this project. Though he was not a member of the committee, his scientific experience and input helped to work out some of the protocols I needed to develop for this project. My sincerest thanks go out to my family—my parents, Jerry and Linda Byrd; my brothers, Collin, Chad, and Tony Smerk; and my grandparents, Dan and Kay Klonk. Without the constant love and support of my family, I would have never lasted six years at BGSU and become the person that I am today, both academically and personally. I am so grateful to all of you for being there for me when I needed advice, support, or just the unconditional love that you have always given me. Thank you, Mom and Dad, for attending my defense, one of the most v important academic moments in my life thus far. It meant more to me than you will know to have both of you sitting in the audience and waiting to hear what happened afterwards. I never would have survived the endless frustration of failed experiment after failed experiment if not for the humor and wisdom provided by my labmates. Dr. Kerry Brinkman and Dr. Kimberly Keller have been a part of my research experience from day one, and without the love, advice, and lunches had at B-dubs, this degree would have never come to fruition. To all of the friends that I have made while in Bowling Green, Ohio, I thank you. To Tori Comstock, Luann Carpenter, and Chad Green, I appreciate most of all that you all listened to all the complaints over the last four summers we have worked together. Though I am moving on to bigger and (maybe) better employment ventures, I hope that we will remain friends. To Erica Hertzfeld, even though we never get to sit and talk, when we do, I know that you will be an ear to listen and a shoulder to cry on, if need be. This masters thesis was funded by the National Science Foundation (MCB-0315983) and the Center for Biomolecular Sciences at Bowling Green State University, and Dr. Ray Larsen. Thank you to BGSU, the BGSU Department of Biological Sciences, and to the Graduate College for allowing me to perform and complete this research project. vi TABLE OF CONTENTS INTRODUCTION TO BACTERIOPHAGE CHAPTER ONE: INTRODUCTION …………………………………………………………..1 CHAPTER TWO: SPECIFIC AIM ONE VERIFY THE ESSENTIAL ROLE OF THE TOLQ PROTEIN IN THE P1 LYTIC CYCLE…………………………………………………………….……………...…12 Introduction……………………………………………………………………………...12 Materials and Methods…………………………………………………………………..13 Results……………………………………………………………………………………17 Discussion………………………………………………………………………………..26 CHAPTER TWO: SPECIFIC AIM TWO USE MORE SENSITIVE AND SPECIFIC ASSAYS TO CONFIRM THE ROLE OF TOLQ AND VARIOUS REGIONS OF THE TOLQ PROTEIN IN THE PRODUCTION OF VIABLE P1……………………………………….………………30 Introduction………………………………………………………………………………30 Materials and Methods…………………………………………………………………...31 Results……………………………………………………………………………………33 vii Discussion………………………………………………………………………………..40 CHAPTER FOUR: BROADER IMPLICATIONS, FUTURE DIRECTIONS, AND CHARACTERIZATION OF P1 AND THE ROLE OF TOLQ IN ITS LYTIC CYCLE…………………………………………….......43 REFERENCES…………………………………………………………………………………..50 viii LIST OF FIGURES Figure Page 1 The Tol system of Escherichia coli. …………………...………………………….. 8 2 Growth profile of wild type bacterial cells (W3110) incubated with P1 at various dilutions. …………………………………………………………… 20 3 Growth profile for tolQRA deletion strain (RA1017) incubated with P1 at various dilutions……………………………………………………………… 21 4 Preliminary growth curve showing RA1004 (W3110 tolR::cm)…………………… 22 5 Growth profile of an E. coli tolA- strain (RA1009) incubated with P1……………. 23 6 Growth profile of TPS66 (tolQ-G181D)…………………………………………… 24 7 Example of a one step growth curve for Yersinia enterocolitica phage φYeO3-12… 31 8 One step growth curve of the wild type strain, W3110……………………………… 34 9 One step growth curve of RA1051 (ΔtolQRA, exbBD)……………………………… 35 10 One step growth curve of RA1035 (ΔtolQR)………………………………………… 36 11 One step growth curve of TPS66 (tolQG181D)……………………………………… 38 12 One step growth curve of RA1034 (ΔtolR, exbBD)……………………………… 39 13 One step growth curve of RA1044 (ΔtolR, exbB)………………………………… 40 ix LIST OF TABLES Table Page 1 Host range of P1……………………………………………… 5 2 Description of strains used in all experiments, including relative genotypic deletions from the chromosome and phenotypic protein expression………….………………… ………………………...... 13 3 Results of colicin and phage assays indicate the level of activity as compared to the wild type strain for the TolA system (ColA) and the TonB system (φ80)……………………………………………… 19 4 Phage and cell quantification……………………………………………. 26 1 CHAPTER ONE INTRODUCTION Viruses are infectious agents that are obligate intracellular parasites, taking over a host cell in order to replicate. Bacteriophages are viruses that infect hosts of the kingdoms Bacteria and Archaea. Consistent with the differences between their hosts, bacteriophages are very distinct from viruses of eukaryotes. Within bacteriophages, different life cycles, morphologies, and host ranges exist, and most bacteria have bacteriophages that are capable of infecting them. Despite their number and diversity, very few bacteriophages have been studied in great detail. Those that have interested researchers, however, have provided many insights into fundamental processes in nature. Since their discovery in the early 1900s, researchers have found that bacteriophages make many contributions to various aspects of biology, from playing a key role in the ecological impact of nutrient cycling, to their evolutionary influence on bacterial genomes (Chennoufi et al., 2004). From an ecological perspective, bacteriophages appear to play a substantial role in the growth and evolution of bacteria in their natural habitats, and it is likely that selection for phage resistance, as well as the development of anti-phage defenses, has shaped the bacterial properties we see today (Campbell, 1994). Bacteriophages have also been presented as a potential substitute for antibiotics, and in recent years, “phage therapy” has grown in popularity. In the years following their discovery, phage research focused mainly on the idea of using bacteriophages to combat bacterial disease, however, their most direct application to medical bacteriology has been to use them as reagents for typing bacterial strains (Campbell, 1994; Summers, 2005). This importance in the medical field has played a crucial role in the advancement of the knowledge gained about phages in the past century. 2 A select few bacteriophages have played an integral part of the development of molecular biology. Early work with the Escherichia coli phage T4 provided a fundamental understanding of the nature of mutations and mechanisms of genetic recombination (Benzer, 1955; Benzer, 1959).