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Identification and Characterisation of Endogenous Inducible Promoters in Mycobacterium Tuberculosis Schuessler, Dorothée Laura

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Identification and Characterisation of Endogenous Inducible Promoters in Mycobacterium Tuberculosis Schuessler, Dorothée Laura Identification and characterisation of endogenous inducible promoters in Mycobacterium tuberculosis Schuessler, Dorothée Laura The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author For additional information about this publication click this link. https://qmro.qmul.ac.uk/jspui/handle/123456789/595 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] “Identification and characterisation of endogenous inducible promoters in Mycobacterium tuberculosis” Dorothée Laura Schuessler Bart’s and the London School of Medicine and Dentistry, Queen Mary University of London Thesis submitted for the degree of Doctor of Philosophy of the University of London January 2010 1 The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. 2 Declaration I declare that this thesis is my original work and has not been submitted in any form for another degree or diploma at any university or other institution of tertiary education. Wherever contributions of others are involved, every effort is made to indicate this clearly, with due reference to the literature and acknowledgement of collaborative research and discussions. Dorothée Laura Schuessler 3 Acknowledgements I would like to thank the European Union and the NM4TB project for funding. I would like to express my thanks and utmost gratitude towards my supervisors Prof. Tanya Parish and Prof. Mike Curtis for their guidance and unlimited advice. I would also like to thank Dr. Renan Goude and Dr. Amanda Brown for their help and the members of the TB Group (Paul, Jade, Melanie, Julian, Lise and Yoann) for their support. Special thanks go to my family (Manfred, Gudrun, Gregor, Serge, Doris and Frank) and friends (especially Alan, Kate, Martin, Elana and Christopher) without whom this work would not have been possible. Thank you. This thesis is dedicated to my grandmother Magdalene, who suffered from tuberculosis. 4 Abstract Mycobacterium tuberculosis is one of the world’s most devastating pathogens. Despite completion of the genome sequence in 1998, research progress has been hampered by a lack of genetic tools and the difficulty of working with the organism. Existing genetic systems are limited by their lack of tight regulation or genetic instability. The aim of this study was to characterise and utilise a range of promoters to express mycobacterial genes in a controllable fashion by generating knockdown strains of a number of target genes, using both sense and antisense approaches. This would help to elucidate the function of a particular gene of interest and identify or validate new drug targets. Sets of genes shown to be inducible by certain stimuli such as tetracycline (Rv0277c, Rv0608, Rv0748, Rv1015c, Rv2487c and Rv3898c), streptomycin (whiB7), sodium dodecyl sulphate or ethanol (whiB6), hypoxia, nitric oxide and stationary phase (Rv2625c, Rv2626c, Rv2627c and hspX), or salicylate (Rv0560c) were selected from the literature. The upstream region of each gene was cloned in front of a reporter gene and activity was tested in M. smegmatis and/or M. tuberculosis. No inducible promoter activity was found for the upstream regions of the tetracycline, streptomycin, sodium dodecyl sulphate or ethanol­responsive genes. Inducible promoter activity was found for some of the hypoxia­responsive genes and was monitored in relation to growth phase in M. tuberculosis wild type, a dosR deletion mutant and a Rv2625c deletion mutant. The upstream region of Rv0560c was found to contain a salicylate­inducible promoter. Promoter elements of this promoter were identified and characterised in M. tuberculosis. Attempts to use the most promising promoters in an antisense setting using the reporter gene lacZ or the mycobacterial gene rpoB were unsuccessful. 5 Table of Contents Declaration....................................................................................... 3 Acknowledgements......................................................................... 4 Abstract............................................................................................ 5 Table of Contents ............................................................................ 6 List of abbreviations and units .................................................... 11 List of figures................................................................................. 15 List of tables .................................................................................. 19 1. General Introduction............................................................... 21 1.1 The genus mycobacterium ...........................................................21 1.1.1 Mycobacterium tuberculosis complex........................................21 1.2 Tuberculosis ..................................................................................22 1.2.1 History and current global implications......................................22 1.2.2 Pathogenesis and immune response ........................................23 1.2.3 Models of mycobacterial persistence (in vivo and in vitro).........25 1.2.4 Vaccination strategies and treatment ........................................26 1.3 Drug discovery ..............................................................................28 1.3.1 Identification of essential genes ................................................28 1.3.2 Drug target validation ................................................................29 1.4 Current research tools ..................................................................30 1.4.1 Construction of targeted deletion mutants .................................31 1.4.2 Antisense oligonucleotides........................................................32 1.4.3 Inducible promoter systems ......................................................32 1.5 Mycobacterial promoters ..............................................................44 1.6 Aims and objectives......................................................................45 2. Materials and methods........................................................... 46 2.1 Bacterial growth ............................................................................46 2.1.1 Media........................................................................................46 2.1.2 Growth of M. smegmatis ...........................................................47 2.1.3 Growth of M. tuberculosis..........................................................47 6 2.2 Extraction, purification and quantification of genomic DNA, RNA and complementary DNA....................................................................50 2.2.1 Extraction and purification of genomic DNA ..............................50 2.2.2 Extraction of mycobacterial DNA for colony PCR ......................51 2.2.3 Extraction and purification of mycobacterial RNA......................51 2.2.4 Preparation of complementary DNA (cDNA) .............................52 2.3 Polymerase chain reaction (PCR).................................................52 2.3.1 PCR reactions using different polymerases...............................52 2.3.2 PCR amplification programmes.................................................53 2.4 Preparation of DNA fragments for cloning, digests and gel electrophoresis....................................................................................54 2.4.1 Cloning PCR products into pSC­A vector..................................54 2.4.2 Blunting of sticky­end DNA fragments.......................................54 2.4.3 A­tailing of DNA fragments........................................................54 2.4.4 Dephosphorylation of DNA fragments .......................................54 2.4.5 Restriction enzyme digests .......................................................55 2.4.6 Gel electrophoresis and DNA extraction from agarose gels ......55 2.4.7 Cleaning and concentrating of DNA ..........................................56 2.4.8 Ligation of DNA fragments ........................................................56 2.5 Transformation of E. coli, preparation of plasmid DNA and electroporation of mycobacteria ........................................................56 2.5.1 Transformation of E. coli ...........................................................56 2.5.2 Preparation of plasmid DNA......................................................57 2.5.3 Site directed mutagenesis (SDM)..............................................58 2.5.4 Construction of plasmids for testing promoter activity................58 2.5.5 Construction of promoter probe vectors containing unstable LacZ ..........................................................................................................59 2.5.6 Construction of antisense plasmids...........................................59 2.5.7 Electroporation of mycobacteria................................................60 2.6 Reporter gene assays ...................................................................61 2.6.1 Preparation of cell­free extracts.................................................61 2.6.2 Determination of protein concentration in cell­free extracts .......61 2.6.3 ß­galactosidase activity assay...................................................61
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