Study of the properties of channel forming proteins of the cell walls of different Corynebacteriae [Elektronische Ressource] / vorgelegt von Enrico Barth

Enrico Castelnuovo

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 38
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

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 ΔporH ΔporA ............................. 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 ΔporH ΔporA 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 ....................................... 75
3.3.7 5' Rapid amplification of cDNA ends .......................................... 75
10
3.3.8 Isolation of the channel-forming proteins................................................................... 76
2+3.3.9 Affinity purification with immobilized Ni -ions ....................................................... 77
3.3.10 Digestion with Factor Xa protease ............................................... 77
3.3.11 Protein electrophoresis, immunoblotting and antibodies ......................................... 77
3.3.12 Black lipid bilayer assay .................................. 78
3.4 Results ..............................................................................