Open Research Online The Open University’s repository of research publications and other research outputs The Construction and Use of a Francisella tularensis DNA Microarray Thesis How to cite: LeButt, Helen (2008). The Construction and Use of a Francisella tularensis DNA Microarray. PhD thesis The Open University. For guidance on citations see FAQs. c 2007 Helen LeButt https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.0000fd6f Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk U a ) % £ST*l/C-ri£'(> The Construction and Use of a Francisella tularensis DNA Microarray A thesis submitted for the degree of Doctor of Philosophy to the Open University by Helen LeButt BSc. (Hons.) December 2007 72 0/7 /art’ /3 -Zocq bA-Tf. a r Ah^/ATSh,: 3 o A P & L . -2 0 & 2 - ProQuest N um ber: 13889945 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 13889945 Published by ProQuest LLC(2019). 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 Abstract Abstract A DNA microarray was designed and constructed using the genome sequence of the highly virulent obligate intracellular pathogen Francisella tularensis strain Schu S4. The microarray was optimised and then tested by performing a comparative genomics study on Francisella strains. The microarray was used to distinguish between Francisella strains at the subspecies level, detecting differences between the genomes of the subspecies at a similar rate to differences previously published from Francisella comparative genomics studies. Further analysis of the genomic differences identified between subspecies using the microarray has provided some suggestions as to the genetic basis for the relative attenuation of one subspecies, and similarly, differences identified between the F. tularensis live vaccine strain and its progenitor strain provided some clues as the genetic basis for the attenuation of the vaccine strain. The microarray was also used to carry out functional genomics studies on Francisella novicida cultured under in vitro stress conditions: iron starvation, oxidative stress, elevated temperature, and acidic pH. A number of genes were regulated in response to each of these conditions, and a detailed analysis of the data has provided insights into the stress response ofFrancisella, and some of the mechanisms that it may employ upon encountering similar stresses in vivo. Table of contents Table of contents Title page ................................................................................................................... i Abstract..................................................................................................................... ii Table of contents .................................................................................................... iii List of figures............................................................................................................x List of tables.......................................................................................................... xiii List of symbols and abbreviations................................................................ xv Acknowledgements .............................................................................................. xxi Author’s declaration.................... xxiii Publications.........................................................................................................xxiv 1. Introduction.................................................................................. .1 1.1. Francisella tularensis and tularemia ...............................................2 1.1.1. Francisella ..........................................................................................2 1.1.2. Tularemia ........................................................................................... 8 1.1.3. The intracellular lifestyle of Francisella ........................................ 16 1.2. DNA microarrays............................................................................ 24 1.2.1. Microarray formats......................................................................... 25 1.2.2. A microarray experiment using fluorescent labeling ..................26 1.2.3. Comparative genomics ...................................................................28 1.2.4. Functional genomics.......................................................................36 1.2.5. Aim.................................................................... 38 2. Methods.......................................................................................40 2.1. Bacterial strains and cultivation.....................................................41 2.2. Isolation and quantification of DNA from Francisella ................ 41 2.3. PCR.................................................................................................. 46 iii Table of contents 2.4. Culture of F. novicida under in vitro stress conditions................48 2.5. RNA isolation................................................................................. 50 2.6. Microarrays..................................................................................... 51 2.7. Quantitative real time PCR (QPCR). ......................................58 3. Design and construction of a F. tularensis microarray 62 3.1. Introduction..................................................................................... 63 3.2. Methods...........................................................................................65 3.2.1. Oligonucleotide design .................................................................. 65 3.2.2. Microarray printing......................................................................... 67 3.2.3. Preparation of Cy3-labelled DNA and hybridisation ................ 70 3.2.4. Microarray wash protocols..........................................................70 3.2.5. Scanning and image quantification .............................................71 3.3. Results............................................................................................72 3.3.1. Microarrays printed at FOI........................................................... 72 3.3.2. Microarrays printed at HPA..........................................................72 3.4 Discussion......................................................................................83 3.4.1. Oligonucleotide probes were selected for the F.tularensis microarray..................................................................................... 83 3.4.2. Aminosilane was used to attach the DNA probes to the slides.................. ......................................................................... 84 3.5. Conclusion..................................................................................... 85 4. Comparative genomics of Francisella ..................................86 4.1. Introduction.................................................................................... 87 4.2. Methods..........................................................................................91 4.2.1. Bacterial strains and genomic DNA isolation.............................91 Table of contents 4.2.2. Microarray hybridisation.............................................................. 91 4.2.3. Data acquisition and analysis ........................................ 91 4.2.4. Confirmation of microarray data by PCR.................................... 93 4.2.5. Strain- and subsp.-specific RD.....................................................93 4.3. Results............................................................................................. 95 4.3.1. RD identified by aCGH compared to those predicted by genome sequence ........................................................................ 95 4.3.2. Confirmation of results by PCR....................................................97 4.3.3. Hybridisations using DNA fromF. tularensis Schu S4.............. 97 4.3.4. Genomic differences identified using the F. tularensis microarray................................................................................... 101 4.3.5. RD/jo/arcf/ca...................................................................................... 103 4.3.6. RDlvs........................................................................................... 129 4.4. Discussion......................................... 131 4.4.1. aCGH studies on Francisella .......................................................131 4.4.2. RD/7 o/arcf/ca...................................................................................... 136 4.4.3. RDlvs..............................................................................................142 4.5. Conclusion.....................................................................................147