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Genetics and Molecular Biology of the Streptomyces Lividans Plasmid Pulol

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Genetics and Molecular Biology of the Streptomyces Lividans Plasmid Pulol I Genetics and molecular biology of the Streptomyces lividans plasmid pUlOl A thesis submitted for the degree of Doctor of Philosophy of the University of London Sara Zaman Department of Biology University College London 1991 ProQuest Number: 10610049 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10610049 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 z Abstract. \ ^ v JLu* >aC( ~>S> pVOj>^e\ The 0.5kb Spel(53)-Bcll(49) fragment containing the^pIJlO 1 coplkorB gene was cloned into the E. coli plasmid pUC8 under the control of the lacZ promoter, creating pQR206. pQR206 produces a lOkDa LacZ-Cop/KorB fusion protein in vitro and in vivo in E. coli. Cop/KorB crude protein is able to retard a 0.5kb Spel(53)-Bcll(49) coplkorB fragment, a 0.5kb Sau3A coplkorB fragment, and a 0.65kb Stf/GI(19)-Ss/II(16) kilB fragment in band-shift assays; however no retardation was observed with a 0.7kb Spel(53)-Bcll(51) sti fragment. DNA footprinting analysis showed that the Cop/KorB protein protects overlapping 33b sequences on the coplkorB coding and non-coding strands respectively. Furthermore, a hypersensitive site present on each strand divides these 33b regions into two equal 16b regions. The kilB protected region covers 60b and 52b on the coding and non-coding strands respectively. Both these defined kilB and coplkorB operator sites for Cop/KorB binding show strong sequence homology and also correspond with the location of their respective promoters. Competition assays suggest that the Cop/KorB protein may have a higher affinity for the kilB operator than for the coplkorB operator. A 0.7kb Bcl\(51)-Spel(53) sti fragment inserted in its correct orientation into pIJ702 (a sti'coplkorB' pIJlOl derivative) and into pIJ702::pUC8 shuttle vectors prevented them from accumulating ssDNA and structurally rearranging respectively. Constructs which contained sti in the reverse orientation were found to accumulate ssDNA. Thus, sti is only active as the site for second-strand synthesis in its natural orientation with respect to the basic replicon. The sti determinant was further defined to a 0.53kb SstU(55)-Spel(53) fragment which contains a potential stem-loop structure with a AG of -64.00 kcal/mol. Cop/KorB does not inhibit the conversion of ssDNA to the double-stranded form and does not significantly alter copy number. The minimal replicon was further defined to a 2.0kb Ball(\5)-Sstll(63) fragment encompassing only the rep ORF and a non-coding region which may either contain the plus origin or the rep promoter. The 1.4kb A^6>rl( 12)-S^rII(63) rep ORF was cloned into pBGS19- under the control of the lacZ promoter, creating pQR431a. pQR431a produces a 50kDa LacZ-Rep fusion protein in vitro. Acknowledgements. I am indebted to Dr John Ward and Dr Hilary Richards for their constant supervision and encouragement throughout the project. I am also indebted to Alex Chapman for proof-reading and formatting the text, and for assisting with the figures. I would like to thank Dr. Rita Barallon for her generosity and invaluable assistance in the lab during the early years of the project. Thanks also to the rest of the group especially to Carol for organising the numerous parties and Sunil for supplying endless amounts of diet cokes and crunch bars. A very special thanks to Sami (my best friend, husband, and lab partner) for being so good at all three jobs. Finally, I would like to dedicate this thesis to my father who has supported me throughout my education. TABLE OF CONTENTS CHAPTER ONE. Introduction........................................................ 9 1.1 Prologue ......................................................................................................9 1.2 The biology of Streptomyces plasmids ................................ ........................12 1.2.1 Plasmid maintenance and replication ..................................................12 1.2.2 Plasmid transfer and pock formation .................................................. 13 1.2.3 Plasmid-mediated fertility and site-specific integration ...................... 15 1.3 The Streptomyces plasmid, pU 101 ...............................................................18 1.3.1 Background ........................................................................................18 1.3.2 Replication and stability determinants ................................................18 1.3.3 Transfer and spread determinants .......................................................22 1.3.4 Sti (strong incompatibility) and Cop (copy number) determinants.... 25 1.3.5 Identification of pUlOl promoters and terminators ............................28 1.4 Streptomyces plasmids as cloning vectors ....................................................29 1.4.1 Construction of cloning vectors based on pUlOl ................................ 29 1.4.2 Plasmid structural instability ...............................................................30 1.5 Aims of the project .......................................................................................31 CHAPTER TWO. Materials and Methods......................................33 2.1 Materials...................................................................................................... 33 2.2 Bacterial strains and plasmids .......................................................................33 2.3 Culture conditions ........................................................................................ 33 2.4 Maintenance of bacterial strains ................................................................... 39 2.5 Preparation and transformation of E. coli JM107 competent cells ................ 39 2.6 Small-scale isolation of plasmid DNA from E. coli......................................40 2.7 Large-scale isolation of plasmid DNA from E. coli......................................40 2.8 Preparation and transformation of Streptomyces protoplasts ........................41 2.9 Small-scale isolation of Streptomyces total DNA......................................... 42 2.10 Small-scale isolation of plasmid DNA from Streptomyces ........................ 42 2.11 Large-scale isolation of plasmid DNA from Streptomyces ........................ 43 2.12 Restriction enzyme digestions, ligations, and agarose gel electrophoresis. .43 2.13 Recovery of DNA from agarose gels .........................................................44 2.14 Radioactive-labelling of DNA ...................................................................45 2.15 Southern blotting.......................................................................................46 J 2.16 Isolation of total mRNA from E. colilStreptomyces ................................... 47 2.17 Northern blotting ....................................................................................... 47 2.18 High resolution SI mapping ...................................................................... 48 2.19 Double-stranded DNA sequencing .............................................................49 2.20 SDS-polyacrylamide gel electrophoresis (PAGE) ...................................... 50 2.21 In vitro coupled transcription/translation ....................................................51 2.22 Protein extraction from E. coli...................................................................52 2.23 Gel retardation assay ..................................................................................52 2.24 Competition assay ....................... :............................................................52 2.25 DNasel protection assay (DNA footprinting) ............................................ 53 CHAPTER THREE........................................................................... 55 3.1 Construction of pQR200 (coplkorB plus sti in pUC8) ................................. 55 3.2 Construction of pQR206 (coplkorB in pUC8) ............................................. 56 3.3 Sequencing of coplkorB (pQR206) ............................................................. 58 3.4 Expression of Cop/KorB protein in vitro .....................................................61 3.5 Expression of Cop/KorB protein in vivo in E. coli.......................................66 3.6 Cloning of coplkorB into other expression vectors ...................................... 72 3.7 Analysis of Cop/KorB binding ....................................................................77 3.7.1 Gel retardation assay .........................................................................78 3.7.2 Competition assay ............................................................................ 89 3.7.3 Identification of coplkorB and kilB recognition sequences ..............93 3.8 DNasel protection analysis (DNA footprinting) ...........................................99 3.9 Summary .................................................................................................... 114 CHAPTER FOUR............................................................................. 116 4.1 Cloning of
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