Iron regulation in the myxobacterium Myxococcus xanthus DK1622 [Elektronische Ressource] / von Matthias O. Altmeyer

Iron regulation in the Myxobacterium
Myxococcus xanthus DK1622






Dissertation
zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät III
Chemie, Pharmazie, Bio- und Werkstoffwissenschaften
der Universität des Saarlandes



von Matthias O. Altmeyer

Saarbrücken, November 2010
1

















Tag des Kolloquiums: 08. 02. 2011

Dekan: Prof. Dr. Stefan Diebels

Berichterstatter: Prof. Dr. Rolf Müller
Prof. Dr. Claus Jacob

Vorsitz der Prüfungskommission: Prof. Dr. Alexandra Kiemer
Akad. Mitarbeiter: Dr. Carsten Volz


2Vorveröffentlichungen der Dissertation
Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Naturwissenschaftlich-
Technischen Fakultät, vertreten durch den Mentor, in folgenden Beiträgen vorab
veröffentlicht:


Publications

1. Schley C, Altmeyer MO, Swart R, Müller R, Huber CG. Proteome analysis of
Myxococcus xanthus by off-line two-dimensional chromatographic separation using
monolithic poly-(styrene-divinylbenzene) columns combined with ion-trap tandem
mass spectrometry. J Proteome Res. 2006 Oct;5(10):2760-8.

2. Schneiker S, Perlova O, Kaiser O, Gerth K, Alici A, Altmeyer MO, Bartels D, Bekel
T, Beyer S, Bode E, Bode HB, Bolten CJ, Choudhuri JV, Doss S, Elnakady YA, Frank
B, Gaigalat L, Goesmann A, Groeger C, Gross F, Jelsbak L, Jelsbak L, Kalinowski J,
Kegler C, Knauber T, Konietzny S, Kopp M, Krause L, Krug D, Linke B, Mahmud T,
Martinez-Arias R, McHardy AC, Merai M, Meyer F, Mormann S, Muñoz-Dorado J,
Perez J, Pradella S, Rachid S, Raddatz G, Rosenau F, Rückert C, Sasse F, Scharfe M,
Schuster SC, Suen G, Treuner-Lange A, Velicer GJ, Vorhölter FJ, Weissman KJ,
Welch RD, Wenzel SC, Whitworth DE, Wilhelm S, Wittmann C, Blöcker H, Pühler
A, Müller R. Complete genome sequence of the myxobacterium Sorangium
cellulosum. Nat Biotechnol. 2007 Nov; 25(11):1281-9.

3. Bode HB, Ring MW, Schwär G, Altmeyer MO, Kegler C, Jose IR, Singer M, Müller
R. Identification of Additional Players in the Alternative Biosynthesis Pathway to
Isovaleryl-CoA in the Myxobacterium Myxococcus xanthus. Chembiochem. 2009
Jan;10(1):128-40

4. Hoiczyk E, Ring MW, McHugh CA, Schwär G, Bode E, Krug D, Altmeyer MO, Lu
JZ, Bode HB. Lipid body formation plays a central role in cell fate determination
during developmental differentiation of Myxococcus xanthus. Mol Microbiol. 2009
Sep [Epub ahead of print]

5. Altmeyer MO, Weissman KJ, Elnakady YA, Bode HB, Klefisch T and Müller R.
Cellular Response of the Myxobacterium Myxococcus xanthus DK1622 to Iron
Limitation, in preparation


Conference contribution / Tagungsbeiträge

1. Altmeyer MO, Schley C, Huber CG, Müller R. Iron regulation of protein expression
and secondary metabolite production in Myxococcus xanthus, - a proteomics approach
- (Poster), Intern. VAAM Workshop: Biology of Bacteria Producing Natural Products,
Dresden 2005

2. Altmeyer MO, Perlova O, Müller R. Analysis of the phosphoproteome of the
thmyxobacterium Sorangium cellulosum So ce56 (Poster), 34 Intern. Conference on
the Biology of the Myxobacteria, Granada (Spain) 2007

3. Altmeyer MO, Müller R. Effects of Iron Limitation on Myxococcus xanthus DK1622
Growth, Proteome Profile and Secondary Metabolite Production (Short presentation
and poster), Intern. VAAM Workshop: Biology of Bacteria Producing Natural
Products, Nonnweiler 2007






4 Table of contents

Table of contents

List of abbreviations ............................................................................................................. 5
List of figures ......................................................................................................................... 7
List of tables ........................................................................................................................... 8


1. Introduction ........................................................................................... 9
1.1 Role of iron in microbial metabolism ................................................................... 10
1.1.1 High-affinity iron uptake .......................................................................................... 10
1.1.2 Low-affinity iron uptake .......................................................................................... 14
1.1.3 Iron homeostasis control by the ferric uptake regulator (Fur) protein ..................... 14
1.1.4 Iron storage and overload ......................................................................................... 18
1.2 Myxobacteria .......................................................................................................... 20
1.2.1 Life cycle .................................................................................................................. 21
1.2.2 Myxococcus xanthus DK1622 as a model strain ...................................................... 24
1.2.3 Myxobacterial secondary metabolism ...................................................................... 26
1.2.4 Natural products from M. xanthus DK1622 ............................................................. 35
1.3 Comparative proteomics ........................................................................................ 40
1.3.1 Proteome analysis by 2D-PAGE .............................................................................. 40
1.3.2 Proteome analysis by 2D-DIGE ............................................................................... 41
1.3.3 Mass spectrometry based protein identification ....................................................... 42
1.4 Metabolite analysis by HPLC-MS ........................................................................ 46
1.5 Protein-D.A interactions (D.A pull-down assay) ............................................. 47
1.6 Target-oriented gene inactivation by homologous recombination .................... 49
1.7 Goal of this study .................................................................................................... 53

2. Material and Methodology ................................................................. 55
2.1 Chemicals ................................................................................................................ 55
2.2 Commercial ´kits´, enzymes and markers ............................................................ 57
2.3 Buffers and solutions .............................................................................................. 58
2.3.1 Antibiotic solution .................................................................................................... 58
2.3.2 Buffers and solutions for 2D-gel electrophoresis ..................................................... 58
2.3.3 Buffers and solutions MALDI mass spectrometry ................................................... 59
2.3.4 Buffers and solutions for DNA pull-down assay ..................................................... 60
2.3.5 Buffers and solutions for biomolecular work ........................................................... 62
2.4 Equipment and instrumentation ........................................................................... 63
2.5 Bioinformatic tools and analysis ........................................................................... 65
2.6 Bacterial strains ...................................................................................................... 67
2.6.1 The strain M. xanthus DK1622 ................................................................................ 67
2.6.2 The strain E. coli DH10B ......................................................................................... 67
2.7 Cultivation media ................................................................................................... 68
2.7.1 Cultivation media for M. xanthus DK1622 .............................................................. 68
Estimation of iron concentrations and iron uptake rates .......................................... 68
2.7.2 Cultivation medium for E. coli DH10B ................................................................... 69
2.8 Cultivation conditions and conserving of microbial strains ............................... 70
2.8.1 Growth conditions of M. xanthus cultures ............................................................... 70
Application of Amberlite XAD 16 absorber resin to M. xanthus cultures ............... 70
a) Growth of M. xanthus wild type cells on different iron concentrations ................... 70
b) Growth of M. xanthus mutant cells on CTT medium .............................................. 71
1Table of contents

2.8.2 Growth conditions of E. coli cultures ....................................................................... 71
2.8.3 Preparation of stock cultures and microbial conserving .......................................... 72
a) Stoc