Computational Genome and Pathway Analysis of Halophilic Archaea

191 Pages·2006·6.07 MB·English

by  Michaela Falb

Computational
Genome and Pathway Analysis of
Halophilic Archaea

Dissertation
Michaela Falb

aus
Heiligenstadt
(Eichsfeld)

2005

Dissertation

zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München

Computational
Genome and Pathway Analysis of
Halophilic Archaea

Michaela Falb

aus
Heiligenstadt
(Eichsfeld)

2005

Erklärung

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 selbständig, ohne unerlaubte Hilfe erarbeitet.

München, den 31. August 2005

Michaela Falb

Dissertation eingereicht am: 01.09.2005

1. Gutachter: Prof. Dr. Dieter Oesterhelt
2. Gutachter: Prof. Dr. Erich Bornberg-Bauer

Mündliche Prüfung am: 22.12.2005

CONTENTS

Summary 1

1 Introduction to Halophilic Archaea 3
1.1 Hypersaline environments 3
1.2 Taxononomy of halophilic archaea 5
1.3 Information processing in archaea 7
1.4 Physiology and metabolism of halophilic archaea 9
1.4.1 Osmotic adaptation 9
1.4.2 Nutritional demands, nutrient transport and sensing 10
1.4.3 Energy metabolism 11
1.5 Genomes of halophilic archaea 13
1.6 Motivation 15

2 Gene Prediction and Start Codon Selection in Halophilic Genomes 17
2.1 Introduction 17
2.2 Post-processing of gene prediction results by expert validation 19
2.3 Intrinsic features of haloarchaeal proteins and gene context analysis 22
2.3.1 Isoelectric points and amino acid distribution of halophilic proteins 22
2.3.2 Development of a pI scanning tool 24
2.3.3 Gene distance analysis of haloarchaeal genomes 26
2.4 Validation of gene starts using proteomics data 27
2.4.1 Definition of a proteomics-verified gene and start codon set 27
2.4.2 Analysis of post-translational modifications in N-terminal peptides 28
2.5 Performance of microbial gene finders for GC-rich genomes 31
2.5.1 Gene prediction in genomes with different GC contents 31
2.5.2 Gene finder assessment using the validated Natronomonas pharaonis gene set 34
2.6 Conclusions 36
2.7 Methods 38
2.7.1 Post-processing of gene prediction results 38
2.7.2 Intrinsic features of haloarchaeal proteins and gene distance analysis 39
2.7.3 Validation of gene starts using proteomics data by expert validation 39
2.7.4 Performance of microbial gene finders for GC-rich genomes 40
2.8 Supplemental material 41
Michaela Falb ▪ 2005
3 Living with two Extremes: Conclusions from the Genome Sequence of
Natronomonas pharaonis 45
3.1 Introduction 45
3.2 Results and discussion 46
3.2.1 Genome and gene statistics 46
3.2.2 Function analysis 48
3.2.3 Central metabolism and transport 49
3.2.4 Nitrogen metabolism 49
3.2.5 Respiratory chain 50
3.2.6 Secretion and membrane anchoring 53
3.2.7 Cell envelope 55
3.2.8 Motility and signal transduction 55
3.3 Conclusions 58
3.4 Materials and methods 58
3.4.1 Genome sequencing and assembly 58
3.4.2 Gene prediction and annotation 59
3.4.3 Motif searches 60
3.4.4 ATP and pH measurements 60
3.4.5 Synthetic medium 61
3.5 Supplemental material 61
3.5.1 Transposases, plasmids, and regions with reduced GC content 62
3.5.2 Physiological capabilities 63
3.5.3 Motility and signal transduction cluster 65

4 Characterisation of Halophilic Secretomes 67
4.1 Introduction 67
4.2 Secretion proteins 72
4.2.1 Utilization of Sec and Tat protein translocation pathways 72
4.2.2 Proteins with flagellin-like cleavage sites 74
4.3 Membrane-anchored proteins 76
4.3.1 Lipobox-containing proteins 76
4.3.2 Proteins with a C-terminal membrane anchor 80
4.4 Identification of secreted proteins by proteomics 82
4.5 Conclusions 84
4.6 Methods 86
4.6.1 Analysis of secreted proteins 86
4.6.2 Analysis of membrane-anchored proteins 88
4.6.3 Identification of secreted proteins by proteomics 89
4.7 Supplemental material 90
CONTENTS
5 Metabolic Pathway Reconstruction for Halobacterium salinarum 93
5.1 Introduction 93
5.2 Enzyme assignment 96
5.2.1 Enzyme classification 96
5.2.2 Enzyme assignment by similarity-based function transfer 98
5.2.3 Development of an enzyme assignment routine 101
5.2.4. Enzyme classification in the KEGG database 102
5.3 Database structure and implementation of a metabolic database 103
5.4 Reconstructing metabolic pathways of Halobacterium salinarum 108
5.5 Graphical representation of metabolic pathways 110
5.6 The metabolism of Halobacterium salinarum 111
5.6.1 Central intermediary metabolism 112
5.6.2 Biosynthesis of nucleotides, lipids, and amino acids 116
5.6.3 Coenzyme biosynthesis 120
5.7 Conclusions 124
5.7 Methods 125
5.8.1 Enzyme assignment 125
5.8.2 Database structure and implementation of the metabolic database 128
5.8.3 Pathway reconstruction procedure 129
5.8.4 Graphical representation of metabolic pathways 130
5.8 Supplemental material 131

6 Pathway Comparison of Selected Metabolic Pathways in Archaea 137
6.1 Introduction 137
6.2 Amino acid metabolism in Natronomonas and Halobacterium 138
6.2.1 Glutamate family 140
6.2.2 Aspartate family 141
6.2.3 Serine family 142
6.2.4 Biosynthesis of branched chain amino acids 145
6.2.5 Biosynthesis of aromatic amino acids 146
6.3 Respiratory chains of haloarchaea and other archaea 148
6.3.1 Respiratory chains of haloarchaea 148
6.3.2 Respiratory chains of archaea 151
6.4 Variations in the metabolism of haloarchaea 153
6.4.1 Sugar and central metabolism 153
6.4.2 Nucleotide and lipid metabolism 155
6.4.3 Amino acid and nitrogen metabolism 156
6.4.4. Cofactor metabolism 156
6.5 Conclusions 159
Michaela Falb ▪ 2005
6.6 Methods 162
6.6.1 Amino acid metabolism in Natronomonas and Halobacterium 162
6.6.2 Respiratory chains of haloarchaea and other archaea 162
6.6.3 Variations in the metabolism of haloarchaea 162
6.7 Supplemental material 163

Abbreviations 167
Species abbreviations 168

References 169
Web links 176
Publications 177

Acknowledgements 179

Curriculum Vitae 181