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Proquest Dissertations The Structure, Function and Regulation of Mycobacterial Porin-Encoding Genes Richard Alan Speight A thesis submitted in partial fulfilment of the requirements of University College London for the degree of Doctor of Philosophy Division of Mycobacterial Research National Institute for Medical Research Mill Hill London October 2000 ProQuest Number: U642571 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 complete 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 U642571 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Index of Contents INDEX OF FIGURES..............................................................................................................................V INDEX OF TABLES______________________________________________________________ VII ABBREVIATIONS...............................................................................................................................VIII ABSTRACT.............................................................................................................................................. X 1. INTRODUCTION...............................................................................................................................1 1.1 T h e M ycobacteria ........................................................................................................ 1 1.2 T a x o n o m y o f th e mycobacteria .................................................................................................................2 1.3 T h e Staining P roperties o f M ycobacteria ............................................................................................ 5 1.4 H o st r espo n se to tuberculosis ................................................................................................................... 5 1.5 T h e M ycobacterial Ce ll E n v e lo pe ...........................................................................................................7 1.5.1 The Plasma Membrane ...................................................................................................................................8 1.5.2 The Cell Wall.....................................................................................................................................................8 1.5.3 The Capsular layer ......................................................................................................................................... 10 1.6 P ublication o f t h e M ycobacterial g en o m e ........................................................................................ 13 1.7 B ACTERIAL PERMEAB ILITY................................................................................................................................13 1.7.1 Permeability in Gram positive bacteria .................................................................................................... 13 1.7.2 Permeability in Gram negative bacteria ...................................................................................................14 1.7.3 Permeability in the Mycobacteria ..............................................................................................................16 1.8 M ethods o f transpo rt across th e ba cteria l ou ter e n v e l o p e ...................................................17 1.8.1 Specific Diffusion Channels ........................................................................................................................17 1.8.2 Active Efflux Pumps ......................................................................................................................................19 1.8.3 Non-Specific Channels, The Porins ..........................................................................................................20 1.9 P orins fro m G ra m P ositive B a c t e r ia ..................................................................................................... 23 1.10 P orins fro m th e M ycobacteria ................................................................................................................ 24 1.10.1 Porins in the cell wall of Mycobacterium tuberculosis............................................................ 25 1.11 Voltage G ating o f P ordm C h a n n e l s ......................................................................................................... 27 1.12 D yna m ic alteration o f bacterial m em bra n e permeability ........................................................28 1.13 Modulation of Porin levels in E scherichia c o u - a bacterial two-component REGULATORY SYSTEM..........................................................................................................................................................29 1.14 O th er tw o -c o m po n en t system s in b a c ter ia .........................................................................................32 1.15 Two-component systems in M ycobacterium tuberculosis ............................................................32 1.16 D isruption o f g en e expression in th e mycobacteria .......................................................................34 1.16.1 Mobile genetic elements ........................................................................................................................ 34 1.16.2 Use of antisense RNA ............................................................................................................................. 37 1.16.3 Targeted homologous recombination ..................................................................................................37 i AIMS OF THE PROJECT.......................................................................................................................... 41 2. MATERIALS AND METHODS ............................................................................................ 42 2.1 B acteria l strains and grow th CONDITIONS u s e d ..............................................................................42 2.2 PGR AMPLIFICATION OF D N A .......................................................................................................................... 43 2.3 A g a ro se g el electrophoresis o f D N A .................................................................................................. 43 2.4 E xcisio n o f D N A bands from aga ro se g els .........................................................................................43 2.5 L igation o f plasm id and insert D N A ......................................................................................................44 2.6 Preparation of competent E scherichia c o u ....................................................................................... 44 2.7 Transformation of E scherichia c o u by heat shock ........................................................................ 45 2.8 Extraction of plasmids from E scherichia c o u ................................................................................ 45 2.9 P reparation o f electrocompetent mycobacteria ...........................................................................45 2.10 E lectroporation o f mycobacteria ........................................................................................................ 46 2.11 Au tom ated sequencing o f p l a s m id s ....................................................................................................... 46 2.12 PGR PROBING OF M ycobacterial species for ompAT b .......................................................................47 2.13 W estern b lo t analysis o f mycobacterial cell ly sa t e s ...............................................................47 2.13.1 Preparation of mycobacterial cell lysates ..........................................................................................47 2.13.2 Determination of protein concentration .......................................................... 48 2.13.3 Polyacrylamide gel electrophoresis .....................................................................................................48 2.13.4 Western transfer of protein gels ...........................................................................................................49 2.13.5 Antibody based detection of OmpATb in Western blotted cell lysates .................................... 49 2.14 Identification o f a putative regulatory s y s t e m ...............................................................................50 2.14.1 Gene Rv0903c is transcribed in mycobacteria .................................................................................50 2.15 E xpressio n and purification o f R v 0903 p r o t e in ................................................................................50 2.15.1 Construction of plasmid pRASl ..........................................................................................................50 2.15.2 Expression of recombinant Rv0903 protein ......................................................................................51 2.15.3 Purification
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