Investigations on halobacterial transducers with respect to membrane potential sensing and adaptive methylation [Elektronische Ressource] / Matthias Koch

Matthias K. Koch

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Investigations on halobacterial transducers
with respect to membrane potential sensing
and adaptive methylation
Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
Matthias Koch
aus Eschwege
2005Erklärung:
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der
Promotionsordnung vom 29. Januar 1998 von Prof. Dr. Dieter Oesterhelt betreut.
Ehrenwörtliche Versicherung:
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe angefertigt.
München, 25.08.2005
.......................................
(Matthias Koch)
Dissertation eingereicht am 9.9.2005
1. Gutachter: Prof. Dr. Dieter Oesterhelt
2. Gutachter: Prof. Dr. Karl-Peter Hopfner
Mündliche Prüfung am 14.12.2005This dissertation was generated between October 1999 and August 2005 at the
Max-Planck Institute of Biochemistry in Martinsried, in the Department of
Membrane Biochemistry under the guidance of Prof. Dr. Dieter Oesterhelt.
Parts of this work were previously published:
Poster presentation at the International Conference on Halophilic
Microorganisms, Sevilla, Spain, September 23 - 27, 2001
Poster presentation at the Gordon Research Conference on Sensory Transduction
In Microorganisms, Ventura, CA, USA, January 11 - 16, 2004
Koch, M.K., and Oesterhelt, D. (2005) MpcT is the transducer for membrane
potential changes in Halobacterium salinarum. Molecular Microbiology 55:
1681-1694.Sprüche des Konfuzius
Dreifach ist des Raumes Maß:
Rastlos fort ohn Unterlaß
Strebt die Länge; fort ins Weite
Endlos gießet sich die Breite;
Grundlos senkt die Tiefe sich.
Dir ein Bild sind sie gegeben:
Rastlos vorwärts mußt du streben,
Nie ermüdet stillestehn,
Willst du die Vollendung sehn;
Mußt ins Breite dich entfalten,
Soll sich dir die Welt gestalten;
In die Tiefe mußt du steigen,
Soll sich dir das Wesen zeigen.
Nur Beharrung führt zum Ziel,
Nur die Fülle führt zur Klarheit,
Und im Abgrund wohnt die Wahrheit.
Friedrich Schiller (1759-1805)TABLE OF CONTENTS
Table of Contents
1 SUMMARY...............................................................................................................................1
2 INTRODUCTION........................................................................................5
2.1 The halophilic archaeon Halobacterium salinarum............................................................5
2.1.1 Taxonomy, ecology and metabolic properties..................................................................5
2.1.2 Morphology and swimming behavior...............................................................................7
2.2 Signal transduction and taxis in bacteria and archaea......................................................8
2.2.1 Histidine-aspartate phosphorelay (HAP) systems are an integral part of most
common prokaryotic sensory pathways......................................................................................9
2.2.2 The molecular machinery mediating enterobacterial signal transduction as a
paradigm of prokaryotic signaling systems...............................................................................10
2.2.3 Transducers relay external stimuli to the cell interior, and changes in their
methylation status are involved in adaptation to a given stimulus intensity.............................14
2.3 Halobacterial signal transduction at a molecular level....................................................18
2.3.1 The halobacterial Che protein machinery displays similarities to that of B. subtilis......18
2.3.2 Halobacterial transducers are class III transducers involved in sensing
a variety of stimuli....................................................................................................................20
2.3.3 Methylation of Htrs.........................................................................................................21
2.4 Bacteriorhodopsin-dependent phototaxis in H. salinarum..............................................22
2.4.1 The proton motive force (pmf)........................................................................................23
2.4.2 Hints towards an involvement of the signaling chain.....................................................23
2.5 Objectives of the thesis........................................................................................25
3 RESULTS AND DISCUSSION.............................................................................................26
3.1 Identification of MpcT (Htr14) as the transducer for ∆Ψ changes in H. salinarum.....26
3.1.1 Construction of retinal-protein knockout strains ............................................................26
3.1.2 Transducer inventory of H. salinarum............................................................................29
3.1.3 Generation of htr-deletions in strains MKK101 and MKK102......................................31
3.1.4 Red-blue colony selection as a new method facilitating the screening of
halobacterial transformants.......................................................................................................32
3.1.5 BR-dependent phototaxis is mediated by Htr14 and involves stimulus-induced
methanol release........................................................................................................................34
3.1.6 Htr14 is also responsible for HR-dependent photoresponses.........................................36
3.1.7 Htr14 is bound to the plasma membrane and can exist in differently
methylated forms.......................................................................................................................37
3.1.8 Mass spectrometrical analysis identified four methylatable residues in Htr14
and suggests a set of Htr14 species consistent with that seen on immunoblots........................39
iTABLE OF CONTENTS
3.1.9 The buffering capacity of the H. salinarum cytoplasm argues for a change in ∆Ψ
as the stimulus that is sensed via Htr14....................................................................................42
3.1.10 MpcT (Htr14) by itself probably serves as the ∆Ψ sensor............................................46
3.1.11 Structural and clustering model for E. coli Tsr and MpcT............................................50
3.1.12 Summary of MpcT (Htr14) action and outlook............................................................59
3.2 Mass spectrometrical identification of methylation sites in Htrs....................................62
3.2.1 Starting point and strategy for the investigation of Htr methylation...............................62
3.2.2 An evaluation of MALDI-TOF MS experiments provided first hints towards the
detectability of methylated peptides via MS.............................................................................65
3.2.3 LC ESI Q-TOF MS(/MS) after in-gel Asp-N digestion of the respective Htrs
is the method of choice to investigate Htr-methylation............................................................67
3.2.4 Mascot MS/MS ions searches with the detected peptide fragment masses
identified differently methylated forms of certain Htr peptides................................................70
3.2.5 For a number of Htrs either the methylation sites or the individual methylated
residues could be identified via tandem MS (MS/MS).............................................................74
3.2.6 MS/MS spectra can provide different levels of information concerning the
methylation of transducer peptides...........................................................................................77
3.2.7 Deamidations at position 2 of heptad -11 of Htr4 and Htr15 only in wild-type
but not in the ∆cheB strain identify CheB as the deamidase of H. salinarum..........................82
3.2.8 Methylation sites within the methylatable regions of membrane-bound Htrs were
identified in heptads -13, -12, +13, +15 and +17......................................................................83
3.2.9 Transducer evolution could be explained by a scenario different from the
insertion/deletion (indel)-hypothesis.........................................................................................86
3.2.10 An additional methylatable site was identified in the signaling region of Htr14
(MpcT) in heptad +2.................................................................................................................89
3.2.11 Summary of Htr methylation and outlook.....................................................................90
4 MATERIALS AND METHODS...........................................................................................94
4.1 Chemicals and enzymes.......................................................................................................94
4.1.1 Chemicals........................................................................................................................94
4.1.2 Kits and Enzymes............................................................................................................95
4.2 Microbiological materials and methods.............................................................................96
4.2.1 Strains and c