Study of the properties of channel-forming proteins of the cell walls of different Corynebacteriae Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Fakultät für Biologie an der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Enrico Barth Würzburg Würzburg 2008 Eingereicht am: ...................................................................................... Prüfungskommission: Vorsitzender: Prof. Dr. Martin Müller, Dekan der Fakultät für Biologie 1. Gutachter: Prof. Dr. Dr. h.c. Roland Benz 2. Gutachter: Prof. Dr. Roy Gross Tag der mündlichen Prüfung: ................................................................ Promotionsurkunde ausgehändigt am: ................................................. Ich versichere, dass ich diese Dissertation selbständig angefertigt und keine anderen, als die von mir angegeben Quellen und Hilfsmittel benutzt habe. Die von mir vorgelegte Dissertation hat noch in keinem früheren Prüfungsverfahren in ähnlicher oder gleicher Form vorgelegen. Ich habe zu keinem früheren Zeitpunkt versucht, einen akademischen Grad zu erwerben. .......................................... Contents Contents................................................................................................................................ 9 Chapter 1 .............................................................................................................................. 15 1.1 Introduction ............................................................................................................. 15 1.2 Bacterial cell wall structures .................................................................................... 16 1.2.1 The cytoplasmic membrane ........................................................................................... 16 1.2.2 The periplasmic space ..................................................................................................... 17 1.2.3 The peptidoglycan layer .................................................................................................. 18 1.2.4 The cell wall of Gram-positive bacteria ....................................................................... 21 1.2.5 The cell wall of Gram-negative bacteria ...................................................................... 23 1.2.6 The cell wall of mycolic acid containing bacteria ....................................................... 26 1.2.6.1 The arabinogalactan moiety ........................................................................................... 29 1.2.6.2 The mycolic acid moiety................................................................................................. 30 1.2.6.3 Lipoarabinomannan ........................................................................................................ 31 1.2.6.4 Extractable lipids ............................................................................................................. 33 1.2.6.5 Porins penetrate the mycolic acid layer ........................................................................ 35 1.3 Aims of the work ...................................................................................................... 38 Chapter 2 ............................................................................................................................. 39 Corynebacterium diphtheriae: identification and characterization of a channel- forming protein in the cell wall ........................................................................................... 39 2.1 Summary .................................................................................................................. 39 2.2 Introduction ............................................................................................................. 40 2.3 Materials and Methods ............................................................................................ 42 2.3.1 Bacterial strains and growth conditions ....................................................................... 42 2.3.2 Construction of C. glutamicum strain ATCC 13032 ΔporHΔporA ............................. 42 2.3.3 Preparation of the cell wall, plasma membrane and cytosol fractions .................... 43 2.3.4 Isolation and purification of the channel-forming protein from the cell wall fraction .............................................................................................................................. 44 2.3.5 Digestion of the polypeptide ......................................................................................... 44 2.3.6 SDS-polyacrylamide gel electrophoresis ...................................................................... 44 2.3.7 Immunological techniques ............................................................................................. 45 2.3.7.1 Western blot analysis ...................................................................................................... 45 2.3.7.2 Enzyme-linked immunosorbent assay .. ...................................................................... 45 2.3.8 Polymerase chain reaction and construction of the expression plasmid ................ 46 2.3.9 Lipid bilayer experiments ............................................................................................... 47 9 2.4 Results ..................................................................................................................... 47 2.4.1 Isolation and purification of the channel-forming protein ........................................ 47 2.4.2 Interaction of the cell wall protein with lipid bilayer membranes ............................ 49 2.4.3 Single-channel analysis .................................................................................................... 49 2.4.4 Selectivity of the cell wall channel of C. diphtheriae ..................................................... 52 2.4.5 The cell wall channel of C. diphtheriae is voltage dependent ...................................... 53 2.4.6 Immunological detection of the channel-forming protein of C. diphtheriae............. 55 2.4.7 Identification of the gene coding for the cell wall channel of C. diphtheriae ATCC 11913 ..................................................................................................................... 56 2.4.8 Expression of CdPorA in C. glutamicum ATCC 13032 ΔporHΔporA and study of its channel-forming ability ............................................................................... 57 2.5 Discussion ............................................................................................................... 58 2.5.1 The cell wall of C. diphtheriae contains a channel-forming oligomer ........................ 58 2.5.2 The cell wall channel of C. diphtheriae is wide and water-filled and does not contain point charges ...................................................................................................... 59 2.5.3 The cell wall channel of C. diphtheriae shows immunological homology with CgPorA of C. glutamicum ................................................................................................. 60 2.5.4 Cloning and sequencing of cdporA................................................................................. 60 2.5.5 CdporA can be expressed in C. glutamicum and complements for CgPorA and CgPorH deficiency ........................................................................................................... 63 Chapter 3 ............................................................................................................................. 65 Study of the major cell wall channel of Corynebacterium glutamicum, Corynebacterium efficiens and Corynebacterium diphtheriae in dependence of PorH and PorA proteins ..................................................................................................... 65 3.1 Summary .................................................................................................................. 65 3.2 Introduction ............................................................................................................ 66 3.3 Materials and methods ............................................................................................ 68 3.3.1 Bacterial strains and growth conditions........................................................................ 68 3.3.2 Plasmids and DNA manipulations ................................................................................ 68 3.3.2.1 Cloning of C. glutamicum porins...................................................................................... 69 3.3.2.2 Cloning of C. efficiens porins ............................................................................................ 70 3.3.2.3 Cloning of C. diphtheriae porins ...................................................................................... 70 3.3.2.4 Cloning of the porin domain of C. callunae .................................................................. 73 3.3.3 Extraction of RNA .......................................................................................................... 73 3.3.4 DIG-labelled DNA probes ............................................................................................ 74 3.3.5 Northern blot analysis ..................................................................................................... 74 3.3.6 Reverse transcription ......................................................................................................