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Bacteriophage T4 Lysis and Lysis Inhibition BACTERIOPHAGE T4 LYSIS AND LYSIS INHIBITION: MOLECULAR BASIS OF AN ANCIENT STORY A Dissertation by TRAM ANH THI TRAN Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2007 Major subject: Biochemistry BACTERIOPHAGE T4 LYSIS AND LYSIS INHIBITION: MOLECULAR BASIS OF AN ANCIENT STORY A Dissertation by TRAM ANH THI TRAN Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Ryland F. Young Committee Members, Deborah Siegele Paul Fitzpatrick Michael Polymenis Head of Department, Gregory D. Reinhart May 2007 Major subject: Biochemistry iii ABSTRACT Bacteriophage T4 Lysis and Lysis Inhibition: Molecular Basis of an Ancient Story. (May 2007) Tram Anh Thi Tran, B.S., Stephen F. Austin State University; M.S., Stephen F. Austin State University Chair of Advisory Committee: Dr. Ryland F. Young T4 requires two proteins: holin, T (lesion formation and lysis timing) and endolysin, E (cell wall degradation) to lyse the host at the end of its life cycle. E is a cytoplasmic protein that sequestered away from its substrate, but the inner membrane lesion formed by T allows E to gain access to the cell wall. T4 exhibits lysis inhibition (LIN), a phenomenon in which a second T4 infection occurs ≤ 3 min after primary infection results a delay in lysis. Mutations that abolish LIN mapped to several genes but only rV encoding the holin, T, and rI, encoding the antiholin, RI, are required for LIN in all hosts which support T4 replication. Antiholin RI inhibits T-mediated lysis by direct interaction with the holin. T has at least one transmembrane domain with its N- terminus (TNTD) in the cytoplasm and C-terminus in the periplasm (TCTD). In contrast, the N-terminus of RI (RINTD) is predicted to function as a cleavable signal sequence allowing the secretion of the RI C-terminal domain (RICTD) into the periplasm. Most of RI mutations which abolish LIN occur in the RICTD, suggesting RI inhibits T-mediated lysis by interacting with T via RICTD. Topological analysis of RI and T showed that iv ss fusion of PhoA signal sequence ( PhoA) to RICTD is necessary and sufficient for LIN ss and PhoAΦTCTD interferes with RI-mediated LIN, indicating T and RI interact via periplasmic C-terminal domains. In T4 infection, LIN is observed only when superinfection takes place, indicating either the antiholin or the LIN signal must be unstable. Both RI and RINTDΦPhoA are localized to both the inner membrane and the periplasm suggesting that the RINTD is a Signal-Anchor-Release (SAR) domain. Protein stability studies indicated that the SAR domain is the proteolytic determinant of RI, and DegP is the protease that is responsible for RI degradation. To date, how TNTD participates in lysis and LIN is not known. Modifications and deletion of the N-terminus of T change the lysis time, indicating this domain is involved the in timing of lysis. GFP fusion to holin T allowed microscopic visualization of fluorescent patches on the membrane at the time of lysis. v DEDICATION This dissertation is dedicated to my dearest grandma and to my parents. vi ACKNOWLEDGEMENTS I would like to thank my mom, grandparents and aunts who raised me and gave me a wonderful childhood in Vietnam. I would like to thank my dad, who drives me crazy with his expectations, but I guess his strategy worked in some way. I also want to express my gratitude to Mr. and Mrs. Thornhill (I call them my American parents) who have helped me a lot, especially when I first started school here in the U. S. I especially wish to thank Dr. Douglas K. Struck for his almost daily discussions and supervision. Without his moral support and genius strategies, I do not think I would get this far for this project. Thanks, Dr. Struck! I would also like to thank all the members of the Young lab, past and present. Dr. Min Xu, a friend and a teacher, taught me so much about cloning. Dr. John Deaton taught me about protein purification, specifically membrane protein purification. Brenley McIntosh let me stay with her while I wrote my dissertation. It would be difficult to do this on the street. Carrie Langlais listened to all of my complaints. These are just some examples. The list goes on. Their friendship made the lab a fun place to work. Last, I want to thank my PI, Dr. Ry Young, for giving me the opportunity to study in his laboratory. The “SWIM” with him every other week was fun, helpful and fruitful. He is serious about science and also very considerate about each student. Thanks, Ry! vii TABLE OF CONTENTS Page ABSTRACT............................................................................................................ iii DEDICATION ........................................................................................................ v ACKNOWLEDGEMENTS .................................................................................... vi TABLE OF CONTENTS........................................................................................ vii LIST OF FIGURES................................................................................................. x LIST OF TABLES .................................................................................................. xii CHAPTER I INTRODUCTION........................................................................... 1 The discovery of bacteriophages......................................... 1 Bacteriophage life cycle ...................................................... 5 Strategies of progeny release from infected host ................ 6 Lysis by small phages ............................................. 7 Lysis by large and double stranded DNA phages ... 9 T4 biology ........................................................................... 15 T4 life cycle............................................................. 15 T4 lysis .................................................................... 21 T4 lysis inhibition.................................................... 31 Goals and specific aims....................................................... 47 I. Determination of the interacting domains of antiholin RI and holin T .......................................... 48 II. Characterization of antiholin RI ......................... 48 III.Characterization of holin T................................. 49 II PERIPLASMIC DOMAINS DEFINE HOLIN-ANTIHOLIN INTERACTIONS IN T4 LYSIS INHIBITION.............................. 50 Introduction ......................................................................... 50 Materials and methods ........................................................ 54 Bacterial strains, bacteriophages, plasmids and culture growth ......................................................... 54 viii CHAPTER Page Standard DNA manipulations, PCR and DNA sequencing............................................................... 54 Construction of plasmids......................................... 61 Subcellular fractionation ......................................... 66 SDS-PAGE and Western blotting ........................... 66 Immunoprecipitation of RI-T complexes................ 68 Phage accumulation during LIN ............................. 69 Results ................................................................................. 69 Domain analysis of the RI antiholin........................ 69 RI-dependent LIN requires binding to the C-terminal domain of T........................................... 72 T and RI form a complex ........................................ 76 Discussion ........................................................................... 77 III THE T4 RI ANTIHOLIN HAS AN N-TERMINAL SAR-DOMAIN THAT TARGETS IT FOR DEGRADATION BY DEGP........................................................................................ 83 Introduction ......................................................................... 83 Materials and methods ........................................................ 85 Bacterial strains, bacteriophages, plasmids and culture growth ......................................................... 85 Standard DNA manipulations, PCR and DNA sequencing............................................................... 86 Subcellular fractionation, SDS-PAGE and Western blotting ...................................................... 86 Construction of plasmids and T4 mutants............... 87 Complementation of T4∆rI with rI alleles expressed from plasmids ......................................... 90 Stability of RI protein.............................................. 92 Phage released assays.............................................. 92 Azide inhibition of secretion ................................... 93 Results ................................................................................. 93 The N-terminal TMD of RI is a SAR domain......... 93 RI is an unstable protein.......................................... 96 The SAR domain is a major determinant of RI’s instability................................................................. 98 The DegP protease is required for the rapid turnover of RI .......................................................... 101 ss Y42am Lysis inhibition of PhoAΦRICTD in T4 infection................................................................... 102 Discussion ........................................................................... 102 ix CHAPTER Page IV CHARACTERIZATION
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