Investigations on flagellar biogenesis, motility and signal transduction of Halobacterium salinarum [Elektronische Ressource] / Wilfried Staudinger

Wilfried Franz Staudinger

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 11 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
Investigations on Flagellar Biogenesis,
Motility and Signal Transduction of
Halobacterium salinarum
Wilfried Staudinger
aus
Heilbronn
2007Erklärung
Diese Dissertation wurde im Sinne von §13 Abs.3 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Dieter Oesterhelt betreut.
Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig und ohne unerlaubte Hilfe angefertigt.
München, am 22. April 2008
. . . . . . . . . . . . . . . . . .
Wilfried Staudinger
Dissertation eingereicht am: 09.11.2007
1. Gutachter: Prof. Dr. Dieter Oesterhelt
2. Gutachter: Prof. Dr. Wolfgang Marwan
Mündliche Prüfung am: 17.03.2008This dissertation was generated at the Max Planck Institute of Biochemistry, in the De-
partment of Membrane Biochemistry under the guidance of Prof. Dr. Dieter Oesterhelt.
Parts of this work were published previously or are in preparation for publi-
cation:
Poster presentation at the Gordon Conference on Sensory Transduction In Micro-
organisms, Ventura, CA, USA, January 22-27, 2006. Behavioral analysis of chemotaxis
gene mutants from H. salinarum.
delRosario, R.C., Staudinger, W.F., Streif, S., Pfeiffer, F., Mendoza, E., andOesterhelt
(D10K,Y100W)D. (2007). Modeling the CheY H. salinarum mutant: sensitivity analysis al-
lows choice of parameter to be modified in the phototaxis model. IET Systems Biology,
1(4):207-221.
Koch, M. K., Staudinger, W. F., Siedler, F., and Oesterhelt, D. (2007). Physiological
sites of deamidation and methyl esterification in sensory transducers of H. salinarum. In
preparation.
Streif, S., Staudinger, W. F., Joanidopoulos, K., Seel, M., Marwan, W., and Oesterhelt,
D. (2007). Quantitative analysis of signal transduction in motile and phototactic archaea
by computerized light stimulation and tracking. In preparation.Meinen Eltern
“Die beste und sicherste Tarnung ist immer noch die blanke und nackte
Wahrheit. Die glaubt niemand!”
Max Frisch
schweizer Schriftsteller (1911 - 1991)Contents
1 Summary 1
2 Introduction 5
2.1 The organism Halobacterium salinarum and its lifestyle . . . . . . . . . . 5
2.1.1 Taxonomic classification . . . . . . . . . . . . . . . . . . . . . . . 5
2.1.2 The habitat of H. salinarum . . . . . . . . . . . . . . . . . . . . . 6
2.1.3 Adaptation to hypersaline conditions . . . . . . . . . . . . . . . . 7
2.1.4 Energy metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.1.5 Morphology and swimming behavior of H. salinarum . . . . . . . 8
2.2 Structure and biogenesis of archaeal flagella . . . . . . . . . . . . . . . . 10
2.2.1 Archaeal and bacterial flagella are only superficially similar . . . . 10
2.2.2 Structure, function and assembly of bacterial flagella . . . . . . . 11
2.2.3 Sre and morphology of archaeal flagella . . . . . . . . . . . 11
2.2.4 Archaeal flagellar and type IV pili biogenesis share similarities . . 13
2.3 Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3.1 Two-component systems in prokaryotic signal transduction . . . . 19
2.3.2 Bacterial chemotaxis as a paradigm of signal transduction . . . . 20
2.3.3 Principles of prokaryotic taxis . . . . . . . . . . . . . . . . . . . . 20
2.3.4 Chemotaxis in E. coli . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3.5otaxis in B. subtilis . . . . . . . . . . . . . . . . . . . . . . . 23
2.3.6 Chemo- and phototaxis of H. salinarum shares similarities with
E. coli and B. subtilis chemotaxis . . . . . . . . . . . . . . . . . . 29
2.3.7 Phototaxis in H. salinarum . . . . . . . . . . . . . . . . . . . . . 30
2.3.8 A model for the H. salinarum motor switch and its photosensory
control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.9 Bacterial and Archaeal Chemoreceptors . . . . . . . . . . . . . . . 33
2.3.10 Structure of transducers and transmembrane signaling . . . . . . 36
2.3.11 Receptor clustering and the sensitivity paradox . . . . . . . . . . 38
2.4 Objectives of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3 Results and Discussion 43
3.1 Gene deletion as an approach to elucidate protein function in H. salinarum 43
3.1.1 General strategy to create in-frame deletions with subsequent com-
plementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.2 Investigations on flagellar biogenesis and motility of H. salinarum . . . . 48
3.2.1 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.2.2 The fla gene cluster of H. salinarum . . . . . . . . . . . . . . . . 49
IContents
3.2.3 Bioinformatic analysis of the proteins encoded in the fla gene cluster 51
3.2.4 Construction and genotypic analysis of fla gene knockout mutants 54
- - -3.2.5 FlaH and flaJ mutants are devoid of flagella while flaD and
-flaCE mutants have less flagella . . . . . . . . . . . . . . . . . . 55
3.2.6 Deletion of flgXXX has neither an effect on flagellar biosynthesis
nor on motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.2.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3.2.8 Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . 76
3.3 Studies on phototaxis of H. salinarum wild type cells . . . . . . . . . . . 78
3.3.1 The response of H. salinarum cells to blue light pulses obeys the
Bunsen Roscoe law of reciprocity . . . . . . . . . . . . . . . . . . 78
3.4 Behavioral studies of H. salinarum cells deleted for chemotaxis genes . . 82
3.4.1 The H. salinarum che operon . . . . . . . . . . . . . . . . . . . . 82
3.4.2 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.4.3 Analysis of already available H. salinarum che mutants . . . . . . 85
3.4.4 Bioinformatic analysis of the H. s CheC proteins . . . . 87
3.4.5 Generation and genotypic analysis of che gene knockout mutants 93
D10K,Y100W3.4.6ationandgenotypicanalysisofacheY doublemutant 93
3.4.7 Chemotaxis proteins influence the rotational bias of the H. sali-
narum flagellar motor . . . . . . . . . . . . . . . . . . . . . . . . 93
3.4.8 Phototaxis and spontaneous motor switching of H. salinarum che
mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
3.4.9 Chemotaxis of H. salinarum che mutants . . . . . . . . . . . . . . 102
3.4.10 H. salinarum CheR is a methyltransferase and CheB is a
methylesterase and glutamine deamidase . . . . . . . . . . . . . . 104
3.4.11 Comparison of the phototactic and chemotactic responses of the
che mutantssuggeststheexistenceofalternativeHtr-mediatedsig-
naling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
3.4.12 Is H. salinarum ParA1 involved in partitioning and localization of
cytoplasmic transducer clusters? . . . . . . . . . . . . . . . . . . . 108
3.4.13 Interpretation of the switching frequencies and rotational biases
and introduction of a modified motor model . . . . . . . . . . . . 110
3.4.14 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4 Materials and Methods 119
4.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
4.2 Kits and Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
4.3 Microbiological materials and methods . . . . . . . . . . . . . . . . . . . 120
4.3.1 Strains and culture conditions . . . . . . . . . . . . . . . . . . . . 120
4.3.2 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.3.3 Media and antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.3.4 Transformation of E. coli . . . . . . . . . . . . . . . . . . . . . . . 124
4.3.5 Trmation of H. salinarum . . . . . . . . . . . . . . . . . . . 124
4.4 Molecularbiological Methods . . . . . . . . . . . . . . . . . . . . . . . . . 127
4.4.1 Preparation of unpurified (“crude”) DNA from H. salinarum . . . 127
IIContents
4.4.2 Preparation of plasmid DNA from E. coli . . . . . . . . . . . . . 127
4.4.3 Isolation of DNA fragments from agarose gels . . . . . . . . . . . 127
4.4.4 Determination of DNA concentration . . . . . . . . . . . . . . . . 128
4.4.5 Sequencing of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 128
4.4.6 Generation of PCR fragments and plasmid construction . . . . . . 129
4.4.7 Southern Blot analysis . . . . . . . . . . . . . . . . . . . . . . . . 131
4.5 Behavioral studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
4.5.1 Swarm plate assay . . . . . . . . . . . . . . . . . . . . . . . . . . 135
4.5.2 Computerized cell tracking (Motion Analysis) . . . . . . . . . . . 135
4.5.3 Dark-field microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 141
4.6 Electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4.6.1 Growth, concentration and washing of the cells . . . . . . . . . . 142
4.6.2 Preparation of grids and electron microscopy . . . . . . . . . . . . 142
5 Appendix 143
5.1 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
5.2 Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
5.3 Raw data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
References 150
Curriculum v