Bacterial Chromosome Organisation and Transcription by Laura Sellars A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences The University of Birmingham April 2014 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract The bacterial chromosome has to be condensed to fit inside the cell, forming a compact structure called the nucleoid, which is confined to a particular region of the cell without constriction by a membrane. Originally, the nucleoid was thought to be packed into the cell in a disordered way, unlike the highly organised chromatin of eukaryotic cells. More recently, the bacterial nucleoid has been shown to be far more structured than previously thought, with DNA present in topologically distinct loops, which are then arranged into macrodomains. Some of the proteins involved in structuring the E. coli chromosome are also known to have important roles in regulating transcription, and at least one transcription factor is known to cause distant DNA sites to cluster upon binding. These factors lead to the idea that chromosome structure could be affected by local gene expression. To investigate the possible link between chromosome structure and gene expression, Fluorescent Reporter/Operator Systems (FROS) were used to study the positions of different inducible promoters, with and without induction. The FROS method was adapted to use a smaller insert, therefore causing less disruption to the chromosome structure. The transcription factor MalI was also developed as a novel FROS reporter. Of the five promoters studied, only araFGH showed any movement upon induction, moving away from the cell pole. In cells at the point of division, induction of the araFGH promoter caused segregation of sister chromatids adjacent to araFGH. These results suggest that induction of promoters can cause a change in local chromosome structure, although this is not seen at all promoters. The diffusion of fluorescently tagged transcription factors, used as reporters in FROS, can also be studied using super resolution microscopy methods. Acknowledgements Over the 4 years it has taken to complete this thesis I have had help and advice from many people for which I am very grateful. Firstly I would like to thank my supervisor, Prof Steve Busby for giving me this opportunity and for his support and guidance over the past few years. I would also like to thank Dr Dave Lee, Rita Godfrey and Dr Doug Browning for sharing their experience and wisdom with me and helping me with all my practical problems. Thank you to Dr Jack Bryant for introducing me to the lab and spending a lot of his time helping me and discussing our projects. Thanks also to Dr Maritoñi Sánchez Romero for her patience and assistance in training me on the microscope, without which this project would never have got started. A massive thank you to Dr Eugenio Sanchez-Moran and his lab for allowing me to use their microscope and helping me with my problems, as well as making me feel very welcome. For the PALM experiments in Chapter 5, I thank Dr Achillefs Kapanidis and Federico Garza de Leon for carrying out the experiments. I look forward to continuing this collaboration. I would like to thank Stephen Bevan for constructing the strains for studying MntR controlled promoters used in Chapter 4, and also for being an excellent student and causing me very little trouble. I thank all members of the Busby and Cole labs, past and present, for their support and friendship. It has made the past 4 years a lot more pleasant than it otherwise would have been. I would like to thank my family, especially my parents and Lindsey for all of their encouragement through the whole of my life. I hope I’ve made you proud. Finally, I thank Andy, for getting me through this especially stressful year. Your turn next, then we will have a rest! Table of Contents 1. Introduction .......................................................................................................................... 1 1.1 Escherichia coli ................................................................................................................. 2 1.2 Chromosome Structure in Escherichia coli ...................................................................... 4 1.2.1 The Nucleoid .............................................................................................................. 5 1.2.2 Macrodomains ............................................................................................................ 8 1.2.3 Compaction of DNA ................................................................................................ 11 1.3 DNA replication and chromosome segregation in Escherichia coli ............................... 14 1.3.1 DNA replication ....................................................................................................... 14 1.3.2 Chromosome Segregation ........................................................................................ 18 1.4 Regulation of transcription in Escherichia coli .............................................................. 25 1.4.1 Bacterial RNA polymerase....................................................................................... 25 1.4.2 Promoter recognition ................................................................................................ 27 1.4.3 Transcription factors ................................................................................................ 30 1.4.4 Link between nucleoid structure and transcription regulation ................................. 34 1.5 Aims of the project ......................................................................................................... 35 2. Materials and Methods ...................................................................................................... 38 2.1 Buffers, Solutions and Reagents ..................................................................................... 39 2.2 Growth Media ................................................................................................................. 39 2.2.1 Solid Media .............................................................................................................. 39 2.2.2 Liquid Media ............................................................................................................ 39 2.2.3 Antibiotics and other supplements ........................................................................... 40 2.3 Bacterial Strains .............................................................................................................. 41 2.4 Plasmids .......................................................................................................................... 41 2.5 Gel electrophoresis of DNA ........................................................................................... 63 2.5.1 Agarose gel electrophoresis ..................................................................................... 63 2.5.2 Polyacrylamide gel electrophoresis .......................................................................... 64 2.6 Extraction and purification of nucleic acids ................................................................... 64 2.6.1 Purification of DNA using QIAquick PCR purification kit ..................................... 64 2.6.2 Extraction of DNA from agarose gels ...................................................................... 65 2.6.3 Preparation of plasmid DNA .................................................................................... 65 2.7 Transformations .............................................................................................................. 65 2.7.1 Preparation of competent cells using the calcium chloride method ......................... 65 2.7.2 Transformation ......................................................................................................... 66 2.8 DNA Manipulations ........................................................................................................ 66 2.8.1 Standard PCR ........................................................................................................... 66 2.8.2 Colony PCR ............................................................................................................. 73 2.8.3 Restriction Digestion of DNA .................................................................................. 73 2.8.4 DNA Ligation ........................................................................................................... 75 2.8.5 DNA Sequencing...................................................................................................... 75 2.9 Gene Doctoring ............................................................................................................... 75 2.9.1 Using homologous recombination to make chromosomal modifications ................ 75 2.9.2 Gene doctoring protocol ..........................................................................................