Cold Adaptation in the Antarctic Archaeon Methanococcoides burtonii: The Role of the Hydrophobic Proteome and Variations in Cellular Morphology
Thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy (Ph.D.)
Dominic W. Burg
School of Biotechnology and Biomolecular Sciences
University of New South Wales
2009
THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet
Surname or Family name: Burg
First name: Dominic Other name/s: William
Abbreviation for degree as given in the University calendar: PhD
School: Biotechnology and Biomolecular sciences Faculty: Science
Title: Cold adaptation in the Antarctic archaeon Methanococcoides burtonii: The role of the hydrophobic proteome and variations in cellular morphology
Abstract
Very little is known about the hydrophobic proteins of psychrophiles and their roles in cold adaptation. In light of this situation, methods were developed to analyse the hydrophobic proteome (HPP) of the model psychrophilic archaeon Methanococcoides burtonii. Central to this analysis was a novel differential solubility fractionation procedure, which resulted in a significant increase in the efficiency of resolving the HPP. Over 50% of the detected proteins were not identified in previous whole cell extract analyses, and these underwent an intensive manual annotation process producing high quality functional assignments. Utilising the functional assignments, biological context analysis of the HPP was performed, revealing novel and often unique biology. The analysis acted as a platform for differential proteomics of the organism’s response to both temperature and substrate using stable isotope labelling. The results of which revealed that low temperature growth was associated with an increase in the abundance of surface and secreted proteins, and translation apparatus. Conversely, growth at a higher temperature was associated with an increase in the abundance of general protein folding machinery and indications of an oxidative stress response, emphasising that the temperature for maximum growth rate is stressful. Through investigation of the response of M. burtonii to substrate it was found that growth on methanol was stressful, and its low energy yield resulted in an increase in the abundance of energy conserving systems. The extracellular polymeric substance (EPS) and morphology of M. burtonii was also investigated with respect to both temperature and substrate, using a number of techniques in microscopy. It was found that the EPS was comprised of proteins, sugars and RNA, and that growth at different temperatures resulted in the production of EPS that displayed significantly different properties on dehydration, thus indicating compositional variation. When cells were grown on methanol they took on highly irregular shapes and had electron transparent inclusions. The observations from the ultrastructural analysis were contemplated with respect to the proteomic findings, revealing novel avenues of research. This study has highlighted the roles of hydrophobic proteins in cold adaptation biology, and the value of comprehensive proteomics for the examination of adaptation in microorganisms.
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I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).
Dominic Burg Iona Williams 16/ 03/10 …………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Date
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II D. Burg UNSW
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I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation
Abstract International.
I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.
Signed ……Dominic Burg………………………………………......
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AUTHENTICITY STATEMENT
I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.
Signed ………… Dominic Burg …………………………………......
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D. Burg UNSW III
Declaration of originality
I, Dominic W. Burg, hereby declare that this submission is my own work and to the best of my knowledge contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.
Signature: Dominc Burg . Date: 16 / 03 /10
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Abstract Very little is known about the hydrophobic proteins of psychrophiles and their roles in cold adaptation. In light of this situation, methods were developed to analyse the hydrophobic proteome (HPP) of the model psychrophilic archaeon Methanococcoides burtonii. Central to this analysis was a novel differential solubility fractionation procedure, which resulted in a significant increase in the efficiency of resolving the HPP. Over 50% of the detected proteins were not identified in previous whole cell extract analyses, and these underwent an intensive manual annotation process producing high quality functional assignments. Utilising the functional assignments, biological context analysis of the HPP was performed, revealing novel and often unique biology. The analysis acted as a platform for differential proteomics of the organism’s response to both temperature and substrate using stable isotope labelling. The results of which revealed that low temperature growth was associated with an increase in the abundance of surface and secreted proteins, and translation apparatus. Conversely, growth at a higher temperature was associated with an increase in the abundance of general protein folding machinery and indications of an oxidative stress response, emphasising that the temperature for maximum growth rate is stressful. Through investigation of the response of M. burtonii to substrate it was found that growth on methanol was stressful, and its low energy yield resulted in an increase in the abundance of energy conserving systems. The extracellular polymeric substance (EPS) and morphology of M. burtonii was also investigated with respect to both temperature and substrate, using a number of techniques in microscopy. It was found that the EPS was comprised of proteins, sugars and RNA, and that growth at different temperatures resulted in the production of EPS that displayed significantly different properties on dehydration, thus indicating compositional variation. When cells were grown on methanol they took on highly irregular shapes and had electron transparent inclusions. The observations from the ultrastructural analysis were contemplated with respect to the proteomic findings, revealing novel avenues of research. This study has highlighted the roles of hydrophobic proteins in cold adaptation biology, and the value of comprehensive proteomics for the examination of adaptation in microorganisms.
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Acknowledgements
I would like to acknowledge a number of people that provided help, advice, and support during the duration of my candidature and in the writing of this dissertation. First and foremost, my supervisor Rick Cavicchioli; without your initial ideas and continuing support, I would not have been able to complete this project. Thank you for letting me follow my nose, and for reeling me in when I went off on too large a tangent. To my co-supervisor Mark Raftery; thanks for the advice, for keeping the machines running smoothly, and for introducing me to the world of proteomics. Special acknowledgement must be given to Jenny Norman and the staff at the EMU. Your help and advice, and the assistance provided in learning and performing the techniques of EM was much appreciated. Thanks for listening when things went bad, and for passing on the ‘EM bug’. I must also acknowledge: Tim Williams, for continuing collaboration, useful discussions on metabolism, and for reading and offering advice on draft chapters; Kevin Barrow for providing advice on detergent selection; Sohail Siddiqui for discussions on protein function and adaptation; Federico Lauro for help with the bioinformatics and for writing custom scripts; and especially Bill O’Sullivan for expert advice and reading of this dissertation, thanks for helping me cut out the waffle. To the past and present members of the Cavicchioli laboratory, especially Davide, Maine, Nico, Neil, and Sabine, thanks for helping me along the way and for joining me for the occasional beer (or three) after a long week, and after those incubator failures. To my friends and family, thanks for being there for me. I appreciate everything that you have done for me. I must especially thank my parents who instilled a love of science and the natural world in me from a very young age, and for the support provided over these long years. Thanks to Gareth and the lads at Mischief Moon, our conversations helped to keep me grounded.
This work was supported by the Australian Postgraduate Award, the Australian Research Council, and the US Air Force Office of Scientific Research. Mass spectrometric analysis for the work was performed at the Bioanalytical Mass Spectrometry Facility UNSW, and was supported in part by grants from the Australian Government Systemic Infrastructure Initiative and Major National Research Facilities Program (UNSW node of the Australian Proteome Analysis Facility), and by the UNSW Capital Grants Scheme. Electron microscopy was performed at the Electron Microscope Unit, Analytical Centre UNSW.
Finally, to Iona; your support, patience, love, and understanding have kept me going through these tough years. Thank you for being there.
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Table of contents Page Dissertation sheet ………………………………………………………………….II Copyright and authenticity statement……………………………………………III Declaration of originality i Abstract ii Acknowledgements iii Chapter index iv List of Figures xii List of Tables xiv Appendix index xv Supplementary material index xvi Common Abbreviations Used xvii Publications xix
Chapter index Chapter 1. General Introduction 1 1.1 The Cold Biosphere 1 1.2 Psychrophiles 1 1.2.1 Psychrophilic eukaryotes 3 1.2.2 Psychrophilic bacteria 3 1.3 Psychrophilic Archaea: Ubiquitous and Elusive 4 1.4 The Methanogens 9 1.5 Isolated Psychrophilic Archaea 12 1.6 Molecular Mechanisms of Cold Adaptation 13 1.6.1 Membrane and membrane protein changes in psychrophiles 15 1.6.2 Properties of psychrophilic enzymes and proteins 16 1.6.3 Genomics of psychrophiles 17 1.6.4 Compatible solutes 18 1.6.5 Identifying molecular mechanisms of cold adaptation using high throughput methods 19
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1.6.5.1 Membrane proteins identified in high throughput analyses 20 1.6.5.2 Cold shock responses in non-psychrophiles 21 1.6.6 Extracellular polymeric substance production by Psychrophiles 21 1.7 Methanococcoides burtonii 22 1.7.1 Methanococcoides burtonii in the environment 22 1.7.2 Cold Adaptation in Methanococcoides burtonii 24 1.7.2.1 Genomics 24 1.7.2.2 Proteomics 25 1.7.2.3 Molecular adaptation in M. burtonii 27 1.7.2.4 Transcription 28 1.7.2.5 Translation 29 1.7.2.6 Protein folding and post-translational modification 30 1.7.2.7 Compatible solutes 30 1.7.2.8 Energy generation and metabolism 31 1.7.2.9 Extracellular polymeric substance 32 1.7.2.10 Membrane lipids of Methanococcoides burtonii 32 1.7.2.11 The role of membrane proteins in the cold adaptation of Methanococcoides burtonii 33 1.8 Project Introduction 33
Chapter 2 Analysing the Hydrophobic Proteome of Methanococcoides burtonii 36 2.1 Summary 36 2.2 Introduction 37 2.3 Materials and Methods 41 2.3.1 Culture media preparation 41 2.3.1.1 Vitamin and mineral stock solutions 41 2.3.1.2 MFM preparation 42 2.3.2 Culture inoculation and growth conditions 45 2.3.3 Cell harvest 45 2.3.4 Hydrophobic protein extraction 46
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2.3.4.1 Cell disruption 46 2.3.4.2 Carbonate extraction and ultracentrifugation 47 2.3.4.3 Protein concentration and buffer exchange 48 2.3.5 Differential solubility fractionation 48 2.3.6 Protein concentration measurement 49 2.3.7 Assessment of protein separation using SDS-PAGE 50 2.3.7.1 Sample preparation 50 2.3.7.2 Electrophoresis 50 2.3.7.3 Gel staining and visualisation 51 2.3.8 Preparation of hydrophobic proteins for mass spectrometry 51 2.3.8.1 Protein reduction and alkylation 51 2.3.8.2 Protein solubilisation and digestion 52 2.3.8.3 Sample clean up 52 2.3.8.3.1 Strong cation exchange chromatography 53 2.3.8.3.2 Reverse phase chromatography 54 2.3.9 Mass spectrometry 54 2.3.9.1 Sample preparation 55 2.3.9.2 Sample evaluation by LC-MS/MS 55 2.3.9.2.1 LC-MS/MS performed on QTof Instrument 56 2.3.9.2.2 LC-MS/MS performed on QStar Instrument 57 2.3.9.3 Analysis of samples using LC/LC-MS/MS 57 2.3.9.3.1 LC/LC-MS/MS performed on QTof Instrument 58 2.3.9.3.2 LC/LC-MS/MS performed on QStar Instrument 59 2.3.9.4 Data processing 60 2.3.10 Protein parameter annotation 62 2.3.11 Statistics 63 2.4 Results and Discussion 64 2.4.1 Hydrophobic fraction separation 64 2.4.2 Trial of solubilisation and extraction strategies 66 2.4.3 Identification trends 70 2.4.4 Differential solubility fractionation 71 2.4.5 Peptide and protein identification statistics 82
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2.5 Conclusions 86 Chapter 3. Biological Context Analysis of the Hydrophobic Proteome 87 3.1 Summary 87 3.2 Introduction 88 3.3 Materials and Methods 90 3.3.1 Protein parameter annotation 90 3.3.2 Protein identity annotation 91 3.3.3 Statistics 91 3.4 Results and Discussion 93 3.4.1 Grouping of identified proteins 93 3.4.2 Biology inferred from proteomics 98 3.4.2.1 Energy production and conversion 98 3.4.2.1.1 Methanogenesis 98 3.4.2.1.2 Energy conservation 102 3.4.2.2 Cell cycle control, cell division, chromosome partitioning 105 3.4.2.3 Amino acid and nucleotide transport and metabolism 105 3.4.2.4 Carbohydrate transport and metabolism 107 3.4.2.5 Co-enzyme transport and metabolism 109 3.4.2.6 Translation, ribosomal structure, and biogenesis 112 3.4.2.7 Lipid transport and metabolism 114 3.4.2.8 Transcription 116 3.4.2.9 Replication, recombination, and repair 118 3.4.2.10 Cell wall/membrane/envelope biogenesis and motility 119 3.4.2.11 Post translational modification, protein turnover, chaperones 124 3.4.2.12 Inorganic ion transport and metabolism 133 3.4.2.12.1 Maintenance of ion balance 138 3.4.2.13 Proteins with general function prediction only, and hypothetical proteins 139 3.4.2.14 Signal transduction mechanisms 139 3.4.2.15 Intracellular trafficking, secretion, and vesicular
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transport 142 3.4.2.16 Defence mechanisms 145 3.4.3 Highly abundant proteins 146 3.4.4 Differentially abundant proteins 148 3.4.4.1 Heat stress 150 3.4.4.2 Metabolic proteins 153 3.4.4.3 Miscellaneous and hypothetical proteins 154 3.5 Conclusions 157
Chapter 4. Thermal and Metabolic Adaptation in the Hydrophobic Proteome of Methanococcoides burtonii 158 4.1 Summary 158 4.2 Introduction 160 4.2.1 Cold adaptation in Methanococcoides burtonii 160 4.2.2 Metabolic aspects of Methanococcoides burtonii growth 161 4.2.3 Experimental approach 162 4.3 Materials and Methods 166 4.3.1 Culture media preparation 166 4.3.1.1 M-media preparation 166 4.3.2 Assessment of growth of M. burtonii in M-media on methanol 167 4.3.3 Culture conditions 168 4.3.4 Hydrophobic protein extraction and protein measurement 168 4.3.5 Preparation of samples for iTRAQ labelling 168 4.3.5.1 iTRAQ experimental design 169 4.3.5.2 iTRAQ labelling 169 4.3.6 Sample clean up 170 4.3.7 Mass spectrometry 170 4.3.7.1 Data processing and visualisation 170 4.3.8 Data annotation and analysis 171 4.4 Results and Discussion 172 4.4.1 Growth response of M. burtonii to different substrates 172
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4.4.2 Differentially abundant proteins identified through iTRAQ 176 4.4.3 The thermal response in M. burtonii 178 4.4.3.1 Transporters and integral membrane proteins 179 4.4.3.2 Surface and secreted proteins 183 4.4.3.3 Translation and related processes 186 4.4.3.4 Protein folding, modification, and turnover 192 4.4.3.5 Transcription, replication, and DNA binding proteins 196 4.4.3.6 Heat induced stress in M. burtonii 198 4.4.4 The response of Methanococcoides burtonii to different substrates and media 202 3.4.4.1 Methanogenesis 203 3.4.4.2 Energy conservation and generation 209 3.4.4.3 General metabolic differences 211 3.4.4.4 Methanol as a stressor 213 4.5 Conclusions 217
Chapter 5. Characterisation of Methanococcoides burtonii cells and EPS with Respect to Temperature and Substrate 219 5.1 Summary 219 5.2 Introduction 220 5.3 Materials and Methods 223 5.3.1 Culture Media and conditions 223 5.3.2 Histochemical examination of EPS composition 223 5.3.2.1 Sample preparation 224 5.3.2.2 Alcian blue-PAS and Aldehyde fuchsin-Alcian blue 224 5.3.2.3 Methyl green-Pyronin 225 5.3.2.4 Other stains 225 5.3.3 Fixative preparation 226 5.3.4 Scanning electron microscopy 227 5.3.4.1 Sample preparation 227 5.3.4.2 Sample imaging 228 5.3.4.3 EDS Analysis 229
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5.3.4.4 Image analysis 229 5.3.5 Environmental scanning electron microscopy 230 5.3.5.1 Sample preparation 231 5.3.5.2 Sample imaging 231 5.3.6 Transmission electron microscopy 232 5.3.6.1 Sample preparation 232 5.3.6.2 Ultramicrotomy 233 5.3.6.3 Grid staining 235 5.3.6.4 TEM imaging 235 5.3.6.5 TEM image analysis 236 5.3.7 Statistical analysis 236 5.4 Results 237 5.4.1 Histochemistry 237 5.4.1.1 Stains acting on sugars 237 5.4.1.2 Stains acting on nucleic acids 238 5.4.1.3 Other stains performed 239 5.4.2 Scanning electron Microscopy 240 5.4.2.1 Cells and EPS from different growth temperatures were remarkably different 240 5.4.2.2 Energy dispersive X-ray spectroscopy of media precipitate 246 5.4.2.2 Statistical analysis 247 5.4.3 Environmental scanning electron microscopy 249 5.4.3.1 Parallel SEM 255 5.4.4 Transmission electron microscopy 257 5.4.4.1 Assessment of sample preparation procedures 257 5.4.4.2 TEM image analysis 259 5.5 Discussion 266 5.5.1 The extracellular polymeric substance of M. burtonii 266 5.5.1.1 The EPS produced by M. burtonii has different properties with respect to temperature 266 5.5.1.2 The EPS of M. burtonii contains RNA 268 5.5.1.3 EPS and cells associate with granular material 269
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5.5.2 Cells grown at low temperatures form large aggregates 269 5.5.3 Cell size differed with respect to temperature 270 5.5.4 The effects of methanol on cell morphology 272 5.5.4.1 Cells grown on methanol have irregular shapes 272 5.5.4.2 Methanol grown cells have cellular inclusions 273 5.6 Conclusions 274
Chapter 6. General Discussion, Conclusions, and Research Significance 276 6.1 The Differential Solubility Fractionation Procedure 276 6.2 The Hydrophobic Proteome 277 6.3 Thermal Adaptation in M. burtonii 281 6.3.1 The low temperature response 281 6.3.2 The high temperature response 283 6.3.3 The global approach 284 6.4 The Response of M. burtonii to Different Substrates and Nutrient Levels 284 6.4.1 The global approach 286 6.5 Growth Conditions Induced Significant Phenotypic Changes in M. burtonii 287 6.6 Combining the Theoretical and the Physical: A Union of Proteomics and Electron Microscopy 289 6.7.1 The DEAD-box helicase, an enigmatic cold adaptation protein 291 6.7 Conclusion and Significance of the Research 292
Chapter 7. Reference List 295
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List of figures Page
Figure 1.1. Ace lake and the Vestfold hills 23 Figure 1.2. Electron micrograph of M. burtonii 24 Figure 2.1 SDS-PAGE of HPP separation 64 Figure 2.2 SDS-PAGE of 4 and 23 C soluble and insoluble fractions 65 Figure 2.3 Comparison of solvents and extraction conditions (23 C cells) 67 Figure 2.4 Comparison of solvents and extraction conditions (4 C cells) 69 Figure 2.5 Progressive identifications 71
Figure 2.6 SDS-PAGE of 23 C DSF fractions 72 Figure 2.7 Minitab output for DSF hydrophobicity 74 Figure 2.8 Comparison of 23 C DSF fractions 75 Figure 2.9 Comparison of 4 C DSF fractions 75 Figure 2.10 Comparison of DSF proteins types with previous runs 77 Figure 2.11 Normalised peptide spectral counts for a soluble high and low abundance proteins 79 Figure 2.12 Normalized peptide spectral counts for membrane and hydrophobic proteins 80 Figure 2.13 Unexpected solubility trends 81 Figure 2.14 Properties of identified proteins 83 Figure 2.15 Proteins with predicted transmembrane domains 84 Figure 2.16 Number of runs in which each protein was identified 84 Figure 3.1 arCOG distribution comparisons 95 Figure 3.2 Annotated arCOG categories of identified proteins 97 Figure 3.3 Methanogenesis in M. burtonii 103 Figure 3.4 The mevalonate pathway in M. burtonii 115 Figure 3.5 dTDP-L-rhamnose synthesis in M. burtonii 121 Figure 3.6 UDP-GlcNAc and UDP-ManNAc synthesis in M. burtonii 122 Figure 3.7 Predicted membrane topology of HtpX-like proteins 127
Figure 3.8 Phylogeny of metazoan, bacterial and M. burtonii A2M 131 Figure 3.9 Domain architecture of sensor proteins 141
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Figure 3.10 The secretion apparatus of M. burtonii 144 Figure 3.11 J-domain of Mbur_1212 155 Figure 4.1 The iTRAQ labelling system 164 Figure 4.2 Growth media and substrate comparison 172 Figure 4.3 Differentially abundant proteins across all experiments 176 Figure 4.4 Overview of thermal responses in the M. burtonii hydrophobic proteome 179 Figure 4.5 Putative membrane anchoring site 186 Figure 4.6 Methanogenesis in M. burtonii 204 Figure 5.1 M. burtonii cell aggregates 221 Figure 5.2 Sampling and measurement procedure 230 Figure 5.3 Alcian blue-PAS stain results 237 Figure 5.4 Methyl green-pyronin stain results 239 Figure 5.5 Coomassie stain of EPS 240 Figure 5.6 SEM of cell clump as seen at 4˚C 241 Figure 5.7 Magnification of cell aggregate surface 241 Figure 5.8 High magnification of cell aggregate surface 242 Figure 5.9 SEM of cells from 4˚C and 23˚C grown in MFM 243 Figure 5.10 SEM of cells from 4˚C and 23˚C grown in M-media 244 Figure 5.11 High resolution SEM of cells from 4˚C and 23˚C 245 Figure 5.12 EDS spectra of media precipitate 246 Figure 5.13 Quantification of micrograph features 248 Figure 5.14 Differences in cell size 249 Figure 5.15 ESEM of 4˚C sample 250-251 Figure 5.16 ESEM of 23˚C sample 252-253 Figure 5.17 ESEM of cell aggregate from 4˚C grown cells 254 Figure 5.18 ESEM of EPS from 23˚C grown cells 255 Figure 5.19 Parallel SEM of ESEM samples 256 Figure 5.20 Semi then sections of TEM sample preparations 258 Figure 5.21 Ultra-thin sections of TEM sample preparations 259 Figure 5.22 TEM micrograph features 261 Figure 5.23 Differences in cell morphology with respect to media 262 Figure 5.24 Frequency distributions of cellular cross sectional areas 263
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Figure 5.25 Peripheral electron density in 4˚C cells 265
List of Tables Page Table 1.1 Cold marine archaea diversity 8 Table 1.2 Isolated psychrophilic archaea 14 Table 3.1 Energy production and conversion 99 Table 3.2 Cell cycle control, cell division, chromosome partitioning 105 Table 3.3 Amino acid and nucleotide transport and metabolism 107 Table 3.4 Carbohydrate transport and metabolism 108 Table 3.5 Coenzyme transport and metabolism 110 Table 3.6 Translation, ribosomal structure and biogenesis 113 Table 3.7 Lipid transport and metabolism 116 Table 3.8 Transcription 117 Table 3.9 Replication, recombination and repair 119 Table 3.10 Cell wall/membrane/envelope biogenesis 121 Table 3.11 Cell motility 124 Table 3.12 Posttranslational modification, protein turnover, chaperones 125 Table 3.13 Inorganic ion transport and metabolism 135 Table 3.14 Signal transduction mechanisms 142 Table 3.15 Intracellular trafficking, secretion, and vesicular transport 145 Table 3.16 Defence mechanisms 146 Table 3.17 High abundance proteins in M. burtonii 147 Table 3.18 Differentially abundant proteins also identified using labelling strategies 149 Table 3.19 Differentially abundant proteins related to those previously identified 151 Table 3.20 Differentially abundant proteins not identified by other methods 153 Table 4.1 Differentially abundant transporters and integral membrane proteins 180 Table 4.2 Differentially abundant surface and secreted proteins 185 Table 4.3 Differentially abundant proteins involved in translation 188
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Table 4.4 Reported roles for differentially abundant rproteins 189
Table 4.5 Differentially abundant proteins involved in protein folding, modification, and turnover 194 Table 4.6 Differentially abundant proteins involved in transcription, replication and DNA binding 196 Table 4.7 Differentially abundant proteins involved in the heat stress Response 198 Table 4.8 Differentially abundant proteins involved in methanogenesis 206 Table 4.9 Proteins with differential abundance involved in energy generation and conservation 211 Table 4.10 Differentially abundant proteins involved in general metabolism 212 Table 4.11 Differentially abundant proteins indicative of the stressful effects of methanol 215
Appendices Page
Appendix A Chapter 2 and 3 Appendices 325 Appendix A.1 Simplified flowchart of methodology 325 Appendix A.2 Rnf operon alignments 326 Appendix A.3 Na+/H+ antiporter alignments 329 Appendix A.4 Polysaccharide gene loci 331 Appendix A.4 a Polysaccharide locus 1 331 Appendix A.4 b Polysaccharide locus 2 332 Appendix A.4 c Polysaccharide locus 3 333 Appendix A.4 d Polysaccharide locus 4 334 Appendix A.4 e Polysaccharide locus 5 334 Appendix A.5 HtpX alignments 335 Appendix A.6 Lon catalytic domain alignments 335 Appendix A.7 KOD-1 Lon alignment 336 Appendix A.8 Stromatin alignment 336
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Appendix A.9 Alpha-2-macroglobulin alignment 337 Appendix A.10 GI numbers of proteins used in the phylogenetic
analysis of A2M proteins 342 Appendix A.11 FKBP type PPIase alignment 343 Appendix A.12 SuDH protein alignments 343 Appendix A.13 YidC protein alignments 344 Appendix A.14 YidC phylogenetic tree 345 Appendix A.15 Hydrophobicity profile of Mbur_2063 346
Appendix B Chapter 4 appendices 347 Appendix B.1 iTRAQ experimental design (thermal adaptation experiments) 347 Appendix B.2 iTRAQ labelling protocol (substrate experiments) 348 Appendix B.3 Formulas used for calculating weighted mean and standard deviation 348 Appendix B.4 Methanogenesis operon organisation in M. burtonii 349 Appendix B.4.1 Gene arrangement of methylamine locus 1 349 Appendix B.4.2 Gene arrangement of methylamine locus 2 349 Appendix B.4.3 Gene arrangement of methylamine locus 3 350 Appendix B.4.4 Gene arrangement of methylamine locus 4 350 Appendix B.4.5 Gene arrangement of methylamine locus 5 352 Appendix B.4.6 Gene arrangement of methanol locus 352
Supplementary material (available online via UNSW library)
Supplementary Table 1 All identified proteins Supplementary Table 2 All differentially abundant proteins
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Common abbreviations used
2DE: Two-dimensional electrophoresis A2M: Alpha-2-Macroglobulin ACN: Acetonitrile Ambic: Ammonium bicarbonate ANOVA: Analysis of variance arCOG: Archaeal cluster of orthologous genes ATP: Adenosine triphosphate BMSF: Bioanalytical mass spectrometry facility BSA: Bovine serum albumin COG: Cluster of orthologous genes CPD: Critical point dried CSP: Cold shock protein Da: Dalton DDA: Data dependant acquisition dH2O: Ultra-pure water (Milli-Q) DSF: Differential solubility fractionation DTT: Dithiothreitol EDS: Energy dispersive X-ray spectroscopy EDTA: Ethylenediaminetetraacetic acid EF: Evidence factor EF2: Elongation factor 2 EMU: Electron microscope unit EPS: Extracellular polymeric substance ER: Evidence rating ESEM: Environmental scanning electron microscopy ESI: Electrospray ionization FDR: False discovery rate FP: False positive GDH: Glutamate dehydrogenase Glufib: Glufibrinopeptide GRAVY: Grand average of hydrophobicity GO: Gene ontology H4SPT: Tetrahydrosarcinapterin Hdr: CoB-CoM heterodisulfide reductase HFBA: Heptafluorobutyric acid HPP: Hydrophobic proteome ICAT: Isotope coded affinity tags IDA: Iodoacetamide IDAM: Information dependant acquisition mode IEF: Isoelectric focusing IEP: Isoelectric point IMG: Integrated microbial genomes iTRAQ: Isobaric tags for relative and absolute quantitation JGI: Joint genome institute
D. Burg UNSW xvii kDa: Kilodalton LC/LC – MS/MS: Tandem liquid chromatography – tandem mass spectrometry LC – MS/MS: Liquid chromatography – tandem mass spectrometry M – media: Marine methanogen minimal media (defined, methanol as substrate) Mcr: Methyl-coenzyme M reductase MeOH: Methanol MFM: Methanococcoides trimethylamine media (rich, complex) MPa: Megapascals Mtr: ` Tetrahydrosarcinapterin–S –methyltransferase MW: Molecular weight MudPIT: Multi-dimensional protein identification technology m/z: Mass to charge ratio NADP: Nicotinamide adenine dinucleotide phosphate NIW: Not identified in whole-cell-extract OD: Optical density OGP: n-Octyl- -D-glucopyranoside PAS: Periodic acid Schiff’s PMF: Proton motive force PMSF: Phenylmethanesulphonylfluoride PPase: Proton translocating inorganic pyrophosphatase PPi: Pyrophosphate PPIase: Peptidyl-prolyl cis-trans isomerase ppm: Parts per million QTOF: Quadrupole time-of-flight RP: Reverse phase rproteins: Ribosomal proteins RT-PCR: Reverse transcriptase – polymerase chain reaction SCX: Strong cation exchange SDS: Sodium dodecyl sulfate SDS-PAGE: Sodium dodecyl sulfate - polyacrylamide gel electrophoresis SEM: Scanning electron microscopy SMF: Sodium motive force TEM: Transmission electron microscopy TEMED: Tetramethylethylenediamine TMA: Trimethylamine Tmax: Maximum growth temperature TMD(s): Transmembrane domain(s) TMHMM: Transmembrane hidden Markov model Tmin: Minimum growth temperature TOF: Time-of-flight TOF MS: Time-of-flight mass spectrum Topt: Temperature for maximum growth rate TP: True positive Tris: Tris(hydroxymethyl)aminomethane v/v: Volume per volume WCE: Whole cell extract w/v: Weight per volume
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Publications
Papers Thomas, T., Egan, S., Burg, D., Ng, C., Ting, L. and Cavicchioli, R. (2007). Integration of Genomics and Proteomics into Marine Microbial Ecology. Marine ecology progress series 332: 291 - 299.
In relation to Chapter 2 and 3 (available on supplementary disk)
Allen, M. A., Lauro, F. M., Williams, T. J., Burg, D., Siddiqui, K. S., De Francisci, D., Chong, K. W., Pilak, O., Chew, H. H., De Maere, M. Z., Ting, L., Katrib, M., Ng, C., Sowers, K. R., Galperin, M. Y., Anderson, I. J., Ivanova, N., Dalin, E., Martinez, M., Lapidus, A., Hauser, L., Land, M., Thomas, T. and Cavicchioli, R. (2009). The Genome Sequence of the Psychrophilic Archaeon, Methanococcoides burtonii: The Role of Genome Evolution in Cold Adaptation. ISME J 3: 1012-1035. (Paper 2)
Burg, D.W., Lauro, F. M., Williams, T. J., Raftery, M. J., Guilhaus, M. and Cavicchioli, R. (2010). Analyzing the Hydrophobic Proteome of the Antarctic Archaeon Methanococcoides burtonii Using Differential Solubility Fractionation. J.Proteome Res 9: 664 - 676 (Paper 1)
In relation to Chapter 4 (available on supplementary disk)
Williams, T. J., Burg, D. W.*, Ertan, H., Raftery, M. J., Guilhaus, M. and Cavicchioli, R. (2010). A Global Proteomic Analysis of the Insoluble, Soluble and Supernatant Fractions of the Psychrophilic Archaeon Methanococcoides burtonii Part II: The Effect of Different Methylated Growth Substrates J.Proteome Res 9: 653 - 663 (Paper 4)
Williams, T. J., Burg, D. W.*, Raftery, M. J., Guilhaus, M., Pilak, O. and Cavicchioli, R. (2010). A Global Proteomic Analysis of the Insoluble, Soluble and Supernatant Fractions of the Psychrophilic Archaeon Methanococcoides burtonii Part I: The Effect of Growth Temperature. J.Proteome Res 9: 640 - 652 (Paper 3)
*Equally contributing primary author
Conference proceedings Burg, D., Raftery, M., and Cavicchioli, R. (2008). Cold adaptation in the Archaeon Methanococcoides burtonii: Proteomic and phenotypic characterization. Oral presentation: 3rd International conference on Polar and Alpine Microbiology. Banff, Canada, May 2008.
Burg, D., Raftery, M., and Cavicchioli, R. (2007) The Role of Hydrophobic Proteins in the Cold Adaptation of Archaea. Poster presentation: Gordon research conferences; Archaea – Physiology, ecology and Molecular biology. Andover, NH. 19 - 24 August 2007
Cavicchioli, R., Raftery, M., Curmi, P., Williams, T., Pilak, O. and Burg, D. 2007. Uncovering Mechanisms for Repair and Protection in Cold Environments through Studies of Cold Adapted Archaea. US Air Force Office of Scientific Research. Biophysical Mechanisms Program Review. Arlington, VA, 15-17 August 2007
D. Burg UNSW xix
Chapter 1. General Introduction
1.1. The Cold Biosphere
The Earth is dominated by regions that are permanently cold (<5 C). These cold environments include alpine regions, terrestrial caves, the deep ocean (which covers
~70% of the Earth’s surface), the upper atmosphere, and polar regions (Cavicchioli
2006; Cowan et al., 2007). The polar regions can be divided into three environments: the terrestrial environment, which includes lakes, and is dominated by tundra
(permafrost) which covers 24% of land surface in the northern hemisphere (Zhang et al., 1999); polar oceanic regions; and seasonal sea ice. Together these cold regions account for ~75% of the Earth’s biosphere. They are found around the globe and contribute significantly to primary production, nutrient cycling, and biomass. Therefore, they are vital for global ecology. With the mounting evidence supporting climate change, which has already been documented as having an impact on the Arctic (IPCC
2008), it is important that these regions and the organisms which thrive in these ecosystems are understood. The study of these regions and organisms will not only allow us to monitor the changes occurring in these environments, but also help us to understand the global ramifications of climate change, as these cold areas will be affected more profoundly than others.
1.2. Psychrophiles
The cold regions of the Earth are biologically dominated by the microscopic psychrophiles (organisms at thermal equilibrium with their cold environment).
Psychrophiles can be divided into two main groups. The stenopsychrophiles (sometimes
D. Burg UNSW 1 referred to as psychrophiles or ‘true’ psychrophiles) are restricted to growth within a narrow range of temperatures, generally having a maximum growth rate (referred to as
Topt) at < 16 to 18 C, and a maximum temperature for growth (referred to as Tmax) of <
25 C. The eurypsychrophiles (sometimes referred to as psychrotrophs), generally have a larger range of growth temperatures, with Topt 18 – 25 C, and Tmax > 25 C (sometimes above 35 C), i.e. the organisms display mesotolerance (Bakermans and Nealson 2004;
Cavicchioli 2006). The eurypsychrophiles are isolated from cold environments more often than stenopsychrophiles (Russell et al., 1990; Bakermans and Nealson 2004), and the ability of these organisms to cope with large temperature ranges is related to the variability of temperature (e.g. due to seasonal changes) in the environments from which the organisms were isolated (reviewed in Russell et al., 1990). The semantics of the definition of psychrophiles does not hold great significance however, as many organisms which are defined as eurypsychrophiles have lower minimal growth temperatures and faster growth rates at low temperatures than stenopsychrophiles
(Cavicchioli 2006). Some eurypsychrophiles have also been shown to have a Tcritical
(maximum growth yields) at much lower temperatures than their reported Topt
(Bakermans and Nealson 2004). Psychrophiles, as a whole, should be viewed as a dynamic and diverse group of organisms, which are able to thrive at low temperatures.
For this reason the demarcation between the eury- and stenopsychrophiles will not be further explored in this review, and the organisms will be referred to as a whole group as psychrophiles.
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1.2.1 Psychrophilic eukaryotes
The psychrophilic eukaryotes include both large multi-cellular organisms and microorganisms. The large multi-cellular organisms include: plants, for example grasses
(Sandve et al., 2008), crucifers (Griffith et al., 2007), orchids (Yang et al., 2007), and seaweeds (Raven et al., 2002); many species of polar fish, including the Atlantic Cod
(Gudmundsdottir and Palsdottir 2005); and marine mollusks and invertebrates
(reviewed in Johnston 1990).
Of the eukaryotic microorganisms, the fungi tend to be the largest group studied, with diverse isolates from the Arctic (Sonjak et al., 2007), Antarctic (Duncan et al., 2006), and alpine regions (Schmidt et al., 2008). Psychrophilic yeasts have also become a focus of interest, especially from the perspective of bioremediation of polluted sites
(Bergauer et al., 2005). Other microscopic eukaryotic isolates include ciliates (Alimenti et al., 2002) and algae (Morgan-Kiss et al., 2008).
1.2.2 Psychrophilic bacteria
The scientific world has known of the existence of psychrophilic bacteria for close to
125 years, after bacterial activity was monitored in cold sediments by Certes (Russell et al., 1990). Psychrophilic bacteria represent the largest and most diverse group of microorganisms found in cold environments. Phylogenetic studies have shown that organisms from diverse bacterial lineages are found in cold environments, and that the population structure varies significantly with respect to sampling site.
Studies of the Southern Ocean have shown that bacteria dominate surface waters and that there is seasonal variation in abundance patterns as daylight hours, and sea ice coverage changes (Murray et al., 1998; Church et al., 2003; Murray and Grzymski
D. Burg UNSW 3
2007). A similar pattern has been observed in the Arctic ocean (Kirchman et al., 2007).
In studies of the deep ocean it has been found that while there are a few species of bacteria that tend to dominate numerically, there are thousands of low abundance populations representing extensive phylogenetic diversity (Sogin et al., 2006). Such oceanic studies have highlighted the complex, dynamic and crucial nature of oceanic psychrophilic bacteria.
Coastal polar environments also show enormous seasonal and spatial variability in bacterial diversity, following the same trends as the oceanic samples (Murray et al.,
1998; Wells and Deming 2003; Garneau et al., 2006). Other sampling sites show less diverse but equally dynamic populations of bacteria; they include recent studies of alpine and arctic soil (Nemergut et al., 2005), ice shelf microbial mats (Bottos et al.,
2008), perennial springs (Perreault et al., 2007), and sub-glacial environments
(Skidmore et al., 2000).
One of the most interesting and increasingly understood groups of psychrophilic bacteria are the eutectophiles. These organisms represent some of the most extreme psychrophiles, surviving in the brine veins of crystalline ice where water temperatures can drop to -20 C (Deming 2002). Several studies have shown that these organisms are diverse and are actively metabolising at -20 C (Junge et al., 2004; Junge et al., 2006).
1.3 Psychrophilic Archaea: Ubiquitous and Elusive
The archaea are recognised as the third domain of life, a division that was proposed following ground breaking comprehensive rRNA gene analysis by Woese et al.,(1990).
The archaea are distinct from the bacteria and eukaryotes, with some of the
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distinguishing features being: the structure of phospholipids (Koga et al., 1998); the cell envelopes (including S-layers), which are diverse in terms of structure and composition
(Forterre et al., 2002); and the unique metabolism shared by some members
(methanogens) (Ferry and Kastead 2007; Gao and Gupta 2007). The archaea share some features with both the bacteria (e.g. certain metabolic pathways) (Danson et al., 2007) and the eukarya (transcriptional machinery) (Forterre et al., 2002).
The domain archaea is divided into four kingdoms, two of which were first identified as the euryarchaeota and the crenarchaeota by Woese and Olsen (1986), and were subsequently classified as unique divisions within the domain archaea by Woese et al.,(1990). These two kingdoms make up the largest groups of archaea, with diverse members such as the halophiles and methanogens from the euryarchaeota, and the hyperthermophiles and marine planktonic group from the crenarchaeota (Schleper et al.,
2005). Recently, two other kingdoms of the archaea have been characterised: The korachaeota first described by Barns et al.,(1996) with the first genome sequence of this kingdom recently completed (Elkins et al., 2008); and the nanoarchaeota, containing only a single characterised member to date (Huber et al., 2002). However, nanoarchaeal
16S rRNA signatures have been identified in a variety of environmental samples, suggesting worldwide distribution (Huber et al., 2003).
The archaea were once thought of as being exclusively extremophilic organisms generally cultivated from hyperthermophilic, highly saline, or acidic environments. This may be due to the fact that most of the archaea are not amenable to culture in the laboratory, and those that were cultured came from these extreme environments where archaea typically show high diversity. With the emergence of molecular phylogenetic techniques, the archaea are increasingly being viewed as contributors to all
D. Burg UNSW 5 environments (reviewed in Stein and Simon 1996; Robertson et al., 2005; Schleper et al., 2005). To highlight this point, archaea have been found in high diversity in the ocean where they contribute significantly to biomass. It is estimated that the pelagic waters of the Pacific Ocean harbor 1.3 x 1028 archaeal cells. When compared to the predicted number of bacteria (3.1 x 1028), it is clear that the archaea are significant contributors to the world’s largest ecosystem (Karner et al., 2001).
Increasingly, the archaea are being seen as major contributors to the world’s cold habitats with many diversity studies showing them as key members of cold environments. The actual proportion of the consortia that archaea represent tends to be geographically and seasonally dynamic, with many studies reporting varying figures with regard to community composition. These are discussed below and are displayed in
Table 1.1.
In samples of Antarctic surface waters in the austral winter, the archaea were initially shown to be highly abundant members of the microbial community representing 34% of the prokaryotic biomass (DeLong et al., 1994). Similar results were gained by Murray et al., (1998), who found that archaea represented 24% of picoplankton in Antarctic waters. In separate Antarctic sampling experiments it was found that they were present in much lower numbers. Massive inter-seasonal variations were recorded, with archaea representing 1% of picoplankton in summer to 10% in the winter months (Church et al.,
2003). The reported archaeal population in polar coastal waters is also variable, with figures dramatically changing with respect to sampling site. In Arctic coastal waters, the numbers of recorded archaea vary, with figures reported ranging from 0.2 to 13 % of total cells (Wells and Deming 2003), to 1.5 – 6% of total cells (Garneau et al., 2006).
6 D. Burg UNSW
Reported numbers of archaea ranged from 1 – 17% of total plankton (Murray et al.,
1998; Church et al., 2003).
In Antarctic coastal waters, the archaea have been reported to be seasonally variable with the highest numbers recorded in the winter months and interannual variations observed (DeLong et al., 2004; Murray et al., 1998). Variations in the reported population of archaea may be due to differences in site of sampling (archaea were shown to be more abundant in water containing particulate matter associated with river outflows (Wells and Deming 2003)), or seasonal changes caused by competition from photosynthetic plankton during summer months. As no samples were gathered in the
Arctic winter, this may account for lower recorded maximum counts. Primer and probe design, as well as experimental technique variation, may have contributed to the outcome of many of these diversity studies (Bano et al., 2004). Some research group’s archaeal primers or molecular probes may have been more effective than others at targeting archaea, or phylogenetic groups within the archaea, which could have lead to varying figures.
In samples taken from the ocean (with sampling starting at 100m depth), the archaea have been shown to become increasingly abundant as depth increases, accounting for a large proportion of biomass (up to 40% of the total cells) (Herndl et al., 2005; Kirchman et al., 2007). Bano et al.,(2004) suggested that the deep Arctic ocean showed little seasonal variation in population composition and that Arctic and Antarctic ribotypes were remarkably similar. However, Kirchman et al., (2007) showed the composition within the archaeal community reported in oceanic samples varies, with Arctic samples displaying a lack of euryarchaeota (group II marine) and an abundance of crenarchaeota
(group I marine) not seen in Antarctic samples. This may be due to the differences in
D. Burg UNSW 7
sampling sites, depths, and quantitative methods, or seasonal and annual variations
mentioned above.
numbers outflows) ribotypes with depth Comments (Atlantic deep) High archaeal diversity Archaeal contribution to variation in deep waters differences in suface water with photosynthetic plankton Seasonal variation. Negative ribotypes similar. Significant nepheloid (particle rich) layer Seasonal bacterial variation in 84% (Labrador sea), 10 - 20% Archaea more associated with Archaea more associated with particle rich samples (e.g. river Archaeal abundance increases water. Arctic and Antarctic deep correlation of Archaeal numbers prokaryotic production varies. 13 - surface vaters. Archaeal seasonal 20% (oxygen minimum layer), 41 - Archaeal richness greatest in deep
FISH FISH FISH FISH DGGE DGGE probes technique Measuring CARD FISH Group specific Group specific Group specific MICRO CARD oligonucleotide oligonucleotide 16S rRNA PCR CARD FISH and Archaeal specific Archaeal specific polyribonucleotide polyribonucleotide hybridization, RNA
Group II
archaea Euryarchaeota - Phylogeny probes used probes used Dominace of crenarchaeota marine archaea marine archaea marine archaea marine archaea No difference in Complex. Large Complex. respect to depth No group specific No group specific Coastal: Coastal: and euryarchaeota and group II marine Group I and group II Group I and group II Dominance of group I metabolic activity with (dominant) and group I River: between crenarchaeota numbers of both group I Rice cluster V and LDS.
: 1%
Summer (deep) prokaryotes picoplankton picoplankton 10% (surface), 13%
cells^ total 13% Archaeal diversity Archaeal 1.3* to 6^% total cells total 6^% to 1.3* (surface), 9 - 39% (deep). (average) of picoplankton^ (surface) to 40% (200m) of Not measured against total total against measured Not total against measured Not Crenarchaeota 17% of total Winter 0.1 - 2.6% total cells*. 2.3 - Archaea increase from 10% counts (increase with depth) 34% of prokaryotic biomass) % picoplankton 18 - 30% of picoplankton (21 - counts (no change with depth) euryarchaeota 18 - 26% of total
1 - 17% of picoplankton*. 24%
early round) early - spring Late winter spring (somespring late summer. Late summer Late summer Late summer early summer sampling year Sampling time Sampling Antarctic:
Late winter to early Spring and Summer Summer and Winter Arctic: - mid summerspring
Antarctic: Site depth) 500m) column mid-oceanic (5 - unknown) (offshore^). Palmer basin (surface* and Canada basin 3000m depth) surface waters) esturine^ waters Chukchi sea and throughout water River and coastal nepheloid^ layers) 235 m). Northwest passage island (nearshore*). Coastal surface* and (samples up to 200 m (exact sampling depth Arthur harbor (Coastal 4000 km transect (200 - Arctic: oceanic and coastal (5 - Surface waters. Anvers Palmer basin. Samples
et et et et et et et et al., (1994) (2006) (2003) (2007) by (2005) (2006) (1998) Cold marine archaea diversity Cold marine archaea
(2004) (2003) Herndl Demming Demming Murray al., Wells and Church Galand Sampling Sampling al., al., al., al., al., al., DeLong Bano Garneau Kirchman and Deep Table 1.1 Arctic Arctic Arctic Arctic Arctic Ocean Atlantic Region Western Antarctic Antarctic Antarctic Antarctic
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Archaea are also being increasingly identified in a variety of cold non-marine environments. Archaea have been shown to be present in high abundance and with high species diversity in a number of cold lakes (Koch et al., 2006; Auguet and Casamayor
2008; Lliros et al., 2008). Group II marine archaea have also been isolated in abundance from the intestinal tracts of North Sea fishes (van der Maarel et al., 1998). In contrast to this, in a 16S rRNA gene clone library from an Arctic ice shelf microbial mat, only a single archaeal clone was identified (Junge et al., 2004; Bottos et al., 2008). In sea ice only 3% of active cells were found to be archaea (Junge et al., 2004). However these low numbers could be influenced by archaeal primer problems as mentioned above, which would result in underestimation of sample diversity.
The aforementioned studies describe the diversity and variability of biological systems in cold environments. The archaea are being increasingly identified as important members of these communities. Figures reporting the abundance of archaea in cold environments tend to vary, with differences occurring spatially and temporally (seasonal and interannual variation). As techniques for identifying archaea in the environment evolve and become more universal, it is likely that the true abundances of these organisms in cold regions will be established.
1.4 The Methanogens
The methanogens are a diverse group of organisms, which form the largest known cohort (in terms of characterised species) within the archaea. All of the methanogens are strict anaerobes (requiring highly reduced conditions for metabolic function) and, collectively, are able to metabolise numerous substrates to methane, producing cellular
D. Burg UNSW 9 energy and biomass. To do this, they utilise a variety of enzymes and co-factors found almost exclusively within the methanogens (Ferry and Kastead 2007). The methanogens are found in a number of environments on Earth, from the guts of ruminant animals, to hyperthermal vents and cold sediments (Ferry and Kastead 2007).
There are three main modes of methanogenic metabolism: CO2-reducing; acetoclastic; and methylotrophic. The first two are found in the competitive methanogens (CO2- reducing and acetoclastic), which form complex syntrophic relationships with other organisms in the environment in their competition for substrate and substrate dynamics
(Ferry and Kastead 2007). The CO2-reducing methanogens are able to convert carbon from its oxidised low energy state CO2 (produced as waste from other organisms), into the reduced high energy CH4, utilising H2 and generating cellular energy in the process.
The acetoclastic methanogens are able to use the acetate fermentation pathway, using acetate and H2 to form CH4, in a process that accounts for the highest proportion of methane produced by methanogens in the environment (Ferry and Kastead 2007). The organisms utilising this pathway generally thrive in a complex relationship with homoacetogenic bacteria (Kotsyurbenko 2005; Ferry and Kastead 2007; Metje and
Frenzel 2007). The third group of methanogenic pathways are those utilised by non- competitive methanogens (also known as the methylotrophic methanogens), which are able to use the waste products of other organisms in the form of simple carbon compounds, for example methanol and methylamines, as substrates for methanogenesis.
Because other organisms do not readily utilise these substrates, the methylotrophic methanogens play important roles in global carbon cycling. Another rare form of methanogenesis involves the use of methylated compounds and hydrogen for methanogenesis and is only known to be used by Methanosarcina stadtmanae (Ferry
10 D. Burg UNSW
and Kastead 2007). Many methanogens, particularly members of the
Methanosarcinaceae, have the capability of utilising a variety of substrates and their consequent biochemical pathways, highlighting the versatility of these important organisms and their ability to cycle carbon in the environment (Ferry and Kastead
2007).
The methanogens make up the most comprehensively studied and most frequently isolated of the psychrophilic archaea. The large number of psychrophilic methanogens identified and classified could, however, be due to the fact that these organisms (along with the halophiles) are relatively easy to grow in the laboratory compared to other archaea. Therefore, the diversity of these organisms in the environment, compared to the number isolated, may be skewed (Kendall and Boone 2006).
There are many reasons for interest in the psychrophilic methanogens, from the obvious perspective of fuel production under cold conditions, through to low temperature waste digestion (Collins et al., 2006). There is also interest in these organisms from a global warming perspective. When the cold habitats of these organisms warm, the methane output is likely to increase, resulting in an increase of the potent greenhouse gas into the atmosphere (Hoj et al., 2008). The potential ramification of global warming, in terms of carbon cycling in currently cold environments, remains unknown.
The methanogens have also come under recent scrutiny from an astrobiological point of view. Methanogens thrive under cold, anoxic conditions, with simple substrates (which can be of inorganic origin), which makes them prime candidates as Earth analogues of potential extraterrestrial life (Cavicchioli 2002). With the discovery of evidence indicating water ice on the surface of Mars (Boynton et al., 2002) and of methane in the
D. Burg UNSW 11
Martian atmosphere (Formisano et al., 2004), Earth’s methanogens have become a major interest as analogues of potential Martian life. Methanogens have been shown to grow in a Mars soil simulant (Kral et al., 2004), and under Mars-like conditions of temperature (Reid et al., 2006; Morozova et al., 2007), UV irradiation, and desiccation stress (Morozova and Wagner 2007).
Psychrophilic methanogens have been discovered in, and isolated from, a variety of cold habitats including: coastal sediments (Purdy et al., 2003; Singh et al., 2005;
Kendall and Boone 2006; Kendall et al., 2007); anoxic waters and sediments of lakes
(Franzmann et al., 1992; Franzmann et al., 1997; Simankova et al., 2001; Purdy et al.,
2003); from glacial ice cores, where active metabolism has been proposed (Tung et al.,
2005); and as important members of permafrost soils which cover 24% of the Northern
Hemisphere’s land surface (Zhang et al., 1999; Wagner et al., 2005; Metje and Frenzel
2007) and account for 14% of global organic soil carbon (Post et al., 1982).
A number of psychrophilic methanogens have been isolated in pure culture and have been described; several have genome sequences resolved. These and other characterised psychrophilic archaea are discussed below.
1.5 Isolated Psychrophilic Archaea
Despite the diversity of psychrophilic archaea in the environment, very few have been isolated or characterised, and even fewer have been the subjects of extensive investigation. The isolated species are summarised in Table 1.2. The majority of isolates are methanogens, cultivated from sediments, permafrost and anoxic lake depths. The other species of isolated psychrophilic archaea are: the sponge symbiont Crenarchaeum
12 D. Burg UNSW
symbiosum, the only characterised psychrophilic crenarchaeon; the halophile
Halorubrum lucusprofundi, isolated from an Antarctic lake; and the SM1 euryarchaeon, isolated from the cold sulfurous waters of Sippenauer Moor in Germany. Of these isolated/characterised psychrophilic archaea, very few have been studied extensively in terms of cold adaptation with the exception of Methanococcoides burtonii, which is discussed in depth in part 1.7 of this review.
1.6 Molecular Mechanisms of Cold Adaptation
As is evident from Table 1.1, there are very few psychrophilic archaeal isolates. Of these psychrophilic isolates, even fewer have been characterised and very few genomes have been sequenced. Studies on the molecular mechanisms of cold adaptation in psychrophilic archaea are limited to only a few species. However, molecular mechanisms of cold adaptation have been investigated in a variety of bacteria and, to some extent, eukaryotes. As molecular cold adaptation appears to be relatively universal across the psychrophiles (Cavicchioli 2006), the mechanisms of cold adaptation will be discussed with respect to all studied organisms, to gain insight into possible mechanisms of cold adaptation in the archaea.
D. Burg UNSW 13
of M. M. opt
species species burtonii archaea Comments charaterised The only isolated The only isolated Closely related to Lowest known T psychrophilic archaea psychrophilic halophile Has not been completely Cold biotype of classified Cold biotype of classified psychrophilic crenarchaea studied of all psychrophilic The most comprehensively been classified but are isolated et et
et , 1998; , (2006) , (2003); , (2003) , (2003) , (2003) , (2003) , (1997); , (2001); , 2007) et al. , 2006) et al. , (2002) , (2008) , (2005) et al. , (1988); Gibson , (1988); Gibson , (2006) , (2007) et al. , (2002); Singh et al. et al. et al. et al. , (2007); Morozova et al. , (2003); Giaquinto , (2003); Giaquinto , (2001); Moissl et al. , 1996; Schleper , (2005) et al. et al. et al. et al. , (2005); Henneberger al.
et al. References et al. et al. et al. et al. et al. et al. et al. Singh Hallam Zhang and Wagner, (2007) Chong (Kendall , (2005); Reid et al. Simankova Simankova Simankova Simankova Simankova Franzmann See part 1.7 of this review , 1997; Schleper , (2002); Moissl al. al. Rudolph et al. von Klein (Preston Franzmann Morozova Saunders Moissl Indicates organisms that have not ‡
only only only only only only Minimal Minimal Minimal Studies and lipids genomics Numerous, Cold shock polymerase polymerase proteomics, proteomics, EPS formation proteins, some characterisation Genomics, DNA DNA Genomics, Stress tolerance Characterisation Characterisation Characterisation Characterisation Characterisation Characterisation homology only. Biofilm formation, bacterial partners, filamentous ‘Hami’ Lipid unsaturation, genomics, enzymes
McrD No No No No No No No No No No No Yes Yes Yes Draft Genome Genome available ? +12 (°C) 1 - 6 1 - 2 4 - 6 1 - 6 1 - 2 1 - 6 5 - 6 1 - 6 4 - 6 5 - 6 temp 8 - 18 -18 to 1 - 16 10 - 11 In situ
on the basis of 16S rRNA and on the basis Russia plateau symbiont Germany Baltic Sea Switzerland Soppen Lake, Tundra, Russia sponge Ocean, Baldegger Lake, Wetland, Tibetan Skan Bay, Alaska Skan Bay, Alaska Skan Bay, Alaska Sippenauer Moor, Skan Bay, Alaska. Isolation location anaerobic digestor pond, Russia. Cold Permafrost, Siberia Switzerland. Waste Ace Lake, Antarctica Ace Lake, Antarctica Deep Lake, Antarctica Paper mill waste pond, Isolated psychrophilic archaea ‡
* ‡ sp* * SMA-21 Table 1.2 Organism hollandica Euryarchaeon SM1 Methanosarcina baltica Methanomethylovorans Methanosarcina mazei Methanogenium boonei Methanogenium frigidum Methanosarcina lacustris Methanocorpusculum Methanogenuim marinum Methanosarcina Halorubrum lacusprofundi Methanococcoides burtonii Crenarchaeum symbiosum Methanolobus psychrophilus Methanococcoides alaskense * denotes organisms that were classified in pure culture
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In psychrophilic microorganisms all aspects of cellular biology must be suitably adapted to the cold, as these organisms are at thermal equilibrium with their habitats
(Cavicchioli 2006). These adaptations include: changes in membrane fluidity and membrane proteins; alterations of thermodynamic properties of proteins and enzymes; genome arrangement and content; intracellular solute concentration; changes in protein levels and expression of specific cold associated proteins; and the production of extracellular polymeric substances (EPS).
1.6.1 Membrane and membrane protein changes in psychrophiles
As temperatures decrease, the membranes of organisms tend to become more rigid
(increased gel phase). This rigidity leads to decreased membrane permeability to small molecules, decreased membrane protein function due to a restriction of their ability to undergo conformational change, and an increased susceptibility to cellular damage by ice crystal formation (Yamada et al., 2002; Russell 2007). The psychrophiles are able to overcome any potential increase in membrane rigidity by increasing its amount of unsaturated lipids. This maintains the membrane in a liquid crystalline state and has been reported for not only psychrophilic bacteria (Tarpgaard et al., 2006) but also psychrophilic archaea. In studies of both H. lacusprofundi and M. burtonii, it was found that cells grown in cold conditions have a much higher level of lipid unsaturation than cells grown at high temperatures (Nichols and Franzmann 1992; Nichols et al., 2004;
Gibson et al., 2005). In contrast to this, in cold adapted fishes, membrane fluidity does not play a major role in membrane physiology, but alterations in the membrane proteins themselves play a role (Romisch et al., 2003). At cold, rigidifying temperatures, membrane proteins display decreased function and reduced affinity for substrates
(Nedwell 1999). It is therefore highly probable that changes in membrane proteins
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(either through protein modification or increases in abundance) is also a feature of membrane adaptation in psychrophilic microorganisms. There is some evidence to suggest that this is the case (see 1.6.5.1), however, due to the paucity of studies of membrane proteins in psychrophilic bacteria and archaea, knowledge in this area is limited.
1.6.2 Properties of psychrophilic enzymes and proteins
Protein adaptations are the most studied aspect of cold adaptation physiology. In recent years several comprehensive reviews have been written in the area (D'Amico et al.,
2002; Siddiqui and Cavicchioli 2006; Feller 2008). This interest is somewhat driven by the impetus to exploit the favourable properties of psychrophilic proteins for use in industry and technology (Cavicchioli et al., 2002). Examples include: exocellular peptides from the bacterium Pseudoalteromonas haloplanktis (Mitova et al., 2005); a pheromone from the ciliate Euplotes nobilii (Alimenti et al., 2002); and the screening of secreted enzymes of psychrophilic yeasts (Brizzio et al., 2007). The ability to recombinantly express active psychrophilic enzymes that retain their intrinsic properties in mesophilic hosts (Feller et al., 1998b), has allowed many potentially useful enzymes from a number of psychrophilic organisms to be mass-produced (Schleper et al., 1997;
Feller et al., 1998a; Tsigos et al., 1998; Camardella et al., 2002; Papanikolau et al.,
2005; Kawakami et al., 2007; Tsuruta et al., 2007).
The properties of cold adapted proteins which make them able to function at low temperatures (where thermodynamic constraints make reaction rates slow), and make them so attractive from an industrial point of view, are high activity and high catalytic efficiency at low temperatures, resulting from low activation energy and high flexibility
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(D'Amico et al., 2002; Siddiqui and Cavicchioli 2006). Low activation energies have been observed in enzymes from psychrophilic yeasts (Petrescu et al., 2000), bacteria
(Georlette et al., 2000) and archaea (Siddiqui et al., 2002). High flexibility is also seen in psychrophilic proteins, and is proposed to facilitate function under cold rigidifying conditions. Flexibility is achieved through extended surface loops, a decrease in proline residues in loops (Gerike et al., 1997), amino acid substitutions (less arginine and lysine, which form stabilising hydrogen bonds) (Feller et al., 1997), and a decrease in the number of buried hydrophobic residues, which increases the plasticity of the active site (Huston et al., 2008). The flexibility of these proteins intrinsically imparts a low thermal stability, as is apparent when psychrophilic proteins are compared to their thermophilic and mesophilic counterparts (Chen and Berns 1978).
The functions of psychrophilic proteins can be highlighted by gene complementation studies; the insertion of genes for certain psychrophilic proteins, determined to be key for cold biology, into cold sensitive hosts can confer the ability to grow in cold conditions (Giaquinto et al., 2007; Yang et al., 2007). These types of studies may prove useful for biotechnology, as engineering lower temperature requirements for key organisms can reduce energy consumption through reduced required incubation temperatures.
1.6.3 Genomics of psychrophiles
The genomes of several psychrophiles have been sequenced. Comparative analysis of these genomes with respect to genomes of non-psychrophiles has revealed several distinguishing features. Genomic analysis performed on the archaea M. burtonii and M. frigidum compared to non-psychrophilic archaea, found that the proteins from the psychrophiles had a higher content of non-charged polar amino acids and a lower
D. Burg UNSW 17 content of hydrophobic amino acids (Saunders et al., 2003). This observation was supported by genomic analysis of the bacteria Psychromonas ingrahamii, with many proteins from this organism showing a reduced hydrophobic character (Riley et al.,
2008). Such changes are thought to be involved in increasing the flexibility of the proteins.
Other genomic analyses have found that cold adapted organisms have high capacities to take up and produce osmolytes (Methe et al., 2005; Riley et al., 2008), possibly as cryoprotectants, and a high number of unique hypothetical proteins (Riley et al., 2008;
Allen et al., 2009), which may be involved in cold adaptation. Comparative genomic analyses of psychrophilic archaea have indicated that tRNA flexibility is not conferred by the GC content (in contrast to thermophiles which have a high GC content, conferring stability) and must be maintained by other means (Saunders et al., 2003).
Several psychrophiles are also predicted to have a relatively large proportion of the genome involved with the production and export of extracellular polymeric substance
(EPS) (Methe et al., 2005; Allen et al., 2009).
1.6.4 Compatible solutes
Organisms can accumulate compatible solutes, such as glycine betaine and other amino acid derivatives, as protein protectants when under stress. This is well characterised as a response to salt stress and high temperatures in archaea (reviewed in Muller et al.,
2005). There is evidence to suggest that compatible solutes play a role in the cold stress response in non-psychrophilic plants (Wanner and Junttila 1999), algae (Nagao et al.,
2008), thermophilic archaea (Weinberg et al., 2005) and mesophilic bacteria (Ko et al.,
1994). However, very little is known about compatible solutes in psychrophiles. The genome of Colwellia psychrerythraea suggests that this organism is able to accumulate
18 D. Burg UNSW
compatible solutes (Methe et al., 2005). As C. psychrerythraea is a sea ice bacterium, which inhabits brine veins within the ice, the capacity to produce and transport compatible solutes might be a reflection of the changing saline conditions of its natural environment.
1.6.5 Identifying molecular mechanisms of cold adaptation using high throughput methods
Apart from the fundamental changes in the components of proteins related to the cold, cells can overcome the kinetic effects of low temperature by employing a strategy of increasing the concentration of enzymes within the cell, which acts to increase the number of reactions that can occur at any given time (Siddiqui and Cavicchioli 2006).
Another strategy involves the production of accessory proteins, which aid in protein stability and folding (Relina and Gulevsky 2003). Proteomics and transcriptomics are methods by which these changes in a cell can be assessed with respect to temperature.
These techniques also allow the detection of changes in processes that are temperature related, e.g. transport of certain molecules, lipid unsaturation processes, and the use of accessory pathways. Such investigations can also identify regulatory elements and other cold specific gene products. At the time of writing this thesis, very few studies had been published on the proteomics and transcriptomics of psychrophiles, with the exception of the extensive proteomic studies of M. burtonii (to be discussed in part 1.7 of this review) and those described below.
Proteomic studies of several psychrophilic species of bacteria have revealed several common cellular adaptations to the cold. In proteomic studies of Psychrobacter cryohalolentis (Bakermans et al., 2007), Exiguobacterium sibiricum (Qiu et al., 2006)
D. Burg UNSW 19 and Shewanella livingstonensis (Kawamoto et al., 2007), it was found that under cold conditions there is an increase in enzymes involved in protein synthesis, indicating a drive to counteract thermally slow rates of transcription. Increases in molecular chaperones, chaperonins and peptidyl prolyl cis-trans isomerases were also identified, indicating the particular importance of protein folding at low temperatures. A large proportion of cold abundant proteins identified in all studies were hypothetical, which indicates these proteins have important roles in psychrophilic physiology. Bakermans et al.,(2007) also noted that there was an increase in proteins involved in energy production when cells were grown under cold conditions. This may be reflective of slow rates of reaction in the cold, and a drive to increase these processes to meet energy demands.
1.6.5.1 Membrane proteins identified in high throughput analyses.
As would be anticipated, membrane proteins have been identified as playing important roles in cold adaptation in a small number of studies (Nevot et al., 2006; Bakermans et al., 2007; Kawamoto et al., 2007). These studies utilised gel based separation protocols prior to mass spectrometric analysis. However, the use of gel based methods for identifying differential abundance in membrane proteins is notoriously difficult and prone to underestimation (Wu and Yates 2003). This is reflected in the results of
Kawamoto et al.,(2007), as even though an enrichment strategy for membrane proteins was utilised, a relatively low number of membrane proteins were identified. In order to fully appreciate the important role of membrane and other hydrophobic proteins in cold adaptation, alternative enrichment methods need to be used prior to high throughput mass spectrometric analysis.
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1.6.5.2 Cold shock responses in non-psychrophiles
Cold shock responses in non-psycrophiles, including several members of the archaea, have also been studied using proteomics and transcriptomics (Beckering et al., 2002;
Kaan et al., 2002; Mihoub et al., 2003; Polissi et al., 2003; Boonyaratanakornkit et al.,
2005; Lang et al., 2005; Weinberg et al., 2005; Zhang et al., 2005; Coker et al., 2007).
These studies draw several interesting parallels, in terms of the cold response, with the studies of psychrophiles. Non-psychrophiles under cold shock conditions show lipid unsaturation, an increase in protein synthesis and protein folding machinery, RNA stabilisation, and transport. Although the types of proteins identified in cold adapted psychrophiles and cold shock responses in non-psychrophiles are similar, it does not necessarily indicate that the psychrophiles are in a state of stress. Such observations are more indicative of, for example in the case of the increase in protein folding machinery, the intrinsic flexibility of psychrophilic proteins requiring folding vs. cold induced denaturation of non-psychrophilic proteins. It reflects temperature critical areas of cell biology and the diverse mechanisms all cells have to impart the ability to survive under cold conditions.
1.6.6 Extracellular polymeric substance production by psychrophiles
Many psychrophiles, including the archaeons M. burtonii, H. lacusprofundi (Reid et al.,
2006) and the euryarchaeon SM1 (Rudolph et al., 2001), produce EPS. The roles and chemical compositions of psychrophilic EPS remain largely unknown, and this is an area of expanding research. Characterised EPS of non-psychrophiles, which consists of polysaccharide, lipid, protein, DNA, and RNA or combinations of elements, has been shown to be widely variable with respect to individual species (Kachlany et al., 2001;
Whitechurch et al., 2002; Ando et al., 2006).
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Many psychrophiles produce an increased amount of EPS under cold conditions
(Nichols et al., 2005; Nevot et al., 2008), and the addition of crude EPS has been shown to increase freezing tolerance in a number of species (Tamaru et al., 2005; Junge et al.,
2006). The exact role of EPS in psychrophiles is not known, however, possible roles in nutrient accumulation and enzyme stabilisation have been proposed (Qin et al., 2007), as well as the experimentally verified cryoprotecting properties.
1.7 Methanococcoides burtonii
Methanococcoides burtonii is the most intensively studied of the psychrophilic archaea.
The large body of work performed on this organism and several of its proteins also makes it one of the most studied of all psychrophilic organisms. This section of the review describes M. burtonii and its natural habitat. Following this description, the known mechanisms of cold adaptation in the organism are considered.
1.7.1 Methanococcoides burtonii in the environment
Methanococcoides burtonii was isolated from the anaerobic, methane and H2S rich depths of Ace Lake, which is located in the Vestfold Hills region, close to Davis
Station, in the Australian Antarctic Territory (Franzmann et al., 1992) (Figure 1.1).
Ace Lake is meromictic (stratified), with upper layers that undergo seasonal mixing due to ice cover melt, a distinct halocline (increasing salinity as depth increases), and an anoxic monimolimnion that occurs at 12 metres depth, to the lake bottom at 25 metres.
The lake has a permanent temperature of 1 – 2 C and is covered with ice for ~11 months of the year. The lake has a complicated Holocene history. It is proposed that it was initially fresh water, formed by an ice cap melt ~ 11000 years ago. Approximately
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9400 years ago, rising sea levels caused the lake to be flooded with seawater. Changes in sea level occurring over the next ~ 4000 years caused it to change from an open marine basin to a seasonally flooded basin. A subsequent drop in sea levels (or an isostatic rebound event) ~ 5100 years ago caused the lake to be permanently isolated from the ocean (Rankin et al., 1996; Cromer et al., 2005). The suite of organisms in the lake is therefore of marine origin, and has formed an isolated community that has developed relatively recently. The organisms of Ace Lake have been studied for a number of years, and form a complex community with many species limited to certain areas of the lake based upon its stratified nature (Rankin et al., 1996). Recent and current expeditions to Ace Lake aim to further chemically analyse it, as well as investigate the Ace Lake community using metagenomic and metaproteomic approaches (R. Cavicchioli, personal communication).
a) b)
Figure 1.1 a) Ace Lake, and b) the Vestfold Hills region in summer. Photographs courtesy of Anthony Hull (a) and Kevin Burg (b)
M. burtonii (Figure 1.2) was isolated from the depths of the lake and described by
Franzmann et al., (1992). M. burtonii is an obligately methylotrophic methanogen, utilising only methylamines and methanol as substrates for methanogenesis. The
D. Burg UNSW 23 organism is flagellated, motile, and stains Gram variable. The viable temperature range of the organism is -2 C to 29 C, with maximum growth rate occurring at 23 C, leading to the classification of this organism as a eurypsychrophile. The ability of this M. burtonii to grow at sub-zero temperatures makes it unique amongst the isolated psychrophilic archaea, with no other characterised archaeon capable of doing so
(Cavicchioli 2006).
1.7.2 Cold adaptation in Methanococcoides burtonii
Cold adaptation in M. burtonii has been the subject of extensive investigation.
Genomics (1.7.2.1), proteomics (1.7.2.2), and focused protein studies have uncovered a variety of mechanisms of cold adaptation, which will be discussed below (1.7.2.3 –
1.7.2.1).
Figure 1.2. Electron micrograph of M. burtonii. Scale bar represents 1 m
1.7.2.1 Genomics
The genome of M. burtonii has recently been closed and this has allowed for comprehensive genomic analysis (Allen et al., 2009). The analysis highlighted the immense adaptive potential of the organism, evidenced by an over-representation of signal transduction genes, several bacteria-like central metabolism genes, likely the
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result of horizontal gene transfer from ε- and δ-proteobacteria, and a large complement of transposases. Proteomic analyses have also identified two of these transposases as being expressed in the cell, indicating active genome rearrangement events under cold conditions (Goodchild et al., 2004a). This ability for genome re-arrangement may be reflective of the relatively recent geographical isolation of Ace Lake, and hence M. burtonii.
As well as adaptive potential and genome plasticity, it was found that while codon usage was similar to mesophilic Methanosarcina spp., amino acid usage was skewed, with M. burtonii preferring those amino acids associated with cold adaptation. It was proposed that genes which are needed to be efficiently expressed in the genome, e.g. ribosomal proteins, had a higher degree of amino acid skew, and this may be reflective of streamlining or incomplete evolution of the genome (Allen et al., 2009).
Other noteworthy findings following genomic analysis include the identification of a number of putative novel ABC transporters, suggesting uncharacterised active transport processes, and a large number of unique hypothetical proteins, which are of interest as potential cold adaptation related proteins.
1.7.2.2 Proteomics
One of the major research techniques that has been applied to M. burtonii is proteome analysis, which is considered complementary to genomic analysis (Goodchild et al.,
2004a). Past analyses have been performed on whole cell extracts and secreted proteins using gel-based methods, stable isotope labelling, and high throughput mass spectrometry.
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Comprehensive analysis of the M. burtonii proteome was achieved by Goodchild et al.,
(2004a) by utilising tandem liquid chromatography – tandem mass spectrometry
(LC/LC-MS/MS) to analyse whole cell extracts of cells grown under cold conditions
(4 C). This analysis enabled the identification of 528 cellular proteins. The biology inferred from this analysis highlighted many metabolic features of M. burtonii (see
1.7.2.3 – 1.7.2.11). However, proteomic analysis of WCE was only able to identify a small number of proteins with transmembrane domains (29), representing just 5% of total identifications, and a small fraction of the proteins in the M. burtonii genome predicted by TMHMM to have transmembrane domains (473). The proteomic analysis therefore would be complemented by a more focused analysis of these and other hydrophobic proteins.
Proteomic analysis of a single condition, while useful in identifying proteins present (a protein ‘snapshot’ of the cell), cannot identify processes or proteins that are directly related to cold adaptation. In M. burtonii, comparative proteomics was achieved through the use of two-dimensional electrophoresis (2DE), stable isotope labelling using isotope coded affinity tags (ICAT), and through one-dimensional electrophoresis of the secreted proteins.
Goodchild et al., (2004b) used 2DE to identify 54 spots with > 2 fold changes in abundance at 4 C vs. 23 C. Following mass spectrometry, 43 differentially abundant proteins were identified. Many of these proteins were identified as differentially abundant at the transcript level in complementary work using RT-PCR. As a complementary analysis to the 2DE performed, Goodchild et al., (2005) also performed differential proteomic analysis using ICAT. This method identified a further 11 proteins
(not found in 2DE) as differentially abundant.
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Secretion of proteins from cells is an important component of cellular physiology, and has been implicated in cold adaptation, including the production of EPS (see 1.6.6). To determine whether secreted proteins play a role in the cold adaptation of M. burtonii, proteomic analysis of the secreted proteins at 4 C and 23 C was performed by Saunders et al., (2006). The authors found several cell surface proteins and a serine protease, which were exclusively expressed at 4 C.
These proteomic analyses highlighted the complexity of cold adaptation processes in M. burtonii. However, proteomics had not been used to analyse membrane and other hydrophobic proteins. Such an analysis would greatly complement the studies already performed, and could lead to the identification of novel processes involved in cold adaptation.
The proteomic analyses described above utilised cells grown on a complex media with trimethylamine as a substrate. Therefore, there is the opportunity to grow cells on a completely defined media and utilise proteomics to compare the ability of the cell to actively synthesise amino acids, and other molecules, with respect to temperature. This type of analysis could give further insight into regulation of processes with respect to the trade-off between energy generation and biosynthesis. Analysis of cells grown on an alternative substrate (methanol) may also complement such an analysis.
1.7.2.3 Molecular adaptation in M. burtonii
The molecular adaptation of the EF2 protein from M. burtonii has been extensively investigated (reviewed in Thomas and Cavicchioli (2002)). EF2 proteins are essential for translation and are relatively conserved amongst organisms. Through analyses of
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EF2 proteins from M. burtonii, it was found that M. burtonii EF2 was likely to be more flexible due to the presence of a low number of predicted salt bridges, less packed hydrophobic cores and a reduction of proline residues in surface loops (Thomas and
Cavicchioli 1998). The EF2 also showed higher activity at low temperatures due to a decreased activation energy, which was accompanied by an increase in thermolability as a result of low activation enthalpy. It was proposed that this resulted from low solvent interactions and a low number of non-covalent bonds resulting in higher entropy
(Thomas and Cavicchioli 2000; Siddiqui et al., 2002). These studies demonstrated that the cold adaptation of an archaeal protein was remarkably similar to that of bacterial proteins.
The stability of tRNA is influenced by temperature. M. burtonii has been used as a model to characterise how psychrophilic archaea cope with the rigidifying effect of cold temperature Noon et al., (2003). They found that M. burtonii had the lowest levels of modification of any organism studied, and that there was a high content of dihydrouridine, which would confer flexibility to the tRNA molecules. There was no significant difference in the nucleosides when cells were grown at 4 and 23 C indicating that the cells probably constitutively produce flexible RNA. Further investigations of tRNA closer to the maximum and minimum growth temperature of M. burtonii may establish whether the organism is capable of modifying tRNA with respect to temperature.
1.7.2.4 Transcription
Transcription and transcriptional regulation have been identified as especially important processes in cold adaptation in M. burtonii. Through differential proteomic analyses,
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RNA polymerase subunit E (transcriptional accessory function), a possible response regulator for a two component regulatory system (Goodchild et al., 2004b), as well as a
TATA binding protein (Goodchild et al., 2005), were found to be increased in abundance at low temperatures. This differential proteomics is supported by proteome analysis, where several regulatory elements were identified in high abundance under cold conditions (Goodchild et al., 2004a). These studies provide evidence of cold induced gene regulation and possible RNA polymerase modulation.
1.7.2.5 Translation
Translation and the proteins related to this process have been identified as some of the major cellular contributors to the cold adaptation of M. burtonii. Proteomics has shown that many of the proteins involved in this process are found in high abundance under cold conditions (Goodchild et al., 2004a).
A DEAD box helicase from M. burtonii was found to be expressed exclusively under cold conditions through mRNA analysis (Lim et al., 2000). DEAD box helicases have a variety of roles in the cell including the stabilising of RNA secondary structures and transcriptional and translational accessory functions. Further attempts at characterising this protein, however, have been thwarted by the inability to recombinantly express this protein in a soluble form (R. Cavicchioli personal communication). This protein may require co-expression with a partner, or has an as yet unidentified membrane association.
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1.7.2.6 Protein folding and post-translational modification
Under cold conditions, protein folding and post-translational modification have been identified as especially important processes in M. burtonii. Through the use of differential proteomic techniques a peptidyl-prolyl cis/trans isomerase was shown to have increased abundance at low temperatures, indicating that this post translational process is an especially important step in the production of mature proteins under cold conditions (Goodchild et al., 2004a). At higher temperatures (23 C - Topt), M. burtonii expresses chaperones (Goodchild et al., 2004b) and a chaperonin in high abundance when compared to low temperature conditions (Goodchild et al., 2005). The results indicate not only that diverse protein folding processes are important under different thermal conditions, but also that growth at Topt is stressful for the organism.
1.7.2.7 Compatible solutes
Additional to the structural protein changes observed in the EF2 protein from M. burtonii (see 1.7.2.1), Thomas and Cavicchioli (2000) also suggested that solutes also had an effect on the in vivo activity and stability of the EF2 protein. Subsequently it was shown that solutes increased the low temperature activity of the protein while decreasing the thermolability (Siddiqui et al., 2002). In contrast, Thomas et al., (2001) showed that there was no appreciable changes in known compatible solute concentration in M. burtonii cells grown at 8 and 23 C. While these analyses lead to the discovery of a previously unidentified solute in the archaea, -alanine betaine, it is surprising that there was no indication of compatible solute involvement in cold adaptation. The lack of identified thermally induced solute accumulation could be due to: the temperatures M. burtonii was grown at may not have been low (or different)
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enough to stimulate appreciable changes in solute concentration (M. burtonii is capable of growing at temperatures over 10 C lower); solute metabolism may be constitutive and not under thermal control; or M. burtonii may also be accumulating novel solutes not examined in the work.
1.7.2.8 Energy generation and metabolism
The efficient generation of energy at cold temperatures is crucial for psychrophiles.
Methanogens are able to generate ATP through the use of ion transporting ATPases, which use electrochemical gradients as the driving force behind ATP production.
Goodchild et al., (2004b), found several methanogenesis proteins related to generation of proton motive force (PMF) as differentially abundant in the cell. The authors argued that the cells prefer to generate energy via a PMF rather than a sodium motive force
(SMF) at cold temperatures. However, there is no evidence to suggest that M. burtonii uses a SMF for ATP production under any situation. As the use of SMF for energy production is poorly characterised in many methanogens (Majernik et al., 2003), it is an area of possible future research.
Goodchild et al., (2004b), also indicate that pathways leading from acetyl-CoA to amino acid metabolism are enhanced in the cold, possibly as a result of cold regulation of carbon and nitrogen metabolism. Thus is appears that that M. burtonii fine-tunes these process in terms of energy generation and biosynthesis under kinetically unfavourable conditions (Goodchild et al., 2005)
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1.7.2.9 Extracellular polymeric substance
As described in 1.6.6, EPS production is a feature of many psychrophiles. Reid et al.,
(2006), showed that M. burtonii cells grown under cold conditions produce EPS.
Genomic analysis of M. burtonii has uncovered over-representations of genes involved in polysaccharide biosynthesis and cell wall / envelope biogenesis (Allen et al., 2009), and proteomics identified several surface proteins expressed exclusively at 4 C
(Saunders et al., 2006). However, the comparison of the production of this substance in cells grown in the cold vs. cells grown at warmer temperatures has not been investigated. The basic composition of the EPS produced by M. burtonii has not been investigated, although Reid et al., (2006) indicated a weakly positive reaction to the
PAS staining method, which could indicate the presence of sugars.
1.7.2.10 Membrane lipids of Methanococcoides burtonii
M. burtonii produces unsaturated lipids in its cell membrane, with the degree of unsaturation increasing as temperature decreases (Nichols et al., 2004). In contrast to bacteria, the method that this archaeon uses to achieve the unsaturation involves the incomplete reduction of precursors (a plant-like mechanism) rather than a bacteria-like desaturase mechanism (Nichols et al., 2004). All of the components required for this proposed mechanism have been identified using genomics (Nichols et al., 2004; Allen et al., 2009). Several of the components of the lipid biosynthetic pathway were identified using proteomics, despite their probable membrane-bound nature. However, the identification of many membrane bound components has yet to be achieved.
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1.7.2.11 The role of membrane proteins in the cold adaptation of Methanococcoides burtonii
Despite thorough proteomic analysis of WCE and secreted proteins of M. burtonii, very few membrane and hydrophobic proteins have been identified. While a small number of membrane bound methanogenesis proteins, transporters, and mevalonate pathway proteins have been identified, there remains an extensive cohort of unidentified membrane, membrane associated, and hydrophobic proteins, which could have crucial roles in the cold adaptation of the organism and reveal novel biology. The analysis of this untapped reservoir could greatly increase understanding of archaeal biology and cold adaptation.
1.8 Project Introduction
As described above, cold environments dominate the Earth. Until recently, the organisms that survived in these environments were poorly characterised. With the development of culture independent techniques, the abundance of microorganisms in these cold environments is beginning to be understood. Cold environments display ecological complexity with the population dynamics varying both geographically and spatially. The archaea constitute a significant proportion of biomass in cold environments and play significant roles in global carbon cycling, yet very few of the psychrophilic archaea have been characterised, and even fewer have been the subject of intensive study. Proteomic analyses of psychrophiles have indicated that cold adaptation is a complex process involving many cellular adaptations to the cold. However, very little is known about the role of membrane and hydrophobic proteins in cold adaptation.
Many psychrophiles produce EPS, the function of which is as yet unknown.
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M. burtonii is the most intensively studied of the psychrophilic archaea. Studies of its proteins, genome, and lipids, reflect findings in other psychrophilic organisms and have provided insight into cold adaptation in the archaea. Proteomic analyses have provided valuable insight into processes important for M. burtonii in cold conditions and have indicated that cold adaptation results from synergistic changes in ‘whole cell’ biology.
The role of membrane and other hydrophobic proteins in the cold adaptation of M. burtonii (as with other psychrophiles) has not been elucidated despite genomic evidence suggesting their importance (Allen et al., 2009). The production of EPS by M. burtonii in cold temperatures has been documented; however, this has not been fully investigated.
The aim of this current study was to utilise proteomics to determine the roles of membrane and other hydrophobic proteins in the cold adaptation of M. burtonii (and by inference other psychrophiles). The changes in these membrane proteins were investigated with respect to temperature, substrate, and nutrient conditions (which was accompanied by parallel work on soluble and secretory proteins by other investigators in the Cavicchioli laboratory). In addition, the production of EPS by M. burtonii was examined at both high and low temperatures and under different substrate conditions.
Changes in cell morphology were also examined and basic characterisation of EPS was performed.
Chapter 2 of this dissertation describes the development of proteomic methods to analyse the hydrophobic proteome of M. burtonii. Chapter 3 describes comprehensive analysis of this proteome, which revealed novel biology and also acted as a platform for further analysis. Chapter 4 describes the growth of M. burtonii on defined media with
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alternative substrate (methanol) and analysis of the hydrophobic proteome of cells grown at 4 C and 23 C for both defined and complex media using a stable isotope labelling method. The production of EPS by M. burtonii, as well as any temperature induced cellular changes were investigated using electron microscopy and are described in Chapter 5, where the results of basic histochemical characterisation of EPS are also presented. Chapter 6 presents an overview of the main conclusions of the work performed, suggests areas for future research, and highlights the significance of the work.
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Chapter 2. Analysing the Hydrophobic Proteome of Methanococcoides burtonii
2.1 Summary
Methods were developed to analyse the hydrophobic proteome (HPP), and the coverage of this HPP was maximised through implementation of a novel differential solubility fractionation (DSF) procedure. Through the application of these procedures a large number of proteins (667) were identified. Over 50% (375) of the identified proteins were not identified in previous proteomic analyses, 30% (201) were predicted to contain transmembrane domains or be associated with the membrane through a complex, and
31% (210) were predicted to be hydrophobic. The DSF procedure enabled the identification of more hydrophobic, membrane, and previously unidentified proteins in far fewer runs than by analysis of the non-fractionated samples alone. The procedure increased the efficacy (up to 169%) of identifying membrane proteins and was economical, requiring far fewer runs (12% of the machine time) to analyse the hydrophobic proteome than for samples without DSF. The implementation of this efficient method in other organisms will reduce temporal and monetary costs usually associated with comprehensive proteomic analyses.
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2.2 Introduction
Genomic (Allen et al., 2009) and lipid analyses (Nichols and Franzmann 1992; Nichols et al., 2004) of M. burtonii have identified potentially crucial roles for membrane and surface proteins in the cold adaptation of the organism. However, despite comprehensive analysis of the proteome of the organism (Goodchild et al., 2004a;
Goodchild et al., 2004b; Goodchild et al., 2005; Saunders et al., 2006), very few membrane and hydrophobic proteins have been identified. For example, only 25 proteins with transmembrane domains (TMD) were revealed through previous proteomic experiments. Therefore, an approach that enriched for these types of proteins was needed in order to assess their roles in the cold adaptation of the organism.
The hydrophobic proteome (HPP) consists of integral membrane proteins, membrane associated proteins, and proteins that form the hydrophobic core of protein complexes.
These proteins perform crucial roles in the cell including: signal transduction
(Whitelegge et al., 1998); transport of various substances; secretion of proteins
(Pohlschroder et al., 2005); and energy generating electron transport (Deppenmeier
2004). The analysis of a hydrophobic proteome is challenging and limited by the intrinsic nature of the target proteins. These proteins need to be converted to a soluble form prior to mass spectrometry, while remaining relatively chemically inert so as not to interfere with instrumentation. In recent years a number of methods have been developed to allow for the comprehensive analysis of the hydrophobic proteome, several of which have been applied to extremophiles and archaea (Zhu et al., 2004;
Barry et al., 2006; Bisle et al., 2006; Graham et al., 2006). The methods include: Phase partitioning with detergents (Everberg et al., 2008); and other detergent based methods
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(Zheng et al., 2007), which are often coupled with 2D electrophoresis (2DE). Such methods give rise to problems of removing MS interfering detergents from samples and problems relating to 2DE. Denaturing chaotropic reagents, which also must be removed prior to analysis and tend to be coupled with gel based methods, have been used for the analysis of hydrophobic proteomes (Graham et al., 2007). The gel-based methods have been shown to be a less powerful alternative for analysis of hydrophobic proteins, due to the tendency for hydrophobic proteins to run as streaks on gels, and problems with post-solubilisation precipitation of these proteins in isoelectric focusing (IEF) strips
(Washburn and Yates 2000; Klein et al., 2005). Other methods include affinity labelling with lipid soluble probes (Tang et al., 2007), and a tube digestion method that allows for removal of interfering detergents (Lu and Zhu 2005). Washburn et al., (2001) developed a method utilising high pH and CNBr, which has been used with a number of modifications in several studies (Wu et al., 2003; Chong et al., 2005; Blackler et al.,
2008). The method developed by Blonder et al (2002), which utilises the organic solvent methanol, combined with thermal denaturation and in-solvent digestion, has proven to be successful in a number of studies (Blonder et al., 2004a; Blonder et al.,
2004b; Blonder et al., 2004c). This method of solubilisation has been shown to be superior to detergent based solubilisation (Mitra et al., 2007; Zhang et al., 2007), and is compatible with LC/LC–MS/MS (Zhang et al., 2007). With slight modifications, this method was used in this study.
High throughput proteomic analysis is often hampered by the complexity of the samples. A given sample from a microorganism may contain several thousand proteins.
When digested, this sample will contain tens of thousands of individual peptides, and when post-translational modifications are taken into account an even higher level of
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complexity is present in a sample (Stasyk and Huber 2004; Righetti et al., 2005). In addition to this difficulty of sample complexity, proteins in a sample have a range of cellular concentrations. Some proteins are highly abundant in the cell, (e.g. metabolic proteins), while others may be up to 7 orders of magnitude less abundant, (e.g. transcriptional regulatory proteins) (Corthals et al., 2000; Issaq et al., 2005). As MS works on intensity based ion selection, the presence of peptides from high abundance proteins (therefore highly intense), masks the selection of the low abundance (low intensity) peptides, and hence their detection. In order to counteract this problem, fractionation strategies have been employed. The introduction of LC/LC-MS/MS (or
MudPIT) analysis revolutionised proteomic analyses (Washburn et al., 2001) however, investigators have recently been striving to further fractionate samples in order to identify more cellular proteins. The fractionation is aimed at the identification of less abundant proteins, which can have crucial roles in the cell despite low copy number
(Issaq et al., 2005). While many off-line and pre-digestion methods are suitable for use with soluble proteins, the application of these pre-fractionation strategies to membrane proteins is more challenging. This type of analysis will not only increase the number of protein identifications, but also decrease the influence of high abundance soluble proteins, which are often contaminate hydrophobic fractions despite separation procedures (Klein et al., 2005). With the exception of a study by Ramos et al., (2008), fractionation strategies have not been developed for hydrophobic and membrane proteins. Ramos et al., (2008) utilised a combination of detergents and chaotropic reagents to separate an insoluble fraction based on the principle of differential solubility, and were able to achieve good separation by using this method with a gel- based desalting step and LC-MS/MS.
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In this section of the project, the method for the isolation and characterisation of the M. burtonii HPP was developed, and conditions for hydrophobic protein extraction, solubilisation, digestion and analysis were optimised. In addition, coverage of the HPP was maximised through the development of pre-fractionation procedures. A novel differential solubility fractionation (DSF), utilising increasing concentrations of a single powerful non-ionic detergent followed by LC/LC-MS/MS, was used to separate the hydrophobic proteins.
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2.3 Materials and Methods
All chemicals and media were prepared, unless otherwise noted, in MilliQ dH2O
(Millipore, Cork, Ireland) with a conductivity of 18.2 m /cm. Reagents, unless otherwise noted, were made with Univar analytical grade chemicals (sourced from APS,
Seven Hills, NSW, or Ajax Finechem, Taren Point, NSW). All mass spectrometry was carried out at the Bioanalytical Mass Spectrometry Facility (BMSF), UNSW.
2.3.1 Culture media preparation
Anaerobic culture media was prepared using equipment and techniques described in
Sowers and Noll (1995).
2.3.1.1 Vitamin and mineral stock solutions
Vitamin and mineral stock solutions (100x concentration) were prepared as described by Goodchild (2004).
Mineral salts were prepared by first dissolving 1.5 g nitrilotriacetic acid in 500mL of dH2O, which was then adjusted to a pH of 6.75 with KOH pellets. This solution acted a chelating agent, aiding the solubility of the following salts, which were added in sequential order: 3g MgSO4.7H2O; 0.5g MnSO4 (Sigma, St Louis, MO, USA); 1g NaCl;
0.1g FeSO4.7H2O (ICN, Aurora, OH, USA); 0.1g CoSO4 (Sigma, St Louis, MO, USA);
0.116g CaCl2.2H2O; 1g ZnSO4 (AnalaR, BDH, Poole, England); 250 l 4% w/v CuSO4
(Sigma, St Louis, MO); 0.01g AlK(SO4)2 (AnalaR, BDH, Poole, England); 0.01g
H3BO3 (AnalaR, BDH, Poole, England); 0.01g Na2MoO4.2H2O (Sigma, St Louis, MO,
USA). The solution was made up to 1000ml, placed on a magnetic stirrer for 20
D. Burg UNSW 41 minutes, filter sterilised using 0.22 m syringe filters (Millipore, Cork, Ireland) and stored in 50mL aliquots at -20 C until needed.
Vitamins were prepared by preparing, in 1L of dH2O: 2mg biotin; 2mg folic acid; 2mg pyridoxine HCl; 10mg thiamine.HCl; 5mg riboflavin; 5mg nicotinic acid; 5mg DL-Ca pantothenate; 0.1mg vitamin B12; 5mg para-aminobenzoic acid; and 5mg lipoic acid. All vitamins were sourced from Sigma (St Louis, MO, USA) except vitamin B12, which was sourced from ICN (Aurora, OH, USA). The solution was sterilised using 0.22 m syringe filters (Millipore, Cork, Ireland) and stored in 10 or 15 mL aliquots at -20 C until required.
2.3.1.2 MFM preparation
A modification of MGM (Franzmann et al., 1992) known as MFM, was prepared as described by Thomas and Cavicchioli (2000). Firstly 1.2L of dH2O was heated in a microwave on 100% power for 3 minutes. The dH2O was poured into a sidearm flask and a Teflon coated magnetic stir-bar inserted. The upper flask opening was plugged with a rubber bung, the solution placed onto a magnetic stirrer and de-gassed using a
Biorad HydroTech vacuum pump (BioRad, Hercules, CA, USA) until gas bubbles were no longer being evacuated. While the dH2O was being prepared, two Schott bottles (a
500mL and a 2L) were thoroughly washed with dH2O to remove any traces of residual detergents. These bottles were flushed with high purity nitrogen gas (BOC gases, North
Ryde, NSW) (the 500mL bottle with a straight unbeveled needle, and the 2L with a gassing stone) using a manifold assembled by UNSW technical staff as described by
Sowers and Noll (1995). A 500mL volume of the de-gassed dH2O was poured into the
2L bottle, which was placed onto a magnetic stirrer and a Teflon coated stir-bar added,
42 D. Burg UNSW
while continually bubbling with nitrogen gas. The remainder of the de-gassed dH2O was added to the 500mL bottle and was bubbled with nitrogen. The following salts were then added to the 2L bottle: 0.335g KCL; 6g MgCl2.6H2O; 1g MgSO4.7H2O; 0.25g
NH4Cl (AnalaR, BDH, Poole, England); 0.14g CaCl2.2H2O; 23.37g NaCl; 2mg Ferrous ammonium sulfate (Sigma, St Louis, MO, USA); and 1mg of the redox indicator rezazurin (Sigma, St Louis, MO, USA). The solution, which now had a purple colour, was then left to stir while being bubbled with nitrogen for 10 –15 minutes. The following chemicals were added in order, making sure each was dissolved before adding the next: 5g trimethylamine HCl (ICN, Aurora, OH, USA); 2g yeast extract
(Oxoid, Hampshire, England); 10mL of the vitamin solution prepared as described in
2.3.1.1; 10mL of the mineral salts solution prepared as described in 2.3.1.1; 0.1g sodium acetate; and 0.14g K2HPO4. Food grade CO2 gas (BOC gases, North Ryde,
NSW) was introduced to a ratio of 80:20, N2:CO2 as measured using a gas proportioner.
The solution was bubbled for 10 minutes or until the colour of the changed from purple to pink indicating a change in pH. Next, 0.5g of the reducing agent cysteine HCl
(Sigma, St Louis, MO, USA) was pre-dissolved in 1mL dH20 and added to the solution followed by 2.52g Na2CO3, which with the CO2 gas, acted to buffer the solution. The volume was adjusted to 1L using the water from the 500mL flask. The solution was left to stir, with continuous gas bubbling, for 30 minutes, or until the solution was pale yellow in colour (indicating reduced conditions). The pH of the solution was adjusted to
6.8 using 10.1M HCl and the stir-bar turned off. Serum bottles (120mL) (Wheaton,
Millville, NJ, USA), were rinsed with dH2O and flushed with the N2:CO2 gas mixture using straight un-beveled syringes for 20 – 30 minutes. The media was transferred to the serum bottles using a 60mL syringe (BD Scientific, Singapore), and a large gauge,
25cm needle. The syringe was first filled with gas from the headspace of the 2L bottle
D. Burg UNSW 43 containing the media solution, ejected and re-filled with gas three times. The media was transferred to a serum bottle, and was repeated until all serum bottles contained 100mL of media solution. The serum bottles were bubbled with the gas mixture for 45 minutes or until the solution was pale yellow in colour. The bottles were plugged with butyl rubber stoppers (Bellco Glass, Vineland, NJ, USA), sealed with aluminium crimp seals
(Bellco Glass, Vineland, NJ, USA), and autoclaved at 121 C for 15 minutes. After autoclaving the media was placed on a shaker overnight to dissolve any precipitate formed. 1mL of 2.5% Na2S solution (a reducing agent, prepared as described below) was injected into each of the serum bottles and these were allowed to equilibrate for 2 hours before inoculation, or if not needed immediately, were stored at 4 C for up to 2 months.
A stock solution of 2.5% Na2S was prepared by first heating and degassing 120mL of dH2O as described above. A 100mL aliquot of this de-gassed dH2O was transferred to a serum bottle and bubbled with nitrogen using the gas manifold for 30 minutes. The serum bottle was temporarily plugged with a butyl rubber stopper and set aside.
Working in a fume hood, slightly more than 2.5g of Na2S crystals (Sigma, St Louis,
MO, USA) were weighed out, washed with dH2O, dried with a paper towel and re- weighed for a final mass of 2.5g. The stopper was removed from the serum bottle and the crystals rapidly transferred before re-inserting the rubber stopper and crimp sealing the serum bottle. This 2.5% Na2S solution was autoclaved at 121 C for 15 minutes, and stored at 4 C until needed.
44 D. Burg UNSW
2.3.2 Culture inoculation and growth conditions
M. burtonii cultures were prepared from laboratory stock cultures at 4 C and 23 C.
Culture media was equilibrated to the required growth temperature and then inoculated with 1mL of culture using the following procedure. A 1mL syringe (BD Scientific,
Singapore) with a fitted 23-gauge 25mm needle (BD Scientific, Singapore) was first filled with nitrogen gas from the manifold and emptied at least three times. 1mL of M. burtonii culture was withdrawn through the rubber stopper of a serum bottle that had been previously relieved of gas pressure by piercing with a needle. The culture was transferred to a fresh bottle of culture media, and were incubated at 4 C in a constant temperature room or at 23 C in a water bath (Custom made, UNSW workshop), which was housed in a constant temperature room to avoid temperature fluctuations. Cultures were passaged at least once through the required growth temperature before harvesting.
2.3.3 Cell harvest
Cells were harvested when the cultures reached an optical density (OD) of 0.250 at
620nm. Optical density was measured by removing a small amount of culture with a
1ml syringe, purged of oxygen as described previously to avoid oxygen contamination of the culture. The culture material was transferred to a plastic cuvette (Sarsdedt,
Numbrect, Germany) and the absorbance measured using a Biochrom Libra S11 spectrophotometer (Biochrom, Cambridge, England). Cultures to be harvested were first relieved of methane build-up by piercing the rubber stopper with a syringe. The crimp seals were removed with a de-crimping tool (Wheaton, Millville, NJ, USA), stoppers removed, and the culture divided into two 50mL tubes (Greiner Bio One,
Frickenhausen, Germany). The tubes were centrifuged at 3200xg for 35 minutes at 4 C
D. Burg UNSW 45 using a Hettich universal 32R bench top centrifuge with swing-out rotor (Hettich,
Germany). The supernatant was discarded and the cell pellets stored at -80 C until needed. When planning experiments, it had to be taken into account that culture times varied according to temperature and state of the inoculating culture. At 23 C, a culture inoculated from an actively growing culture had a harvest time of 1-2 weeks. However, from a culture in stationary phase, the culture time increased to 5-6 weeks. At 4 C, a culture inoculated from an actively growing culture had a harvest time of 1.5-2 months, however, from a culture in stationary phase the culture time increased to 4-6 months.
2.3.4 Hydrophobic protein extraction
Hydrophobic proteins were extracted from cell pellets using a method modified from
Blonder et al. (2002). A simplified flowchart of methodology is presented in Appendix
A.1.
2.3.4.1 Cell disruption
Cell pellets (~500 - 1000 L) were first transferred to a 2mL tube (Axygen Scientific,
Union City, CA, USA) and suspended in 1mL disruption buffer (50mM Tris-HCl pH
7.2, with 2mM of the metalloprotease inhibitor EDTA). To this 20 L of 150mM PMSF
(serine protease inhibitor) (Sigma, St Louis, MO, USA) was added (final concentration
2mM). PMSF was prepared in advance in absolute methanol and stored in 25 L aliquots at -20 C.
The cellular material was mixed using a vortex (Scientific Industries, MA, USA) and then ultrasonically disrupted using a Branson digital sonifier 250 (Branson Ultrasonics,
Danbury, CT, USA). Cells were disrupted at 30% amplitude, with a 0.5 second pulse for
46 D. Burg UNSW
2 minutes in an ice bath, with a 30 second rest every 30 seconds to prevent thermal damage to the cellular proteins. In order to remove any media precipitates from the protein, lysates were centrifuged in a Heraeus Multifuge 1S R (Thermo Fisher
Scientific, Waltham, MA, USA) with a fixed angle rotor at 1500xg for 5 minutes at 4 C.
The supernatant was collected and the pellet discarded.
2.3.4.2 Carbonate extraction and ultracentrifugation
In order to promote the formation of lipid rafts and to dissociate any non-specific protein-membrane and protein-protein interactions, a carbonate extraction was performed as described by Fujiki et al. (1982). Cellular proteins, as prepared above, were diluted to 10mL in ice-cold, 0.1M NaCO3 pH 11.2 (a pH of 12 was also used for comparative purposes) and agitated on a shaker, in an ice-water bath for 1 hour. The carbonate-extracted material was transferred to 5mL OptiSeal ultracentrifuge tubes
(Beckman Coulter, Palo Alto, CA, USA. The centrifuge tubes were placed in a fixed angle rotor (NVTi 90, Beckman, Ireland) and spun at 115 000xg for 90 minutes at 4 C in a Sorvall discovery 100 ultracentrifuge (Thermo Fisher Scientific, Waltham, MA,
USA). The supernatant was collected and stored at 4 C for comparison and assessment of separation. The insoluble pellets were washed twice with dH2O, re-suspended in 5mL freshly made 40mM ammonium bicarbonate (ambic) (Sigma, St Louis, MO, USA), and ultracentrifuged again at 115000xg for 30 minutes. The supernatant was discarded. The insoluble pellet was re-suspended in 2mL 40mM ambic and briefly homogenised using a Branson digital sonifier at 15% amplitude for 10-20 seconds.
D. Burg UNSW 47
2.3.4.3 Protein concentration and buffer exchange
Both hydrophobic and soluble proteins were concentrated prior to storage.
Concentration and buffer exchange was performed in 4ml Amicon 5kDa centrifugal concentration units (Millipore, Billerica, MA, USA), which were preconditioned and washed in fresh 40mM ambic by spinning in a Hereus 1S-R centrifuge with a swing-out rotor (Thermo Fisher Scientific, Waltham, MA, USA) at 5000xg for 30 minutes at 6 C.
The protein material was added and concentrated by centrifugation at 5000xg for 45 minutes at 6 C. The protein concentrate was re-suspended in 40mM ambic and centrifuged again at 5000xg for 45 minutes at 6 C. This process was repeated once more and a final volume of ~250 L of concentrated protein in 40mM ambic was recovered. Recovery was enhanced by briefly placing the concentration apparatus in a sonicating bath, and by repeated flushing with a pipette. Protein samples were stored at
-80 C until needed.
2.3.5 Differential solubility fractionation
As a means of augmenting the mass spectrometry of the hydrophobic proteins, differential solubility fractionation (DSF) was trialed as a solubility-based separation method to reduce sample complexity. The non-ionic detergent n-octyl- -D- glucopyranoside (OGP) (Sigma, St Louis, MO, USA) was chosen due to its ease of removal from samples and known ability to solubilise membrane proteins (Gould et al.,
1981; Lorber et al., 1990). Whole hydrophobic fractions were prepared in 40mM ambic as described above. The sample, in a 1.5mL tube (Axygen Scientific, Union City CA,
USA), was pelleted by centrifugation in a Hereus 1S-R centrifuge with fixed angle rotor at 20 000xg for 15 minutes at 4 C, and the supernatant discarded. The sample was
48 D. Burg UNSW
washed, by re-suspension in 1mL 40mM ambic and spinning at 20 000xg for 15 minutes at 4 C. The supernatant was discarded. The sample was re-suspended in 1mL
0.5mM OGP made in 40mM ambic, vortexed for 5s, placed in a sonicating bath
(Edwards Instruments, Thebarton, South Australia) for 20s, and vortexed for 5s. The sample was spun at 20 000xg for 15 minutes at 4 C and the supernatant collected. The supernatant was concentrated and de-salted as described above with five buffer exchanges performed. This procedure was repeated, gradually increasing the detergent concentration applied to the protein pellet in the following steps: 0.5mM; 1.25mM;
2.5mM; 5mM; 12.5mM; 20mM; 30mM; 100mM; 500mM. After buffer exchange, protein fractions were stored at -20 C until required for SDS-PAGE and mass spectrometry.
2.3.6 Protein concentration measurement
The concentration of protein was measured using the Bradford microassay method, reagents were prepared as described in Kruger (2002). Standard curves were generated using bovine serum albumin (BSA) as the reference protein. Aliquots of increasing concentration of insoluble protein were prepared in 1.5mL centrifuge tubes. Bradford agent was added, absorption was measured against a blank at 595nm and results recorded. The concentration of protein in the sample in g/ L was calculated using the
B value from the standard curve by the formula: C = (A/B)/V, where C = the concentration of protein, A = the absorbance, B = the slope of the standard curve, and V
= the volume of sample added in L. The average concentration of all volumes added was calculated, and this was taken to be the final concentration of the protein sample.
D. Burg UNSW 49
2.3.7 Assessment of protein separation using SDS-PAGE
To determine the quality of protein separation, the cellular proteins were visualised using SDS-PAGE. Gels and reagents were prepared as described in Walker (2002).
2.3.7.1 Sample preparation
Samples were denatured and prepared for separation by mixing the required concentrations of sample in 2x sample buffer such that the final volume was always
10 L and each sample always contained 5 L, 2x sample buffer. For example, 5 L of a sample was added to 5 L sample buffer. If a greater dilution was required, for example in cases of a highly concentrated sample or for contrasting purposes, sample was added to 5 L sample buffer which was made up to 10 L with dH2O. For every electrophoresis experiment, a standard high range molecular weight ladder (BioRad, Hercules, CA,
USA) and a BSA standard were also prepared. Samples were denatured by heating at
95 C in a heating block for 5 minutes prior to loading.
2.3.7.2 Electrophoresis
Samples were loaded into the wells of the gel, and a voltage of 50V was applied via a
BioRad power pack 300 (BioRad, Hercules, CA, USA) for ~ 15 minutes or until the dye band had reached the resolving gel. The voltage was increased to 150V and current applied until the dye band had reached the bottom of the gel. The current was switched off and the plates removed and allowed to cool for ~ 5 minutes. The gels were removed from the glass plates, and placed into plastic boats (Sarsdedt, Numbrect, Germany) for staining.
50 D. Burg UNSW
2.3.7.3 Gel staining and visualisation
Gels were stained in the dark with Coomassie staining solution for ~ 45 minutes to 1 hour in the dark on a shaker. The stain was discarded, the gels washed in tap water and covered with de-staining solution for ~ 45 minutes to 1 hour on a shaker. The de- staining solution was then changed and the gel de-stained for a further hour, or until the gel background was clear. The gels were washed in dH2O and visualised using an
Amersham image scanner (GE Healthcare, Buckinghamshire, England) and Umax
Magicscan software version 4.3 (Umax, Dallas, TX, USA).
2.3.8 Preparation of hydrophobic proteins for mass spectrometry
To successfully process the hydrophobic proteins using ESI LC-MS/MS and ESI
LC/LC-MS/MS, they had to be digested, in soluble form, to peptides using a modified method developed from Blonder et al. (2002), Russell et al. (2001), and Park and
Russell (2000) . Samples containing 50 g of protein were first diluted to 90 L with
40mM ambic.
2.3.8.1 Protein reduction and alkylation
In order to reduce protein secondary structure, they were treated with 5 L 0.1M
Dithiothreitol (DTT) (final concentration 5mM) and incubated at 60 C in the dark for
45 minutes. After this treatment, the proteins were treated with 5 L 0.3M iodoacetamide (IDA) (Sigma, St Louis, MO, USA) (final concentration 15mM) at 60 C for 45 minutes in the dark, in order to alkylate cysteine residues, preventing disulfide bridges, and hence secondary structure, from re-forming.
D. Burg UNSW 51
2.3.8.2 Protein solubilisation and digestion
The reduced and alkylated proteins were thermally denatured at 90 C for 2 minutes followed by solubilisation, achieved by adding 150 L HPLC grade methanol to a final concentration of 60%. The protein solvent mixture was cooled and further solubilised in a sonicating bath (Edwards Instruments, Thebarton, South Australia). If insoluble material was still visible, the samples were briefly sonicated using a Branson digital sonifier for 5-10s at 15% amplitude. This procedure was also performed with a variety of solvents to compare the quality of solubilisation and results. The solvents used were:
HPLC grade acetonitrile (ACN) to a final concentration of 40%; Acetone to a final concentration of 50%; and isopropanol to a final concentration of 50%.
The solubilised proteins were digested to peptides at 37 C for 5 hours, in solvent, with sequencing grade porcine modified trypsin (Promega, Madison, WI, USA), with a trypsin to protein ratio of 1:20 ( g). The solution was vacuum dried using a Savant
Speed-Vac SC10A (Savant, Farmingdale, NY, USA) with the drying control set to medium. Dried samples were stored at -20 C until needed.
2.3.8.3 Sample clean up
Samples prepared for mass spectrometry, especially LC/LC-MS/MS, need to be free of any salts and other contaminants which could interfere with any chromatography steps, or become charged during ESI creating a high level of background in mass spectra, and/or saturate detectors leading to reduced sensitivity. Sample clean up was achieved through off-line chromatography. Most samples were cleaned-up using a C18 reverse phase (RP) cartridge. Samples with higher levels of contaminants, for example those from DSF, were first treated with strong cation exchange (SCX), and followed by RP.
52 D. Burg UNSW
2.3.8.3.1 Strong cation exchange chromatography
Protein samples with high levels of contaminants were cleaned via SCX chromatography using an Applied Biosystems cation exchange system with an Opti-
Lynx cartridge holder (Applied Biosystems, Forster City, CA, USA). All steps were performed at a flow rate of 9.5mL/hour using a 1mL glass needle (Alltech, Baulkham
Hills, NSW) and a syringe pump (KD Scientific, Holliston, MA, USA). All flow- through was collected and stored at 4 C until the success of the procedure was verified.
Vacuum-dried sample pellets were re-suspended in 5ml of load buffer (10mM KH2PO4 in 25% ACN pH 3.0) and centrifuged in a Hereus 1S-R centrifuge with a swing out rotor at 5000xg for 5 minutes at 4 C. The supernatant was collected in a 15mL tube and the pellet containing any insoluble peptide remnants and salt precipitate discarded. The sample was adjusted to a pH between 2.5 and 3.3 using glacial acetic acid. The SCX cartridge was assembled, and washed with 2mL of load buffer. The sample was loaded onto the column and washed with a further 1mL of load buffer. Peptides were eluted with 0.5mL of elution buffer (10mM KH2PO4, 350mM KCl in 25% ACN pH 3.0), and samples collected in a 1.5mL tube. The column was then washed with 1mL cleaning buffer (10mM KH2PO4, 1M KCl in 25% ACN pH 3.0), followed by 1mL of storage buffer (10mM KH2PO4, 0.1% sodium azide in 25% ACN pH 3.0) and then stored at 4 C for further use. The eluted sample collected was vacuum dried in a Speed-Vac with the drying speed set to medium. Dried samples underwent immediate RP chromatography
(2.3.8.3.2).
D. Burg UNSW 53
2.3.8.3.2 Reverse phase chromatography
Protein samples with low levels of expected contaminants, and samples that had previously undergone SCX, were purified by C18 RP chromatography. All steps were performed at a flow rate of 9.5mL/hour using a 1mL glass needle and syringe pump as described above. All flow-through was collected and stored at 4 C until the success of the procedure was verified. Dried samples were suspended in 500 L 0.2M heptafluorobutyric acid (HFBA) (Pierce Biotechnology, Rockford, IL, USA) (an ion pairing agent) and mixed by vortexing. An RP macrotrap (Microm Bioresources,
Auburn, CA, USA) was fitted into a steel column holder, making sure the cartridge was in the correct flow orientation. The column was washed with 0.5mL ACN, followed by
0.5mL 50% 0.2M HFBA: 50% ACN. An equilibration step was carried out by injecting
1.5mL 0.2M HFBA. The sample was injected, and the column containing bound peptides washed with 1.5mL 0.2M HFBA. The sample was eluted by injecting 250 L
50% 0.2M HFBA: 50% ACN, followed by 250 L ACN. The eluent was collected in a
1.5 ml tube. Finally the column was washed in 1mL ACN and stored at room temperature for further use. The eluted sample was vacuum dried in a Speed-Vac with the drying speed set to medium. Dried samples then stored at -80 C until needed.
2.3.9 Mass spectrometry
Purified peptides from digested hydrophobic protein samples were analysed and proteins identified by high throughput tandem liquid chromatography – tandem mass spectrometry (LC/LC-MS/MS). In all experiments, buffer A refers to; H2O:CH3CN
(98:2, 0.1 % formic acid), while buffer B refers to; H2O:CH3CN (80:20, 0.1 % formic acid).
54 D. Burg UNSW
2.3.9.1 Sample preparation
Samples were prepared by dissolving dried peptide pellets in 50 L 0.05% heptafluorobutyric acid/ 1% formic acid (Pierce Biotechnology, Rockford, IL, USA).
The formic acid provides protons and improves peak shape during separation. HFBA is used as an ion pairing reagent, which binds with charged peptides and renders them electrically neutral, increasing their affinity for the RP column and increasing resolution, while not affecting the UV absorption of the sample (Roberts and Hughes
1998). Samples: formic acid/HFBA solution (1:20) were prepared for analysis.
2.3.9.2 LC-MS/MS
The instruments were calibrated prior to all runs, using the LC-MS/MS procedures described in 2.3.9.2.1 and 2.3.9.2.2, by running 1 L of 50fmol/ L Glufibrinopeptide
(Glufib) standard (Sigma, St Louis, MO, USA). Spectra were visualised using instrument software, and the spectra generated by the Glufib standard inspected. The liquid chromatogram was analysed for correct retention time of 22-25 minutes, and an approximate peak width at half height of 0.4 minutes. Fragmentation spectra were inspected and compared to the known fragmentation spectra of Glufib, and mass error inspected to ensure an error of less than 50ppm. If any of the parameters did not meet expected values, the standard was repeated, and if these values did not meet the criteria,
BMSF staff manually calibrated the instruments.
Due to the sensitivity of the instruments used, all samples were diluted as described above and subjected to a preliminary evaluation using LC-MS/MS. Samples (1:20 dilution) were loaded and analysed as described in 2.3.9.2.1 and 2.3.9.2.2. The resulting spectra were analysed for LC, MS and MS/MS signal intensity, ensuring that intensity counts for most abundant peaks were in the range of 1000 – 1500 cps. The
D. Burg UNSW 55 concentration of sample required for LC/LC-MS/MS was then estimated, based on a
10 L injection.
2.3.9.2.1 LC-MS/MS performed on QTof Instrument
Sample peptides were separated by nano-LC using a Cap-LC autosampler system
(Waters, Milford, MA, USA). Samples (1µL) were concentrated and desalted onto a micro C18 precolumn (500µm x 2mm, Michrom Bioresources, Auburn, CA) with
H2O:CH3CN (98:2, 0.05 % HFBA) at 15uL/min. After a 4 min wash, the pre-column was automatically switched using a 10 port valve (Valco, Houston, TX, USA) into line with a fritless nano-column manufactured according to (Gatlin et al., 1998). Peptides were eluted using a linear gradient of buffer A to 45% buffer B at ~300nL/min over 30 minutes. The precolumn was connected via a fused silica capillary (10cm, 25µm) to a low volume tee (Upchurch Scientific, Oak Harbour, WA, USA) where High voltage
(2400 V) was applied and the column tip positioned ~ 1cm from the Z-spray inlet of a
QTof Ultima API hybrid quadrupole time-of-flight tandem mass spectrometer
(Micromass, Manchester, England). Positive ions were generated by electrospray (ESI) and the QTof operated in data dependent acquisition mode (DDA). A TOF MS survey scan was acquired (m/z 350-1700, 1s) and the 2 largest multiply charged ions (counts >
20) were sequentially selected by the quadrupole for MS/MS analysis. Argon (BOC gases, North Ryde, NSW) was used as the collision gas and an optimum collision energy chosen (based on charge state and mass). Tandem mass spectra were accumulated for up to 2s (m/z 50-2000). Peak lists were generated by MassLynx
(version 4.0 SP4, Micromass, Manchester, England), using the Mass Measure program.
56 D. Burg UNSW
2.3.9.2.1 LC-MS/MS performed on QStar Instrument.
Sample peptides were separated by nano-LC using an Ultimate HPLC and Famos autosampler system (LC-Packings, Amsterdam, Netherlands). Samples (1µL) were concentrated and desalted onto a micro C18 precolumn (500µm x 2mm, Michrom
Bioresources, Auburn, CA) with H2O:CH3CN (98:2, 0.05 % HFBA) at 20µl/min. After a 4 min wash the pre-column was switched (Switchos, LC Packings Amsterdam,
Netherlands) into line with a fritless nano-column manufactured according to (Gatlin et al., 1998). Peptides were eluted using a linear gradient of buffer A to 45% buffer B at
~300nL/min over 30 minutes. High voltage (2300 V) was applied to a low volume tee
(Upchurch Scientific, Oak Harbour, WA, USA) and a column tip positioned ~ 1 cm from the orifice of an API QStar Pulsar I hybrid tandem mass spectrometer (Applied
Biosystems, Foster City CA). Positive ions were generated by ESI and the QStar operated in information dependent acquisition mode (IDAM). A TOF MS survey scan was acquired (m/z 350-1700, 1s). The 2 largest multiply charged ions (counts > 15) were sequentially selected by the quadrupole for MS-MS analysis. Nitrogen (BOC gases, North Ryde, NSW) was used as collision gas and an optimum collision energy chosen (based on charge state and mass). Tandem mass spectra were accumulated for
2.5s (m/z 65-2000). Peak lists were generated using Mascot Distiller (Matrix Science,
London, England) using the default parameters.
2.3.9.3 Analysis of samples using LC/LC-MS/MS
Peptide samples were analysed with LC/LC-MS/MS for the purpose of protein identification. Dilutions of sample were chosen based upon LC-MS/MS experiments and machines were calibrated as described in 2.3.9.2.
D. Burg UNSW 57
2.3.9.3.1 LC/LC-MS/MS performed on Qtof instrument
Peptides were separated by nano-LC using a Cap-LC autosampler system (Waters,
Milford, MA, USA) (Link et al., 1999). 10 L was loaded onto a SCX micro column
(0.76 x ~15 mm) containing Poros S10 or S20 resin (Applied Biosystems, Foster City,
CA, USA). Peptides were eluted sequentially using 5, 10, 15, 20, 25, 30, 40, 50, 100,
250, 500, and 1000 mM ammonium acetate (20µL). The unbound load fraction and each salt step were concentrated and desalted onto a micro C18 precolumn (500µm x
2mm, Michrom Bioresources, Auburn, CA, USA) with buffer A at 15 µl/min. After a
10 minute wash the pre-column was switched into line with a fritless analytical column
(75µm x ~10 cm) containing C18 reverse phase packing material (Magic, 5 μ, 200Å,
Michrom Bioresources, Auburn, CA, USA) as described by (Gatlin et al., 1998).
Peptides were eluted using a linear gradient of buffer A to 45% buffer B at ~300 nL/min over 90 minutes. The precolumn was connected via a fused silica capillary (10cm,
25 m) to a low volume tee (Upchurch Scientific, Oak Harbour, WA, USA) where high voltage (2400V) was applied and the column tip positioned ~ 0.5cm from the Z-spray inlet of an Ultima API hybrid QTof tandem mass spectrometer (Micromass,
Manchester, England). Positive ions were generated by ESI and the QTof operated in
DDA. A TOF MS survey scan was acquired (m/z 350-1700, 1s) and the 3 largest multiply charged ions (counts > 20) were sequentially selected by the quadrupole for
MS-MS analysis. Argon (BOC gases, North Ryde NSW) was used as collision gas and an optimum collision energy chosen (based on charge state and mass). Tandem mass spectra were accumulated for up to 3s per precursor (m/z 50-2000). Peak lists were generated by MassLynx (Micromass, Manchester, England), using the Mass Measure program.
58 D. Burg UNSW
2.3.9.3.2 LC/LC-MS/MS performed on QStar instrument
Sample peptides were separated by automated online strong cation exchange (SCX) and nano C18 LC using an Ultimate HPLC, Switchos and Famos autosampler system (LC-
Packings, Amsterdam, Netherlands) (Link et al., 1999). A sample volume of 10 L was loaded onto a SCX micro column (0.75 x ~15mm, Poros S10, Applied Biosystems,
Foster City, CA, USA). Peptides were eluted sequentially using 5, 10, 15, 20, 25, 30,
40, 50, 100, 250, 500 and 1000 mM ammonium acetate (20µl). The unbound load fraction and each salt step was concentrated and desalted onto a micro C18 precolumn
(500µm x 2mm), (Michrom Bioresources, Auburn, CA, USA) with buffer A at
20µl/min. After a 10 minute wash the pre-column was switched (Switchos) into line with a fritless analytical column (75 µm x ~12 cm) containing C18 reverse phase packing material (Magic, 5 μ, 200Å) prepared as described by Gatlin et al.,(1998).
Peptides were eluted using a linear gradient of buffer A to 45% buffer B at ~300 nL/min over 90 minutes. High voltage (2300 V) was applied through a low volume tee
(Upchurch Scientific, Oak Harbour, WA, USA) at the column inlet, and the outlet positioned ~ 1 cm from the orifice of an API QStar Pulsar I hybrid tandem mass spectrometer (Applied Biosystems, Foster City CA). Positive ions were generated by
ESI and the QStar operated in IDAM. A TOF MS survey scan was acquired (m/z 350-
1700, 0.75 s) and the 3 largest multiply charged ions (counts > 20, charge state 2 and
4) sequentially selected by the quadrupole for MS-MS analysis. Nitrogen (BOC gases,
North Ryde, NSW) was used as collision gas and an optimum collision energy automatically chosen (based on charge state and mass). Tandem mass spectra were accumulated for 2s (m/z 65-2000) with up to 2 repeated spectra. Peak lists were
D. Burg UNSW 59 generated using Mascot Distiller (Matrix Science, London, England) using the default parameters.
2.3.9.4 Data processing
Data generated by the mass spectrometers were processed to a format suitable for searching by the database search program Mascot (Matrix science London, UK; version
2.1) by MasLynx (QTof) and Mascot distiller (QStar). Sequential salt elutions from
LC/LC-MS/MS experiments were combined into a single file using Mergefile (Matrix
Science, London, England). This was then searched using Mascot against an in silico digestion of the local M. burtonii protein FASTA database created in Mascot, with the following parameters: tryptic digestion; variable modifications of carbamidomethylation
(caused by alkylation with IDA) and oxidation of methionine; a peptide mass tolerance of 0.25 Da; a fragment mass tolerance of 0.2 Da; maximum number of missed cleavages set to 1, and a peptide rank of 1. A decoy database, created Mascot by randomising the M. burtonii local FASTA database, was also searched with the same parameters. All spectra, which matched the databases with a Mascot score of less than
30, were rejected (based upon calibration of several searches to give the maximum number of identifications with the minimum number of false positives). All matching spectra with Mascot scores > 30 were manually inspected to ensure ion progressions of
4 or more consecutive ions of a single class (e.g. 4 consecutive y-type ions). Any matches that did not meet these criteria were rejected. From the identifications in both the M. burtonii database and the decoy database it was possible to calculate the false discovery rate (FDR) for any given experimental run. This was calculated using the formula,
60 D. Burg UNSW
(Kall et al., 2008) where: FP = the number of peptides passing the above criteria in the decoy database (false positives); and TP = the number of peptides passing the above criteria in the M. burtonii database (true positives). Any experiment that had a FDR >
0.02 or (2%) was rejected. Identifications were then moved to a database for further processing.
Data from the DSF experiments also underwent secondary searching using the program
Scaffold 2 (version Scaffold_2_00_06, Proteome Software Inc., Portland, OR, USA).
The Scaffold searches were used to identify differences in identification between fractions and also, using normalised peptide data, were used to identify trends within the dataset. All MS/MS samples were analysed using Mascot (version 2.2) and X! Tandem
(www.thegpm.org; version 2007.01.01.1). Both programs were set up to search the M. burtonii database assuming digestion with trypsin. X! Tandem was searched with a fragment ion mass tolerance of 0.10 Da and a parent ion tolerance of 0.25 Da. Mascot was searched with a fragment ion mass tolerance of 0.20 Da and a parent ion tolerance of 0.25 Da. Oxidation of methionine and iodoacetamide derivatives of cysteine were specified in the programs as variable modifications.
Scaffold was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 1 identified peptide for each condition for the purpose of trend identification. Protein probabilities were assigned by the Protein Prophet
D. Burg UNSW 61 algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
2.3.10 Protein parameter annotation
Annotation of individual protein properties was carried out using a variety of platforms.
The IMG expert review platform was used to predict transmembrane domains (TMD) using TMHMM. ConPred2 (http://bioinfo.si.hirosaki-u.ac.jp/~ConPred2/) (Arai et al.,
2004) was also used to validate TMD prediction based on consensus between the two programs and to generate topology maps. Proteins with a predicted transmembrane domain at the C terminus were searched to determine the presence of a signal peptide, which would indicate the protein is secreted by the cell. SignalP
(www.cbs.dtu.dk/services/SignalP/) (Bendtsen et al., 2004) was used to identify signal peptides based on consensus between Gram positive, Gram negative, and eukaryotic criteria using both hidden Markov models and neural networks, as described in
(Saunders et al., 2006), due to the lack of archaeal prediction models. ConPred2 was used in a similar manner to identify signal peptides based on consensus between the prokaryotic and eukaryotic criteria. Agreement between the two programs was used as criteria to identify a protein as containing a signal peptide. The parameters of molecular weight (MW), iosoelectric point (IEP), and hydrophobicity (GRAVY), were generated for all identified proteins by in-house developed PERL scripts (Neil Saunders, UNSW).
Proteins were classified as: Hydrophobic if GRAVY values are positive; membrane if
TMD were present; membrane associated if they were non-TMD containing members of an integral membrane complex; NIW (not identified in WCE) if they were not previously identified in proteomic analyses of WCE (Goodchild et al., 2004a;
62 D. Burg UNSW
Goodchild et al., 2004b; Goodchild et al., 2005); and secreted if they contained a signal peptide and no TMD, identified as described above.
2.3.11. Statistics
All statistical analyses were performed using Minitab 13 statistical software (Minitab,
State College, PA, USA)
D. Burg UNSW 63
2.4 Results and Discussion
The aims of the work performed were: To optimise the conditions for analysis of the
HPP of M. burtonii; to develop methods to maximise the protein identifications; and to define the HPP of M. burtonii, to complement the analyses performed previously on M. burtonii WCE and secreted proteins. These aims were achieved and are discussed below.
2.4.1 Hydrophobic protein separation
Hydrophobic proteins were enriched through carbonate extraction and ultracentrifugation. To determine the quality of the separation, the proteins were visualised on a 12% SDS-PAGE gel (Figure 2.1). Equal concentrations of hydrophobic and soluble protein were loaded onto the gel, along with an aliquot of crude whole cell extract.
Size (kDa) 1 2 3 4 5 6 7 200
116.3 97.4
66.2
45
31
21.5 14.4 6.5 L W S I W S I
Figure 2.1 SDS-PAGE of HPP separation. L – MW marker, W – whole cell extract, S – soluble fraction,
I – insoluble fraction. Lanes 5, 6, & 7 represent a 1:2 dilution of lanes 2, 3, & 4.
64 D. Burg UNSW
As is evident from Figure 2.1, good separation of protein was achieved, with both the soluble and insoluble fractions showing distinct banding. Both fractions displayed distinct separation from the WCE, which is a good indication that proteomics performed on these fractions would be complimentary to previous WCE studies, as sample complexity has decreased. This would allow for the enhancement of identification of low abundance proteins.
To determine whether the extraction protocol was suitable for proteins for both 4 and
23 C grown cells, SDS-PAGE was performed on both soluble and insoluble fractions
(Figure 2.2). Hydrophobic and soluble protein at equal concentrations from 4 and 23 C, were loaded onto the gel, along with an aliquot of BSA to aid in MW determination.
Size (kDa) 1 2 3 4 5 6 7 8 9 10
200 116.3 97.4 66.2
45
31
21.5
14.4
6.5 L S23 S4 I23 I4 S23 S4 I23 I4 B
Figure 2.2 SDS-PAGE of 4 and 23 C soluble and insoluble fractions. L – MW marker, S23 – soluble fraction 23 C, S4 – soluble fraction 4 C, I23 – insoluble fraction 23 C, I4 – insoluble fraction 4 C, B –
BSA (66.2 kDa). Lanes 6, 7, 8, and 9 are 1:2 dilutions of lanes 2, 3, 4, and 5.
D. Burg UNSW 65
The extraction procedure worked equally well for both 4 and 23 C grown cells, with satisfactory separation achieved for proteins for both growth conditions. From inspection of the gel differences in protein profiles, in both band intensity and presence, between the growth temperatures were also evident. These differences are further explored in Chapter 4 using stable isotope techniques.
2.4.2 Trial of solubilisation and extraction strategies
To determine the most effective solvent for use in the ‘in solvent digestion’, several solvents were trialed using extracts from M. burtonii cells grown at 23 C. During the digestion procedure, the hydrophobic proteins are rendered soluble, which allows trypsin to cleave their soluble domains (e.g. loops of membrane proteins). The soluble domains can then be analysed via LC/LC-MS/MS. To determine the best solvent for this procedure in M. burtonii several were tried, and the results compared. The solvents chosen were the protic solvents methanol (60%) and isopropanol (50%), and the aprotic solvents acetone (50%) and acetonitrile (ACN, 40%). The hydrophobic proteins for these experiments were extracted in 0.1M NaHCO3 at a pH of 11.
As well as the comparison of solvents, the optimum pH (11 or 12) of the carbonate extraction was investigated, as well as the addition of a reduction and alkylation step prior to solubilisation (with methanol) and digestion. The results are summarised in
Figure 2.3. As the total number of identifications in any LC/LC-MS/MS experiment varied, a percentage of total identifications was chosen for comparison, as it gave a better representation of the distribution of protein parameters between runs. In many of the analyses depicted in this section, a distinction was made between membrane proteins and hydrophobic proteins. This was made to illustrate the effectiveness of the analyses however, ‘membrane’ and ‘hydrophobic’ are not mutually exclusive, as
66 D. Burg UNSW
although many membrane proteins are hydrophobic, hydrophobic proteins are not necessarily membrane proteins. Many membrane proteins are also classified as hydrophilic as they contain large surface loops, which exert a hydrophilic influence on the average of hydrophobicity
Figure 2.3 Comparison of solvents and extraction conditions (23 C cells). Coloured bars represent: the % of proteins per run with predicted transmembrane domains (membrane, dark grey); the % of proteins with positive GRAVY scores (hydrophobic, pale grey); and the percentage of proteins which were NIW
(black)
Statistical analysis of the data could be performed using ANOVA, as the data were normally distributed (measured by the Anderson-Darling test), and had equal variances
(measured by Bartlett’s and Levene’s tests). Significant differences were found in the
D. Burg UNSW 67 membrane (p = 0.01) and hydrophobic (p = 0.009) categories. To determine where those differences lay, Tukey’s pairwise comparison was implemented, and was able to determine that for both the membrane and hydrophobic categories, there was a larger proportion of identifications when the proteins were reduced and alkylated prior to digestion. No significant differences were found between the solvents used and the pH of extraction.
A similar comparison was performed on M. burtonii extracts from cells grown at 4 C
(Figure 2.4). All runs were performed with an extraction pH of 11.5. The distribution of protein types was similar to the 23 C grown cells. However, within the 4 C dataset there were no differences detected between the solvents and the addition of reduction and alkylation (membrane p = 0.5, hydrophobic p = 0.2, NIW p = 0.2).
When the results from the 4 C extraction were compared to the 23 C extractions, no significant differences were found between the solvents used or with the addition of reduction and alkylation to 4 C grown cells (membrane p = 0.09, hydrophobic p = 0.3,
NIW p = 0.6). However there was a difference between the reduced and alkylated 23 C samples with the rest of the dataset (membrane p < 0.0001, and hydrophobic p = 0.001).
It is unclear why this trend occurred, although it could be influenced by the lack of repeats in the 4 C dataset.
68 D. Burg UNSW
Figure 2.4 Comparison of solvents and extraction conditions (4 C cells). Coloured bars represent: the % of proteins per run with predicted transmembrane domains (membrane, grey); the % of proteins with positive GRAVY scores (hydrophobic, light grey); and the percentage of proteins which were NIW
(black)
All solvents trialed were equally effective when solubilising the hydrophobic proteins of
M. burtonii. This may have been facilitated by the intrinsic nature of proteins from psychrophiles, which impart a reduced hydrophobicity and result in loosely packed protein cores (Saunders et al., 2003). The addition of a reduction and alkylation step, acted to increase the efficacy of the analyses at 23 C. In the method used, the proteins were solubilised in organic solvent-aqueous mixtures, then digested in-solution with trypsin. This procedure allows for the digestion of the soluble domains of the hydrophobic and membrane proteins, which could be analysed with LC/LC-MS/MS. By reducing and alkylating the proteins, the influence of disulfide bridges is eliminated, so that the proteins are presented to trypsin in a linear manner. This is especially useful when digesting membrane proteins, as their surface loops have turns mediated by
D. Burg UNSW 69 disulfide bonds (Nelson and Cox 2000). This is reflected in the results, with a larger proportion of membrane and hydrophobic proteins identified when reduction and alkylation was performed.
For all subsequent experiments the proteins were extracted at a pH of 11.5, were reduced and alkylated prior to solubilisation and digestion, and methanol was chosen as the solvent.
2.4.3 Identification trends
As the experiments progressed, the number of new identifications and membrane identifications tended to plateau (Figure 2.5). It indicated that the limit of detection under these conditions was being approached, and any new identifications with subsequent runs would be minimal. Increases in the number of identifications could be seen where conditions differed (e.g. temperature or instrument), however the overall trend was one of diminishing novel and membrane identifications as the number of runs increased. Thus, in order to achieve a more thorough coverage of the proteome, fractionation procedures needed to be performed.
70 D. Burg UNSW
23 C 4 C Qstar
600
500
400
300
200
Identifications Cumulative
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Run Number
Figure 2.5 Progressive identifications. Bars indicate the number of protein identifications, expressed as a cumulative total. Black indicates all identifications, pale grey indicates NIW proteins, and grey indicates proteins with predicted transmembrane domains. The divider at the top of the graph indicates differences in conditions of cell growth and the instrument used. Runs 1 – 21 were performed on the QTOF instrument, runs 21 – 26 were performed on the Qstar instrument. Runs 1 – 15, and 22 – 23 were performed on 23 C grown cells and include the runs represented in Figure 2.3. Runs 15 – 21, and 24 – 26 were performed on 4 C grown cells and include the runs represented in Figure 2.4
2.4.4 Differential solubility fractionation
To separate hydrophobic proteins prior to digestion, a differential solubility fractionation procedure was implemented. It involved treating washed insoluble extracts with increasing concentrations of the non-ionic detergent OGP. Hydrophobic extracts, from both 23 C and 4 C cells, were separated, underwent buffer exchange and concentration, and fractions were analysed using SDS-PAGE (Figure 2.6).
D. Burg UNSW 71
MW (kDa) 1 2 3 4 5 6 7 8 200 116 97.4
66.2
31
14.4
L F1 F2 F3 F4 F5 F6 B
Figure 2.6 SDS-PAGE of 23 C DSF fractions. L = MW ladder; F1 – F6 = Fractions 1 – 6; B – BSA standard. OGP concentration were: F1 = 0.5mM; F2 = 1.25mM; F3 = 2.5mM; F4 = 5mM; F5 = 12.5mM;
F6 = 20mM. The insoluble pellet was completely dissolved by 20mM OGP with subsequent additions of detergent showing no protein content by Bradford assay. Fractions 4 and 5 had minimal protein content.
The detergent fractions appear to have different protein contents. Indistinct banding and streaking is a feature of membrane / hydrophobic proteins on SDS-PAGE
Separation occurred between 0.5 and 20mM OGP, with the insoluble pellet completely dissolving at the higher detergent concentration, evidenced by no detectable protein in following fractions. Very low protein levels were seen in fractions 4 and 5, and of these two fractions, only fraction 4 contained enough protein for LC/LC-MS/MS. A similar pattern was observed for the 4 C sample; however, fractions 4 and 5 contained appreciable amounts of protein and were able to be analysed by mass spectrometry.
Samples were prepared and initially analysed by LC-MS/MS. For the 23 C sample, fraction 4 gave poor results, so fractions 1-3 and 6 were chosen for analysis with
LC/LC-MS/MS. For the 4 C sample, all fractions could be analysed by LC/LC-MS/MS.
All samples were run were run with SCX10 or SCX20 resin. From the resulting protein
72 D. Burg UNSW
identifications the hydrophobicity (measured in GRAVY) of the fractions was compared (Figure 2.7). The data were not normally distributed, and displayed unequal variances. Due to difficulties in transforming the data as parametric, non-parametric analysis was performed using the Kruskal-Wallace test to determine any significant differences in the hydrophobicity of the samples, and Mood’s median test to determine where those differences occurred.
The Mood’s median test was able to identify clear differences in the fractions from the
23 C sample, and this was confirmed by the Kruskal-Wallace test (p = 0.022). Fractions
2 and 6 had the greatest hydrophobicity (by GRAVY index) while fraction 1 had the most hydrophilic proteins. There may be a threshold for the solvation, allowing for the solubilisation of more proteins between fractions 3 and 5 rather than 2 and 3. For the
4 C sample, a trend was observable up to fraction 4, where hydrophobicity peaks, then drops off to fraction 6.
When the samples were compared in terms of protein types (which could be performed using standard parametric analyses) similar trends to the solubility profiles are seen
(Figure 2.8 and Figure 2.9). For the 23 C sample significant, differences were found when protein types were analysed by ANOVA, with significant differences occurring in proportion of membrane proteins (p < 0.001), hydrophobic proteins (p = 0.004), and
NIW identifications (p = 0.049) following the same trend as described for the Mood’s median test (Figure 2.8).
D. Burg UNSW 73
a) Mood median test for 23 C GRAVY
Chi-Square = 8.55 DF = 3 P = 0.036
Individual 95.0% CIs Fraction N<= N> Median Q3-Q1 ------+------+------+------1 205 165 -0.147 0.479 (------+------) 2 178 190 -0.097 0.530 (------+------) 3 290 263 -0.135 0.563 (----+------) 6 267 306 -0.081 0.553 (-----+------) ------+------+------+------0.150 -0.100 -0.050 Overall median = -0.107
b) Mood median test for 4 C Gravy
Chi-Square = 3.53 DF = 5 P = 0.619
Individual 95.0% CIs Fraction N<= N> Median Q3-Q1 -+------+------+------+----- 1 147 120 -0.160 0.467 (------+----) 2 196 190 -0.137 0.592 (------+------) 3 178 183 -0.124 0.596 (------+------) 4 173 189 -0.108 0.614 (------+------) 5 89 90 -0.124 0.615 (------+------) 6 158 155 -0.136 0.638 (------+------) -+------+------+------+------0.200 -0.150 -0.100 -0.050 Overall median = -0.136
Figure 2.7 Minitab output for DSF hydrophobicity. For 23 C fractions (a) and 4 C fractions (b). Dashed graphs on the right of the data represent the inter-quartile range of the data (bounded by brackets) and the median identified by a +. Clear differences can be seen in the 23 C data while a clear trend towards hydrophobicity as fractions increase is observable in the 4 C data, with a maximum occurring in fraction
4.
When the proportions of protein types in the 4 C sample were compared with ANOVA, significant differences were found between the proportion of membrane, hydrophobic and NIW proteins in each fraction (P < 0.05 for all categories), and an obvious trend of increasing identifications as detergent concentration increased.
74 D. Burg UNSW
50
40
30
20
% of Identifications
10
0 F1 (0.5 mM) F2 (1.25 mM) F3 (2.5 mM) F6 (20 mM)
Fraction (OGP concentration)
Integral Membrane Hydrophobic NIW
Figure 2.8 Comparison of 23 C DSF fractions. Data were pooled and averaged for each fraction. A clear trend can be seen with the proportion of NIW identifications. The proportion of membrane and hydrophobic proteins follows the same trend as seen in the Moods median test
50
40
30
20
% of identifications
10
0 F1 (0.5mM) F2 (1.25mM) F3 (2.5mM) F4 (5mM) F5 (12.5mM) F6 (20mM) Fraction (OGP concentration)
Figure 2.9 Comparison of 4 C DSF fractions. Data were pooled and averaged for each fraction. Legend follows from Figure 2.8. A clear trend can be seen with the proportion of NIW identifications. Fraction 1 had significantly less proteins from all categories.
D. Burg UNSW 75
When the results of the DSF experiments were compared to previous results (Figure
2.10) it is clear that the fractionation procedure resulted in a much larger proportion of membrane, hydrophobic and previously unidentified proteins. Statistically, the results showed significance when proportions of protein types for all runs were compared using a 2-sample t-test (unequal variances) for both 23 and 4 C data sets: membrane proteins,
23 C p < 0.001, 4 C p < 0.001; hydrophobic proteins, 23 C p < 0.001, 4 C p < 0.001;
NIW proteins, 23 C p = 0.001, 4 C p < 0.001.
While the total number of proteins identified from any fractionated sample was similar to those identified from an equal number of non-fractioned runs, the procedure was able to identify far more membrane, hydrophobic and NIW proteins from far fewer runs. The improvements achieved by DSF are illustrated by the identification of 137 membrane proteins from one DSF experiment (with 4 LC/LC-MS/MS runs), compared to a total of
51 membrane proteins from 4 LC/LC-MS/MS runs without DSF treatment. Across all experiments performed, the DSF treatment led to an efficacy gain (number of membrane, hydrophobic and NIW proteins divided by the number of LC/LC-MS/MS runs, expressed as a percentage increase) of 112 – 169% for membrane proteins, 76 –
106% for hydrophobic proteins, and 71 – 117% for NIW proteins. The procedure was also much more economical in terms of machine time required to identify proteins. For example, a DSF experiment with 4 LC/LC-MS/MS runs (96 hrs) was able to identify more membrane proteins than from 31 LC/LC-MS/MS runs (744 hrs) of samples without DSF (12% of the machine time). Across all experiments the DSF procedure enabled the identification of: Membrane proteins in 12 – 19% of the machine time;
76 D. Burg UNSW
hydrophobic proteins in 15 – 24% of the machine time; and NIW proteins in 25 – 40% of the machine time required for samples without DSF.
As a whole the DSF experiments added a further 115 proteins to the cumulative total identifications (74 from the 23 C experiments and 41 from the 4 C experiments), which included 106 NIW proteins, and 51 proteins with predicted TMD. There was significant overlap between experimental results, with many proteins that were first identified in the 23 C DSF experiments also being identified in the subsequent 4 C experiments.
60
50
40
30
20 % of identifications
10
0
DSF worst DSF best Pre-DSF worst DSF averagePre-DSF best Pre-DSF average
Integral membrane Hydrophobic NIW
Figure 2.10 Comparison of DSF proteins types with previous runs. Worst best and average runs for individual DSF fractions are compared to the worst, the best and the average of all previous runs (no
DSF). The proportion of proteins seen for each type is higher than previous runs.
D. Burg UNSW 77
The use of the SCX20 resin had little positive effect. The runs where this resin was used resulted in a lower total number of identifications, and those proteins identified were no different to those from the same fraction processed with SCX10 resin (data not shown).
The DSF procedure resulted in a vast increase in the efficiency of identification of desirable protein types. However, there tended to be overlaps in protein identification between the detergent fractions. This overlap however, involved progressive increases in the number of peptides of proteins with hydrophobic properties, and a corresponding progressive decrease in high abundance hydrophilic proteins as detergent concentration increased. The effect is clear when normalised peptide spectral counts across fractions are examined. The presence of high abundance proteins in samples is a continuing problem in the field of proteomics, as these proteins mask the identification of low abundance proteins (Stasyk and Huber 2004). When protein data were processed using
Scaffold2, normalised peptide spectral counts could be obtained, allowing for the identification of the fractions where the majority of peptides for a given protein were identified. This overcomes the problem of protein identification overlap between fractions, and allows for the identification of trends in the dataset.
In the DSF method, peptides from soluble high abundance proteins are mainly identified in the first fractions (Figure 2.11i). This decreases the influence of the peptides from these proteins in later fractions. These proteins were unable to be removed by repeated water washes, possibly due to non-specific interaction with other proteins. Treatment with a low concentration of detergent was able to remove the majority of these high abundance proteins, which decreased their influence across the remainder of the samples. The removal of the influence of peptides from soluble high abundance proteins
78 D. Burg UNSW
facilitated the identification of hydrophobic low abundance proteins (Figure 2.11ii).
Many of these were unidentified in any run prior to the application of the DSF method.
i ii
18 2.0
16
14 1.5 12
10 1.0 8 6 0.5 4 Normalized peptide count peptide Normalized Normalized peptide count peptide Normalized 2 0 0.0 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM OGP concentration OGP concentration
Figure 2.11 Normalised peptide spectral counts for high and low abundance proteins. As fraction number increased (left to right), the peptide spectral counts for high abundance soluble proteins tended to decrease (i), and the identification of low abundance proteins increased (ii). (i) Mbur_1364
Dimethylamine methyltransferase, 0 TMD, GRAVY -0.036. (ii) Mbur_1074 Small-conductance mechanosensitive ion channel, 3 TMD, GRAVY 0.028.
The analysis of normalised peptide counts also allowed for the identification of solubility trends in hydrophobic proteins (Figure 2.12). This illustrates the fraction where the majority of peptides from the protein were identified. For hydrophobic proteins, this tended to peak in the later fractions, with very few or no identifications in earlier fractions. These profiles tended to vary with the hydrophobicity of the protein, as well as with the number of TMD.
D. Burg UNSW 79
i ii 5 18
16 4 14
12 3
10
8 2 6
4 count peptide Normalized count peptide Normalized 1 2
0 0 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM OGP concentration OGP concentration iii 18 14 iv 16 12 14
10 12
8 10 6 8
6 4 Normalized peptide count peptide Normalized 4 Normalized peptide count peptide Normalized 2 2
0 0 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM OGP concentration OGP concentration
Figure 2.12 Normalised peptide spectral counts for membrane and hydrophobic proteins. As fraction number increased (left to right), the peptide spectral counts for hydrophobic and membrane proteins increased. This varied according to number of TMD and GRAVY index. (i) Mbur_1367 Trimethylamine permease, 10 TMD, GRAVY 0.842. (ii) Mbur_1579 Transmembrane oligosaccharyl transferase,13 TMD,
GRAVY 0.182. (iii) Mbur_0024 Preprotein translocase SecY subunit, 4 TMD, GRAVY 0.695. (iv)
Mbur_1689 Hypothetical protein (ER5). 0 TMD, GRAVY 0.24
An unexpected outcome of utilising the DSF method was the identification of hydrophobicity trends in proteins that were not predicted to be hydrophobic or have
TMD’s (Figure 2.13i, ii). The proteins displaying these trends are predicted to be strongly membrane associated. The trend is contrasted by proteins that are non-TMD containing members of membrane complexes, which were identified in earlier fractions
(Figure 2.13iii, iv).
80 D. Burg UNSW
i ii 50 14
12 40
10
30 8
6 20
4 Normalized peptide count peptide Normalized Normalized peptide count peptide Normalized 10 2
0 0 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM OGP concentration OGP concentration iii iv 8 25 7
20 6
5 15 4
3 10 2 Normalized peptide count peptide Normalized Normalized peptide count peptide Normalized 5 1 0 0 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM 0.5 mM 1.25 mM 2.5 mM 5 mM 12.5 mM 20 mM OGP concentration OGP concentration
Figure 2.13 Unexpected solubility trends. For several proteins not predicted to be hydrophobic or membrane bound, as fraction number increased (left to right), the peptide spectral counts increased (i, ii).
Contrasting this, are proteins that are associated with the membrane through other proteins as non-TMD members of membrane bound complexes (iii, iv) which may suggest a close membrane association of i and ii. (i) Mbur_1950 DEAD box RNA helicase, 0 TMD, GRAVY -0.681. (ii) Mbur_2256 FKBP-type peptidyl-prolyl cis-trans isomerase, 0 TMD, GRAVY -0.05. (iii) Mbur_1243 V-type ATP synthase alpha chain, 0 TMD, GRAVY -0.237. (iv) Mbur_2371 F420H2 dehydrogenase subunit F, 0 TMD, GRAVY -
0.137.
The DEAD-box RNA helicase (Mbur_1950) of M. burtonii (peptide counts displayed in
Figure 2.13), was unable to be identified in WCE studies (Goodchild et al., 2004a), and was shown to be exclusive to 4 C at a transcript level (Lim et al., 2000). The DEAD box helicase was however, found to be one of the most abundant proteins in the
D. Burg UNSW 81 hydrophobic fractions at both temperatures (4 and 23 C), which does not reflect its predicted hydrophilic nature (GRAVY of –0.681). Compounding this finding is the DSF data, where the protein showed an increase in abundance as the detergent concentration increased. The data indicate that the protein has an uncharacterised membrane association or is strongly associated with highly hydrophobic proteins, which is not unusual for a DEAD-box helicase. For example, a cold induced DEAD-helicase from an
Anabaena species has been shown to be membrane localised (El-Fahmawi and Owttrim
2003), and be associated with a protein complex (Yu and Owttrim 2000). The DSF data suggest that this could also be the case with Mbur_1950.
2.4.6 Peptide and protein identification statistics
The combination of all runs performed resulted in the processing of 304383 spectra;
21576 of these spectra resulted in a positive match to the M. burtonii protein database in silico digestion, based on the strict criteria outlined in 2.3.9.4. The processing of the spectra through the decoy M. burtonii database resulted in the identification of 121 positive matches. The FDR for all peptides is therefore 0.0058 or 0.6%. For individual runs the FDR varied between 0 and 0.016, indicating high quality identifications throughout the runs, which led to high confidence in the data. The combination of all of the runs resulted in the identification of a large number of proteins. In total 667 were identified (Figure 2.14): 375 (56%) of these were NIW; 165 (25%) of these proteins were predicted to have TMDs (with numbers of domains ranging from 1 – 19 (Figure
2.15)). Along with these proteins with obvious TMDs a further 36 are predicted to be associated with the membrane through a complex, bringing the number of membrane proteins to 201 (30%). 210 (31%) of the proteins identified were predicted to be hydrophobic based on GRAVY scores. Of the 667 identified proteins 65 were identified
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on only one occasion, 60 of these were identified with only 1 peptide and of those 60,
39 had peptide scores of less than 40 indicating low confidence. The remaining proteins were identified in several LC/LC-MS/MS runs (with at least two peptides) including proteins identified in all runs (36 proteins) (Figure 2.16). The above statistics describe a high quality, high confidence dataset, which is significantly different to the dataset obtained through analysis of M. burtonii WCE.
1.4 1.2 1.0 0.8 0.6 0.4 1.4 0.2 0.0
1.2 -0.2 GRAVY 1.0 -0.4 0.8 -0.6 0.6 -0.8 -1.0 pI 0.4 -1.2 0.2 -1.4 0.0 0.0 50.0k100.0k150.0k200.0k250.0k300.0k -0.2
GRAVY -0.4 MW (Da) -0.6 1.4 -0.8 14 1.2 1.0 -1.0 12 0.8 -1.2 10 0.6 -1.4 0.4 0.0 8 0.2 50.0k 0.0 -0.2 100.0k 6 pI GRAVY -0.4 150.0k -0.6 MW (Da)200.0k 4 -0.8 250.0k -1.0 300.0k -1.2 2 -1.4 New ID Prevoius ID MW (Da) 2 4 6 81012 14 pI
Figure 2.14 Properties of identified proteins. Solid red circles indicated NIW proteins. Solid blue triangles represent proteins identified in HPP experiments that were identified in previous WCE studies.
The smaller inset graphs represent rotations of the larger graph. The distribution of identifications covered the range of pI and molecular weight for M. burtonii proteins indicating no bias in these areas (as would be seen in 2D gels). A large number of identified proteins had positive GRAVY values indicating hydrophobicity. The large bias towards new identification in this area is reflective of the enrichment for hydrophobic proteins performed.
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Figure 2.15 Proteins with predicted transmembrane domains. 165 proteins with predicted TMDs were identified, with up to 19 TMDs predicted in a single protein. Proteins with single transmembrane domains were searched for the presence of a signal peptide. Proteins that were positive for the presence of a signal peptide were not included in this graph.
Figure 2.16 The number of runs in which each protein was identified. The majority of proteins were identified in a number of LC/LC-MS/MS runs. Many proteins were identified in all experiments performed. The number of times a protein was identified relates directly to the relative abundance of that protein in the cell.
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A significant proportion of proteins identified were not identified in previous WCE studies of M. burtonii. However, there were a large number of proteins in the dataset that were identified in previous work (Goodchild et al., 2004a; Goodchild et al., 2004b;
Goodchild et al., 2005). These proteins included: the high abundance cellular proteins; high abundance membrane proteins that could be identified without enrichment; and proteins which are members of large complexes. The HPP enrichment procedure involved an ultra-centrifugation step. In this step large complexes, due to their density, were pelleted with the hydrophobic proteins, resulting not only in the contamination of the insoluble proteins with proteins from these complexes, but a subsequent enrichment of these in the sample. This allowed for the identification of many proteins from these complexes which were previously unidentified.
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2.5 Conclusions
The implementation of the DSF procedure greatly increased the efficacy and economy of HPP analysis. The combination of the results of this technique and other analyses of the HPP led to a significant increase the identified proteome of M. burtonii. Over 50% of the proteins identified were NIW, and a large number of proteins identified had multiple transmembrane domains, and/or were hydrophobic. The DSF procedure was also useful in identifying solubility trends in proteins, and was able to suggest membrane associations in proteins that could not be predicted by other means. The DSF procedure for the analysis of the HPP, which was highly successful for M. burtonii, could be implemented in other organisms. This will decrease time and monetary costs generally associated with comprehensive analysis. The analysis of the biological context of the HPP proteins (Chapter 3) follows from the above proteomics.
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Chapter 3. Biological Context Analysis of the Hydrophobic Proteome
3.1 Summary
A significant body of work has been performed on the cold adapted archaeon
Methanococcoides burtonii, including genomics, proteomic analyses and studies of individual proteins. However, little is known about the hydrophobic and membrane proteins of this organism and the corresponding biology. The proteogenomic analysis of the biological context of the proteins identified in Chapter 2 revealed many new aspects of the biology of M. burtonii. The novel findings came from diverse areas of cell biology and include: methanogenesis; maintenance of osmotic balances; protein turnover; coenzyme metabolism; glycosylation and surface proteins; inorganic ion metabolism; and thermal adaptation. Within these areas, many proteins were identified that were unique to M. burtonii and are presumably crucial in the psychrophilic lifestyle of the organism. The identification of an -2-macroglobulin protein in M. burtonii (a unique finding among the characterised archaea) has raised questions about gene loss scenarios or horizontal gene transfer. The analysis of peptide spectral counts from large numbers of experiments across two temperatures resulted in the identification of thermally important proteins in M. burtonii. This type of analysis proved to be complementary to previous differential proteomic analyses of M. burtonii, and resulted in the identification of low abundance proteins unable to be quantified using labelling strategies.
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3.2 Introduction
Genomic and proteomic analyses of M. burtonii have revealed a variety of cold adaptation strategies, ranging from genome content (Saunders et al., 2003), to transcriptional and metabolic adaptations (Thomas et al., 2001; Goodchild et al.,
2004b). However very little is known about the roles of hydrophobic and membrane proteins in the cold adaptation of this organism, despite genomic evidence that they may fulfill crucial roles (Allen et al., 2009). This is also true for all psychrophiles, as no focused analyses of membrane and hydrophobic protein adaptations have been performed, with the exception of minimal and largely unsuccessful work by Kawamoto et al., (2007) where only low numbers of hydrophobic and membrane proteins were identified. Hydrophobic proteins have however, been implicated in cold adaptation through whole cell extract proteomics of psychrophiles (Goodchild et al., 2004b;
Bakermans et al., 2007).
The comprehensive approach for the identification of HPP proteins described in Chapter
2, complemented previous analyses of whole cell extracts (Goodchild et al., 2004a;
Goodchild et al., 2004b; Goodchild et al., 2005). Over 50% of the proteins identified through HPP analysis were not identified in these WCE studies; this represented a significant increase in the identifiable proteome of the organism. The biological context analysis of these NIW proteins therefore, represented an opportunity to reveal many new aspects of the biology of this Antarctic archaeon, specifically, the biology of membrane and surface proteins. In this light, comprehensive proteogenomic analysis of the HPP proteins of M. burtonii was performed. Central to this analysis was the intensive manual annotation of the genome of M. burtonii, which resulted in high
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quality functional annotations, from which the biology of the organism could be explored.
One of the limitations of isotope labelling strategies for differential proteomics is that in these techniques, peptides must be present in sufficient abundance from all conditions tested for quantitation. That is, if proteins/peptides from a single condition are of very low abundance or absent, then that protein cannot be confidently quantified. The development of methods for the analysis of the HPP of M. burtonii required a large number of LC/LC-MS/MS to be performed with cells grown at high (23˚C) and low
(4˚C) temperatures. Utilising Scaffold2, proteins that are at very low abundance or absent in one condition, while remaining at higher abundance in the other, can be identified. The analysis of peptide spectral counts with Scaffold2 allowed for the determination of any differences in peptide abundance in a semi-quantitative manner and, given the large numbers of runs performed at both temperatures, was statistically viable. The results of this analysis proved to be complementary to labelling based and
2DE proteomics previously performed in M. burtonii (Goodchild et al., 2004b;
Goodchild et al., 2005; Saunders et al., 2006) (and also Chapter 4).
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3.3 Materials and Methods
All proteins were identified through the mass spectrometry described in Chapter 2.
3.3.1 Protein parameter annotation
Proteins were assigned arCOG (archaeal COG) categories using in-house developed
PERL scripts (Federico M. Lauro, UNSW) by assigning corresponding genome ORFs to particular arCOG with BLASTP at a minimum e-value of 1e-5 and 30% identity.
Gene ontology was assigned using the program Blast2Go (http://www.blast2go.de/)
(Conesa et al., 2005). These automated annotations were manually verified for accuracy against the proteins manually annotated as described below.
Proteins were classified according to the presence of TMD, hydrophobicity (GRAVY), and whether the protein was NIW. Other parameters such as the presence of a signal peptide were also investigated as described in 2.3.10. Secondary structure of selected proteins was predicted using the program GOR IV (Garnier et al., 1996), and membrane topology maps were generated in Conpred2 (http://bioinfo.si.hirosaki- u.ac.jp/~ConPred2/) (Arai et al., 2004). Multiple protein sequence alignments were performed using ClustalW (Thompson et al., 2002). Maximum likelihood phylogenetic trees were constructed using PhyML (Tamura et al., 2007), with the LG substitution model (Saitou and Nei 1987). Topological rearrangements were performed using SPR
(Felsenstein 1985). Branch positions were evaluated with the approximate likelihood ratio test (aLRT) using the SH-like model (Schwarz and Dayhoff 1979).
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3.3.2 Protein identity annotation
All protein identifications were manually annotated using the joint genome institute’s
(JGI) integrated microbial genomes (IMG) expert review platform (http://imgweb.jgi- psf.org/cgi-bin/img_er_v260/main.cgi) and assigned an evidence rating (ER) as described in Allen et al. (2009). ER1 indicates that the protein from M. burtonii had been experimentally characterised (a self match); ER2, the most closely related functionally-characterised homologue is not from M. burtonii but the BLAST alignments share ≥ 35% sequence identity along the entire length of the protein; ER3, the most closely related functionally-characterised homolog shares <35% sequence identity along the length of the protein, but all required motifs/domains for function are present and complete; ER4, an experimentally characterised full-length homolog is not available but conserved protein motifs or domains can be identified; ER5 (hypothetical protein), no functionally characterised homolog can be found, and no characterised protein domains above the Pfam and InterProScan cut-off thresholds can be identified.
The author of this dissertation, in this manner, manually annotated a majority of the proteins identified by mass spectrometry. However, a small proportion of identified
HPP proteins were annotated by other members of the Cavicchioli research group
(Allen et al., 2009).
3.3.3 Statistics
Comparisons of statistically over-represented arCOG categories were performed with the resampling method of Rodriguez-Brito et al. (2006) at a confidence level of 99% using custom PERL scripts developed by Federico M. Lauro (UNSW). To determine differences in relative peptide spectral counts between categories (4˚C and 23˚C), t-tests
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3.4 Results and Discussion
3.4.1 Grouping of identified proteins
All positively identified proteins were manually annotated and grouped into categories using in house generated scripts (arCOG classifier) against the arCOG database
(Makarova et al., 2007) and Blast2GO. Statistical analysis was performed with the arCOG assignments (displayed in Figure 3.1) to determine whether any categories had over or under-representation in the datasets: (a) whole genome vs. all identifications; (b) genome vs. NIW identifications; and (c) all identifications vs. NIW identifications.
Statistical differences (99% CI) were found in all datasets.
In dataset a) genome vs. all identifications: The genome had a higher representation of proteins in the category replication, recombination, and repair; while the proteomic dataset had higher representations in the categories: energy production and conversion; and translation, ribosomal structure, and biogenesis. This over representation in the total proteomic dataset is reflective of the importance of the proteins in these categories for the general biology of M. burtonii and was influenced by the relative abundance of these proteins in the cell. This is evident in Table 3.17 as many of the proteins in this table are members of these categories.
In dataset (b) genome vs. NIW identifications: the genome had a higher representation in the categories carbohydrate transport and metabolism; replication, recombination and repair; and transcription. The NIW identifications dataset had an over-representation of proteins in the categories: cell wall/membrane/envelope biogenesis; inorganic ion transport and metabolism; intracellular trafficking, secretion and vesicular transport; and signal transduction mechanisms. These categories contain large numbers of membrane
D. Burg UNSW 93 and membrane associated proteins and the enrichment for hydrophobic proteins facilitated their identification.
In the dataset (c) all identifications vs. NIW identifications, the total proteomic dataset had a higher representation in the categories: coenzyme transport and metabolism; translation, ribosomal structure and biogenesis; and transcription. Again reflecting the high abundance of these proteins in the cell, and hence their ease of identification in previous studies. The NIW identifications dataset had a higher representation of the arCOG category; function unknown. The analysis of the hydrophobic fraction enabled the identification of over 180 NIW proteins with no known or only general function predicted. The large number of newly identified proteins in the unknown function categories reflects the lack of knowledge in the area of membrane and hydrophobic protein biology; as indicated in the 70 proteins of unknown function with predicted transmembrane domains. The methanogens have large numbers of hypothetical proteins in their genomes (Ferry and Kastead 2007) and this is compounded by the relative lack of knowledge regarding hydrophobic proteins in archaea and organisms in general.
Some of these hypothetical proteins are highly abundant in the cell, with 7 of the top 50 most abundant proteins in the hydrophobic fraction falling into this category (Table
3.17).
The arCOG category annotations were manually curated, which resulted in high quality functional category assignments for all proteins identified. For information on annotation of proteins and changes to functional assignments from the auto-annotation pipelines see Allen et al. (2009). The arCOG classifier mis-annotated 200 (out of 670 or
30%) of the proteins from this study, which had to be manually assigned to different functional groups (See supplementary Table 1 available on accompanying disk). It is
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worthwhile to note that while this number is high, the majority of changes made were minor, e.g. changing a protein from the category ‘general function prediction only’ to
‘function unknown’. In many cases the assignments were ambiguous so any changes made in these cases were purely cosmetic.
a
b c
[C] Energy production and conversion [D] Cell cycle control, cell division, chromosome partitioning [E] Amino acid transport and metabolism [F] Nucleotide transport and metabolism [G] Carbohydrate transport and metabolism [H] Coenzyme transport and metabolism [I] Lipid transport and metabolism [J] Translation, ribosomal structure and biogenesis [K] Transcription [L] Replication, recombination and repair [M] Cell w all/membrane/envelope biogenesis [N] Cell motility [O] Posttranslational modification, protein turnover, chaperones [P] Inorganic ion transport and metabolism [Q] Secondary metabolites biosynthesis, transport, and catabolism [R] General function prediction only [S] Function unknow n [T] Signal transduction mechanisms [U] Intracellular trafficking, secretion, and vesicular transport [V] Defense mechanisms
Figure 3.1 arCOG distribution comparisons. Differences can be seen in the distribution of COG categories between the genome (a), and total identifications (b). The distribution of COG categories also differs between the total identifications (b) and the proteins unidentified in previous WCE studies (c).
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In total 60 proteins were moved to different categories (e.g. coenzyme transport and metabolism to energy production), 94 proteins were given a less specific category (e.g. function assigned when no function is known), and 24 were given a more specific category. 26 proteins were not assigned to arCOGs; these mainly consisted of proteins unique to M. burtonii. 75 proteins (11%) had truly incorrect assignments. Many of these were methanogenesis proteins incorrectly assigned to the coenzyme transport and metabolism arCOG instead of the energy production and conversion arCOG.
Blast2GO performed remarkably well when annotating the M. burtonii proteins, however the program was unable to assign functions to 190 proteins, the majority of these were hypothetical or general function prediction only. 16 classifications were given a less specific assignment and 8 proteins were given a more specific assignment
(the majority of these were methanogenesis proteins). 17 classifications by Blast2GO were incorrect and had to be changed to a more specific function.
When the curated arCOG distributions were further analysed for the presence of newly identified proteins and proteins with predicted TMDs (Figure 3.3) it is clear that the newly identified proteins are present in all areas of cell biology. The proteins that are predicted to contain TMDs are also present in many categories further highlighting the importance of these proteins in all areas of cell biology, and the importance of this type of HPP analysis.
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ons, NIW identifications, had members identified had with members s in the format: total identificati s in the format: eas of cell biology. Most categories eas of cell biology. y names are followed by number TMDs. NIW proteins were identified from all ar from TMDs. NIW proteins were identified oteins. Shortened COG categor
arCOG categories of identified pr and number of identifications with predicted identifications of and number predicted domains. transmembrane Figure 3.2
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3.4.2 Biology inferred from proteomics
Proteins that were identified in the HPP that were NIW are analysed and discussed below, separated into curated functional categories. Included in the identification tables are previously identified proteins that are predicted to be hydrophobic, contain TMDs or be associated to the membrane through a membrane bound complex. A complete list of identifications is available in Supplementary Table 1 (available on accompanying disk)
3.4.2.1 Energy production and conversion
Identified proteins from this category are collated in Table 3.1. M. burtonii, as a methylotrophic methanogen, generates energy, and carbon and nitrogen for biomass, from the dismutation of trimethylamine (methanol can also be used as a carbon source, to be discussed in Chapter 4) via a methanogenic pathway. While many protein members of this pathway could be identified in WCE studies, the analysis of the HPP has revealed membrane related aspects of this biology previously not identified.
3.4.2.1.1 Methanogenesis
Methanogenesis is a complex process involving a number of membrane bound and hydrophobic components, many of which are involved in electron transport, oxidation and reduction of intermediates, and the formation of electrochemical gradients that drive
ATP production. In the experiments performed on the HPP of M. burtonii almost all membrane bound components relating to this process were identified. A cartoon depicting this process is presented in Figure 3.3.
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Table 3.1 Energy production and conversion
Locus tag GRAVY Id'd Type TMD Comment Annotation Plasma-membrane proton-efflux P-type ATPase (EC Mbur_0068 0.333 3 nm 10 3.6.3.6) (ER2) Formate dehydrogenase family accessory protein FdhD Mbur_0280 -0.076 1 n (ER3) Pyrophosphate-energised proton pump / Pyrophosphate- energised inorganic pyrophosphatase (H(+)-PPase) (EC Mbur_0994 0.826 57 m 16 3.6.1.1) [hppA] (ER2) Mbur_1237 -0.781 4 p membrane complex H(+)-transporting ATP synthase, subunit H (ER2) Mbur_1238 0.226 57 m 6 Putative 116kDa V-type ATPase subunit (ER4) H(+)-transporting ATP synthase, subunit K (EC 3.6.3.14) Mbur_1239 1.163 57 m 2 (ER2) V-type ATP synthase subunit E [atpE] (EC 3.6.3.14) Mbur_1240 -0.437 5 p membrane complex (ER2) V-type ATP synthase subunit C [atpC] (EC 3.6.3.14) Mbur_1241 -0.294 41 p membrane complex (ER2) V-type ATP synthase subunit F [atpF] (EC 3.6.3.14) Mbur_1242 -0.081 1 p membrane complex (ER2) V-type ATP synthase alpha chain (atpA) (EC 3.6.3.14) Mbur_1243 -0.237 29 p membrane complex (ER2) V-type ATP synthase beta chain [atpB] (EC 3.6.3.14) Mbur_1244 -0.256 57 p membrane complex (ER2) V-type ATP synthase subunit D [atpD] (EC 3.6.3.14) Mbur_1245 -0.283 5 n membrane complex (ER2)
Mbur_1286 -0.191 20 p membrane complex F420H2 dehydrogenase subunit O (fpoO) (ER2)
Mbur_1287 0.976 15 nm 14 F420H2 dehydrogenase subunit N (ER3)
Mbur_1288 1.042 38 nm 6 F420H2 dehydrogenase subunit M (ER2) Mbur_1289 1.085 28 nm 7 F420H2 dehydrogenase subunit L (ER2)
Mbur_1290 0.798 6 nm 2 F420H2 dehydrogenase subunit K (ER2)
Mbur_1291 0.564 47 nm 2 F420H2 dehydrogenase subunit J (ER2)
Mbur_1292 -0.245 36 p membrane complex F420H2 dehydrogenase subunit I (fpoI) (ER2)
Mbur_1293 0.969 41 nm 8 F420H2 dehydrogenase subunit H (ER2)
Mbur_1294 -0.111 41 p membrane complex F420H2 dehydrogenase subunit D (fpoD) (ER2)
Mbur_1295 -0.376 23 n membrane complex F420H2 dehydrogenase subunit C (ER2)
Mbur_1296 -0.21 17 p membrane complex F420H2 dehydrogenase subunit B (fpoB) (ER2)
Mbur_1297 0.748 23 nm 3 F420H2 dehydrogenase subunit A (ER2) Mbur_1365 0.09 28 p Dimethylamine corrinoid protein (mtbC) (ER2) Mbur_1367 0.842 46 m 10 Trimethylamine permease (MttP) (ER4) Mbur_1368 0.229 21 p Trimethylamine corrinoid protein (mttC) (ER2) Mbur_1378 0.982 20 nm 3 UPF0132 family protein (ER3) Mbur_1379 0.228 35 sec sec membrane complex Electron transport complex protein rnfB Mbur_1380 1.217 21 nm 6 Electron transport complex protein rnfA (ER2) Mbur_1381 1.007 19 nm 6 Electron transport complex protein rnfE (ER2) Mbur_1382 0.174 42 nm 1 Electron transport complex protein rnfG (ER3) Mbur_1383 0.913 18 nm 6 Electron transport complex protein rnfD (ER3) Mbur_1384 0.006 31 p membrane complex Electron transport complex protein rnfC [rnfC] (ER3) Protein containing signal peptide and multi-heme region Mbur_1385 -0.208 16 secn sec membrane complex (ER4)
Tetrahydrosarcinapterin S-methyltransferase subunit H Mbur_1518 -0.136 57 p membrane complex (mtrH) (EC 2.1.1.86) (ER2)
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Table 3.1 cont’d energy production and conversion
Locus tag GRAVY Id'd Type TMD Comments Annotation
Tetrahydrosarcinapterin S-methyltransferase subunit G Mbur_1519 0.433 36 m 1 (mtrG) (EC 2.1.1.86) (ER2) Tetrahydrosarcinapterin S-methyltransferase subunit F Mbur_1520 0.661 45 m 1 (mtrF) (EC 2.1.1.86) (ER2) Tetrahydrosarcinapterin S-methyltransferase subunit A Mbur_1521 0.136 57 m 1 (mtrA) (EC 2.1.1.86) (ER2)
Tetrahydrosarcinapterin S-methyltransferase subunit B Mbur_1522 0.537 49 m 1 (mtrB) (EC 2.1.1.86) (ER2) Tetrahydrosarcinapterin S-methyltransferase subunit C Mbur_1523 0.977 35 nm 7 (mtrC) (EC 2.1.1.86) (ER2)
Tetrahydrosarcinapterin S-methyltransferase subunit D Mbur_1524 0.972 42 nm 6 (mtrD) (EC 2.1.1.86) (ER2)
Tetrahydrosarcinapterin S-methyltransferase subunit E Mbur_1525 0.527 57 m 5 (mtrE) (EC 2.1.1.86) (ER2)
Formylmethanofuran--tetrahydrosarcinapterin Mbur_1728 0.031 6 p formyltransferase (ftr) (EC 2.3.1.101) (ER2) Methylamine-specific methylcobamide:CoM Mbur_2082 0.086 16 p methyltransferase (mtbA) (EC 2.1.1.-) (ER2) Coenzyme F420 hydrogenase subunit beta (EC 1.12.98.1) Mbur_2261 -0.177 1 n membrane complex (FRH) (ER3) Mbur_2288 0.071 2 p Dimethylamine corrinoid protein (mtbC) (ER2) Mbur_2310 0.206 38 p Trimethylamine corrinoid protein (mttC) (ER2) Mbur_2311 0.928 20 m 9 Trimethylamine transporter (ER4)
Mbur_2371 -0.137 41 p membrane complex F420H2 dehydrogenase subunit F (fpoF) (ER2) Coenzyme F420-dependent N(5),N(10)- methylenetetrahydrosarcinapterin reductase (mer) (EC Mbur_2372 0.017 5 p 1.5.99.11) (ER2)"" Methyl-coenzyme M reductase, subunit beta (mcrB) (EC Mbur_2421 0.096 57 p 2.8.4.1) (ER2) CoB-CoM heterodisulfide reductase, subunit E (EC Mbur_2436 0.742 36 nm 5 1.8.98.1) (ER2) CoB-CoM heterodisulfide reductase, subunit E (EC Mbur_2436 0.742 8 nm 5 1.8.98.1) (ER2) CoB-CoM heterodisulfide reductase, subunit D (EC Mbur_2437 -0.178 14 p membrane complex 1.8.98.1) (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d –
the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW; nm – NIW
identification with TMDs; m – protein also identified in WCE studies but containing TMD; p – protein
also identified in previous WCE studies but either hydrophobic in nature or part of a membrane complex;
sec – protein also identified in previous WCE studies that contains a signal peptide; secn – NIW protein
with signal peptide. Comments indicate whether the protein is part of a membrane complex.
A key finding in the area of energy production in M. burtonii is the expression in high
abundance of a complex with high identity to the Methanosarcina acetivorans Ma-Rnf
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complex (Li et al., 2006) including the flanking UPF0132 and multi-haem containing hypothetical proteins (see Appendix A.2 for alignments). The Rnf complex is thought to replace the Ech complex in acetate utilising methanogens, and is used to oxidise reduced ferredoxins produced during the production of CO2 from formylmethanofuran by formylmethanofuran dehydrogenase (Fmd). The electrons gained are then passed in the membrane through methanophenazine to CoB-CoM heterodisulfide reductase (Hdr).
This is the first evidence of the expression of this complex in another methanogen, and the first example of this electron and ion-translocating complex in an organism utilising methylamines as opposed to acetate.
Along with the identification of the Rnf complex, all membrane components of the Fpo,
Hdr and Mtr complexes were identified in this study. Several identified proteins were also annotated as DUF6 proteins. These are predicted to be trimethylamine permeases based on their position in the genome, and their alignments to other predicted trimethylamine transporters in Methanosarcina spp.. M. burtonii expresses multiple copies of the putative methylamine transporters, methyltransferases and corrinoid proteins; these are discussed further in Chapter 4.
The terminal step in the M. burtonii reverse methanogenesis pathway is mediated by the formylmethanofuran dehydrognease complex (FD), which produces CO2 from formylmethanofuran. The CO2 is then condensed with a methyl group from methyltetrahydrosarcinapterin by the CODH/ACS complex to form Acetyl-CoA. In a similar fashion to M. barkeri, M. burtonii only expresses a molybdenum FD, organised in the genome in a (FmEFACBD) operon structure (Vorholt et al., 1996). In a separate putative operon elsewhere in the genome, M. burtonii codes for subunits of the tungsten FD in a similar (FwGBD) operon structure to Methanopyrus kandleri
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(Vorholt et al., 1997) but containing the cysteine (fwcB) (rather than selenocysteine,
FwuB) subunit. However, as tungsten was not included in the growth media of M. burtonii, and to our knowledge the organism has never been grown with tungsten included in the media, it remains unclear whether the organism has the ability to express, as a protein, the functional tungsten FD. As other tungsten containing FD are constitutively expressed (Hochheimer et al., 1996) it may be that M. burtonii produces transcripts of the tungsten FD, but does not produce the metalloprotein when tungsten is not available.
3.4.2.2 Energy conservation
Methanogenesis is an energetically poor process, therefore in order to conserve energy many methanogens utilise energy conserving mechanisms (Deppenmeier and Muller
2008). From the analysis of the HPP of M. burtonii, several membrane bound energy conserving apparatus have been identified. Similar to the work performed by Li et al.
(2006) on M. acetivorans, M. burtonii expresses a mulit-subunit Na+/H+ antiporter
(Mbur_0132 – 138) homologous to the Ma-Mrp antiporter (see Appendix A.3 for alignments). The antiporter was thought to specific for M. acetivorans, and has been related to the marine, high sodium environment of this organism (Li et al., 2006). In M. acetivorans the Mrp antiporter is predicted to conserve energy by utilising the sodium gradient across the cell membrane to export protons for use in ATP generation. Given the marine derived environment of M. burtonii, it is likely that this complex is fulfilling the same role, and is being utilised to bolster the PMF for ATP generation.
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Figure 3.3 Methanogenesis in M. burtonii. Figure legend is on following page
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Figure 3.3 legend. Abbreviations used: CODH/ACS – CO dehydrogenase/Acetyl-CoA synthase complex; Fd – ferredoxin; FdhD – Formate dehydrogenase accessory protein; Fmd – Molybdenum formylmethanofuran dehydrogenase; Fpo – F420H2 dehydrogenase; Ftr – formylmethanofuran- tetrahydrosarcinapterin formyltransferase; GDH – glutamate dehydrogenase; GLUL – glutamine synthetase; GS-GOGAT – two-step glutamine synthetase, glutamate synthase system; H4SPT – tetrahydrosarcinapterin; Hdr – CoB-CoM heterodisulfide reductase; Mch – methenyltetrahydrosarcinapterin cyclohydrolase; Mcr – methyl-coenzyme M reductase; Mer –
Coenzyme F420-dependent N(5),N(10)-methylenetetrahydrosarcinapterin reductase; MFR –
Methanofuran; MP – methanophenazine; MtbA methylamine-specific methylcobamide:CoM methyltransferase; MtbB – dimethylamine methyltransferase; MtbC – dimethylamine corrinoid protein;
Mtd – methylenetetrahydrosarcinapterin dehydrogenase; MtmB –monomethylamine methyltransferase;
MtmC – monomethylamine corrinoid protein; Mtr – Tetrahydrosarcinapterin S-methyltransferase; MttB
– trimethylamine methyltransferase; MttC – trimethylamine corrinoid protein; MttP – trimethylamine permease; RamA – methylamine:CoM methyl transfer reductive activation protein; Rnf – Rhodobacter nitrogen fixation-like complex; TMA – trimethylamine. Gene locus tags in red indicate proteins that were not identified in this study. Ellipses are representative of protein complexes. Red arrows indicate where a pathway splits and can proceed in two directions.
The HPP data also shows that M. burtonii expresses in high abundance, a single subunit proton pumping inorganic pyrophosphatase (H+-PPase, Mbur_0994), with high identity to the Mvp1 pyrophosphatase from M. mazei. Pyrophosphatases have been proposed to play two crucial roles in the metabolism of methylotrophic methanogens (Baumer et al.,
2002). H+-PPases reduce the amount of free PPi in the cytoplasm which pulls biosynthetic reactions to completion. In addition, the energy from the bonds in pyrophosphate are used, in an energy conserving manner, to transport protons against the gradient, which helps to establish and maintain the PMF used by the H+-ATPases to generate ATP (Baumer et al., 2002).
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3.4.2.2 Cell cycle control, cell division, chromosome partitioning
Very few new proteins were identified in this category (Table 3.2). As no relevant new biology could be inferred from these proteins they are not discussed in this dissertation.
Table 3.2 Cell cycle control, cell division, chromosome partitioning
Locus tag GRAVY Id'd Type TMD Annotation Mbur_0991 -0.254 2 n Putative ATPase of the PP-loop superfamily (ER3) Mbur_1684 -0.165 2 n Putative tubulin/FtsZ protein (ER4) Mbur_1942 0.001 49 p Cell division protein FtsZ (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – new identification; p
– protein also identified in previous WCE studies but either hydrophobic in nature or part of a membrane complex.
3.4.2.3 Amino acid and nucleotide transport and metabolism
Proteins from three membrane bound transporting process were identified (Table 3.3).
Subunits of glycine betaine transporters (ProU-like solute binding region Mbur_0489, and opuA solute binding and permease region Mbur_0502, 0503) were identified. The
ProU solute-binding region had high identity (57%) with the experimentally characterised solute binding subunit from Archaeoglobus fulgidus (Schiefner et al.,
2004). In A. fulgidus this solute binding protein has been shown to bind to glycine betaine, proline betaine and trimethylammonium (Schiefner et al., 2004). The expression of this protein in M. burtonii may be indicative of the cell utilising compatible solutes as cryoprotectants or osmoprotectants, or may be a strategy for high affinity methylamine uptake in periods of substrate scarcity.
The ABC transporter opuA, from M. burtonii was identified (permease and solute binding protein). This protein had sequence similarity to regions from the crystallised
D. Burg UNSW 105 glycine betaine/proline betaine solute binding protein from Bacillus subtilis (Horn et al.,
2006). It also had high identity with other proteins from other Methanosarcinaceae
(>60% identity). This transporter is putatively classed as a glycine/proline betaine transporter however, due to its only partial alignment with the crystallised protein. The exact specificity is yet to be elucidated.
Along with the glycine/proline/methylamine ABC transporters, a putative amine/amino acid permease was identified. This protein is annotated as a methylamine permease in
Methanosarcina sp.. Its specific function is uncertain.
As well as the membrane bound components, several novel non-membrane identifications were made, many of which were hydrophobic enzymes. Components were identified from the biosynthesis of: branched chain amino acids (Mbur_0709); aromatic amino acids (Mbur_2000); serine and glycine (Mbur_0935, 2385); arginine and lysine; histidine (Mbue_1331); and leucine (Mbur_1781). Components were identified that are involved in the biosynthesis of nucleotides including: purine and pyrimidine (Mbur_1877, 2179); uracil (Mbur_1985); and a phosphoribosyltransferase of unknown specificity (Mbur_1806)
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Table 3.3 Amino acid and nucleotide transport and metabolism
Locus tag GRAVY Id'd Type TMD Comment Annotation Mbur_0056 0.075 1 p Acetylglutamate kinase (EC 2.7.2.8) (ER2) Mbur_0148 0.061 6 p Dihydrodipicolinate reductase (EC 1.3.1.26) (ER2) ABC ProU-like glycine betaine transporter, solute-binding subunit Mbur_0489 -0.063 14 n membrane complex (ER2) Mbur_0502 0.919 16 nm 6 Glycine betaine ABC transporter permease protein opuAB (ER2) Mbur_0503 -0.206 33 n membrane complex Glycine betaine ABC transporter solute binding protein opuAC (ER2) Mbur_0628 -0.225 3 n Orn/DAP/Arg decarboxylase family / TabA-like (ER3) Mbur_0709 0.111 4 n Acetolactate synthase, small subunit (EC 2.2.1.6) (ER2) Mbur_0838 0.682 7 nm 11 Amino acid permease (ER4) Mbur_0935 -0.039 5 n Phosphoserine phosphatase (EC 3.1.3.3) (ER2) Mbur_1331 0.204 12 n ATP phosphoribosyltransferase (EC 2.4.2.17) (ER3) Mbur_1781 0.088 3 n 2-isopropylmalate synthase (EC 2.3.3.13) (ER2)
Mbur_2000 -0.154 10 n 3-dehydroquinate synthase (EC 4.6.1.3) (ER3) D-3-phosphoglycerate dehydrogenase (serA) (PGDH) (EC 1.1.1.95) Mbur_2385 0.114 13 p (ER2) Mbur_1806 0.048 1 n Phosphoribosyltransferase (ER3) Ribose-phosphate pyrophosphokinase (RPPK) (Phosphoribosyl Mbur_1877 0.09 2 n pyrophosphate synthetase) (PRPP synthetase) (EC 2.7.6.1) (ER2) Mbur_1985 -0.132 2 n dCMP deaminase (EC 3.5.4.12) (ER2) Amidophosphoribosyltransferase precursor (Glutamine phosphoribosylpyrophosphate amidotransferase) (purF) (EC Mbur2179 -0.125 2 n 2.4.2.14) (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d –
the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification;
nm – NIW identification with TMDs; p – protein also identified in previous WCE studies but either
hydrophobic in nature or part of a membrane complex. Comments indicate whether the protein is part of a
membrane complex or a large complex.
3.4.2.4 Carbohydrate transport and metabolism
Very few proteins were annotated to the carbohydrate transport and metabolism
category in this study (Table 3.4). This is partially due to the fact that many proteins in
this category also fall into the membrane/envelope biogenesis category (see 3.4.2.10).
The lack of new identifications in this category may also relate to the solubility of the
proteins involved in these processes (cytoplasmic and hydrophilic) and the possibility
D. Burg UNSW 107 that M. burtonii produces many as yet uncharacterised proteins for polysaccharide biosynthesis (Allen et al., 2009).
Table 3.4 Carbohydrate transport and metabolism
Locus tag GRAVY Id'd Type TMD Annotation Mbur_1503 -0.27 2 n Phosphoglucose isomerase (ER3) Fae/hps bifunctional enzyme (Includes: Formaldehyde- activating enzyme (Fae) (EC 4.3.-.-) & 3-hexulose-6- Mbur_1994 0.181 27 n phosphate synthase (HPS) (EC 4.1.2.-)) (fae-hps) (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n - NIW identification.
There was a single hydrophobic protein identification in this category; the formaldehyde activating enzyme (Fae)/3-hexulose-6-phosphate synthase (Hps) bifunctional enzyme.
The M. burtonii genome encodes three copies of Fae, two present as Fae proteins
(Mbur_0367 and Mbur_1447) and the Fae/Hps bifunctional enzyme (Mbur_1994). In many methylotrophic bacteria, Fae catalyses the condensation of formaldehyde and tetrahydromethanopterin (H4MPT) to form methylene-H4MPT, detoxifying formaldehyde produced by methanol dehydrogenase (Goenrich et al., 2005).
Methanogens do not utilise a methanol dehydrogenase, but utilise methyltransferases and proportional dismutation; processes that produce no free formaldehyde (Welander and Metcalf 2008). It is unlikely that the Fae enzyme is fulfilling this role in the utilisation of C1 compounds by M. burtonii. Methanosarcina barkeri expresses both
Fae and Fae/Hps when grown on methanol, and the genomes of other Methanosarcina sp. code for the Fae and the Fae/Hps enzymes (Goenrich et al., 2005). Conversely, M. burtonii has been found to express only the bifunctional enzyme when grown on TMA
(this study). The bifunctional enzyme is involved in ribulose-5-phosphate synthesis
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from fructose-6-phosphate. When hexulose-6-phosphate is converted to ribulose-5- phosphate in the pathway, formaldehyde is produced, the Fae portion of the enzyme then combines this with H4SPT producing Methylene-H4SPT, effectively detoxifying the formaldehyde. It is possible that this enzyme is playing the same role in M. burtonii.
As with M. barkeri, the M. burtonii genome does not encode NAD(P) dependant glucose-6-phosphate dehydrogenase, 6-phospogluconate lactonase, transketolase, transaldolase, and ribulose-5-phosphate epimerase. But does however, contain the proteins thought to be involved in the production of ribulose-5-phosphate in M. barkeri;
D-Ribose-5-phosphate isomerase (Mbur_0773), 6-phospho-3-hexuloisomerase
(Mbur_1899), Fructose 1-6-bisphosphatase (Mbur_1354), and class II fructose- bisphosphate aldolase family protein (Mbur_1969). It is therefore, probable that this pathway occurs in M. burtonii. However, only Mbur_1969 has been identified in proteomic studies.
3.4.2.5 Coenzyme transport and metabolism
A number of enzymes involved in the biosynthesis and transport of Co-enzymes, were identified as NIW or hydrophobic in this study (Table 3.5).
The enzymes involved in the pathways of methanogenic coenzyme biosynthesis are poorly characterised (reviewed in White (2001)). However, several enzymes have been characterised in M. jannaschii and other methanogenic archaea that are involved in the biosynthesis of: F420 (Graupner and White 2001; Graupner et al., 2002a, b; Graham et al., 2003; Graupner and White 2003; Li et al., 2003a; Forouhar et al., 2008); coenzyme
B (Howell et al., 2000); coenzyme M (Graupner et al., 2000; Graham et al., 2001;
Graham et al., 2002); and methanopterin/sarcinapterin (Rasche and White 1998; Scott and Rasche 2002). The genome of M. burtonii codes for homologues of these enzymes;
D. Burg UNSW 109 with the exception of the ComA, ComB, and ComC enzymes in CoM biosynthesis, and the AskA enzyme in CoB biosynthesis. Of the known enzymes, only an AskD homologue (CoB biosynthesis) (Mbur_2263, 3-isopropylmalate dehydratase, large subunit), was previously shown to be expressed in M. burtonii (Goodchild et al.,
2004a). In this HPP study the hydrophobic Coenzyme F420 gamma-glutamyl ligase
(Mbur_1849), and the 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) synthase (F420 biosynthesis) subunits (Mbur_1303, 1304) were identified.
Table 3.5 Coenzyme transport and metabolism
Locus tag GRAVY Id'd Type TMD Annotation Pyridoxal 5'-phosphate (Vitamin B6) synthase, lyase subunit Mbur_0421 0.017 15 p pdxS (ER2) 6,7-dimethyl-8-ribityllumazine synthase (ribH) (EC 2.5.1.9) Mbur_0429 0.233 26 p (ER3) Mbur_0515 -0.197 6 n Riboflavin synthase (ER2) NifH class IV protein homolog (putative carbon monoxide dehydrogenase/acetyl-CoA synthase complex, nickel-inserting Mbur_0861 -0.317 1 n subunit) (ER3) Mbur_0940 0.157 1 nm 8 UbiA prenyltransferase-family protein (ER4) Mbur_1035 0.157 3 p Sirohydrochlorin cobaltochelatase (CbiXS) (EC 4.99.1.3) (ER2) Mbur_1303 -0.183 3 n FO synthase (ER3) Mbur_1304 -0.204 2 n FO Synthase (ER3) Mbur_1387 -0.099 9 nm 1 Thiamine biosynthesis lipoprotein apbE (apbE) (ER3) Mbur_1700 0.98 15 nm 6 CbiM-like protein (ER3) Mbur_1793 1.048 13 nm 6 CbiM-like protein (ER3) Mbur_1815 0.06 1 n Dihydropteroate synthase (DHPS) (folP) (EC 2.5.1.15) (ER3) Mbur_1849 0.155 2 n Coenzyme F420-0 gamma-glutamyl ligase (EC 6.3.2.-) (ER2) Adenosylcobinamide-phosphate synthase (cbiB) (EC 6.3.1.10) Mbur_2090 0.643 9 nm 6 (ER2) Alpha-ribazole-phosphate phosphatase (cobZ) (EC 3.1.3.73) Mbur_2091 -0.252 3 n (ER2) Mbur_2092 0.961 12 nm 7 Cobalamin-5'-phosphate synthase CobS (EC 2.7.8.26) (ER3)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification; nm – NIW identification with TMDs; p – protein also identified in previous WCE studies but either hydrophobic in nature or part of a membrane complex.
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Several other enzymes possibly involved in methanogenic coenzyme biosynthesis have been identified. Including a dihidropteroate synthase like protein (ER3) (Mbur_1815).
In bacteria dihidropteroate synthase catalyses the condensation of 6-hydroxymethyl-7,8- dihydropteridine pyrophosphate with para-aminobenzoic acid to form 7,8- dihydropteroate in folate biosynthesis. In the biosynthesis of methanopterin, condensation of 6-hydroxymethyl-7,8-dihydropterin pyrophosphate occurs with 4-(-D- ribofuranosyl) aminobenzene 5'-phosphate (RFA-P) (White 2001), in an analogous step.
At the time of White’s review (2001), no archaeal analogues of dihidropteroate synthase were identified. The M. burtonii dihidropteroate synthase-like protein has several high identity homologues (on BLAST search) among the Methanosarcinaceae, M. barkeri
(63%), M. mazei (62%), M. acetivorans (62%), also to Methanosaeta thermophila
(41%) and to a korarchaeon Candidatus Korarchaeum cryptofilum (35%). The dihidropteroate synthase identified may be involved in the biosynthesis of sarcinapterin/methanopterin.
Two proteins possibly involved in factor III biosynthesis were also identified. Factor III is a cofactor of methyltransferases and has a similar structure to vitamin B12
(cobalamin) (Ferry and Kastead 2007). Two putative membrane spanning cobalamin biosynthesis-like proteins (CbiM-like) (Mbur_1700, 1793) were identified, which on
BLAST, have highest similarities to Methanosarcina spp.. CbiM has no known function in bacteria and eukarya (Raux et al., 2000) and the genes for these proteins in M. burtonii are separated from the rest of the cobalamin biosynthesis proteins. For example, three proteins that were identified in this study (CbiB - Mbur 2090, CobZ -
Mbur_2091, and CobS - Mbur_2092) are organised in a putative operon with other cobalamin biosynthesis proteins. The CbiM-like proteins however, are both situated
D. Burg UNSW 111 separate from the above proteins in the genome and are next to two predicted cobalt
ABC transporters (Mbur_1697 – 1699, and Mbur_1794 – 1795). It is possible that these proteins are factor III biosynthetic enzymes.
3.4.2.6 Translation, ribosomal structure, and biogenesis
In the course of analysing the HPP of M. burtonii, a large number of ribosomal and ribosome associated proteins were identified (Table 3.6). There are two reasons why this has occurred. Firstly ribosomes are dense and are concentrated with the hydrophobic pellet on ultra-centrifugation. Secondly, ribosomes associate with the membrane in archaea at SecYE based sites (Ring and Eichler 2004b), mediated by the signal recognition particles (SRP) (Egea et al., 2008a), during secretion of proteins and insertion of proteins into the membrane. These two factors led to the concentration of the ribosomes in the hydrophobic samples, resulting in a high number of identifications.
Along with the structural ribosomal proteins, many proteins that are transiently associated with the ribosome were identified (e.g. tRNA synthetases). Two DEAD box helicases were also identified, one of which was discussed in 2.4.4, and will be further discussed in chapters 4 and 5.
A SelB-like protein was also identified in HPP extracts. M. burtonii does not incorporate selenocysteine into proteins, unlike several other methanogens (Wilting et al., 1997; Rother et al., 2003; Leibundgut et al., 2005). This is surprising from an evolutionary perspective, as selenocysteine forms highly reactive centres of proteins
(much more so than cysteine) and could provide an advantage to the cell under kinetically unfavourable cold conditions. The exact function of this protein in M. burtonii is unclear.
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Table 3.6 Translation, ribosomal structure and biogenesis
Annotation Locus tag GRAVY Id'd Type TMD Comment
Mbur_0005 0.121 14 n Complex SSU ribosomal protein S19P (rps19p) (ER2) Mbur_0006 -0.209 14 n Complex LSU ribosomal protein L22P (rpl22p) (ER2) Mbur_0007 -0.292 12 n Complex SSU ribosomal protein S3P (rps3p) (ER2) Mbur_0010 0.022 1 n Complex SSU ribosomal protein S17P (rps17p) (ER2) Mbur_0014 -0.272 5 n Complex LSU ribosomal protein L5P (rpl5p) (ER2) Mbur_0016 -0.151 5 n Complex SSU ribosomal protein S8P (rps8p) (ER2) Mbur_0187 0.154 2 n Complex RNA-binding protein, containing PUA domain (ER3) Mbur_0192 -1.08 57 n Complex LSU ribosomal protein L15E (rpl15e) (ER2) Archaeal exosome complex RNA-binding protein 4 (rrp4) (EC Mbur_0198 -0.351 11 n Complex 3.1.13.-) (ER2) Mbur_0245 -0.551 38 n DEAD-box RNA helicase (ER2) Glutamyl-tRNA (Gln) amidotransferase subunit E (EC 6.3.5.7) Mbur_0391 -0.327 5 n Complex (ER2) Phenylalanyl-tRNA synthetase, beta subunit (EC 6.1.1.20) Mbur_0447 -0.093 1 n Complex (ER2) Mbur_0456 -0.642 57 n Complex Translation initiation factor aIF-1A (ER2) Mbur_0772 -0.288 2 n Complex Aspartyl-tRNA synthetase (EC 6.1.1.12) (ER2) Mbur_0923 -0.379 2 n Complex O-phosphoseryl-tRNA(Cys) synthetase (EC 6.1.1.-) (ER2) Mbur_0971 0.072 2 n Diphthine synthase (EC 2.1.1.98) (ER2) Threonyl/alanyl tRNA synthetase second additional domain, Mbur_1041 -0.471 6 n Complex SAD (ER3) Mbur_1175 0.517 7 p Complex LSU ribosomal protein L30E (rpl30e) (ER2) Ribosomal RNA large subunit methyltransferase RrmJ/FtsJ (EC Mbur_1190 -0.368 2 n Complex 2.1.1.-) (ER2) Mbur_1281 -0.492 5 n Complex Pseudouridine synthase (ER3) Mbur_1396 -0.375 35 n Complex Translation initiation factor a/eIF-2 alpha subunit (ER2) Mbur_1397 -1.045 1 n Complex Ribosome biogenesis protein Nop10 (Nop10p) (ER2) Mbur_1423 -0.33 26 n Complex Translation initiation factor a/eIF-2 beta subunit (ER2) Mbur_1562 -0.267 2 n Complex Pre-mRNA processing ribonucleoprotein (ER3) Mbur_1738 -0.378 15 n Complex Peptide chain release factor eRF/aRF, subunit 1 (ER2) Mbur_1757 0.044 2 n Complex Translation elongation factor SELB (SelB) (ER3) Phosphoadenosine phosphosulfate reductase fused to RNA- Mbur_1929 -0.232 2 n binding PUA and 4Fe-4S binding domains (ER4) Mbur_1950 -0.601 57 n DEAD-box RNA helicase (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d –
the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification; p
– protein also identified in previous WCE studies but either hydrophobic in nature or part of a membrane
complex. Comments indicate whether the protein is associated with a large complex, in the case of the
proteins in the table above, the ribosome.
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3.4.2.7 Lipid transport and metabolism
One of the distinguishing features of the archaea is their unique lipids, which have a glycerol ether configuration rather than the glycerol ester configuration found in bacteria and eukarya (Koga et al., 1993). NIW proteins that are involved in the synthesis of these lipids are displayed in Table 3.7.
M. burtonii produces two major types of phospholipids: archaeol phosphatidylglycerol; and archaeol phosphatidylinositol, which have higher degrees of unsaturation at lower temperatures brought about by selective retention of double bonds, mediated by a plant- like geranylgeranyl reductase (Nichols et al., 2004).
A method of lipid biosynthesis using a modified mevalonate pathway, similar to that found in M. jannaschii (Grochowski et al., 2006), was proposed for M. burtonii, with previous proteomic analyses identifying several components despite the membrane bound nature of the lipid biosynthetic apparatus (Goodchild et al., 2004a; Nichols et al.,
2004; Allen et al., 2009). All components from this proposed pathway identified in
WCE analyses (bar one) were found in the HPP analysis, and several further proteins were identified including two integral membrane components (Mbur_2209 and 2406)
(Figure 3.4).
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Acetyl-CoA
Acetoacetyl-CoA thiolase Mbur_1933 (ER3)
Acetoacetyl-CoA + Acetyl-CoA
HMG-CoA synthase Mbur_1932 (ER3)
HMG-CoA HMG-CoA reductase Mbur_1098 (ER2)
Mevalonate
Mevalonate kinase Mbur_2395 (ER3)
Mevalonate-P Mevalonate phosphate decarboxylase Mbur_2394 (ER4) Isopentenyl-P
Isopentenyl phosphate kinase Mbur_2396 (ER2) DHAP
IPP G-1-P dehydrogenase Mbur_2397 (ER2) IPP isomerase Mbur_1032 (ER2) DMAPP GPP FPP GGPP + sn-Glycerol-1-P
GGPP synthase FPP synthase GGPP synthase GGGP synthase Mbur_1652 (ER3)
Mbur_1235 (ER2) Mbur_2399 (ER3) Mbur_1235 (ER2) GGGP
Mbur_2381 (ER2) Myo-inositol-1 phosphate synthase DGGGP synthase Mbur_1679 (ER3)
D-glucose 6-phosphate myo-inositol DGGGP Glycerol-3-P Mbur_2209 (ER4) Mbur_2406 (ER4) CDP-DGGGP cytidyltransferase CDP-glycerol transferase Phosphatidylinositol Phosphatidylglycerol
Geranylgeranyl reductase Mbur_1077 (ER3) Archaetidic acid
Figure 3.4 The mevalonate pathway in M. burtonii. Enzyme names in grey boxes are those identified in
both this study and WCE analyses. Black boxes represent NIW proteins. Grey hatched boxes represent
proteins that were not identified in this work but were identified in WCE proteomics. Mbur_2406 and
2209 are integral membrane components of this pathway and were identified in this study. Abbreviations
used: 3-hydroxy-3-methylglutaryl CoA (HMG-CoA); phosphate (P); isopentyl diphosphate (IPP);
dimethylallyl diphosphate (DMAPP); geranyl diphosphate (GPP); farnesyl diphosphate (FPP);
geranylgeranyl diphosphate (GGPP); geranylgeranylglyceryl phosphate (GGGP);
digeranylgeranylglyceryl phosphate (DGGGP). Figure was taken and modified, with permission, from
(Allen et al., 2009).
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Table 3.7 Lipid transport and metabolism
Comment Locus tag GRAVY Id'd Type TMD Annotation Mbur_1933 0.059 1 p complex Acetoacetyl-CoA thiolase (EC 2.3.1.9) (ER3) Mbur_2209 0.737 14 nm 4 complex CDP-diacylglycerol alcohol/inositol phosphatidyltransferase (ER4) Mbur_2397 0.079 13 n complex Isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2) (ER2) CTP:2,3-di-O-geranylgeranyl-sn-glycero-1-phosphate Mbur_2406 0.745 2 nm 6 complex cytidyltransferase (EC 2.7.7.-) (ER3)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification; nm – NIW identification with TMDs; p – protein also identified in previous WCE studies but either hydrophobic in nature or part of a membrane complex.
3.4.2.8 Transcription
A eukaryote-like RNA polymerase complex, including promoter elements and transcription factors, mediates archaeal transcription (Werner 2007). Regulation of this transcription however, tends to be more bacterial in nature, mediated by repressor proteins which bind promoter elements preventing the attachment of the TATA binding protein (TBP) and Transcription factor B (TFB). The bacteria-like regulation of a eukayotic-like apparatus is not the sole type of regulation in the archaea, with several eukaryotic-like positive regulators also identified (Bell 2005; Geiduschek and
Ouhammouch 2005). The knowledge of archaeal transcriptional regulation remains poorly understood. In previous WCE studies of M. burtonii, many components of the transcription apparatus were identified, including several transcriptional regulators. The current work on the hydrophobic fraction was able to identify NIW members of this machinery (Table 3.8). The promoter element TFB (Mbur_0963) and the bacteria-like terminator NusA (Mbur_1174) were identified in this study.
Several regulatory elements were also identified. An iron dependant repressor protein was identified (Mbur_0776), located in the genome adjacent to ferrous iron uptake
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proteins, and possibly involved in iron homeostasis or uptake regulation (3.4.2.12). Two
members of the ArsR family of regulatory proteins were also identified (Mbur_0946,
1148). In bacteria, these winged helix DNA binding proteins are involved in protection
against heavy metals by repressing metal resistance genes in times of low metal
concentration, and conversely detaching from the promoter elements of these genes
during times of metal stress (Xu and Rosen 1997; Busenlehner et al., 2003; Pennella
and Giedroc 2005). Similar functions, in terms of metal resistance, have been found in
archaea (Gihring et al., 2003; Schelert et al., 2006; Baker-Austin et al., 2007). Members
of the ArsR family however, have also been shown to be involved in thermal stress
induced gene regulation as well as having proposed chaperone accessory functions (Liu
et al., 2007; Itou et al., 2008). These proteins may play a similar role in M. burtonii.
Table 3.8 Transcription
Annotation Locus tag GRAVY Id'd Type TMD Comment Mbur_0039 0.143 14 p complex DNA-directed RNA polymerase subunit D, rpoD (ER2) Mbur_0776 -0.571 1 n Iron dependent repressor (ER3) Mbur_0946 -0.612 2 n Transcriptional regulator (ArsR family) (ER3) Mbur_0963 -0.496 46 n complex Transcription initiation factor B (TFB) (ER2) Mbur_1148 -0.125 5 nm 3 Transcriptional regulator (ArsR family) (ER4) Mbur_1174 -0.029 2 n complex Transcription termination factor NusA (ER3) Mbur_1176 0.033 15 p complex DNA-directed RNA polymerase subunit A, rpoA2 (ER2) Mbur_1439 0.027 4 p complex DNA-directed RNA polymerase subunit L, rpoL (ER2) Mbur_1496 0.098 20 p TATA-box binding protein (ER3) Helix-turn-helix (HTH) 3-containing transcriptional regulator, XRE Mbur_2130 0.046 4 p family (ER4)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification; nm – NIW identification
with TMDs; p – protein also identified in previous WCE studies but either hydrophobic in nature or part of a
membrane complex. Comments indicate whether the protein is associated with a large complex (RNAP)
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3.4.2.9 Replication, recombination, and repair
Many novel proteins were identified in the replication, recombination and repair category (Table 3.9). Such proteins tend to be hydrophilic but associate in large complexes or with hydrophobic chromatin, and hence concentrate during centrifugation.
Notable identifications included several proteins involved in DNA recombination and repair (excinuclease, Mbur_1158, 1467; a RecJ-like phosphoesterase, Mbur_2340; and
ATP dependant DNA ligase, Mbur_1088), and a eukaryotic-like minichromosome maintenance protein (MCM, Mbur_2432) involved in control of cell division. Several proteins previously annotated as KaiC (circadian clock) proteins were also found.
The KaiC proteins have been recently been characterised as RadA family proteins in archaea (Haldenby et al., 2009), which are involved in recombination and repair. Based on this assignment Mbur_1691 is now classified aRadC, with exact function unknown.
Along with these proteins, 3 transposases (involved in genome rearrangement) were identified. The M. burtonii genome encodes a large number of transposases, a fact that distinguishes it from other archaea (Allen et al., 2009). Transposases have been implicated in cold sensitivity in Photobacterium profundum (Lauro et al., 2008), and have been identified as over represented in deep oceanic metagenomic samples
(DeLong et al., 2006). The fact that these transposases are expressed indicates an actively evolving genome in M. burtonii.
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Table 3.9 Replication, recombination and repair
Locus tag GRAVY Id'd Type TMD Comment Annotation Mbur_0095 -0.442 10 n complex DNA topoisomerase (ER3) Mbur_0162 -0.598 3 n Transposase (ER4) Mbur_0419 -0.353 12 n complex DNA gyrase subunit B (ER2) Mbur_1088 -0.122 2 n DNA ligase 1, ATP-dependent (dnl1) (ER2) Mbur_0758 -0.311 7 n RadA family protein (ER4) Mbur_0822 -0.204 2 n RadA family protein (ER4) Mbur_1158 -0.282 2 n complex Excinuclease ABC, subunit A (ER2) Mbur_1377 -0.146 22 n complex Replication factor C small subunit (ER3) Mbur_1392 -0.392 2 n complex DNA primase, small subunit (ER3) Mbur_1466 -0.48 3 n Putative exonuclease (ER4) Mbur_1467 -0.413 3 n complex Excinuclease ABC, C subunit (ER2) Mbur_1688 -0.344 4 n complex DNA polymerase B, delta subunit with exonuclease activity (ER3) Mbur_1691 -0.305 16 n aRadC protein (ER3) Mbur_1733 -0.173 5 n complex ORC complex protein Cdc6/Orc1(ER3) Mbur_1755 -0.048 2 n RadA family protein (ER4) Mbur_2095 0.012 13 n RadA family protein (ER4) Mbur_2242 -0.571 4 n complex Replication factor C large subunit (ER3) Mbur_2248 -0.428 1 n Transposase (ER4) Mbur_2297 -0.571 14 n Transposase (ER4) Single-stranded DNA-specific exonuclease (RecJ)-like Mbur_2340 0.026 2 n phosphoesterase (ER3) Mbur_2432 -0.381 1 n complex MCM (minichromosome maintenance protein) (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d –
the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification.
Comments indicate whether the protein is associated with a large complex.
3.4.2.10 Cell wall/membrane/envelope biogenesis, and motility
Most archaeal species do not synthesise classical cell walls (Konig et al., 2007), many
produce only a glycoprotein S-layer (Engelhardt 2007). Among the archaea, the
Methanosarcina spp. produce an outer cell wall of methanochondroitin as well as an S-
layer (Sowers et al., 1993). It is not known, whether M. burtonii produces a
methanochondriotin outer layer as well as an S-layer, and the exact composition of the
S-layer is not known. Through the analysis of the HPP of M. burtonii in this project,
several clues have been uncovered in relation to: the sugars involved in the S-layer
D. Burg UNSW 119 composition of the organism; possible indicators of a methanochondriotin-like cell wall; and a gene arrangement suggesting an extensive array of putative novel processes in relation to glycosylation and EPS. Novel, hydrophobic, and membrane identifications are shown in Table 3.10.
M. burtonii is a motile organism utilising a flagellum. As glycosylation of the flagella occurs in a similar manner to the surface sugars in archaea, and occurs as a surface structure, this apparatus will also be discussed in this section.
The archaea possess more diversity in linking sugars and glycan composition for post- translational modification than any other domain of life (Eichler and Adams 2005), where diversity in sugars can occur in an antigen like manner within a single species
(Mayerhofer et al., 1998). Archaeal S-layer and flagella proteins undergo N-linked glycosylation prior to insertion into the array, in a bacteria-like process that remains poorly understood, although recent advances have increased the knowledge of this process in several species (Yurist-Doutsch et al., 2008).
In the archaea UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetyl-D- mannosamine (UDP-ManNAc), are known components of glycoprotein glycans involved in S-layers and flagella (Voisin et al., 2005). The bacteria of the genus’
Geobacillus and Aneurinibacillus are known to produce S-layers containing rhamnose, which is utilised via the molecule dTDP-L-rhamnose (Graninger et al., 2002; Novotny et al., 2004; Steiner et al., 2008). Proteins and processes involved in the biosynthesis and N-linked glycosylation of these sugar entities (glucose, mannose, and rhamnose) were identified in this study and in the M. burtonii genome (Figures 3.5 and 3.6). The
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inclusion of rhamnose in Geobacillus stearothermophilus S-layer is growth temperature
dependant, a phemomenon which may be mirrored in M. Burtonii.
Table 3.10 Cell wall/membrane/envelope biogenesis
Locus tag GRAVY Id'd Type TMD Annotation Mbur_0049 -0.201 19 m 1 UDP-glucose/GDP-mannose dehydrogenase (ER3) Mbur_0124 0.838 17 nm 9 Glycosyl transferase, family 4 (AlgH-like) (ER3) Mbur_0725 0.031 5 n Glycosyl transferase, group 1 (ER3) Protein containing Mur ligase middle domain-like central region Mbur_1036 -0.021 3 n (ER4) Mbur_1495 0.115 4 nm 2 Glycosyl transferase, family 2 (ER4) Mbur_1581 -0.104 2 n Glycosyl transferase, group I (ER3) Mbur_1585 -0.337 2 n N-acylneuraminate cytidylyltransferase protein (ER2) Mbur_1607 -0.251 7 n Glycosyl transferase, family 2 (ER4) Mbur_1608 0.195 32 nm 4 Glycosyl transferase, family 2 (ER4) Dolichyl-phosphate beta-D-mannosyltransferase like protein with Mbur_1615 0.126 5 nm GtrA-like C-terminal domain (ER3) Mbur_2029 -0.141 10 n UDP-N-acetyl-D-mannosaminuronate dehydrogenase (ER3) Mbur_2231 -0.403 2 n dTDP-4-dehydrorhamnose reductase (EC:1.1.1.133) (ER2) Mbur_2234 -0.329 5 n UDP-glucoronic acid decarboxylase (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d –
the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification;
nm – NIW identification with TMDs.
Mbur_2230 Mbur_2233
dTDP-4- Glucose-1-phosphate dehydrorhamnose 3,5- thymidylyltransferase epimerase dTDP-6-deoxy-D- dTDP-6-deoxy-L- dTDP-L- D-Glucose-1-phosphate dTDP-D-Glc xylo-4-hexulose xylo-4-hexulose rhamnose
dTDP-glucose dTDP-4- 4,6-dehydratase dehydrorhamnose reductase Mbur_2232 Mbur_2231
Figure 3.5 dTDP-L-rhamnose synthesis in M. burtonii. dTDP-L-rhamnose appears to be synthesised
in a manner similar to the thermophilic Gram positive bacteria Aneurinibacillus thermoaerophilus
(Graninger et al., 2002). Boxes indicate enzymes involved, grey boxes indicate proteins identified in this
study.
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UDP-ManNAc Mbur_2029 UDP-N-acetyl-D- mannosaminuronate dehydrogenase
Mbur_2343 Mbur_2341 UDP-N-acetylglucosamine 2- glucosamine-fructose-6- epimerase phosphate aminotransferase Bifunctional UDP-GlcNAc pyrophosphorylase (GlmU) Mbur_2022 Glucosamine-fructose-6-phosphate α-D-glucosamine-1-phosphate UDP-GlcNAc
phosphoglucosamine mutase N-acetylglucosamine-1-phosphate uridyltransferase ????? Mbur_2342 Mbur_2344
Figure 3.6 UDP-GlcNAc and UDP-ManNAc synthesis in M. burtonii. UDP-GlcNAc and UDP-
MaNAc appear to be synthesised in a similar manner to M. maripaludis and M. Jannaschii (Yurist-
Doutsch et al., 2008). Boxes indicate enzymes involved, grey boxes indicate proteins identified in this
study.
Several aspects of the process of N-linked glycosylation have been shown for Haloferax
volcanii (Abu-Qarn and Eichler 2006; Abu-Qarn et al., 2007; Abu-Qarn et al., 2008)
and Methanococcus voltae (Voisin et al., 2005; Chaban et al., 2006; Shams-Eldin et al.,
2008; Chaban et al., 2009). The M. burtonii genome encodes distant homologs of
several of the enzymes involved, in a mosaic of both species, and several were
identified in this study of the HPP. From H. volcanii, AglF (Mbur_2431), AglD
(Mbur_2237), and AglB (Mbur_1579), and from M. voltae AglH (Mbur_0124) and
AglK (Mbur_2018). As the composition of the S-layer oligosaccharides in M. burtonii
is not known, the specificity and order in which these enzymes act is uncertain.
The M. burtonii genome encodes for many more polysaccharide biosynthesis genes than
its mesophilic methanogenic counterparts (3.3% of genome compared to only 0.6% in
M. acetivorans) (Allen et al., 2009). This could be reflective of the organism’s capacity
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to produce EPS (Chapter 5) and a variety of glycosylations, which have been implicated in thermal adaptation (Chapter 4). The proteins involved in polysaccharide biosynthesis appear to be organised into 5 loci (Appendix A.4a-e), with only a small number predicted to be involved in this process found elsewhere. These loci contain many hypothetical proteins, with several identified in this HPP analysis, some of which are unique, as well as members of several families of glycosyltransferases and characterised enzymes. A large number of these proteins were identified as expressed in the cell and have also been identified in WCE proteomics and secretome investigations. The combination of these data indicates an active and extensive potential for polysaccharide biosynthesis in M. burtonii for S-layer, EPS and cell wall biosynthesis.
The cell walls of Methanosarcina spp. are made of methanochondroitin, a polymer of glucuronic and galacturonic acids and N-acetyl-D-galactosamine that resembles the eukaryotic chondriotin (Hartmann and Konig 1991; Sowers et al., 1993). The biosynthesis of this heteropolysaccharide is poorly characterised. Several potentially relevant proteins were identified as expressed in the HPP analysis. These proteins include a UDP-glucuronic acid decarboxylase and a protein containing a Mur ligase-like region (bacterial cell wall biosynthesis) (Smith 2006).
As flagella are highly abundant in proteomic samples, many of the proteins involved in the flagella complex and in the regulation of chemotaxis were identified in WCE studies despite their membrane localisation (Goodchild et al., 2004a). Several of these flagella proteins were identified in this study, as well as components of the Che chemotaxis system and flagella elements (Table 3.11).
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Table 3.11 Cell motility
Annotation Locus tag GRAVY Id'd Type TMD Comment Mbur_0104 0.105 57 m 1 Flagellin (ER2) Mbur_0346 0.188 9 m 1 Flagellin (ER2) Mbur_0347 0.087 9 m 1 Flagellin (ER3) Mbur_0359 0.075 3 p Chemotaxis (CheY) protein (ER2) Mbur_0361 -0.323 3 n Chemotaxis protein CheA (ER2) Mbur_0878 -0.087 3 n CheY-like response regulator receiver (ER3) Mbur_1570 -0.528 2 n membrane complex Putative archaeal flagellar protein D/E (ER3)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in; Type: n – NIW identification; m
– protein also identified in WCE studies but containing TMD; p – protein also identified in previous
WCE studies but either hydrophobic in nature or part of a membrane complex. Comments indicate whether the protein is part of a membrane complex.
3.4.2.11 Post translational modification, protein turnover, chaperones
Many chaperones and other proteins related to polypeptide folding were identified in
WCE proteomics of M. burtonii, and were also identified in this study. This is reflective of their relative cellular abundance and importance to the cell. However, through the analysis of the HPP, many membrane-bound proteins that play important roles in protein folding, turnover and cellular protection were also identified. These proteins include proteases, protease inhibitors, and cis-trans isomerases. Many of the proteins are homologues to characterised enzymes with dual or multiple functions, for example, acting as proteases as well as chaperones. The NIW and hydrophobic identifications are shown in Table 3.12.
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Table 3.12 Posttranslational modification, protein turnover, chaperones
Locus tag GRAVY Id'd Type TMD Annotation Mbur_0529 -0.238 7 secn sec Family I4 proteinase inhibitor, serpin (ER3) Zn-dependent protease (M48 family) with putative chaperone (HtpX) Mbur_0716 0.498 14 nm 3 function (ER4) Mbur_0880 -0.147 57 m 1 Lon family peptidase with N terminal transmembrane domain (ER3) Mbur_1082 0.193 19 nm 4 Peptidase, M48 family (ER4) Mbur_1185 0.028 57 nm 1 SPFH domain / Band 7 family-like protein (ER4) Mbur_1212 -0.814 15 n Atypical type III J-domain family protein (ER3) Mbur_1255 0.415 14 nm 4 Peptidase, M48 family (ER4) Mbur_1707 -0.079 4 secn Protease inhibitor, alpha(2)-macroglobulin -like protein (ER3) Mbur_1742 -0.309 19 secn sec Putative secreted proteinase inhibitor I4 (ER3) Mbur_1804 0.211 51 p Putative stomatin, band 7 family protein (ER2) FKBP-type peptidyl-prolyl cis-trans isomerase with putative C- Mbur_2255 -0.186 8 n terminal chaperone domain (ER2) FKBP-type peptidyl-prolyl cis-trans isomerase with putative C- Mbur_2256 -0.05 35 n terminal chaperone domain (ER2) Proteasome-activating nucleotidase (Proteasome regulatory subunit) Mbur_2301 -0.161 7 n (pan) (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification; nm – NIW identification with TMDs; m – protein also identified in WCE studies but containing TMD; p
– protein also identified in previous WCE studies but either hydrophobic in nature or part of a membrane complex; secn – NIW identifications that contains a signal peptide.
M. burtonii expresses three M48 family metalloproteases with homology to the heat shock induced HtpX protein of E. coli (see Appendix A.5 for alignments). This membrane bound protein is involved in the thermal stress response where it is thought to act to control the quality of membrane proteins in conjunction with other regulatory elements (Kornitzer et al., 1991; Shimohata et al., 2002; Sakoh et al., 2005). In
Streptococcus gondii, a HtpX-like protein has been shown to not be involved in a heat shock response but is involved in the surface structures of the organism, as mutants lacking the gene for this protein displayed changes in adhesiveness, morphology and expression of surface antigens (Vickerman et al., 2002). Due to the difficulties in
D. Burg UNSW 125 purifying and expressing these proteins (due to their toxic nature to the expression system), the elucidation of their exact function has not been established. However several recent advances have allowed for the expression, purification and preliminary characterisation of HtpX and HtpX-like proteins (Sakoh et al., 2005; Siddiqui et al.,
2007).
Of the three proteins expressed by M. burtonii two (Mbur_1082, 1255) match well with the E. coli HtpX protein in terms of both sequence and predicted membrane topology
(Figure 3.7), and may have similar roles in M. burtonii. The third protein
(Mbur_0716), also aligned well with the HtpX and HtpX-like proteins. However, there were several crucial differences. The M48 family of proteases is characterised by the
HExxH motif, which forms part of the zinc binding active site (Rawlings and Barret
1995). Mbur_0716 contains an arginine in the first histidine position of this motif. The guanidinium group in arginine cannot complex any metal as it retains a delocalised positive charge, even at high pH (Sohail Siddiqui, personal communication), so it is unlikely that this site remains active unless a novel active center is formed with an unknown molecule. Mbur_0716 is also predicted to have a different membrane topology to the other characterised HtpX and HtpX-like proteins with the majority of this protein predicted to be on the outside face of the cell membrane. On inspection of the genome context of this protein, it was observed that the orientation is immediately upstream of polysaccharide locus 1 (Figure 2.32a). This, taken with the unusual membrane topology and the function of the HtpX-like protein of S. gordonii, suggests that Mbur_0716 plays a role in the surface morphology of M. burtonii, or EPS production, degradation, or modification in a role similar to S. gondii.
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a b
c Figure 3.7 predicted membrane topology of HtpX-like
proteins. Legend is below left, where TMS abbreviated
transmembrane segment. Catalytic domains are bordered
by red boxes. The predicted membrane topology of the E.
Coli HtpX (a) and Mbur_1082 (b) are very similar. A similar topology is predicted for other Mbur_1255 and
other HtpX-like proteins. Mbur_0716 (c) however, differs
in predicted topology with the majority of the protein
predicted to be on the outside face of the cell membrane
(below membrane representation)
M. burtonii also expresses a Lon family peptidase (Mbur_0880) with a short N-
terminal region that is characteristic of the LonB family (Rotanova et al., 2006), and a
single transmembrane domain. The catalytic domain of this protein aligns well with the
type II Lon catalytic domains (proposed Ser-Lys-Asp triad) as described in (Maupin-
Furlow et al., 2005b) (See Appendix A.6 for alignments). The entire protein has high
identity (61%) to the characterised membrane bound Lon protease from Thermococcus
kodakarensis KOD-1 (See Appendix A.7 for alignment). The T. kodakarensis Lon
D. Burg UNSW 127 protease displays ATP independent hydrolysis of unfolded proteins. However, for proteins with secondary structure the ATPase acts in a chaperone like manner, unfolding proteins prior to degradation (Fukui et al., 2002). The M. burtonii Lon protease could behave in a similar manner.
HPP analysis has also revealed two proteins of the SPFH (stromatin, prohibitin, flotillin,
HflK) family, one of which is predicted to be hydrophobic and may be membrane associated, as is the case with most proteins of this family (Tavernarakis et al., 1999).
The exact function of this protein is unclear, as the SPFH family contains a diverse range of proteins with an equally diverse range of functions. The other protein identified in this family was a stromatin homologue with high identity to eukaryotic stromatin and the structurally characterised stromatin from Pyrococcus horikoshii
(Yokoyama et al., 2008) (See Appendix A.8 for alignment). Stromatins play a role in eukaryotic membrane physiology, where they are thought to regulate ion conductance, be involved in mechano-sensation, have proteolytic functions, and are possibly involved in cell growth. The function of these proteins in archaea is as yet unknown (Yokoyama et al., 2008).
The archaeal proteasome, which undergoes complicated regulation of changes in structure and function (Reuter et al., 2004), is an essential large complex involved in the degradation of proteins. The proteasome activating nucleotidase (PAN) has not only been implicated in the function of the proteasome complex, but also has an ATP independent/dependant chaperone-like function where it prevents the aggregation of unfolded proteins and promotes refolding (Benaroudj and Goldberg 2000). This HPP analysis has identified a PAN subunit, homologous to H. volcanii PanA, as expressed in
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M. burtonii. This protein may have been identified due to the dense nature of the proteasomes, which would be concentrated during sample preparation. Interestingly, in
H. volcanii, knockouts of the PanA gene result in increased thermotolerance by an unknown mechanism (Zhou et al., 2008). It would be interesting to study the effects of these PAN proteins in the psychrotolerance of M. burtonii, either due to the chaperone- like function or through the unknown mechanism identified in H. volcanii.
The analysis of the HPP of M. burtonii, several expressed protease inhibitors including two predicted surface serine proteases (Mbur_0509, 1742) and an alpha-macroglobulin- like (A2M-like) protein, which is predicted to be secreted from the cell and/or surface localised, were detected. A2M proteins are a family of large (1500aa) non-specific protease inhibitors usually found as members of the innate immune systems of metazoans. These proteins act by entrapping proteases, or specific binding partners, in a cage like structure, formed through a structural change in the A2M following proteolytic attack of a ‘bait’ region in the protein. The A2M then presents a receptor-binding region of the protein to cells, which then internalise and degrade the A2M-protease complex and/or initiate more specific immune responses (Borth 1992; Armstrong and Quigley
1999). The M. burtonii A2M-like protein contained homologous regions to all functional domains of metazoan A2M proteins, including the receptor-binding domain
(See Appendix A.9). While homologues of A2M are known to occur in bacteria, no other sequenced archaea are known to harbor this protein (Budd et al., 2004; Doan and
Gettins 2008). This was confirmed in this study by independent BLAST searches with the M. burtonii A2M against archaeal genomes where no other archaeal homologues were found. In bacteria, the presence of A2M-like proteins was thought to be a result of horizontal gene transfer events from metazoan hosts either by pathogenic or symbiotic
D. Burg UNSW 129 organisms (Budd et al., 2004; Doan and Gettins 2008). These organisms are thought utilise the A2M-like proteins as a protection against host defenses, and the A2M-like genes are often coupled with those involved in surface and cell wall repair. To determine the evolutionary relationships of metazoan, bacterial, and the M. burtonii
A2M proteins a phylogenetic tree was constructed (Figure 3.8). The constructed tree revealed that the M. burtonii A2M clusters independently from any other sequence, and that the presence of A2M genes appears to be widespread. It is found not only among pathogenic and symbiotic species as previously described (Budd et al., 2004; Doan and
Gettins 2008), but also amongst environmental bacterial isolates. Searches against metagenomic data for A2M like sequences also identified a number of candidate reads in several environmental datasets (data not shown). The most likely explanation for the evolution of A2M is that it is an ancient gene that has been lost from many species.
However, the possibility of horizontal gene transfer (HGT) from a metazoan to M. burtonii cannot be ruled out. The importance of HGT in evolution of cold adaptation in
M. burtonii (e.g. polysaccharide biosynthesis genes), including gene exchange between specific lineages (e.g. ε and γ-Proteobacteria) has been described (Allen et al., 2009)
The retention (or possibly HGT) of A2M in M. burtonii may reflect the advantage that
A2M (and the other protease inhibitors) offers towards proteolytic attack of surface structures and extracellular polymeric material, as a mechanism of maintaining surface and cell wall integrity.
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.
0.8 0.9 0.9 1.0 0.9 1.0 1.0 1.0 0.9 1.0 0.9
0.9 1.0 0.8
1.0 0.9
Figure 3.8 Phylogeny of metazoan, bacterial and M. burtonii A2M. Caption is on following page
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Figure 3.8 Phylogeny of metazoan, bacterial and M. burtonii A2M contd.
Bacterial A2M-like proteins were chosen based upon bacterial families outlined in (Budd et al., 2004)
(blue), and BLAST searches against bacterial genomes including environmental isolates (green). The metazoan A2M family proteins were chosen from complement proteins which are marked on the tree in pink (including a predicted early evolution sea Strongylocentrotus C3 protein (Al-Sharif et al., 1998)), and specific A2M proteins marked in red (including marine species), and the settlement inducing factor
SIPC from the barnacle Balanus amphitrite (Dreanno et al., 2006). Abbreviated sequences are, A: Rattus norvegicus, B: Mus musculus, C: Pongo abelii, D: Homo sapiens, E: Thermotoga petrophila, F:
Thermotoga neapolitana, G: Thermotoga maritima, H: Thermotoga sp RQ2, I: Marinitoga piezophila. GI numbers for each protein are available in Appendix A.10. The evolutionary history was inferred using the maximum likelihood method. The optimal tree is shown. aLRT probabilities are shown next to major nodes. The tree was drawn using FigTree v1.2.3 (Rambaut 2008).
In the HPP analysis performed, a J-domain protein with no known homologues in cultured organisms (see 3.4.4.3), and two very similar FKBP-type peptidyl-prolyl cis- trans isomerases (PPIases) were identified (Mbur_2255, 2256). PPIase proteins accelerate cis-trans isomerisation of the proline imide bond in polypeptides, which is a rate limiting step in the protein folding process. The two highly similar proteins identified in M. burtonii are homologous to the FKBP type proteins from the thermophiles Thermococcus sp KS-1 (TcFKBP18) and Methanothermococcus thermolithotrophicus (formerly known as Methanococcus thermolithotrophicus and
Methanobacterium thermoautotrophicum) (MtFKBP17) (see Appendix A.11 for alignment). These proteins are known not only to perform cis-trans isomerisation, but prevent denatured proteins from aggregating and then inducing refolding in a chaperone like manner (Ideno et al., 2000). This action is independent from the PPIase activity
(Furutani et al., 2000). The chaperone activity is maintained by an ‘insert in the flap’ domain (Suzuki et al., 2003), which is present in both of the M. burtonii homologues.
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The FKBP type PPIase from Thermococcus sp KS-1 is a cold shock induced protein, implicated in enhancing the survival of these cells at sub-optimum temperatures (Ideno et al., 2001). Both homologues of this protein in M. burtonii were identified on numerous occasions in the HPP analysis at both 4 and 23 C. Although no TMD were detected in this protein, and the protein was not predicted to be hydrophobic, analysis of normalised peptide counts for Mbur_2256 (the most often identified of the pair) from the DSF procedure show that as the fractions become more hydrophobic, there is a trend of identifying more peptides from the protein (Figure 2.13). This could indicate that the protein is membrane localised.
3.4.2.12 Inorganic ion transport and metabolism
The inorganic ion transport functional category makes up one of the largest groups of identified proteins from the HPP analysis. 43 proteins, 34 of which were NIW were identified in this category. The majority of these proteins (39) were predicted either to contain TMDs or be associated to the membrane through their contacts with other proteins (Table 3.13). Inorganic ion transporters from all major transporter classes were identified. These included: (a) several sodium/solute symporters (Mbur_0408, 0425,
1952) which act by using a sodium motive force (SMF) to initiate solute co-transport into the cell (Reizer et al., 1994; Jung 2002). The sodium/solute symporter family is responsible for the transport of a large number of solutes including proline, glucose, nucleosides, and pantothenate. The specific solutes of the symporters of M. burtonii could not be classified bioinformatically; (b) Several potassium uptake proteins, one of which is a calcium gated potassium channel, similar to the MthK protein from
Methanothermobacter thermautotrophicus (30% identity) (Jiang et al., 2002), and
D. Burg UNSW 133 another a NAD+ dependant potassium transporter homologous to TrkA (Schlosser et al.,
1993); (c) three divalent cation transporters were identified, two with uncharacterised substrates, and a CorA protein. CorA proteins are Mg2+ transporters which are ubiquitous amongst the bacteria and archaea (Kehres et al., 1998), and transport Mg2+ by a unique mechanism that involves stripping tightly bound water molecules from the very large hydration shell of Mg2+ in order to transport it in a dehydrated form (Smith et al., 1998; Moomaw and Maguire 2008); (d) several ATPase-type transporters were identified, including a copper transporting ATPase, ABC transporters for molybdenum, cobalt, zinc, and iron complex/vitamin B12, and a putative nitrate/aliphatic sulphonate/bicarbonate ABC transporter (based upon similarity of the binding domain with non-ABC substrate binders). Several other members of the ABC transport family were identified in these proteomic analyses, but were unable to be bioinformatically characterised based on their solute binding domains.
The implementation of the DSF procedure facilitated the identification of four small conductance mechanosensitive ion channels (Msc), which were NIW and not identified pre-DSF due to their low cellular abundance. Msc transporters act in times of hypo- osmotic stress where stretching of the cell membrane is followed by opening of a central pore, by an unknown mechanism (Hamill and Martinac 2001). This allows the influx of ions, which acts to re-establish the osmotic balance. Msc have also been implicated in maintaining cell turgor, which is important for cell division and growth
(Martinac and Kloda 2003).
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Table 3.13 Inorganic ion transport and metabolism
Annotation Locus tag GRAVY Id'd Type TMD Comment Mbur_0132 0.789 34 nm 19 Na+/H+ antiporter subunit (ER2) Mbur_0135 0.962 5 nm 13 Na+/H+ antiporter subunit (ER2) Mbur_0136 0.387 9 nm 3 Na+/H+ antiporter subunit (ER2) Mbur_0138 0.85 16 nm 3 Na+/H+ antiporter subunit (ER2) Mbur_0247 0.048 1 nm 3 Calcium-gated potassium channel (ER3) Mbur_0408 0.11 7 nm 4 Sodium/solute symporter (ER3) Mbur_0425 0.951 9 n membrane complex Sodium/solute symporter (ER3) Mbur_0474 0.272 2 nm 4 Small-conductance mechanosensitive ion channel (ER4) Mbur_0476 0.495 1 nm 3 Small-conductance mechanosensitive ion channel (ER3) Mbur_0612 0.188 3 m 8 Copper-transporting P-type ATPase (EC 3.6.3.4) (ER2) Mbur_0646 0 3 nm 2 Divalent metal ion transporter CorA (ER2) Mbur_0778 0.183 13 p membrane complex FeoA-domain protein (ER4) Mbur_0779 0.393 49 nm 8 Ferrous iron transport protein B (ER2) Mbur_0791 1.018 4 nm 5 Divalent cation transporter (ER4) Mbur_0792 0.908 18 nm 5 Divalent cation transporter (ER4) Mbur_0885 0.192 22 nm 10 P-type cation transporting ATPase (ER2) Mbur_1016 -0.073 1 n membrane complex Zinc ABC transporter ATPase subunit (ER2) Mbur_1038 0.002 4 n Nitrogenase iron protein (EC:1.18.6.1) (ER2) Iron complex/Vitamin B12 ABC transporter substrate binding protein Mbur_1047 -0.158 24 n membrane complex (ER3) Mbur_1074 0.028 5 nm 3 Small-conductance mechanosensitive ion channel (ER3) Mbur_1120 -0.242 3 nm 1 Ferritin (ER2) Mbur_1310 0.174 2 n membrane complex Potassium uptake protein TrkA (ER2) Mbur_1327 -0.141 4 n Phosphoadenosine phosphosulfate reductase (ER3) Sulfide dehydrogenase (flavoprotein) subunit SudB (EC 1.97.-.-) Mbur_1329 0.01 12 p (ER2) Mbur_1363 0.775 19 nm 13 Sodium/hydrogen antiporter (ER4) Molybdenum ABC transporter, molybdate-binding protein (modA) Mbur_1443 -0.038 16 nm 1 (ER3) Mbur_1461 0.63 7 m 10 Monovalent cation/proton antiporter family protein (ER3) Iron complex/Vitamin B12 ABC transporter substrate binding protein Mbur_1547 -0.117 2 secn sec membrane complex (ER3) Molybdenum ABC transporter, molybdate-binding protein (modA) Mbur_1553 0.156 13 nm 1 (ER2) Mbur_1692 0.343 5 m 8 Cation-transporter, P-type ATPase (EC 3.6.3.-) (ER2) Mbur_1794 -0.022 9 n membrane complex Putative cobalt ABC transporter ATP-binding protein cbiO (ER3) Mbur_1795 0.596 10 nm 5 Putative cobalt ABC transporter permease protein cbiQ (ER4) Mbur_1872 0.529 2 nm 10 Ferrous iron transport protein B (ER3) Mbur_1952 0.7 42 m 13 Sodium/solute symporter (ER3) Mbur_2131 0.052 56 n membrane complex Putative ABC transporter, substrate binding protein (ER4) Mbur_2132 0.908 3 nm 7 ABC transporter permease protein (ER2) Mbur_2133 -0.275 4 n membrane complex ABC transporter ATPase subunit (ER2) Mbur_2189 0.718 30 nm 10 Sulphate transporter (ER3) Mbur_2273 0.437 1 nm 3 Small-conductance mechanosensitive ion channel (ER3) Mbur_2435 0.081 3 n Sulphite reductase, assimilatory-type (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification; nm – NIW identification with TMDs; m – protein also identified in WCE studies but containing TMD; continues on following page
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Table legend continued: p – protein also identified in previous WCE studies but either hydrophobic in nature or part of a membrane complex; secn – NIW identifications that contains a signal peptide.
Comment – indicates the protein is associated with the membrane through a complex
Very few archaeal Msc have been characterised, with the only examples coming from
M. jannaschii (Kloda and Martinac 2001b) and Thermoplasma volcanium (Kloda and
Martinac 2001a). Patch clamping techniques have also identified the presence of mechanosensitive channels in H. volcanii (Le Dain et al., 1998). Proteins of this class are generally characterised through patch clamping and hence the protein sequences of
MscS, at this stage, do not hold any clues as to the specificity or relative conductance of these enigmatic transporters.
In anaerobic environments iron is available in the soluble reduced ferrous (Fe2+) form
(Kammler et al., 1993). M. burtonii expresses two transporters (FeoB, Mbur_0778,
1872) for the uptake of ferrous iron. The FeoB Mbur_0779 is accompanied by the expression of an FeoA domain protein (Mbur_0778) with unknown function. These proteins are arranged in the genome with three hypothetical genes (Mbur_0780 – 0782), and an iron-dependant repressor protein (Mbur_0783). The other expressed FeoB protein (Mbur_1872) is located immediately downstream of an iron dependant repressor protein (Mbur_1871) in the genome. These data suggest that iron uptake is a regulated process in M. burtonii, and that each Feo transporter could be discreetly regulated.
In the HPP analysis performed, M. burtonii was found to express a sulphate transporter
(Mbur_2189), and the ABC transporter ATPase-like protein SufC (Mbur_0491). Similar to characterised SufC proteins, the M. burtonii SufC contains the atypical FSGGE motif
136 D. Burg UNSW
in the ABC signature region (usually LSGGQ). The SufC protein works in an ATP dependant manner with other components of the Suf system for the pre-assembly of iron-sulphur clusters (Watanabe et al., 2005).
The most interesting proteins identified in the analysis of sulphur metabolic proteins from the HPP studies however, are the sulphide dehydrogenases SudA and SudB
(mbur_1328 and 1327). Immediately upstream of these in the genome, is a protein annotated as a ferredoxin containing phosphoadenosine phosphosulfate reductase-like protein (PAPS reductase-like, Mbur_1329) with some similarity to sulfur metabolism related proteins, however the exact function of this protein is unknown. The SudA and
SudB proteins are homologous to the characterised SuDH proteins from P. furiosus
(Hagen et al., 2000), both matching with 53% Identity (See Appendix A.12 for alignments), including all proposed active residues. The SuDH complex has been shown to function as a reduced ferredoxin:NADP oxidoreductase with high affinity for reduced ferredoxin (Ma and Adams 1994). The SuDH complex utilises reduced ferredoxin as the electron donor for the reduction of NADP+ to NADPH. The expression of the SuDH complex in M. burtonii may be related to the assimilation of ammonia by
GDH, which would produce excess NADP+, which could then be recycled to NADPH by SuDH. The SuDH complex has also been shown to reduce polysulphides to H2S for cellular use when operating as a reduced ferredoxin:NADP oxidoreductase, however it is unclear whether M. burtonii uses it in this manner. The sulphate depleted depths of
Ace Lake, although containing sediments rich in polysulphides (Kok et al., 2000), are also rich in H2S (Coolen et al., 2004a). It is therefore unlikely that M. burtonii requires this activity to provide H2S as a biologically available sulfur source in its natural environment. However, in the laboratory, the complex media used to grow the organism could contain metal polysulphides formed upon autoclaving, which could be used for
D. Burg UNSW 137 sulphur reduction. Despite this, it is most likely the SuDH complex in M. burtonii acts as a NADP+ recycling complex, independent of sulfur reducing activity, utilising reduced ferredoxins produced in methanogenesis and anaerobic metabolism. Several closely related methanogens that also produce reduced ferredoxin from reverse methanogenesis (Methanosarcina spp.) have homologues of these SuDH proteins in their genomes; however, none are in close proximity to PAPS reductase-like proteins.
3.4.2.12.1 Maintenance of ion balance
M. burtonii utilises the high extracellular sodium provided in its natural environment as a SMF to drive the actions of the tetrahydrosarcinapterin–S–methyltransferase (Mtr) complex (which imports sodium during the dismutation of C1 compounds (Welander and Metcalf 2005), and the sodium solute symporters. As well as the use of the SMF for these processes, it is also proposed that the Na+/H+ antiporters utilise this SMF to export protons in an energy conserving mechanism (see 3.4.2.1.2). While the maintenance of the PMF is well understood in the methylotrophic methanogens, and is mediated by the Methyl CoM reductase, F420H2 dehydrogenase complexes (Baumer et al., 2000) and PPases (Baumer et al., 2002), there remains no evidence as to how M. burtonii maintains the SMF required for the aforementioned processes. In light of this, it is proposed that the Rnf complex, which operates in electron transport (see 3.4.2.1.2), acts to export and hence reduce the intracellular Na+ concentration, helping to maintain a sodium motive force. This is more in line with the predicted sodium ion transporting function of the complex in the bacterial species where it is generally found (Masepohl and Klipp 1996; Kumagai et al., 1997; Bruggemann et al., 2003), than the H+ transporting model proposed by Li et al., (2006). The proposed sodium exporting action of the Rnf complex may help to maintain a SMF, but it may not be able to do this alone.
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The expression of several types of energy conserving Na+/H+ antiporters (Mbur_0132 –
0137, 1363 and the possibly the putative antiporter Mbur_1461) as mentioned in
2.4.8.1, may hold some clues to this process. It could be that these have different orientations in the membrane, i.e. some import Na+ and export H+, and others act in the opposite direction. Transporters functioning as such could act to maintain the delicate balance between the intracellular and extracellular ion concentrations, and sustain the low intracellular Na+ and H+ needed for PMF and SMF driven processes. It has been suggested that SMF is difficult to maintain at low temperatures (Goodchild et al.,
2004b). If this is the case, then the actions and subtle variations in these transporters may be crucial for the metabolism of this organism under cold conditions.
3.4.2.13 Hypothetical proteins, and proteins with only general function prediction.
M. burtonii expresses a large number of hypothetical proteins and proteins with no predictable specific function (3.4.1). Of the expressed hypothetical proteins, 21 of these are unique to M. burtonii as classified and discussed in Allen et al.,(2009). The complete list of these proteins is available in Supplementary Table 1 (supplementary disk).
3.4.2.14 Signal transduction mechanisms.
All organisms need to sense and respond to changes in the environment through signal transduction pathways. In prokaryotes, signal transduction can be effected by one or two-component regulatory systems. In the one component system, which has recently been identified as the major prokaryotic signal transduction mechanism (Ulrich et al.,
2005), a single protein senses an environmental cue and facilitates a response to this cue through an effector domain. In the two-component system, a sensory protein with a
D. Burg UNSW 139 protein kinase domain phosphorylates a response regulator receiver, which then effects the change by DNA binding or protein interaction (Kennelly 2007). In this study, several sensory proteins from two-component systems were identified (Table 3.14).
These were identified following HPP analysis, as most histidine kinases are membrane bound or associated (Ulrich et al., 2005). Very few response regulators were identified, and only one was NIW. As most response regulators are cytoplasmic, this is not a surprising result.
Sensor histidine kinases have a huge variety of architectures and respond to a large range of signals from the environment (Galperin et al., 2001). This is evident in the identified proteins of M. burtonii, where no two proteins had the same domain configuration (Figure 3.9). These proteins were re-annotated according to the guidelines set out in Galperin and Nikolskaya (2007).
Many of these protein contained PAS domains, which are usually located in the cytoplasm and can sense a variety of environmental conditions including redox potential, oxygen, and energy levels (Taylor and Zhulin 1999). Most identified proteins contained histidine kinase domains and can be inferred to be part of two-component systems. Examples of these sensory proteins include: Mbur_0083 and 0842, which contain both an external sensor domain and an internal PAS sensor domain identifying these proteins as sensors of both the internal and external environment; Mbur_0312 and
1264, which appear to be internal sensor histidine kinases and are relatively unique to
M. burtonii along the entire length of the proteins; and Mbur_0694, which contains a unique external sensor domain.
Examples of these expressed sensory proteins, which do not fit into the classical two- component sensor mould include: Mbur_0217, annotated as a multi-sensor protein containing three PAS domains and a GAF domain (involved in binding the second
140 D. Burg UNSW
messenger, cyclic GMP (Ho et al., 2000)), this protein contains no domains known to interact with other proteins and may be a novel one component regulatory system; and
Mbur_2108 which contains both an N-terminal response receiver domain and histidine kinase domains.
Mbur_0083 Mbur_0217
Mbur_0312 Mbur_0694
Mbur_0842 Mbur_1023
Mbur_1264 Mbur_2108
Mbur_2300
Legend TMD Putative external sensor PAS domain
Histidine kinase HATPase_c GAF domain
CACHE domain HAMP linker Regulator receiver
Figure 3.9 Domain architecture of sensor proteins. All identified sensor proteins had unique domain architecture
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Table 3.14 Signal transduction mechanisms
Locus tag GRAVY Id'd Type TMD Annotation Multisensor signal transduction histidine kinase, with external sensor Mbur_0083 -0.199 1 nm 1 domain and cytoplasmic PAS domain (ER4) Mbur_0217 -0.232 2 n Multi-sensor protein with a GAF and three PAS domains (ER4) Mbur_0312 -0.248 6 n PAS domain signal transduction histidine kinase (ER3) Signal transduction histidine kinase with external sensor domain Mbur_0694 -0.182 30 nm 2 (ER3) Multisensor signal transduction histidine kinase, with external Mbur_0842 -0.148 2 m 1 CACHE domain and cytoplasmic PAS domain (ER4) Mbur_0947 -0.312 2 n Serine kinase RIO1 (ER2) Signal transduction histidine kinase with external sensor domain Mbur_1023 -0.3 9 nm 2 and cytoplasmic HAMP domain (ER4) Mbur_1264 -0.209 1 n PAS domain signal transduction histidine kinase (ER4) Unusual signalling protein with regulatory receiver domain, PAS Mbur_2108 -0.126 4 n domain and histidine kinase domain (ER4) Mbur_2300 -0.335 17 n PAS domain signal transduction histidine kinase (ER3) Putative alkaline phosphatase synthesis transcriptional regulatory Mbur_2445 0.233 3 n protein (phoP)-like response regulator receiver (ER2)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification; nm – NIW identification with TMDs; m – protein also identified in WCE studies but containing TMD; secn – NIW identifications that contains a signal peptide
As well as the sensor histidine kinases a response receiver (Mbur_2445), with high N- terminal homology to E. coli PhoP was identified. However this protein contained no output domain, suggesting that it may be part of a multi protein complex. Also identified in this study was the RIO1 atypical protein kinase, an essential protein involved in regulation of ribosome biogenesis and cell cycle progression.
3.4.2.15 Intracellular trafficking, secretion, and vesicular transport
While the proteomics of secreted proteins (Saunders et al., 2006) and WCE of M. burtonii have been previously investigated, proteins of the secretion apparatus were not identified due to their membrane bound/associated nature. In this work all components
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(bar one) of the putative Sec secretion apparatus of M. burtonii were identified, as well as other proteins putatively involved in secretion (Figure 3.10 and Table 3.15).
Most components of the Sec system in archaea are fairly well characterised ((Egea et al., 2008a; Egea et al., 2008b) and have been reviewed (Eichler and Moll 2001; Ring and Eichler 2004a; Pohlschroder et al., 2005; Albers et al., 2006)). Subunits identified in M. burtonii all displayed high homology to characterised components.
The secretion accessory protein YidC is not well characterised in Archaea, and its function is proposed based upon distant homology (Yen et al., 2001). The protein
Mbur_0026, annotated as a putative YidC-like protein, only displayed low homology to the proteins described in (Yen et al., 2001) (Appendix A.13) and is annotated as a
DUF106 protein. The protein displays closest phylogeny to hypothetical proteins in the
Methanosarcinaceae (Appendix A.14) and its function as an accessory protein in the Sec pathway in M. burtonii is only speculative.
M. burtonii expresses two putative signal peptidases. Mbur_1734 had been previously implicated as a signal sequence peptidase (Ng et al., 2007) and is similar to Sec11a and b from H. volcanii (Fine et al., 2006), while Mbur_1937 had a C terminal homologous to a characterised signal peptidase from E. coli (Kim et al., 2008).
Also identified were two type II secretion system F domain proteins. The genome codes for six of these F domain proteins and also for six type II secretion system E domain proteins, most frequently occurring side by side in the genome sequence. These type II proteins may be implicated in flagella synthesis (Peabody et al., 2003).
The analysis of the HPP of M. burtonii also identified a MotA/TolQ/ExbB-like proton channel protein. Proteins of this family have been implicated in transport of proteins
D. Burg UNSW 143 and large molecules in association with proton transport in bacteria (Postle and Kadner
2003). The function of this protein in archaea is unknown, although several close homologues among the methanogens were identified from BLAST searches.
a
b, c j in e k l
d f h
i out g Figure 3.10 The secretion apparatus of M. burtonii . Most components of the secretion apparatus of
M. burtonii were identified and are labeled: a - Ribosome; b, c - Signal recognition particles, SRP19
(Mbur_2116), and SRP54 (Mbur_0141); d - signal recognition particle docking protein (FtsY)*
(Mbur_0110); e - nascent polypeptide; f - SecYE pore, SecY (Mbur_0024), SecE (Mbur_1943); g -
SecDF accessory subunits, SecD (Mbur_1987), SecF (Mbur_1986), h - putative YidC accessory subunit
(Mbur_0026); i - signal sequence peptidases (Mbur_1734, 1937); j - type ii secretion protein E*#
(Mbur_0047, 0254, 0352, 0448, 1574, 2400); k - type ii secretion protein F#(Mbur_0252, 0253*, 0353*,
0449*, 1575, 2401); and l – MotA/TolQ/ExbB-like proton channel protein# (Mbur2258).
* indicates proteins that were not identified in this study, # indicates proteins that are not components of the Sec secretory system.
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Table 3.15 Intracellular trafficking, secretion, and vesicular transport
Annotation Locus tag GRAVY Id'd Type TMD Comment Mbur_0024 0.695 48 nm 4 Preprotein translocase SecY subunit (ER2) Mbur_0141 -0.241 3 p membrane complex signal recognition particle SRP54 (ER2) Mbur_0252 0.177 6 nm 3 type II secretion system protein F domain (ER3) Mbur_1575 0.498 2 nm 9 type II secretion system protein F domain (ER3) Mbur_1734 -0.022 20 nm 1 putative S24-like signal peptidase (ER3) Peptidase family S49 (putative signal peptide peptidase SppA, Mbur_1937 -0.182 2 nm 2 36K type) (ER3) Mbur_1943 0.675 24 nm 1 Preprotein translocase subunit secE (ER2) Mbur_1986 0.615 55 nm 6 Preprotein translocase SecF subunit (ER3) Mbur_1987 0.293 34 nm 6 Protein-export membrane protein secD (ER3) Mbur_2116 -0.589 6 n complex Ribonucleoprotein complex SRP, Srp19 subunit (ER2) Mbur_2258 0.286 2 nm 3 MotA/TolQ/ExbB like proton channel protein (ER4)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification; nm – NIW identification with TMDs; m – protein also identified in WCE studies but containing TMD; p
– protein also identified in previous WCE studies but either hydrophobic in nature or part of a membrane complex. Comments indicate whether the protein is part of a large complex, or indicate the protein is associated with the membrane through a complex.
3.4.2.16 Defence mechanisms
Very few proteins were identified in the defence mechanisms functional category (Table
3.16). Proteins that were identified had low confidence. These proteins were a restriction endonuclease, possibly involved in protection of the genome from foreign
DNA, a MatE family protein, which may be involved in the export of potentially damaging solutes such as antimicrobials, and a universal stress family protein, which may have a variety of functions in the cell (Nachin et al., 2005).
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Table 3.16 Defence mechanisms
Locus tag GRAVY Id'd Type TMD Comment Annotation Mbur_0704 0.128 3 n Protein similar to MJ0577 (Usp family) (ER3) Mbur_1841 -0.548 1 n Type-1 site specific restriction enzyme R subunit (ER2) Mbur_2307 0.949 1 nm 12 MatE family protein (ER4)
Table legend: GRAVY – hydrophobicity values, positive values indicate a hydrophobic protein. Id’d – the number of LC/LC-MS/MS experiments the protein was identified in. Type: n – NIW identification; nm – NIW identification with TMDs; m – protein also identified in WCE studies but containing TMD.
3.4.3 Highly abundant proteins
Throughout the experiments performed, many proteins were identified in large numbers of LC/LC-MS/MS runs, several in every experiment performed. The frequency of identification can be related directly to relative cellular abundance, which can be used to identify important aspects of cellular biology. The top 50 proteins identified in M. burtonii are displayed in Table 3.17.
When the abundant proteins are analysed for function it is clear that proteins involved in translation and energy production make up the largest categories. This is reflective of the crucial nature of these processes to the cell. Flagellin and DUF1608 (S-layer related) proteins were also identified in high abundance, which is not surprising given the hydrophobic nature of the extracts analysed, which facilitates the identification of membrane and membrane related proteins. Other interesting proteins identified as abundant were the chaperone DnaK and the Lon family peptidase with chaperone function (3.4.2.11).
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Table 3.17 High abundance proteins in M. burtonii
Locus tag Id'd Annotation Locus tag Id'd Annotation Mbur_0001 LSU ribosomal protein L3P (rpl3p) V-type ATP synthase beta chain [atpB] (EC 57 (ER2) Mbur_1244 57 3.6.3.14) (ER2) LSU ribosomal protein L4P (rpl4p) Mbur_0002 57 (ER2) Mbur_1312 53 Chaperone DnaK (Hsp70) (ER2) LSU ribosomal protein L32E (rpl32e) Trimethylamine methyltransferase (mttB) Mbur_0018 51 (ER2) Mbur_1369 57 (EC 2.1.1.-) (ER2) LSU ribosomal protein L19E (rpl19e) LSU ribosomal protein L10AE (rpl10ae) Mbur_0019 57 (ER2) Mbur_1422 57 (ER2) SSU ribosomal protein S5P (rps5p) Protein of unknown function DUF1699 Mbur_0021 57 (ER2) Mbur_1508 (b) 57 (ER4) Tetrahydromethanopterin S- LSU ribosomal protein L30P (rpl30p) methyltransferase subunit H (mtrH) (EC Mbur_0022 51 (ER2) Mbur_1518 57 2.1.1.86) (ER2) Tetrahydromethanopterin S- LSU ribosomal protein L15P (rpl15p) methyltransferase subunit A (mtrA) (EC Mbur_0023 57 (ER2) Mbur_1521 (a,d) 57 2.1.1.86) (ER2) Tetrahydromethanopterin S- SSU ribosomal protein S4P (rps4p) methyltransferase subunit E (mtrE) (EC Mbur_0037 57 (ER2) Mbur_1525 (a,d) 57 2.1.1.86) (ER2) SSU ribosomal protein S11P (rps11p) Mbur_0038 57 (ER2) Mbur_1689 (b,e) 57 Hypothetical protein (ER5) Mbur_0104 (a) 57 Flagellin (ER2) Mbur_1690 57 DUF 1608 protein (ER4) LSU ribosomal protein L31E (rpl31e) Mbur_0114 57 (ER2) Mbur_1718 (c) 52 Hypothetical protein (ER5) SSU ribosomal protein S19E (rps19e) Mbur_0118 57 (ER2) Mbur_1769 56 Putative nucleic acid binding protein (ER4) LSU ribosomal protein L15E (rpl15e) Putative stomatin, band 7 family protein Mbur_0192 (b) 57 (ER2) Mbur_1804 (d) 51 (ER2) Mbur_0343 (c,f) 54 Hypothetical protein (ER5) Mbur_1892 53 GTP-binding domain protein (ER4) Translation initiation factor aIF-1A Mbur_0456 (b) 57 (ER2) Mbur_1950 57 Dead box RNA helicase (ER2) Monomethylamine methyltransferase Mbur_0839 52 mtmB (EC 2.1.1.-) (ER2) Mbur_1986 (c,d) 55 Preprotein translocase SecF subunit (ER3) Lon family peptidase with N terminal Mbur_0880 (a) 57 transmembrane domain (ER3) Mbur_2063 (b,f) 51 Hypothetical protein (ER5) Pyrophosphate-energised proton pump / Pyrophosphate-energised inorganic pyrophosphatase (H(+)- Mbur_0994 (a) 57 PPase) (EC 3.6.1.1) [hppA] (ER2) Mbur_2101 57 SSU ribosomal protein S8E (rps8e) (ER2) SSU ribosomal protein S10P (rps10p) Mbur_1169 57 (ER2) Mbur_2129 (a) 56 DUF1608 containing protein (ER4) Translation elongation factor EF-1, Putative ABC transporter, substrate binding Mbur_1170 57 subunit alpha (EF-1A, EF-Tu) (ER2) Mbur_2131 (b,d) 56 protein (ER4) SSU ribosomal protein S7P (rps7p) Trimethylamine methyltransferase (mttB) Mbur_1172 57 (ER2) Mbur_2308 55 (EC 2.1.1.-) (ER2) SSU ribosomal protein S12P (rps12p) Mbur_1173 57 (ER2) Mbur_2336 57 SSU ribosomal protein S3Ae (rps3Ae) (ER2) SPFH domain / Band 7 family-like Methyl-coenzyme M reductase, subunit Mbur_1185 (c,d) 57 protein (ER4) Mbur_2417 57 alpha (mcrA) (EC 2.8.4.1) (ER2) Putative 116kDa V-type ATPase Methyl-coenzyme M reductase, subunit Mbur_1238 (a,d) 57 subunit (ER4) Mbur_2418 57 gamma (mcrG) (EC 2.8.4.1) (ER2) H(+)-transporting ATP synthase, Methyl-coenzyme M reductase, subunit beta Mbur_1239 (a,d) 57 subunit K (EC 3.6.3.14) (ER2) Mbur_2421 (d) 57 (mcrB) (EC 2.8.4.1) (ER2)
Table legend: a) protein with TMD; b) NIW protein; c) NIW protein with TMD; d) hydrophobic protein;
e) protein with signal peptide; f) protein unique to M. burtonii
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Interestingly many remaining proteins have unknown or unclear functions, including two unique hypothetical proteins. The presence of these proteins in high abundance in the cell is indicative of their importance and is also reflective of how little is known regarding many aspects of archaeal physiology, and cold adapted organisms. Specific examples of these proteins are: a nucleic acid binding protein (Mbur_1769); the unique hypotheticals including Mbur_2063, which displays a trend towards hydrophobicity in normalised peptide counts despite being predicted as hydrophilic (See Appendix A.15); a GTP binding protein (Mbur_1892); and the Band 7 family stromatin or SPFH-like proteins (Mbur_1185, 1804).
3.4.4 Differentially abundant proteins
The large number of runs performed across two growth temperatures (4˚C and 23˚C) allowed for the utilisation of Scaffold2 to determine any differences in protein abundance levels. Significant differences in peptide spectral counts between the temperatures tested were identified in 58 proteins (p < 0.05). Of these proteins 30 had been previously identified as differentially abundant using traditional differential proteomic techniques, (see Chapter 4 and: Goodchild et al., 2004b; Goodchild et al.,
2005; Williams et al., submitted-b) (Table 3.18) and a further 16 could be related directly to previous findings as members of protein complexes, or processes previously identified as thermally influenced (Table 3.19). The remaining 12 proteins were not previously identified as differentially abundant (Table 3.20). The large overlap between this method of identifying differential abundance and other methods, indicates that the techniques are complementary. The proteins newly identified as differentially abundant fell into three general categories: Heat stress, general metabolism, and miscellaneous and hypothetical.
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Table 3.18 Differentially abundant proteins also identified using labelling strategies.
Increased at Locus tag Annotation (˚C) Mbur_0002 LSU ribosomal protein L4P (rpl4p) (ER2) 4 Mbur_0018 LSU ribosomal protein L32E (rpl32e) (ER2) 4 Mbur_0022 LSU ribosomal protein L30P (rpl30p) (ER2) 4 Mbur_0023 LSU ribosomal protein L15P (rpl15p) (ER2) 4 Mbur_0104 Flagellin (ER2) 4 Mbur_0268 Protein with duplicated DUF1608 (ER4) 4 Mbur_0779 Ferrous iron transport protein B (ER2) 23 Mbur_0847 Monomethylamine corrinoid protein (mtmC) (ER2) 23 Translation elongation factor EF-1, subunit alpha (EF-1A, EF-Tu) Mbur_1170 (ER2) 23 Mbur_1173 SSU ribosomal protein S12P (rps12p) (ER2) 4 Mbur_1177 DNA-directed RNA polymerase subunit A', rpoA1 (ER2) 23
Mbur_1244 V-type ATP synthase, beta chain (atpB) (EC 3.6.3.14) (ER2) 23 Mbur_1312 Chaperone DnaK (Hsp70) (ER2) 23 Mbur_1349 Protein containing trypsin-like serine/cysteine protease domain (ER4) 4 Mbur_1350 Hypothetical protein (ER5) 4 Mbur_1494 Thiamine biosynthesis protein thiC (thiC) (ER2) 23 Mbur_1608 Glycosyl transferase, family 2 (ER4) 23 Mbur_1942 Cell division protein FtsZ (ER2) 23 Mbur_1950 DEAD box RNA helicase (ER2) 4 Mbur_2063 Hypothetical protein (ER5) 4 Mbur_2129 Protein with duplicated DUF1608 (ER4) 4
Mbur_2131 ABC transporter, solute-binding protein (nitrate/sulfonate?) (ER4) 23 Mbur_2146 Thermosome subunit (Chaperonin) (ER2) 23 Mbur_2308/ Mbur_2309 Trimethylamine methyltransferase (mttB) (EC 2.1.1.-) (ER2) 23 Mbur_2310 Trimethylamine corrinoid protein (mttC) (ER2) 23 Mbur_2312 MttQ (function unknown) (ER4) 23 Mbur_2390 SSU ribosomal protein S9P (rps9p) (ER2) 4 Methyl-coenzyme M reductase, subunit gamma (mcrG) (EC 2.8.4.1) Mbur_2418 (ER2) 23 Methyl-coenzyme M reductase, subunit beta (mcrB) (EC 2.8.4.1) Mbur_2421 (ER2) 23
Mbur_2345 SSU ribosomal protein S15p (ER2) 4
A large number of proteins were identified as differentially abundant that were also identified using labelling strategies
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3.4.4.1 Heat stress
Growth at 23˚C (Topt) is stressful to M. burtonii, with signs of both oxidative and protein denaturation occurring at this temperature (Chapter 4; Goodchild et al., 2004b;
Goodchild et al., 2005; Williams et al., submitted-b). In this study the DNA repair and recombination protein RadA (Mbur_2033), and the chaperones ClpB (Mbur_2199) and
DnaJ (Mbur_1311) were identified as associated with growth at 23˚C.
The Repair and recombination protein RadA, a RecA family protein with similar function to eukaryotic Rad51 (McRobbie et al., 2009) is known to play a pivotal role in the repair of double stranded DNA breaks (Haldenby et al., 2009). This type of damage can occur as a result of oxidative stress (Demple and Harrison 1994). Increased metabolic activity at 23˚C in M. burtonii has been proposed to result in the formation of non-oxygen radicals (Chapter 4 and; Williams et al., submitted-b) , including
5’adenosyl radicals, which are potentially highly reactive (Ugulava et al., 2003). The increased abundance of RadA at 23˚C may be acting to counteract any oxidative damage to DNA, along with the other oxidative stress proteins identified in other studies (Williams et al., submitted-b).
In previous studies of thermal adaptation in M. burtonii, only the chaperone DnaK was identified as displaying increased abundance at 23˚C (Williams et al., submitted-b). In this study the co-chaperones DnaJ and ClpB were identified as differentially abundant at 23˚C. At high temperatures, proteins become denatured, corresponding to a loss of functional configuration, often accompanied by aggregation (Macario et al., 1999). This can be prevented and corrected by the chaperones and the chaperonins, which are often
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seen at elevated levels in cells undergoing heat stress (Boonyaratanakornkit et al., 2005;
Coker et al., 2007).
Many archaea have a bacteria-like ATP-dependant chaperone system (DnaK/Hsp70,
DnaJ/Hsp40, GrpE), which catalyses the refolding of denatured proteins, and assists in the folding of nascent polypeptides (Macario et al., 1999; Zmijewski et al., 2004). The identification of DnaJ in this study, along with the identification of DnaK in previous studies is indicative of this process occurring at 23˚C in M. burtonii.
Table 3.19 Differentially abundant proteins related to those previously identified
Increased abundance Locus tag Annotation at Comment Mbur_0006 LSU ribosomal protein L22P (rpl22p) (ER2) 4˚C Ribosomes increased at 4˚C Mbur_0012 LSU ribosomal protein L24P (rpl24P) (ER2) 4˚C Ribosomes increased at 4˚C Mbur_0013 SSU ribosomal protein S4E (rps4e) (ER2) 4˚C Ribosomes increased at 4˚C Mbur_0014 LSU ribosomal protein L5P (rpl5p) (ER2) 4˚C Ribosomes increased at 4
Mbur_0502 Glycine betaine ABC transporter permease protein 4˚C Substrate binding protein opuAB (ER2) increased at 4˚C Mbur_0905 LSU ribosomal protein L21E (rpl21e) (ER2) 4˚C Ribosomes increased at 4˚C
Mbur_0929 Methylenetetrahydromethanopterin dehydrogenase 23˚C Methanogenesis components (mtd) (EC 1.5.99.9) (ER2) increased at 23
Mbur_1179 DNA-directed RNA polymerase subunit B', rpoB2 23˚C RNAPA (co-core subunit) (ER2) increased at 23˚C
Mbur_1239 H(+)-transporting ATP synthase, subunit K (EC 23˚C Other members of complex 3.6.3.14) (ER2) increased at 23˚C
Mbur_1293 23˚C Other members of complex F420H2 dehydrogenase subunit H (ER2) increased at 23˚C MttQ isoenzyme increased at Mbur_1366 23˚C MttQ (function unknown) (ER4) 23˚C
Mbur_1379 23˚C Other members of complex Electron transport complex rnfB protein (ER3) increased at 23˚C
Mbur_1384 23˚C Other members of complex Electron transport complex rnfC protein (ER3) increased at 23˚C
Mbur_1519 Tetrahydromethanopterin S-methyltransferase 23˚C Methanogenesis components subunit G (mtrG) (EC 2.1.1.86) (ER2) increased at 23˚C
Mbur_1902 Methylcoenzyme M reductase system component 23˚C Methanogenesis components A2 (McrA)(ER2) increased at 23˚C MttP isoenzyme increased at Mbur_2311 23˚C TMA permease (MttP) (ER4) 23˚C
Several proteins that were identified as differentially abundant were members of complexes, or involved in processes, that have previously been identified as thermally influenced in M. burtonii.
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In contrast the ATP dependant chaperone system, which enhances folding and prevents aggregation (Lee et al., 2003), the molecular chaperone ClpB rescues proteins from an aggregated state, often acting with DnaK as a co-chaperone (Kim et al., 1998; Kim et al., 2003; Mogk et al., 2003). ClpB chaperones are rare in the archaea and apart from
M. burtonii, ClpB chaperones are only found in a few isolated mesophilic methanogens
(Candidatus Methanoregula boonei 6A8, Methanospirillum hungatei JF-1,
Methanohalophilus mahii and Methanohalophilus portucalensis, identified through
IMG genome BLAST and NCBI BLAST). The presence of ClpB in these organisms is thought to have resulted from horizontal gene transfer events from low G + C Gram positive bacteria (Shih and Lai 2007). A close homologue of the M. burtonii ClpB has been shown to be associated with heat and osmotic stress in M. portucalensis (Shih and
Lai 2007). The identification of the chaperone ClpB as increased in abundance at 23 C, adds evidence to the observation that growth at this temperature (Topt) is stressful to the organism (Goodchild et al., 2004b; Goodchild et al., 2005), and is indicative of high temperature induced protein aggregation in M. burtonii. The identification of the chaperones DnaJ and ClpB in this study, and not previous work (and including Chapter
4) may be indicative of their low abundance. It contrasts DnaK which is identified in high abundance in all previous proteomic investigations of M. burtonii. The high abundance of DnaK, and hence its ease of identification in other studies, indicates a central multi-functional role for this protein in the heat stress response in M. burtonii, highlighted by inferred interactions with different chaperone systems involving DnaJ and ClpB.
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Table 3.20 Differentially abundant proteins not identified by other methods
Increased Locus tag Annotation abundance at Mbur_0078 Protein of unknown function DUF214, possible permease (ER4) 4˚C Mbur_0308 DUF124-domain protein (ER4) 4˚C Mbur_0441 Hypothetical protein (ER5) 4˚C Mbur_0686 Ribosomal protein S6 modification protein (RimK) (ER2) 4˚C Mbur_1212 Atypical type iii J-domain protein (ER3) 4˚C Mbur_2059 Hypothetical protein (ER5) 4˚C Mbur_1311 Chaperone DnaJ (ER3) 23˚C Mbur_1602 Radical SAM protein (ER4) 23˚C Mbur_1892 GTP-binding domain protein (ER4) 23˚C Mbur_2001 2-amino-3,7-dideoxy-D-threo-hept-6-ulosonate synthase (ER2) 23˚C Mbur_2033 DNA repair and recombination protein RadA (ER2) 23˚C Mbur_2199 Chaperone ClpB (ER2) 23˚C
Several proteins were identified through the use of peptide counts that were not able to be identified through traditional methods.
3.4.4.2 Metabolic proteins
Three proteins that have possible roles in the central metabolism of the organism were identified as having increased abundance at 23˚C. These were a radical SAM protein
(Mbur_1602), a GTP-binding domain protein (Mbur_1892), and 2-amino-3,7-dideoxy-
D-threo-hept-6-ulosonate synthase (Mbur_2001). The increase in abundance of metabolic proteins at 23˚C is consistent with previous findings in where the increased rate of growth at 23˚C is associated with an increase in metabolic proteins (Williams et al., submitted-b). This is especially true for Mbur_2001, which is involved in amino acid biosynthesis, reflects an increased demand for the biosynthesis of amino acids at the faster growth rate. The functions of Mbur_1602 and Mbur_1892, are less easily defined. However an increase in the activity of radical SAM proteins at 23˚C has been related to the production of non-oxygen radicals, which contribute to the oxidative stress response seen at this temperature in M. burtonii (Williams et al., submitted-b).
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2.4.10.3 Miscellaneous and hypothetical proteins
Several proteins with unspecified function were identified as associated with growth at different temperatures. Only two of these were able to have putative function inferred.
A ribosomal protein S6 modification protein (RimK-like protein) (Mbur_0686) had increased abundance at 4˚C. In bacteria, this protein is involved in the post translational addition of glutamic acid to ribosomal protein S6 (Kang et al., 1989). However, the function of this protein in archaea is unknown, as the archaea do not have a bacteria-like
S6 ribosomal protein (Galperin and Koonin 1997; Li et al., 2003b). RimK-like proteins have been shown to be tetrahydromethanopterin:α-L-glutamate ligases (MptN) in tetrahydrosarcinapterin biosynthesis (Li et al., 2003b). While M. burtonii possesses a homologue of characterised archaeal MptN proteins (Mbur_1276), Mbur_0686 has higher sequence identity with bacterial RimK. The role of RimK in M. burtonii is therefore difficult to determine. However, it is highly likely that the protein is an amino acid ligase, (probably an α-L-glutamate ligase as is E.coli RimK), with a possible role in low temperature induced posttranslational modification.
The most interesting protein associated with growth at 4 C was an atypical J-domain protein (Mbur_1212) that is unique to M. burtonii. This protein falls into the type III category in the J-domain family. Type I proteins have full domain conservation with the chaperone DnaJ (HSP40), type II contain N terminal J and G/F domains, and type III only contain a J-domain (Cheetham and Caplan 1998). The J-domain is classified as atypical as it does not follow the conserved helix structure, does not have the dominance of basic residues in helix II of typical J-domains (Walsh et al., 2004), and lacks helix
IV. The protein however, has a pair of basic residues in helix II, the highly conserved
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HPD motif, essential in the interaction with DnaK (HSP70), and conserved hydrophobic residues in Helix III (Figure 3.11).
101 EDKDIHDKRKEAREFFDCAHDENDFEAINKKYKEMAKELHPDKPTGDTEKFKQLNVAHKILKRELT 177 cchhhhhhhhhhhhhhcccccccchhhhhhhhhhhhhhcccccccccchhhhhhhhhhhhhhceec
Figure 3.11 J-domain of Mbur_1212. The domain spans residues 101 to 177 (C-terminus) and lacks helix IV. Pink indicates basic residues, red indicates conserved HPD motif and blue indicates conserved hydrophobic residues. Secondary structure was predicted with GOR IV, helixes are highlighted in magenta. h – helix, c – random coli, e – extended strand
The function of DnaJ is well characterised as a co-chaperone to DnaK. It can also:
Stimulate ATP hydrolysis; aid DnaK in protein folding; assist in protein degradation and protein complex disassembly; guide translocation; and also has regulatory functions
(Wild et al., 1992; Bukau and Horwich 1998; Wiedemann et al., 2004). However, the function of J-family proteins is less clear. It has been shown that type III J-domain proteins also interact with DnaK and accelerate ATP hydrolysis (Barouch et al., 1997), but the J-domain alone is insufficient to stimulate this activity (usually provided by the
G/F domain) (Karzai and McMacken 1996). It is hypothesised that J-domain proteins target and bind specific protein substrates, and recruit these to DnaK. Once the pre- protein is recruited to DnaK, the protein itself, along with the J-domain, stimulates APT hydrolysis and hence the function of DnaK (Karzai and McMacken 1996). This helps to promote kinetically unfavorable reactions by an increase in local concentrations of the protein substrate, DnaK, and the J-domain protein (Kelley 1998).
The exact cellular function of Mbur_1212 is cryptic. Among all characterised organisms, no homologs of Mbur_1212 could be identified through BLAST. This
D. Burg UNSW 155 protein represents a strong candidate for future research and may provide valuable insight into protein folding under kinetically unfavorable cold conditions, and may be responsible for specific targeting of a select group of proteins to DnaK for folding and processing.
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2.5 Conclusions
Through the analysis of the biological context of the proteins identified through HPP analysis, many novel aspects of the biology of M. burtonii were uncovered. This has provided a novel insight into membrane and membrane related biology in M. burtonii, and can be related to other archaea and psychrophiles. The biology came from diverse metabolic areas including: the methanogenesis pathway; cofactor biosynthesis; glycosylation and surface modifications; proteases; chaperones; and the delicate ion balance that M. burtonii has to maintain in order for optimum cell function. As a direct result of biological context analysis many interesting candidates for future research have been identified. The analysis of proteins that were associated with high or low temperatures using Scaffold2, was complementary to previous analyses, and has uncovered new thermal adaptation related proteins.
Because of the thorough HPP analysis, a platform for future work on M. burtonii has been established. It is from this platform that the analysis of data from differential proteomic experimentation can be approached (Chapter 4), and new HPP related aspects of the cold adaptation biology of the organism revealed.
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Chapter 4. Thermal and Metabolic Adaptation in the Hydrophobic Proteome of Methanococcoides burtonii
4.1 Summary
Proteomics has previously been utilised to identify cellular processes important for cold adaptation in Methanococcoides burtonii. However, very little is known about the role that hydrophobic proteins, for example membrane bound proteins, play in the cold adaptation of this model organism. The central metabolism and dynamics of substrate utilisation in M. burtonii is also poorly understood. In order to address these issues, the general method which enriched for hydrophobic proteins, described in Chapter 2, was employed with iTRAQ labelling. Using this approach proteins and processes were identified that are important to the cell when grown at different temperatures, and also when grown under different substrate and nutrient conditions.
Cold adaptation was linked to a number of processes. Translation was identified as an important process in cold adaptation, with important roles identified for: a membrane associated DEAD-box RNA helicase and ribosomes, which appear to be membrane localised under cold conditions. This may be a cellular adaptation to efficiently secrete proteins. This idea is strengthened by the identification of a large number of secreted and surface proteins that displayed increased abundance under cold growth conditions
(4ºC). Variations in protein modification and turnover were also identified as important during low temperature growth, with important roles identified for peptidyl-prolyl cis- trans isomerases, the proteasome, and a membrane bound SPFH-domain protein.
Growth of M. burtonii at a higher temperature (23ºC) resulted in an increased abundance of proteins involved in surface glycosylation, and increases in a number of hypothetical integral membrane proteins. Stress proteins were also identified, providing
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evidence for oxidative stress and thermal denaturation of proteins at elevated temperatures.
When M. burtonii was grown under different substrate and nutrient conditions, very few proteins showed differential abundance with respect to substrate alone. Many proteins also displayed differential abundance with respect to temperature, presumably reflecting effects of temperature on rates of growth and subsequent demand on metabolism.
Methanogenesis protein complexes (tetrahydrosarcinapterin S methyltransferase complex, and the methyl coenzyme-M reductase) had different subunit compositions when grown on different substrates (trimethylamine vs. methanol). Growth on methanol was also associated with an increase in abundance of energy conserving mechanisms, possibly related to difficulties in transmembrane diffusion of this solvent and the lower energy yield per mol of substrate provided by methanol. Methanol was also identified as a stressful agent to the organism, requiring the cell to modify its surface to counteract the detrimental effect of the solvent on surface proteins and membranes. This phase of the work highlighted the complexities of adaptation strategies employed by M. burtonii, not only in terms of the thermal response but also in terms of response to substrate and nutrient levels.
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4.2 Introduction
4.2.1 Cold adaptation in Methanococcoides burtonii
The major approach used to analyse the cold adaptation of M. burtonii has been proteomics. This approach utilises cells that have been adapted for a large number of generations at specific temperatures; a low temperature, 4˚C; and a high temperature,
23˚C; as opposed to the analysis of a cold shock response, where cells are rapidly cooled prior to analysis. The proteomic techniques of two dimensional electrophoresis
(2DE) (Goodchild et al., 2004b), and ICAT (isotope coded affinity tags) (Goodchild et al., 2005), have been used to analyse the difference in the protein expression profiles of whole cell extracts (WCE) at these two temperatures. These two approaches led to the detection of a variety of mechanisms of cold adaptation in the organism including: the importance of protein isomerisation, transcription and transcriptional regulation. Cells grown at higher temperatures were also found to show signs of heat stress, with evidence of protein denaturation at what is conventionally described as the optimum growth temperature (Topt) of the organism.
While there is no experimental data inferring the roles of proteins from the hydrophobic proteome in cold adaptation in M. burtonii, genomic analyses have inferred potentially important roles for these hydrophobic proteins (Allen et al., 2009). Important roles for secreted/surface proteins have also been identified using the low resolution technique of one dimensional SDS PAGE combined with LC-MS/MS in M. burtonii (Saunders et al.,
2006). Despite the above analyses identifying various mechanisms of cold adaptation in
M. burtonii, they do not represent a truly global view. There remains scope to analyse the roles in cold adaptation of proteins in the hydrophobic proteome (membrane proteins, membrane associated proteins, surface, and hydrophobic proteins), as well as
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those proteins which are secreted from the cell. Thus, in order to facilitate a truly global view of cold adaptation in M. burtonii, an approach utilising the separation of cellular material into soluble, hydrophobic and secreted proteins was implemented. This chapter of the dissertation describes the analysis of the hydrophobic portion of this global approach. The hydrophobic proteome was analysed with respect to both temperature effects on cellular protein levels, as well as analysis of metabolic aspects of
M. burtonii growth (see below). Pooled global results are available in the manuscripts
(Williams et al., Submitted-a; Williams et al., submitted-b), which are included in the supplementary material.
4.2.2 Metabolic aspects of Methanococcoides burtonii growth
The previous analyses of cold adaptation in M. burtonii identified a large number of metabolic proteins that showed differential abundance with respect to temperature
(Goodchild et al., 2004a; Goodchild et al., 2004b; Saunders et al., 2006). Whether the changes in these proteins are truly thermally related, or more related to differences in growth rate and nutrient conditions, is not well understood. M. burtonii, unlike the closely related metabolically diverse Methanosarcina spp. can only utilise a limited number of substrates for growth. Methanogenesis in M. burtonii is restricted to the use of methanol and methylamines (Franzmann et al., 1992). While these substrates have very similar pathways of utilisation, there are expected to be differences in the initial de-methylation of these substrates, with pathways converging after methyltransfer to
Co-enzyme M (Bose et al., 2006). Growth on methylamines is also expected to provide a higher energy yield per mole of substrate than methanol (Bose et al., 2006), as well as providing a nitrogen as well as a carbon source. Cells grown on methanol alone have to source nitrogen externally, which may severely limit the availability of nitrogen to the
D. Burg UNSW 161 cell, as there is a lack of identifiable ammonia transporters and nitrogenases in the M. burtonii genome (Allen et al., 2009). Methanol may also act as a stressor to the cells, as it can have detrimental effects on cellular structures (Sikkema et al., 1995; Sardessai and Bhosle 2002). It has been shown to induce stress-like responses in Methanosarcina acetivorans with cellular chaperone levels increasing in the presence of this substrate
(Li et al., 2005b).
Genetic analysis of M. burtonii has identified a distinct lack of identifiable amino acid and large molecule transporters (Allen et al., 2009), suggesting that the organism either utilises novel mechanisms of nutrient uptake, or is intrinsically geared to biosynthesise, regardless of the composition of the surrounding media. To assess this finding, cells grown in a complex (rich) media (MFM, TMA substrate), were compared to cells grown in a simple (defined) media (M-media) with methanol as a substrate. The objective was to address the issue of nutrient uptake, identify any components of methanogenesis that differ with respect to substrate, and identify signs of surface and membrane stress as a result of solvent effects.
4.2.3 Experimental approach
Proteomics has been utilised to examine cold adaptation in a number of organisms
(examples include; Seo et al., 2004; Qiu et al., 2006). However, few studies have utilised proteomics to address the roles of hydrophobic proteins in cold adaptation.
Several studies of psychrophilic bacteria, aiming at analysing hydrophobic proteome elements involved in cold adaptation or shock, have taken an approach using 2DE
(Mihoub et al., 2003; Kawamoto et al., 2007; Hongsthong et al., 2008). The gel-based methods have been shown to be low power approaches for analysis of hydrophobic proteins, due to the tendency for hydrophobic proteins to run as streaks on gels, and
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problems with post-solubilisation precipitation in isoelectric focusing (IEF) strips
(Washburn and Yates 2000; Klein et al., 2005). Similarly, there have been no focused hydrophobic proteome analyses of substrate and metabolic adaptation in the methanogens, despite the large number of membrane bound proteins that are known to be involved in methanogenesis (Ferry and Kastead 2007). The general technique for analysis of hydrophobic proteins described in Chapter 2 of this dissertation, has been described as suitable for use with affinity labelling systems (e.g. iTRAQ, ICAT) for proteome analysis (Goshe et al., 2003; Mitra and Goshe 2009). Labelling systems that target N-termini and lysine side-chains (as does iTRAQ) appear to be better suited than cysteine labelling strategies (e.g. ICAT) for analysis of membrane proteins (Bisle et al.,
2006). Therefore, the utilisation of the HPP analysis technique with the iTRAQ labelling system would provide a thorough analysis of cold adaptation in the hydrophobic proteome of M. burtonii, and represented the first truly comprehensive view of cold adaptation processes with respect to hydrophobic proteins of any cold adapted organism.
The iTRAQ method utilises a series of isobaric tags that attach to N-termini and lysine side chains of peptides (Figure 4.1a). Samples from different conditions are labeled with different tags and then combined. The tags are isotopically balanced (Figure 4.1a), so that labeled peptides are indistinguishable in liquid chromatography and the first dimension of MS. However, on collision induced dissociation the tag is fragmented, such that a reporter ion of specific mass is released (Figure 3.1b). The relative intensities of the reporter ions can be directly correlated to the relative abundance of the labeled peptide so that relative quantitation can be achieved (Ross et al., 2004).
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a b RDAE DLVLI EVDLD G
Figure 4.1 The iTRAQ labelling system. a) iTRAQ tags are balanced such that they exert no effect on
LC, and first dimension of MS (diagram from Ross et al., 2004). b) On CID, the reporter group
dissociates from the tag resulting in singly charged reporter ions. The relative intensities of these relates
to the relative abundance of the fragmented peptides.
As iTRAQ allows for a multiplex-type analysis, two samples from each test condition
were labeled simultaneously (e.g. 2 samples from 4˚C from MFM media were
compared to two samples from 23˚C from MFM media; see Appendix B.1). Technical
replicates were also performed, i.e. repeat injections. This experimental design therefore
utilised both biological and technical replicates, which have been shown to increase
proteome coverage and accuracy (Chong et al., 2006; Gan et al., 2007), resulting in a
high quality dataset.
Utilising the platform of the comprehensive analysis of the hydrophobic proteome
described in Chapter Two and Three, the hydrophobic proteome was analysed with
respect to temperature and substrate/nutrient conditions. From this analysis, the
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mechanisms of cold and substrate/nutrient adaptations were identified in the hydrophobic proteome of M. burtonii.
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4.3 Materials and Methods
All chemicals and media were prepared, unless otherwise noted, in MilliQ dH2O
(Millipore, Cork, Ireland) with a conductivity of 18.2 m /cm. Reagents, unless otherwise noted, were made with Univar, analytical grade chemicals (sourced from
APS, Seven Hills, NSW, or Ajax Finechem, Taren Point, NSW). All mass spectrometry was carried out at the Bioanalytical Mass Spectrometry Facility (BMSF), UNSW.
4.3.1 Culture media preparation
Vitamin and mineral solutions and MFM were prepared as described in 2.3.1.
4.3.1.1 M-media preparation
Parallel with the growth of M. burtonii on MFM (complex media) cultures were grown in M-medium (defined medium), prepared with several modifications to a formulation kindly provided by Prof. Kevin Sowers (Centre for Marine Biotechnology, Baltimore,
MD, USA). Preparation vessels and dH2O were prepared as described in 2.3.1.2. The following ingredients were added to the prepared dH2O in order: 23.38g/L NaCl;
12.32g/L MgSO4.6H2O (Fluka, Steinheim, Germany); 0.76g/L KCl; 0.14g/L
CaCl2.2H2O; 0.5g/L NH4Cl; 1mg/L resazurin (Sigma, St Louis, MO, USA); 10mL vitamin solution (as prepared in 2.3.1.1); and10mL mineral solution (as prepared in
2.3.1.1). The gas was changed to 80:20 N2: CO2 using a gas proportioner and bubbled for 15 minutes, after which the media should have been pink in colour. The following ingredients were added in order, making sure each was dissolved before adding the next: 0.2mL/L 10.2M HCl; 0.75g/L thioglycolic acid (Sigma, St Louis, MO, USA);
1.12g/L Na2HPO4.7H2O (ICN, Aurora, OH, USA); 0.25g/L cysteine.HCl.H2O (MP
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Biomedicals, Solon, OH, USA); and 3g/L Na2CO3 added very slowly, ~ 0.25 - 0.5g at a time, ensuring turbidity cleared before adding the next portion. The media was bubbled with gas until it appeared colourless, and the pH adjusted to 6.8 using 10.2M
HCl. Media was dispensed into serum bottles as described in 2.3.1.2, autoclaved at
121 C for 15 minutes, and placed on a shaker overnight to dissolve any precipitates formed. After the precipitate had dissolved and the media cooled, 1mL per 100mL 2.5%
Na2S (prepared as described in 2.3.1.2) was added anaerobically, followed by the required amount of methanogenesis substrate (prepared as described below). The media was allowed to equilibrate for 4 hours before inoculation.
Anaerobic absolute methanol was prepared by bubbling nitrogen for 1 hour through a serum bottle containing solvent which had been degassed using procedures described previously for dH2O (2.3.1.2). The serum bottles were sealed and capped and stored at
4 C until needed. Anaerobic 5M TMA (ICN, Aurora, OH, USA) was prepared in degassed dH2O, and bubbled with nitrogen for 30 minutes. The solution was sealed, capped, autoclaved at 121 C for 15 minutes, and stored at 4 C until needed.
4.3.2 Assessment of growth of M. burtonii in M-media on methanol
As M. burtonii had not previously been grown on methanol or in M-medium in the
Cavicchioli research laboratory, an assessment of its growth was carried out. Media were prepared as described in 3.3.1. Cultures were prepared in triplicate in M-medium with the following substrate concentrations: 150mM methanol and 50mM TMA;
150mM methanol and 5mM TMA; 150mM methanol; 300mM methanol; 25mM methanol; 50mM TMA. Cultures were also prepared in triplicate in MFM as a control.
The culture bottles were inoculated as described in 2.3.2, and incubated at 10 C (to
D. Burg UNSW 167 avoid influence of heat stress occurring at 23 C, and to avoid the decrease in culture time experienced at 4 C) in a Sanyo MIR 153 benchtop incubator (Sanyo Electric,
Japan). Absorbance readings were taken at 620nm every 48-72 hours until growth was apparent, then regularly (~every 12-24 hours), until stationary phase was reached.
Curves were then plotted using Origin 6 (Microcal Software, Northampton, MA, USA).
4.3.3 Culture conditions
Cultures to be used in the comparative proteomics experiments were prepared in both
MFM and M-media (25mM methanol, a further 25mM added at onset of visible growth) at 4 C and 23 C. Cultures were inoculated anaerobically as described in 2.3.2 and incubated to an optical density of 0.25 at 620nm. Cultures were harvested as described in 2.3.3 and stored at -80 C.
4.3.4 Hydrophobic protein extraction and protein measurement
Hydrophobic proteins were extracted from the cells as described in 2.3.4. However as ambic interferes with iTRAQ labelling (which reacts with amines), all ambic steps were replaced with 25mM NaHCO3. Protein concentration was measured using the Bradford assay as described in 2.3.6.
4.3.5 Preparation of samples for iTRAQ labelling
Samples containing 50 g of protein were reduced, alkylated, solubilised and digested as described in 2.3.8.1 and 2.3.8.2, with the addition of 1 L 2% SDS prior to reduction as described in the iTRAQ kit manufacturer’s instructions (Applied Biosystems Forster
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City, CA, USA). All other reagents provided with the kit were not used as they interfered with the mass spectrometry (as recommended by BMSF staff).
4.3.5.1 iTRAQ experimental design
The iTRAQ system (Applied Biosystems Forster City, CA, USA) enables samples to be labelled in a 4-plex manner (with tags of (m/z) 114, 115, 116, 117). To achieve maximum output from the experimentation, samples from M-media and MFM were prepared in separate parallel labelling experiments, with two samples from each temperature prepared per labelling run (Appendix B.1). This approach provided a biological replicate for each condition in each labelling experiment, and discrete data sets for MFM and M-media cultures.
In order to assess the effect of substrate on the growth of M. burtonii, the above labelling protocol was repeated with the following changes; two cultures grown in M- media at 4˚C were compared with two cultures grown in MFM at 4 ˚C. Cultures were also compared in a similar manner for cells grown at 23˚C (Appendix B.2).
4.3.5.2 iTRAQ labelling
Samples were labelled with the isobaric tags as described in the iTRAQ kit handbook
(Applied Biosystems, Forster City, CA, USA). Dried peptide samples were resuspended in 30 L of 25mM NaHCO3. Each iTRAQ reagent was then dissolved in
70 L ethanol and the tubes mixed by vortexing. Each reagent was then added to its corresponding sample tube, the tubes mixed and the samples were incubated at room temperature for 1 hour. After incubation the contents of the tubes were transferred to a single 15 mL tube mixed and prepared for ‘clean up’.
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4.3.6 Sample clean up
In order to remove any unbound iTRAQ labels, trypsin, SDS, solvents and other substances interfering with LC/LC-MS/MS, SCX and RP chromatography clean up steps were performed as described in 2.3.8.3. The only variation on the methods described was that samples were diluted to 6mL with cation exchange load buffer prior to loading onto the column.
4.3.7 Mass spectrometry
Mass spectrometry was performed as described in 2.3.9. When performing iTRAQ experiments, initial LC-MS/MS was inspected for the presence of the iTRAQ reporter ions as an initial measure of labelling success. All experiments were performed with two LC/LC-MS/MS runs, providing a technical replicate. Six labelling experiments were performed for each culture media, which resulted in 12 biological replicates and a total of 12 LC/LC-MS/MS runs.
4.3.7.1 Data processing and visualisation
Data were processed, spectra manually verified and FDR assigned as described in
2.3.9.4. iTRAQ abundance data were processed using Analyst QS software (Build
7051) (Applied Biosystems, Foster City, CA, USA) and searched against the local M. burtonii interrogator database with settings of: MS tolerance of 0.2Da; MS/MS tolerance of 0.15Da; store hits with confidence > 99%; charge range for intermediate precursors of ≥ 2, ≤ 4; maximum number of missed cleavages of 1; modifications of, iTRAQ reagents, acetamidomethyl-cysteine, methionine sulfoxide, with maximum number of modifications set to 8; and correction factors entered from the iTRAQ certificate of analysis.
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Data were visualised using ProGroup Viewer version 1.0.5 (Applied Biosystems, Foster
City, CA, USA), with ProQuant search settings as with the Analyst QS program described above. Data were inspected for variation in abundance (p < 0.05), with error factor (EF) values less than 2. In order for a protein to be classified as differentially abundant it had to pass the above criteria with a variation of > 1.5 fold (Yu et al., 2007), in three or more individual experiments (including technical replicates). In certain instances, where ratio values were borderline (e.g 1.45 fold), previous results across all experiments were cross referenced. If variation had occurred in the protein at a > 1.5 fold level in other experiments (e.g. in different media), then the borderline result was included in the dataset. This was useful in the comparison of variation across all conditions tested. The mean and standard deviation of differential abundance ratios between experimental sets were calculated as described by Zhou et al., (2007) using iTRAQ error factors to weight individual iTRAQ ratios (see Appendix B.3). A weighted
SD of > 0.1 indicated significant variation in the sample.
4.3.8 Data annotation and analysis
All protein identification and quantitation data were analysed and annotated as described in 2.3.10 and 3.3.
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4.4 Results and Discussion
4.4.1 Growth response of M. burtonii to different substrates
To determine the growth responses of M. burtonii to culture in M-media and on methanol, growth curves for various substrate compositions were compared (Figure
4.2). Growth was measured at 10˚C to reduce the effects of heat stress (at 23˚C) and to decrease the time required for the experiment (due to slow growth of cultures at 4˚C)
0.40
0.35
0.30
0.25
0.20
0.15
OD 620nM 0.10
0.05
0.00
-0.05 0 500 1000 1500 2000 2500 3000 3500 Time (Hours)
150mM MeOH 50mM TMA 150mM MeOH + 50mM TMA 150mM MeOH + 5mM TMA 25mM MeOH, spiked to 50mM at T=1520 hrs MFM
Figure 4.2 Growth media and substrate comparison. Cultures were grown in triplicate and OD620 measured periodically. High concentrations of methanol retarded the growth of cells, no growth was observed at a concentration of 300mM methanol. At 10˚C a low concentration of methanol produced a similar growth curve to cells grown on TMA and MFM media.
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The growth curves indicated several interesting features of the growth of M. burtonii on methanol. High concentrations of methanol had an inhibitory (toxic) effect on the growth of the organism; M. burtonii appeared to be unable to switch substrates from
TMA to methanol; and at 10˚C the growth of M. burtonii on low concentrations of methanol was on par with cells grown on TMA.
The toxic effects of methanol on the cell were evident, as cells innoculated in media with high concentrations (300mM) of this substrate failed to grow. At lower concentrations (150mM alone) cells grew at a slower rate, and reached a lower maximum OD than cells grown on TMA or 25mM methanol. This inhibitory effect is also evident when cells were grown on 150mM methanol + 50mM TMA; cells reached a much lower final OD than cells grown on 50mM TMA alone, suggesting that despite the presence of adequate carbon and nitrogen sources growth was suppressed by the
MeOH present.
The growth curves also indicated that M. burtonii was unable to rapidly switch substrates from TMA to methanol. It appears that M. burtonii utilises TMA preferentially, evidenced by the length of the lag phase and an initial rate of growth similar to TMA utilisation alone. However, it appears that that methanol is not utilised in later stages of growth, if at all. When cells were grown on 5mM TMA + 150mM methanol and 50mM TMA + 150mM methanol, the cultures produced growth curves of similar shapes. However, cells grown on the lower concentration of TMA reached a lower maximum OD than cells grown on 50mM TMA. In Methanosarcina spp., TMA is used preferentially to methanol in early stages of growth followed by simultaneous utilisation of TMA and methanol in later growth (Bose et al., 2006). In these species
D. Burg UNSW 173 growth on methanol and substrate switching is governed by the presence of multiple isoenzymes for the methanol corrinoid proteins and methyltransferases, which are proposed to have differing roles with respect to substrate switching and utilisation (Bose et al., 2006). Three known types of the methanol methyltransferase/ methanol corrinoid gene pairs are present in the M. acetivorans genome. Of these the MtaCB3 gene pair is thought to be involved in the switching of substrates from TMA to methanol (Bose et al., 2006). The M. burtonii genome has only two copies of the MtaCB gene pairs
(Appendix B.4). While the lack of a third MtaCB gene pair in M. burtonii may be responsible for the apparent inability of this organism to switch substrates ‘mid-growth’ from TMA to methanol, it is more likely to be due to a regulatory issue. The methanol methyltransferase/corrinoid protein pairs in M. acetivorans are regulated by a set of transcriptional activators and repressors located in close proximity to the MtaCB1 and
MtaCB2 gene pairs (Bose and Metcalf 2008). The M. burtonii genome does not encode any homologues of these regulators, with the exception of several within the methylamine gene loci (Appendix B.4), which may be involved in the regulation of the methylamine methyltransfer / corrinoid isoenzymes. The apparent lack of genes for the regulation of methanol metabolism and substrate switching may reflect a possible lack
(or an extremely low concentration) of methanol in Ace Lake, which would result in the loss of these genes. In general, the major source of environmental methanol is from the degradation of pectin and lignin by bacterial methyl esterases (Schink and Zeikus 1980).
These polymeric substances are produced by plants and higher algae, none of which are found in Ace Lake (Burch 1988; Coolen et al., 2004b). Methanol can also be produced through methane monooxygenase by aerobic metanotrophic bacteria, however this activity has not been reported in Ace Lake (Bowman et al., 1997; Coolen et al., 2004a).
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When M. burtonii was initially described, growth on 44mM methanol at 20˚C was accompanied by a long lag phase (Franzmann et al., 1992). The experiments described above, indicated that growth on 25mM methanol at 10˚C had very similar lag times and growth rates to cells grown on TMA. At higher temperatures, the difference in growth times between MeOH and TMA becomes apparent; growth at 23˚C on methanol took approximately three fold longer than growth on TMA. M. burtonii appears to have a lower Topt when grown on methanol than TMA, and this value is proposed to be between 10˚C and 16˚C (D. Burg and T. Williams, unpublished data).
There was very little difference in the growth curves between cells grown on defined media and complex (yeast extract enriched) media (MFM); it appears that the cells receive little nutritional benefit from the inclusion of amino acids, peptides, and other complex components in the media, reflecting the lack of identifiable amino acid and large molecule transporters in M. burtonii (Allen et al., 2009).
As a result of these observations, all cultures grown in methanol M-media for proteomic analysis were incubated with 25mM MeOH, until the onset of visible growth (~ OD
0.060 – 0.1), after which the methanol concentration was increased to 50mM. Culture times (1:100 inoculation from actively growing cultures to an OD620 of 0.25) on this regime were; in M-medium 23˚C, ~20 days (480 hours), and 4°C ~ 65 days (1560 hours). In contrast, M. burtonii cultures grown in MFM at 23°C took ~ 6 days (144 hours); 4°C growth in MFM took ~ 60 days (1440 hours).
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4.4.2 Differentially abundant proteins identified through iTRAQ
The experiments performed resulted in a complicated data set, where many proteins varied not only according to temperature, but also to substrate and nutrient conditions
(Figure 4.3).
Figure 4.3 Differentially abundant proteins across all experiments. Very few proteins displayed variation solely with respect to substrate. The large overlap between temperature and substrate experiments is expected to be due to thermal effects on rate of growth and metabolism, as well as the stressful effects of methanol. The temperature experiments alone, accounted for 77 differentially abundant proteins, 34 of these were common to both media conditions and are considered to be the core cold adaptation proteins.
Likewise the substrate experiments alone accounted for 59 differentially abundant proteins, 22 of these were common to both temperatures, and are considered to be the key proteins in the metabolic response.
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Overall 100 proteins were identified as differentially abundant. Of these, 40 showed variation exclusively in temperature experiments; within this thermally associated cohort, 28 proteins showed increased abundance at 23˚C, while 12 had increased abundance at 4˚C. Very few (13) proteins displayed variation according to media conditions alone, and of these 4 had increased abundance in MFM, while 9 had increased abundance in M-media. There was significant overlap between the experimental sets, with many proteins varying according to both temperature and media conditions. The large overlap was attributed to: the combination of thermal effects on rates of growth and metabolism; as well as substrate effects, resulting from either utilisation of different methyltransfer reactions, or the lower energy yield of methanol per mol of substrate (Bose et al., 2006). For example many proteins that differed according to temperature and substrate were methanogenesis proteins (see 4.4.4.1), and energy conserving mechanisms (see 4.4.4.2). As well as thermal and metabolic effects, the stressful effects of methanol on the cell (see 4.4.4.4) also accounted for some of the variation of proteins according to both temperature and substrate. Responses related to thermal stress and surface modification were also found to be associated with growth on methanol, as was observed in methanol grown M. acetivorans (Li et al., 2007).
Of the proteins that were differentially abundant as a function of temperature, 34 displayed differential abundance across both experimental sets and are considered the core cold adaptation proteins. However, other proteins that showed confident variation in only one of the experimental data sets may also be relevant, and were also considered. The samples tested through LC/LC-MS/MS were complex, containing many thousands of peptides, which may have resulted in subtle variations in each experimental run; by chance, proteins may not have been identified confidently.
Additionally, many proteins that displayed differential abundance according to only one
D. Burg UNSW 177 condition in the hydrophobic proteome, also varied in the other or both conditions in other sub-cellular fractions in the global analysis (Williams et al., Submitted-a;
Williams et al., submitted-b).
The enrichment of the samples for hydrophobic and membrane proteins, allowed the identification of many proteins and processes not identified in previous experiments
(4˚C vs. 23˚C, MFM media using ICAT (Goodchild et al., 2005), 2DE (Goodchild et al., 2004b), and 1DE analysis of secreted proteins (Saunders et al., 2006)). Of the identified proteins, 78 (out of 100) proteins were not identified in previous experiments; of these 68 varied with respect to temperature; and of these 48 varied in MFM media
(which was used in previous analyses). The enrichment strategy allowed for the identification of: surface and/or secreted proteins (15 identified, 14 NIW); integral membrane proteins and those which are associated to the membrane through a complex
(27 identified, 25 NIW); and proteins and processes with previously unidentified membrane associations (for example DEAD-box RNA helicase, ribosomes).
3.4.3 The thermal response in Methanococcoides burtonii
From the proteomic dataset, a range of proteins were identified as important to the thermal response of M. burtonii; either as cold adaptation proteins or proteins involved in a heat stress response. The relevant biological categories considered were: transporters and integral membrane proteins; surface and secreted proteins; translation and related processes; protein folding, modification and turnover; transcription, replication and DNA repair; and heat induced stress. An overview of thermal adaptation with respect to the hydrophobic proteome is shown in Figure 4.4.
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Figure 4.4 Overview of thermal responses in the M. burtonii hydrophobic proteome. The left side of the diagram represents proteins with increased abundance at 4˚C, while the right represents proteins with increased abundance at 23˚C
4.4.3.1 Transporters and integral membrane proteins
A number of integral membrane proteins and transporter components were identified as differentially abundant with respect to temperature. The majority of these, including a number of hypothetical proteins, had increased abundance at 23˚C (Table 4.1).
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Table 4.1 Differentially abundant transporters and integral membrane proteins
Ratio 4˚C/23˚C MFM M-media Gene locus Annotation TMD (TMA) (MeOH) Glycine betaine ABC transporter substrate binding Mbur_0503 - 1.961 1.718 protein opuAC (ER2) ABC transporter, solute-binding protein Mbur_2131 - - 0.525 (sulfonate/nitrate) (ER4) Mbur_0779 Ferrous iron transport protein B (ER2) 8 0.26 0.274 Mbur_1579 Transmembrane oligosaccharyl transferase (ER3) 13 0.519 - Mbur_1608 Glycosyl transferase, family 2 (ER4) 4 0.342 - von Willebrand factor-domain membrane protein Mbur_0801 2 - 0.414 (ER4) von Willebrand factor-domain membrane protein Mbur_0802 3 - 0.482 (ER4) Mbur_0140 Hypothetical protein (ER5) Unique to M. burtonii 1 0.517 - Mbur_0319 Hypothetical protein (ER5) 2 0.279 - Mbur_0343 Hypothetical protein (ER5) 1 0.47 - Mbur_1718 Hypothetical protein (ER5) 1 0.502 - Mbur_1853 Hypothetical protein (ER5) 3 0.339 - Mbur_1970 Hypothetical protein (ER5) 5 0.411 -
Bold gene loci and annotations indicate proteins that display differential abundance in both media tested.
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C.
The only identified transporter component with increased abundance at 4˚C was the
‘periplasmic’ binding protein (Mbur_0503) of an ABC transporter similar to those
involved in the uptake of glycine betaine. As M. burtonii does not have a canonical
periplasmic space, it is assumed that this protein operates in the region between the cell
membrane and S-layer; the ‘quasi-periplasmic space’ (Konig et al., 2007).
Glycine betaine is a major compatible solute of methanogens (Muller et al., 2005).
However, it cannot be synthesised and has to be transported (Sowers and Gunsalus
1995). As glycine betaine generally occurs in low abundance in the environment, its
transport is mediated by high affinity ABC transport systems (Pfluger and Muller 2004).
The compatible solute is generally associated with salt tolerance (Sowers and Gunsalus
1995; Pfluger and Muller 2004), and operates by maintaining the intracellular osmotic
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potential and constant turgor pressure. Compatible solutes also have a role in maintaining stability of proteins under high salt conditions, an effect that could be explained by the preferential exclusion model. This hypothesis predicts that compatible solutes are excluded from the immediate hydration shell of proteins. The resulting disequilibrium provides a thermodynamic force to minimise the surface of the protein and reduces the amount of hydration water, stabilising the native structure of proteins and favouring the formation of protein assemblies. It has been suggested that the stabilising activity may also be involved in the cold adaptation/shock response of many organisms (Schiefner et al., 2004). Glycine betaine is known to be involved in cold responses in a number of organisms, including the psychrotolerant bacteria: Bacillus subtilis (Budde et al., 2006); and Listeria monocytogenes (Ko et al., 1994). In L. monocytogenes, accumulation of glycine betaine was demonstrated following the addition of the solute to the growth media, and was associated with increased growth rates at low temperatures (Ko et al., 1994). Glycine betaine transport is increased in E. coli following low temperature acclimatisation (Polissi et al., 2003), as well as in plants
(Zhang et al., 2005). It is also thought to be utilised in eutectophiles (1.2). However, this may be a salt tolerance mechanism, utilised to enhance survival in brine veins rather than a cold response (Riley et al., 2008).
In M. burtonii only the solute binding protein of the glycine betaine ABC transport system was identified as differentially abundant. This is consistent with findings following cold shock in Pyrococcus furiosus, where ABC substrate binding proteins
(and not other components) were increased in abundance following temperature downshift (Weinberg et al., 2005). It indicated that increased uptake was mediated by increased solute capture rather than through an increase in the total number of complete transporter complexes.
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The ‘periplasmic’ binding protein of a putative sulfonate/nitrate ABC transport system
(Mbur_2131) was increased in abundance at 23˚C. A similar finding has been observed following temperature up-shift in Yersinia pestis (Pieper et al., 2008). The increase in abundance of this protein at high temperatures may reflect increased growth rate and subsequent demand for the inorganic ions captured by this binding protein.
Also associated with growth at 23˚C, was the ferrous ion transport protein B
(Mbur_0779). In anaerobic environments iron is available in the soluble, reduced, ferrous (Fe2+) form (Kammler et al., 1993). The ferrous iron is transported into the cell where is captured and oxidised (to Fe3+) by ferritins. The role of these ferritins in a high temperature related oxidative stress response is discussed in 4.4.3.6.
Two proteins identified as increased in abundance at 23˚C are predicted to have functions in glycolsylation. Mbur_1579 (oligosaccharyl transferase), which is a homologue of the characterised AlgB protein from Haloferax volcanii (Abu-Qarn et al.,
2007), presumably has a direct role in surface protein / cell wall modification, by addition of oligosaccharides via N-linked glycosylation. Also linked to this process is
Mbur_1608 (glycosyltransferase family 2). Glycosylation has been suggested to have an important role in rigidifying the S-layer of thermophiles, stabilising them at high temperatures (Engelhardt and Peters 1998; Konig et al., 2007). Increased glycosylation has also been reported after growth at higher than optimum temperatures in the bacteria
Geobaccillus stearothermophilus (Egelseer et al., 2001). In M. burtonii the evidence of glycosylation occurring at high temperatures may be reflective of a response to correct or minimise thermal damage to the S-layer.
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Two von Willebrand factor A (VWA) domain containing proteins were identified as having increased abundance at 23˚C (Mbur_0801, 0802). Also associated with these proteins is a MoxR AAA+ ATPase (Mbur_0804) which also had increased abundance at 23˚C (Williams et al., submitted-b), and is encoded in the genome in a putative operon with the VWA domain proteins. VWA domain proteins are widespread among the prokaryotes (Ponting et al., 1999), and are commonly associated with MoxR AAA+
ATPases (Snider and Houry 2006). Common attributes of these proteins include chaperone functions for protein complex assembly, metal insertion, and protein binding roles (Ponting et al., 1999; Snider and Houry 2006). In M. burtonii the VWA domain proteins may work in association with the MoxR AAA+ ATPase as chaperones, acting to aid in the correct insertion of thermally compromised proteins into the membrane and
/ or the assembly of heat effected integral membrane protein complexes.
A striking feature of growth of M. burtonii at 23˚C is the increased abundance of a number of integral membrane proteins with no known or predictable function. Of these
Mbur_0140 is predicted to be unique to M. burtonii (Allen et al., 2009).
4.4.3.2 Surface and secreted proteins
In direct contrast to the number of integral membrane proteins and transporters identified as having increased abundance at 23˚C, the identified surface and secreted proteins of M. burtonii almost exclusively displayed increased abundance at 4˚C; though many of these had no predictable function (Table 4.2).
Many archaea do not have bacteria-like cell walls, but rather produce an S-layer that is composed of proteins and/or glycoproteins, which form a regular, highly stable protective array (Engelhardt and Peters 1998; Claus et al., 2002). The methanogenic
D. Burg UNSW 183 archaea have the ability to alter the composition of their S-layers with respect to growth phase and conditions, displaying heterogeneity that is associated with adaptation to environmental change (Mayerhofer et al., 1995; Mayerhofer et al., 1998; Claus et al.,
2002). While there has been extensive research performed on the S-layers of mesophiles, thermophiles and hypothermophiles (Engelhardt and Peters 1998; Akca et al., 2002; Claus et al., 2002), very little is known about the S-layers of psychrophiles.
The rigid S-layers of thermophiles are thought to provide protection to cells growing under these very hot conditions (Engelhardt and Peters 1998). Lipid unsaturation, providing membrane flexibility, is a feature of many psychrophiles (see 1.6.1) including
M. burtonii. However this can result in loss of membrane integrity as unsaturated lipids do not pack regularly. Modification of S-layer protein composition in cold conditions
(rather than the protein glycosylation seen at high temperatures), therefore may act as a protective measure to compensate for a modified and potentially compromised membrane. A ‘reinforced’ S-layer may also be crucial for protection against potentially damaging ice crystal formation.
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Table 4.2 Differentially abundant surface and secreted proteins
Ratio 4C/23C M-media Gene locus Annotation TMD MFM (TMA) (MeOH) Mbur_0268 Protein with duplicated DUF1608 (ER4) 2 - 1.818 Mbur_1690 Protein with duplicated DUF1608 (ER4) - 1.475 1.835 Mbur_0295 DUF306 protein (ER4) - 1.616 2.89 Mbur_0513 Hypothetical protein (ER5) 1? 1.828 4 Mbur_0660 Hypothetical protein (ER5) - - 1.887 Mbur_1109 Hypothetical protein (ER5) - - 5.682 Mbur_1350 Hypothetical protein (ER5) - - 3.049 Mbur_1403 Hypothetical protein (ER5) - - 0.219 Hypothetical protein (ER5) unique to M. Mbur_2063 burtonii - 3.311 3.3
Bold gene loci and annotations indicate proteins that display differential abundance in both media tested.
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C
Two DUF1608 (S-layer related) proteins were increased in abundance at 4˚C. These proteins may be involved in ‘reinforcing’ the S-layer, or alternatively have a role in cell- cell interactions (Jing et al., 2002). At low temperatures M. burtonii forms extensive clumps in the growth media (see Chapter 4 and; Reid et al., 2006), an interaction that could be mediated by S-layer related, and other surface proteins.
As well as the DUF1608 proteins identified in this study, a hypothetical protein containing a putative hydrophobic anchor site (Mbur_0513) was found to be associated with growth at 4˚C. The predicted anchor site, found at the C-terminus of the protein is highly conserved among several other M. burtonii proteins, all of which are functionally predicted to be membrane anchored, and several of which are associated with growth at 4˚C (Williams et al., submitted-b) (Figure 4.5). This moiety may be a lipid anchoring, or sorting sequence site in these M. burtonii proteins. Lipid anchoring has been reported in haloarchaea as a post translational modification following protein translocation (Konrad and Eichler 2002).
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Mbur_0060 DDTEVILTDN----EIPEFPTVALPIAAIIGLAFFFQRRKNE 468 Mbur_0059 ---EVVGNGNGNGNEIPEFPTVALPIAAIIGLAFFFQRRKNE 444 mbur_0886 ----QFGYDRCTSEEIPEFPTVALPIVAIIGLTFIFQRRKEE 208 Mbur_0513 -IEFPVIVRTCQEEEIPEFPTIALPVAAIIGLAFFMQRRKD- 340 Mbur_0714 --EVDSASRTI---EVPEFPTIALPVAAIIGLAFFLQRRKEE 167 mbur_0649 -VEANPMHPVN-TEEIPEFPTVALPVAAIIGIAFFFQHRKE- 193 Mbur_2441 -GASVKVADGGCGQEIPEFPTIALPVVAILGLAFIFMRRKE- 193 Mbur_1273 DIKFDMLNAYLP-EEIPEFPTIALPILSVLGLMFFLQRRK-- 275 Mbur_2003 ---NEVTKKWIVSEEIPEFPTIALPVMAVLGLMFLTMRRREE 462 *:*****:***: :::*: *: :*:
Figure 4.5 Putative membrane anchoring site. A highly conserved C-terminal sequence was found in several M. burtonii proteins that are predicted to be membrane anchored. Many of these display increased abundance at 4˚C (bold gene loci). In this highly conserved region a stretch of hydrophobic amino acids (red) is followed by a short (2 – 3 aa) stretch of basic residues (pink)
Several other secreted hypothetical proteins displayed increased abundance at 4˚C. Of these, Mbur_2063 is unique to M. burtonii (Allen et al., 2009), and Mbur_0660 appears to contain a copper binding domain.
4.4.3.3 Translation and related processes
A striking feature of growth at 4˚C in M. burtonii is the number of proteins related to translation that display increased abundance across both media tested (Table 4.2).
The archaeal ribosome is an evolutionary mosaic that has both bacterial and eukaryotic features, with ribosomal proteins having similar primary structure to the eukarya, while the genes encoding these proteins are organised in a similar fashion to bacteria (Sano et al., 1999; Yang et al., 1999). Archaeal ribosomes are organised into two subunits (large and small), with sizes similar to their bacterial counterparts (50s and 30s respectively).
A large number of ribosomal subunits were found to have increased abundance in the insoluble fraction at 4˚C. Despite ribosome composition modification being described
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as a feature of M. burtonii, (Thomas and Cavicchioli 2000; Thomas et al., 2001), previous proteomic experiments on WCE identified very few ribosomal proteins
(Goodchild et al., 2004b; Goodchild et al., 2005), and few were identified in soluble fraction analysis that accompanied this work (Williams et al., submitted-b). However, given that there is a marked increase in abundance of surface and secreted proteins at
4˚C (see 3.4.3.2) it could be that this is due to an increased membrane association of the ribosomal complex at 4˚C. During protein secretion or membrane insertion, archaeal ribosomes have been demonstrated to be membrane-associated with the Sec-based translocation complex functioning as the ribosome receptor via the signal recognition particle (Ring and Eichler 2004b; Egea et al., 2008a; Egea et al., 2008b).
Only certain ribosomal proteins (rproteins) consistently displayed increased abundance at 4˚C. A similar phenomenon has been observed at the transcript level following cold shock in B. subtilis (Kaan et al., 2002). Therefore, in order to determine any possible additional functions, apart from the proposed role in secretion, these were further investigated (Table 4.4). The rproteins L4P, S19E and S8E are known to be involved in the early assembly of the ribosome complex (Yeh and Lee 1998; Malygin et al., 2000;
Tishchenko et al., 2001; Yang et al., 2005). The increased abundance of these proteins suggests a drive towards ribosome assembly at low temperatures. The rproteins L15P and S13E, are known to be associated with cold adaptation and tolerance, with L15P and S13E increased in plants in cold conditions (Kim et al., 2004; Lang et al., 2005;
Zhang et al., 2005), and S13E from an algae able to enhance freezing tolerance in E. coli (Tanaka et al., 2001). Another rprotein involved in thermal adaptation is S8E, which is thought to modulate flexibility (or rigidity) of the SSU and LSU interactions with respect to temperature (Gruber et al., 2003).
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Table 4.3 Differentially abundant proteins involved in translation
Ratio 4˚C/23˚C M-media Gene locus Annotation MFM (TMA) (MeOH) Mbur_0002 LSU ribosomal protein L4P (rpl4p) (ER2) 2.203 1.715 Mbur_0018 LSU ribosomal protein L32E (rpl32e) (ER2) 3.003 0.459 Mbur_0019 LSU ribosomal protein L19E (rpl19e) (ER2) 2.132 1.701 Mbur_0023 LSU ribosomal protein L15P (rpl15p) (ER2) 1.531 1.715 Mbur_0115 LSU ribosomal protein L39E (rpl39e) (ER2) 0.556 0.262 Mbur_0192 LSU ribosomal protein L15E (rpl15e) (ER2) - 2.024 Mbur_1422 LSU ribosomal protein L10AE (rpl10ae) (ER2) - 0.538 Mbur_1947 LSU ribosomal protein L10E (rpl10e) (ER2) 2.457 - Mbur_2388 LSU ribosomal protein L18E (rpl18e) (ER2) 2.899 1.603 Mbur_0036 SSU ribosomal protein S13P (rps13p) (ER2) 2.309 1.712 Mbur_0118 SSU ribosomal protein S19E (rps19e) (ER2) 1.61 1.701 Mbur_1172 SSU ribosomal protein S7P (rps7p) (ER2) 1.621 - Mbur_1173 SSU ribosomal protein S12P (rps12p) (ER2) 2.283 - Mbur_2101 SSU ribosomal protein S8E (rps8e) (ER2) 1.488 3.003 Mbur_2336 SSU ribosomal protein S3Ae (rps3Ae) (ER2) - 0.412 Mbur_2345 SSU ribosomal protein S15P (rps15p) (ER2) 2.128 0.31 Mbur_1897 translation initiation factor aIF-5A (ER2) - 0.398 Mbur_2186 Alanyl-tRNA synthetase (EC 6.1.1.7) (ER3) 2.183 - Mbur_0245 DEAD-box RNA helicase (ER2) 2.564 - Mbur_1950 DEAD box RNA helicase (ER2) 2.155 3.534
Bold gene loci and annotations indicate proteins that display differential abundance in both media tested.
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C.
Ribosomal protein annotation follows the following pattern: subunit (L – large, S – small); ribosomal protein number; protein type (E – eukayotic-like; B – Bacteria like; A – archaeal)
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Table 3.4 Reported roles for differentially abundant rproteins
rProtein associated with subunits L4P, S19E, Early assembly S8E Cold tolerance and L15P, S13E adaptation Flexibility S8E L15P, L18E, Increase in growth rate S19E Increase in rate and L19E, L15E, efficiency of translation L18E, S13P Associates with DExD L4P helicase
The rproteins L15P, L18E and S19E are involved in the acceleration of growth rate
(Faliks and Meyuhas 1982; Franceschi and Nierhaus 1990; Miyake et al., 2008). The increased abundance of these in low temperatures may be indicative of an attempt to increase growth rate through protein synthesis under kinetically unfavourable conditions. Similarly, several rproteins increased abundance at low temperatures in M. burtonii, are also known to be involved with an increase in the rate and/or efficiency of protein synthesis. Several of these are known to be increased in abundance in cancer cell lines, a feature of which is increased protein synthesis. These rproteins include
L19E, L15E, S13P and L18E (Henry et al., 1993; Davies and Fried 1995; Kumar et al.,
1999; Cukras et al., 2003; Cukras and Green 2005; Bee et al., 2006; Wang et al., 2006).
The rprotein L4P is also known to interact with a DExD helicase (Yang et al., 2005), two of which are increased in abundance at 4˚C and will be discussed below.
Several ribosomal proteins however, displayed increased abundance at 23˚C, of these
L39E is known to be involved in maintaining accuracy of translation (Dresios et al.,
2000). The increased abundance of this protein at 23˚C could reflect heat induced errors during the translation process.
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Overall there is complicated role for rproteins in the cold adaptation of M. burtonii under thermodynamically unfavourable conditions. An increase in the assembly of ribosomes would act to increase the net number of these macromolecules in the cell, counteracting slow rates of protein synthesis at low temperatures (Sahara et al., 2002).
The modulation of the composition of the ribosome may also be involved in increasing the efficiency and rate with which the ribosome is able to synthesise proteins, counteracting kinetically unfavourable conditions. Proteins involved in the flexibility of the interaction of the ribosomal subunits may also be important for cold adaptation counteracting the rigidifying effects of cold temperatures on proteins and rRNA.
Ribosomal proteins may also have accessory roles separate from translation, for example as RNA chaperones (Jones et al., 1996).
While the composition of the ribosome tended mainly to be influenced by temperature, several subunits varied according to the growth media (e.g. L32E, S15P). This phenomenon has been identified in other methanogens (Li et al., 2005a; Li et al., 2007).
The translation initiation factor aIF-5A (Mbur_1897) had increased abundance at 23˚C.
The identification of increased amounts of this protein, which is sensitive to heat stress
(thermally unstable) (Takeuchi et al., 2002; Gosslau et al., 2009), in 23˚C extracts of M. burtonii could reflect an increased pool of thermally compromised (denatured) protein in the cell, or an increase in the synthesis of this protein to counteract a thermally induced decrease in protein half life (Gosslau et al., 2009).
Two DEAD-box RNA helicases had increased abundance at 4˚C (Mbur_0245, 1950).
Of these Mbur_1950 was previously implicated in the cold adaptation of M. burtonii, as transcripts of this gene were found to be expressed exclusively at 4˚C (Lim et al., 2000).
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Mbur_1950 was, however, not identified in any previous proteomic investigations of cold adaptation in M. burtonii. The DEAD-box helicase was one of the most abundant proteins in hydrophobic fractions, and DSF analysis indicated a strong membrane association (see 2.4.4).
RNA helicases unwind double-stranded RNA, affecting the rearrangement of RNA secondary structure, traditionally associated with activation of RNA function (Owttrim
2006). The DEAD-box family of RNA helicases has an extremely wide range of cellular functions in all three domains of life, with roles in all cellular process involving RNA metabolism including: maturation; ribosome biogenesis; RNA splicing; transport and turnover; transcription; translation initiation, RNA interference; and RNA editing
(Cordin et al., 2006; Owttrim 2006). Multiple roles have also been observed for individual DEAD-box helicases, including transcriptional regulation, in addition to the central helicase activity (Fuller-Pace 2006). Many DEAD-box helicases, despite having similar sequences, display very little functional complementarity, leading to the proposal that each unique helicase has a different function (Owttrim 2006). This suggests that the two identified DEAD-box helicases in M. burtonii have different roles in the cell.
DEAD box helicases are associated with a cold shock response in a number of organisms, including: E. coli (Jones et al., 1996; Polissi et al., 2003); Vibrio parahaemolyticus (Yang et al., 2009); Bacillus subtilis (Beckering et al., 2002; Hunger et al., 2006); cyanobacteria (Chamot et al., 1999); and in the archaeons Pyrococcus furiosus (Weinberg et al., 2005), and Methanococcus jannaschii (Boonyaratanakornkit et al., 2005). Under cold conditions, mRNA forms stable secondary structures which inhibit translation initiation. These structures are thought to be de-stabilised by cold induced DEAD-box helicases maintaining proper translation initiation (Jones et al.,
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1996; Chamot et al., 1999; Hunger et al., 2006). DEAD-box helicases also associate with cold shock proteins (CSP), where they act as RNA co-chaperones (Jones et al.,
1996; Jiang et al., 1997; Hunger et al., 2006). Although the M. burtonii genome does not encode any traditional CSP, two proteins with cold shock domain folds have been identified as increased in abundance at 4˚C, and are thought to be structural homologues of CSP (Goodchild et al., 2004b). These cold shock domain proteins have been shown to complement Csp knockouts in E. coli (Giaquinto et al., 2007). A similar role in mRNA chaperoning may be occurring in M. burtonii. However, it has also been proposed that small TRAM domain proteins may be functioning as RNA chaperones in
M. burtonii in concert with the DEAD-box helicase(s) (Williams et al., submitted-b).
The apparent membrane localisation of the DEAD-box helicase in M. burtonii is similar to the cold shock induced helicase in Anabaena sp. (El-Fahmawi and Owttrim
2003). Given that the ribosome has been implicated as membrane associated in M. burtonii grown at 4˚C, and that ribosomal proteins are known to associate with DEAD- box helicases (Jones et al., 1996; Yang et al., 2005), it may be that these proteins are positioned together at specific membrane sites as a measure to increase the efficiency of translation and secretion at low temperatures.
4.4.3.4 Protein folding, modification and turnover
Protein folding has been previously shown to be an especially important process in the thermal adaptation of M. burtonii (Goodchild et al., 2004b; Goodchild et al., 2005).
Protein cis-trans isomerisation has been implicated as important at cold temperatures, while heat shock-like responses involving chaperones and chaperonins are found at higher temperatures. In this study of the HPP of M. burtonii similar results were uncovered, with NIW proteins identified as performing these functions. Those proteins
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involved in the low temperature response will be discussed below, while proteins involved in the heat stress response will be discussed in 4.4.3.6.
Two peptidyl-prolyl cis-trans isomerases (PPIase), an FKBP-type (Mbur_2256) and a cyclophilin-type (Mbur_1485), were identified as important for growth at low temperature. PPIases associate with the ribosome, as a primary step in the folding of the nascent polypeptide (Kandror and Goldberg 1997). PPIases catalyse the cis-trans isomerisation of aa-proline peptide bonds in both small peptides and polypeptides. Thus accelerating the speed of protein folding which is rate-limited, especially at low temperatures, by the intrinsically slow isomerisation of aa-proline bonds (Stoller et al.,
1995; Furutani et al., 2000; Maruyama et al., 2004).
PPIases are important in cold shock responses in bacteria and archaea. Members of this protein class have been identified following temperature downshift in E. coli (trigger factor) (Kandror and Goldberg 1997), M. jannaschii (Boonyaratanakornkit et al.,
2005), and Thermococcus sp. KS-1 (Ideno et al., 2001). PPIases are classified into three distinct, structurally unrelated families, according to their sensitivity to inhibitors.
FK506 (tacrolimus)-binding protein family (FKBP) members are sensitive to the immunosuppressant FK506, the Cyclophilin family is sensitive to another immunosuppressant, cyclosporin A, while the Parvulin family is insensitive to either
FK506 or cyclosporine A (Ideno et al., 2001).
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Table 4.5 Differentially abundant proteins involved in protein folding, modification, and turnover
Ratio 4C/23C M-media Gene locus Annotation MFM (TMA) (MeOH)
Mbur_0196 Proteasome alpha subunit (psmA) (EC 3.4.25.1) (ER2) - 2.358 Secreted hypothetical protein with PepSY-like domain Mbur_1112 (possible protease inhibitory function) (ER4) - 1.538 Mbur_1185 SPFH domain / Band 7 family-like protein (ER4) 1.712 1.383* Protein containing trypsin-like serine/cysteine protease Mbur_1349 domain (ER4) - 2.268 Mbur_1742 Secreted proteinase inhibitor I4 (serpin) (ER3) - 0.465 FKBP-type peptidyl-prolyl cis-trans isomerase with Mbur_2256 putative C-terminal chaperone domain (ER2) - 2.058 Cyclophilin-type peptidyl-prolyl cis-trans isomerase Mbur_1485 (ER2) 1.832 1.769
Bold gene loci and annotations indicate proteins that display differential abundance in both media tested.
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C. * value does not pass the > 1.5 fold change criteria.
The FKBP-type PPIase identified as important for cold growth in M. burtonii
(Mbur_2256) is homologous to FKBP type proteins from the thermophiles
Thermococcus sp KS-1 (cold induced) and Methanothermococcus thermolithotrophicus
(MtFKBP17), which not only perform cis-trans isomerisation, but prevent denatured proteins from aggregating and then induce refolding in a chaperone like manner (Ideno et al., 2000). This action is independent from the PPIase activity (Furutani et al., 2000), and is maintained by an ‘insert in the flap’ (IF) domain (Suzuki et al., 2003). The M. burtonii homologue contains this IF domain (3.4.2.11), so it is highly likely that
Mbur_2256 has a similar dual function. The protein is implicated as being associated with the membrane (see 3.4.2.11). As the ribosomes and other components of translation in M. burtonii appear to be membrane associated (4.4.3.3), and as PPIases have been identified in other organisms as ribosome associated (Kandror and Goldberg
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1997), it is conceivable that the PPIase Mbur_2256 is anchored to, or associated with the membrane in close proximity to this translation complex in a similar fashion to the
DEAD-box RNA helicase. Thus, Mbur_2256 could rescue any cold induced structures or aggregates of the nascent polypeptide(s), and perform isomerisation as the peptide(s) emerges from the ribosome.
The exact function of the cyclophilin-type PPIase (Mbur_1485), is less able to be inferred, as all cyclophilin-type PPIases have relatively similar sequences, and can perform a variety of functions apart from PPIase activity, including cell signalling and acting as chaperones (Wang and Heitman 2005).
As well as protein isomerisation, protein degradation and turnover seems to be especially important for the growth of M. burtonii at 4˚C. The proteasome alpha subunit (Mbur_0196) was identified as increased in abundance in the cold. Proteasomes provide a critically important service in protein quality control by catalysing the ATP- dependent degradation of irretrievably misfolded, denatured or aggregated proteins
(Maupin-Furlow et al., 2005a). The increased abundance of the proteasome, along with the putative protease Mbur_1349 may be indicative of increased protein misfolding or aggregation, which can occur at cold temperatures (Phadtare 2004). An increased tendency towards the degradation of misfolded proteins may also be a mechanism of amino acid scavenging.
Also involved in protein turnover is Mbur_1185, a SPFH family protein, which was identified as increased in abundance at 4˚C. SPFH domain proteins are integral membrane proteins with unclassified roles in membrane physiology. It is thought that they play a role in membrane associated protein degradation, as they form complexes with membrane associated proteases (Tavernarakis et al., 1999).
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Two secreted proteins with predicted protease inhibitory functions were identified as displaying differential abundance according to temperature. A PepSY domain protein
(Mbur_1112) was increased at 4˚C, and may have a role in the inhibition of proteases
(Yeats et al., 2004) as a protective measure against proteolytic attack of surface proteins increased at 4˚C. Conversely, a serine protease inhibitor (Mbur_1742) was identified as increased in abundance at 23˚C.
4.4.3.5 Transcription, replication, and DNA binding proteins
From the iTRAQ experiments performed, several proteins with roles in replication, transcription, and DNA maintenance (Table 4.6) were increased at 23˚C.
Table 4.6 Differentially abundant proteins involved in transcription, replication, and DNA binding
Ratio 4C/23C M-media Gene locus Annotation MFM (TMA) (MeOH) Mbur_0055 Chromosomal protein MC1 (ER2) 0.459 - Mbur_1769 Toprim domain protein (ER4) 1.792 - Mbur_1942 Cell division protein FtsZ (ER2) 0.37 - Mbur_0963 Transcription initiation factor IIB (TFB) (ER2) - 0.425 DNA-directed RNA polymerase subunit A', Mbur_1177 rpoA1 (ER2) 0.654 0.44
Bold gene loci and annotations indicate proteins that display differential abundance in both media tested.
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C.
The basal transcription machinery in archaea closely resembles that of eukaryotes.
Transcription is initiated in archaea by the binding of the TATA-box binding protein
(TBP) to the TATA box, an interaction that is stabilised by the association of transcription initiation factor B (TFB). The resulting pre-initiation complex then acts to recruit RNA polymerase (RNAP) for transcription (Reeve 2003). The M. burtonii
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genome encodes single genes for TBP and TFB, consistent with most archaeal genomes
(Reeve 2003), and 12 genes encode the multisubunit RNAP. In the analysis of the HPP of M. burtonii via iTRAQ, both the TFB and RNAP-A subunit, which forms the major catalytic centre of RNAP (Bartlett 2005; Werner 2007), were found to be increased at
23˚C. Thus, suggesting increased transcription at high temperatures, which would reflect the high rate of cellular growth at 23˚C.
The high rate of growth could also be the reason that the cell division protein FtsZ was identified as increased in abundance at 23˚C. FtsZ is crucial for cell division, where it is present early at the division site to form a ring shaped septum involved in constriction of the cell membrane (Lowe and Amos 1998). It is this membrane localisation that facilitated the identification of the protein in this study, as has been reported in other proteomic analyses of membranes (Ohtsu et al., 2005).
Two DNA binding proteins were identified as differential abundance with respect to temperature. Of these, a toprim domain protein had increased abundance at 4˚C. This protein falls into a class of toprim proteins with no known function (toprim domain only), although a role as nucleotidyl transferases or nucleases has been proposed
(Aravind et al., 1998).
The chromosomal protein MC1 was increased in M. burtonii cells at 23˚C. Almost all of the euryarchaeota, but none of the crenarchaeota, have archaeal histones (Reeve
2003). M. burtonii, in a similar fashion to Methanosarcina species, only encodes one archaeal histone. However this has not been identified in any proteomic analysis. It is the MC1 family proteins that are the predominant nucleoid proteins in Methanosarcina
(Reeve 2003), and given the close phylogenetic relationship of M. burtonii to the
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Methanosarcinaceae, this may occur in M. burtonii. The binding if MC1 to DNA results in bending and negative supercoiling (Le Cam et al., 1999), and protects DNA against radiolysis and heat denaturation (Chartier et al., 1988; Reeve 2003). The increased abundance of the chromosomal protein MC1 at 23˚C could protect DNA against heat denaturation.
4.4.3.6 Heat induced stress in M. burtonii
The response of M. burtonii to growth at 23˚C is associated with proteins that are related to either oxidative stress or heat stress effects on proteins (Table 4.7).
Table 4.7 Differentially abundant proteins involved in the heat stress response
Ratio 4C/23C M-media Gene locus Annotation MFM (TMA) (MeOH) Mbur_1494 Thiamine biosynthesis protein thiC (thiC) (ER2) 0.268 0.43 Mbur_2183 Universal stress protein A (ER4) - 0.314 Mbur_1120 Ferritin (ER2) 0.276 0.307 Mbur_1312 Chaperone DnaK (Hsp70) (ER2) 0.357 0.492 Mbur_2146 Thermosome subunit (Chaperonin) (ER2) - 0.353 translation elongation factor EF-1, subunit alpha (EF- Mbur_1170 1A, EF-Tu) (ER2) - 0.47
Bold gene loci and annotations indicate proteins that display differential abundance in both media tested.
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C
At 23˚C, M. burtonii grows at a maximum rate. The high rate of growth is associated with a high rate of metabolism and would lead to an increased production of potentially damaging by-products (e.g. radicals). In the metabolism of aerobic organisms, oxygen radicals arise via the tendency for molecular oxygen to gain single electrons, forming reactive oxygen species, which damage proteins, lipids, and DNA (Sweetlove et al.,
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2002). Despite M. burtonii being an obligate anaerobe, potentially damaging non- oxygen radicals could be formed from the anaerobic metabolism of the organism.
Growth at 23˚C is associated with an increase in the abundance of the enzyme ThiC
(Mbur_1494), required for providing the cofactor thiamine pyrophosphate (TPP) to TPP dependant enzymes with increased activity/abundance at 23˚C (Goodchild et al., 2004a,
2005). Mbur_1494 is an iron-sulphur protein belonging to the radical SAM family, which produce 5’adenosyl radicals, generated by the reductive cleavage of S- adenosylmethionine (SAM) (Wang and Frey 2007; Chatterjee et al., 2008). As a consequence of increased activity of this (and related) enzymes at 23˚C, some of the potent oxidising 5’adenosyl radicals may ‘escape’ into the cytoplasm, with subsequent oxidative damage. The DNA oxidising activity of these species comparable to hydroxyl radicals (the most damaging to DNA of the reactive oxygen species) (Ugulava et al.,
2003). An increase in methanogenesis at 23˚C (see 4.4.4.1) could also result in the formation of radicals. For example, the reduction of methyl-CoM generates S-centred radicals (thiyl, sulfuranyl) (Buckel and Golding 2006) which can also cause oxidative damage to proteins, lipids and DNA (Schoneich and Asmus 1990; Ferreri et al., 2005;
Schoneich 2008)
Growth at 23˚C in M. burtonii is associated with an increase in abundance of two proteins which may provide protection against these non-oxygen radicals, a ferritin
(Mbur_1120) and universal stress protein A (UspA, Mbur_2183). The ferritin
(Mbur_1120) found to be increased at 23˚C is a member of the Dps (DNA-binding proteins from starved cells) (Almiron et al., 1992) or mini-ferritin family; it is predicted to have a membrane association facilitating its identification in the HPP. Dps proteins are known to protect DNA from oxidative damage (Carrondo 2003; Chiancone et al.,
2004). While the activity of these proteins against peroxide and other reactive oxygen
D. Burg UNSW 199 species is known, their activity against non-reactive oxygen species had not been reported. If they are protecting the anaerobic cell from non-oxygen radical damage, it would represent a novel function.
The UspA family of proteins are involved in a variety of functions in bacteria and archaea, many of which have not been characterised (Kvint et al., 2003), and are usually associated with a stress response (Jones et al., 1992; Nachin et al., 2005). A central role for UspA in the protection of DNA against oxidative damage has been described in bacteria (Nachin et al., 2005). The UspA protein Mbur_2183 may be performing a similar role in M. burtonii.
At high temperatures, proteins are denatured, corresponding to a loss of functional configuration, which is often accompanied by aggregation (Macario et al., 1999). It can be prevented and corrected by the chaperones and the chaperonins, which are often elevated in cells undergoing heat stress (Boonyaratanakornkit et al., 2005; Coker et al.,
2007).
Many archaea have a bacteria-like ATP-dependant chaperone system (DnaK/Hsp70,
DnaJ/Hsp40, GrpE), which catalyses the refolding of denatured proteins, and assists in the folding of nascent polypeptides (Macario et al., 1999; Zmijewski et al., 2004).
DnaK binds unstructured peptides in an ATP dependent manner, which is enhanced by the action of DnaJ (Karzai and McMacken 1996; Suh et al., 1998). This binding maintains the polypeptide in an extended form and prevents aggregation. The protein
GrpE then binds the DnaK/DnaJ complex, causing a loss of affinity with the peptide, stimulating release. The polypeptide can then be folded by chaperonins (Langer et al.,
1992; Macario et al., 1999).
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In contrast to the bacteria-like chaperone system, the archaea possess a eukayotic-like chaperonin system (Macario et al., 1999), which acts in an ATP-dependent manner
(Yoshida et al., 2002), in conjunction with prefoldins (Whitehead et al., 2007), to direct the folding of proteins. Non-native proteins are captured by the chaperonin complex, where they enter a central ring and fold, protected from aggregation with other non- native proteins (Hartl and Hayer-Hartl 2002).
Growth at 23˚C in M. burtonii was associated with increased abundance of both the chaperone DnaK, and a subunit of the chaperonin complex. The identification of the chaperonin in the hydrophobic fraction indicates a membrane association, as has been observed in Methanosarcina spp. (Francoleon et al., 2009). While an increase in abundance of these proteins at 23˚C has been observed previously in M. burtonii, and has been related to a heat stress response (Goodchild et al., 2004b; Goodchild et al.,
2005), it is interesting to note that only one member of the chaperone machine (DnaK), had ever been identified as displaying differential protein abundance (Goodchild et al.,
2004b; Goodchild et al., 2005; Williams et al., submitted-b). It would be expected that all members of the chaperone machine would be present in elevated abundance, if this process was occurring at 23˚C. Mirroring these results in M. burtonii, only the DnaK transcript was found to be induced in a heat shock response in Halobacterium sp. Nrc-1
(Coker et al., 2007), suggesting an alternative function, separate to DnaJ and GrpE.
DnaK can operate separately from the traditional ‘chaperone machine’, in concert with the chaperone ClpB. These proteins act together with small heat shock proteins
(sHSPs), to rescue aggregated proteins (Kim et al., 1998; Kim et al., 2003; Mogk et al.,
2003). While the chaperone ClpB was found to be associated with growth at 23˚C (see
3.4.4.1), no sHSPs have ever been identified in the proteomics of M. burtonii
(Goodchild et al., 2004a; Goodchild et al., 2004b; Goodchild et al., 2005; Williams et
D. Burg UNSW 201 al., submitted-b), suggesting these are not involved in the biology of the organism, or are expressed at very low levels. There is however an alternative function for DnaK. It has been found to work separately from other components of the ‘chaperone machine’
(i.e. DnaJ), participating in the assembly of iron sulphur clusters (Wu et al., 2005). As maximum rate of growth of M. burtonii occurs at 23˚C, iron sulphur proteins would be in high demand (especially for methanogenesis proteins), so this function for DnaK cannot be ruled out.
Growth at 23˚C in M. burtonii was also associated with an increase in abundance of the protein elongation factor 1 alpha. This protein is known to have a chaperone-like function (Caldas et al., 1998; Caldas et al., 2000), an may be fulfilling a similar role in the heat stress response in M. burtonii.
4.4.4 The response of Methanococcoides burtonii to different substrates and media
The combined proteomic analyses of the response of M. burtonii to different substrate/nutrient and temperature conditions, revealed that many proteins not only varied according to temperature, but also with respect to the media conditions tested.
Very few showed differences solely with respect to media/substrate. The proteins for which media conditions effected a change in abundance are discussed below, in combination with temperature effects on metabolism. Areas where these effects were most prominent were: the methanogenesis proteins; energy generation and conservation; response to nutrient levels; and the effects of methanol as a stressor.
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4.4.4.1 Methanogenesis
M. burtonii can only utilise methylamines and methanol, and not H2:CO2, formate, or acetate, as substrates for methanogenesis (Franzmann et al., 1992). The catabolism of these substrates for biosynthesis and energy generation proceeds via methyltransfer reactions, resulting in the transfer of a methyl group to Coenzyme M (CoM). From that point a common disproportionate dismutation pathway is utilised, resulting in the oxidation of one methyl group to CO2 for every three methyl groups reduced to methane
(Figure 4.6) (Paul et al., 2000; Ferry and Kastead 2007).
The catabolism of TMA is initiated after transport of methylamine into the cell via the putative TMA permease (MttP) (Paul et al., 2000). TMA is serially demethylated by a set of methyltransferases (Tri-, di, and monomethylamine), and their cognate corrinoid proteins (Hippe et al., 1979; Ferguson and Krzycki 1997; Burke et al., 1998; Paul et al.,
2000). The methyl groups are transferred to CoM via a methylamine specific CoM methylase (MtbA), which operates with all methylamine methyltransferase: corrinoid protein pairs (Ferguson et al., 1996). The ammonia remaining following demethylation can be utilised in nitrogen metabolism.
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Figure 4.6 Methanogenesis in M. burtonii. Dotted box a encloses the methanol specific reactions. Dotted box b encloses the methylamine specific reactions. Colour coding: Purple, proteins increased in abundance in TMA grown cells; green, proteins increased in abundance in methanol grown cells; blue, no differential abundances. Cont’d on following page.
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Figure 4.6 legend cont’d. Pale proteins indicate a component not identified in the hydrophobic analysis, but was been identified in the global proteomics of M. burtonii (Williams et al., Submitted-a).
Black arrows indicate reactions involving the flow of carbon. Red arrows indicate reactions involved in the flow of electrons. White (clear) arrows indicate reactions involving water. Hatched arrows indicate passive diffusion across a membrane. Abbreviations used: Fd – ferredoxin; Fmd – Molybdenum formylmethanofuran dehydrogenase; Fpo – F420H2 dehydrogenase; Ftr – formylmethanofuran- tetrahydrosarcinapterin formyltransferase; GDH – glutamate dehydrogenase; GS glutamine synthetase;
H4SPT – tetrahydrosarcinapterin; Hdr – CoB-CoM heterodisulfide reductase; Mch – methenyltetrahydrosarcinapterin cyclohydrolase; Mcr – methyl-coenzyme M reductase; Mer –
Coenzyme F420-dependent N(5),N(10)-methylenetetrahydrosarcinapterin reductase; MFR –
Methanofuran; MP – methanophenazine; MtaA – methanol-specific methylcobalamin:CoM methyltransferase; MtaB – methanol methyltransferase; MtaC – methanol corrinoid protein; MtbA methylamine-specific methylcobamide:CoM methyltransferase; MtbB – dimethylamine methyltransferase; MtbC – dimethylamine corrinoid protein; Mtd – methylenetetrahydrosarcinapterin dehydrogenase; MtmB –monomethylamine methyltransferase; MtmC – monomethylamine corrinoid protein; Mtr – tetrahydrosarcinapterin S-methyltransferase; MttB – trimethylamine methyltransferase;
MttC – trimethylamine corrinoid protein; MttP – trimethylamine permease; RamA – methylamine:CoM methyl transfer reductive activation protein; RamM – methanol:CoM methyl transfer reductive activation protein.
The catabolism of methanol proceeds in a similar manner following its passive diffusion across the cell membrane. The methanol specific methyltransferase:corrinoid protein pair transfers a methyl group to CoM via a methanol specific CoM methyltransferse, generating the key intermediate methyl-CoM (Ferguson et al., 1996). From this point the pathway converges. It can proceed to tetrahydrosarcinapterin-S- methyltransferase, from where a series of reactions leads to CO2, which can be used as a
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Table 4.8 Differentially abundant proteins involved in methanogenesis
Temperature Substrate experiments. experiments. Ratio MFM/MeOH Ratio 4˚C/23˚C Cells Cells MFM M-media Gene locus Annotation grown at grown at (TMA) (MeOH) 4˚C 23˚C Methanol specific Mbur_0813 Methanol corrinoid protein (mtaC-1) (ER2) - 0.301 0.244 0.101 Methanol--corrinoid methyltransferase Mbur_0814 - 0.178 - 0.176 (MtaB-1) (EC 2.1.1.90) (ER2) TMA specific Monomethylamine methyltransferase Mbur_0846 0.302 - - - (mtmB-2) (EC 2.1.1.-) (ER2) Mbur_1366 MttQ (function unknown) (ER4) (MttQ-1) - - 3.186 - Mbur_1367 TMA permease MttP (ER4) (MttP-1) 0.351 - 2.284 - Trimethylamine corrinoid protein (mttC-1) Mbur_1368 - 0.188 - - (ER2) Trimethylamine methyltransferase (mttB-1) Mbur_1369 0.416 0.145 3.683 3.322 (EC 2.1.1.-) (ER2) Methylamine-specific methylcobamide:CoM Mbur_2082 0.388 0.398 - - methyltransferase (mtbA) (EC 2.1.1.-) (ER2) Trimethylamine methyltransferase (mttB-2) Mbur_2308 0.463 - - 5.747 (EC 2.1.1.-) (ER2) Mbur_2312 MttQ (function unknown) (ER4) (MttQ-2) 0.569 2.874 - - Common to both substrates Mbur_1288 F420H2 dehydrogenase subunit M (ER2) 0.38 - - - Mbur_1291 F420H2 dehydrogenase subunit J (ER2) 0.391 - - - Electron transport complex protein rnfA Mbur_1380 0.444 - - - (Nitrogen fixation protein rnfA ) (ER2) Electron transport complex protein rnfE Mbur_1381 0.338 - - - (Nitrogen fixation protein rnfE) (ER2) Tetrahydrosarcinapterin S-methyltransferase Mbur_1518 - - 2.234 - subunit H (mtrH) (EC 2.1.1.86) (ER2) Tetrahydrosarcinapterin S-methyltransferase Mbur_1525 - - 0.522 0.581 subunit E (mtrE) (EC 2.1.1.86) (ER2) Methyl-coenzyme M reductase, subunit Mbur_2417 0.449 0.715* 0.647 1.473* alpha (mcrA) (EC 2.8.4.1) (ER2) Methyl-coenzyme M reductase, subunit Mbur_2418 0.381 0.459 - - gamma (mcrG) (EC 2.8.4.1) (ER2) Conserved methyl-coenzyme M reductase Mbur_2420 1.631 0.191 1.746 2.941 operon protein D (mcrD) (ER4) Methyl-coenzyme M reductase, subunit beta Mbur_2421 0.422 0.292 - - (mcrB) (EC 2.8.4.1) (ER2) CoB-CoM heterodisulfide reductase, subunit Mbur_2437 0.494 - - - D (EC 1.8.98.1) (ER2)
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C. Green
ratios indicate increased abundance in M-Media (MeoH), Pink ratios indicate increased abundance in
MFM (TMA). * value does not pass the > 1.5 fold change criteria.
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carbon source for biosynthesis via an Acetyl CoA synthase complex (Allen et al.,
2009). This series of reactions leads to the generation of six electrons for every methyl group, which can then be used for the conversion of three methyl groups to methane via methyl-CoM reductase and heterodisulfide reductase (Hdr) (Ferry 1999; Ferry and
Kastead 2007).
In the series of reactions in the oxidative branch of this pathway, coenzyme F420 and ferredoxin are the electron carriers, passing electrons to Hdr through methanophenazine via the F420H2 dehydrogenase (Fpo) and the Rnf complex respectively (Baumer et al.,
2000; Li et al., 2006; Ferry and Kastead 2007). The actions of the Hdr and Fpo complexes also export protons, which sets up a gradient for the generation of ATP
(Deppenmeier 2004).
Many of the proteins involved in methanogenesis that were identified as differentially abundant displayed an increase in abundance at 23˚C (Table 3.8). This is consistent with an increased rate of growth and hence energy and carbon demands that corresponds to an increased rate of methanogenesis.
Of the methanol specific proteins, only one MtaCB protein pair was identified, this was also true for global proteome results (Williams et al., Submitted-a). This is consistent with findings in M. acetivorans where MtaCB-1 was expressed at high levels during exponential phase, while the MtaCB-2 tends to be upregulated in stationary phase (Bose et al., 2006).
The data for the methylamine-specific proteins from this insoluble fraction alone may be misleading, as many proteins that appear to show no difference according to substrate, differed in other sub-cellular fractions (Williams et al., Submitted-a). Most methylamine specific proteins had increased abundances in MFM media. The exception
D. Burg UNSW 207 is the Methylamine-specific methylcobamide:CoM methyltransferase (Mtba)
(Mbur_2082), which only showed variation according to temperature. This is consistent with findings in M. acetivorans, where MtbA is expressed regardless of substrate (Bose et al., 2008). Two proteins designated MttQ were also identified with interesting patterns of differential abundance. Mbur_1366 was found to be increased at TMA, while Mbur_2312 varied according to temperature, increasing in abundance at 23˚C in
MFM (TMA), while increasing in abundance at 4˚C on methanol. These results cannot be interpreted biologically as MttQ has, as yet, no determined function, however a role in mediating interactions between the TMA permease and methyltransferases is conceivable (Williams et al., Submitted-a).
Members of all membrane bound components of the pathways common to both TMA and methanol, as well as several other non-membrane bound components, were identified in this HPP analysis. While most of these had increased abundances at 23˚C, a few proteins displayed differential abundance according to substrate. In methanogenesis from methylamines and methanol, the tetrahydrosarcinapterin S methyltransferase complex (Mtr) utilises a sodium ion gradient (Gottschalk and Thauer
2001; Welander and Metcalf 2005) to drive the unfavourable transfer of the methyl group of methyl-CoM to H4SPT. Several subunits of this complex were found to vary according to substrate. The MtrH subunit was increased in TMA grown cells, while the
MtrE subunit was increased in methanol grown cells. These subunits are proposed to play important functions in the Mtr complex. MtrH is thought to be involved in the transfer of a methyl group via the MtrA subunit to H4SPT, while the MtrE subunit is thought to be directly involved with linking ion translocation to the de-methylation of methyl-CoM (Gottschalk and Thauer 2001). The pattern of abundance is quite
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interesting, as both steps are assumedly highly important to the function of the Mtr complex, and could be specific for methanol vs. methylamine metabolism, as acetate vs. methanol gown cells have shown no differences in abundance of any subunits of this complex (Li et al., 2005a; Li et al., 2006).
The Methyl coenzyme-M reductase (Mcr) participates in the last step of methanogenesis, where HS-CoB donates electrons for the production of methane from the demethylation of methyl-CoM, producing the heterodisulfide (CoMS-SCoB).
Previous studies of M. burtonii showed that subunits of this complex were increased at
4˚C (Goodchild et al., 2004b) and subunits have been shown to vary according to substrate in M. acetivorans (Li et al., 2005b). In this work, subunits of Mcr varied not only according to temperature, but also according to substrate in a complicated manner.
For example the McrA subunit had increased abundance in methanol vs. TMA at 4˚C, but had increased abundance in TMA vs. methanol at 23˚C.
4.4.4.2 Energy conservation and generation
Several proteins that are related to energy conservation and generation were identified as differentially abundant (Table 4.9).
A membrane-bound proton-translocating pyrophosphatase (PPase) subunit was found to be differentially abundant according to both temperature and substrate. This membrane- bound PPase has been suggested to have dual roles in methanogenic archaea. One of these is the disposal of cytoplasmically produced pyrophosphate (PPi), which thereby shifts PPi-generating reactions toward product formation. PPases are also involved in salvaging the free energy of PPi hydrolysis for proton translocation and the formation of a proton gradient (Baumer et al., 2002). Both roles could be very important at elevated
D. Burg UNSW 209 growth rates, with DNA and RNA synthesis, and amino acid activation being PPi- generating processes. However, growth on methanol also led to higher PPase at 4˚C compared to growth on TMA at this temperature; one scenario is that the energy- conservation mechanism of PPase is in more demand under these growth conditions, which results in the slowest rate of M. burtonii growth. This presumably occurs as a direct result of the lower available energy yield of methanol vs. TMA, combined with the thermodynamic constraints on rates of growth at this low temperature. Growth at
23˚C on methanol was also associated with the increased abundance of a Na+/H+ antiporter with high identity to the Mrp complex of M. acetivorans. This antiporter was thought to specific for M. acetivorans, and has been related to the marine, high sodium environment of this organism (Li et al., 2006). In M. acetivorans the Mrp antiporter is thought to conserve energy by utilising the sodium gradient across the cell membrane
(external > internal) to export protons for use in ATP generation. Given the marine derived environment of M. burtonii, it is likely that this complex is fulfilling the same role, and is being utilised to bolster the PMF for ATP generation when the cells are grown on the low energy yielding substrate methanol (as opposed to TMA).
Growth at 23˚C on both media was also associated with an increase in the abundance of subunits of a H+ transporting ATPase. The increased abundance of these subunits of the major ATP synthase of M. burtonii at 23˚C is reflective of high rates of growth at this temperature and presumably high energy demands.
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Table 4.9 Proteins with differential abundance involved in energy generation and conservation
Temperature Substrate experiments. experiments. Ratio Ratio MFM/MeOH 4˚C/23˚C Cells Cells MFM M-media Gene locus Annotation grown at grown at (TMA) (MeOH) 4˚C 23˚C Pyrophosphate-energised proton pump / Pyrophosphate-energised inorganic Mbur_0994 0.578 0.423 0.546 - pyrophosphatase (H(+)-PPase) (EC 3.6.1.1) [hppA] (ER2) Putative 116kDa V-type ATPase subunit Mbur_1238 0.529 0.622 - - (ER4) V-type ATP synthase, beta chain (atpB) Mbur_1244 0.399 - - - (EC 3.6.3.14) (ER2) Mbur_1363 Sodium/hydrogen antiporter (ER4) - - - 0.529
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C. Green
ratios indicate increased abundance in M-Media (MeoH), Pink ratios indicate increased abundance in
MFM (TMA)
4.4.4.3 General metabolic differences
Despite the very different compositions of the media tested: Rich (MFM) vs. defined
(M-media); very few differences were seen in proteins involved in non-methanogenic
central metabolism and biosynthesis (Table 4.10). This suggests that M. burtonii
constitutively synthesises all amino acids and large molecules required for growth and
metabolism. Adding further evidence towards this is the genomic analysis of M.
burtonii, where very few transporters for large molecules (none for amino acids) were
identified in the genome of the organism (Allen et al., 2009).
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Table 4.10 Differentially abundant proteins involved in general metabolism
Temperature Substrate experiments. experiments. Ratio Ratio MFM/MeOH 4˚C/23˚C
Cells Cells MFM M-media Gene locus Annotation grown at grown at (TMA) (MeOH) 4˚C 23˚C Mbur_0104 Flagellin (ER2) - 1.541 0.514 0.407 ABC transporter, solute-binding protein Mbur_2131 (sulfonate/nitrate?) (ER4) - 0.525 - 0.593
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C. Green
ratios indicate increased abundance in M-Media (MeoH), Pink ratios indicate increased abundance in
MFM (TMA)
The main difference M. burtonii faces when grown in the different media is nitrogen
availability to the cell. Growth on TMA results in the liberation of ammonia, which can
be combined with 2-oxoglutarate by glutamate dehydrogenase to form glutamate as a
entry point to nitrogen metabolism (Allen et al., 2009). When the cell is grown on
methanol, nitrogen in the form of ammonium or ammonia must be transported into the
cell. The only ammonia transporter in the genome of M. burtonii is predicted to be
truncated and non functional (Allen et al., 2009). The lack of a specific transporter for
ammonia means that M. burtonii can only import it through passive diffusion across the
cell membrane (Allen et al., 2009). Ammonia would favourably exist in ammonium
+ (NH4 ) form under the pH (neutral) conditions of the media. As lipid bi-layers are
highly impermeable to ammonium ions (Antonenko et al., 1997), there is a highly
limited source of nitrogen for the cell in the absence of any identifiable transporters.
While the consequences of this are not readily apparent from the analysis of the HPP,
the global proteomics approach was able to identify several proteins which were
affected by this problem (Williams et al., Submitted-a). The HPP analysis was however,
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able to identify a putative sulfonate/nitrate transporter solute binding protein as increased in abundance in methanol grown cells. It is unclear whether M. burtonii can utilise nitrate as a nitrogen source, as the genome of the organism encodes no nitrate reductase (Allen et al., 2009).
M. burtonii is motile by means of a single polar flagellum (Franzmann et al., 1992), and possesses a bacteria-like Che chemotaxis system (Goodchild et al., 2004a). A flagellar protein was found to have higher abundance in cells grown in M-media. The increased abundance of this protein may be related to nutrient levels in the media (much lower than in MFM), as flagellar abundance in other archaea has been shown to increase under nutrient scarcity (Szabo et al., 2007).
4.4.4.4 Methanol as a stressor
The presence of high concentrations of methanol had a detrimental effect on the growth of M. burtonii. Cells did grow in concentrations of 300mM methanol, and at concentrations of 150mM, exhibit a long lag phase, slower rate of growth, and reach a lower maximum density (4.4.1). Organic solvents such as methanol have detrimental effects on cell membranes, resulting in loss of membrane integrity and function, which can result for example, in the loss of ability to maintain ion gradients (Sikkema et al.,
1995). Organic solvents also denature protein molecules and act as competitive inhibitors of enzyme activity. The presence of a solvent reduces the polarity of the medium surrounding a protein molecule, so that buried hydrophobic residues are energetically favoured for interaction with the solvent, causing conformational changes or unfolding (Ogino 2008).
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The reaction of cells to the presence of organic solvents has been well studied in bacteria, with several mechanisms of response to these damaging agents known. The presence of organic solvents can result in: modification of the cell envelope; increased concentrations of repair enzymes; production of solvent de-activating enzymes; and the induction of a generalised stress response (Sardessai and Bhosle 2002). The response of archaea to the presence of organic solvents has been less well studied however, methanol induces a stress morphotype in M. mazei, with increased aggregation occurring when the solvent is present in the medium (Macario and Conway De Macario
2001).
The hydrophobic proteome of M. burtonii cells grown on methanol (vs. TMA) displayed increased abundances of several proteins, which may relate to the detrimental effects of methanol (Table 4.11). As well as the changes in the abundance levels of these proteins when cells were grown on methanol, the abundances of proteins as a whole were variable. This is indicated by the weighted SD values (available in
Supplementary Table 2 in accompanying disk, where values > 0.1 indicate significant variability). Variability in protein abundance is a feature of cells undergoing stress
(Maytin 1992), and these data may be indicative of this occurring in M. burtonii.
M. burtonii has no identifiable specific transporters for methanol (Allen et al., 2009), therefore it is most likely that it enters the cell through passive diffusion across the S- layer and membrane. During this process, the solvent would come into contact with membrane proteins and surface molecules, where it could exert negative effects.
Several proteins were identified as increased in abundance when grown on methanol, possibly related to these effects; including an increased abundance of the S-layer
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proteins (Mbur_0268, 1690), a stromatin-like protein (Mbur_ 1804), and a VWA
domain protein (Mbur_0801).
Table 4.11 Differentially abundant proteins indicative of the stressful effects of methanol
Temperature Substrate experiments. experiments. Ratio Ratio MFM/MeOH 4˚C/23˚C Cells Cells MFM M-media Gene locus Annotation grown at grown at (TMA) (MeOH) 4˚C 23˚C Transmembrane oligosaccharyl Mbur_1579 0.519 - - 0.62 transferase (ER3) von Willebrand factor-domain Mbur_0801 - 0.414 - 0.404 membrane protein (ER4) Mbur_0268 Protein with duplicated DUF1608 (ER4) - 1.818 0.604 0.158 Mbur_1690 Protein with duplicated DUF1608 (ER4) 1.475 1.835 - 0.558 Putative stomatin, band 7 family protein Mbur_1804 - - - 0.613 (ER2) Thermosome subunit (Chaperonin) Mbur_2146 - 0.353 - 0.283 (ER2)
Blue ratios indicate increased abundance at 4˚C, red ratios indicate increased abundance at 23˚C. Green
ratios indicate increased abundance in M-Media (MeOH), Pink ratios indicate increased abundance in
MFM (TMA)
While the differential abundance of the S-layer proteins may be indicative of S-layer
modification induced by the presence of methanol, the stromatin like protein and the
VWA-domain protein are more likely to be involved in the degradation and
modification of damaged surface and membrane proteins. Stromatins play an
incompletely characterised role in membrane physiology, and may act as proteases for
protein quality control (Yokoyama et al., 2008), while VWA domain proteins are
predicted to have a variety of functions, including chaperone functions for protein
complex assembly, metal insertion, and protein binding (Ponting et al., 1999; Snider
and Houry 2006). In M. burtonii, the VWA domain proteins could work in association
D. Burg UNSW 215 with the MoxR AAA+ ATPase as chaperones, acting to aid in the correct insertion of solvent compromised proteins into the membrane and / or the assembly of solvent affected integral membrane protein complexes. A subunit of the chaperonin complex was also identified as increased in abundance in methanol grown cells. This protein could have been induced in the membrane, as is known to occur in the methanosarcina
(Francoleon et al., 2009), as a response to the protein denaturing effects of methanol.
The occurrence of the oligosaccharyl transferase and the S-layer domain proteins at higher abundance in methanol, suggest some level of S-layer modification. It could indicate a cellular response to repair or minimise solvent induced damage to the S-layer of the organism. In bacteria solvents can induce the modification of outer membrane lipopolysaccharides so as to stabilise the structure through increasing the hydrophobic character of the outer membrane (Aono and Kobayashi 1997). Glycosylation has been shown to increase the hydrophobic character of proteins (Spiriti et al., 2008), and glycosylation events have been shown to be increased in mouse neuronal cells in the presence of ethanol (Braza-Boils et al., 2006). This could be occurring in M. burtonii in a similar way to which lipopolysaccharide modification is protective against solvents in
Gram negative organisms.
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4.5 Conclusions
Through the utilisation of a strategy that enriched for hydrophobic proteins, aspects related to cold adaptation, and substrate utilisation and subsequent metabolism were observed. Cold adaptation in M. burtonii involves a drive towards translation and an increased expression of surface and secreted proteins. Translation is hypothesised as being especially important for cells at low temperatures, as this could be a process that where slow rates of reaction may have the most profound effect. Alternatively, the high abundance of ribosomes in the hydrophobic fraction could be related to efficient transport of the large number of secreted and surface proteins which displayed increased abundance at 4˚C. Similarly a DEAD-box RNA helicase and a FKBP-type PPIase were also identified as membrane associated, and increased at low temperatures. They are likely involved in RNA chaperoning and rate-limiting protein isomerisation respectively. These proteins may be membrane associated in proximity to the ribosome, as an adaptation to increase the efficiency of the translation process. Subunits of the proteasome and a membrane associated SPFH-domain protein also displayed increased abundance at 4˚C; these proteins are likely to be involved in the quality control of cold affected cytoplasmic and membrane proteins respectively.
When cells were grown at 23˚C many of the proteins identified could be directly related to heat stress. Processes identified as important in protecting the cell from heat induced stress include surface glycosylation, and the presence of chaperone/chaperonin systems in high abundance for the rescue denatured or aggregated proteins. Indications of oxidative stress were also identified at 23˚C; this is possibly due to the production of non-oxygen radicals by thermally influenced high rates of metabolism.
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The high rate of metabolism and growth at 23˚C influenced the abundance of many metabolic proteins. Most of which had differential abundance not only according to temperature but also with respect to substrate. The compounding effects of growth temperature and difference in substrate energy yield, had an effect on the abundance of energy conserving proteins. There was very little effect of growth media on the central metabolism of M. burtonii, despite wide differences in nitrogen and nutrient availability. This suggests M. burtonii intrinsically synthesises all its biochemical building blocks, regardless of nutrient availability.
The organism was unable to switch between TMA and methanol in growth experiments, possibly related to the lack of identifiable methanol gene locus transcriptional regulators, or the lack of a third MtaCB gene pair. Methanol was also found to have inhibitory effects on the growth of cultures and proteomics provided evidence for this.
Several surface and membrane localised elements were identified an increased in abundance in methanol grown cells. Many of these could have functions as countermeasures to the damaging effects of methanol on proteins and membranes.
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Chapter 5. Characterisation of Methanococcoides burtonii Cells and EPS with Respect to Temperature and Substrate
5.1 Summary
The morphology and extracellular polymeric substance (EPS) of M. burtonii was investigated with respect to both temperature and nutrient conditions. The use of histochemical staining, scanning electron microscopy (SEM), environmental scanning electron microscopy (ESEM), and transmission electron microscopy (TEM) revealed significant differences in the EPS and morphology of cells with respect to temperature and substrate. The EPS of M. burtonii was found to be comprised of protein, polysaccharides and RNA. The evidence that RNA is produced in the EPS of M. burtonii is the first evidence of this phenomenon in the archaea. It was also found that growth at high temperatures was associated with the presence of acidic residues in EPS.
Electron microscopy revealed that cells grown at different temperatures produced EPS with different dehydration properties, which indicated differences in composition with respect to temperature. This appears to be the first time that dehydration (normally considered an artefact of preparation) has been used in a qualitative manner in the analysis of EPS. Cells grown at low temperature were also found to be smaller than cells grown at high temperatures, and evidence of surface modification was observed.
TEM revealed that cells grown on methanol took on highly irregular shapes and had circular electron transparent inclusions in their cytoplasm. The phenotypic analysis revealed avenues for future research that will help to resolve the roles of EPS and ultrastructural changes in the adaptation of the organism.
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5.2 Introduction
A variety of microorganisms produce EPS, which can be comprised of combinations of polysaccharides (Vu et al., 2009), proteins (Nevot et al., 2006), DNA (Whitechurch et al., 2002; Steinberger and Holden 2005), and RNA (Watanabe et al., 1998; Nishimura et al., 2003). EPS production is a feature of many characterised psychrophiles and occurs in the psychrophilic bacteria (Poli et al., 2004; Nichols et al., 2005; Nevot et al.,
2008) and the archaea (Rudolph et al., 2001; Moissl et al., 2002; Reid et al., 2006;
Kruger et al., 2008; Wrede et al., 2008). Genomic analyses of several psychrophiles also indicates a high capacity to produce EPS (Lauro et al., 2008; Riley et al., 2008), though its exact function is unknown. Several investigators have found that EPS production increases at low temperature (Nichols et al., 2005; Qin et al., 2007; Nevot et al., 2008), is protective under very cold or freezing conditions (Tamaru et al., 2005;
Knowles and Castenholz 2008; Marx et al., 2009) and can enhance the activity of extracellular enzymes (Vetter et al., 1998; Deming 2007). Under circumstances where ice crystals can potentially damage cells, EPS has also been shown to maintain a gel- like barrier around cells, enhancing their survival (Junge et al., 2006).
Many organisms that produce EPS also form cellular aggregates. Aggregate formation is an advantageous microbial feature for biotechnological applications, including waste treatment, as aggregating organisms naturally settle in bioreactors, facilitating the retention of biomass and the separation of liquids and solids (Fukuzaki et al., 1991;
Watanabe et al., 1998). Methanogens are known to form aggregates, and are important in anaerobic waste digestion. Multi-cellular structure formation in the Methanosarcina spp. (closely related to M. burtonii) is well characterised; aggregation in these species
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is known to be strongly affected by growth conditions (Sowers et al., 1993; Macario and Conway De Macario 2001).
M. burtonii is known to produce EPS (Reid et al., 2006), and forms aggregates in liquid culture at low temperatures (Figure 5.1). The genome of the organism also suggests an extensive capability to produce EPS (Allen et al., 2009). However, the comparison of
EPS production has never been investigated with respect to growth temperature and nutrient conditions. The composition of EPS in M. burtonii is unknown, and possible changes in cellular morphology have never been examined with respect to temperature.
4˚C 23˚C
Figure 5.1 M. burtonii cell aggregates. M. burtonii forms cell aggregates when grown at 4˚C (left).
Growth at high temperatures (right) is associated with planktonic growth
Electron microscopy is a powerful tool for use in the analysis of cellular morphology and extracellular features. However, the various types of electron microscopy have
D. Burg UNSW 221 associated strengths and weaknesses. SEM is a powerful method of visualising cells, cell surfaces and extracellular material. This method, however, requires the complete dehydration of samples prior to imaging. The dehydration can cause significant cellular shrinking (Schmid-Schonbein et al., 1980; Forge et al., 1992), and can cause artefacts in
EPS, which in its natural state is extensively hydrated (Kachlany et al., 2001; Baum et al., 2009). ESEM operates in low vacuum, and can be used to image hydrated samples with minimal sample preparation. However, the resolution of ESEM is much lower than other types of electron microscope (Stokes 2003; Bergmans et al., 2005). TEM is a very powerful method for the high resolution analysis of ultra-thin sections of microbial cells. However, this method involves extensive sample preparation and optimisation of contrasting agents (Bergmans et al., 2005). Artefacts can be introduced during ultramicrotomy (Mandarim-de-Lacerda 2003), and the analysis and quantification of images is complex (Gundersen and Osterby 1981; Mandarim-de-Lacerda 2003;
Vanhecke et al., 2008)
The EPS and cellular morphology of M. burtonii was examined with respect to temperature and media composition. This was achieved through the use of a combination of SEM, ESEM and TEM. The composition of the EPS was also investigated at a general level through the use of a number of histological staining techniques.
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5.3 Materials and Methods
All chemicals and media were prepared, unless otherwise noted, in MilliQ dH2O
(Millipore) with a conductivity of 18.2 m /cm. Reagents, unless otherwise noted, were made with Univar, analytical grade chemicals (sourced from APS, Seven Hills, NSW, or Ajax Finechem, Taren Point, NSW). All electron microscopy was carried out at the
Electron Microscope Unit (EMU), UNSW
5.3.1 Culture media and conditions
Cultures of M. burtonii were grown at 4 C and 23 C, as described in 2.3.2, in both
MFM and M-media prepared as described in 2.3.1 and 3.3.1, to an OD620 of between
0.075 and 0.1 (early – mid log phase). To assess the production of EPS at different stages of growth, cells were also grown in MFM at 4 C to OD’s of 0.05 and 0.3 respectively, prior to analysis by SEM.
5.3.2 Histochemical examination of EPS composition
To determine the general chemical composition of the EPS produced by M. burtonii, basic histological techniques were applied, using a variety of specific stains. After staining slides were visualized on an Olympus BX51 microscope (Olympus, Tokyo,
Japan) with attached Leica DFC420 camera (Leica, Wetzlar, Germany). Slides were examined using x50 and x100 (oil emersion) objective lenses. Images were recorded using Photoshop Creative suite software (Adobe, San Jose, CA, USA)
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5.3.2.1 Sample preparation
Slides were prepared from 4 C and 23 C cultures, by anaerobically removing a small amount of culture from serum bottles with a 1mL syringe, and spotting onto glass slides. The slides were dried under a stream of nitrogen from the gassing manifold and, once dried, immediately heat fixed. After fixation, they were stored at 4 C until needed. Prior to staining, they were rinsed in dH2O to remove any traces of salt
5.3.2.2 Alcian blue-PAS and Aldehyde fuchsin-Alcian blue
The alcian blue-periodic acid Shiff’s (PAS) and Gomori’s aldehyde fuchsin-alcian blue staining protocols were performed in order to determine whether the EPS of M. burtonii consisted of polysaccharides (e.g. chains of sugars attached to protein) (Smith and
Dixon 2003).
Neutral polysaccharides were differentiated from acid or acetic polysaccharides using the alcian blue-PAS method. Slides were stained by covering with alcian blue staining solution (10g/L alcian blue (AnalaR, BDH, Poole, England) in 3% acetic acid pH 3) for
30 minutes followed by a 2 minute rinse in tap water and a 1 minute rinse in dH2O.
Slides were oxidized with 0.5% periodic acid (Sigma, St Louis, MO, USA) for 5 minutes to form Schiff’s bases on those polysaccharides not stained by alcian blue. The slides were rinsed in dH2O, flooded with PAS reagent (Sigma, St Louis, MO, USA) for
30 minutes, followed by a rinse in warm tap water, and air-dried.
Acid polysaccharides were differentiated from sulphated polysaccharides using the aldehyde fuchsin-alcian blue method. Aldehyde fuchsin was prepared by dissolving 1g of basic fuchsin (Sigma, St Louis, MO, USA) in 200mL 70% ethanol, followed by the addition of 1mL paraldehyde (Sigma, St Louis, MO, USA) and 1mL 32% HCl. The stain was mixed on a magnetic stirrer for 2 hours then ripened at room temperature for
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48 hours before storage at 4 C until required. Slides were stained with aldehyde fuchsin for 15 minutes, and rinsed in 70% ethanol for 1 minute, followed by dH2O for 1 minute.
Slides were stained with alcian blue for 15 minutes, rinsed in tap water followed by dH2O and air-dried.
5.3.2.3 Methyl green-pyronin
To determine whether the EPS contained any nucleic acids, reported by Nishimura et al., (2003) (RNA) and Steinberger and Holden (2005) (DNA) , slides were stained with methyl green-pyronin, a differential stain for DNA and RNA (Taft 1951; Chayen 1952).
Slides were immersed in methyl-green pyronin stain (Sigma, St Louis, MO, USA) for 5 minutes and rinsed in tap water followed by air-drying. As a control, prior to staining, separate slides were prepared by treating smears with 0.1mg/ml RNase A (Roche,
Manheim, Germany) for 1 hour at 37 C followed by a dH2O wash. Slides were stained as above.
5.3.2.4 Other stains
A variety of simple stains were also performed on the M. burtonii smears. To resolve any protein in the EPS slides were stained with Coomassie staining solution for 10 minutes followed by a quick rinse in de-stain (stains were as described in 2.3.6). To resolve any lipids, slides were stained in 10g/L Sudan black (AnalaR BDH, Poole,
England) in 70% ethanol for 10 minutes followed by a quick 70% ethanol rinse and a tap water rinse. Gram stains were also performed using reagents prepared by UNSW technical staff. Slides were flooded with crystal violet for 2 minutes, rinsed with concentrated iodine and stained with concentrated iodine for a further 2 minutes. Slides
D. Burg UNSW 225 were then de-colourised with alcohol-iodine. Slides were then counter-stained with carbol-fuchsin for 1 minute, rinsed in tap water and air-dried.
5.3.3 Fixative preparation
To avoid osmotic and oxidative stress to the cells, which could cause alteration of cellular and EPS morphology, glutaraldehyde fixative was prepared in anaerobic culture media. Anaerobic concentrated culture media (50mL) was prepared such that when
10mL of 25% glutaraldehyde solution (Electron Microscopy Sciences, Hatfield, MA,
USA) was added, salt concentrations would be similar to those in the culture media, with a final glutaraldehyde concentration of 4%. Culture media was prepared as described previously in 2.3.1 and 3.3.1 with the following alterations.
MFM: 0.0201g KCL; 0.36g MgCl2.6H2O; 0.06g MgSO4.7H2O; 0.015g NH4Cl;
0.0084g CaCl2.2H2O; 1.40g NaCl; 0.1mg ferrous ammonium sulfate; 0.06mg
resazurin; 0.3g TMA; 0.12g yeast extract; 0.6mL vitamin solution; 0.6mL
mineral solution; 0.006g sodium acetate; 0.0084g K2HPO4; 0.03g
cysteine.HCl; and 0.015g Na2CO3.
M-media: 1.39g NaCl; 0.74g MgSO4.6H2O; 0.046g KCl; 0.008g CaCl2.2H2O;
0.03g NH4Cl; 0.06mg resazurin; 0.6mL vitamin solution; 0.6mL mineral
solution; 0.03mL HCl; 0.045g thioglycolic acid: 0.067g Na2HPO4.7H2O;
0.015g cysteine.HCl.H2O; 0.018g Na2CO3; and 12 L methanol.
Both media concentrates were autoclaved, allowed to cool and anaerobically injected with 0.06mL 2.5% Na2S. The glutaraldehyde (10mL 25%) was injected anaerobically under a fume hood and the fixative was stored at 4 C for a maximum of 3 months.
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5.3.4 Scanning electron microscopy
The structure and appearance of the M. burtonii cells, grown at 4 C and 23 C in MFM and M-media, along with any EPS present was assessed using scanning electron microscopy.
5.3.4.1 Sample preparation
Anaerobic culture tubes (20mL) (Wheaton, Millville, NJ, USA) were plugged with butyl rubber stoppers and crimp sealed. The tubes were made anaerobic by flushing with nitrogen for 45 minutes by placing two, 23 gauge 25mm needles through the seal, and attaching one of the needles to the gassing manifold. To avoid thermally shocking the cells, the fixative and anaerobic tube were bought to the specific cultures growth temperature. 2mL of culture was then anaerobically transferred to the anaerobic tube using a 3mL syringe and a wide gauge needle (19 gauge 39mm) to avoid mechanically disrupting cells and EPS. A 2mL volume of fixative was slowly introduced to the cells
(final glutaraldehyde concentration of 2%) and the contents of the tube gently mixed.
Cells were fixed at the growth temperature of the culture for 1hour, then overnight at
4 C.
Approximately 1mL of fixed cellular material was removed from the anaerobic tube and gently passed onto a 0.22 m Isopore membrane (Millipore, Cork, Ireland), fitted into a
Swinnex filter holder with gasket (Millipore, Cork, Ireland). The cells were then washed three times in filtered growth media for 10 minutes, followed by a 5 minute dH2O wash.
SEM operates under high vacuum, it therefore requires completely dried samples. Air- drying samples in a hydrated state is destructive to cells and cellular features, as forces caused by surface tension of water during this process are very high. Critical point
D. Burg UNSW 227 drying makes use of the critical point of carbon dioxide (where gaseous and liquid phases are indistinguishable), which occurs at relatively low temperature and pressure.
By drying cells in this manner, cellular morphology and delicate features were preserved. In order to achieve this, the water in samples must first be gradually replaced by a volatile solvent prior to critical point drying. The membranes containing the sample were transferred into a critical point dryer specimen rack, and immersed in 30% ethanol for 10 minutes. The sample was dehydrated in 10 minute steps through 50, 70,
80, 90, 95, then through three washes of 100% ethanol. The sample basket was transferred to the sample reservoir of a Bal-Tec CPD030 critical point drier (Bal-Tec,
Liechtenstein). The instrument was sealed and the ethanol gradually replaced with liquid CO2 (under high pressure from a CO2 cylinder) by successive partial flushing and re-filling of the sample chamber. Once the ethanol was completely replaced by CO2, the chamber was heated past the critical point of CO2 (31 C, 72bar) and the pressure slowly relieved through a metering valve.
After critical point drying, membranes were mounted onto 13mm SEM sample stubs with carbon tape and chromium coated with a K575X Peltier cooled high resolution sputter coater (Emitech, Kent, England), for high resolution imaging. Samples were stored in a sealed container with desiccating material at room temperature until required.
5.3.4.2 Sample imaging
Samples were imaged on a Hitachi s3400 automated variable pressure SEM (Hitachi,
Tokyo, Japan). They were measured and size parameters entered to the SEM software.
Samples were introduced to the chamber and the chamber brought to vacuum. The sample stub was brought to within 10mm of the detector. An accelerating voltage of
228 D. Burg UNSW
15kV was applied, with probe current set to 10. Random images were taken at a magnification of 12.5k from areas of medium cell density by first zooming out to 500 x magnification, moving the stage and zooming in to 12.5k x magnification, focusing and recording the image using the microscope software. This was repeated until the required number of images (50 for each condition) was recorded. Images were also recorded of any interesting features. High resolution SEM imaging was performed by Dr Karen
Privatt, using a Hitachi S4500II field emission SEM (Hitachi, Tokyo, Japan).
5.3.4.3 EDS Analysis
Energy dispersive X-ray spectroscopy (EDS) was performed on amorphous non-cellular and non-EPS material to determine its elemental composition. The analysis was performed by Ms Jenny Norman (EMU), using an energy-dispersive X-ray microanalysis system attached to the S3400 instrument, with corresponding NORAN
System SIX software (Thermo Fisher Scientific, Waltham, MA, USA).
5.3.4.4 Image analysis
Quantitation of micrograph features was achieved through the use of an unbiased sampling regime utilising a 36 point counting grid (Figure 5.2a). The grid was transposed over the micrographs such that the distance between the points was 1μm.
Features were counted if they crossed any of the grid points. Features counted were
EPS, cells with association (either by contact or through EPS) with > 3 other cells, and cells with association with < 4 other cells. Cellular association was measured to determine the level of cellular aggregation in the samples and the thresholds of identifying an associated cell were chosen to minimise the influence of cells that were associated by chance or as a consequence of sample preparation.
D. Burg UNSW 229
The size of cells was also measured from the micrographs. 100 cells were randomly chosen, based upon point intercepts with the unbiased sampling grid. Any cells that were obviously dividing (bi-lobar) were disregarded. Cell size was measured along the longest axis of the cell, then at 90˚ to that measurement (Figure 5.2b).
5.3.5 Environmental scanning electron microscopy
To determine whether any features seen in the SEM micrographs were artifacts of dehydration, which has been reported for EPS (Little et al., 1991; Priester et al., 2007), environmental scanning electron microscopy (ESEM) was performed. ESEM has the advantage of operation with samples in a hydrated state. However, resolution of images is not as high as with dehydrated samples and SEM.
b a
Figure 5.2 Sampling and measurement procedure. a) Micrograph features were quantified using a 36 point counting grid, calibrated such that the distance between points was 1μm. Features were counted if they crossed the centre of a point. b) Cells were measured along their longest axis, and then at 90˚ to that axis
230 D. Burg UNSW
5.3.5.1 Sample preparation
M. burtonii cultures grown on MFM at 4 C and 23 C were processed for ESEM.
Samples were prepared in a similar manner to those for SEM, with the following changes: During fixation, ruthenium red (Sigma, St Louis, MO, USA) was added to the sample to a concentration of 0.075% w/v; Samples were introduced to the membrane and washed as with SEM, which was followed by a 2 hour stain with 2% OsO4
(ProSciTech, Kirwan, Queensland) prepared by EMU staff. The Ruthenium red-OsO4 combination has been reported as an excellent contrasting agent for ESEM studies of
EPS (Priester et al., 2007). Following the OsO4 staining, samples were subjected to three 10-minute washes with dH2O, then stored in a humidified, sealed, petri dish at 4 C until imaging the same day.
5.3.5.2 Sample imaging
Hydrated M. burtonii samples were imaged on a FEI Quanta 200 ESEM (FEI,
Hillsboro, OR, USA) with the assistance of Ms Jenny Norman (EMU). After several cycles of vapor pressure change (4 Torr – 9 Torr) to equilibrate the chamber, samples were imaged using the microscope software, with an accelerating voltage of 15kV, at
5 C, 4 Torr (60% relative humidity). Samples were gradually taken to ~100% relative humidity (5 C, 7 Torr) and sequential images taken. Samples were then gradually taken to 15% relative humidity (5 C, 1 Torr) and sequential images taken. This process was performed in order to evaluate whether surface features seen in the images were water droplets or pools, and also to see if any dehydration effects could be monitored in real- time.
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5.3.6 Transmission electron microscopy
In order to evaluate any differences in cellular morphology, membrane and internal structure, as well as any changes in EPS in two-dimensional space, TEM was performed.
5.3.6.1 Sample preparation
Samples for TEM were initially fixed and prepared in the same manner as those for
SEM. After the samples were transferred to the membranes and washed, the membranes were removed from their holders, placed in a petri dish, and the cellular material gently scraped into small piles. These were overlaid with a drop of cool ‘just-liquid’ 4% low melting point agar (Progen, Darra, Queensland). It was important that the agar be as cool as possible without setting, to avoid any thermal shock to the cells. The samples were cooled at 4 C for 20 minutes to allow the agar to set. The solidified agar was cut to remove any material not containing sample, and placed into 2mL centrifuge tubes
(Axygen Scientific, Union City, CA, USA). As an evaluation of preparation methods, several membranes were prepared omitting the agar steps. The samples were stained with 2% OsO4 for 1 hour, followed by three 10-minute washes in dH2O. The agar pieces containing samples were sequentially brought to 100% ethanol as for SEM preparation, and passed through three successive changes of 100% acetone. Spurr’s resin was prepared by adding, in order, to a 50mL tube: 10g ERL 4221 (vinyl cyclohexene dioxide) (ProSciTech, Kirwan, Queensland); 6g DER 732 (diglicidyl ether of polypropylene glycol) (ProSciTech, Kirwan, Queensland), followed by mixing; 26g
NSA (nonenyl succinic anhydride) (ProSciTech, Kirwan, Queensland), followed by mixing; and 0.4g DMAE (dimethylamine ethanol) (ProSciTech, Kirwan, Queensland) followed by a final mix. Samples were infiltrated with Spurr’s resin in 24 hour steps.
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Initially, samples were infiltrated by a 1:1 mixture of Acetone:Spurr’s resin, followed by a 1:9 mixture of Acetone:Spurr’s resin, followed by two infiltrations with 100%
Spurr’s resin. After a final 1 hour infiltration with 100% Spurr’s resin samples were cut into small pieces and placed, as close to the apex as possible, in silicone embedding moulds half-filled with Spurr’s resin, and containing a small label identifying the sample. The moulds were completely filled with Spurr’s resin and cured for 48 hours at
60 C. The resin blocks were removed, released from the silicone moulds, trimmed of any excess resin and stored at room temperature.
5.3.6.2 Ultramicrotomy
All ultramicrotomy was performed on a Leica EM UC6 ultramicrotome (Leica, Wetzlar,
Germany). Resin blocks were fixed into ultramicrotome chucks and prepared by trimming with a razor under a Leica L2 dissecting microscope (Leica, Wetzlar,
Germany). The blocks were trimmed such that a ‘mesa’ was formed around the sample.
The sides leading down towards the chuck away from the face to be cut were trimmed at a ~ 45 angle to the face, the leading edge and top edges were trimmed to be parallel and the side edges trimmed at a slight angle, such that the leading edge was slightly more narrow than the top edge.
Glass cutting knives were prepared in a Leica EM KMR2 knife maker (Leica, Wetzlar,
Germany). Following scoring and breaking, the cutting edges were inspected and any defective knives discarded.
The sample chuck was clamped into the ultramicrotome, and the orientation of the block noted for future reference. A glass knife was clamped into position in the ultramicrotome, the cutting edge manually brought to within 5mm of the block face, and the knife locked into place. The knife was advanced with the knife advancement knob to
D. Burg UNSW 233 within 1mm of the block face and the knife and the block face were aligned. The knife was brought as close as possible to the knife face and the block trimmed at 50mm/s in
1000nm sections until samples could be seen in the sections (identified by OsO4 fixed black material). The block was re-trimmed under the dissecting microscope, as described above, to minimise the area of the block face while retaining maximum amount of sample.
A plastic water bath was attached to a glass knife with molten wax prepared in a
Multiplate (Reichert-Jung, Depew, NY, USA), sealing any gaps and taking care not to touch the cutting edge. The sample chuck and knife were attached to the ultramicrotome as described above, the knife advanced to the block and the knife and block aligned.
Semi-thin sections were then cut from the block with settings of 2.5 mm/s, 1000nm advancement. Sections were retrieved from the water bath with a glass rod, and floated onto a drop of dH2O on a glass slide. The slides were air-dried, stained with 1%
Toluidine blue/1% Sodium Borate (ProSciTech, Kirwan, Queensland) for 5 minutes and rinsed with tap water. The slides were allowed to air dry and inspected with an Olympus
BX51 microscope (Olympus, Tokyo, Japan). Images were recorded as described in
4.3.2. Suitable blocks were then chosen for ultra thin sectioning.
Knives with water-baths attached were prepared as described above. The knife was advanced to the block face and aligned as described above, with the addition of a tilt alignment to ensure the block face passed the knife edge evenly throughout the cutting stroke. Ultra-thin sections were cut from the block with settings of 1mm/s, 70nm section thickness, changing the area of the knife used and re-aligning with every 10-20 cut sections. Good sections, identified by appearing silver to pale gold, were moved into close proximity to one another using an eyelash tool (made by attaching an eyelash to a toothpick using super-glue) and chloroform was wafted over their surface using a
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bamboo rod to spread out the section surface. Groups of sections were picked up on the surface of 0.25mm, 200 square formvar coated copper grids. Grids were dried by capillary action with filter paper and stored at room temperature prior to staining.
5.3.6.3 Grid staining
To add electron density and contrast to the sections, staining with uranyl acetate – lead citrate was performed. A drop of Reynolds lead citrate (~ 26g/L PbNO3, 35g/L sodium citrate (ProSciTech, Kirwan, Queensland), made alkaline with NaOH) per grid to be stained was placed on a small piece of plastic film. This was placed in a humidified petri dish containing NaOH pellets to remove CO2 and avoid precipitation of PbCO3.
The petri dish was covered and set aside. Small drops of 2% uranyl acetate (ProSciTech,
Kirwan, Queensland) in 50% ethanol were placed onto a piece of plastic film, and the grids containing sections were placed face down onto the drops for 20 minutes. Grids were thoroughly washed with dH2O, dried and placed face down onto the previously prepared drops of lead citrate and stained for a further 4 minutes. The grids were then thoroughly washed with dH2O and dried by capillary action with filter paper. Grids were stored at room temperature until needed.
5.3.6.4 TEM imaging
Samples were imaged using a JEOL JEM-1400 transmission electron microscope
(JEOL USA, Inc., Peabody MA) at 100kV, 75μA. Images were taken at random using the same procedure as with SEM at 15k x magnification. A total of 40 images were taken for each sample condition. High magnification images of areas of interest were also taken
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5.3.6.5 TEM image analysis
TEM images were analysed in a similar manner to SEM images, using an unbiased sampling protocol to quantify extracellular material. Other features quantified were cells that had highly irregular shapes, this did not include bi-lobar cells which were assumed to be dividing. Cells that had circular areas of electron transparency were also counted.
Cross sectional areas of cells were measured in order to enable the application of seterological procedures. Images were first converted to binary representations (solid black and white), using ImageJ (Abramoff et al., 2004), the edges smoothed and the scale calibrated. Cross sectional areas were calculated by the software for 400 cells from each sample condition. Areas were not calculated for cells that were obviously dividing.
Cellular volume was estimated based on the principals outlined in (Hammel 1986) using the equation:
v = β √ā 3 where: v = volume; β = coefficient of configuration, estimated using the principals of
Weibel and Gomez (1962) from the mean axial ratio measured from SEM micrographs
(5.3.4.4); and ā = mean cross sectional area.
5.3.7 Statistical analysis
Statistical analyses and data transformations were performed using the Minitab 13 statistical software package (Minitab, State College, PA, USA)
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5.4 Results
5.4.1 Histochemistry
To determine the composition of the EPS produced by M. burtonii at a general level, and to determine if any differences in that composition occurred with respect to temperature, a variety of staining protocols were performed.
5.4.1.1 Stains acting on sugars
When cell smears were stained with the alcian blue-PAS stain, which differentiates between acidic and neutral protein-sugar complexes, different results were achieved for cells grown at 4˚C and 23˚C (Figure 5.3), indicating that the EPS of cells grown at the two tested temperatures had different properties.
4˚C 23˚C
Figure 5.3 Alcian blue-PAS stain results. The EPS from cells grown at 4˚C (left) stained pink/purple (as above) indicating neutral composition. EPS from cells grown at 23˚C (right) stained blue, which is indicative of acidic composition.
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The EPS of cells grown at 23˚C was able to complex with the alcian blue dye, which only binds to acidic residues on polysaccharides (carboxylated). The EPS of cells grown at 4˚C stained pink/purple, indicative of a neutral polysaccharide.
Smears of cells were also stained with Aldehyde fuchsin-Alcian blue, a staining protocol which distinguishes between sulphated and non-sulfated acidic polysaccharides. When the smears from 23˚C grown cells (which were shown to contain acidic residues) were stained with this protocol, EPS remained blue (as in
Figure 5.3), indicative of a non-sulfated polysaccharide.
5.4.1.2 Stains acting on nucleic acids
In order to determine whether the EPS produced by M. burtonii had any nucleic acid content, smears of cells grown at 4˚C and 23˚C were stained with the methyl green- pyronin stain (Figure 5.4). EPS material stained red, which is indicative of the presence of RNA. In order to determine if these results were experimental artefacts, smears were treated with RNase prior to application of the stain (Figure 5.4). Following this treatment, no colour was evident after application of the stain, a good indication that
RNA is in fact present in the EPS produced by M. burtonii.
To gain further evidence for this finding, cell suspensions, containing aggregated cell clumps in the case of 4˚C cells, were treated with RNase in order to determine whether
RNase was able to disaggregate any cell clumps, as described by Watanabe et al.,(1998) for EPS containing RNA in Rhodovulum sp.. When cells were incubated with RNase at
30˚C, all samples, including controls, disaggregated. Subsequent repeats at lower temperatures with low salt concentrations (to facilitate RNase activity at lower temperatures), also showed disaggregation in all samples, including controls. While no clear results on RNase activity against cellular aggregates could be determined from
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these experiments, the data did indicate that aggregation, and following from this the
EPS, of M. burtonii is thermally labile, and is disrupted by low salt concentration.
a b
c d
Figure 5.4 Methyl green-pyronin stain results. In cells smears stained with methyl green-pyronin, EPS stained pink/red for both 4˚C (a) and 23˚C (b) samples, indicating the presence of RNA. When cell smears were treated with RNase prior to staining, EPS did not stain red for both 4˚C (c) and 23˚C (d) samples. This is indicative of the removal of the RNA by the RNase enzyme. Cytoplasm of cells stained pale green/blue. This indicated that the DNA present was not affected by the RNase treatment.
5.4.1.3 Other stains performed
A variety of other stains were performed on the samples from both 4˚C and 23˚C. A
Coomassie stain of the EPS indicated the presence of protein (Figure 5.5). Staining with
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Sudan black indicated that lipids were not present in the EPS (data not shown), and both
4˚C and 23˚C cells stained Gram variable (data not shown).
4˚C 23˚C
Figure 5.5 Coomassie stain of EPS. Cell preparations from both 4˚C (left) and 23˚C (right) had extracellular material that stained positive for protein.
5.4.2 Scanning electron microscopy
5.4.2.1 Cells and EPS from different temperatures were remarkably different
When cells from both 4˚C and 23˚C were examined under SEM very apparent differences were seen in the morphology of the cells, the EPS produced and the level of cellular association. At low magnifications, large clumps were seen in 4˚C samples
(Figure 5.6), but not seen in 23˚C samples. These clumps were complex in structure, irregular in shape, and consisted of many thousands of individual cells (Figure 5.7), bound together by apparently filamentous EPS (Figure 5.8).
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Figure 5.6 SEM of a cell clump as seen at 4˚C. Samples of cells grown at 4˚C contained large aggregates of cells. These aggregates were irregular in size and morphology
Figure 5.7 Magnification of a cell aggregate surface. The cell aggregates seen at 4˚C were comprised of many thousands of individual cells.
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Figure 5.8 High magnification of a cell aggregate surface. The individual cells were connected by apparently filamentous EPS.
Examination at high magnifications away from any cellular clumps, indicated very apparent differences between 4˚C cells and 23˚C cells. Cells from both MFM (Figure
5.9) and M-media (Figure 5.10) displayed differences according to temperature, with
4˚C cells appearing smaller and were surrounded by filamentous EPS material. Cells grown at 23˚C appeared larger, and were not surrounded by filamentous EPS. Any EPS present appeared as sheet like material. This was observed for cells at all growth phases tested.
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a
b
Figure 5.9 SEM of cells from 4˚C and 23˚C grown in MFM. Both micrographs were gathered at 12k magnification. Cells grown at 4˚C (a) appeared smaller and were surrounded by filamentous EPS. Cells grown at 23˚C (b) appeared larger and were often associated with sheet-like EPS (solid arrow). The dotted arrow shows media precipitate.
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a
b
Figure 5.10 SEM of cells from 4˚C and 23˚C grown in M-media. Both micrographs were gathered at 12k magnification. Cells grown at 4˚C (a) appeared smaller and were surrounded by filamentous EPS. Cells grown at 23˚C (b) appeared larger and were often associated with sheet-like EPS (arrow). The dotted arrow shows media precipitate.
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Another apparent feature of the cells, was differences in the appearance of the cell
surface at both 4 and 23˚C. At high magnifications (Figure 5.11), cells from 4˚C took
on a ‘pimpled’ appearance, while cells from 23˚C had smooth surfaces.
4˚C MFM 23˚C MFM
4˚C M -media 23˚C M-media
Figure 5.11 High resolution SEM of cells from 4˚C and 23˚C. Images from MFM were taken using a
high resolution instrument. Cells from 4˚C appeared to have surface modifications, taking on a ‘pimpled’
appearance. Cells grown at 23˚C had smooth surfaces.
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5.4.2.2 Energy dispersive X-ray spectroscopy of media precipitate
From inspection of the SEM micrographs, it was apparent that clusters of cells and EPS
were often associated with granular material that was most likely precipitate from the
media (see Figures 5.9 and 5.10). Whether this was an artefact of sample preparation,
or a real association is unclear. However, as this phenomenon could be a feature of
cellular growth (i.e. association of cells with a nutrient source), EDS analysis of
granular material was performed (Figure 5.12).
Fe S
Na
Z Fe Cr Cr
Figure 5.12 EDS spectra of media precipitate. The media precipitate consisted of high proportions of
iron and sulphur. Some zinc and sodium peaks were also detected. The chromium detection was due to
the ~50nm chromium coating utilised in sample preparation.
The largest peaks detected from the EDS analysis corresponded to the X-ray energies of
iron and sulphur. The possibility that these precipitates are polysulfide salts (as
discussed in 2.4.8.12), could not be determined from this analysis.
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5.4.2.3 Statistical analysis
Quantitation of differences seen in micrographs of cells at different temperatures was achieved through an unbiased sampling protocol coupled with statistical analysis.
Statistical differences were observed, in several measurement categories, between cultures grown under different conditions. Micrographs (50 from each condition) were examined. The features quantified were: EPS; cells associated with other cells (via contact or EPS) in groups of > 3; and cells associated with other cells (via contact or
EPS) in groups of < 4. The results of this quantitation are displayed in Figure 5.13.
Statistical differences were observed (ANOVA, p < 0.001 in all cases) between both the cultures grown at different temperatures and in different media. Where those differences occurred was determined using Tukey’s pairwise comparisons. Cultures grown at 4˚C had a higher level of EPS than cells grown at 23˚C, and cells grown in M- media had higher counts of EPS than cells grown in MFM. Measurements of cellular association also showed statistical differences, with cells grown at 4˚C having higher numbers of cells in association with surrounding cells. Samples from 4˚C also showed a difference between growth media, with cells grown in M-media displaying much greater cellular association than those grown in MFM. Conversely, the results indicate that cells grown at 23˚C are more ‘free living’; that is, cells from 23˚C grown cells tended not to be associated in large groups.
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EPS Cell association < 4
18 Cell association > 3
16
14
12
10
8
6
Point intercepts per grid intercepts Point 4
2
0 4C MFM 4C M 23C MFM 23C M
Sample
Figure 5.13 Quantification of micrograph features. Sample types are in the format: temperature; media,
M media or MFM. Features of micrographs were measured using unbiased sampling techniques and compared using ANOVA. Statistical differences were observed in the amount of EPS, and the level of cellular association. As a whole 4˚C cells had more EPS and cellular association. Cells from M-media had more EPS than cells from the corresponding temperature in MFM
The size of the cells was also measured as a function of temperature and media. As
SEM micrographs are only 2D representations of 3D features, and as the M. burtonii cells are irregular in shape, this had to be approached carefully. From each condition,
100 cells were chosen, based upon point intercepts of sampling grids, and measured.
Measurement was achieved by determining the longest vector across the cell surface. A further measurement was taken at 90˚ to that first measurement. The measurement of the cells from all conditions resulted in a non-parametric dataset that was Box-Cox
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transformed to normality, and to equalise variance. ANOVA, with Tukey’s pairwise comparisons, was then performed, and statistical differences were found (p < 0.001) between the size of cells. Cells from 4˚C cultures were significantly smaller than those from 23˚C cultures (Figure 5.14). No significant differences were observed between cells grown in different media (from the same temperature).
Maximum
Perpendicular 1.2
1.0
m) u 0.8
0.6
0.4
Cell axis ( length
0.2
0.0 4C MFM 4C M 23C MFM 23C M
Sample
Figure 5.14 Differences in cell size. Sample types are in the format: temperature; media, M media or
MFM. Cells grown at 4˚C were significantly smaller than cells grown at 23˚C in both media and along both axes measured. No significant differences were observed between cells grown in different media.
5.4.3 Environmental scanning electron microscopy
The appearance of EPS is often altered by the sample dehydration required for imaging using SEM (Little et al., 1991). In order to determine whether the features of EPS
D. Burg UNSW 249 observed following SEM were true representations of the morphology of the EPS, or alternatively were artefacts of dehydration, ESEM of hydrated samples was performed.
Samples were imaged through various stages of humidity/pressure for both 4˚C and
23˚C samples (MFM grown samples were used, as features of EPS on SEM were consistent across media types). Time series micrographs of this progressive dehydration are illustrated in figures 5.15 (A and B) and 5.16 (A and B). This passage through levels of hydration was performed for two reasons. Firstly, water pooling on the surface of the sample is indistinguishable from EPS, so samples were taken from a humidity and pressure where water was obviously pooling on the surface of the samples, through to relative dryness (low humidity and pressure). Secondly, samples were imaged after an extended period at very low humidity and pressure to see if changes in the morphology of EPS could be induced through dehydration.
x
x x
Figure 5.15 A: ESEM of 4˚C sample. Sample at high humidity and pressure. Pooled water is marked by x. Dehydration steps (figure 5.15 B) continue on following page.
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a b
c d
Figure 5.15 A: ESEM of 4˚C sample. Sample at high humidity and pressure. Pooled water is marked by
x. Dehydration steps (figure 5.15 B) continue on following page.
e Figure 5.15 B: ESEM of 4˚C sample. Progressive
sample dehydration. The sample was taken to low
pressure and humidity, with humidity decreasing left to
right (a-c) and was kept at low pressure for an extended
period (d). EPS appeared as confluent material even
after extended periods of drying (e). Arrows mark the frame of reference.
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x x
x x
Figure 5.16 A: ESEM of 23˚C sample; Sample in a hydrated state. Samples were imaged at high pressure and humidity, with humidity decreasing left to right. Examples of pooled water are marked with
X. The arrow indicates point of reference
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Figure 5.16 B: ESEM of 23˚C sample A: Sample in a dehydrated state. Samples were imaged at low pressure and humidity with humidity decreasing left to right. The arrow indicates point of reference. EPS appeared very similar to that from 4˚C samples
The EPS from 4˚C and 23˚C samples appeared remarkably similar when imaged with
ESEM. No string-like structures were seen in 4˚C samples, even following extended
D. Burg UNSW 253 drying. 23˚C samples had high levels of EPS not seen in SEM samples, and a much higher level of cellular association than previously recorded.
A lack of string like structures between cells was also noted in cellular aggregates at
4˚C in the ESEM micrographs. These appeared to be bound by a confluent mass of EPS
(Figure 5.17). The cell aggregates were irregular in size and shape, as seen in SEM analyses.
Figure 5.17 ESEM of cell aggregate from 4˚C grown cells. No string like EPS was observed. Aggregates appeared irregular in shape and structure.
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ESEM of 23˚C samples revealed that high levels of EPS were present in samples grown at this temperature. The EPS had similar morphology as of that seen in 4˚C samples
(Figure 5.18).
Figure 5.18 ESEM of EPS from 23˚C grown cells. EPS appeared very similar to that observed in 4˚C samples (see Figure 5.15 B(e)). Some cracking was observed (arrow), this was not seen in EPS from 4˚C.
5.4.3.1 Parallel SEM
In order to determine whether the features seen in the ESEM were due to unexpected changes in sample morphology, i.e. whether the differences between ESEM and SEM were real, samples were prepared in parallel. These samples came from the same cultures used for ESEM, and were imaged via SEM as in described 5.2.2.
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a
b
Figure 5.19 Parallel SEM of ESEM samples. Following SEM samples from 4˚C (a) and 23 ˚C (b) appeared as did previous SEM samples. String like structures were seen in 4˚C samples (arrow), and very little EPS was seen connecting cells in 23˚C samples. Where EPS was seen in 23˚C samples (micrograph for display of EPS) it appeared in a sheet-like form (arrow).
The SEM images taken in parallel with the ESEM, were as seen in previous SEM analyses (Figure 5.19), indicating the samples were similar, and displayed the same
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behaviour on dehydration. This showed that the ESEM was a true representation of the samples from 4˚C and 23˚C.
The information from the images gathered with ESEM, therefore shows that EPS production by samples at 4˚C and 23˚C is similar. However, the behaviour of the EPS on dehydration was significantly different. Dehydration of 23˚C samples resulted destruction or extensive dehydration of EPS to sheet-like material, while dehydration of
4˚C samples resulted in dehydration of EPS into string like material that remained attached to cells. This indicated that the EPS from 4˚C and 23˚C samples has different properties, and hence different composition. Large cell aggregates were only seen at
4˚C, however, a feature that is macroscopically apparent when examining cell cultures.
5.4.4 Transmission electron microscopy
In order to assess changes in cellular size and morphology seen in SEM analyses, TEM was performed on cells grown at 4˚C and 23˚C, for both media types. The presence of
EPS was also assessed using this method.
5.4.4.1 Assessment of sample preparation procedures
Before any TEM analyses were performed, a procedure for embedding the cellular material and EPS of M. burtonii had to be developed. Two methods were assessed; one involved embedding a membrane, that was prepared in a similar fashion to SEM samples, in resin prior to sectioning; the other method, involved the capture of cellular material into agarose prior to embedding in resin. Samples were embedded in resin, polymerised and semi- thin sections analysed under a light microscope (Figure 5.20).
Samples prepared in agar had a good dispersion of cells over a relatively large area.
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Conversely samples prepared on membranes had cells in high density at the membrane interface.
Figure 5.20 Semi then sections of TEM sample preparations. Samples prepared in agarose prior to embedding in resin (left) displayed good dispersion of cells. Samples prepared on membranes (right) had cells clustered over a small area at the membrane interface (arrow). Defects in sections were also apparent as sections tended to contain several folds (dashed arrow).
When the samples were ultra-thin sectioned and examined under TEM, the membrane samples displayed marked folding, while the agarose samples were clear and free of defects (Figure 5.21). The defects seen in the membrane samples were due to sections tearing and separating along the membrane interface. This occurred as the membrane material did not become infiltrated with resin resulting in an area of weakness in the sections. From this assessment it is clear that membrane preparations had clumps of cells at the membrane interface, which may not reflect the true nature of cellular association. The preparations were also difficult to section without defects. Therefore, the procedure utilising agarose was chosen as the best method for preparing samples.
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Sections from this preparation displayed good dispersal of cells and an ease of obtaining good sections.
Figure 5.21 Ultra-thin sections of TEM sample preparations. Samples prepared using agarose (left) displayed good dispersal of cells and were free of defects. Striations seen in the section are due to knife resonance. Samples prepared from membranes (right) tended to tear at the membrane interface (arrow), and had marked rippling (dashed arrow).
5.4.4.2 TEM image analysis
Following method evaluation, samples were prepared using the agar method and 70 –
90 nm (silver appearance) ultra-thin sections were prepared for all conditions ( 4˚C,
23˚C, MFM, M-media). On random inspection of these sections, clear differences were observable between cells from all conditions. Attempts were made to quantify differences in cells and micrograph features. These followed the same principles used for SEM inspection, with counting parameters as outlined in (5.3.4.4 and 5.3.6.5). The features quantified by point intercepts were: granular extracellular material (ECM); filamentous ECM; cells with circular electron transparent inclusions; and irregularity in cell shape. Statistical differences were observed in all of these categories except in the
D. Burg UNSW 259 presence of filamentous ECM. Representative micrographs from all conditions are displayed in Figure 5.22. Cells from 4˚C grown cells from both media had more granular ECM (ANOVA p = 0.032) than 23˚C grown cells, with 4˚C MFM cells displaying the most (Tukey’s pairwise comparisons). Cells grown in M-media
(methanol) tended to have circular electron transparent inclusions (ANOVA p < 0.001), while the staining of MFM grown cells was relatively uniform (see below and figure
5.25 for exceptions) (Figure 5.23). Cells grown in M-media were also highly irregular in shape (ANOVA p < 0.001) when compared to MFM grown cells, with cells grown in
M-media at 23˚C having the highest incidence of irregularity (Tukey’s pairwise comparisons) (Figure 5.23).
Calculations of cellular parameters from polydispersed cells, visualised in random sections, are achievable through the use of stereology and morphometry. The term polydispersed refers to the fact that a section taken from cellular material in a resin block will contain cross sections from all levels of individual 3D cells, from extremities
(smallest cross section) to maximum cross section, for all cellular orientations.
However, these stereological calculations rely on the basic assumption that cells are of relatively equal size (Schmid-Schonbein et al., 1980; Tian et al., 2005). Further, the stereological principals rely on the application of calculations for regular 3D objects, for example: the volume and surface area of spheroids or cylinders (Schmid-Schonbein et al., 1980; Tian et al., 2005).
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a b
c d
e f g
Figure 5.22 TEM micrograph features. Micrographs features were quantified for all samples: (a) 4˚C
MFM; (b) 23˚C MFM; (c) 4˚C M-media; (d) 23˚C M-media. Features quantified were: cells with
irregular shapes (arrows in d) - cells in M- media were more irregular (p < 0.001); (e) cells with circular
electron transparent inclusions - cells grown in M- media tended to have inclusions (p < 0.001); (f) the
presence of granular ECM - sections from samples grown at 4˚C had more of this substance (p = 0.032);
and (g) filamentous ECM - no differences were observed in the presence of this material between
samples.
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60
50
40
30
20 % of cell intercepts
10
0 MFM 23 M 23 MFM 4 M 4 Culture conditions (media, temperature) Cells with Irregular shape Cells with circular electron transparent zones
Figure 5.23 Differences in cell morphology with respect to media. Cells grown in M-media (methanol, defined) had highly irregular cross sectional shapes, and tended to have circular electron transparent zones
The calculation of volumes and surface areas of irregular shapes can be achieved using stereological principals, however, these rely on applying Cavalieri’s principal to serial sections through entire cells, with measurement of, for example, 1 in every 5 sections
(Mandarim-de-Lacerda 2003). In an attempt to quantify any changes in cell size across all conditions, cross sectional areas were measured from binary transformed micrographs (Figure 5.24). Frequency distributions of these sorts of measurements from random sections have been shown to reflect stereological measurements (Tian et al., 2005), and can be indicative of regularity of a population.
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a b
25 25
20 20
15 15
% of areas 10 % of areas 10
5 5
0 0
<0.1 >0.9 <0.1 >0.9 0.1 - 1.90.2 - 0.290.3 - 0.390.4 - 0.490.5 - 0.590.6 - 0.690.7 - 0.790.8 - 0.89 0.1 - 1.90.2 - 0.290.3 - 0.390.4 - 0.490.5 - 0.590.6 - 0.690.7 - 0.790.8 - 0.89 Area Area
c d 25 25
20 20
15 15 % of areas % of areas 10 10
5 5
0 0
<0.1 >0.9 <0.1 >0.9 0.1 - 1.90.2 - 0.290.3 - 0.390.4 - 0.490.5 - 0.590.6 - 0.690.7 - 0.790.8 - 0.89 0.1 - 1.90.2 - 0.290.3 - 0.390.4 - 0.490.5 - 0.590.6 - 0.690.7 - 0.790.8 - 0.89 Area Area
Figure 5.24 Frequency distributions of cellular cross sectional areas. Area is in μm2. Distributions
differed across cell growth conditions: (a) 4˚C M-media; (b) 4˚C MFM; (c) 23˚C M-media; (d) 23˚C
MFM. Cells grown at 23˚C in MFM had a tendency to be larger in cross sectional area, with a distribution
pattern suggesting a polydisperse population of similarly sized cells. M- media grown cells had relatively
large proportions of cells at the lower area range, reflective of sectioning cells of irregular shape.
Population means: M-media - 0.33μm2 for 23˚C cells and 0.34 μm2 for 4˚C cells; MFM - 0.53μm2 for
23˚C cells and 0.36 μm2 for 4˚C cells.
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Cells grown at in MFM displayed a pattern of distribution that would be expected if measuring samples from a spheroid/ellipsoid population, with relatively low variation in cell size (Schmid-Schonbein et al., 1980). The frequency distribution generated displayed roughly equal (normal) distribution around a central point that corresponds to the mean measurement (0.53μm2 for 23˚C cells and 0.36 μm2 for 4˚C cells). The differences in the spread (width) and shape (skew) of these distributions can also be attributed to cell size. Cross sections from a larger spheroid ‘mother cell’ population will display a larger spread of frequency distribution, as cross sections can occur across a larger range of values due to the larger maximum diameter. The converse is also true for smaller cells. The 23˚C cell area frequency distribution is skewed towards the right and the 4˚C distribution is skewed towards the left, a direct result of the first and last categories encompassing a range of measurements i.e. < 0.1 and > 0.9. If further area categories were included in these samples the distributions appear roughly symmetrical.
Statistics were applied to the area measurements and the 23˚C cells from MFM had a larger population mean (p < 0.001) than the 4˚C grown cells; indicative that cells grown at 4˚C were smaller. As the population of MFM grown cells appeared to have similar features to documented spheroid populations (Schmid-Schonbein et al., 1980), cell volume was estimated. Utilising the cell measurements taken in SEM analysis (5.3.4.4) as the axial ratio, the volume of cells grown on MFM was calculated as 0.509 μm3 for
23˚C cells, and 0.289 μm3 for 4˚C grown cells. Cells grown at 23˚C had a 76% larger volume.
Comparative measurement and volume estimation of the cells grown in M-media was unable to be achieved due to the irregular shapes of the cells. This was reflected by a relatively large population of cells with small cross sectional areas, resulting in obvious frequency distribution skews (Figure 5.24). Intensive stereological studies would be
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required in order to measure the relative volumes of these cells, and would require the serial sectioning of hundreds, if not thousands of cells, a process that would be highly intensive and is beyond the scope and time frame of this investigation.
Another feature noted in the samples was that 4˚C cells sometimes appeared more electron dense at the perimeter (Figure 5.25), this observation was not recorded in 23˚C cells. The phenomenon was only seen in ~ 1 in 20 cells so could not be quantified from the limited number of samples examined. Intensive TEM, and possibly TEM tomography, is needed to determine the validity of this observation.
Figure 5.25 Peripheral electron density in 4˚C cells. Some cells grown at 4˚C were more electron dense towards the perimeter of the cell.
In direct contrast to SEM measurements, no differences in the surface of the cells were seen between the cells grown at 4˚C and 23 ˚C. However, the implementation of higher resolution TEM, or cryo-techniques may be better suited to observe this phenomenon.
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5.5 Discussion
5.5.1 The extracellular polymeric substance of M. burtonii
5.5.1.1 The EPS produced by M. burtonii has different properties with respect to temperature
The visualisation and quantitation of SEM micrographs, showed that cells grown at different temperatures produced EPS that had different properties when dehydrated. The
EPS of cells grown at 4˚C dehydrated into string like structures, while the EPS of cells grown at 23˚C dehydrated into sheet-like material, and/or was destroyed by critical point drying. This occurred regardless of the growth media. When the cells were visualised using ESEM, no differences were observed in the appearance of the EPS. It suggests that while cells grown at different temperatures produce EPS, there is a significant difference in its composition. This appears to be the first time that a combination of these two techniques has been used in a qualitative manner to examine differences in dehydration of EPS, which has normally been considered an experimental artefact. Some psychrophiles are known to increase their EPS production at low temperature (Nichols et al., 2005; Marx et al., 2009), however different quantitative techniques need to be performed to determine if modulation of EPS quantity, as well as composition, is a feature of thermal adaptation in M. burtonii.
Visualisation of the EPS of M. burtonii using TEM was more difficult, as much material that could be EPS tended to stain poorly. However, a difference in the amount of extracellular granular material was observed, with more of this material seen at 4˚C.
EPS is known to take on this appearance (Reese and Guggenheim 2007), however there remains the possibility that the electron dense granular material seen around the cells was inorganic precipitate. Granular material was often seen around cells in the SEM
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micrographs (Figures 5.9 and 5.10), and the increased amount of this material could be a real association or may have occurred by chance. String-like extracellular material was also seen around the cells. EPS can also take on this appearance (Baum et al.,
2009). However, no differences in the amount of this material were seen between cells grown at different temperatures. Alternative staining protocols (for example the use of ruthenium red as in ESEM) might enhance contrast and the electron density of EPS.
Another possible explanation of why EPS was difficult to visualise using this technique, is that any EPS was disrupted by heat during sample preparation. During sample preparation cells are suspended in low melting point agarose. While this material was at its lowest temperature before setting (~37˚C), it was shown that cell aggregation is disrupted in M. burtonii at 30˚C (5.4.1.2). The contact of the cells and EPS with this material could have resulted in its alteration or destruction. Therefore, an alternative preparation procedure could be considered, for example using gelatine, if any further
TEM is to be carried out on M. burtonii and its EPS.
Staining techniques, provided evidence that the EPS produced by M. burtonii was contained protein, sugars and nucleic acid (5.4.1), as previously reported for EPS in other organisms (Watanabe et al., 1999; Nishimura et al., 2003; Nevot et al., 2006). It was also found that EPS of cells grown at 4˚C contained neutral polysaccharides, while the EPS of cells grown at 23˚C contained acidic polysaccharides. Psychrophiles are known to change the acidic properties of their EPS, however this has been observed to occur at low temperatures (Nichols et al., 2005), rather than at high temperatures as seen in M. burtonii. Acidic residues in EPS, for example glucuronic acid, can bind cations (Kawaguchi and Decho 2002; Nichols et al., 2005), and dissolved organic matter (Decho and Herndl 1995). One possible explanation of why M. burtonii increases the acidic residues on EPS at high temperatures is related to increased nutrient
D. Burg UNSW 267 demand. As described in Chapter 4, the high growth rate of M. burtonii at 23˚C is associated with an overall increased demand for nutrients. By increasing the acidity of the EPS produced at 23˚C, the organism would be able to trap more effectively nutrients, and essential cations, required for growth.
5.5.1.2 The EPS of M. burtonii contains RNA
The results of the methyl green-pyronin stain indicated that the EPS produced by M. burtonii contained RNA. Nucleic acids, especially DNA, are known to be essential for biofilm formation (Whitechurch et al., 2002; Vilain et al., 2009), EPS stability
(Steinberger and Holden 2005), and have roles in antibiotic resistance (Mulcahy et al.,
2008). Extracellular DNA is also known to be an important structural element in EPS, serving as a scaffold for the attachment of other EPS components and cells
(Bockelmann et al., 2007; Smalyukh et al., 2008). The high stability of extracellular
DNA is imparted through these interactions (Vlassov et al., 2007). Extracellular RNA in
EPS of microorganisms is less common than extracellular DNA, and so far has only been identified in a limited number of bacterial species (Watanabe et al., 1998;
Watanabe et al., 1999; Nishimura et al., 2003; Ando et al., 2006). Extracellular RNA
(composed of tRNA and rRNA-like elements) has an important role in the flocculation of cells (Nishimura et al., 2003), may have a structural role as with extracellular DNA
(Ando et al., 2006), and has also been shown to act as a cofactor for extracellular enzymes (Nakazawa et al., 2005). The role of the extracellular RNA in M. burtonii is unclear, and the occurrence of this material needs to be confirmed. However, the detection of extracellular RNA in this organism is the first evidence of this phenomenon in the archaea.
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5.5.1.3 EPS and cells associate with granular material
Cells and EPS were often found associated with granular material. EDS analysis showed that this material was composed principally of iron and sulphur. Insoluble iron and sulphur compounds are often associated with methanogenic consortia (Yamaguchi et al., 2001). However, the material seen in these analyses most likely corresponds to black precipitate found in the culture media, as a consortium of organisms is generally required for mineralisation. In the case of laboratory grown M. burtonii, precipitation of these inorganic substances could lead to the reduction of their free ionic forms in the media, and the organism may then need to closely associate with these iron and sulphur precipitates in order to metabolise them. Several organisms are known to do this with insoluble iron and sulphur compounds (Kachlany et al., 2001; Qin et al., 2007).
5.5.2 Cells grown at low temperatures form large aggregates
Multi-cellular aggregates are known to occur in the archaea, in a phenomenon that is generally modulated by media composition and stress (Sowers et al., 1993; Macario and
Conway De Macario 2001; Frols et al., 2008). Aggregates of organisms are often more resistant to environmental stressors (Klebensberger et al., 2006), and proteolytic attack
(Baum et al., 2009). However, in M. burtonii, large aggregates of cells formed at low temperatures, which are not associated with a general stress response (Chapter 4). A higher level of cellular association was also seen in methanol grown cells at 4˚C, compared to cells grown on TMA at 4˚C. The methanol related aggregation may be related to the stressful effect of methanol, as is seen in the Methanosarcina spp
(Macario and Conway De Macario 2001). Aggregation of cells also facilitates gene transfer events (Frols et al., 2008). As there are indications of recent genome evolution in M. burtonii, which is related to its recent geographical isolation in Ace Lake (Allen et
D. Burg UNSW 269 al., 2009), the aggregation of cells under the in situ cold conditions may have facilitated the transfer of altered genetic material between individual organisms, or from other species trapped in the cell aggregate. Another scenario where aggregation is likely to be an advantage in cold conditions involves the exchange of nutrients (Reid et al., 2006).
Large multicellular aggregates, encased in EPS, could effectively trap nutrients, either from the external environment or from lysed cells within the structure. A communal extracellular pool of enzymes could then assimilate these nutrients increasing the efficiency of foraging (Vetter et al., 1998). An increase in efficiency of nutrient assimilation would be advantageous under cold conditions where general rates of reaction are slow.
The cell aggregates of M. burtonii appeared to have structure, with cracks and channels visible in both dehydrated (SEM) and hydrated (ESEM) samples. Biofilms are often structured to allow the passage of nutrients (Baum et al., 2009), and methanogenic granules often have channels and cracks which allow methane generated at the centre of the aggregation to escape (Fukuzaki et al., 1991). Further investigations of the granules are required to determine if these aggregates are truly organised
5.5.3 Cell size differed with respect to temperature
Differences in cell size were observed when cells were analysed using both SEM and
TEM. Cells appeared smaller when grown at low temperatures. While this appears to be a thermally related phenomenon, some differences were also seen when cells were grown on methanol. There are two possible reasons why cells size would be altered with respect to temperature. Psychrophilic organisms are known to increase the concentration of intracellular solutes at very cold temperatures as a protection against ice crystal damage. This phenomenon is generally only seen in the eutectophiles
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(Rodrigues and Tiedge 2008) however, M. burtonii may use this strategy to increase internal solute concentrations as a means of stabilising proteins (4.4.3.1). An alternative explanation could be that cells grown at high temperatures are decreasing their internal salt (Na+) concentration. M. burtonii utilises sodium gradients for a number of cellular processes (3.4.2.12.1). At high temperatures, and hence fast growth rates, there would be a rapid accumulation of intracellular sodium that could be detrimental to the cell.
This accumulation would decrease the electrochemical potential gradient required for methanogenesis and transport processes. To counteract this, M. burtonii could take on water as a means to maintain low intracellular sodium.
Differences in the surfaces of cells were also seen following SEM, with cells grown at
4˚C taking on a ‘pimpled’ appearance. Differences in the surface of cells were not seen however, using TEM. It is unclear whether the differences seen were thermally related or artefactual. During dehydration the membranes, which are not fixed by glutaradehyde
(Cook 1998), might behave differently in cells grown at different temperatures. Cells grown at low temperature have a higher degree of membrane lipid unsaturatiuon
(Nichols et al., 2004) resulting in looser packing. During dehydration the membrane may have collapsed/shrunk to a greater extent resulting in surface changes. Any EPS on the surface of the cells may also have dehydrated differently to cells grown at a higher temperature.
Several cells grown at 4˚C were observed to have electron dense areas close to the periphery of the cell on TEM. These electron dense areas at the periphery of the cells could be related to the number of cellular components (e.g. ribosomes) that are predicted to be membrane associated under cold conditions (4.4.3.3). This phenomenon was not observed on a large number of cells. However, this may be related to the
D. Burg UNSW 271 position from where sections were taken from the cell. That is, sections taken through the centre of a cell are more likely to show this phenomenon than sections taken through the cellular periphery. A larger number of cells need to be analysed to validate this observation.
5.5.4 The effects of methanol on cell morphology
5.5.4.1 Cells grown on methanol have irregular shapes
When the cells from methanol grown cells were inspected with TEM they clearly displayed irregular shapes; either due to the cell increasing membrane surface area to facilitate the diffusion of nutrients, or due to the effects of methanol on cell structures.
Cells grown on methanol have to diffuse both ammonium (4.2.2) and methanol (4.4.4.4) across the cell membrane. Both processes would occur at a slow rate. Cells are known to increase nutrient uptake efficiency by increasing their surface area to volume ratio
(Lovdal et al., 2008; Marchetti and Cassar 2009). Increasing the amount of membrane without increasing cell volume could be a means by which M. burtonii accomplishes this. This is illustrated by the observation that while cells grown on methanol at 23˚C appeared similar in size to those grown on TMA in profile (SEM), the cross sectional areas were significantly different. This was due to folds in the membranes, seen on
TEM, which were not able to be appreciated from SEM visualisation, where superficially, cells appeared the same size.
Methanol has a detrimental effect on cell membranes. This could cause loss of membrane integrity and subsequent alteration of the membrane and S-layer (4.4.4.4). S- layer modification proteins are known to be increased in methanol grown M. burtonii
(4.4.4.4) and S-layer modification is known to result in change of shape in
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Methanocorpusculum sinese (Pum et al., 1991); a phenomenon that is possibly being reflected in methanol grown M. burtonii.
5.5.4.2 Methanol grown cells have cellular inclusions
Electron transparent cellular inclusions were seen in cells grown on methanol. The function and composition of these inclusions was not determined, however there are several possible explanations. A variety of organisms, including halophiles (Walsby
1972; Offner et al., 1998) and methanogens (Kamagata et al., 1992), produce gas vacuoles. These could be being formed in methanol grown M. burtonii as a means to aid cellular dispersal as part of a low nutrient response that also includes the increased production of flagellar proteins (4.4.4.3). Several organisms also form electron transparent inclusion bodies for storage of nutrients (Scott and Finnerty 1976; Alvarez et al., 1996), however this scenario is unlikely given the nutrient depleted nature of growth on methanol in M. burtonii. Stress responses also result in the formation of inclusions in cells, however these are generally protein aggregates which would appear electron dense (Lee et al., 2008).
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5.6 Conclusions
The use of a combination of techniques in electron microscopy was advantageous in the qualitative and quantitative analysis of the EPS produced by M. burtonii. EPS was produced by M. burtonii at both high and low temperatures, however it displayed different behaviour on dehydration, indicating fundamental differences in the composition of this substance at different temperatures. Staining techniques established that EPS at high temperatures contained acidic residues, which could act to bind cations and organic nutrients, an advantage for rapidly growing cells. Evidence also suggests the EPS of M. burtonii contains RNA, the first indication of this in an archaeon.
A feature of growth at 4˚C was the formation of large multicellular aggregates. These have possible functions in enhancing cell survival and nutrient pooling and acquisition.
As they appeared to have structure, further analysis should be carried out to determine if there is any organisation within these aggregates. The formation of aggregates by M. burtonii at low temperatures identifies this organism as a candidate for use, with a consortium of other organisms, in cold anaerobic digestion of waste of marine origin
(e.g. from aquaculture).
The cells of M. burtonii were found to alter their morphology with respect to both temperature and growth media. It is unclear why this occurs, although modulation of internal solute concentration with respect to temperature, and the detrimental effects of methanol may have roles in this phenomenon.
This analysis of the morphology of M. burtonii appears to be the first application of a number of electron microscopy techniques on an EPS producing psychrophile. The biological interpretation of the results of the microscopic analysis was also aided by the extensive proteomics performed on M. burtonii, which enabled several possible
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explanations for observed cellular changes to be proffered. While the phenotypic analysis was not exhaustive, it proved to be extremely useful in identifying areas in which future research could be focused.
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Chapter 6. General Discussion, Conclusions, and Research Significance
6.1 The Differential Solubility Fractionation Procedure
While several procedures have been developed for the analysis of the hydrophobic proteome (2.2), the comprehensive analysis of samples is often hampered by the presence of high abundance soluble proteins. These proteins are often difficult to remove from samples despite repeated washes of hydrophobic pellets. The DSF procedure developed in this work was effective in removing high abundance soluble proteins in early fractions. This facilitated the identification of membrane and hydrophobic proteins in later fractions. The effect of utilising this procedure is highlighted by the significant increase in the number of membrane proteins identified when compared with an equal number of LC/LC-MS/MS experiments without fractionation (2.4.4). The application of this procedure with the implementation of more powerful, next-generation mass spectrometers, for example orbitrap or quadrupole ion trap Fourier transform mass spectrometers, would be expected to achieve even greater coverage of the hydrophobic proteome.
The overlap in identifications between fractions provided the possibility of resolving solubility trends in proteins. It proved especially useful for the biological context analysis of a number of proteins from M. burtonii, including the DEAD-box helicase
(Mbur_1950) and PPIase (Mbur_2256), which exhibited fractionation profiles that provided evidence for an unexpected membrane association (Figure 2.13).
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There appears to be no reason why this DSF procedure cannot be applied to other microorganisms or cell lines. It would decrease the instrument time required for hydrophobic protein analysis (and hence decrease costs), and facilitate increased membrane protein identifications. The application of this procedure in other organisms could also be conducive to relative quantitation using isotopic labels (either metabolic or tag-based).
6.2 The Hydrophobic Proteome
The hydrophobic proteome analysis resulted in a significant increase in the identifiable proteome of M. burtonii, with over 50% of the proteins identified being NIW. From these NIW identifications, previously unknown aspects of the biology of M. burtonii were revealed. Significant findings include: evidence of the expression of the Rnf complex in a methylamine utilising methanogen (3.4.2.1.1); indications that M. burtonii utilises rhamnose in surface glycosylation (3.4.2.10); and the expression of a number of proteins from five identified glycosylation gene loci (Appendix A.4).
M. burtonii also expresses a large number of proteins with unknown or unclear functions (3.4.2.13), including 111 hypothetical proteins, 44 of which were predicted to be membrane proteins, and 21 of which were unique to M. burtonii as classified and discussed in Allen et al., (2009). The large number of expressed hypothetical proteins highlights the lack of knowledge with respect to the membrane proteins of psychrophiles and methanogens.
As many of the proteins with unknown function have the possibility of being important for M. burtonii physiology and cold adaptation, it is recommended that the
D. Burg UNSW 277 aforementioned hypothetical proteins undergo regular checks and homology searches.
This will ensure that as new functions are assigned to proteins in other organisms, homologues can be identified in M. burtonii. A case in point for this recommendation is the recent characterisation of the RamA protein from M. barkeri (Ferguson et al., 2009), which is required for reductive activation of methylamine:CoM methyl transfer from all three methylamines. M. burtonii expresses a homologous protein to RamA
(Mbur_1370), which was previously annotated as ‘conserved methanogen protein with ferredoxin domains (ER4)’. This protein is located on the genome near the methyltransferase genes as described by (Ferguson et al., 2009) and is now assigned a specific function. Additionally, based upon sequence similarity and genome positioning, a putative function for the protein Mbur_0809 can be inferred as RamM, involved in a similar process as RamA but acting with methanol corrinoid proteins (Ferguson et al.,
2009).
The unique hypotheticals identified also represent an area of future study, especially in light of their presumably important role in the cold adaptation physiology of M. burtonii. The most important targets for research are the unique hypothetical proteins
Mbur_0343 and Mbur_2063, which evidence suggests are highly abundant in the cell
(3.4.3).
Apart from the unique hypothetical proteins, several other important potential targets for future study were revealed. M. burtonii was shown to express a SuDH complex
(3.4.2.12). While this complex in M. burtonii is thought to be a reduced ferredoxin:NADP oxidoreductase, recycling NADP+ produced by GDH with reduced ferredoxin as the electron donor, it is unclear whether this complex is also reducing sulphur (or polysulphides). Electron microscope studies showed that cells associated
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with precipitates of iron and sulphur (Figure 5.12). However, it was unable to be determined whether they were polysulphides, and the association with this material seen in EM may have been incidental. The potential of M. burtonii to reduce sulphur utilising the SuDH complex should be investigated, applying the methods outlined in
Ma and Adams (1994).
The HtpX-like protein Mbur_0716 is an interesting candidate for future research. The membrane topology of this protein (orientated towards the cell exterior) is predicted to be different to other members of the HtpX family, and the functional domain of this protein appears to have an amino acid substitution that has the potential to affect its function (Figure 3.7 and Appendix A.5). The protein is coded immediately upstream of polysaccharide locus 1 in the genome. As a similar HtpX protein in S. gordonii is known to be involved in surface modification (Vickerman et al., 2002), and as surface modification has been identified as an important thermal response in M. burtonii,
Mbur_0716 is a likely candidate for future study in the organism. Any research into this protein will be facilitated by recent advances in the expression and purification of these types of proteins, which are generally toxic to the expression system (Sakoh et al.,
2005; Siddiqui et al., 2007).
One of the more intriguing findings of this work was the identification of an A2M family protein. Members of this family are not present in the genomes of any other characterised archaea (Figure 3.8). The phylogenetic analysis performed suggested that the presence of the A2M gene was likely the result of a gene loss scenario. The presence and expression of this protein in M. burtonii raises questions as to why this gene was retained in M. burtonii, that is, what evolutionary advantage does the protein bestow on
D. Burg UNSW 279 the organism? Given that surface structures and EPS are important in the cold adaptation of the organism, a role in the protection of these structures and substances from proteolytic attack is likely. Focused analysis of this protein, including added confirmation of expression by western blot, is recommended given the unique presence of this gene, amongst the archaea, in M. burtonii and the possible role of this protein in the psychrophilic lifestyle of the organism.
The processes related to the maintenance of ion balance (especially sodium) were highlighted as an important area of future research (3.4.2.12.1). Changes in cell size observed in electron microscopy also suggested that cells could be modulating internal ion concentration with respect to temperature (6.6). However, the mechanisms behind the maintenance of the balance of sodium ions in the cell are unclear, and may be especially important in cold adaptation given that sodium motive force is difficult to maintain at low temperatures (Goodchild et al., 2004b). The Rnf complex and the
Na+/H+ antiporters may have a role in ion balance maintenance, and the relative ion conductance of these proteins should be investigated.
Other important targets for future research include: the mechanosensitive ion channels
(3.4.2.12), which among the archaea, have only been characterised in thermophiles (Le
Dain et al., 1998; Kloda and Martinac 2001c, a); the unique sensory proteins expressed by M. burtonii (3.4.2.14), including the putative novel one component system protein
Mbur_0217; the CbiM proteins and their possible role in Factor III biosynthesis
(3.4.2.5); and the tungsten formylmethanofuran dehydrogenase, which was not expressed in M. burtonii (3.4.2.1.1). As there was no tungsten included in the growth
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media in this study, the expression of this protein following the addition of tungsten should be tested.
6.3 Thermal Adaptation in M. burtonii
The labelling technique iTRAQ and the semi-quantitative approach utilising Scaffold2 peptide spectral counts proved to be complementary approaches in the analysis of cold adaptation in M. burtonii. These approaches highlighted processes related to low temperature adaptation, and the stress responses seen when M. burtonii is grown at Topt.
6.3.1 The low temperature response
The low temperature response of M. burtonii was dominated by increased expression of surface and secreted proteins with unclear function, and proteins involved in translation
(4.4.3.2). The majority of the secreted proteins are thought to be involved in modulating cellular adhesion, and or the protection of surface structures and EPS against proteolytic attack. The identification of ribosomal proteins in relatively high abundance at low temperature in the hydrophobic fraction (4.4.3.3) is likely to be related to the increased expression of the secreted and surface proteins, as ribosomes would be associated with the secretion apparatus. The membrane localisation of the ribosomes may also correspond to the identification of membrane localised PPIases and the DEAD-box helicase, as localisation of these together at membrane sites could act to increase the efficiency of the translational process at low temperatures. The membrane localised PPIases were shown to be important proteins in the cold adaptation of the organism, acting to accelerate the rate limiting isomerisation aa-proline peptide bonds.
These low temperature related PPIases have sequence identity with dual-functional
D. Burg UNSW 281 proteins from other organisms (3.4.2.11 and Appendix A.11), suggesting that these proteins are not only important in isomerisation but also have chaperone functions and could be important in preventing and reversing cold induced protein denaturation.
Novel subjects for focused investigation were identified from the differential proteomic analyses of the HPP. Of these proteins, the unique hypothetical protein Mbur_2063 and the SPFH family protein are important targets for future research as these proteins presumably have important roles at the surface of the cell, with the SPFH protein likely to be involved in quality control of membrane proteins and surface structures. The
PPIases, especially Mbur_2256, are also important targets for future research. Given the roles of these proteins in low temperature isomerisation and as low temperature chaperones, their potential for exploitation in biotechnological applications is high.
The unique atypical J-domain protein identified through Scaffold2 analysis (3.4.4.3) is also a target for future research. As many of the type III J-family proteins are predicted to recruit specific polypeptides to the chaperone machinery, the M. burtonii J-domain protein could have a crucial role in modulating the folding of specific low temperature induced proteins. Investigations into the as yet unknown binding partners of this protein, are likely to provide critical insight into the low temperature adaptation of this organism.
The low temperature related DEAD-box helicase Mbur_1950, is one of the more important targets for future research identified in this study. It is discussed in 6.7.1.
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6.3.2 The high temperature response
The response of M. burtonii to high temperature growth indicated that the cells were stressed at what is considered Topt. It was not only the effects of high temperature on proteins (presumably causing denaturation or miss-folding) that was observed, as has been previously described (Goodchild et al., 2004b; Goodchild et al., 2005), but also the effects of an increased metabolic rate. Increased metabolism is proposed to be associated with an increase in the concentration of non-oxygen radicals which have the potential to be highly damaging to the cell.
Chaperones and chaperonins were identified in relatively high abundance at high temperatures (4.4.3.6), which is indicative of heat denaturation or mis-folding of the cellular proteins. Central to this response is the chaperone DnaK, which is predicted to have a critical role in controlling the rescue of denatured and aggregated proteins, in both the traditional chaperone machine (DnaK, DnaJ, GroEL) and the ClpB based chaperone. The central function of this protein is based on its preferential identification compared to other members of the chaperone machines, which indicates a potentially crucial role for this protein in high temperature survival, and possibly in the modulation of the chaperone response.
High temperature growth (accompanied by high growth rate) in M. burtonii is likely to promote the formation of non-oxygen radicals (4.4.3.6). Evidence of a cellular response to minimise the potential damage of these radicals was observed after high temperature growth. The response included the expression of ferritin (Dps) proteins (and accompanying FeoB transporters), and the universal stress protein A.
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Evidence of surface modification was observed at 23˚C; through glycosylation, as well as through VWA domain proteins (4.4.3.1). The expression of these may have roles in strengthening the S-layer or EPS modification, and degradation of thermally denatured surface proteins respectively.
6.3.3 The global approach
This analysis of the hydrophobic proteome only represented a portion of the global analysis of cold adaptation in M. burtonii. Many of the ideas presented in this dissertation are reinforced by the results of the global analysis, which provided a more comprehensive view of the oxidative and heat stress related responses at 23˚C
(Williams et al., submitted-b). However, the most important of the findings was the increased abundance of a much larger number of surface and secreted proteins at 4˚C.
Many of these are predicted to be involved in adhesion, and are thought to be involved in the distinct cold phenotype (see 6.7)
6.4 The Response of M. burtonii to Different Substrates and Nutrient
Levels
When the HPP of M. burtonii was analysed with respect to media conditions it was found that many metabolic proteins displayed differential abundance, not only according to substrate, but also with respect to temperature (4.4.4). The increase in abundance of many metabolic proteins is likely to be related to increased growth rates and hence increased metabolic demand at the higher growth temperature.
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Evidence that the organism is unable to switch between the substrates TMA and methanol (4.4.1) could be related to low levels of methanol in Ace Lake, which could have led to loss of specific genes for methanol methyltransferase isoenzymes and regulatory proteins. A phenomenon could be reflected by the presence of only two
MtaCB isoenzyme pairs in the genome, and lack of regulatory genes within the methanol utilisation gene locus (Appendix B.4).
One of the major findings of the HPP analysis of substrate utilisation was that growth on methanol was stressful to the cells (3.4.4.4), and toxic at higher concentrations
(4.4.1). The stressful effects of methanol manifested at the surface and membranes of the cell, with many proteins displaying increased abundance on methanol predicted to be involved in surface modification and repair. Growth on methanol was also associated with an increase in abundance of energy conserving mechanisms, which are likely related to the low energy yield of the substrate (3.4.4.2).
An interesting finding of the substrate utilisation analysis was that specific subunits of the tetrahydrosarcinapterin S methyltransferase complex (Mtr) varied according to substrate (3.4.4.1). The MtrH subunit was increased in TMA grown cells, while the
MtrE subunit was increased in methanol grown cells. These subunits are proposed to play important functions in the Mtr complex. MtrH is thought to be involved in the transfer of a methyl group via the MtrA subunit to H4SPT, while the MtrE subunit is thought to be directly involved with linking ion translocation to the de-methylation of methyl-CoM (Gottschalk and Thauer 2001). The pattern of abundance seen is quite interesting as both steps are assumedly highly important to the function of the Mtr complex. The finding certainly warrants further investigation, and may be specific for
D. Burg UNSW 285 methanol vs. methylamine metabolism, as acetate vs. methanol gown cells have shown no differences in abundance of any subunits of this complex (Li et al., 2005a; Li et al.,
2006).
A surprising observation from the substrate/nutrient utilisation analysis was that cells grown in rich versus defined media had very little differences in the abundances of proteins involved in biosynthesis of amino acids and nucleotides (3.4.4.3). This suggests that despite their presence in the growth media, M. burtonii constitutively synthesises these molecules. This could be related to the lack of corresponding transporters in the
M. burtonii genome (Allen et al., 2009).
6.4.1 The global approach
In terms of the metabolic adaptation, the global analysis (Williams et al., Submitted-a) presented a more comprehensive picture of the metabolism of the organism, especially in terms of methanogenesis and nitrogen utilisation.
When the soluble and secreted fraction data were combined with the hydrophobic fraction results, subcellular localisation could be inferred. For example, the preferential localisation of one of the expressed TMA methyltransferases in the membrane fraction is associated with a possible shift to associate with the TMA transporter. Likewise the identification of the methyl-CoM reductase subunits in the insoluble fraction as compared to the soluble fraction could indicate an association of this complex with the heterodisulfide reductase complex. It could increase the efficiency of the steps in the methanogenesis pathway mediated by these two important complexes.
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Growth on TMA was associated with the abundance of glutamate dehydrogenase
(GDH), while growth on methanol was associated with the increased abundance of glutamine synthetase/glutamate synthase (GS-GOGAT), presumably directly related to nitrogen availability. M. burtonii is predicted to have ammonium transport systems that have diminished function or are non-functional, so cellular levels of ammonia would be restricted when cells were grown on methanol. Due to this, the cell favours the high affinity GS-GOGAT over the lower affinity GDH under these conditions.
There was a general lack of differences in biosynthetic processes, as described for the insoluble fraction, suggesting intrinsic biosynthesis of amino acids and other metabolic essentials despite available nutrient levels.
6.5 Growth Conditions Induced Significant Phenotypic Changes in M. burtonii
Investigation of the cellular morphology and EPS of M. burtonii revealed significant differences with respect to both temperature and substrate. Histochemical analysis provided evidence that the EPS produced by the organism contains sugars, protein and
RNA, and that growth at high temperatures is associated with acidification (most probably carboxylation) of polysaccharides (5.4.1). A modification that is likely to be involved in trapping of cations for use in metabolism, which is occurring at a higher rate at increased temperatures. The identification of RNA in the EPS of M. burtonii is the first evidence of this in an archaeon. This finding should be validated, and the extracellular RNA characterised as described by Ando et al., (2006), to determine
D. Burg UNSW 287 whether it plays a structural role within the EPS (e.g. ribosomal RNA-like function) or has a role in extracellular enzyme function (Nakazawa et al., 2005).
The use of a combination of techniques in electron microscopy (EM) proved critical in identifying differences in the EPS produced with respect to temperature. It was found that while EPS is produced at both high and low temperatures, the substance shows significantly different behaviour on dehydration (5.4.2 and 5.4.3). EPS from cells at 4˚C dehydrated to string like structures, while the substance produced by cells grown at
23˚C dehydrated into sheets and/or was completely destroyed by the critical point drying process. The difference was seen regardless of the growth media. This fundamental difference in behaviour suggests that the composition of the EPS changes with respect to temperature, and is presumably highly important in the thermal adaptation of the organism. It is recommended that detailed chemical analysis of the
EPS of M. burtonii be performed, in order to gain further insight into its role at both high and low temperatures in M. burtonii.
Differences in cell size were also observed in cells grown at different temperatures, in both SEM (5.4.2.3) and TEM (5.4.4.2, measurements achievable for MFM cells only), indicating that this was not an artefact due to procedure induced cellular shrinkage . The reason why this occurs is unclear, however, an increase in internal solute concentration as a cold adaptation strategy, or a reduction of internal salt concentrations as a strategy to maintain electrochemical potentials during rapid growth, are plausible scenarios.
This phenomenon should be investigated further, with internal solute analyses likely to provide evidence as to why this phenomenon is occurring.
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An unexpected finding of the TEM analysis was that cells grown on methanol were highly irregular in shape, and had electron transparent inclusions (5.4.4.2). The reason why this significant change in morphology is occurring is unclear, although the availability of nutrients to the cell, and the detrimental effects of methanol on cell membranes, may have roles in the phenomenon. These findings warrant further investigation, including the determination of whether these electron transparent areas are bounded by membranes, as is seen in gas vesicles (Walsby 1972) and storage inclusions (Alvarez et al., 1996).
6.6 Combining the Theoretical and the Physical: A Union of
Proteomics and Electron Microscopy
The consideration of the physical manifestations of growth conditions, in light of the theoretical processes inferred from proteomic data, has provided insight into the adaptation of the organism. The complementary nature of these two techniques highlights the value of multifaceted analyses when investigating microbial adaptation.
It was observed through electron microscopy that cells grown at 4˚C produce EPS that forms string like structures on dehydration, while growth at 23˚C resulted in the formation of sheet like material and/or the destruction of EPS on dehydration (5.4.2.1).
Differential proteomic analysis indicated that cells grown at 4˚C were geared towards protein secretion (4.4.3.2, and 4.4.3.3), and the global proteomic analysis revealed that cells were secreting a variety of adhesion-like molecules (Williams et al., submitted-b).
Conversely, cells grown at 23˚C had indications that glycosylation was occurring
(4.4.3.1). A scenario that can be inferred from these data is that the EPS of M. burtonii
D. Burg UNSW 289 at 4˚C contains long chains of proteins, which on dehydration, maintained some structural integrity, due to the actions of the glutaraldehyde fixative, and dehydrated into strings. Cells grown at 23˚C alternatively produce a highly hydrated EPS matrix, which consists of larger amounts of sugars. The protein cross linking action of glutaraldehyde would be less profound in this case, leading to dehydration into thin brittle material that was subject to destruction during critical point drying.
Low temperature growth was associated with an increase in the abundance of surface proteins, and indicators of S-layer protein modification (4.4.3.2). SEM analysis revealed that cells grown at 4˚C had ‘pimpled’ surfaces, presumably as a result of this surface modification (5.4.2.1). However, TEM analysis did not show any differences in the surface of the cells (5.4.4.2). The disagreement between the two techniques could be due to a number of reasons, including the appearance of surface morphology (lumps) as an artefact of dehydration, with the differences seen occurring due to the EPS or S-layer at the immediate surface of the cell having different properties. Alternatively, the contrasting agents used in TEM may not have provided sufficient electron density to the surface structures. This matter should be further investigated using alternative methods; with cryo-techniques including freeze fracture representing the best option, as EPS is likely to foul atomic force microscope probe tips.
The issue of maintenance of SMF was highlighted through proteomics (3.4.2.12.1), and no intimation of how this occurs was able to be proffered. The cell size differences seen in electron microscopy could be directly related to this ionic balance maintenance. It could involve cells grown at 23˚C, a temperature which is associated with high rate of metabolic activity and associated sodium uptake, taking on water (with corresponding
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increase in cell volume) as a means to maintain low intracellular sodium. However, the mechanism by which this would occur is unclear, and would have to involve active transport of water against a gradient, or symport with other ions.
Growth at 4˚C was associated with an increase in a number of membrane localised components, including the ribosomes and associated machinery (4.4.3.3). A number of cells visualised through TEM at this temperature had electron dense areas at their periphery, which could provide further evidence to this observation (Figure 5.25).
Electron tomographic reconstructions from tilt series as described in Robertson (2007), would provide a good platform for comparative analysis of ribosome localisation in cells grown at different temperatures.
Another parallel drawn between the proteomics and EM was that when M. burtonii was grown on methanol, proteomics indicated a general stress response, and indicated the solvent had a detrimental effect on the membranes and surface structures of the cell
(3.4.4.4). EM showed that cells grown in these conditions had highly irregular shapes, and had electron transparent inclusions (Figure 5.22), presumably as a direct result of membrane and cellular stress.
6.6.1 The DEAD-box helicase, an enigmatic cold adaptation protein
The DEAD-box helicase (Mbur_1950) has been previously nominated as an important protein in the cold adaptation of M. burtonii (Lim et al., 2000). The identification of
Mbur_1950 in this study confirms it as a key cold adaptation protein. The exact function of Mbur_1950 remains enigmatic, and the protein is yet to be characterised due to difficulties in recombinant expression. The identification of this protein exclusively
D. Burg UNSW 291 in insoluble fractions, and the trends in peptide spectral counts, has indicated a membrane association of this protein. This observation should be validated using immuno-electron microscopy as described by El-Fahmawi and Owttrim (2003). Any recombinant expression and subsequent characterisation can then be approached in light of this knowledge.
Despite the lack of characterisation of this protein, roles in unwinding cold stabilised mRNA secondary structures, and as an RNA co-chaperone with TRAM domain proteins have been proffered (Lim et al., 2000; Williams et al., submitted-b). The evidence provided by microscopy that RNA is present in the EPS of M. burtonii, infers another possible role for this protein. DEAD-box family helicases are known to be important for the export of RNA across the nuclear membrane in eukaryotes (reviewed in: Linder and Stutz 2001; Tanner and Linder 2001; Cole and Scarcelli 2006). It is therefore possible, that the DEAD-box helicase is performing a role in the export of
RNA for incorporation into EPS.
6.7 Conclusion and Significance of the Research
The methods developed for the analysis of the hydrophobic proteome appear to be broadly applicable to other organisms and cell lines. They led to the identification of a large number of proteins in M. burtonii which in turn has led to new insight into the general physiology of the organism and into thermal adaptation. The comprehensive analyses represent an excellent platform for the comparative analysis of the cold adaptation response in M. burtonii with other organisms.
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Prior to this research the significance of membrane proteins in cold adaptation could only be inferred from genomic studies (for example Allen et al., (2009)). This study has confirmed that membrane and other hydrophobic proteins play important roles in cold adaptation. However, the lack of knowledge regarding the function of many membrane proteins has made concise biological interpretation of the roles of these proteins difficult. Such difficulty is reflected in the large number of hypothetical proteins (and hypothetical membrane proteins) identified as expressed and differentially abundant with respect to growth conditions. The identification of expression of these proteins, which are often present in large numbers in the genomes of psychrophiles (Riley et al.,
2008; Allen et al., 2009) and methanogens (Ferry and Kastead 2007), provides evidence that these proteins have functions within the cell, many of which are presumably important for thermal and metabolic adaptation.
Additionally, the analysis of the HPP with respect to WCE and global approaches (e.g. against soluble and secreted fractions) has proven valuable in inferring sub-cellular localisation of proteins. Similar analyses in other organisms are likely to prove as effective in providing information that is crucial when predicting functions of proteins and for directing efforts at further characterisation.
The phenotypic analysis of M. burtonii has led to the identification of a number of novel avenues of research. The analysis of this information with respect to the comprehensive proteomics of the organism was not only extremely valuable in identifying these areas of future research, but also in inferring function; thus highlighting the value of a comprehensive proteomic dataset as a reference platform for the examination of cellular adaptation in microorganisms. Any research proceeding from this analysis will not only
D. Burg UNSW 293 help to elucidate the role of EPS in cold adaptation, but also provide further insight into the physiology and thermal response of this model cold adapted archaeon.
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Chapter 7. Reference List
Abramoff, M. D., Magelhaes, P. J. and Ram, S. J. (2004). Image processing with Imagej. Biophotonics Int 11: 36-42. Abu-Qarn, M. and Eichler, J. (2006). Protein N-glycosylation in archaea: Defining Haloferax volcanii genes involved in S-layer glycoprotein glycosylation. Mol Microbiol 61: 511-525. Abu-Qarn, M., Giordano, A., Battaglia, F., Trauner, A., Hitchen, P. G., Morris, H. R., Dell, A. and Eichler, J. (2008). Identification of AglE, a second glycosyltransferase involved in N glycosylation of the Haloferax volcanii S-layer glycoprotein. J Bacteriol 190: 3140-3146. Abu-Qarn, M., Yurist-Doutsch, S., Giordano, A., Trauner, A., Morris, H. R., Hitchen, P., Medalia, O., Dell, A. and Eichler, J. (2007). Haloferax volcanii AglB and AglD are involved in N-glycosylation of the S-layer glycoprotein and proper assembly of the surface layer. J Mol Biol 374: 1224-1236. Akca, E., Claus, H., Schultz, N., Karbach, G., Schlott, B., Debaerdemaeker, T., Declercq, J. P. and Konig, H. (2002). Genes and derived amino acid sequences of S-layer proteins from mesophilic, thermophilic, and extremely thermophilic methanococci. Extremophiles 6: 351-358. Al-Sharif, W. Z., Sunyer, J. O., Lambris, J. D. and Smith, L. C. (1998). Sea urchin coelomocytes specifically express a homologue of the complement component C3. J Immunol 160: 2983-2997. Albers, S., Szaba, Z. and Driessen, A. J. M. (2006). Protein secretion in the archaea: Multiple paths towards a unique cell surface. Nat Rev Microbiol 4: 537-547. Alimenti, C., Ortenzi, C., Carratore, V. and Luporini, P. (2002). Structural characterization of a protein pheromone from a cold-adapted (antarctic) single-cell eukaryote, the ciliate Euplotes nobilii. FEBS Letters 514: 329-332. Allen, M. A., Lauro, F. M., Williams, T. J., Burg, D., Siddiqui, K. S., De Francisci, D., Chong, K. W., Pilak, O., Chew, H. H., De Maere, M. Z., Ting, L., Katrib, M., Ng, C., Sowers, K. R., Galperin, M. Y., Anderson, I. J., Ivanova, N., Dalin, E., Martinez, M., Lapidus, A., Hauser, L., Land, M., Thomas, T. and Cavicchioli, R. (2009). The genome sequence of the psychrophilic archaeon, Methanococcoides burtonii: The role of genome evolution in cold adaptation. ISME J 3: 1012-1035. Almiron, M., Link, A. J., Furlong, D. and Kolter, R. (1992). A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli. Genes Dev 6: 2646- 2654. Alvarez, H. M., Mayer, F., Fabritius, D. and Steinbuchel, A. (1996). Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain pd630. Arch Microbiol 165: 377-386. Ando, T., Suzuki, H., Nishimura, S., Tanaka, T., Hiraishi, A. and Kikuchi, T. (2006). Characterisation of extracellular RNAs produced by the marine photosynthetic bacterium Rhodovulum sulfidophilum. J. Biochem 139: 805-811. Antonenko, Y. N., Pohl, P. and Denisov, G. A. (1997). Permeation of ammonia across bilayer lipid membranes studied by ammonium ion selective microelectrodes. Biophys J 72: 2187-2195. Aono, R. and Kobayashi, H. (1997). Cell surface properties of organic solvent-tolerant mutants of Escherichia coli K-12. Appl Environ Microbiol 63: 3637-3642. Arai, M., Mitsuke, H., Ikeda, M., Xia, J. X., Kikuchi, T., Satake, M. and Shimizu, T. (2004). Conpred II: A consensus prediction method for obtaining transmembrane topology models with high reliability. Nucleic Acids Res 32: W390-393.
D. Burg UNSW 295
Aravind, L., Leipe, D. D. and Koonin, E. V. (1998). Toprim--a conserved catalytic domain in type Ia and II topoisomerases, DnaG-type primases, old family nucleases and RecR proteins. Nucleic Acids Res 26: 4205-4213. Armstrong, P. B. and Quigley, J. P. (1999). Alpha2-macroglobulin: An evolutionarily conserved arm of the innate immune system. Dev Comp Immunol 23: 375-390. Auguet, J. C. and Casamayor, E. O. (2008). A hotspot for cold crenarchaeota in the neuston of high mountain lakes. Environ Microbiol 10: 1080-1086. Baker-Austin, C., Dopson, M., Wexler, M., Sawers, R. G., Stemmler, A., Rosen, B. P. and Bond, P. L. (2007). Extreme arsenic resistance by the acidophilic archaeon 'Ferroplasma acidarmanus' Fer1. Extremophiles 11: 425-434. Bakermans, C. and Nealson, K. H. (2004). Relationship of critical temperature to macromolecular synthesis and growth yield in Psychrobacter cryopegella. J Bacteriol 186: 2340-2345. Bakermans, C., Tollaksen, S. L., Giometti, C. S., Wilkerson, C., Tiedje, J. M. and Thomashow, M. F. (2007). Proteomic analysis of Psychrobacter cryohalolentis K5 during growth at subzero temperatures. Extremophiles 11: 343-354. Bano, N., Ruffin, S., Ransom, B. and Hollibaugh, J. T. (2004). Phylogenetic composition of Arctic ocean archaeal assemblages and comparison with Antarctic assemblages. Appl Environ Microbiol 70: 781-789. Barns, S. M., Delwiche, C. F., Palmer, J. D. and Pace, N. R. (1996). Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences. Proc Natl Acad Sci U S A 93: 9188-9193. Barouch, W., Prasad, K., Greene, L. and Eisenberg, E. (1997). Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets. Biochemistry 36: 4303-4308. Barry, R. C., Young, M. J., Stedman, K. M. and Dratz, E. A. (2006). Proteomic mapping of the hyperthermophilic and acidophilic archaeon Solfolobus solfataricus P2. Electrophoresis 27: 2970-2983. Bartlett, M. S. (2005). Determinants of transcription initiation by archaeal RNA polymerase. Curr Opin Microbiol 8: 677-684. Baum, M. M., Kainovic, A., O'Keeffe, T., Pandita, R., McDonald, K., Wu, S. and Webster, P. (2009). Characterization of structures in biofilms formed by a Pseudomonas fluorescens isolated from soil. BMC Microbiol 9: 103. Baumer, S., Ide, T., Jacobi, C., Johann, A., Gottschalk, G. and Deppenmeier, U. (2000). The F420H2 dehydrogenase from Methanosarcina mazei is a redox-driven proton pump closely related to NADH dehydrogenases. J Biol Chem 275: 17968-17973. Baumer, S., Lentes, S., Gottschalk, G. and Deppenmeier, U. (2002). Identification and analysis of proton-translocating pyrophosphatases in the methanogenic archaeon Methansarcina mazei. Archaea 1: 1-7. Beckering, C. L., Steil, L., Weber, M. H., Volker, U., Marahiel, M. A., Yamada, T., Kuroda, K., Jitsuyama, Y., Takezawa, D., Arakawa, K. and Fujikawa, S. (2002). Genomewide transcriptional analysis of the cold shock response in Bacillus subtilis. J Bacteriol 184: 6395-402. Bee, A., Ke, Y., Forootan, S., Lin, K., Beesley, C., Forrest, S. E. and Foster, C. S. (2006). Ribosomal protein L19 is a prognostic marker for human prostate cancer. Clin Cancer Res 12: 2061-2065. Bell, S. D. (2005). Archaeal transcriptional regulation--variation on a bacterial theme? Trends Microbiol 13: 262-265. Benaroudj, N. and Goldberg, A. L. (2000). PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone. Nat Cell Biol 2: 833-839. Bendtsen, J. D., Nielsen, H., von Heijne, G. and Brunak, S. (2004). Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340: 783-795.
296 D. Burg UNSW
Bergauer, P., Fonteyne, P.-A., Nolard, N., Schinner, F. and Margesin, R. (2005). Biodegradation of phenol and phenol-related compounds by psychrophilic and cold-tolerant alpine yeasts. Chemosphere 59: 909-918. Bergmans, L., Moisiadis, P., Van Meerbeek, B., Quirynen, M. and Lambrechts, P. (2005). Microscopic observation of bacteria: Review highlighting the use of environmental SEM. Int Endod J 38: 775-788. Bisle, B., Schmidt, A., Scheibe, B., Klein, C., Tebbe, A., Kellermann, J., Siedler, F., Pfeiffer, F., Lottspeich, F. and Oesterhelt, D. (2006). Quantitative profiling of the membrane proteome in a halophilic archaeon. Mol Cell Proteomics 5: 1543-1558. Blackler, A. R., Speers, A. E., Ladinsky, M. S. and Wu, C. C. (2008). A shotgun proteomic method for the identification of membrane-embedded proteins and peptides. J Proteome Res 7: 3028-3034. Blonder, J., Conrads, T. P., Yu, L. R., Terunuma, A., Janini, G. M., Issaq, H. J., Vogel, J. C. and Veenstra, T. D. (2004a). A detergent- and cyanogen bromide-free method for integral membrane proteomics: Application to halobacterium purple membranes and the human epidermal membrane proteome. Proteomics 4: 31-45. Blonder, J., Goshe, M. B., Moore, R. J., Pasa-Tolic, L., Masselon, C. D., Lipton, M. S. and Smith, R. D. (2002). Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry. J Proteome Res 1: 351-360. Blonder, J., Goshe, M. B., Xiao, W., Camp, D. G., 2nd, Wingerd, M., Davis, R. W. and Smith, R. D. (2004b). Global analysis of the membrane subproteome of Pseudomonas aeruginosa using liquid chromatography-tandem mass spectrometry. J Proteome Res 3: 434-444. Blonder, J., Terunuma, A., Conrads, T. P., Chan, K. C., Yee, C., Lucas, D. A., Schaefer, C. F., Yu, L. R., Issaq, H. J., Veenstra, T. D. and Vogel, J. C. (2004c). A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry. J Invest Dermatol 123: 691-699. Bockelmann, U., Lunsdorf, H. and Szewzyk, U. (2007). Ultrastructural and electron energy- loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate. Environ Microbiol 9: 2137-2144. Boonyaratanakornkit, B. B., Simpson, A. J., Whitehead, T. A., Fraser, C. M., El-Sayed, N. M. A. and Clark, D. S. (2005). Transcriptional profiling of the hyperthermophilic methanarchaeon Methanococcus jannaschii in response to lethal heat and non- lethal cold shock. Environ Microbiol 7: 789-797. Borth, W. (1992). Alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics. Faseb J 6: 3345-3353. Bose, A. and Metcalf, W. W. (2008). Distinct regulators control the expression of methanol methyltransferase isozymes in Methanosarcina acetivorans C2a. Mol Microbiol 67: 649-661. Bose, A., Pritchett, M. A. and Metcalf, W. W. (2008). Genetic analysis of the methanol- and methylamine-specific methyltransferase 2 genes of Methanosarcina acetivorans C2a. J Bacteriol 190: 4017-4026. Bose, A., Pritchett, M. A., Rother, M. and Metcalf, W. W. (2006). Differential regulation of the three methanol methyltransferase isozymes in Methanosarcina acetivorans C2a. J Bacteriol 188: 7274-7283. Bottos, E. M., Vincent, W. F., Greer, C. W. and Whyte, L. G. (2008). Prokaryotic diversity of Arctic ice shelf microbial mats. Environ Microbiol 10: 950-966. Bowman, J. P., McCammon, S. A. and Skerratt, J. H. (1997). Methylosphaera hansonii Gen. Nov., sp. Nov., a psychrophilic, group I methanotroph from Antarctic marine- salinity, meromictic lakes. Microbiology 143: 1451-1459. Boynton, W. V., Feldman, W. C., Squyres, S. W., Prettyman, T. H., Bruckner, J., Evans, L. G., Reedy, R. C., Starr, R., Arnold, J. R., Drake, D. M., Englert, P. A., Metzger, A. E.,
D. Burg UNSW 297
Mitrofanov, I., Trombka, J. I., D'Uston, C., Wanke, H., Gasnault, O., Hamara, D. K., Janes, D. M., Marcialis, R. L., Maurice, S., Mikheeva, I., Taylor, G. J., Tokar, R. and Shinohara, C. (2002). Distribution of hydrogen in the near surface of mars: Evidence for subsurface ice deposits. Science 297: 81-85. Braza-Boils, A., Tomas, M., Marin, M. P., Megias, L., Sancho-Tello, M., Fornas, E. and Renau- Piqueras, J. (2006). Glycosylation is altered by ethanol in rat hippocampal cultured neurons. Alcohol Alcohol 41: 494-504. Brizzio, S., Turchetti, B., de Garcia, V., Libkind, D., Buzzini, P. and van Broock, M. (2007). Extracellular enzymatic activities of basidiomycetous yeasts isolated from glacial and subglacial waters of northwest Patagonia (Argentina). Can J Microbiol 53: 519- 525. Bruggemann, H., Baumer, S., Fricke, W. F., Wiezer, A., Liesegang, H., Decker, I., Herzberg, C., Martinez-Arias, R., Merkl, R., Henne, A. and Gottschalk, G. (2003). The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc Natl Acad Sci U S A 100: 1316-1321. Buckel, W. and Golding, B. T. (2006). Radical enzymes in anaerobes. Annu Rev Microbiol 60: 27-49. Budd, A., Blandin, S., Levashina, E. A. and Gibson, T. J. (2004). Bacterial alpha2- macroglobulins: Colonization factors acquired by horizontal gene transfer from the metazoan genome? Genome Biol 5: R38. Budde, I., Steil, L., Scharf, C., Volker, U. and Bremer, E. (2006). Adaptation of Bacillus subtilis to growth at low temperature: A combined transcriptomic and proteomic appraisal. Microbiology 152: 831-853. Bukau, B. and Horwich, A. L. (1998). The Hsp70 and Hsp60 chaperone machines. Cell 92: 351-366. Burch, M. D. (1988). Annual cycle of phytoplankton in Ace Lake, an ice covered, saline meromictic lake. Hydrobiologica 165: 59-75. Burke, S. A., Lo, S. L. and Krzycki, J. A. (1998). Clustered genes encoding the methyltransferases of methanogenesis from monomethylamine. J Bacteriol 180: 3432-3440. Busenlehner, L. S., Pennella, M. A. and Giedroc, D. P. (2003). The SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural insights into prokaryotic metal resistance. FEMS Microbiol Rev 27: 131-143. Caldas, T., Laalami, S. and Richarme, G. (2000). Chaperone properties of bacterial elongation factor EF-G and initiation factor IF2. J Biol Chem 275: 855-860. Caldas, T. D., El Yaagoubi, A. and Richarme, G. (1998). Chaperone properties of bacterial elongation factor EF-tu. J Biol Chem 273: 11478-11482. Camardella, L., Di Fraia, R., Antignani, A., Ciardiello, M. A., di Prisco, G., Coleman, J. K., Buchon, L., Guespin, J. and Russell, N. J. (2002). The Antarctic Psychrobacter sp. Tad1 has two cold-active glutamate dehydrogenases with different cofactor specificities. Characterisation of the NAD+-dependent enzyme. Comp Biochem Physiol - Part A: Mol Integrat Physiol 131: 559-567. Carrondo, M. A. (2003). Ferritins, iron uptake and storage from the bacterioferritin viewpoint. EMBO J 22: 1959-1968. Cavicchioli, R. (2002). Extremophiles and the search for extraterrestrial life. Astrobiology 2: 281-292. Cavicchioli, R. (2006). Cold-adapted archaea. Nat Rev Microbiol 4: 331-43. Cavicchioli, R., Siddiqui, K. S., Andrews, D. and Sowers, K. R. (2002). Low-temperature extremophiles and their applications. Curr Op Biotech 13: 253-261. Chaban, B., Logan, S. M., Kelly, J. F. and Jarrell, K. F. (2009). AglC and AglK are involved in biosynthesis and attachment of diacetylated glucuronic acid to the N-glycan in Methanococcus voltae. J Bacteriol 191: 187-195.
298 D. Burg UNSW
Chaban, B., Voisin, S., Kelly, J., Logan, S. M. and Jarrell, K. F. (2006). Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: Insight into N-linked glycosylation pathways in archaea. Mol Microbiol 61: 259-268. Chamot, D., Magee, W. C., Yu, E. and Owttrim, G. W. (1999). A cold shock-induced cyanobacterial RNA helicase. J Bacteriol 181: 1728-1732. Chartier, F., Laine, B. and Sautiere, P. (1988). Characterization of the chromosomal protein MC1 from the thermophilic archaebacterium Methanosarcina sp. Chti 55 and its effect on the thermal stability of DNA. Biochim Biophys Acta 951: 149-156. Chatterjee, A., Li, Y., Zhang, Y., Grove, T. L., Lee, M., Krebs, C., Booker, S. J., Begley, T. P. and Ealick, S. E. (2008). Reconstitution of thic in thiamine pyrimidine biosynthesis expands the radical SAM superfamily. Nat Chem Biol 4: 758-765. Chayen, J. (1952). The methyl green-pyronin method. Exper. Cell Res 3: 652-655. Cheetham, M. E. and Caplan, A. J. (1998). Structure, function and evolution of DnaJ: Conservation and adaptation of chaperone function. Cell Stress Chaperones 3: 28- 36. Chen, C.-H. and Berns, D. S. (1978). Comparison of the stability of phycocyanins from thermophilic, mesophilic, psychrophilic and halophilic algae. Biophys Chem 8: 203- 213. Chiancone, E., Ceci, P., Ilari, A., Ribacchi, F. and Stefanini, S. (2004). Iron and proteins for iron storage and detoxification. Biometals 17: 197-202. Chong, P. K., Gan, C. S., Pham, T. K. and Wright, P. C. (2006). Isobaric tags for relative and absolute quantitation (iTRAQ) reproducibility: Implication of multiple injections. J Proteome Res 5: 1232-1240. Chong, P. K., Wright, P. C., Gan, C. S. and Pham, T. K. (2005). Identification and characterization of the Sulfolobus solfataricus P2 proteome. J Proteome Res 4: 1789-1798. Church, M. J., DeLong, E. F., Ducklow, H. W., Karner, M. B., Preston, C. M. and Karl, D. M. (2003). Abundance and distribution of planktonic archaea and bacteria in the waters west of the Antarctic Peninsula. Limnol Oceanog 48: 1893-1902. Claus, H., Akca, E., Debaerdemaeker, T., Evrard, C., Declercq, J. P. and Konig, H. (2002). Primary structure of selected archaeal mesophilic and extremely thermophilic outer surface layer proteins. Syst Appl Microbiol 25: 3-12. Coker, J. A., Dassarma, P., Kumar, J., Muller, J. A. and Dassarma, S. (2007). Transcriptional profiling of the model archaeon Halobacterium sp. NRC-1: Responses to changes in salinity and temperature. Saline Systems 3: 6. Cole, C. N. and Scarcelli, J. J. (2006). Transport of messenger RNA from the nucleus to the cytoplasm. Curr Opin Cell Biol 18: 299-306. Collins, G., Mahony, T. and O'Flaherty, V. (2006). Stability and reproducibility of low- temperature anaerobic biological wastewater treatment. FEMS Microbiol Ecol 55: 449-458. Conesa, A., Gotz, S., Garcia-Gomez, J. M., Terol, J., Talon, M. and Robles, M. (2005). BLAST2GO: A universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674-3676. Cook, D. J. (1998). Cellular pathology. Butterworth Heinemann, Oxford, UK. Coolen, M. J. L., Hopmans, E. C., Rijpstra, W. I. C., Muyzer, G., Schouten, S., Volkman, J. K. and Sinninghe Damsté, J. S. (2004a). Evolution of the methane cycle in Ace Lake (Antarctica) during the holocene: Response of methanogens and methanotrophs to environmental change. Organic Geochem 35: 1151-1167. Coolen, M. J. L., Muyzer, G., Rijpstra, W. I. C., Schouten, S., Volkman, J. K. and Sinninghe Damste', J. S. (2004b). Combined DNA and lipid analyses of sediments reveal changes in holocene haptophyte and diatom populations in an Antarctic lake. Earth Planet Sci Lett 223: 225–239.
D. Burg UNSW 299
Cordin, O., Banroques, J., Tanner, N. K. and Linder, P. (2006). The DEAD-box protein family of RNA helicases. Gene 367: 17-37. Corthals, G. L., Wasinger, V. C., Hochstrasser, D. F. and Sanchez, J. C. (2000). The dynamic range of protein expression: A challenge for proteomic research. Electrophoresis 21: 1104-1115. Cowan, D. A., Casanueva, A. and Stafford, W. (2007). Ecology and diversity of cold-adapted microorganisms. Physiology and biochemistry of extremophiles. C. Gerday and N. Glansdorff. Washington D.C., ASM press: 119 -132. Cromer, L., Gibson, J. A. E., Swadling, K. M. and Ritz, D. A. (2005). Faunal microfossils: Indicators of holocene ecological change in a saline Antarctic lake. Palaeogeo, Palaeoclim, Palaeoecol 221: 83-97. Cukras, A. R. and Green, R. (2005). Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation. Journal of Molecular Biology 349: 47-59. Cukras, A. R., Southworth, D. R., Brunelle, J. L., Culver, G. M. and Green, R. (2003). Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex. Molecular Cell 12: 321-328. D'Amico, S., Claverie, P., Collins, T., Georlette, D., Gratia, E., Hoyoux, A., Meuwis, M. A., Feller, G. and Gerday, C. (2002). Molecular basis of cold adaptation. Philos Trans R Soc Lond B Biol Sci 357: 917-25. Danson, M. J., Lamble, H. J. and Hough, D. W. (2007). Central metabolism. Archaea: Molecular and cellular biology. R. Cavicchioli. Washington D. C, ASM Press. Davies, B. and Fried, M. (1995). The L19 ribosomal protein gene (RpL19): Gene organization, chromosomal mapping, and novel promoter region. Genomics 25: 372-380. Decho, A. W. and Herndl, G. J. (1995). Microbial activities and the transformation of organic matter within mucilaginous material. Sci Tot Environ 165: 33-42. DeLong, E. F., Preston, C. M., Mincer, T., Rich, V., Hallam, S. J., Frigaard, N., Martinez, A., Sullivan, M. B., Edwards, R. and Brit, B. R. (2006). Community genomics among stratified microbial assemblages in the Ocean's interi or Science 311: 496-503. DeLong, E. F., Wu, K. Y., Prezelin, B. B. and Jovine, R. V. (1994). High abundance of archaea in antarctic marine picoplankton. Nature 371: 695-697. Deming, J. W. (2002). Psychrophiles and polar regions. Curr Op Microbiol 5: 301-309. Deming, J. W. (2007). Life in iceformations at vey cold temperatures. Physiology and biochemistry of extremophiles. C. Gerday and N. Glansdorff. Washington D.C, ASM press: 133-155. Demple, B. and Harrison, L. (1994). Repair of oxidative damage to DNA: Enzymology and biology. Annu Rev Biochem 63: 915-948. Deppenmeier, U. (2004). The membrane-bound electron transport system of Methanosarcina species. J Bioenerg Biomembr 36: 55-64. Deppenmeier, U. and Muller, V. (2008). Life close to the thermodynamic limit: How methanogenic archaea conserve energy. Results Probl Cell Differ 45: 123-152. Doan, N. and Gettins, P. G. (2008). Alpha-macroglobulins are present in some gram- negative bacteria: Characterization of the alpha2-macroglobulin from Escherichia coli. J Biol Chem 283: 28747-56. Dreanno, C., Matsumura, K., Dohmae, N., Takio, K., Hirota, H., Kirby, R. R. and Clare, A. S. (2006). An alpha2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite. Proc Natl Acad Sci U S A 103: 14396-14401. Dresios, J., Derkatch, I. L., Liebman, S. W. and Synetos, D. (2000). Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves
300 D. Burg UNSW
translational accuracy, while mutants lacking both remain viable. Biochemistry 39: 7236-7344. Duncan, S. M., Farrell, R. L., Thwaites, J. M., Held, B. W., Arenz, B. E., Jurgens, J. A. and Blanchette, R. A. (2006). Endoglucanase-producing fungi isolated from Cape Evans historic expedition hut on Ross Island, Antarctica. Environ Microbiol 8: 1212-1219. Egea, P. F., Napetschnig, J., Walter, P. and Stroud, R. M. (2008a). Structures of SRP54 and SRP19, the two proteins that organize the ribonucleic core of the signal recognition particle from pyrococcus furiosus. PLoS ONE 3: e3528. Egea, P. F., Tsuruta, H., de Leon, G. P., Napetschnig, J., Walter, P. and Stroud, R. M. (2008b). Structures of the signal recognition particle receptor from the archaeon Pyrococcus furiosus: Implications for the targeting step at the membrane. PLoS ONE 3: e3619. Egelseer, E. M., Danhorn, T., Pleschberger, M., Hotzy, C., Sleytr, U. B. and Sara, M. (2001). Characterization of an S-layer glycoprotein produced in the course of S-layer variation of Bacillus stearothermophilus ATCC 12980 and sequencing and cloning of the SbsD gene encoding the protein moiety. Arch Microbiol 177: 70-80. Eichler, J. and Adams, M. W. (2005). Post-translational modification in the archaea. Microbiol Mol Biol Rev 69: 393 - 425. Eichler, J. and Moll, R. (2001). The signal recognition particle of archaea. Trends in Microbiology 9: 130-136. El-Fahmawi, B. and Owttrim, G. W. (2003). Polar-biased localization of the cold stress- induced RNA helicase, CrhC, in the cyanobacterium Anabaena sp. Strain PCC 7120. Mol Microbiol 50: 1439-1448. Elkins, J. G., Podar, M., Graham, D. E., Makarova, K. S., Wolf, Y., Randau, L., Hedlund, B. P., Brochier-Armanet, C., Kunin, V., Anderson, I., Lapidus, A., Goltsman, E., Barry, K., Koonin, E. V., Hugenholtz, P., Kyrpides, N., Wanner, G., Richardson, P., Keller, M. and Stetter, K. O. (2008). A korarchaeal genome reveals insights into the evolution of the archaea. Proc Natl Acad Sci U S A 105: 8102-8107. Engelhardt, H. (2007). Are S-layers exoskeletons? The basic function of protein surface layers revisited. J Struct Biol 160: 115-24. Engelhardt, H. and Peters, J. (1998). Structural research on surface layers: A focus on stability, surface layer homology domains, and surface layer-cell wall interactions. J Struct Biol 124: 276-302. Everberg, H., Gustavsson, N. and Tjerneld, F. (2008). Enrichment of membrane proteins by partitioning in detergent/polymer aqueous two-phase systems. Methods in molecular biology. A. Posch. Totowa, NJ, Humana press. 424. Faliks, D. and Meyuhas, O. (1982). Coordinate regulation of ribosomal protein mRNA level in regenerating rat liver. Study with the corresponding mouse cloned cDNAs. Nucleic Acids Res 10: 789-801. Feller, G. (2008). Enzyme function at low temperature in psychrophiles. Protein adaptation in extremophiles. K. S. Siddiqui and T. Thomas. Nova Science Publishers, Hauppauge, NY. Feller, G., D'Amico, S., Benotmane, A. M., Joly, F., Van Beeumen, J. and Gerday, C. (1998a). Characterization of the C-terminal propeptide involved in bacterial wall spanning of alpha-amylase from the psychrophile Alteromonas haloplanctis. J Biol Chem 273: 12109-12115. Feller, G., Le Bussy, O. and Gerday, C. (1998b). Expression of psychrophilic genes in mesophilic hosts: Assessment of the folding state of a recombinant alpha-amylase. Appl Environ Microbiol 64: 1163-1165. Feller, G., Zekhnini, Z., Lamotte-Brasseur, J. and Gerday, C. (1997). Enzymes from cold- adapted microorganisms. The class C beta-lactamase from the antarctic psychrophile Psychrobacter immobilis A5. Eur J Biochem 244: 186-191.
D. Burg UNSW 301
Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791. Ferguson, D. J., Jr. and Krzycki, J. A. (1997). Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri. J Bacteriol 179: 846-852. Ferguson, D. J., Jr., Krzycki, J. A. and Grahame, D. A. (1996). Specific roles of methylcobamide:Coenzyme M methyltransferase isozymes in metabolism of methanol and methylamines in Methanosarcina barkeri. J Biol Chem 271: 5189- 5194. Ferguson, T., Soares, J. A., Lienard, T., Gottschalk, G. and Krzycki, J. A. (2009). RamA, a protein required for reductive activation of corrinoid-dependent methylamine methyltransferase reactions in methanogenic archaea. J Biol Chem 284: 2285-2295. Ferreri, C., Kratzsch, S., Landi, L. and Brede, O. (2005). Thiyl radicals in biosystems: Effects on lipid structures and metabolisms. Cell Mol Life Sci 62: 834-847. Ferry, J. G. (1999). Enzymology of one-carbon metabolism in methanogenic pathways. FEMS Microbiol Rev 23: 13-38. Ferry, J. G. and Kastead, K. A. (2007). Methanogenesis. Archaea: Molecular and cellular biology. R. Cavicchioli. Washington, D. C., ASM press: 288-314. Fine, A., Irihimovitch, V., Dahan, I., Konrad, Z. and Eichler, J. (2006). Cloning, expression, and purification of functional Sec11a and Sec11b, type I signal peptidases of the archaeon Haloferax volcanii. J Bacteriol 188: 1911-1919. Forge, A., Nevill, G., Zajic, G. and Wright, A. (1992). Scanning electron microscopy of the mammalian organ of corti: Assessment of preparative procedures. Scanning Microsc 6: 521-534. Formisano, V., Atreya, S., Encrenaz, T., Ignatiev, N. and Giuranna, M. (2004). Detection of methane in the atmosphere of Mars. Science 306: 1758-1761. Forouhar, F., Abashidze, M., Xu, H., Grochowski, L. L., Seetharaman, J., Hussain, M., Kuzin, A., Chen, Y., Zhou, W., Xiao, R., Acton, T. B., Montelione, G. T., Galinier, A., White, R. H. and Tong, L. (2008). Molecular insights into the biosynthesis of the F420 coenzyme. J Biol Chem 283: 11832-11840. Forterre, P., Brochier, C. and Philippe, H. (2002). Evolution of the archaea. Theor Popul Biol 61: 409-422. Franceschi, F. J. and Nierhaus, K. H. (1990). Ribosomal proteins L15 and L16 are mere late assembly proteins of the large ribosomal subunit. Analysis of an Escherichia coli mutant lacking L15. J Biol Chem 265: 16676-16682. Francoleon, D. R., Boontheung, P., Yang, Y., Kim, U., Ytterberg, A. J., Denny, P. A., Denny, P. C., Loo, J. A., Gunsalus, R. P. and Ogorzalek Loo, R. R. (2009). S-layer, surface- accessible, and concanavalin a binding proteins of Methanosarcina acetivorans and Methanosarcina mazei. J Proteome Res 8: 1972-1982. Franzmann, P. D., Liu, Y., Balkwill, D. L., Aldrich, H. C., Conway de Macario, E. and Boone, D. R. (1997). Methanogenium frigidum sp. Nov., a psychrophilic, H2-using methanogen from Ace Lake, Antarctica. Int J Syst Bacteriol 47: 1068-1072. Franzmann, P. D., Springer, N., Ludwig, W., De Macario, E. C. and Rohde, M. (1992). A methanogenic archaeon from Ace Lake, Antarctica: Methanococcoides burtonii sp. Nov. System App Microbiol 15: 573-581. Frols, S., Ajon, M., Wagner, M., Teichmann, D., Zolghadr, B., Folea, M., Boekema, E. J., Driessen, A. J., Schleper, C. and Albers, S. V. (2008). UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation. Mol Microbiol 70: 938-952. Fujiki, Y., Hubbard, A., Fowler, S. and Lazarow, P. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: Application to endoplasmic reticulum. J. Cell Biol. 93: 97-102.
302 D. Burg UNSW
Fukui, T., Eguchi, T., Atomi, H. and Imanaka, T. (2002). A membrane-bound archaeal Lon protease displays ATP-independent proteolytic activity towards unfolded proteins and ATP-dependent activity for folded proteins. J Bacteriol 184: 3689-3698. Fukuzaki, S., Nishio, N., Sakurai, H. and Nagai, S. (1991). Characteristics of methanogenic granules grown on propionate in an upflow anaerobic sludge blanket reactor. J. Ferm Bioeng 71: 50-57. Fuller-Pace, F. V. (2006). DExD/H box RNA helicases: Multifunctional proteins with important roles in transcriptional regulation. Nucleic Acids Res 34: 4206-4215. Furutani, M., Ideno, A., Iida, T. and Maruyama, T. (2000). FK506 binding protein from a thermophilic archaeon, Methanococcus thermolithotrophicus, has chaperone-like activity in vitro. Biochemistry 39: 2822. Galperin, M. Y. and Koonin, E. V. (1997). A diverse superfamily of enzymes with ATP- dependent carboxylate-amine/thiol ligase activity. Protein Sci 6: 2639-2643. Galperin, M. Y. and Nikolskaya, A. N. (2007). Identification of sensory and signal- transducing domains in two-component signaling systems. Methods Enzymol 422: 47-74. Galperin, M. Y., Nikolskaya, A. N. and Koonin, E. V. (2001). Novel domains of the prokaryotic two-component signal transduction systems. FEMS Microbiol Lett 203: 11-21. Gan, C. S., Chong, P. K., Pham, T. K. and Wright, P. C. (2007). Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). J Proteome Res 6: 821-7. Gao, B. and Gupta, R. S. (2007). Phylogenomic analysis of proteins that are distinctive of archaea and its main subgroups and the origin of methanogenesis. BMC Genomics 8: 86. Garneau, M., Vincent, W. F., Alonso-Saez, L., Gratton, Y. and Lovejoy, C. (2006). Prokaryotic community structure and heterotrophic production in a river- influenced coastal arctic ecosystem. Aquatic microbial ecology 42: 27 - 40. Garnier, J., Gibrat, J.-F. and Robson, B. (1996). GOR secondary structure prediction method version iv. Methods Enzymol 266: 540-553. Gatlin, C. L., Kleemann, G. R., Hays, L. G., Link, A. J. and Yates, J. R., 3rd (1998). Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal Biochem 263: 93-101. Geiduschek, E. P. and Ouhammouch, M. (2005). Archaeal transcription and its regulators. Mol Microbiol 56: 1397-407. Georlette, D., Jonsson, Z. O., Van Petegem, F., Chessa, J., Van Beeumen, J., Hubscher, U. and Gerday, C. (2000). A DNA ligase from the psychrophile Pseudoalteromonas haloplanktis gives insights into the adaptation of proteins to low temperatures. Eur J Biochem 267: 3502-3512. Gerike, U., Danson, M. J., Russell, N. J. and Hough, D. W. (1997). Sequencing and expression of the gene encoding a cold-active citrate synthase from an Antarctic bacterium, strain DS2-3r. Eur J Biochem 248: 49-57. Giaquinto, L., Curmi, P. M., Siddiqui, K. S., Poljak, A., DeLong, E., DasSarma, S. and Cavicchioli, R. (2007). Structure and function of cold shock proteins in archaea. J Bacteriol 189: 5738-5748. Gibson, J. A. E., Miller, M. R., Davies, N. W., Neill, G. P., Nichols, D. S. and Volkman, J. K. (2005). Unsaturated diether lipids in the psychrotrophic archaeon Halorubrum lacusprofundi. System Appl Microbiol 28: 19-26. Gihring, T. M., Bond, P. L., Peters, S. C. and Banfield, J. F. (2003). Arsenic resistance in the archaeon "Ferroplasma acidarmanus": New insights into the structure and evolution of the ars genes. Extremophiles 7: 123-130.
D. Burg UNSW 303
Goenrich, M., Thauer, R. K., Yurimoto, H. and Kato, N. (2005). Formaldehyde activating enzyme (Fae) and hexulose-6-phosphate synthase (Hps) in Methanosarcina barkeri: A possible function in ribose-5-phosphate biosynthesis. Arch Microbiol 184: 41-48. Goodchild, A. (2004). Insight into the biology of a cold adapted archaeon from genomic and proteomic studies. School of Biotechnology and Biomolecular sciences. University of New South Wales, Sydney,. Goodchild, A., Raftery, M., Saunders, N. F., Guilhaus, M. and Cavicchioli, R. (2004a). Biology of the cold adapted archaeon, Methanococcoides burtonii determined by proteomics using liquid chromatography-tandem mass spectrometry. J Proteome Res 3: 1164-1176. Goodchild, A., Raftery, M., Saunders, N. F., Guilhaus, M. and Cavicchioli, R. (2005). Cold adaptation of the antarctic archaeon, Methanococcoides burtonii assessed by proteomics using icat. J Proteome Res 4: 473-480. Goodchild, A., Saunders, N. F., Ertan, H., Raftery, M., Guilhaus, M., Curmi, P. M. and Cavicchioli, R. (2004b). A proteomic determination of cold adaptation in the antarctic archaeon, Methanococcoides burtonii. Mol Microbiol 53: 309-421. Goshe, M. B., Blonder, J. and Smith, R. D. (2003). Affinity labelling of highly hydrophobic integral membrane proteins for proteome-wide analysis. J Proteome Res 2: 153- 161. Gosslau, A., Jao, D. L., Butler, R., Liu, A. Y. and Chen, K. Y. (2009). Thermal killing of human colon cancer cells is associated with the loss of eukaryotic initiation factor 5A. J Cell Physiol 219: 485-93. Gottschalk, G. and Thauer, R. K. (2001). The NA+-translocating methyltransferase complex from methanogenic archaea. Biochim Biophys Acta 1505: 28-36. Gould, R. J., Ginsberg, B. H. and Spector, A. A. (1981). Effects of octyl beta-glucoside on insulin binding to solubilized membrane receptors. Biochemistry 20: 6776-6781. Graham, D. E., Graupner, M., Xu, H. and White, R. H. (2001). Identification of coenzyme m biosynthetic 2-phosphosulfolactate phosphatase. A member of a new class of mg2+- dependent acid phosphatases. Eur J Biochem 268: 5176-5188. Graham, D. E., Xu, H. and White, R. H. (2002). Identification of coenzyme M biosynthetic phosphosulfolactate synthase: A new family of sulfonate-biosynthesizing enzymes. J Biol Chem 277: 13421-13429. Graham, D. E., Xu, H. and White, R. H. (2003). Identification of the 7,8-didemethyl-8- hydroxy-5-deazariboflavin synthase required for coenzyme F420 biosynthesis. Arch Microbiol 180: 455-464. Graham, R. L., O'Loughlin, S. N., Pollock, C. E., Ternan, N. G., Weatherly, D. B., Jackson, P. J., Tarleton, R. L. and McMullan, G. (2006). A combined shotgun and multidimensional proteomic analysis of the insoluble subproteome of the obligate thermophile, Geobacillus thermoleovorans T80. J Proteome Res 5: 2465-2473. Graham, R. L., Pollock, C. E., O'Loughlin, S. N., Ternan, N. G., Weatherly, D. B., Tarleton, R. L. and McMullan, G. (2007). Multidimensional analysis of the insoluble sub- proteome of Oceanobacillus iheyensis HTE831, an alkaliphilic and halotolerant deep-sea bacterium isolated from the Iheya ridge. Proteomics 7: 82-91. Graninger, M., Kneidinger, B., Bruno, K., Scheberl, A. and Messner, P. (2002). Homologs of the RmL enzymes from Salmonella enterica are responsible for DTDP-L-rhamnose biosynthesis in the Gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155. App Environ Microbiol 68: 3708-3715. Graupner, M. and White, R. H. (2001). Biosynthesis of the phosphodiester bond in coenzyme F420 in the methanoarchaea. Biochemistry 40: 10859-71082. Graupner, M. and White, R. H. (2003). Methanococcus jannaschii coenzyme F420 analogs contain a terminal alpha-linked glutamate. J Bacteriol 185: 4662-4665.
304 D. Burg UNSW
Graupner, M., Xu, H. and White, R. H. (2000). Identification of an archaeal 2-hydroxy acid dehydrogenase catalyzing reactions involved in coenzyme biosynthesis in methanoarchaea. J Bacteriol 182: 3688-3692. Graupner, M., Xu, H. and White, R. H. (2002a). Characterization of the 2-phospho-l-lactate transferase enzyme involved in coenzyme F420 biosynthesis in Methanococcus jannaschii. Biochemistry 41: 3754-3761. Graupner, M., Xu, H. and White, R. H. (2002b). The pyrimidine nucleotide reductase step in riboflavin and F420 biosynthesis in archaea proceeds by the eukaryotic route to riboflavin. J Bacteriol 184: 1952-1957. Griffith, M., Timonin, M., Wong, A. C., Gray, G. R., Akhter, S. R., Saldanha, M., Rogers, M. A., Weretilnyk, E. A. and Moffatt, B. (2007). Thellungiella: An arabidopsis-related model plant adapted to cold temperatures. Plant Cell Environ 30: 529-538. Grochowski, L. L., Xu, H. and White, R. H. (2006). Methanocaldococcus jannaschii uses a modified mevalonate pathway for biosynthe sis of isopentenyl diphosphate. J. Bacteriol 188: 3192-3198. Gruber, T., Kohrer, C., Lung, B., Shcherbakov, D. and Piendl, W. (2003). Affinity of ribosomal protein S8 from mesophilic and (hyper)thermophilic archaea and bacteria for 16S rrna correlates with the growth temperatures of the organisms. FEBS Letters 549: 123-128. Gudmundsdottir, A. and Palsdottir, H. M. (2005). Atlantic cod trypsins: From basic research to practical applications. Mar Biotechnol (NY) 7: 77-88. Gundersen, H. J. and Osterby, R. (1981). Optimizing sampling efficiency of stereological studies in biology: Or 'do more less well!'. J Microsc 121: 65-73. Hagen, W. R., Silva, P. J., Amorim, M. A., Hagedoorn, P. L., Wassink, H., Haaker, H. and Robb, F. T. (2000). Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with asp(cys)3 ligands. J Biol Inorg Chem 5: 527-534. Haldenby, S., White, M. F. and Allers, T. (2009). RecA family proteins in archaea: RadA and its cousins. Biochem Soc Trans 37: 102-107. Hamill, O. P. and Martinac, B. (2001). Molecular basis of mechanotransduction in living cells. Physiol Rev 81: 685-740. Hammel (1986). Progression errors in morphometry: Estimation of particle number density. J Histochem Cytochem 34: 941-944. Hartl, F. U. and Hayer-Hartl, M. (2002). Molecular chaperones in the cytosol: From nascent chain to folded protein. Science 295: 1852-1858. Hartmann, E. and Konig, H. (1991). Nucleotide-activated oligosaccharides are intermediates of the cell wall polysaccharide of Methanosarcina barkeri. Biol Chem Hoppe Seyler 372: 971-974. Henry, J. L., Coggin, D. L. and King, C. R. (1993). High-level expression of the ribosomal protein L19 in human breast tumors that overexpress erbB-2. Cancer Res 53: 1403- 1408. Herndl, G. J., Reinthaler, T., Teira, E., van Aken, H., Veth, C., Pernthaler, A. and Pernthaler, J. (2005). Contribution of archaea to total prokaryotic production in the deep Atlantic Ocean. Appl Environ Microbiol 71: 2303-2309. Hippe, H., Caspari, D., Fiebig, K. and Gottschalk, G. (1979). Utilization of trimethylamine and other N-methyl compounds for growth and methane formation by Methanosarcina barkeri. Proc Natl Acad Sci U S A 76: 494-498. Ho, Y. S., Burden, L. M. and Hurley, J. H. (2000). Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor. EMBO J 19: 5288-5299. Hochheimer, A., Linder, D., Thauer, R. K. and Hedderich, R. (1996). The molybdenum formylmethanofuran dehydrogenase operon and the tungsten formylmethanofuran dehydrogenase operon from Methanobacterium
D. Burg UNSW 305
thermoautotrophicum. Structures and transcriptional regulation. Eur J Biochem 242: 156-162. Hoj, L., Olsen, R. A. and Torsvik, V. L. (2008). Effects of temperature on the diversity and community structure of known methanogenic groups and other archaea in high Arctic peat. ISME J 2: 37-48. Hongsthong, A., Sirijuntarut, M., Prommeenate, P., Lertladaluck, K., Porkaew, K., Cheevadhanarak, S. and Tanticharoen, M. (2008). Proteome analysis at the subcellular level of the cyanobacterium Spirulina platensis in response to low- temperature stress conditions. FEMS Microbiol Lett 288: 92-101. Horn, C., Sohn-Bosser, L., Breed, J., Welte, W., Schmitt, L. and Bremer, E. (2006). Molecular determinants for substrate specificity of the ligand-binding protein OpuAC from Bacillus subtilis for the compatible solutes glycine betaine and proline betaine. J Mol Biol 357: 592-606. Howell, D. M., Graupner, M., Xu, H. and White, R. H. (2000). Identification of enzymes homologous to isocitrate dehydrogenase that are involved in coenzyme B and leucine biosynthesis in methanoarchaea. J Bacteriol 182: 5013-6. Huber, H., Hohn, M. J., Rachel, R., Fuchs, T., Wimmer, V. C. and Stetter, K. O. (2002). A new phylum of archaea represented by a nanosized hyperthermophilic symbiont. Nature 417: 63-67. Huber, H., Hohn, M. J., Stetter, K. O. and Rachel, R. (2003). The phylum nanoarchaeota: Present knowledge and future perspectives of a unique form of life. Res Microbiol 154: 165-171. Hunger, K., Beckering, C. L., Wiegeshoff, F., Graumann, P. L. and Marahiel, M. A. (2006). Cold-induced putative DEAD box RNA helicases CshA and CshB are essential for cold adaptation and interact with cold shock protein B in Bacillus subtilis. J Bacteriol 188: 240-248. Huston, A. L., Haeggström, J. Z. and Feller, G. (2008). Cold adaptation of enzymes: Structural, kinetic and microcalorimetric characterizations of an aminopeptidase from the arctic psychrophile Colwellia psychrerythraea and of human leukotriene A4 hydrolase. BBA - Proteins & Proteomics 1784: 1865-1872. Ideno, A., Yoshida, T., Furutani, M. and Maruyama, T. (2000). The 28.3 kDa FK506 binding protein from a thermophilic archaeum, Methanobacterium thermoautotrophicum, protects the denaturation of proteins in vitro. Eur J Biochem 267: 3139-3149. Ideno, A., Yoshida, T., Iida, T., Furutani, M. and Maruyama, T. (2001). FK506-binding protein of the hyperthermophilic archaeum, Thermococcus sp. KS-1, a cold-shock- inducible peptidyl-prolyl cis-trans isomerase with activities to trap and refold denatured proteins. Biochem J 357: 465-71. IPCC. (2008). "Intergovernmental panel on climate change." 2008, from http:/www.ipcc.ch. Issaq, H. J., Chan, K. C., Janini, G. M., Conrads, T. P. and Veenstra, T. D. (2005). Multidimensional separation of peptides for effective proteomic analysis. J Chromatography B 817: 35-47. Itou, H., Yao, M., Watanabe, N. and Tanaka, I. (2008). Crystal structure of the Ph1932 protein, a unique archaeal ArsR type winged-HTH transcription factor from Pyrococcus horikoshii OT3. Proteins 70: 1631-1634. Jiang, W., Hou, Y. and Inouye, M. (1997). CSPa, the major cold-shock protein of Escherichia coli, is an RNA chaperone. J Biol Chem 272: 196-202. Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B. T. and MacKinnon, R. (2002). Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417: 515- 522. Jing, H., Takagi, J., Liu, J. H., Lindgren, S., Zhang, R. G., Joachimiak, A., Wang, J. H. and Springer, T. A. (2002). Archaeal surface layer proteins contain beta propeller, PKD, and beta helix domains and are related to metazoan cell surface proteins. Structure 10: 1453-64.
306 D. Burg UNSW
Johnston, I. A. (1990). Cold adaptation in marine organisms. Philos Trans R Soc Lond B Biol Sci 326: 655-667. Jones, P. G., Cashel, M., Glaser, G. and Neidhardt, F. C. (1992). Function of a relaxed-like state following temperature downshifts in Escherichia coli. J Bacteriol 174: 3903- 3914. Jones, P. G., Mitta, M., Kim, Y., Jiang, W. and Inouye, M. (1996). Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in Escherichia coli. Proc Natl Acad Sci U S A 93: 76-80. Jung, H. (2002). The sodium/substrate symporter family: Structural and functional features. FEBS Lett 529: 73-77. Junge, K., Eicken, H. and Deming, J. W. (2004). Bacterial activity at -2 to -20 degrees C in Arctic wintertime sea ice. Appl Environ Microbiol 70: 550-557. Junge, K., Eicken, H., Swanson, B. D. and Deming, J. W. (2006). Bacterial incorporation of leucine into protein down to -20 °C with evidence for potential activity in sub- eutectic saline ice formations. Cryobiology 52: 417-429. Kaan, T., Homuth, G., Mader, U., Bandow, J., Schweder, T., Beckering, C. L., Steil, L., Weber, M. H., Volker, U., Marahiel, M. A., Yamada, T., Kuroda, K., Jitsuyama, Y., Takezawa, D., Arakawa, K. and Fujikawa, S. (2002). Genome-wide transcriptional profiling of the cold-shock response in Bacillus subtilis. Microbiology 148: 3441-3455. Kachlany, S. C., Levery, S. B., Kim, J. S., Reuhs, B. L., Lion, L. W. and Ghiorse, W. C. (2001). Structure and carbohydrate analysis of the exopolysaccharide capsule of Pseudomonas putida G7. Environ Microbiol 3: 774-784. Kall, L., Storey, J. D., MacCoss, M. J. and Noble, W. S. (2008). Assigning significance to peptides identified by tandem mass spectrometry using decoy databases. J Proteome Res 7: 29-34. Kamagata, Y., Kawasaki, H., Oyaizu, H., Nakamura, K., Mikami, E., Endo, G., Koga, Y. and Yamasato, K. (1992). Characterization of three thermophilic strains of Methanothrix ("Methanosaeta") thermophila sp. Nov. And rejection of Methanothrix ("Methanosaeta") thermoacetophila. Int J Syst Bacteriol 42: 463-468. Kammler, M., Schon, C. and Hantke, K. (1993). Characterization of the ferrous iron uptake system of Escherichia coli. J Bacteriol 175: 6212-6219. Kandror, O. and Goldberg, A. L. (1997). Trigger factor is induced upon cold shock and enhances viability of Escherichia coli at low temperatures. Proc Natl Acad Sci U S A 94: 4978-4981. Kang, W. K., Icho, T., Isono, S., Kitakawa, M. and Isono, K. (1989). Characterization of the gene RimK responsible for the addition of glutamic acid residues to the C-terminus of ribosomal protein S6 in Escherichia coli K12. Mol Gen Genet 217: 281-288. Karner, M. B., DeLong, E. F. and Karl, D. M. (2001). Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature 409: 507-510. Karzai, A. W. and McMacken, R. (1996). A bipartite signaling mechanism involved in DnaJ- mediated activation of the Escherichia coli DnaK protein. J Biol Chem 271: 11236- 11246. Kawaguchi, T. and Decho, A. W. (2002). In situ analysis of carboxyl and sulfhydryl groups of extracellular polymeric secretions by confocal laser scanning microscopy. Anal Biochem 304: 266-267. Kawakami, R., Sakuraba, H. and Ohshima, T. (2007). Gene cloning and characterization of the very large NAD-dependent L-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil. J Bacteriol 189: 5626-5633. Kawamoto, J., Kurihara, T., Kitagawa, M., Kato, I. and Esaki, N. (2007). Proteomic studies of an Antarctic cold-adapted bacterium, Shewanella livingstonensis AC10, for global identification of cold-inducible proteins. Extremophiles 11: 819-826. Kehres, D. G., Lawyer, C. H. and Maguire, M. E. (1998). The CorA magnesium transporter gene family. Microb Comp Genomics 3: 151-69.
D. Burg UNSW 307
Keller, A., Nesvizhskii, A. I., Kolker, E. and Aebersold, R. (2002). Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74: 5383-5392. Kelley, W. L. (1998). The J-domain family and the recruitment of chaperone power. Trends Biochem Sci 23: 222-227. Kendall, M. M. and Boone, D. R. (2006). Cultivation of methanogens from shallow marine sediments at hydrate ridge, Oregon. Archaea 2: 31-38. Kendall, M. M., Wardlaw, G. D., Tang, C. F., Bonin, A. S., Liu, Y. and Valentine, D. L. (2007). Diversity of archaea in marine sediments from Skan Bay, Alaska, including cultivated methanogens, and description of Methanogenium boonei sp. Nov. Appl Environ Microbiol 73: 407-414. Kennelly, P. J. (2007). Sensing, signal transduction, and posttranslational modification. Archaea: Cellular and moecular biology. R. Cavicchioli. Washington D. C., ASM press. Kim, A. C., Oliver, D. C. and Paetzel, M. (2008). Crystal structure of a bacterial signal peptide peptidase. J Mol Biol 376: 352-366. Kim, K. Y., Park, S. W., Chung, Y. S., Chung, C. H., Kim, J. I. and Lee, J. H. (2004). Molecular cloning of low-temperature-inducible ribosomal proteins from soybean. J Exp Bot 55: 1153-1155. Kim, R., Kim, K. K., Yokota, H. and Kim, S. H. (1998). Small heat shock protein of Methanococcus jannaschii, a hyperthermophile. Proc Natl Acad Sci U S A 95: 9129- 9133. Kim, R., Lai, L., Lee, H. H., Cheong, G. W., Kim, K. K., Wu, Z., Yokota, H., Marqusee, S. and Kim, S. H. (2003). On the mechanism of chaperone activity of the small heat-shock protein of Methanococcus jannaschii. Proc Natl Acad Sci U S A 100: 8151-8155. Kirchman, D. L., Elifantz, H., Dittel, A. I., Malmstrom, R. R. and Cottrell, M. T. (2007). Standing stocks and activity of archaea and bacteria in the western Arctic Ocean. Limnol Oceanog 52: 495-507. Klebensberger, J., Rui, O., Fritz, E., Schink, B. and Philipp, B. (2006). Cell aggregation of Pseudomonas aeruginosa strain PAO1 as an energy-dependent stress response during growth with sodium dodecyl sulfate. Arch Microbiol 185: 417-27. Klein, C., Garcia-Rizo, C., Bisle, B., Scheffer, B., Zischka, H., Pfeiffer, F., Siedler, F. and Oesterhelt, D. (2005). The membrane proteome of Halobacterium salinarum. Proteomics 5: 180-197. Kloda, A. and Martinac, B. (2001a). Mechanosensitive channel of thermoplasma, the cell wall-less archaea: Cloning and molecular characterization. Cell Biochem Biophys 34: 321-347. Kloda, A. and Martinac, B. (2001b). Molecular identification of a mechanosensitive channel in archaea. Biophys J 80: 229-240. Kloda, A. and Martinac, B. (2001c). Structural and functional differences between two homologous mechanosensitive channels of Methanococcus jannaschii. EMBO J 20: 1888-1896. Knowles, E. J. and Castenholz, R. W. (2008). Effect of exogenous extracellular polysaccharides on the desiccation and freezing tolerance of rock-inhabiting phototrophic microorganisms. FEMS Microbiol Ecol 66: 261-270. Ko, R., Smith, L. T. and Smith, G. M. (1994). Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes. J Bacteriol 176: 426- 31. Koch, M., Rudolph, C., Moissl, C. and Huber, R. (2006). A cold-loving crenarchaeon is a substantial part of a novel microbial community in cold sulphidic marsh water. FEMS Microbiol Ecol 57: 55-66. Koga, Y., Kyuragi, T., Nishihara, M. and Sone, N. (1998). Did archaeal and bacterial cells arise independently from noncellular precursors? A hypothesis stating that the
308 D. Burg UNSW
advent of membrane phospholipid with enantiomeric glycerophosphate backbones caused the separation of the two lines of descent. J Mol Evo 46: 54-63. Koga, Y., Nishihara, M., Morii, H. and Akagawa-Matsushita, M. (1993). Ether polar lipids of methanogenic bacteria: Structures, comparative aspects, and biosyntheses. Microbiol Rev 57: 164-182. Kok, M. D., Rijpstra, W. I. C., Robertson, L., Volkman, J. K. and Sinninghe Damstéé, J. S. (2000). Early steroid sulfurisation in surface sediments of a permanently stratified lake (Ace Lake, Antarctica). Geochim Cosmochim Acta 64: 1425-1436. Konig, H., Rachel, R. and Claus, H. (2007). Proteinaceous surface layers of archaea: Ultrastructure and biochemistry. Archaea: Cellular and molecular biology. R. Cavicchioli. ASM Press, Washington DC. Konrad, Z. and Eichler, J. (2002). Lipid modification of proteins in archaea: Attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation. Biochem J 366: 959-964. Kornitzer, D., Teff, D., Altuvia, S. and Oppenheim, A. B. (1991). Isolation, characterization, and sequence of an Escherichia coli heat shock gene, HtpX. J Bacteriol 173: 2944- 2953. Kotsyurbenko, O. R. (2005). Trophic interactions in the methanogenic microbial community of low-temperature terrestrial ecosystems. FEMS Microbiology Ecology 53: 3-13. Kral, T. A., Bekkum, C. R. and McKay, C. P. (2004). Growth of methanogens on a Mars soil simulant. Origins Life Evol. Biosph 34: 615–626 Kruger, M., Blumenberg, M., Kasten, S., Wieland, A., Kanel, L., Klock, J. H., Michaelis, W. and Seifert, R. (2008). A novel, multi-layered methanotrophic microbial mat system growing on the sediment of the Black Sea. Environ Microbiol 10: 1934-1947. Kruger, N. J. (2002). The bradford method for protein quantitation. The protein protocols handbook. J. M. Walker. Humana Press Inc., Totowa, NJ, USA. Kumagai, H., Fujiwara, T., Matsubara, H. and Saeki, K. (1997). Membrane localization, topology, and mutual stabilization of the RnfABC gene products in Rhodobacter capsulatus and implications for a new family of energy-coupling NADH oxidoreductases. Biochemistry 36: 5509-5521. Kumar, K. U., Srivastava, S. P. and Kaufman, R. J. (1999). Double-stranded RNA-activated protein kinase (Pkr) is negatively regulated by 60S ribosomal subunit protein L18. Mol Cell Biol 19: 1116-1125. Kvint, K., Nachin, L., Diez, A. and Nystrom, T. (2003). The bacterial universal stress protein: Function and regulation. Curr Opin Microbiol 6: 140-145. Lang, P., Zhang, C.-k., Ebel, R. C., Dane, F. and Dozier, W. A. (2005). Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display. Gene 359: 111-118. Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, M. K. and Hartl, F. U. (1992). Successive action of dnak, DnaJ and groel along the pathway of chaperone-mediated protein folding. Nature 356: 683-689. Lauro, F. M., Tran, K., Vezzi, A., Vitulo, N., Valle, G., and Bartlett, D. G. (2008). Largescale transposon mutagenesis of Photobacterium profundum SS9 reveals new genetic loci important for growth at low temperature and high pressure. J. Bacteriol. 190: 1699-1709. Le Cam, E., Culard, F., Larquet, E., Delain, E. and Cognet, J. A. H. (1999). DNA bending induced by the archaebacterial histone-like protein MC1. Journal of Molecular Biology 285: 1011-1021. Le Dain, A. C., Saint, N., Kloda, A., Ghazi, A. and Martinac, B. (1998). Mechanosensitive ion channels of the archaeon Haloferax volcanii. J Biol Chem 273: 12116-12119.
D. Burg UNSW 309
Lee, K. K., Jang, C. S., Yoon, J. Y., Kim, S. Y., Kim, T. H., Ryu, K. H. and Kim, W. (2008). Abnormal cell division caused by inclusion bodies in E. coli; increased resistance against external stress. Microbiol Res 163: 394-402. Lee, S., Sowa, M. E., Watanabe, Y. H., Sigler, P. B., Chiu, W., Yoshida, M. and Tsai, F. T. (2003). The structure of ClpB: A molecular chaperone that rescues proteins from an aggregated state. Cell 115: 229-240. Leibundgut, M., Frick, C., Thanbichler, M., Bock, A. and Ban, N. (2005). Selenocysteine tRNA-specific elongation factor selb is a structural chimaera of elongation and initiation factors. EMBO J 24: 11-22. Li, H., Graupner, M., Xu, H. and White, R. H. (2003a). CofE catalyzes the addition of two glutamates to F420-0 in F420 coenzyme biosynthesis in Methanococcus jannaschii. Biochemistry 42: 9771-9778. Li, H., Xu, H., Graham, D. E. and White, R. H. (2003b). Glutathione synthetase homologs encode alpha-L-glutamate ligases for methanogenic coenzyme F420 and tetrahydrosarcinapterin biosyntheses. Proc Natl Acad Sci U S A 100: 9785-90. Li, L., Li, Q., Rohlin, L., Kim, U., Salmon, K., Rejtar, T., Gunsalus, R. P., Karger, B. L. and Ferry, J. G. (2007). Quantitative proteomic and microarray analysis of the archaeon Methanosarcina acetivorans grown with acetate versus methanol. J Proteome Res 6: 759-771. Li, Q., Li, L., Rejtar, T., Karger, B. L. and Ferry, J. G. (2005a). Proteome of Methanosarcina acetivorans part I: An expanded view of the biology of the cell. J Proteome Res 4: 112-128. Li, Q., Li, L., Rejtar, T., Karger, B. L. and Ferry, J. G. (2005b). Proteome of Methanosarcina acetivorans part II: Comparison of protein levels in acetate- and methanol-grown cells. J Proteome Res 4: 129-135. Li, Q., Li, L., Rejtar, T., Lessner, D. J., Karger, B. L. and Ferry, J. G. (2006). Electron transport in the pathway of acetate conversion to methane in the marine archaeon Methanosarcina acetivorans. J Bacteriol 188: 702-710. Lim, J., Thomas, T. and Cavicchioli, R. (2000). Low temperature regulated DEAD-box RNA helicase from the Antarctic archaeon, Methanococcoides burtonii. Journal of Molecular Biology 297: 553-567. Linder, P. and Stutz, F. (2001). mRNA export: Travelling with DEAD box proteins. Curr Biol 11: R961-963. Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., Garvik, B. M. and Yates, J. (1999). Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 17: 676-682. Little, B., Wagner, P., Ray, R., Pope, R. and Scheetz, R. (1991). Biofilms: An ESEM evaluation of artifacts introduced during SEM preparation. J Industrial Microbiol 8: 213-222 Liu, W., Vierke, G., Wenke, A. K., Thomm, M. and Ladenstein, R. (2007). Crystal structure of the archaeal heat shock regulator from Pyrococcus furiosus: A molecular chimera representing eukaryal and bacterial features. J Mol Biol 369: 474-488. Lliros, M., Casamayor, E. O. and Borrego, C. (2008). High archaeal richness in the water column of a freshwater sulfurous karstic lake along an interannual study. FEMS Microbiol Ecol. Lorber, B., Bishop, J. B. and DeLucas, L. J. (1990). Purification of octyl [beta]-- glucopyranoside and re-estimation of its micellar size. BBA - Biomembranes 1023: 254-265. Lovdal, T., Skjoldal, E. F., Heldal, M., Norland, S. and Thingstad, T. F. (2008). Changes in morphology and elemental composition of Vibrio splendidus along a gradient from carbon-limited to phosphate-limited growth. Microb Ecol 55: 152-161. Lowe, J. and Amos, L. A. (1998). Crystal structure of the bacterial cell-division protein FtsZ. Nature 391: 203-206.
310 D. Burg UNSW
Lu, X. and Zhu, H. (2005). Tube-gel digestion: A novel proteomic approach for high throughput analysis of membrane proteins. Mol Cell Proteomics 4: 1948-1958. Ma, K. and Adams, M. W. (1994). Sulfide dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus: A new multifunctional enzyme involved in the reduction of elemental sulfur. J Bacteriol 176: 6509-6517. Macario, A. J. and Conway De Macario, E. (2001). The molecular chaperone system and other anti-stress mechanisms in archaea. Front Biosci 1: D262-283. Macario, A. J., Lange, M., Ahring, B. K. and De Macario, E. C. (1999). Stress genes and proteins in the archaea. Microbiol Mol Biol Rev 63: 923-67. Majernik, A., Cubonova, L., Polak, P., Smigan, P. and Greksak, M. (2003). Biochemical analysis of neomycin-resistance in the methanoarchaeon Methanothermobacter thermautotrophicus and some implications for energetic processes in this strain. Anaerobe 9: 31-38. Makarova, K. S., Sorokin, A. V., Novichkov, P. S., Wolf, Y. I. and Koonin, E. V. (2007). Clusters of orthologous genes for 41 archaeal genomes and implications for evolutionary genomics of archaea. Biology Direct 2: Online. Malygin, A. A., Shaulo, D. D. and Karpova, G. G. (2000). Proteins S7, S10, S16 and S19 of the human 40S ribosomal subunit are most resistant to dissociation by salt. Biochimica et BBA - Gene Structure and Expression 1494: 213-216. Mandarim-de-Lacerda, C. A. (2003). Stereological tools in biomedical research. An Acad Bras Cienc 75: 469-486. Marchetti, A. and Cassar, N. (2009). Diatom elemental and morphological changes in response to iron limitation: A brief review with potential paleoceanographic applications. Geobiology 7: 419-431. Martinac, B. and Kloda, A. (2003). Evolutionary origins of mechanosensitive ion channels. Prog Biophys Mol Biol 82: 11-24. Maruyama, T., Suzuki, R. and Furutani, M. (2004). Archaeal peptidyl prolyl cis-trans isomerases (PPIases) update 2004. Front Biosci 9: 1680-1720. Marx, J. G., Carpenter, S. D. and Deming, J. W. (2009). Production of cryoprotectant extracellular polysaccharide substances (EPS) by the marine psychrophilic bacterium Colwellia psychrerythraea strain 34H under extreme conditions. Can J Microbiol 55: 63-72. Masepohl, B. and Klipp, W. (1996). Organization and regulation of genes encoding the molybdenum nitrogenase and the alternative nitrogenase in Rhodobacter capsulatus. Arch Microbiol165: 80-90. Maupin-Furlow, J. A., Gil, M. A., Humbard, M. A., Kirkland, P. A., Li, W., Reuter, C. J. and Wright, A. J. (2005a). Archaeal proteasomes and other regulatory proteases. Curr Opin Microbiol 8: 720-728. Maupin-Furlow, J. A., Gil, M. A., Humbard, M. A., Kirkland, P. A., Li, W., Reuter, C. J. and Wright, A. J. (2005b). Archaeal proteasomes and other regulatory proteases. Curr Op Microbiol 8: 720-728. Mayerhofer, L. E., Conway de Macario, E. and Macario, A. J. (1995). Conservation and variability in archaea: Protein antigens with tandem repeats encoded by a cluster of genes with common motifs in Methanosarcina mazei S-6. Gene 165: 87-91. Mayerhofer, L. E., Conway de Macario, E., Yao, R. and Macario, A. J. (1998). Structure, organization, and expression of genes coding for envelope components in the archaeon Methanosarcina mazei S-6. Arch Microbiol 169: 339-345. Maytin, E. V. (1992). Differential effects of heat shock and UVB light upon stress protein expression in epidermal keratinocytes. J Biol Chem 267: 23189-23196. McRobbie, A. M., Carter, L. G., Kerou, M., Liu, H., McMahon, S. A., Johnson, K. A., Oke, M., Naismith, J. H. and White, M. F. (2009). Structural and functional characterisation of a conserved archaeal RadA paralog with antirecombinase activity. J Mol Biol 389: 661-673.
D. Burg UNSW 311
Methe, B. A., Nelson, K. E., Deming, J. W., Momen, B., Melamud, E., Zhang, X., Moult, J., Madupu, R., Nelson, W. C., Dodson, R. J., Brinkac, L. M., Daugherty, S. C., Durkin, A. S., DeBoy, R. T., Kolonay, J. F., Sullivan, S. A., Zhou, L., Davidsen, T. M., Wu, M., Huston, A. L., Lewis, M., Weaver, B., Weidman, J. F., Khouri, H., Utterback, T. R., Feldblyum, T. V. and Fraser, C. M. (2005a). The psychrophilic lifestyle as revealed by the genome sequence of Colwellia psychrerythraea 34H through genomic and proteomic analyses. Proc Natl Acad Sci U S A 102: 10913-10918. Metje, M. and Frenzel, P. (2007). Methanogenesis and methanogenic pathways in a peat from subarctic permafrost. Environ Microbiol 9: 954-964. Mihoub, F., Mistou, M. Y., Guillot, A., Leveau, J. Y., Boubetra, A. and Billaux, F. (2003a). Cold adaptation of Escherichia coli: Microbiological and proteomic approaches. Int J Food Microbiol 89: 171-184. Mitova, M., Tutino, M. L., Infusini, G., Marino, G. and De Rosa, S. (2005). Exocellular peptides from antarctic psychrophile Pseudoalteromonas haloplanktis. Mar Biotechnol (NY) 7: 523-531. Mitra, S. K., Gantt, J. A., Ruby, J. F., Clouse, S. D. and Goshe, M. B. (2007). Membrane proteomic analysis of Arabidopsis thalania using alternative solubilisation techniques. J Proteome Res 6: 1933-1950. Mitra, S. K. and Goshe, M. B. (2009). Cysteinyl-tagging of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry. Methods Mol Biol 528: 311-326. Miyake, K., Utsugisawa, T., Flygare, J., Kiefer, T., Hamaguchi, I., Richter, J. and Karlsson, S. (2008). Ribosomal protein S19 deficiency leads to reduced proliferation and increased apoptosis but does not affect terminal erythroid differentiation in a cell line model of diamond-blackfan anemia. Stem Cells 26: 323-329. Mogk, A., Schlieker, C., Friedrich, K. L., Schonfeld, H. J., Vierling, E. and Bukau, B. (2003). Refolding of substrates bound to small HSPs relies on a disaggregation reaction mediated most efficiently by ClpB/DnaK. J Biol Chem 278: 31033-31042. Moissl, C., Rudolph, C. and Huber, R. (2002). Natural communities of novel archaea and bacteria with a string-of-pearls-like morphology: Molecular analysis of the bacterial partners. Appl Environ Microbiol 68: 933-937. Moomaw, A. S. and Maguire, M. E. (2008). The unique nature of Mg2+ channels. Physiology 23: 275-285. Morgan-Kiss, R. M., Ivanov, A. G., Modla, S., Czymmek, K., Huner, N. P., Priscu, J. C., Lisle, J. T. and Hanson, T. E. (2008). Identity and physiology of a new psychrophilic eukaryotic green alga, Chlorella sp., strain BI, isolated from a transitory pond near Bratina Island, Antarctica. Extremophiles 12: 701-711. Morozova, D., Mohlmann, D. and Wagner, D. (2007). Survival of methanogenic archaea from Siberian permafrost under simulated martian thermal conditions. Orig Life Evol Biosph 37: 189-200. Morozova, D. and Wagner, D. (2007). Stress response of methanogenic archaea from siberian permafrost compared with methanogens from nonpermafrost habitats. FEMS Microbiol Ecol 61: 16-25. Mulcahy, H., Charron-Mazenod, L. and Lewenza, S. (2008). Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS Pathog 4: 21. Muller, V., Spanheimer, R. and Santos, H. (2005). Stress response by solute accumulation in archaea. Curr Opin Microbiol 8: 729-736. Murray, A. E. and Grzymski, J. J. (2007). Diversity and genomics of antarctic marine micro- organisms. Philos Trans R Soc Lond B Biol Sci 362: 2259-2271. Murray, A. E., Preston, C. M., Massana, R., Taylor, L. T., Blakis, A., Wu, K. and DeLong, E. F. (1998). Seasonal and spatial variability of bacterial and archaeal assemblages in
312 D. Burg UNSW
the coastal waters near Anvers Island, Antarctica. Appl Environ Microbiol 64: 2585- 2595. Nachin, L., Nannmark, U. and Nystrom, T. (2005). Differential roles of the universal stress proteins of Escherichia coli in oxidative stress resistance, adhesion, and motility. J Bacteriol 187: 6265-6272. Nagao, M., Matsui, K. and Uemura, M. (2008). Klebsormidium flaccidum, a charophycean green alga, exhibits cold acclimation that is closely associated with compatible solute accumulation and ultrastructural changes. Plant Cell Environ 31: 872-885. Nakazawa, F., Kannemeier, C., Shibamiya, A., Song, Y., Tzima, E., Schubert, U., Koyama, T., Niepmann, M., Trusheim, H., Engelmann, B. and Preissner, K. T. (2005). Extracellular RNA is a natural cofactor for the (auto-)activation of factor vii-activating protease (FsaP). Biochem J 385: 831-838. Nedwell, D. B. (1999). Effect of low temperature on microbial growth: Lowered affinity for substrates limits growth at low temperature. FEMS Microbiol Ecol 30: 101-111. Nelson, D. L. and Cox, M. M. (2000). Lehninger principles of biochemistry. Worth publishers, New york Nemergut, D. R., Costello, E. K., Meyer, A. F., Pescador, M. Y., Weintraub, M. N. and Schmidt, S. K. (2005). Structure and function of alpine and Arctic soil microbial communities. Research in Microbiology 156: 775-784. Nesvizhskii, A. I., Keller, A., Kolker, E. and Aebersold, R. (2003). A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75: 4646-4658. Nevot, M., Deroncele, V., Messner, P., Guinea, J. and Mercade, E. (2006). Characterization of outer membrane vesicles released by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3. Environ Microbiol 8: 1523-1533. Nevot, M., Deroncele, V., Montes, M. J. and Mercade, E. (2008). Effect of incubation temperature on growth parameters of Pseudoalteromonas antarctica NF and its production of extracellular polymeric substances. J Appl Microbiol 105: 255-263. Ng, S. Y., Chaban, B., VanDyke, D. J. and Jarrell, K. F. (2007). Archaeal signal peptidases. Microbiology 153: 305-314. Nichols, C. M., Bowman, J. P. and Guezennec, J. (2005). Effects of incubation temperature on growth and production of exopolysaccharides by an antarctic sea ice bacterium grown in batch culture. Appl Environ Microbiol 71: 3519-3523. Nichols, D. S., Miller, M. R., Davies, N. W., Goodchild, A., Raftery, M. and Cavicchioli, R. (2004). Cold adaptation in the antarctic archaeon Methanococcoides burtonii involves membrane lipid unsaturation. J Bacteriol 186: 8508-8515. Nichols, P. D. and Franzmann, P. D. (1992). Unsaturated diether phospholipids in the Antartic methanogen Methanococcoides burtonii. FEMS Microbiology Letters 98: 205-208. Nishimura, S., Tanaka, T., Fujita, K., Itaya, M., Hiraishi, A. and Kikuchi, Y. (2003). Extracellular DNA and RNA produced by a marine photosynthetic bacterium Rhodovulum sulfidophilum. Nucleic Acids Res Suppl: 279-280. Noon, K. R., Guymon, R., Crain, P. F., McCloskey, J. A., Thomm, M., Lim, J. and Cavicchioli, R. (2003). Influence of temperature on tRNA modification in archaea: Methanococcoides burtonii (optimum growth temperature [Topt], 23˚C) and Stetteria hydrogenophila (Topt, 95˚C). J. Bacteriol. 185: 5483-5490. Novotny, R., Schaffer, C., Strauss, J. and Messner, P. (2004). S-layer glycan-specific loci on the chromosome of Geobacillus stearothermophilus NRS 2004/3a and dTDP-L- rhamnose biosynthesis potential of G. stearothermophilus strains. Microbiology 150: 953-965. Offner, S., Ziese, U., Wanner, G., Typke, D. and Pfeifer, F. (1998). Structural characteristics of halobacterial gas vesicles. Microbiology 144: 1331-1342. Ogino, H. (2008). Organic solvent stable enzymes. Protein adaptation in extremophiles. K. S. Siddiqui and T. Thomas. Nova Biomediacal Books, New York.
D. Burg UNSW 313
Ohtsu, I., Nakanisi, T., Furuta, M., Ando, E. and Nishimura, O. (2005). Direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric identification of proteins on membrane detected by Western blotting and lectin blotting. J Proteome Res 4: 1391-1396. Owttrim, G. W. (2006). RNA helicases and abiotic stress. Nucleic Acids Res 34: 3220-3230. Papanikolau, Y., Tsigos, I., Papadovasilaki, M., Bouriotis, V. and Petratos, K. (2005). Crystallization and preliminary X-ray diffraction studies of an alcohol dehydrogenase from the antarctic psychrophile Moraxella sp. TAE123. Acta Crystallogr Sect F Struct Biol Cryst Commun 61: 246-8. Park, Z. Y. and Russell, D. H. (2000). Thermal denaturation: A useful technique in peptide mass mapping. Anal Chem 72: 2667-2670. Paul, L., Ferguson, D. J., Jr. and Krzycki, J. A. (2000). The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons. J Bacteriol 182: 2520- 2529. Peabody, C. R., Chung, Y. J., Yen, M. R., Vidal-Ingigliardi, D., Pugsley, A. P. and Saier, M. H. J. (2003). Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149: 3051-3072. Pennella, M. A. and Giedroc, D. P. (2005). Structural determinants of metal selectivity in prokaryotic metal-responsive transcriptional regulators. Biometals 18: 413-428. Perreault, N. N., Andersen, D. T., Pollard, W. H., Greer, C. W. and Whyte, L. G. (2007). Characterization of the prokaryotic diversity in cold saline perennial springs of the Canadian high arctic. Appl Environ Microbiol 73: 1532-1543. Petrescu, I., Lamotte-Brasseur, J., Chessa, J. P., Ntarima, P., Claeyssens, M., Devreese, B., Marino, G. and Gerday, C. (2000). Xylanase from the psychrophilic yeast Cryptococcus adeliae. Extremophiles 4: 137-144. Pfluger, K. and Muller, V. (2004). Transport of compatible solutes in extremophiles. J Bioenerg Biomem 36: 17-24. Phadtare, S. (2004). Recent developments in bacterial cold-shock response. Curr Issues Mol Biol 6: 125-136. Pieper, R., Huang, S. T., Clark, D. J., Robinson, J. M., Parmar, P. P., Alami, H., Bunai, C. L., Perry, R. D., Fleischmann, R. D. and Peterson, S. N. (2008). Characterizing the dynamic nature of the Yersinia pestis periplasmic proteome in response to nutrient exhaustion and temperature change. Proteomics 8: 1442-1458. Pohlschroder, M., Gimenez, M. I. and Jarrell, K. F. (2005). Protein transport in archaea: Sec and twin arginine translocation pathways. Curr Op Microbiol 8: 713-719. Poli, A., Moriello, V. S., Esposito, E., Lama, L., Gambacorta, A. and Nicolaus, B. (2004). Exopolysaccharide production by a new halomonas strain CRSS isolated from a saline lake in Cape Russell in Antarctica growing on complex and defined media. Biotechnol Lett 26: 1635-1638. Polissi, A., De Laurentis, W., Zangrossi, S., Briani, F., Longhi, V., Pesole, G. and Deho, G. (2003). Changes in Escherichia coli transcriptome during acclimatization at low temperature. Research in Microbiology 154: 573-580. Ponting, C. P., Aravind, L., Schultz, J., Bork, P. and Koonin, E. V. (1999). Eukaryotic signalling domain homologues in archaea and bacteria. Ancient ancestry and horizontal gene transfer. J Mol Biol 289: 729-745. Post, W. M., Emanuel, W. R., Zinke, P. J. and Strangenberger, A. G. (1982). Soil carbon pools and world life zones. Nature 298: 156-159. Postle, K. and Kadner, R. J. (2003). Touch and go: Tying TonB to transport. Mol Microbiol 49: 869-882. Priester, J. H., Horst, A. M., Van de Werfhorst, L. C., Saleta, J. L., Mertes, L. A. and Holden, P. A. (2007). Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy. J Microbiol Methods 68: 577-587.
314 D. Burg UNSW
Pum, D., Messner, P. and Sleytr, U. B. (1991). Role of the S-layer in morphogenesis and cell division of the archaebacterium Methanocorpusculum sinense. J Bacteriol 173: 6865-6873. Purdy, K. J., Nedwell, D. B. and Embley, T. M. (2003). Analysis of the sulfate-reducing bacterial and methanogenic archaeal populations in contrasting Antarctic sediments. Appl Environ Microbiol 69: 3181-3191. Qin, G., Zhu, L., Chen, X., Wang, P. G. and Zhang, Y. (2007). Structural characterization and ecological roles of a novel exopolysaccharide from the deep-sea psychrotolerant bacterium Pseudoalteromonas sp. Sm9913. Microbiology 153: 1566-1572. Qiu, Y., Kathariou, S. and Lubman, D. M. (2006). Proteomic analysis of cold adaptation in a siberian permafrost bacterium--Exiguobacterium sibiricum 255-15 by two- dimensional liquid separation coupled with mass spectrometry. Proteomics 6: 5221- 33. Rambaut, A. (2008). Figtree v1.1.2. Ramos, Y., Garcia, Y., Llopiz, A. and Castellanos-Serra, L. (2008). Selectivity of bacterial proteome fractionation based on differential solubility: A mass spectrometry evaluation. Anal Biochem 377: 134-140. Rankin, L. M., Gibson, J. A. E. and Franzmann, P. D. (1996). The chemical stratification and microbial communities of Ace Lake, Antarctica: A review of the characteristics of a marine-derived meromictic lake. Polarforschung 66: 33-52. Rasche, M. E. and White, R. H. (1998). Mechanism for the enzymatic formation of 4-(beta- d-ribofuranosyl)aminobenzene 5'-phosphate during the biosynthesis of methanopterin. Biochemistry 37: 11343-51. Raux, E., Schubert, H. L. and Warren, M. J. (2000). Biosynthesis of cobalamin (vitamin B12): A bacterial conundrum. Cell Mol Life Sci 57: 1880-1893. Raven, J. A., Johnston, A. M., Kubler, J. E., Korb, R., McInroy, S. G., Handley, L. L., Scrimgeour, C. M., Walker, D. I., Beardall, J., Clayton, M. N., Vanderklift, M., Fredriksen, S. and Dunton, K. H. (2002). Seaweeds in cold seas: Evolution and carbon acquisition. Ann Bot (Lond) 90: 525-536. Rawlings, N. D. and Barret, A. J. (1995). Evolutionary families of metalloproteases. Methods Enzymol 248: 183-228. Reese, S. and Guggenheim, B. (2007). A novel TEM contrasting technique for extracellular polysaccharides in in vitro biofilms. Microsc Res Tech 70: 816-822. Reeve, J. N. (2003). Archaeal chromatin and transcription. Mol Microbiol 48: 587-598. Reid, I. N., Sparks, W. B., Lubow, S., McGrath, M., Livio, M., Valenti, J., Sowers, K. R., Shukla, H. D., MacAuley, S., Miller, T., Suvanasuthi, R., Belas, R., Colman, A., Robb, F. T., DasSarma, P., uuml, ller, J. A., Coker, J. A., Cavicchioli, R., Chen, F. and DasSarma, S. (2006). Terrestrial models for extraterrestrial life: Methanogens and halophiles at martian temperatures. Int J Astrobiol 5: 89-97. Reizer, J., Reizer, A. and Saier, M. H., Jr. (1994). A functional superfamily of sodium/solute symporters. Biochim Biophys Acta 1197: 133-166. Relina, L. I. and Gulevsky, A. K. (2003). A possible role of molecular chaperones in cold adaptation. Cryo Letters 24: 203-12. Reuter, C. J., Kaczowka, S. J. and Maupin-Furlow, J. A. (2004). Differential regulation of the pana and PanB proteasome-activating nucleotidase and 20S proteasomal proteins of the haloarchaeon Haloferax volcanii. J Bacteriol 186: 7763-7772. Righetti, P. G., Castagna, A., Antonioli, P. and Boschetti, E. (2005). Prefractionation techniques in proteome analysis: The mining tools of the third millennium. Electrophoresis 26: 297-319. Riley, M., Staley, J. T., Danchin, A., Wang, T. Z., Brettin, T. S., Hauser, L. J., Land, M. L. and Thompson, L. S. (2008). Genomics of an extreme psychrophile, Psychromonas ingrahamii. BMC Genomics 9: 210.
D. Burg UNSW 315
Ring, G. and Eichler, J. (2004a). Extreme secretion: Protein translocation across the archaeal plasma membrane. J Bioenerg Biomembr 36: 35-45. Ring, G. and Eichler, J. (2004b). Membrane binding of ribosomes occurs at SecYE-based sites in the archaea Haloferax volcanii. J Mol Biol 336: 997-1010. Roberts, J. K. and Hughes, M. J. (1998). The use of heptafluorobutyric acid as a volatile ion- pair reagent in the high-performance liquid chromatographic isolation of sb- 223070. Journal of Chromatography A 828: 297-302. Robertson, C. E. (2007). Electron microscopy of archaea. Methods in cell biology. J. R. McIntosh, Academic Press. Volume 79: 169-191. Robertson, C. E., Harris, J. K., Spear, J. R. and Pace, N. R. (2005). Phylogenetic diversity and ecology of environmental archaea. Curr Op Microbiol 8: 638-642. Rodrigues, D. F. and Tiedge, J. M. (2008). Coping with our cold planet. Appl Environ Microbiol 74: 1677-1686. Rodriguez-Brito, B., Rohwer, F. and Edwards, R. A. (2006). An application of statistics to comparative metagenomics. BMC Bioinformatics 7: 162. Romisch, K., Collie, N., Soto, N., Logue, J., Lindsay, M., Sheper, W. and Cheng, C.-H. C. (2003). Protein translocation accros the endoplasmic reticulum membrane in cold- adapted organisms. J Cell Science 116: 2875-2883. Ross, P. L., Huang, Y. N., Marchese, J. N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A. and Pappin, D. J. (2004). Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3: 1154-1169. Rotanova, T. V., Botos, I., Melnikov, E. E., Rasulova, F., Gustchina, A., Maurizi, M. R. and Wlodawer, A. (2006). Slicing a protease: Structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Sci 15: 1815-1828. Rother, M., Mathes, I., Lottspeich, F. and Bock, A. (2003). Inactivation of the SelB gene in Methanococcus maripaludis: Effect on synthesis of selenoproteins and their sulfur- containing homologs. J Bacteriol 185: 107-114. Rudolph, C., Wanner, G. and Huber, R. (2001). Natural communities of novel archaea and bacteria growing in cold sulfurous springs with a string-of-pearls-like morphology. Appl Environ Microbiol 67: 2336-2344. Russell, N. J. (2007). Psychrophiles: Membrane adaptations. Physiology and biochemistry of extremophiles. C. Gerday and N. Glansdorff. Washington D. C., ASM press, Washington D. C. Russell, N. J., Harrisson, P., Johnston, I. A., Jaenicke, R., Zuber, M., Franks, F. and Wynn- Williams, D. (1990). Cold adaptation of microorganisms [and discussion]. Phil Trans Royal Soc London. Series B, Biological Sciences 326: 595-611. Russell, W. K., Park, Z. Y. and Russell, D. H. (2001). Proteolysis in mixed organic-aqueous solvent systems: Applications for peptide mass mapping using mass spectrometry. Anal Chem 73: 2682-2685. Sahara, T., Goda, T. and Ohgiya, S. (2002). Comprehensive expression analysis of time- dependent genetic responses in yeast cells to low temperature. J Biol Chem 277: 50015-50021. Saitou, N. and Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evo 4: 406-425 Sakoh, M., Ito, K. and Akiyama, Y. (2005). Proteolytic activity of HtpX, a membrane-bound and stress-controlled protease from Escherichia coli. J Biol Chem 280: 33305- 33310. Sandve, S. R., Rudi, H., Asp, T. and Rognli, O. A. (2008). Tracking the evolution of a cold stress associated gene family in cold tolerant grasses. BMC Evol Biol 8: 245.
316 D. Burg UNSW
Sano, K., Taguchi, A., Furumoto, H., Uda, T. and Itoh, T. (1999). Cloning, sequencing, and characterization of ribosomal protein and RNA polymerase genes from the region analogous to the [alpha]-operon of Escherichia coli in halophilic archaea, Halobacterium halobium. Biochemical and Biophysical Research Communications 264: 24-28. Sardessai, Y. and Bhosle, S. (2002). Tolerance of bacteria to organic solvents. Res Microbiol 153: 263-268. Saunders, N. F., Ng, C., Raftery, M., Guilhaus, M., Goodchild, A. and Cavicchioli, R. (2006). Proteomic and computational analysis of secreted proteins with type I signal peptides from the Antarctic archaeon Methanococcoides burtonii. J Proteome Res 5: 2457-2464. Saunders, N. F., Thomas, T., Curmi, P. M., Mattick, J. S., Kuczek, E., Slade, R., Davis, J., Franzmann, P. D., Boone, D., Rusterholtz, K., Feldman, R., Gates, C., Bench, S., Sowers, K., Kadner, K., Aerts, A., Dehal, P., Detter, C., Glavina, T., Lucas, S., Richardson, P., Larimer, F., Hauser, L., Land, M. and Cavicchioli, R. (2003). Mechanisms of thermal adaptation revealed from the genomes of the Antarctic archaea Methanogenium frigidum and Methanococcoides burtonii. Genome Res 13: 1580-1588. Schelert, J., Drozda, M., Dixit, V., Dillman, A. and Blum, P. (2006). Regulation of mercury resistance in the crenarchaeote Sulfolobus solfataricus. J Bacteriol 188: 7141-7150. Schiefner, A., Holtmann, G., Diederichs, K., Welte, W. and Bremer, E. (2004). Structural basis for the binding of compatible solutes by ProX from the hyperthermophilic archaeon Archaeoglobus fulgidus. J Biol Chem 279: 48270-48281. Schink, B. and Zeikus, J. G. (1980). Microbial methanol formation: A major end product of pectin metabolism. Current Microbiology 4: 387-389. Schleper, C., Jurgens, G. and Jonuscheit, M. (2005). Genomic studies of uncultivated archaea. Nat Rev Microbiol 3: 479-488. Schleper, C., Swanson, R. V., Mathur, E. J. and DeLong, E. F. (1997). Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum. J Bacteriol 179: 7803-11. Schlosser, A., Hamann, A., Bossemeyer, D., Schneider, E. and Bakker, E. P. (1993). NAD+ binding to the escherichia coli K(+)-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport. Mol Microbiol 9: 533-543. Schmid-Schonbein, G. W., Shih, Y. Y. and Chien, S. (1980). Morphometry of human leukocytes. Blood 56: 866-875. Schmidt, S. K., Wilson, K. L., Meyer, A. F., Gebauer, M. M. and King, A. J. (2008). Phylogeny and ecophysiology of opportunistic "Snow molds" from a subalpine forest ecosystem. Microb Ecol. 56: 681-687 Schoneich, C. (2008). Mechanisms of protein damage induced by cysteine thiyl radical formation. Chem Res Toxicol 21: 1175-1179. Schoneich, C. and Asmus, K. D. (1990). Reaction of thiyl radicals with alcohols, ethers and polyunsaturated fatty acids: A possible role of thiyl free radicals in thiol mutagenesis? Radiat Environ Biophys 29: 263-271. Schwarz, R. and Dayhoff, M. (1979). Matrices for detecting distant relationships. Atlas of protein sequences. National Biomedical Research Foundation: 353-358. Scott, C. C. and Finnerty, W. R. (1976). A comparative analysis of the ultrastructure of hydrocarbon-oxidizing micro-organisms. J Gen Microbiol 94: 342-350. Scott, J. W. and Rasche, M. E. (2002). Purification, overproduction, and partial characterization of beta-RfaP synthase, a key enzyme in the methanopterin biosynthesis pathway. J Bacteriol 184: 4442-4448.
D. Burg UNSW 317
Seo, J. B., Kim, H. S., Jung, G. Y., Nam, M. H., Chung, J. H., Kim, J. Y., Yoo, J. S., Kim, C. W. and Kwon, O. (2004). Psychrophilicity of Bacillus psychrosaccharolyticus: A proteomic study. Proteomics 4: 3654-3659. Shams-Eldin, H., Chaban, B., Niehus, S., Schwarz, R. T. and Jarrell, K. F. (2008). Identification of the archaeal Alg7 gene homolog (encoding n-acetylglucosamine-1- phosphate transferase) of the N-linked glycosylation system by cross-domain complementation in Saccharomyces cerevisiae. J Bacteriol 190: 2217-2220. Shih, C. J. and Lai, M. C. (2007). Analysis of the AAA+ chaperone ClpB gene and stress- response expression in the halophilic methanogenic archaeon Methanohalophilus portucalensis. Microbiology 153: 2572-2583. Shimohata, N., Chiba, S., Saikawa, N., Ito, K. and Akiyama, Y. (2002). The Cpx stress response system of escherichia coli senses plasma membrane proteins and controls HtpX, a membrane protease with a cytosolic active site. Genes Cells 7: 653-662. Siddiqui, A. A., Jalah, R. and Sharma, Y. D. (2007). Expression and purification of HtpX- like small heat shock integral membrane protease of an unknown organism related to Methylobacillus flagellatus. J Biochem Biophys Methods 70: 539-546. Siddiqui, K. S. and Cavicchioli, R. (2006). Cold adapted enzymes. Ann Review Biochem 75: 403-433. Siddiqui, K. S., Cavicchioli, R. and Thomas, T. (2002). Thermodynamic activation properties of elongation factor 2 (EF-2) proteins from psychrotolerant and thermophilic archaea. Extremophiles 6: 143-150. Sikkema, J., de Bont, J. A. and Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 59: 201-222. Simankova, M. V., Parshina, S. N., Tourova, T. P., Kolganova, T. V., Zehnder, A. J. and Nozhevnikova, A. N. (2001). Methanosarcina lacustris sp. Nov., a new psychrotolerant methanogenic archaeon from anoxic lake sediments. Syst Appl Microbiol 24: 362-367. Singh, N., Kendall, M. M., Liu, Y. and Boone, D. R. (2005). Isolation and characterization of methylotrophic methanogens from anoxic marine sediments in Skan Bay, Alaska: Description of Methanococcoides alaskense sp. Nov., and emended description of Methanosarcina baltica. Int J Syst Evol Microbiol 55: 2531-8. Skidmore, M. L., Foght, J. M. and Sharp, M. J. (2000). Microbial life beneath a high arctic glacier. Appl Environ Microbiol 66: 3214-20. Smalyukh, I.I., Butler, J., Shrout, J. D., Parsek, M. R. and Wong, G. C. (2008). Elasticity- mediated nematiclike bacterial organization in model extracellular DNA matrix. Phys Rev E Stat Nonlin Soft Matter Phys 78: 23. Smith, C. A. (2006). Structure, function and dynamics in the Mur family of bacterial cell wall ligases. J Mol Biol 362: 640-655. Smith, J. L. and Dixon, M. F. (2003). Is subtyping of intestinal metaplasia in the upper gastrointestinal tract a worthwhile exercise? An evaluation of current mucin histochemical stains. British J Biomed Sc. 60: 180-186 Smith, R. L., Szegedy, M. A., Kucharski, L. M., Walker, C., Wiet, R. M., Redpath, A., Kaczmarek, M. T. and Maguire, M. E. (1998). The cora Mg2+ transport protein of salmonella typhimurium. Mutagenesis of conserved residues in the third membrane domain identifies a Mg2+ pore. J Biol Chem 273: 28663-28669. Snider, J. and Houry, W. A. (2006). MoxR AAA+ atpases: A novel family of molecular chaperones? J Struct Biol 156: 200-209. Sogin, M. L., Morrison, H. G., Huber, J. A., Mark Welch, D., Huse, S. M., Neal, P. R., Arrieta, J. M. and Herndl, G. J. (2006). Microbial diversity in the deep sea and the underexplored "Rare biosphere". Proc Natl Acad Sci U S A 103: 12115-12120. Sonjak, S., Frisvad, J. C. and Gunde-Cimerman, N. (2007). Genetic variation among Penicillium crustosum isolates from arctic and other ecological niches. Microb Ecol 54: 298-305.
318 D. Burg UNSW
Sowers, K., Boone, J. E. and Gunsalus, R. P. (1993). Disaggregation of Methanosarcina spp. and growth as single cells at elevated osmolarity. Applied and Environmental Microbiology 59: 3832-3839. Sowers, K. and Noll, M. (1995). Techniques for anaerobic growth. Archaea: A laboratory manual. F. T. Robb, A. R. Place, K. Sowers et al., Cold Spring Harbour Laboratory Press. Sowers, K. R. and Gunsalus, R. P. (1995). Halotolerance in Methanosarcina spp.: Role of Nε- acetyl-β-lysine, α-glutamate, glycine betaine, and K+ as compatible solutes for osmotic adaptation. Appl Environ Microbiol 61: 4382-4388. Spiriti, J., Bogani, F., van der Vaart, A. and Ghirlanda, G. (2008). Modulation of protein stability by O-glycosylation in a designed Gc-MAF analog. Biophys Chem 134: 157- 67. Stasyk, T. and Huber, L. A. (2004). Zooming in: Fractionation strategies in proteomics. Proteomics 4: 3704-3716. Stein, J. L. and Simon, M. I. (1996). Archaeal ubiquity. Proc Natl Acad Sci U S A 93: 6228-30. Steinberger, R. E. and Holden, P. A. (2005). Extracellular DNA in single- and multiple- species unsaturated biofilms. Appl Environ Microbiol 71: 5404-5410. Steiner, K., Novotny, R., Werz, D. B., Zarschler, K., Seeberger, P. H., Hofinger, A., Kosma, P., Schaffer, C. and Messner, P. (2008). Molecular basis of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus. J Biol Chem 283: 21120-33. Stokes, D. J. (2003). Recent advances in electron imaging, image interpretation and applications: Environmental scanning electron microscopy. Philos Transact A Math Phys Eng Sci 361: 2771-2787. Stoller, G., Rucknagel, K. P., Nierhaus, K. H., Schmid, F. X., Fischer, G. and Rahfeld, J. U. (1995). A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor. EMBO J 14: 4939-48. Suh, W. C., Burkholder, W. F., Lu, C. Z., Zhao, X., Gottesman, M. E. and Gross, C. A. (1998). Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaK. Proc Natl Acad Sci U S A 95: 15223-15228. Suzuki, R., Nagata, K., Yumoto, F., Kawakami, M., Nemoto, N., Furutani, M., Adachi, K., Maruyama, T. and Tanokura, M. (2003). Three-dimensional solution structure of an archaeal FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities. J Mol Biol 328: 1149-1160. Sweetlove, L. J., Heazlewood, J. L., Herald, V., Holtzapffel, R., Day, D. A., Leaver, C. J. and Millar, A. H. (2002). The impact of oxidative stress on Arabidopsis mitochondria. Plant J 32: 891-904. Szabo, Z., Sani, M., Groeneveld, M., Zolghadr, B., Schelert, J., Albers, S. V., Blum, P., Boekema, E. J. and Driessen, A. J. (2007). Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus. J Bacteriol 189: 4305- 4309. Taft (1951). The specificity of the methyl green-pyronin stain for nucleic acids. Exp Cell Res 2: 312-326. Takeuchi, K., Nakamura, K., Fujimoto, M., Kaino, S., Kondoh, S. and Okita, K. (2002). Heat stress-induced loss of eukaryotic initiation factor 5A (eiF-5A) in a human pancreatic cancer cell line, MIA PaCa-2, analyzed by two-dimensional gel electrophoresis. Electrophoresis 23: 662-669. Tamaru, Y., Takani, Y., Yoshida, T. and Sakamoto, T. (2005). Crucial role of extracellular polysaccharides in desiccation and freezing tolerance in the terrestrial cyanobacterium Nostoc commune. Appl Environ Microbiol 71: 7327-7333. Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596-1599.
D. Burg UNSW 319
Tanaka, S., Ikeda, K. and Miyasaka, H. (2001). Enhanced tolerance against salt-stress and freezing-stress of Escherichia coli cells expressing algal bbc1 gene. Curr Microbiol 42: 173-177. Tang, X., Yi, W., Munske, G. R., Adhikari, D. P., Zakharova, N. L. and Bruce, J. E. (2007). Profiling the membrane proteome of Shewanella oneidensis MR-1 with new affinity labelling probes. J Proteome Res 6: 724-734. Tanner, N. K. and Linder, P. (2001). DExD/H box RNA helicases: From generic motors to specific dissociation functions. Mol Cell 8: 251-262. Tarpgaard, I. H., Boetius, A. and Finster, K. (2006). Desulfobacter psychrotolerans sp. Nov., a new psychrotolerant sulfate-reducing bacterium and descriptions of its physiological response to temperature changes. Antonie Van Leeuwenhoek 89: 109- 124. Tavernarakis, N., Driscoll, M. and Kyrpides, N. C. (1999). The SPFH domain: Implicated in regulating targeted protein turnover in stomatins and other membrane-associated proteins. Trends Biochem Sci 24: 425-427. Taylor, B. L. and Zhulin, I. B. (1999). PAS domains: Internal sensors of oxygen, redox potential, and light. Microbiol Mol Biol Rev 63: 479-506. Thomas, T. and Cavicchioli, R. (1998). Archaeal cold-adapted proteins: Structural and evolutionary analysis of the elongation factor 2 proteins from psychrophilic, mesophilic and thermophilic methanogens. FEBS Letters 439: 281-286. Thomas, T. and Cavicchioli, R. (2000). Effect of temperature on stability and activity of elongation factor 2 proteins from antarctic and thermophilic methanogens. J. Bacteriol. 182: 1328-1332. Thomas, T. and Cavicchioli, R. (2002). Cold adaptation of archaeal elongation factor 2 (EF- 2) proteins. Curr Protein Pept Sci 3: 223-230. Thomas, T., Kumar, N. and Cavicchioli, R. (2001). Effects of ribosomes and intracellular solutes on activities and stabilities of elongation factor 2 proteins from psychrotolerant and thermophilic methanogens. J. Bacteriol. 183: 1974-1982. Thompson, J. D., Gibson, T. J. and Higgins, D. G. (2002). Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics Chapter 2: Unit 2 3. Tian, J., Sinskey, A. J. and Stubbe, J. (2005). Kinetic studies of polyhydroxybutyrate granule formation in Wautersia eutropha HL6 by transmission electron microscopy. J Bacteriol 187: 3814-3824. Tishchenko, S. V., Vassilieva, J. M., Platonova, O. B., Serganov, A. A., Fomenkova, N. P., Mudrik, E. S., Piendl, W., Ehresmann, C., Ehresmann, B. and Garber, M. B. (2001). Isolation, crystallization, and investigation of ribosomal protein S8 complexed with specific fragments of rRNA of bacterial or archaeal origin. Biochemistry (Mosc) 66: 948-953. Tsigos, I., Velonia, K., Smonou, I. and Bouriotis, V. (1998). Purification and characterization of an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123. Eur J Biochem 254: 356-362. Tsuruta, H., Hayashi, R., Ohkawa, H., Ohkatsu, N., Morimoto, M., Nishimoto, K., Santou, S. and Aizono, Y. (2007). Characterisitics and gene cloning of phospholipase D of the psychrophile, Shewanella sp. Biosci Biotechnol Biochem 71: 2534-2542. Tung, H. C., Bramall, N. E. and Price, P. B. (2005). Microbial origin of excess methane in glacial ice and implications for life on mars. Proc Natl Acad Sci U S A 102: 18292- 18296. Ugulava, N. B., Frederick, K. K. and Jarrett, J. T. (2003). Control of adenosylmethionine- dependent radical generation in biotin synthase: A kinetic and thermodynamic analysis of substrate binding to active and inactive forms of BioB. Biochemistry 42: 2708-2719. Ulrich, L. E., Koonin, E. V. and Zhulin, I. B. (2005). One-component systems dominate signal transduction in prokaryotes. Trends Microbiol 13: 52-56.
320 D. Burg UNSW
van der Maarel, M. J., Artz, R. R., Haanstra, R. and Forney, L. J. (1998). Association of marine archaea with the digestive tracts of two marine fish species. Appl Environ Microbiol 64: 2894-2898. Vanhecke, D., Studer, D. and Ochs, M. (2008). Stereology meets electron tomography: Towards quantitative 3D electron microscopy. J Struct Biol 161: 314-321. Vetter, Y. A., Deming, J. W., Jumars, P. A. and Krieger-Brockett, B. B. (1998). A predictive model of bacterial foraging by means of freely released extracellular enzymes. Microb Ecol 36: 75-92. Vickerman, M. M., Mather, N. M., Minick, P. E. and Edwards, C. A. (2002). Initial characterization of the Streptococcus gordonii HtpX gene. Oral Microbiol Immunol 17: 22-31. Vilain, S., Pretorius, J. M., Theron, J. and Brozel, V. S. (2009). DNA as an adhesin: Bacillus cereus requires extracellular DNA to form biofilms. Appl Environ Microbiol 75: 2861-2868. Vlassov, V. V., Laktionov, P. P. and Rykova, E. Y. (2007). Extracellular nucleic acids. Bioessays 29: 654-667. Voisin, S., Houliston, R. S., Kelly, J., Brisson, J. R., Watson, D., Bardy, S. L., Jarrell, K. F. and Logan, S. M. (2005). Identification and characterization of the unique N-linked glycan common to the flagellins and S-layer glycoprotein of Methanococcus voltae. J Biol Chem 280: 16586-16593. Vorholt, J. A., Vaupel, M. and Thauer, R. K. (1996). A polyferredoxin with eight [4Fe-4S] clusters as a subunit of molybdenum formylmethanofuran dehydrogenase from Methanosarcina barkeri. Eur J Biochem 236: 309-317. Vorholt, J. A., Vaupel, M. and Thauer, R. K. (1997). A selenium-dependent and a selenium- independent formylmethanofuran dehydrogenase and their transcriptional regulation in the hyperthermophilic Methanopyrus kandleri. Mol Microbiol 23: 1033-1042. Vu, B., Chen, M., Crawford, R. J. and Ivanova, E. P. (2009). Bacterial extracellular polysaccharides involved in biofilm formation. Molecules 14: 2535-54. Wagner, D., Lipski, A., Embacher, A. and Gattinger, A. (2005). Methane fluxes in permafrost habitats of the Lena Delta: Effects of microbial community structure and organic matter quality. Environ Microbiol 7: 1582-1592. Walker, J. M. (2002). SDS polyacrilamide gel electrophoresis of proteins. The protein protocols handbook. J. M. Walker. Humana Press Inc. Totowa, NJ, USA. Walsby, A. E. (1972). Structure and function of gas vacuoles. Bacteriol Rev 36: 1-32. Walsh, P., Bursac, D., Law, Y. C., Cyr, D. and Lithgow, T. (2004). The J-protein family: Modulating protein assembly, disassembly and translocation. EMBO Rep 5: 567- 571. Wang, H., Zhao, L. N., Li, K. Z., Ling, R., Li, X. J. and Wang, L. (2006). Overexpression of ribosomal proteinL15 is associated with cell proliferation in gastric cancer. BMC Cancer 6: 91. Wang, P. and Heitman, J. (2005). The cyclophilins. Genome Biol 6: 27. Wang, S. C. and Frey, P. A. (2007). S-adenosylmethionine as an oxidant: The radical SAM superfamily. Trends Biochem Sci 32: 101-110. Wanner, L. A. and Junttila, O. (1999). Cold-induced freezing tolerance in Arabidopsis. Plant Physiol 120: 391-400. Washburn, M. P., Wolters, D. and Yates, J. R., 3rd (2001). Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19: 242-247. Washburn, M. P. and Yates, J. R. (2000). Analysis of the microbial proteome. Curr Op Microbioly 3: 292-297.
D. Burg UNSW 321
Watanabe, M., Sasaki, K., Nakashimada, Y., Kakizono, T., Noparatnaraporn, N. and Nishio, N. (1998). Growth and flocculation of a marine photosynthetic bacterium Rhodovulum sp. Appl Microbiol Biotechnol 50: 682-691. Watanabe, M., Suzuki, Y., Sasaki, K., Nakashimada, Y. and Nishio, N. (1999). Flocculating property of extracellular polymeric substance derived from a marine photosynthetic bacterium, Rhodovulum sp. J Biosci Bioeng 87: 625-629. Watanabe, S., Kita, A. and Miki, K. (2005). Crystal structure of atypical cytoplasmic ABC- ATP ase SufC from thermus thermophilus HB8. J Mol Biol 353: 1043-1054. Weibel, E. R. and Gomez, D. M. (1962). A principal of counting tissue structures on random sections. J. Appl Physiol 17: 343 - 348. Weinberg, M. V., Schut, G. J., Brehm, S., Datta, S. and Adams, M. W. (2005). Cold shock of a hyperthermophilic archaeon: Pyrococcus furiosus exhibits multiple responses to a suboptimal growth temperature with a key role for membrane-bound glycoproteins. J Bacteriol 187: 336-48. Welander, P. V. and Metcalf, W. W. (2005). Loss of the Mtr operon in methanosarcina blocks growth on methanol, but not methanogenesis, and reveals an unknown methanogenic pathway. Proc Natl Acad Sci U S A 102: 10664-10669. Welander, P. V. and Metcalf, W. W. (2008). Mutagenesis of the C1 oxidation pathway in Methanosarcina barkeri: New insights into the Mtr/Mer bypass pathway. J Bacteriol 190: 1928-36. Wells, L. E. and Deming, J. W. (2003). Abundance of bacteria, the cytophaga- flavobacterium cluster and archaea in cold oligotrophic waters and nepheloid alyers of the Northwest Passage, Canadian Archpelago. Aqua microbial ecol 31: 19- 31. Werner, F. (2007). Structure and function of archaeal RNA polymerases. Mol Microbiol 65: 1395-1404. White, R. H. (2001). Biosynthesis of the methanogenic cofactors. Vitam Horm 61: 299-337. Whitechurch, C. B., Tolker-Neilson, T., Ragas, P. C. and Mattick, J. S. (2002). Extracellular DNA required for bacterial biofilm formation. Science 295: 1487. Whitehead, T. A., Boonyaratanakornkit, B. B., Hollrigl, V. and Clark, D. S. (2007). A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii. Protein Sci 16: 626-634. Whitelegge, J. P., Gundersen, C. B. and Faull, K. F. (1998). Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins. Protein Sci 7: 1423-1430. Wiedemann, N., Frazier, A. E. and Pfanner, N. (2004). The protein import machinery of mitochondria. J Biol Chem 279: 14473-14476. Wild, J., Altman, E., Yura, T. and Gross, C. A. (1992). DnaK and DnaK heat shock proteins participate in protein export in Escherichia coli. Genes Dev 6: 1165-1172. Williams, T. J., Burg, D. W., Ertan, H., Raftery, M. J., Guilhaus, M. and Cavicchioli, R. (Submitted-a). A global proteomic analysis of the insoluble, soluble and supernatant fractions of the psychrophilic archaeon Methanococcoides burtonii part II: The effect of different methylated growth substrates Williams, T. J., Burg, D. W., Raftery, M. J., Guilhaus, M., Pilak, O. and Cavicchioli, R. (submitted-b). A global proteomic analysis of the insoluble, soluble and supernatant fractions of the psychrophilic archaeon Methanococcoides burtonii part I: The effect of growth temperature. Wilting, R., Schorling, S., Persson, B. C. and Bock, A. (1997). Selenoprotein synthesis in archaea: Identification of an mRNA element of Methanococcus jannaschii probably directing selenocysteine insertion. J Mol Biol 266: 637-41. Woese, C. R., Kandler, O. and Wheelis, M. L. (1990). Towards a natural system of organisms: Proposal for the domains archaea, bacteria, and eucarya. Proc Natl Acad Sci U S A 87: 4576-4579.
322 D. Burg UNSW
Woese, C. R. and Olsen, G. J. (1986). Archaebacterial phylogeny: Perspectives on the urkingdoms. Syst Appl Microbiol 7: 161-177. Wrede, C., Heller, C., Reitner, J. and Hoppert, M. (2008). Correlative light/electron microscopy for the investigation of microbial mats from black sea cold seeps. J Microb Methods 73: 85-91. Wu, C. C., MacCoss, M. J., Howell, K. E. and Yates, J. R., 3rd (2003). A method for the comprehensive proteomic analysis of membrane proteins. Nat Biotechnol 21: 532- 538. Wu, C. C. and Yates, J. R., 3rd (2003). The application of mass spectrometry to membrane proteomics. Nat Biotechnol 21: 262-267. Wu, S. P., Mansy, S. S. and Cowan, J. A. (2005). Iron-sulfur cluster biosynthesis. Molecular chaperone DnaK promotes IscU-bound [2fe-2s] cluster stability and inhibits cluster transfer activity. Biochemistry 44: 4284-4293. Xu, C. and Rosen, B. P. (1997). Dimerization is essential for DNA binding and repression by the ArsR metalloregulatory protein of Escherichia coli. J Biol Chem 272: 15734- 15738. Yamada, T., Kuroda, K., Jitsuyama, Y., Takezawa, D., Arakawa, K. and Fujikawa, S. (2002). Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing. Planta 215: 770-778. Yamaguchi, T., Yamazaki, S., Uemura, S., Tseng, I.-C., Ohashi, A. and Harada, H. (2001). Microbial-ecological significance of sulfide precipitation within anaerobic granular sludge revealed by micro-electrodes study. Water Research 35: 3411-3417. Yang, D., Kusser, I., Kopke, A. K., Koop, B. F. and Matheson, A. T. (1999). The structure and evolution of the ribosomal proteins encoded in the Spc operon of the archaeon (crenarchaeota) Sulfolobus acidocaldarius. Mol Phylogenet Evol 12: 177-185. Yang, H., Henning, D. and Valdez, B. C. (2005). Functional interaction between RNA helicase II/Guα and ribosomal protein L4. FEBS J 272: 3788-3802. Yang, L., Zhou, D., Liu, X., Han, H., Zhan, L., Guo, Z., Zhang, L., Qin, C., Wong, H. C. and Yang, R. (2009). Cold-induced gene expression profiles of Vibrio parahaemolyticus: A time-course analysis. FEMS Microbiol Lett 291: 50-8. Yang, Z. J., Zhou, J. P., Li, G. R., Zhang, Y. and Ren, Z. L. (2007). Cloning and expression of Gymnadenia conopsea GcSec61beta gene encoding endosplasmic reticulum membrane translocation channel protein. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 33: 354-360. Yeats, C., Rawlings, N. D. and Bateman, A. (2004). The PepSY domain: A regulator of peptidase activity in the microbial environment? Trends Biochem Sci 29: 169-172. Yeh, L. C. and Lee, J. C. (1998). Yeast ribosomal proteins L4, L17, L20, and L25 exhibit different binding characteristics for the yeast 35S precursor rRNA. Biochim Biophys Acta 1443: 139-148. Yen, M. R., Harley, K. T., Tseng, Y. H. and Saier, M. H., Jr. (2001). Phylogenetic and structural analyses of the Oxa1 family of protein translocases. FEMS Microbiol Lett 204: 223-231. Yokoyama, H., Fujii, S. and Matsui, I. (2008). Crystal structure of a core domain of stomatin from Pyrococcus horikoshii illustrates a novel trimeric and coiled-coil fold. J Mol Biol 376: 868-878. Yoshida, T., Kawaguchi, R., Taguchi, H., Yoshida, M., Yasunaga, T., Wakabayashi, T., Yohda, M. and Maruyama, T. (2002). Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin. J Mol Biol 315: 73-85. Yu, E. and Owttrim, G. W. (2000). Characterization of the cold stress-induced cyanobacterial dead-box protein CrhC as an RNA helicase. Nucleic Acids Res 28: 3926-3934.
D. Burg UNSW 323
Yu, L. R., Chan, K. C., Tahara, H., Lucas, D. A., Chatterjee, K., Issaq, H. J. and Veenstra, T. D. (2007). Quantitative proteomic analysis of human breast epithelial cells with differential telomere length. Biochem Biophys Res Commun 356: 942-7. Yurist-Doutsch, S., Chaban, B., VanDyke, D. J., Jarrell, K. F. and Eichler, J. (2008). Sweet to the extreme: Protein glycosylation in archaea. Mol Microbiol 68: 1079-1084. Zhang, C. K., Lang, P., Dane, F., Ebel, R. C., Singh, N. K., Locy, R. D. and Dozier, W. A. (2005). Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata). Plant Cell Rep 23: 764-769. Zhang, N., Chen, R., Young, N., Wishart, D., Winter, P., Weiner, J. H. and Li, L. (2007). Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2D LC-MS/MS. Proteomics 7: 484-493. Zhang, T., Barry, R. G., Knowles, K., Hegnibottom, J. A. and Brown, J. (1999). Statistics and characteristics of permafrost and ground-ice distribution in the Northern Hemisphere. Polar Geography 2: 132-154. Zheng, J., Wei, C., Leng, W., Dong, J., Li, R., Li, W., Wang, J., Zhang, Z. and Jin, Q. (2007). Membrane subproteomic analysis of Mycobacterium bovis bacillus calmette-guerin. Proteomics 7: 3919-3931. Zhou, G., Kowalczyk, D., Humbard, M. A., Rohatgi, S. and Maupin-Furlow, J. A. (2008). Proteasomal components required for cell growth and stress responses in the haloarchaeon Haloferax volcanii. J Bacteriol 190: 8096-8105. Zhou, L., Beuerman, R. W., Huang, L., Barathi, A., Foo, Y. H., Li, S. F., Chew, F. T. and Tan, D. (2007). Proteomic analysis of rabbit tear fluid: Defensin levels after an experimental corneal wound are correlated to wound closure. Proteomics 7: 3194- 3206. Zhu, W., Reich, C. I., Olsen, G. J., Giometti, C. S. and Yates, J. R., 3rd (2004). Shotgun proteomics of Methanococcus jannaschii and insights into methanogenesis. J Proteome Res 3: 538-548. Zmijewski, M. A., Macario, A. J. L. and Lipinska, B. (2004). Functional similarities and differences of an archaeal Hsp70(DnaK) stress protein compared with its homologue from the bacterium Escherichia coli. J Mol Biol 336: 539-549.
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Appendix A. Chapter 2 and 3 Appendices
Appendix A.1 Simplified flowchart of methodology
Cell pellets, 4, 23 C
Sonication
Whole cell extract
Carbonate extraction, pH 11-12 Ultracentrifugation at 115 000xg Soluble protein
De-salt and Hydrophobic pellet concentrate in Wash in dH2O Amicon Ultracentrifugation at 115 000xg
Re-suspend in ambic Store at -80 C Hydrophobic protein Homogenise by gentle sonication SDS-PAGE De-salt and concentrate in Amicon Store at -80 C Differential solubility fractionation Pellet treated with increasing concentrations of OGP De-salt and concentrate in Sample for mass spectrometry Amicon Reduce and Fractions alkylate Denature and dissolve in organic solvent SDS-PAGE Digest with trypsin
Peptides Sample clean up: SCX; RP Vacuum dry LC-MS/MS and sample ‘Clean’ peptides LC/LC-MS/MS evaluation File conversion
Data analysis Data processing
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Appendix A.2 Rnf operon alignments
Alignments were performed using ClustalW
UPF0132 Mbur_1378 MTYKTSIGLNENIVAVLCYLGFWMTGVLFLFVEKENKFVRFHAIQSAMVF 50 MA0665 MSYNTSLGLSENIVAALCYPVGWLSGLFFLLLERKNKFVRFHAMQSVLLF 50 *:*:**:**.*****.*** *::*::**::*::********:**.::*
Mbur_1378 MVLTAIVFLIAWVPYVGWLLADFAGFFSLFLWALFMFMAWRGSRLKIPVI 100 MA0665 MPIALFIFLVAWIPTIGWFIADGAGMTAMLLILIPMYMAFRGSKFKIPII 100 * :: ::**:**:* :**::** **: :::* : *:**:***::***:*
Mbur_1378 GRIAYNYVYQ- 110 MA0665 GNIAYNFAYGE 111 *.****:.*
RnfB Mbur_1379 MSLITLIIQAMATLGGLGLVIGIMLIAASRMFKVETNPLVEEVVEILPGANCGACGYAGC 60 MA0664 --MSSVLINSIAVLAGLGFAVGVMLVIASKVFKIDSNPLIDDVASLLPGANCGGCGFAGC 58 : :::*:::*.*.***:.:*:**: **::**:::***:::*..:*******.**:***
Mbur_1379 ADFAEKVVAEDAPLDGCPVGGFDTAKEIGAIIGQEVSESEKEYPYVRCGGG-IRCVDRFD 119 MA0664 AACAEAIVEQGAPVNSCPVGGFEVAKQIGALLGQEVTESEKEFPFVRCQGGNQHCTTLYD 118 * ** :* :.**::.******:.**:***::****:*****:*:*** ** :*. :*
Mbur_1379 YVGIEDCTAVIMLSDGEKGCNYGCMGRGTCVRACPFGAISINENRLPSVNKNLCTSCGLC 179 MA0664 YHGVENCKVALMLCDSRKGCTYGCLGLGTCVQACQFGALSMGEDGFPVVNKALCTSCGNC 178 * *:*:*...:**.*..***.***:* ****:** ***:*:.*: :* *** ****** *
Mbur_1379 LAACPNDILMFAKDSEQVHVQCNSHDKGKAVKAVCEVGCIGCKICEKNCPEDAIKVTNFL 239 MA0664 IAACPNGVLTFARDSEKVHVLCRSHDKGKDVKAVCEVGCIGCKKCEKECPAGAIRVTEFL 238 :*****.:* **:***:*** *.****** ************* ***:** .**:**:**
Mbur_1379 AEVDQDKCTACGICVEKCPQNCIEMR 265 MA0664 AEIDQEKCTACGACVAICPQKAIELR 264 **:**:****** ** ***:.**:*
RnfA Mbur_1380 -MAADVSLFQIFMDGVFIKNFLIIQFLGLCSFVGVTKDTKSAAGMSGAVIFVMTLAATVS 59 MA0663 MVKMAESLFTIFLEGVFIKNFLLIQFLGLCSFVGVTKDLKSASGMSGAVVFVMAMAATVS 60 : *** **::********:*************** ***:******:***::*****
Mbur_1380 FLIYNFVLVPLKLEFLDLISFIVVIAALVQLVEFVVRKNVPSLYRSLGIYLPLITTNCAV 119 MA0663 FALYNFILVPLKLEFLRTIAFIVVIAALVQLVEFIVRKHVPALYRSLGIYLPLITTNCAV 120 * :***:********* *:**************:***:**:******************
Mbur_1380 LGVVLLNVLNEYSFIQSVVFGVSAGIGYALVMLMMSGIRERTTLVNVPSSM-RGLPHAFL 178 MA0663 LGAVLLNVMNDYDLAQSVVFGVAAGLGYTVAMLMMAAIRERSDLVEVPKSVGRGVTYAFF 180 **.*****:*:*.: *******:**:**::.****:.****: **:**.*: **:.:**:
Mbur_1380 IATMLSMAFVNYFGVIPL- 196 MA0663 IATIMSMSFVNFFGVIPLE 199 ***::**:***:******
RnfE Mbur_1381 ------MDPLSEFIRGITKDNPIFGLVLGLCPTLAVTTSIDNAIGMSAGTAFVLICSN 52 MA0662 MYPHRRADMNPISEFIRGITKDNPTFGLVLGLCPTLAVTTSVENGIGMAMGTLFVLVGSN 60 *:*:************ ****************::*.***: ** ***: **
Mbur_1381 LMVSGLRKQIPASVRLPIFILIIATFVSIVKMVMEAYFPPMYAALGVFIPLIVVNCIIIG 112 MA0662 MMVSAIRKGIPGTVRLPVEIIVIATFVTIVDMVMEAFTPDLYTSLGVFIPLIVVNCIVIG 120 :***.:** **.:****: *::*****:**.*****: * :*::*************:**
Mbur_1381 RAEAYANKNNMFYSLIDALGSSAGFLLVLVLIGGIRELLGTGGIAIFGTQLVSIP--INP 170 MA0662 RAEAYALKNGVFYSIIDALGEGTGFLLVLILIGGIRELLGTGIIDPFGMTLINLSGVINP 180 ****** **.:***:*****..:******:************ * ** *:.:. ***
Mbur_1381 ITFMILSPGAFLTIGILMAIVNHRRAKKLARGG 203 MA0662 AMFMTMSPGAFLTIAVLMTIVNYRRQQKAAKGG 213 ** :********.:**:***:** :* *:**
326 D. Burg UNSW
RnfG Mbur_1382 MTDSNKDIVIIIGKLVLISVIASALLAVAFVPTNEQLKKNYAEARTSTLAEIMPSAAEFE 60 MA0661 MSDS-KEITKVIVTMVVISAVAAALLALTYTPTQAQLKLLQAEQQKEAMKEILPQAADFE 59 *:** *:*. :* .:*:**.:*:****:::.**: *** ** :..:: **:*.**:**
Mbur_1382 AVYGDTVINDEGDKEILYYRAKDSSGNLVGYAFFRKQVGAQGLIEVAGGVDSSFNAITGI 120 MA0661 PVTG-SEVDDDGNPVVLYYKGVDSSGNVVGYVVERNQVGAQGMIQLLAGISSDFSTITGF 118 .* * : ::*:*: :***:. *****:***.. *:******:*:: .*:.*.*.:***:
Mbur_1382 GIMAHTETPGLGSKIADDPFKSQFTNVKVADLSLSKSGGAIDSITGATISSQAVIDALNE 180 MA0661 QVMKHSETPGLGALITTPEFQGQFVDLPVADTSLTKNGGQVDAISGATISSQAVVDALHS 178 :* *:******: *: *:.**.:: *** **:*.** :*:*:*********:***:.
Mbur_1382 QVGDIKVAEA 190 MA0661 AVDYVSAQEG 188 *. :.. *.
RnfD Mbur_1383 -MTFTISAPPHKKENITFKKIMWAKVLALMPVVLLSVYLFGLPAIGLVAASIFAAIATEA 59 MA0660 MTSFTVSPPPHIKKKIFIKNLIWSRIVALLPISAAAVYFFGFAALGNIIASILGAVGIEF 60 :**:*.*** *::* :*:::*::::**:*: :**:**:.*:* : ***:.*:. *
Mbur_1383 TIQKAFGQKITIADGHAVLIGLMVAMIIPPEVPLWIPMIGTVFAVGIGKHAFGGIGSYVF 119 MA0660 VIQKAFNKKLTIMDGNAIYLGLLLALICPPTLPAWMIFIGGAFAVGVGKHAFGGIGSYTF 120 .*****.:*:** **:*: :**::*:* ** :* *: :** .****:***********.*
Mbur_1383 NPVLAAWLFLSLAWGSIMAPASYPQTSALFELVLENGAGVMAGVSPFFILLAGAVLIFRR 179 MA0660 HPSLAAWVFLSLAWAQDMLPGTIPILSSFSDLILENGAGFLTDVSPILVLLAGVILILVK 180 :* ****:******.. * *.: * *:: :*:******.::.***:::****.:**: :
Mbur_1383 YIEWRIPVTFFVTTVLLAFLLGDSLSYVVLGVVVFGVLFIATDTSTSPTTKNGRVIYGIV 239 MA0660 YIEWRIPLSYLLTTVILALVLGDPLAYVVSGTFLLGVFFIATETVTSPVTQNGRIVYGIL 240 *******:::::***:**::***.*:*** *..::**:****:* ***.*:***::***:
Mbur_1383 CGVLVVVYGHFA-NYVDGTFYGLFLANCVASLIDNNTLPASYGTETFLQCKYRSIVSKIP 298 MA0660 CGFLTVIYGYFSGNYVWGTLYALLLSNAVAPFIELKTLPKPMG------GVANE-- 288 **.*.*:**:*: *** **:*.*:*:*.**.:*: :*** . * .:..:
Mbur_1383 FKDRLEVFLDD 309 MA0660 ------
RnfC Mbur_1384 ------MTNVYVMEKIPEKIIIPLKQHDGCLCTPLVNKGDMVSMGQKIGECECY 48 MA0659 MKRSLHSKEVANLSDVIKIDKLPEKAIIPMRQHDGIACAPLVKKGAEVIVGQKLGECEGS 60 :::* ::*:*** ***::**** *:***:** * :***:****
Mbur_1384 DSAAVHSSVCGEVISIEKAANPNGTKVNSVIIKPTEDVECVDFTPVKDVSASKLVDVIKD 108 MA0659 DLAYVHSPFCGTVNSIELMPNPSGKRILSVVLTPSECAQTVDFVPEKDAPPSRLIEIIKE 120 * * ***..** * *** .**.*.:: **::.*:* .: ***.* **...*:*:::**:
Mbur_1384 AGIVEHYGTPTHKVLRPEGKQIDTVLINATSSEWIGGHYDTSAQYASQMIDALKLLMKAA 168 MA0659 AGIVEYYEKPTYLALKP-GKRIDTLLMNATFPLITHAYLSS----LDKVLEGFKLMLEAS 175 *****:* .**: .*:* **:***:*:*** . .: .: .::::.:**:::*:
Mbur_1384 GASKGAVVLRNDDLESINAFDHLRVDGKLLTVAPLMGKRKLSYYFKDMGSDIIVVSQERI 228 MA0659 GISRGVIVLRADDKESIKAFKNAKVDGKPLTVAPIVGMRHADYYLEDVEDQIIVVAAGKI 235 * *:*.:*** ** ***:**.: :**** *****::* *: .**::*: .:****: :*
Mbur_1384 -YGQKILDLLTYNLTGRRVSFCCDPTDVGVAICGVKSAKALYDAVHEGRPYIETVVSVSG 287 MA0659 TYTPTMMNLLSANVMGRKLPLGYEPPDVHVVVCGVKSAKAVYDAINEGKPYLESAVTVTG 295 * .:::**: *: **::.: :*.** *.:********:***::**:**:*:.*:*:*
Mbur_1384 KVNSPKKILVKIGTPFKDVIDACGGYMGDTGKLIANGAVTGVAQYTDDVPVTKMTTSITV 347 MA0659 AVNNPKTVIVKFGTPIKDVIEACGGYKGEPGKVIVNGSMGGVAVYTDEAPVVKNTVGIVV 355 **.**.::**:***:****:***** *:.**:*.**:: *** ***:.**.* *..*.*
Mbur_1384 LSADEVIRDVSIDCTHCARCVDVCPVDLIPSRITAFADQGRFDECRQMNILNCVECGRCS 407 MA0659 QTEAEVLRDEATVCIHCARCVDVCPMNLLPGRIAAMADMGMFDRCREYFALNCIECGECA 415 : **:** : * **********::*:*.**:*:** * **.**: ***:***.*:
Mbur_1384 AVCPSKIHLLQLIRYAKNSIEN-AYEDLSSKESPANLKLGCGCSGGN 453 MA0659 VVCPAKRHLVQLIRYSKLQIMNQKNETVEATE------447 .***:* **:*****:* .* * * :.:.*
D. Burg UNSW 327
Multiheme protein Mbur_1385 ---MARLEIILLGAAIFIFAFAGVFAGMGYSGNEAIATHYMTEGEWSDASCGGCHFTAPD 57 MA0658 MVIMNRLNLLVSGVAVLLLLAAGAYSSLGYSGNDAIASHYMTKGEWSDSVCGGCHFGVYE 60 * **:::: *.*:::: **.::.:*****:***:****:*****: ****** . :
Mbur_1385 HVATNTHIQRDIGEWDPLTNFDIEVEGEDEWVKNFGAYHPGGGELEAYGVDVDCMICHEQ 117 MA0658 NVNNSYHVQVNMSRWSPLTNFDLETSGEEEWVKKFGMYHPGGGPLAKYGIDIDCMMCHEK 120 :* .. *:* ::..*.******:*..**:****:** ****** * **:*:***:***:
Mbur_1385 YGEYGFDARAELFAAGDFENANAAAMEAANIKVQQDTIRKITYVGNAVTPLPLLLLFHDS 177 MA0658 YGLYDFDARAEAIANGDFANANSLAVANFSATAQSDPLHLFVYTANVLTPYPLLIVFHDA 180 ** *.****** :* *** ***: *: . ..*.*.:: :.*..*.:** ***::***:
Mbur_1385 VNGAPVKESCSDNCHVTNVETTAVTWAS-EDYHKFDAHAD--VNCVECH------223 MA0658 VNGAPI--SCAQRCHRIDVETSAVMWADEEDFEESDAHAANGVECTECHHTEAFIITSDH 238 *****: **::.** :***:** **. **:.: **** *:*.***
Mbur_1385 EISQSSMFVKQDLENIHEVEAETKSCDDPGCHKGISHGPIVDAH-ETVACESCHIPALPG 282 MA0658 QIGRGNTSGTPDLPDSH-YDDTMRSCDDAECHAGISHGPFADSHMEFLACEACHTPELPG 297 :*.:.. . ** : * : :****. ** ******:.*:* * :***:** * ***
Mbur_1385 GDLGGVIPLASFDWSNGERVDSYKDDSFEPVLAWSNGIEGHELPATDGRGEDGVLLEPFN 342 MA0658 GDLPGGNVLESFSWQNGEREDVYRDSDFQPALAWYNGNFGDVLPSVDTRNDTDVKVTPFN 357 *** * * **.*.**** * *:*..*:*.*** ** *. **:.* *.: .* : ***
Mbur_1385 VVTGIWWDAGINSEIVSSPDTSNEIGDAIPVSDVKAADSNDDGVVTADEMRSFDGDIDGN 402 MA0658 NITGTWWDAGTDPEVLANPNTSISTGDPIPVQYVKAADANGDGEVTVEEMQAYDADGDGE 417 :** ***** :.*:::.*:** . **.***. *****:*.** **.:**:::*.* **:
Mbur_1385 ADYPNAVLRTVELYYKLSHNIASSEVGLAGPLACADCHGSTANAIDWESIGFISDPAETD 462 MA0658 ADYPNAVLRTVELYYQVAHSIVSSDIGLADPYTCKDCHGNEA-VIDWAALGYEQDPGGES 476 ***************:::*.*.**::***.* :* ****. * .*** ::*: .**. .
Mbur_1385 PPTDFTLKDIGVTIPGAKPPEVEREPAF 490 MA0658 S----AVKSIAVTYDKPRPVEVETEPAL 500 . ::*.*.** .:* *** ***:
328 D. Burg UNSW
Appendix A.3 Na+/H+ antiporter (Mrp) alignments
Mbur_0132 MDSFNAITVAIFLPFIFAGLVPVMEKLLKQRVGWFAAATALASFALIGMAATEVIEGQII 60 MA4572 MDPFNAIVIAVFLPFILAWTLPVLYSVFKQRIGWISALIAFTCFVLNAQIIPYTMAGTPV 60 **.****.:*:*****:* :**: .::***:**::* *::.*.* . . .: * :
Mbur_0132 QGSISWIPSAGVNFTIYADGLATMIGFIASGIGVLIMSYSNGYMSRTEDLTRYYQYLLLF 120 MA4572 TGSFTWLPAAGLSLDFYGDGMAVLLALIVSGVGVIIMSYSNGYMSTKEDLPRYYQWLLLF 120 **::*:*:**:.: :*.**:*.::.:*.**:**:********** .***.****:****
Mbur_0132 MGSMIGMVFSGNTIQLFIFWELTSITSFMLIGYWRHKPESIYGATKSLLLTASGGLAMLA 180 MA4572 MGAMLGIAYTDNTIQMFIFWELTSITSFMLIGYWRERPESVYGATKAFLITAGGGLFMFA 180 **:*:*:.::.****:*******************.:***:*****::*:**.*** *:*
Mbur_0132 GFLLLGNITGSFELAAILNDPQVINAIKEHELFLITLILILIGAAAKSAQGPFYIWLPNA 240 MA4572 GFLMLRVVTGTYGISEVAQSSELLSTLHGSPLYVAVLVLIFIGAASKSAQGPFYIWLPNA 240 ***:* :**:: :: : :..:::.::: *:: .*:**:****:**************
Mbur_0132 MEAPTPVSAFLHSATMVKAGIYLVARIHPIFSGTEAWFILVSGTGIITMLVAGFLAFRQT 300 MA4572 MEAPTPVSAFLHSATMVKAGVYLVARMHPILSGTPEWLILVSGTGMITMVMAGFMAFRQT 300 ********************:*****:***:*** *:*******:***::***:*****
Mbur_0132 DIKGILAYSTISQLAYMMTMYGYTTHHEPGIGVAAATFHLLNHAAFKACLFLVAGIVAHE 360 MA4572 DIKAILAYSTISQLAYLMTMYGYSTAEHPGLGFAAATFHLLNHSTFKATLFLVAGIVAHE 360 ***.************:******:* ..**:*.**********::*** ***********
Mbur_0132 AATRDIRKLGGLRREMPITFIIACIAGLAMAGIPPLNGFLSKEMFYESSIEMG----ASI 416 MA4572 ATTRDIRKLGGLRKEMPKTFIVAVIAAASMAGVPPLNGFLSKEMFYETSLEIGELVSETY 420 *:***********:*** ***:* **. :***:**************:*:*:* :
Mbur_0132 GGIFTFLIPAFAVLGGVFTFAYSIKLIDGIFLGER-DKNAIPHHIHEPPMTMLIPPAFLA 475 MA4572 GGPWAIIFPAVAVAGGVFTLMYSIKLIDGIFLGERTHDHDVPHHVHEAPWVMLAPAVFLV 480 ** :::::**.** *****: ************** ..: :***:**.* .** *..**.
Mbur_0132 FLVILFGVIPSLPTHYIIEPAVSGILLETAHLHVKLWHGFTASLMMTIVTFILGILIYTK 535 MA4572 GLIIFFGLYPTFPISVLIQPAYSGLVPHADHLHVALWHGVTTPLLMTIATFAIGLVLYKF 540 *:*:**: *::* :*:** **:: .: **** ****.*:.*:***.** :*:::*.
Mbur_0132 YDRIAAWQDNFNMKNPRISINYYYDRAVDSAKGNAFRFSSRSQTGNIKLYMSAMLLLMIA 595 MA4572 YDAIAAWQNSFNDKLPWISVNYWYDATVNNAKGITHKFAAFAQPGPIGVYVKMTLLFMIF 600 ** *****:.** * * **:**:** :*:.*** :.:*:: :*.* * :*:. **:**
Mbur_0132 LIAIPAAILATN------IMPSQLNFDIPPYEAILLLLLIVAALSAAILPTYLPSIIAL 648 MA4572 LIFWPVYMFGINLGIGVGDLLPAGIIYDAQPYEIVLYSLMILAALGAALLPRYLPAVISL 660 ** *. ::. * ::*: : :* *** :* *:*:***.**:** ***::*:*
Mbur_0132 SALGYGVSLLFIYLKAPDLALTQILVETLSTIIFLLAITKIPQKYKEKISISVFTRDILI 708 MA4572 SELGFLVALLYVYLKAPDLAMTQVCVETLSTIIFILVLIKIPQKFKEPMPAGKVLVNLLI 720 * **: *:**::********:**: *********:*.: *****:** :. . . ::**
Mbur_0132 AITVAASVFVVLLNATQGIVDPFASLSHYFIDNSFLLAGGHNIVNVIIVDFRGYDTLGEI 768 MA4572 SGVVSFGVLAVMLNANLGVLAPFETFNYYFIEKALAMTGGLNVVNVIVVDFRGYDTIGEI 780 : .*: .*:.*:***. *:: ** ::.:***:::: ::** *:****:********:***
Mbur_0132 SVLCLAALGVYNLIHSRSEE------788 MA4572 SVLSLAALGVYSLILSRAKKVKGGKE 806 Mbur_0133 --MTTIITKTITKICIPLVILFSISLLLAGHNNPGGGFIGGVMFASVIALAYVVFGLKDI 58 MA4571 MADTTVITKTIAKVCLMIDMLYSIDLLLIGGNRPGGGFIGGVLCAAGIGLIYVAYGYDAI 60 **:*****:*:*: : :*:**.*** * *.*********: *: *.* **.:* . *
Mbur_0133 KTFFNPDWAKWFGFGLTLASFTAFSAMMFGHNFFRSAVEFVHLPLFG----EIELVSAGL 114 MA4571 KKIWNPDWHMWFGYGLLFASITAWSPLFAGHKYFRSAFDFVPVEVGGMHLFELELVSSMF 120 *.::**** ***:** :**:**:*.:: **::****.:** : : * *:****: :
Mbur_0133 FDIGVYFVVIGSLLFIFKNVGDDNE------139 MA4571 FDLGVYFVVVGGLLFIATKLGADKGPEGEHE 151 **:******:*.**** .::* *:
Mbur_0134 MNNFILTLTISILFGIGTFLILRRDMIKVIIGLSVLSHAVNLLIVSAGVFENG-KVPIIT 59 MA4570 MNNFFLELTIALLFGIGTFLVLRRDMMKVIIGFGIISHAINLYIVGSGVFTEGTLVPILE 60 ****:* ***::********:*****:*****:.::***:** **.:*** :* ***:
Mbur_0134 -GSGHGGEVTGTIYNDNIAEGILAPIVSAGTHVEFVDPLVQALVLTAIVISLATTAFILI 118 MA4570 HEQALEGLGQGLIYNDNLTSGILGPITTN-PYTEFVDPLVQALVLTAIVIGLATTAFVLT 119 .. * * *****::.***.**.: .:.*****************.******:*
Mbur_0134 LAYRIYEEYGTTDIRELRRLWG 140 MA4570 LCYRVNEEYGTVDVQELRRLRE 141 *.**: *****.*::*****
D. Burg UNSW 329
Mbur_0135 MNISMTHLPILLVAVPILMSALMIFLRSNANVQKWLNVSVSLAMMLISIILLLQVWNGGI 60 MA4569 ----MDNLPMFLIAIPLLMAPVTILIKGNPGLQKVLDIVVGFVLLCLSVLLIITVWNNGI 56 * :**::*:*:*:**:.: *:::.*..:** *:: *.:.:: :*::*:: ***.**
Mbur_0135 QVYEVGEWGKYGIVLVADLLSSGMVVLSSIVSFLALLYSLDYIEGRSLNSTYHSLFNLLV 120 MA4569 IAYDVGEFGKYGIVLVADLLGAGMVLLSCFIGFLSLIYSYDYIEDQSLNQTYHSLYNLMI 116 .*:***:************.:***:**.::.**:*:** ****.:***.*****:**::
Mbur_0135 AGLNGTFLTGDIFNMFVFFEILLLASCGLVIANEKGGVTKSSDKMEATFKYLILNMIGSF 180 MA4569 AGINGIFLTGDIFNMFVFFEVMLLSSAALIVANETSKVTKISDKMEATMKYLVLNILGGN 176 **:** **************::**:*..*::***.. *** *******:***:**::*.
Mbur_0135 VMLIAVASLYATVGTLNMADLSVKISTMSAAGTLPWHIYAIALLFIVVFGNKAAIFPMHY 240 MA4569 VMLIAVASLYASIGSLNMADITLKIRMLAETGAVPWHLYAVGLLFIIVFGGKAAMFPLHY 236 ***********::*:*****:::** :: :*::***:**:.****:***.***:**:**
Mbur_0135 WLPDVHPTAPSPISAMLSGVMIKVGAYGILRIFFLIFRDALFLLQPIIILLALVTIIIGA 300 MA4569 WLPDVHPTAPSPISAMLSGVIIKIGAYGILRVLFMVFAPFQAVYGPVILFTALVTLALGA 296 ********************:**:*******::*::* : *:*:: ****: :**
Mbur_0135 VSAVGQNDVKKLLAYSSVSQIGYVFLGIGIGSVYALA------AALVYMANHAIA 349 MA4569 TSAVGQSDVKRMLAYSSVSQIGYIFLGFGMAAMANVAGETAAATLILGASVVYMINHALA 356 .*****.***::***********:***:*:.:: :* *::*** ***:*
Mbur_0135 KSMLFLTSGGIIHHAGTRDMRHMGGMNKTSPIMSIAFLIGAMSIAGLPPMGGFVAKFILF 409 MA4569 KSMLFLTSGGIIHTADTRDMRQMGGMINTAPMMGVAFLVGAMSIGGVPPMGGFIAKFMLF 416 ************* *.*****:**** :*:*:*.:***:*****.*:******:***:**
Mbur_0135 DAGLRAEYYIPVAIALFFAVFTLFYMFRAWLLMFWGETRDVEEYGEYSSHKPSLMIALPI 469 MA4569 DSGLRAQFYLPIGIALIFAIFTLFYMFRGWMLIFWGE-RD-PQHGEYSHHKLSPLIVAPI 474 *:****::*:*:.***:**:********.*:*:**** ** ::**** ** * :*. **
Mbur_0135 VSLAALVFIFGVYAEPLIALAQAIAEQLIDPQPYIDAVMTRVVR 513 MA4569 LILAAIVLIVGLYPQPVVEFSQTIANQIIDPQPYIDAVIVRVIR 518 : ***:*:*.*:*.:*:: ::*:**:*:**********:.**:*
Mbur_0136 MKRYILYSMFFGLIWCFVHGTVNLNNFLIGALLGPIVIRPFRPLYGFENKISYK-KVIRK 59 MA4568 MKRRTITYMALPVVWCLVSGQITLGSVLLGLIFGVVVVAPFSELYRLDEVVHPTGDWISK 60 *** : * : ::**:* * :.*...*:* ::* :*: ** ** ::: : . . * *
Mbur_0136 IPKLIKFFYVLFIEIIKANIMMAKIILQPKMDIKPGIIAVPIRTKTNTGITAIANTITLT 119 MA4568 IPKKLKYFYVLIKEIIKANIVVAKIVIKPKIDIKPGIIAVPIRTKTNIGITGIANTITLT 120 *** :*:****: *******::***:::**:**************** ***.********
Mbur_0136 PGTLTIDVSDNKEVLYVHAIDASDPQGLCDSIRDDLEEYVLEAFE 164 MA4568 PGTLTVDISDDKSVLYVHSIDSTDPQGVRDSIRDDLEYYVLEAFE 165 *****:*:**:*.*****:**::****: ******** *******
Mbur_0137 -MNSLIFD--ISITFMVIAIIPCIYRVIKGPTIPDRVIAVDAMTTVIIVILGIYSYMQES 57 MA4567 MIITLEQANFLALVVMVVALVPSTYRVLYGPTLPDRIAASDAVGNVLAMIFALYAFQGSS 60 : :* :::..**:*::*. ***: ***:***: * **: .*: :*:.:*:: .*
Mbur_0137 AFFMDVALVLAIISFVGTVTISKYLDEGAVF 88 MA4567 IYLMDVAMLLSIISFVGTVIVAKYLDTGEVL 91 ::****::*:******** ::**** * *:
Mbur_0138 MIEIGLIQDIISTFLLLVGAFFVFLGMVGLIRLPDVYNRLHATTKIGTLGAFGVMLSIVA 60 MA4566 MDIISTVLDILSIISLVIGLLFLCLGMLGLLRLPDVYNRLHATTKVATLGALGVLLSIII 60 * *. : **:* : *::* :*: ***:**:**************:.****:**:***:
Mbur_0138 KLGLEPIGVKAITVGFFILLTAPVAAHMIGRAAHRHGVGLCKESVIDEYG-----KEYNK 115 MA4566 QEGYTPMGVKAFTVAMFILLTAPISGHMIGKAAYSHGVKLCEGTCIDEYGPSRGPQSVSK 120 : * *:****:**.:*******::.****:**: *** **: : ***** :. .*
Mbur_0138 --- MA4566 KKK 123
330 D. Burg UNSW
Appendix A.4 Polysaccharide gene loci
0718 0719 0720 0721 0722 0723 0724 0725 0726 0727 0728 0729
Appendix A.4a Polysaccharide locus 1. This region encodes a number of proteins with unknown or uncharacterized function. Grey indicates protein was expressed in HPP analysis, checks indicate the protein was expressed in WCE analyses (in this case all were also identified in HPP analysis), horizontal stripes indicate a protein that is expressed in secretome (T. Williams, personal communication). Bold and underlined gene numbers indicate a protein with predicted transmembrane domains. Gene locus tags represent: 0718 - hypothetical protein; 0719 - hypothetical protein; 0720 – DUF 354 protein; 0721 - bacterial transferase hexapeptide repeat protein; 0722 – glycosyltransferase family 2; 0723 - hypothetical protein; 0724 - glycosyltransferase group 1; 0725 - glycosyltransferase group 10; 0726 - glycosyltransferase group 1; 0727 – polysaccharide biosynthesis protein; 0728 - serine-rich surface protein (adhesin) with putative Ig domain, two dockerin type 1 domains and a cohesin domain; 0729 - protein with cohesin domain.
D. Burg UNSW 331
1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593
1579 1604 1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1605 1606 1607 1608 1609 1610 1611
1612 1613 1614
1615 1616 1617
Appendix A.4b Polysaccharide locus 2. This region contains proteins that perform a variety of biosynthetic and modification roles. Grey indicates protein was found to be expressed in HPP analysis, stripes indicate a protein that is implicated in N-linked glycosylation, checks indicate the protein has been found as expressed in WCE analyses. Bold and underlined gene numbers indicate a protein with predicted transmembrane domains. Gene locus tags represent:1579 - AglB-like oligosaccharyltransferase. 1580 -
Asparagine synthetase (glutamine-hydrolysing); 1581 - glycosyltransferase group 1; 1582 - NAD- dependent sugar epimerase; 1583 - nucleotide sugar dehydratase; 1584 - Mannose-1-phosphate guanyltransferase; 1585 - N-acylneuraminate cytidylyltransferase protein; 1586 - N-acetylneuraminic acid synthase and/or N-acetylneuraminate-9-phosphate synthase; 1587 - UDP-N-acetylglucosamine 2- epimerase; 1588 - protein with transferase hexapeptide repeat domains; 1589 - aminotransferase; 1590 – methyltransferase; 1591 - Polysaccharide biosynthesis protein CapD; 1592 - hypothetical protein; 1593 -
Polysaccharide biosynthesis protein, membrane-associated; 1594 - N-acylneuraminate cytidylyltransferase; 1595 - 2-dehydro-3-deoxyglucarate aldolase; 1596 - D-3-phosphoglycerate dehydrogenase (serA); 1597 - NAD-dependent sugar epimerase/dehydratase; 1598 - Cyclase family protein; 1599 - Haloacid dehalogenase-like hydrolase; 1600 – methyltransferase; 1601 - Hypothetical protein; 1602 - Radical SAM protein; 1603 - Cytidine diphosphoglucose 4,6-dehydratase; 1604 - dTDP-
4-dehydrorhamnose 3,5-epimerase related protein; 1605 - Glucose-1-phosphate cytidylyltransferase
(rfbF). 1606 – aminotransferase; 1607 - glycosyl transferase, family 2; 1608 - glycosyl transferase, family
2; 1609 – acetyltransferase; 1610 – aminotransferase; 1611 - Oxidoreductase family protein.
Continued on following page.
332 D. Burg UNSW
Appendix A.4b Cont’d Polysaccharide locus 2. 1612 - UDP- glucose pyrophosphorylase; 1613 -
Xanthan-like biosynthesis protein (Includes: Mannose-6-phosphate isomerase (Phosphomannose isomerase) (PMI) (Phosphohexomutase); Mannose-1-phosphate guanylyl transferase (GDP) (GDP- mannose pyrophosphorylase) (GMP)); 1614 - protein of unknown function UPF0104; 1615 - Dolichyl- phosphate beta-D-mannosyltransferase like protein with GtrA-like C-terminal domain; 1616 – methyltransferase; 1617- protein with oligosaccharyltransferase domain.
2018 2020 2021 2022 2023 2024 2025 2026 2027
2019 2028 2029 2030
Appendix A.4c Polysaccharide locus 3. This region contains proteins that perform a variety of functions. Grey indicates protein was found to be expressed in HPP analysis, stripes indicate a protein that is implicated in N-linked glycosylation, checks indicates the protein has been found to be expressed in WCE analyses. Bold and underlined gene numbers indicate a protein with predicted transmembrane domains. Gene locus tags represent: 2018 - AglK like protein; 2019 transposase; 2020 – hypothetical protein; 2021 – DUF 354; 2022 - UDP-N-acetylglucosamine 2-epimerase; 2023 – glycosyltransferase group 1; 2024 - UDP-galactose -4-epimerase; 2025 - hypothetical protein; 2026 - hypothetical protein;
2027 - glycosyltransferase group 1; 2028 - Polysaccharide biosynthesis protein, membrane-associated;
2029 - UDP-N-acetyl-D-mannosaminuronate dehydrogenase; 2030 - Oxidoreductase (NAD- or NADP- binding)-like protein
D. Burg UNSW 333
2222 2223 2224 2225 2226 2227 2228 2229 2230 2231 2232 2233 2234 2236 2237
2235
Appendix A.4d Polysaccharide locus 4. This region contains proteins that perform a variety of functions. Grey indicates protein was found to be expressed in HPP analysis, checks indicates the protein has been found to be expressed in WCE analyses, vertical stripes indicate proteins that are unique to M. burtonii, Mbur_2237 is implicated in N-linked glycosylation. Bold and underlined gene numbers indicate a protein with predicted transmembrane domains. Gene locus tags represent: 2222 - hypothetical protein;
2223 - hypothetical protein; 2224 - hypothetical protein; 2225 - Polysaccharide biosynthesis protein, membrane-associated 2226 - hypothetical protein; 2227 - hypothetical protein; 2228 - hypothetical protein; 2229 – glycosyltransferase group 2; 2230 - Glucose-1-phosphate thymidylyltransferase (dTDP- glucose synthase); 2231 - dTDP-4-dehydrorhamnose reductase; 2232 - dTDP-glucose 4,6-dehydratase;
2233 - dTDP-4-dehydrorhamnose 3,5-epimerase related protein; 2234 - UDP-glucoronic acid decarboxylase; 2235 - hypothetical protein; 2236 - Nucleotidyl transferase with trimeric LpxA-like domain; 2237 - glycosyltransferase family 2 AglD – like
2341 2342 2343 2344
Appendix A.4e Polysaccharide locus 5. This region codes for proteins involved in GlcNAc metabolism.
Stripes indicate a protein that is implicated in N-linked glycosylation, checks indicate the protein has been found to be expressed in WCE analyses. Gene locus tags represent: 2341 - Bifunctional UDP-GlcNAc pyrophosphorylase (GlmU); 2342 - phosphoglucosamine mutase; 2343 - glucosamine-fructose-6- phosphate aminotransferase; 2344 - N-acetylglucosamine-1-phosphate uridyltransferase (GlmU-like).
334 D. Burg UNSW
Appendix A.5 HtpX alignments
Mbur_1082 ------MKN------MLKTTLLLASLTGLLIIVGRLIG------GTTGMFIAFA 36 Mbur_1255 ------MKWKHDRGLEGRMLLTMFLLAAVYLAFLAFLFYAG------APQMFMVLF 44 HtpX_Streptococcus ------MLFEQIAAN---KRRTWFLLVAFFALLALIGAAAGYLWMNSPLGGVIIAFI 48 htpX_E.coli ------MMRIALFLLTNLAVMVVFGLVLSLTGIQSS-----SVQGLMIMALL 41 Mbur_0716 ------MISIPAPVIAFLLLGPVGFVAT------LA 24 . . : :
Mbur_1082 FALMLNFGSYWYSDKIVLKMYHAKEVTESESPQ---LYDIVRNLAMRAQLPMPKVYIVET 93 Mbur_1255 IGAFMGL-QYYYSDRLVLWTMHAKVVTAQEAPD---LHQTITRLCAIADLPMPRVAVVNT 100 HtpX_Streptococcus IGLIYAITMIFQSTEVVMSMNGARQVSEQEAPE---LYHIVQDMAMVAQIPMPRVYIVED 105 htpX_E.coli FGFGGSFVSLLMSKWMALRSVGGEVIEQPRNERERWLVNTVATQARQAGIAMPQVAIYHA 101 Mbur_0716 LFILLSYVLYSYSGKILLKWYKAKKVG------SLENLAVKAGVVTPDMYMFDH 72 : * : : .. : : . * : * : : .
Mbur_1082 SMPNAFATGRDPKHAAVAATTGIMNILTTEELEGVLAHELAHVKNRDTLISAVAATIAGV 153 Mbur_1255 SIPNAFATGRGPKNAVVAVTTGLMDQLNQGELEAVLAHELSHVKNRDMAILTIASFIS-- 158 HtpX_Streptococcus DSPNAFATGSNPENAAVAATTGLLRLMNREELEGVIGHEVSHIRNYDIRISTIAVALASA 165 htpX_E.coli PDINAFATGARRDASLVAVSTGLLQNMSPDEAEAVIAHEISHIANGDMVTMTLIQGVVN- 160 Mbur_0716 HLPMIFTAGTRG-KFDIAVSSGAMGLFDASELEVMLAREIGHILNNDVPMNTMVALFAGS 131 *::* :*.::* : : * * ::.:*:.*: * * :: .
Mbur_1082 ITMLATWARWAAIFGG---IGGRDD---DGGG---NIVGFIALAIVAPLAATIIQFAISR 204 Mbur_1255 -TMAFYIVRYSFYFGGMGGMGGRRK---ESGG----IVAIWIVSLLVWIISFLLIRALSR 210 HtpX_Streptococcus ITMISSVAGRMMWYGGGRRRNDRDD---DSGLGLLMLVFSLIAIILAPLAATLVQLAISR 222 htpX_E.coli -TFVIFISRILAQLAAGFMGGNRDEGEESNGNPLIYFAVATVLELVFGILASIITMWFSR 219 Mbur_0716 LASVSTFALWGALLGGFGQDYDPAP------RFIRFLGMGLVAVPSALIAQLALSP 181 : .. . : :: : : :*
Mbur_1082 SREFGADAEGARISQKPWALASALSKLESGAQNYKPGKNDVQPSQNTAHMFIVNPLRGSS 264 Mbur_1255 YREFAADKGSAVITGQPSNLASALTKISG--IMPRIPKDDLREVEG-MNAFFIFPAVSGS 267 HtpX_Streptococcus QREFLADASSVELTRNPQGMIRALQKLD----NSEPMHRHVDDAS--AALYISDPKKKGG 276 htpX_E.coli HREFHADAGSAKLVGRE-KMIAALQRLKT------SYEPQEATSMMALCINGKSKS 268 Mbur_0716 SREIMVDAVSVELTKEPKLLADTLEYMQK-YVGHYPMSLNPGHAHLFPLNLLSMEEFYDM 240 **: .* .. : . : :* :. :
Mbur_1082 LMKLFSTHPATEERIRRLDEM------285 Mbur_1255 FMSLLSTHPTIEKRIAALEKIQRELEL 294 HtpX_S.gordonii LQKLFYTHPPISERVERLRKM------297 htpX_E.coli LSELFMTHPPLDKRIEALRTGEYLK-- 293 Mbur_0716 HLSLFNTHPDVDIRVKQIMSKVN---- 263 .*: *** . *: :
*Catalytic domain is highlighted red
Appendix A.6 Lon catalytic domain alignments
AF0364 ---VHIQFVGTYEG-VEGDSASISIATAVISAIEGIPVDQSVAMTGSLSVKGEVLPVGGV 56 MA3289 ---VHIQFVGTYEG-VEGDSASVSIATAVISAIERIPVDQTVAMTGSLSVRGDVLPVGGV 56 Mbur_Lon_0880 NHDIHIQFIGTYEG-VEGDSASISIATAVISALENIPIDQTVAMTGSLSVRGDVLPIGGA 59 VNG0303G ---THIQFVQAGEGGVDGDSASITVATAVISALEDIPVAQELAMTGSLSVRGDVLPVGGV 57 MMP1186 ---IYIQFSQSYSK-IDGDSATAAACLSIISALLNIPLKQDFCITGSLDLNGEILAIGGV 56 MJ1417 ---IYIQFSQSYSK-IDGDSATAAVCLAIISALLDIPLKQDFAITGSLDLSGNVLAIGGV 56 :*** : . ::****: : . ::***: **: * ..:****.: *::*.:**.
AF0364 TQKIEAAIQAGLKKVIIPKDNIDDVLLDAEHEGKIEVIPVSRINEVLEHVLEDGKKKNRL 116 MA3289 TYKIEAAAQAGLKKVIIPKANEADVLVEKAYREKIQIIPVSSIAEVMEHSLM-GPKKNTI 115 Mbur_Lon_0880 TYKIEAAVQAGIKKVIIPKSNEADVLIEDYYKDKVEIIPVTTIAEVIEHSLV-GEDKAGI 118 VNG0303G THKIEAAAKAGCERVIIPKANEDDVMIEDEYEEQIEIIPVTHISEVLDVALVGEPEKDSL 117 MMP1186 NEKINAAKEYGFKRVIIPKSNFDDVIDP----ERIEVIAVTRLEEIIP--LAFELNNL-- 108 MJ1417 NEKIEAAKRYGFKRVIIPEANMIDVIET----EGIEIIPVKTLDEIVP--LVFDLDNRGG 110 . **:** . * ::****: * **: :::*.*. : *:: * .:
AF0364 MSKFK------ELELAAV------128 MA3289 IEKLKNITKLSFDIPEVTPASVQAMNLFGCRN- 147 Mbur_Lon_0880 VEKLKNMSN-----KKIDYAARSKK-VAGSDDV 145 VNG0303G VDRLKSITG-----KALDSASDSGTTGGNPSPQ 145 MMP1186 ------MJ1417 AERFN------115
Organism abbreviations are followed by locus tags and are as follows: AF, Archaeoglobus fulgidus;
MA, Methanosarcina acetivorans; Mbur, Methanococcoides burtonii; VNG, Halobacterium sp. NRC-1;
MMP, Methanococcus maripaludis; MJ, Methanocaldococcus jannaschii. Proposed catalytic residues are coloured red
D. Burg UNSW 335
Appendix A.7 KOD-1 Lon alignment
Mbur_0880 MEDEMTAT------EETELFGDDFNTTSSIDVPELMIDQVIGQEHAVEVVKKAASQRRH 53 KOD1_Lon MDEESTKERLIPREYGESLDLGIDFKTTEEIPVPEKLIDQVIGQEHAVEVIKTAANQRRH 60 *::* * *: :* **:**..* *** :*************:*.**.****
Mbur_0880 VMMIGTPGTGKSMLAKAMAELLPKEELEDIMTYPNLEDNNNPKIREVPAGKGREIVLAHK 113 KOD1_Lon VLLIGEPGTGKSMLGQAMAELLPTENLEDILVFPNPEDENMPKIKTVPACQGRRIVENYR 120 *::** ********.:*******.*:****:.:** **:* ***: *** :**.** ::
Mbur_0880 MEARKKAQSRNMMMMFLVLGIVMYSFYVG---QLLWGIIAAIMILMLTRQFMPKEELMIP 170 KOD1_Lon RKAKEQEGIKNYLLMFVIFTVILAIIMEPTATTLLMGMFVVLLSMMVLSNMRFRNTVLVP 180 :*::: :* ::**::: ::: : ** *::..:: :*: :: :: :::*
Mbur_0880 KLIVSNYQKEHAPYIDATGTHAGALLGDVRHDPFQSGGLETPAHDRVESGDIHKSHKGVL 230 KOD1_Lon KLLVDNCGRKKAPFVDATGAHAGALLGDVRHDPFQSGGLGTPAHERVEPGMIHRAHKGVL 240 **:*.* :::**::****:******************* ****:***.* **::*****
Mbur_0880 FIDEINTLRLESQQSLLTALQEKEYAITGQSERSSGALVKTEPVPCDFIMVAAGNLDAVE 290 KOD1_Lon FIDEIATLSLKMQQSLLTAMQEKKFPITGQSEMSSGAMVRTEPVPCDFILVAAGNLDTID 300 ***** ** *: *******:***::.****** ****:*:*********:*******:::
Mbur_0880 KMHPALRSRIKGYGYEVFMRDSMEDTLENRKSLVRFVAQEVVRDGHIPPFDKGAVKEVIR 350 KOD1_Lon KMHPALRSRIRGYGYEVYMRTTMPDTIENRRKLVQFVAQEVKRDGKIPHFTREAVEEIVR 360 **********:******:** :* **:***:.**:****** ***:** * : **:*::*
Mbur_0880 ESRRRAGRKGHLTLKFRDLGGLVRVAGDIAHSENAEITTATHVLAAKKIARSIEQQLADS 410 KOD1_Lon EAQKRAGRKGHLTLRLRDLGGIVRAAGDIAIKKGKKYVEREDVLEAMRMAKPLEKQLADW 420 *:::**********::*****:**.***** .:. : . .** * ::*:.:*:****
Mbur_0880 YLERRKDYQLFGKLGAAVGRVNGLAVMGGDSGIVLPIVAEVTPTQSSAGGHVIATGMLKD 470 KOD1_Lon YIENKKEYQVIKTEGGEIGRVNGLAVIGEQSGIVLPIEAVVAPAASKEEGKIIVTGKLGE 480 *:*.:*:**:: . *. :********:* :******* * *:*: *. *::*.** * :
Mbur_0880 IAKEAVQNVSAVIKKVTGEDISNHDIHIQFIGTYEGVEGDSASISIATAVISALENIPID 530 KOD1_Lon IAKEAVQNVSAIIKRYKGEDISRYDIHVQFLQTYEGVEGDSASISVATAVISALENIPIR 540 ***********:**: .*****.:***:**: *************:*************
Mbur_0880 QTVAMTGSLSVRGDVLPIGGATYKIEAAVQAGIKKVIIPKSNEADVLIEDYYKDKVEIIP 590 KOD1_Lon QDVAMTGSLSVRGEVLPIGGATPKIEAAIEAGIKKVIIPKANEKDVFLSPDKAEKIEIYP 600 * ***********:******** *****::**********:** **::. :*:** *
Mbur_0880 VTTIAEVIEHSLV-GEDKAGIVEKLKNMSNKKIDYAARSKKVAGSDDV 637 KOD1_Lon VETIDQVLEIALQDGPEKDELLRRIR------EALPLYGSS-- 635 * ** :*:* :* * :* ::.::: .: : **.
Appendix A.8 Stromatin alignment
Stomatin_C.elegans MQPSETVEMQEMAQPSGQQRDVEAR--VQSAPANHSHDAGCTEMFCIAMS 48 Stomatin_Human ------MAEKR-HTRDSEAQRLPDSFKDSPSKGLGPCGWILVAFS 38 Mbur_1804 ------MIEEY 5 Stomatin_P.Horikoshii ------
Stomatin_C.elegans YVLIFLTFPVSVFMCIKIVQEYQRAVVFRLGRLVP-DVKGPGIFFIIPCI 97 Stomatin_Human FLFTVITFPISIWMCIKIIKEYERAIIFRLGRILQGGAKGPGLFFILPCT 88 Mbur_1804 IIPILVIAVIILSQSLKMVKEYERVVIFRLGRLSG--VKGPGLFLIIPII 53 Stomatin_P.Horikoshii ------MIF 3
Stomatin_C.elegans DTFLNIDLRVASYNVPSQEILSRDSVTVSVDAVVYFKVFDPITSVVGVGN 147 Stomatin_Human DSFIKVDMRTISFDIPPQEILTKDSVTISVDGVVYYRVQNATLAVANITN 138 Mbur_1804 DSVVKIDLRVVTIDVPKQAVITKDNVTVAVDAVIYYRVLKPAAAVTEVEN 103 Stomatin_P.Horikoshii EKAVIVDLRTQVLDVPVQETITKDNVPVRVNAVVYFRVVDPVKAVTQVKN 53 :. : :*:*. ::* * :::*.*.: *:.*:*::* .. :*. : *
Stomatin_C.elegans ATDSTKLLAQTTLRTILGTHTLSEILSDREKISADMKISLDEATEPWGIK 197 Stomatin_Human ADSATRLLAQTTLRNVLGTKNLSQILSDREEIAHNMQSTLDDATDAWGIK 188 Mbur_1804 YKFATAMLSQTTLRDVIGQIELDDVLSKRDTINKDIQELLDASTDPWGIK 153 Stomatin_P.Horikoshii YIMATSQISQTTLRSVIGQAHLDELLSERDKLNMQLQRIIDEATDPWGIK 103 :* ::***** ::* *.::**.*: : ::: :* :*:.****
Stomatin_C.elegans VERVELRDVRLPSQMQRAMAAEAEATRDAGAKIIAAEGELRASAALAEAA 247 Stomatin_Human VERVEIKDVKLPVQLQRAMAAEAEASREARAKVIAAEGEMNASRALKEAS 238 Mbur_1804 VTAVTLRDVSIDETMLRAIAKQAEAEREKRARIILSEGEFLAAEKMRQAA 203 Stomatin_P.Horikoshii VTAVEIKDVELPAGMQKAMARQAEAERERRARITLAEAERQAAEKLREAA 153 * * ::** : : :*:* :*** *: *:: :*.* *: : :*:
Stomatin_C.elegans TIISKSEGAMQLRYLHTLNAISSEKTSTIIFPFPMEILGGISKVGSGGTS 297 Stomatin_Human MVITESPAALQLRYLQTLTTIAAEKNSTIVFPLPIDMLQGI--IG----- 281 Mbur_1804 QLYQDMPAAIKLREFQTIAEVAREKNLIVISTSSNTAEIAA------244 Stomatin_P.Horikoshii EIISEHPMALQLRTLQTISDVAGDK------SN------180 : . *::** ::*: :: :* .
Stomatin_C.elegans QNFPVQEMMNAALQSIQRQDTVPATASSSGSRL 330 Stomatin_Human ------AKHSHLG--- 288 Mbur_1804 ------LSKAFSQK--- 252 Stomatin_P.Horikoshii ------LEHHHHHH--- 188
336 D. Burg UNSW
Appendix A.9 Alpha-2-macroglobulin alignment
Ixodes ------MDATRLLLLTSAFLPLVAPQSSGIYTIVAPRKLRPN------36 Chlamys ------MLWVGLTTLLLGLAAAKDS--YVVITPKDVRPG------31 Homo ------MGKNKLLHPSLVLLLLVLLPTDASVSGKPQYMVLVP------36 Pongo ------MGKNKLLHPSLVLLLLVLLPTDASVSGKPQYMVLVP------36 Mus ------MRRNQLPTPAFLLLFLLLPRDATTATAKPQYVVLVP------36 Gallus ------MWLKFILAILLLHAAAGKEPEPQYVLMVP------29 Mbur_1707 ------MKYNGVTKKGFCLLVLLSCILLAGCIGQDDGNVQTSSNEALSYDEGGTY----- 49 Drosophila ------MMWHLLRALLVVAAVLDALQPAVGQNDNYYNPNQNQQNPQQPLLPNQQWGN 51 Oncorhynchus ------Eptatretus ------VLVIAPAATSSYDDLAVAILMVD------23
Ixodes ------Chlamys ------Homo ------Pongo ------Mus ------Gallus ------Mbur_1707 ------Drosophila NPQTNQYSNNNQNFGQTNPSDRPPYRTDSGSYNDIAGQDDYNKRVGGGYQDNEEPSLTRG 111 Oncorhynchus ------Eptatretus ------
Ixodes ------LKYHVSTSLSQSASSPPVDLRVTLSGPSDNGSSNRITKQVQLHDPFG 83 Chlamys ------VSLNISVNILQAAGDVHVTAKLIHVADKSVKAFSTGTFQQHVPDTMQ 78 Homo ------SLLHTETTEKGCVLLSYLNETVTVSASLESVRGNRSLFTDLEAENDV 83 Pongo ------SLLHTEAAEKGCVLLSYLNETVTVSASLESVRGNRSLFTDLEAENDV 83 Mus ------SEVYQESLKRPCVSLNHVNETVMLSLTLEYAMQQTKLLTDQAVDKDS 83 Gallus ------AVLQSDSPSQVCLQFFNLNQTISVRVVLEYDTINTTIFEKNTTTSNG 76 Mbur_1707 ------SEEDEYLVLVPKALFSGGESSVTMSAFLDDEPVSRGVEYTLTSAAGD 96 Drosophila KSSYNIKATFLESLHSREPTYFIVASRMVRPGLIYQVSVSILQAQYPITVHASIACDGVQ 171 Oncorhynchus ------FVESQDHVGGPLNVKIMVKNHPTQSKELASKSVVLDQANNFQA 43 Eptatretus ------QKKITEVHVLLVNPHTGATLDEKKVKLQWDNKFIA 58
Ixodes LLREQRWSSQVGDWGPGKYKLSASGSGGLDFFNE------TELTYEHKSYS 128 Chlamys IMIPDMIP------SGTNQLTVEGSNGLTFSGK------TNLHYASKGMS 116 Homo LHCVAFAVPKSSSNEEVMFLTVQVKGPTQEFKKR------TTVMVKNEDSL 128 Pongo LHCVAFAIPKSSSNEEVMFLTVQVKGPTQEFKKR------TTVMVKNEDSL 128 Mus FYCSPFTIS--GSPLPYTFITVEIKGPTQRFIKK------KSIQIIKAESP 126 Gallus LQCLNFMIP-PVTSVSLAFISFTAKGTTFDLKER------RSVMIWNMESF 120 Mbur_1707 IIPLVKAATSESGNNVAKFDVPDVEEGSYTLTATPSGAESG------FTTTVKVMQNNP 149 Drosophila ISGDSKDVKEGIPETLLMRIPPTSVTGSYKLRVEGFYQNVFGGLAFLNETRLDFSQRSMT 231 Oncorhynchus MTQLVIQRGPLVDDPKQKQYVVLQAQFPDRLLEK------VVLVSFQSGY 87 Eptatretus FTKLQVTPKEVEKWKEDFVRLMVKWDGGQHMEID------IPLTSRRGL 101 : :
Ixodes VFVQTDKAVYKPGQKVLFRVIVMDPYLLPTVTGAMNVHVTDAKGNRIHQWDRVLTQ---- 184 Chlamys VFIQTDKAMYKPGQTVNFRAFAIFPNLT-VYSGPLDIEIYDPNSNKIKQWFGMKDS---- 171 Homo VFVQTDKSIYKPGQTVKFRVVSMDENFHPLNELIPLVYIQDPKGNRIAQWQSFQLE---- 184 Pongo VFVQTDKSIYKPAQTVKFRVVSMDENFHPLNELIPLVYIQDPKGNRIAQWQSFQLE---- 184 Mus VFVQTDKPIYKPGQIVKFRVVSVDISFRPLNETFPVVYIETPKRNRIFQWQNIHLA---- 182 Gallus VFVQTDKPIYKPGQSVMFRVVALDFNFKPVQEMYPLIAVQDPQNNRIFQWQNVTSE---- 176 Mbur_1707 IFIETDKPIYKPGQIIHVRLLSLNNNLIPVVQN-TTVEIADAKGVKIYKDDLVTNE---- 204 Drosophila IFVQTDKPLYMQGETVRFRTIPITTELKGFDNP-VDVYMLDPNRHILKRWLSRQSN---- 286 Oncorhynchus IFIQTDKTIYTPASTVHYRVFSMTPGLEPLTREIFEDQEVAKNKEIAVSVEIMTPENITI 147 Eptatretus VFAQTDQPIYTPNNDVNIRLFPVTRQLNPILSS-LVVDIMNPDGVVVDRIEKNAFEV--- 157 :* :**:.:* . : * . : : .
Ixodes ------KGIYSSELQLSDQPVLGDWAIHVDILG---QKYSKNFTVAEYVLPTFEVRVK 233 Chlamys ------SGVITNFMAMDTKPVLGDWKIRVKTYGG--LTKDKMFTVARYVLPKFEVTVD 221 Homo ------GGLKQFSFPLSSEPFQGSYKVVVQKKSG--GRTEHPFTVEEFVLPKFEVQVT 234 Pongo ------GGLKQFSFPLSSEPFQGSYKVVVQKKSG--RRTEHPFTVEEFVLPKFEVQVT 234 Mus ------GGLHQLSFPLSVEPALGIYKVVVQKDSG--KKIEHSFEVKEYVLPKFEVIIK 232 Gallus ------INIVQIEFPLTEEPILGNYKIIVTKKSG--ERTSHSFLVEEYVLPKFDVTVT 226 Mbur_1707 ------YGVAFFDLPLASELNLGTWKVKATSGS---SMSEVDIRVEKYVLPKFDLETS 253 Drosophila ------LGSVSLEYKLSDQPTFGEWTIRVIAQG---QQEESHFTVEEYYQTRFEVNVT 335 Oncorhynchus FREIVNPDKGVKSGQFKLPDIVSFGTWHVVTRFQSTPQKTFSSEFEVKEYVLPSFEVSLT 207 Eptatretus ------EKVMELRPFHVPAITSLGDWKIVSWMKDKPQFNYTSGFKVEEYVLPTFDVSIT 210 : * : : . : * .: . *::
Ixodes LPAYATYNKS-EVVATVSATYTYGKPVKGTVTLTVAP------269 Chlamys LPSYDWTTAT-SILGAVKAKYTYGKPVNGTVKIRAHADFY------260 Homo VPKIITILEE-EMNVSVCGLYTYGKPVPGHVTVSICRKYS------273 Pongo VPKIITILEE-EMNVSVCGLYTYGKPVPGHVTVSICRKYS------273 Mus MQKTMAFLEE-ELPITACGVYTYGKPVPGLVTLRVCRKYSR------272 Gallus APGSLTVMDS-ELTVKICAVYTYGQPVEGKVQLSVCRDFD------265 Mbur_1707 TEKSWFLADE-PITGTVSANYFFGKVVEGTVEVKASRYVG------292 Drosophila MPAYFFTTDP-FIYGRVMANFTSGLPVRGNLTIKATIRPIG-----YFSNQVLNEKYRLG 389 Oncorhynchus PAKAFFYVDDNDLTVDITARYLYGKEVTGTGYVVFGVITTE------248 Eptatretus SEQPYLHVYDKAFTIHIKAMHIYGKPVMGRAYVRYGVKHQS------251 . . . * * * :
D. Burg UNSW 337
Ixodes ------RTRYHQLRPRPYEQYQTKAEI------290 Chlamys ------HYNYYHPAPIPTIELTMDING------281 Homo ------DASDCHGEDSQAFCEKFSGQLN------295 Pongo ------DASNCHGEDSQAFCEKFSGQLN------295 Mus ------YRSTCHNQNSMSICAEFSQQAD------294 Gallus ------SYGRCKKS-PVCQSFTKDLD------284 Mbur_1707 ------VWEEYSTSTATLK------305 Drosophila RSPLEQTNLYNERWRYNNPNQNPQVQYNVPGQLPQDGADLSQDILYRNQYVVERHYQFDE 449 Oncorhynchus ------SEKKSFPASLQRVEIKDGK------267 Eptatretus ------KRTLLSTSSALARFEQGE------269
Ixodes ------DGSVDIPVAVVRDLSLKTDFFRRDIEF 317 Chlamys ------ETKFTLPVSGLTSHTYYTSLNSRNVVV 308 Homo ------SHGCFYQQVKTKVFQLKRKEYEMKLHT 322 Pongo ------SHGCFYQQVKTKVFQLKRKEYEMKLHT 322 Mus ------DKGCFSQVVKTKVFQLSQKGHDMKIEV 321 Gallus ------TDGCLSHILSSKVFELNRIGYKRNLDV 311 Mbur_1707 ------DGNVEFTLPAVGYVAGTFGAGGQGSVM 332 Drosophila EWPFWVRKPEYQDSSYEAWSGTYRKTLPYLRYFNGTFDFKWPLRELELLVPNLANSEVLI 509 Oncorhynchus ------GVACLKKEHITQTFPKIHDLVKQSIFV 294 Eptatretus ------AMHTLRQKHILEQYPDPKLLLGQSLYV 296
Ixodes FALVEERLTGRKYNSTSYLTLHDKEVKVELVKTS-ETFKPGLKYTCFLKVAYQDDTPVHD 376 Chlamys EANVTESLTQITLNGTGKMHFYTHAEKIELLPSNPTTFKPGLQYIAYAKVVQQDDMPLAA 368 Homo EAQIQEEGTVVELTGRQSSEITRTITKLSFVKVD-SHFRQGIPFFGQVRLVDGKGVPIPN 381 Pongo KAQIQEEGTVVELTGRQSSEITRTITKLSFVKAD-SHFRQGIPFFGQVRLVDGKGVPIPN 381 Mus EAKIKEEGTGIELTGIGSCEIANALSKLKFTKVN-TNYRPGLPFSGQVLLVDEKGKPIPN 380 Gallus KAIVTEKEQVCNLTATQSISITQVMSSLQFENVD-HHYRRGIPYFGQIKLVDKDNSPISN 370 Mbur_1707 LNVTVTDTGGNSEESTELLTIAEHPFIMQMIPES-NSIKPGMPLQVLLVTKDPGGEPLEK 391 Drosophila TATVGEKFYDEIISGYSVARVYNSSLRVVFLGDSPQVFKPAMPFTTYLAVEYHDGSPIDP 569 Oncorhynchus SVSVLTEGGGEMVEAEKRGIQIVTSPYSILFKRTPKYFKPGMPFDVSVYITNPDNSPAIG 354 Eptatretus EASVISSDAGEIENSILDDIPIVASPYSIKSKWTVPFFKPGVPYIYKVLVLNPDGSPASG 356 . : .: . *
Ixodes AVN------QLTLYQGFNFNEDLWKTSRHWVPANGVVRLELFPPNDNAT 419 Chlamys GSSKSLTVHTSVTANLPETTTPLYYYGPRTMNYQLPDQSFTITDTGLVQAKIDIPDNATS 428 Homo KVIFIRG------NEANYYSNATTDEHGLVQFSINTTNVMGTSLTVRVNYKDRS 429 Pongo KVIFIRG------NEANYYSNATTDEHGLVQFSINTTNVMGTSLTVRVKYKDRS 429 Mus KNITSVV------SPLGYLSIFTTDEHGLANISIDTSNFTAPFLRVVVTYKQNH 428 Gallus KVIQLFV------NNKNTHN-FTTDINGIAPFSIDTSKIFDPELSLKALYKTSD 417 Mbur_1707 EVTLVAN------FRDDNYNYEEIKETYTTETGIALVTLEIPED 429 Drosophila NLLRQGLMEVS------GFVESRNGGRRDWPAQRLPMSQQSDGIWEVKIDIRNDLNL 620 Oncorhynchus VEVEVTP------DHAKGVTRANGFAKIPLNTVASATELVITVKTKDPGD 398 Eptatretus VPIKVSFS------FDSSGNWITQKRKTMDNGIAMQTINTARNSKKLNIKV 401
Ixodes VVLGLRAEFRGQTHYLEGIYPARSPTRSFLQAWVTTEDPMIG--DLVEVEVNS------470 Chlamys ISLQFKYGQITQYHSVQRSY---SPSDSFIQIFLESNNLQAGHDKVVDFRVVS------478 Homo PCYGYQWVSEEHEEAHHTAYLVFSPSKSFVHLEPMSHELPCGHTQTVQAHYILNGGTL-- 487 Pongo PCYGYQWVSEEHEEAHHTAYLVFSPSKSFVHLEPVSHELPCGQTQTVQAHYILNGGAL-- 487 Mus VCYDNWWLDEFHTQADHSATLVFSPSQSYIQLELVFGTLACGQTQEIRIHYLLNEDIM-- 486 Gallus QCHSEGWIEPSYPDASLSVQRLYSWTSSFVRIEPLWKDMSCGQKRMITVYYILNTEGY-- 475 Mbur_1707 AVSVELSAASEKVKASSTLNSVYSPSASFLHLSQVSEGIPEVG-DVISFKVYS------481 Drosophila DDRPQARDFLNGVQNMRLQANFVDPRGERIQTELLLVSHYSPRNQHIKVTTSTEKPVVGE 680 Oncorhynchus P------RQQTGGGTMKALPYRTSTKNFLHVGVDSNELKIGDPIKIDLNLGP------444 Eptatretus QTEDERLEQSQQAEASFTIASYSSPSGSFIHLNAHREVKSPGEHIVFDVFIKS------454 . :: .
Ixodes ------TQPLDHLVYEVMGRGDIVFA-----QTLPASGVRTYRFSFSTSFRMAPRARV 517 Chlamys ------TSPIDKLVYQVLGRGSIAVS-----GAINGNNAKAFPFNVPLNAKMAPNARI 525 Homo ------LGLKKLSFYYLIMAKGGIVRTGTHGLLVKQEDMKGHFSISIPVKSDIAPVARL 540 Pongo ------QGLKKLSFYYLIMAKGGIVRTGTHGLLVKQEDMKGHFSISIPVKSDIAPVARL 540 Mus ------KNEKTLTFYYLIKARGSIGNLGSHVLSLEQGNMKGVFSLPIQVEPGMAPEAQL 539 Gallus ------EHINIVNFYYVGMAKGKIVLTGEIKVNI-QADQNGTFMIPLVVNEKMAPALRL 527 Mbur_1707 ------TNAGTVFYDVFANGRTVYS------ATSDEPQINILVTPQMSPSAKV 522 Drosophila YIIFHIRTNFYLEEFNYLIMSKGVILVN------DRETITEGIKTIAVVLSSEMAPVATI 734 Oncorhynchus ------TTIPNHDLTYMFLSRGQLVKVG-----RFKRQGNALVTLSVPVSKELLPSFRI 492 Eptatretus ------AAKDHVLHFNYLMISNGKIHNFLQ------EGRKGDTTSVSLLLTPELVPQFRL 502 . * ..* . . : * :
Ixodes LVYYVRKDGELVADAVNFDLGGILRTPVQVQSNLAET--KPGGQVDILVSTRPNAYVGLL 575 Chlamys VAYYVRADGEIVTDSISFDVSGTFENDVSIRFDKSKA--QPGDGINVDVSADPNSIVNLL 583 Homo LIYAVLPTGDVIGDSAKYDVENCLANKVDLSFSPSQS--LPASHAHLRVTAAPQSVCALR 598 Pongo LIYAVLPTGDVIGDSAKYDVENCLANKVDLSFSPSQS--LPALHAHLRVTAAPQSLCALR 598 Mus LIYAILPNEELVADAQNFEIEKCFANKVNLSFPSAQS--LPASDTHLKVKAAPLSLCALT 597 Gallus LVYMLHPAKELVADSVRFSIEKCFKNKVQLQFSEKQM--LTTSNVSLVIEAAANSFCAVR 585 Mbur_1707 VAYLINPNNEVSADSLPFDVKFSTQVDLSTGFDEEMV--APGDAVSVELDAGTKSMIGLS 580 Drosophila VVWKINQQGQVVADSLTFPVNGISRNNFTVYINNRKA--RTGEKVEVAIFGEPGSYVGLS 792 Oncorhynchus VAYYHVGAADLVADSVWVDIKVSCMGSLKVTSTRPKASYEPRRAFSLTITGDPGAKVGLV 552 Eptatretus VAFFILPSGELVADSIIIDVKDSCHAKLSLDVAGGKRLFSPRDNVNFDLSGESDSWVAVG 562 : : :: *: : . . . : . : :
Ixodes GVDQSVLLLKKGNDLSQEQVIEELESFDSGKQAR------VWPPWYR--- 616 Chlamys AVDQSVLLLKSGNDITPAEVVDELKSYDTIVHSN------NNGPIFFGGG 627 Homo AVDQSVLLMKPDAELSASSVYNLLPEKD--LTG------FPGPLNDQDD 639 Pongo AVDQSVLLMKPDAELSASSVYNLLPEKD--LTG------FPGPLNDQGD 639 Mus AVDQSVLLLKPEAKLSPQSIYNLLPGKT--VQGA------FFGVPVYKDH 639 Gallus AVDKSMLLLKSETELSAETIYNLHPIQD--LQGY------IFNGLNLEDD 627 Mbur_1707 IVDESVYALSEG-RLNLKQVFDELEIRF------607 Drosophila GIDSAFYTMQAGNELTYAKIITKMSNFDEQTNGT------YKHIWYSHEG 836 Oncorhynchus AVDKGVYVLNSKHRLTQTKIWDTIEKHDTGCTAGGGADNMGVFYDAGLVFETNTAKGTGI 612 Eptatretus VVDKAAYVLDKKNKLTANKVYKAMEASDLGCSVGSGKTGPLVFRDAGLAIMAKEISGMDD 622 :*.. :. :. :
338 D. Burg UNSW
Ixodes ------RRRRSLWWP-----GSTTAHDLFKDSGMVVLTNGLVYESDD------652 Chlamys GIMPEPMPVGRRKRMIWWPFTTYYGGSDAEQIFQNAGVNVMTDALVYHHVEPHIYLPTFQ 687 Homo --EDCINRHNVYINGITYTPVSSTNEKDMYSFLEDMGLKAFTNSKIR-KPKMCPQLQQYE 696 Pongo --EDCINRHNVYINGITYTPVSSTNEKDMYSFLEDMGLKAFTNSKIR-KPKLCPQLQQYE 696 Mus --ENCISGEDITHNGIVYTPKHSLGDNDAHSIFQSVGINIFTNSKIH-KPRFCQEFQHYP 696 Gallus PQDPCVSSDDIFHKGLYYRPLTSGLGPDVYQFLRDMGMKFFTNSKIR-QPTVCTRETVRP 686 Mbur_1707 ------MEPRVEAHPQYFGYQTTAYDVLDDAGMIVLASGSLEIPRSQTNDAKFID 656 Drosophila ------NPDELVYFPASSFGVDANRTFEYSGLIVFTDGYVPRRQDTCNRTLGFG 884 Oncorhynchus RTDPSCPVSSRRRRAVTISDVITSMASKYHGLAKECCVDGMRDNTMGYTCDRRAQYISDG 672 Eptatretus VKDPGCPNGHTRRKRELVLEIAIEKASTYPAELRKCCRDAAIESPLRLSCEERTKHIHDE 682 . :
Ixodes ------GLFARKQVIRLDTDVLTNPVLPPSDLPEAPPPVPG------687 Chlamys HHGFLGGFGGFESVHALAGVSSAVNSIGMAPGAGSAAIPSHPQPQHIDNHATQDLKE--- 744 Homo MHG---PEGLRVGFYES------DVMGRGHARLVHVEEPH------727 Pongo MHG---PEGLRVGFYES------DVMGRGHARLVHAEEPP------727 Mus AMGGVAPQALAVAASGPGSS-----FRAMGVPMMGLDYSDEINQVVEV------739 Gallus PSY-----FLNAGFTAS------THHVKLSAEVAREERGKRHI------718 Mbur_1707 FAAAVFEMDGMEVMDEAVMEMPREEAAMDDEADSGEQLAEVER------699 Drosophila ECLSGRCYRLEKQCDGLFDCDDGTDEINCHARNDTELLNYRKYR------928 Oncorhynchus DVCVQAFLVCCTEMASKKIESKQDALLLSRSEEDDDDDAYMRSE------716 Eptatretus GEGCQETFLECCKHVEEELLIAMEEEDEDLGRSQGEDFMIQESQ------726
Ixodes --RIRLRQQYPETWLWSNVTASHD------GRVVISSTVPDTITSWVISAFALDSLT 736 Chlamys --PARVRLVFPETWLWTNRTVGAD------GHVTIAATVPDTITSWVASAFAVHPTS 793 Homo --TETVRKYFPETWIWDLVVVNSA------GVAEVGVTVPDTITEWKAGAFCLSEDA 776 Pongo --TETVRKYFPETWIWDLVVVNSS------GVAEVGVTVPDTITEWKAGAFCLSEDA 776 Mus --RETVRKYFPETWIWDLVPLDVS------GDGELAVKVPDTITEWKASAFCLSGTT 788 Gallus --LETIREFFPETWIWDIILINST------GKASVSYTIPDTITEWKASAFCVEELA 767 Mbur_1707 -----VRQFFPETWIWMPEILTDDN------GLATLDLNAPDSITKWRLHAVSSGP-E 745 Drosophila --FNRVLRHYENVWLWKDVNIGPH------GRYIFNVEVPDRPAYWMVSAFSVSPSK 977 Oncorhynchus --DIVSRSQFPESWMWEDTNLPECPAQNKHCESTSVIRNNFLKDSITTWQITAISLSKTH 774 Eptatretus ---VVIRSHFPESFMWEIIKLSRSAE------NGKSRITKKMPDSITTWDIQAVEVSQSK 777 : : ::* * : * *.
Ixodes GLGIAPSQAKVTVFRPFFVTASLPYSILRGESVAIQCVVFNYNNKPVQARVTLENAKSEF 796 Chlamys GLGIAPTSAKVEAFRPFFVSLTLPYSVVRGEQLVLQANVFNYMTTDMDVVVTLE-KNDDL 852 Homo GLGIS-STASLRAFQPFFVELTMPYSVIRGEAFTLKATVLNYLPKCIRVSVQLEASPAFL 835 Pongo GLGIS-STASLRAFQPFFVELTMPYSVIRGEVFTLKATVLNYLPKCIRVSVQLEASPAFL 835 Mus GLGSS-STISLQAFQPFFLELTLPYSVVRGEAFTLKATVLNYMSHCIQIRVDLEISPDFL 847 Gallus GFGMS-VPATLTAFQPFFVDLTLPYSIIHGEDFLVRANVFNYLNHCIKINVLLLESLDYQ 826 Mbur_1707 GIGIS--EAGLTVFQDFFIDPDLPYAVIRGEEFPVQVQVYNYLDMPQNVKLTLSGAEWFE 803 Drosophila GFGMMNKALEYVGVQPFFINVEMPEACRQGEQVGIRVTVFNYMITPIEAIVVLHDSPDYK 1037 Oncorhynchus GICVA-DPFEMIVLKEFFIDLKLPYSAVRNEQLEVKAILHNYSEDPIIVRVELMENGEVC 833 Eptatretus GLCVG-PSLELTVFKQFFLKVHTPYALKQYEQVELRVVIYNYMNQDVKGEIQVKCGDGIC 836 *: .: **: * : : * . :: : ** : :
Ixodes VFTSLSNDVGGEQSKD----RRSKEVTVPAQDGVAVSFLITPTKLGYIDIH----VSATS 848 Chlamys VNVVFDTQGAESYIAQ----TTAKTVHVTAGGSKSVFFPVVPAGLGSVSIN----VKAQS 904 Homo AVPVEKEQAPHCICANG---RQTVSWAVTPKSLGNVNFTVSAEALESQELCGTEVPSVPE 892 Pongo AVPVEKEQAPHCICANG---RQTVSWAITPKSLGNVNFTVSAEALESQELCGTEVASVPE 892 Mus AVPVGGHENSHCICGNE---RKTVSWAVTPKSLGEVNFTRTAEALESQELCGNKLTEVPA 904 Gallus AKLISPEDD-GCVCAKI---RKSYVWNIFPKGTGDVLFSITAETND-DEACEEEALRNIR 881 Mbur_1707 LVG------DDVVEVGVDANSVTHVSFTIRPTRVGVQTVE----LTGQT 842 Drosophila FVHVEEDGIVRSYNPRTSFGEHQFFIYLEAQGTTVVYVPVVPQRLGNVDVT----LHVAT 1093 Oncorhynchus SSASKKG------KYRQEVNMDPMSTRVVPYVIIPMKLGLHSIEVK--ASVKN 878 Eptatretus TDAEQNE------PLKSRFAVEKNSATSFSFMVVPLSSSDSSVS--VLARVFG 881 : . . .
Ixodes SLAGDSILKKLLVKPEGSKQHFNRAVLVDRRN------PSAPPTSTNISIPIPKNAVP 900 Chlamys TLAADAVRRQLLIEAEGVPKEYNIPMLVDLKH------NTN--FAETVDVTLPAGVVA 954 Homo HGRKDTVIKPLLVEPEGLEKETTFNSLLCPS------GGEVSEELSLKLPPNVVE 941 Pongo YGKKDTVIKPLLVEPEGLEKETTFNSLLCPS------GGEVSEELSLKLPPNVVE 941 Mus LVHKDTVVKSVIVEPEGIEKEQTYNTLLCPQ------DTELQDNSSLELPPNVVE 953 Gallus IDYRDTQIRALLVEPEGIRREETQNFLICMK------DDVISQDVAIDLPTNVVE 930 Mbur_1707 TEKADAIRKTIIVEAEGVTREIVDNGILKNG------TVELDATLPDAIVP 887 Drosophila LLGTDTITRTLHVESDGLPQYRHQSVLLDLSNRAYVLEYMHVNVTQTPEIPYQVDRYFVY 1153 Oncorhynchus SGSNDGVKRDLRVVAEGVLVKKETNVLLNPVK------HGGEQTSHIPSGVPRNQVP 929 Eptatretus SDVHDAVEKDLRVMPEGNYEEMSRSWSVQPRR------HGGQQVIVVDNETPQNVVP 932 * : : : .:* : *
Ixodes GSERISVSAVGD------LLGPHVN------NLDQLLVMPHGCGEQNMLDFVPNVVVLDY 948 Chlamys GSQRVRISAIGD------LMGPTVN------GLDKLLRMPTGCGEQTMLGFAPDVFVTNY 1002 Homo ESARASVSVLGD------ILGSAMQ------NTQNLLQMPYGCGEQNMVLFAPNIYVLDY 989 Pongo ESARASVSVLGD------ILGSAMQ------NTQNLLQMPYGCGEQNMVLFAPNIYVLDY 989 Mus GSARATHSVLGD------ILGSAMQ------NLQNLLQMPYGCGEQNMVLFVPNIYVLNY 1001 Gallus GSPRPSFSVVGD------IMGTAIQ------NVHQLLQMPFGNGEQNMVLFAPNIYVLDY 978 Mbur_1707 DSEKVILSFTPS------IVAQTIS------GVDDLLGMPYGCGEQNMMLFSTDVEVLRY 935 Drosophila GSNKARISVVGD------VVGPIFPTMP--VNASSLLSLPMESGEQNAFSFAANLYTIMY 1205 Oncorhynchus NSDADTLISVTAGEQTSVLVEQAISG----DSLGSLIVQPVGCGEQNMIYMTLPVIATHY 985 Eptatretus GTEMSAFLSAQG-----NLVAETIQNTLKGSKISNLLRLPRGCGEQNMMYTSITVMVARY 987 : :: . .*: * ***. . : . *
Ixodes LRRANRLSPA----VRGKALRNLEDGYQRQLTYKRDDNSFSAFGNT---DRSGSTWLTAF 1001 Chlamys LTDTHQLTSS----VEEKAINFMEKGYQRELTFQHKDGSFSAFGDN---DPSGSMWLTAF 1055 Homo LNETQQLTPE----VKSKAIGYLNTGYQRQLNYKHYDGSYSTFGERYGRNQ-GNTWLTAF 1044 Pongo LNETQQLTPE----IKSKAIGYLNTGYQRQLNYKHYDGSYSTFGERYGRNQ-GNTWLTAF 1044 Mus LNETQQLTEA----IKSKAINYLISGYQRQLNYQHSDGSYSTFGNHGGGNTPGNTWLTAF 1057 Gallus LDKTRQLSED----VKSKTIGYLVSGYQKQLSYKHPDGSYSTFGIR---DKEGNTWLTAF 1031 Mbur_1707 LKATGQQNPE----VQAKALTFITTGYQRELTFMHSDGSFSAFGES---DGEGSLWLTSF 988 Drosophila MRLINQRNKT----LEKNAFYHMNIGYQRQLSFMRPDGSFSLFRSDWN-NSDSSVWLTSY 1260 Oncorhynchus LDNTKKWEDIG-LDKRNTAIKYINIGYQRQLAYRKEDGSYAAWVSR-----QSSTWLTAY 1039 Eptatretus LNRSDQWNKMGDPQLKKRSFDFITSGFASQLTYRKPDYSYAAWLHR-----ASSTWLTAF 1042 : : . :: : *: :* : : * *:: : .. ***::
D. Burg UNSW 339
Ixodes VLKSFVQA-----VPYTSVDPAVLENATRWLVE-RQKPDGSFEEPGEVIYKPMQ---SGA 1052 Chlamys VAKSFHQA-----KRHVFIDDETLTRAIDWMIN-RQAANGSFPEPGRIIHKNMQ---GGS 1106 Homo VLKTFAQA-----RAYIFIDEAHITQALIWLSQ-RQKDNGCFRSSGSLLNNAIK---GGV 1095 Pongo VLKTFAQA-----RAYIFIDEAHITQALIWLSQ-RQKDNGCFRSSGSLLNNAIK---GGV 1095 Mus VLKAFAQA-----QSHIFIEKTHITNAFNWLSM-KQKENGCFQQSGYLLNNAMK---GGV 1108 Gallus VYKSFAEA-----SRFIYIDDNVQAQTLIWLAT-KQKTDGCFQSTGILVNNAMK---GGV 1082 Mbur_1707 VLSQFSGA-----RDLTTIDENIVREAAEWIGS-YQKEDGSWEAVGFVIHEDMM---GGV 1039 Drosophila CLRVFQEASFYEWENFIWIDATIIEKNMRWLLQ-HQTPQGSFFEVTWLPDRKMNR--TNF 1317 Oncorhynchus VVKVFAMS-----STLISVQENVLCTAVKWLILNTQQPDGIFNEFAPVIHAEMTGNVRGS 1094 Eptatretus VAKVFSQA-----RQLVFIPVSEICGSVRWLMR-KQDKDGSFLESKPVVHLNMMG---QV 1093 * : : *: * :* : : :
Ixodes GSGAALTAYVLIALLEN---KVGFQHALRFAASAAEEFLLK-----ELRTQSDPYVVAVV 1104 Chlamys ASGASLTAFVLIALLENSDLQGGVHMRIQSAASKAQAYLEG-----EVSAMTDPYGLSIC 1161 Homo EDEVTLSAYITIALLEIP--LTVTHPVVRNALFCLESAWKTAQ---EGDHGSHVYTKALL 1150 Pongo EDEVTLSAYITIALLEIP--LTVTHPVVRNALFCLESAWKTAQ---EGDHGSHVYTKALL 1150 Mus DDEVTLSAYITIALLEMP--LPVTHSAVRNALFCLETAWASIS---QSQE-SHVYTKALL 1162 Gallus ENELSLSAYITIALLEAG--HSMSHTVIRNAFYCLETASE------KNITDIYTQALV 1132 Mbur_1707 SGTYALTAYVTLALDEYG------YAAPIVMENARSYLEA-----ELDKQDDPYALAIG 1087 Drosophila DKNITLTSHVLITLATVKDISGTLGSRVALATQRALAYIERNMD--FLRHQAQPFDVAIT 1375 Oncorhynchus DNDASMTAFVLIAMQEASSVCEQSVNSLPGSMAKAVAYLEK-----RLPHLTNPYAVAMT 1149 Eptatretus TGKVVLTSFVFIALLEARESCINEVEGFTVVVEKAHGYLTS-----QAMNGLEDFPLAIT 1148 :::.: ::: . : ::
Ixodes TYALHLSGHRA-RDGA-FQKLLSLATREDDMVFWKDP----GVAPVNTTDKQSDFFFKA- 1157 Chlamys SYALTLASSQS-SATT-FQKLMAKAVTKDGMTHWHEP----ESAAPSTGHYWSPPHQQS- 1214 Homo AYAFALAGNQDKRKEV-LKSLNEEAVKKDNSVHWERP----QKPKAPVGHFYEPQA---- 1201 Pongo AYAFALAGNQDKRKEV-LQSLHEEAVKKDNSVHWERP----QKPKAPVGHFYEPQA---- 1201 Mus AYAFALAGNKAKRSEL-LESLNKDAVKEEDSLHWQRPG---DVQKVKALSFYQPRA---- 1214 Gallus AYAFCLAGKAEICESF-LRELQKSAKEVDGSKYWEQN----QRSAPEKSHLLDHV----- 1182 Mbur_1707 TLALQKLESDR-ADES-MEKLLELAKEDENGVYWGYDD-VMPEPYEYGGYGFHPI----- 1139 Drosophila AYALQLC-NSPIAEEV-FAILRRQARTIGDFMYWGNQEIPQPPRKLENQKWFSLPRLPY- 1432 Oncorhynchus SYALANAGKLN------KETLLKFASP--QLDHWPVPG------1179 Eptatretus AYALSLWKVSDGAAKVTMHTLKTSGLQTEELIHWGSNK------1186 : *: * . .*
Ixodes -----HFKDVEMTAYALLTLMER------GDVSAAIPVMRWLVSKQNSNGGYSST 1201 Chlamys -----KPVDIEMTSYGLMVFAHN------SQFTEGLPFMKWITKQRNPNGGFSST 1258 Homo -----PSAEVEMTSYVLLAYLTAQPAP-----TSEDLTSATNIVKWITKQQNAQGGFSST 1251 Pongo -----PSAEVEMTSYALLAYLTAQPAP-----TSEDLTSATNIVKWITKQQNAQGGFSST 1251 Mus -----PSAEVEMTAYVLLAYLTSESSRPTRDLSSSDLSTASKIVKWISKQQNSDGGLLLT 1269 Gallus -----QSTDVEITSYVLLALLYK-PNR-----SQEDLTKASAIVQWIIRQQNSYGGFASM 1231 Mbur_1707 -----SSKNVETTAYATLALIET------NDPRASSSLKWIAAQRNSNGGFSST 1182 Drosophila ---EYDSLNIETTAYALLVYVAR------REFFVDPIVRWLNSQRLNDGGWAST 1477 Oncorhynchus ----GYQYTLEATSYALLALVKVK------AFEEAGPIVRWLNKQKKVGGGYGST 1224 Eptatretus ----GKAAAVESTAYGLLAAIQHE------EGEIAEKATNWLSQSATFGGYFQST 1231 :* *:* :. .*: . *
Ixodes QDTVIGIQALARLAA--SVVS-QTIAVDASVKYGDGRKRTLKIHSGNALVLQRIELPSDL 1258 Chlamys QDTVLALQSLSEFAR--IGYS-EHFDMQIGIVAG-QTTHTFSVTRQNALLLQSLELPSIP 1314 Homo QDTVVALHALSKYGA--ATFTRTGKAAQVTIQSSGTFSSKFQVDNNNRLLLQQVSLPELP 1309 Pongo QDTVVALHALSKYGA--ATFTRTGKAAQVTIQSSGTFSNKFQVDNNNRLLLQQVSLPELP 1309 Mus QDTVVALQALSKYGS--ATFTRSQKEVLVTSRSSGTFSKTFHVNSGNRLLLQEVRLPDLP 1327 Gallus QDTVVALQALAAYGA--ATYN-SVTQNVIKINSKNTFEKVFTVNNENRLLLQQTPLPQVP 1288 Mbur_1707 QDTVMAFRALMTAAA--VAGR--DVDATVTVSGDGVEIKGLRITSENYDVVNIIEVPEGV 1238 Drosophila QDTSAALKALVEYTVRSRLREVSSLTVEIEASSQGGKTQTLYIDDTNLAKLQSIEIPDAW 1537 Oncorhynchus QSTIMVFQAVAEYWSHVKDLKDFDLNINLEVAGRASVTK-WSINNKNQFHTRTDKVNSID 1283 Eptatretus QDTVMALQALTGFESCQSRMKKMDLSFKIRAEENGVFDKEFQITNDNAFVQKPFKVP-VH 1290 *.* :::: : * . :
Ixodes KYVEIESSGFGVAIIQVSWSFNLAVSSE------APAFFLNPL------LDKTSTESYLQ 1306 Chlamys SHVTVTGTGSGMGLVEVSVFFNVEQEVE------QPSFEVDVT------IMEETIN-SLK 1361 Homo GEYSMKVTGEGCVYLQTSLKYNILPEKE----EFPFALGVQTLPQT---CDEPKAHTSFQ 1362 Pongo GEYSMKVTGEGCVYLQTSLKYNILPEKE----EFPFALGVQTLPQT---CDEPKAHTSFQ 1362 Mus GNYVTKGSGSGCVYLQTSLKYNILPVADG---KAPFALQVNTLPLN---FDKAEDHRTFQ 1381 Gallus GKYSLTVNGTGCVLIQTALRYNIHLPEG----AFGFSLSVQTSNAS---CPRDQPGK-FD 1340 Mbur_1707 EVLEMELSGSGELNYQLVRRFNVILPEIIEHEQIELDVEYDST------DVEVDDVVKVD 1292 Drosophila GTIKVQAKGAGYAILQMHVQYNVDIEKFQT-KPPVPAFGLHTKA-----IFHGRNQSHIS 1591 Oncorhynchus KDLTVKASGNGEATLSVVTLYYALPEEK-DSDCESFDLSVTLT------KMDKTSHEDAK 1336 Eptatretus GQLTVTASGTGQGILTFVKKYREKVVIKKDCKGFSLEITTNLDNQVK---QRRRQSINPE 1347 .* * : . .
Ixodes LSVCTH----YRGEGEASNMAVMEVGLPSGYLFDFDTLSSIHRT------KEVRRVES 1354 Chlamys VRSCTK----WLKTG-ASGMTVQEVGVPTGFAPDVESIGKIATL------KKT----- 1403 Homo ISLSVS----YTGSRSASNMAIVDVKMVSGFIPLKPTVKMLERS------NHVSR--T 1408 Pongo ISLSVS----YTGSRSASNMAIVDVKMVSGFIPLKPTVKMLERS------NHVSR--T 1408 Mus IRINVS----YTGERPSSNMVIVDVKMVSGFIPMKPSVKRLQDQ------PNIQR--T 1427 Gallus IVLISS----YTGKRSSSNMVIIDVKMLSGFVPVKSSLDQLIDD------HTVMQ--V 1386 Mbur_1707 VRLKYNGMPGIRGVIESSGMMIVDIAVPTGFTPVGSSLEALKED------GTITR--Y 1342 Drosophila YVACQN--WINQNESERSGMAVLDVAIPTGYWIQQQKLDTYVLS-----NRVRNLRR--A 1642 Oncorhynchus ESFMLTIEVLYKNSERDATMSILDIGLLTGFIVDTDDLNQLSKG---RERYIEKFEMDKV 1393 Eptatretus FNVYRFIGCFRYLRNQEPGMVVMDISLPTGFEAKKKDLDDMKNL------VDNYIVQY 1399 . * : :: : :*: :
Ixodes QDSDTNVVIYFDRIGR-EELCVTVPAHREHKVANQ-KPVPVKVYDYYDLARSARMFYSPY 1412 Chlamys ETENRKVILYFDEITT-TPLCVTMNAYRTDQVAKS-QPAPIRVYDYYEPSNQVTKFYQST 1461 Homo EVSSNHVLIYLDKVSN-QTLSLFFTVLQDVPVRDL-KPAIVKVYDYYETDEFAIAEYNAP 1466 Pongo EVSNNHVLIYLDKVSN-QTLSLFFTVLQDVPVRDL-KPAIVKVYDYYETDEFAIAEYNAP 1466 Mus EVNTNHVLIYIEKLTN-QTLGFSFAVEQDIPVKNL-KPAPIKVYDYYETDEFTVEEYSAP 1485 Gallus EYKKNHVLLYLGNILQKRRKEVTFSVEQDFVVTHP-KPAPVQIYDYYETEEYAVAEYMSL 1445 Mbur_1707 EIAGRKIILYIDEMMPGEEMEFSLNMRAMFPVKAM-VQE-SSAYSYYNPEVKAEVKGMNV 1400 Drosophila RYLERKIVFYFDYLDH-EDICVNFTIERWYPVANMSRYLPVRIYDYYAPERFNESIFDAL 1701 Oncorhynchus LSERGSLILYLDKVSHKLEDRISFKIHRVQEVGVL-QPAAVSVYEYYNQKRCVKFYHPQR 1452 Eptatretus EIRPGRVFLYLDKVNKDEKNCVGFRLNQVFESNLV-LPVTATVFEYYEPDFRCSKSYHPK 1458 :.:*: : . . :.**
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Ixodes KTTLCDVCDGVECGNDCNTVKGTKAGTDQLERETEPDA-AADTRASLAAVLALSLTAVLF 1471 Chlamys VLKNSGVCDLCKECGCHGQH------1481 Homo CS--KDLGNA------1474 Pongo CS--KDLGNA------1474 Mus FSDGSEQGNA------1495 Gallus CRGVVEEMG------1454 Mbur_1707 TVV------1403 Drosophila PTYLLNICEVCGSSQCPYCSIYNMGWRASMSMSLLFFS-VFIYLLRSRTHLVLNMMQLLT 1760 Oncorhynchus EGGTLSRLCLGDVCTCAEESCSMQKKGEPDVQRIDKAC-GAGLDYVYKATVVDSKLTTHT 1511 Eptatretus MEVNPDASCHGNICNCLQRHCVELKGMADEDRNADRNGNACRAEYVFIIGVTKVTKTASY 1518
Ixodes VRWW------1475 Chlamys ------Homo ------Pongo ------Mus ------Gallus ------Mbur_1707 ------Drosophila ------Oncorhynchus DTYTVKIDLVIKPGTDEGV-EGKNRDFMGLAYCREALGLMQGKTYMIMGKSEDLHRVEDK 1570 Eptatretus ININAALKTVLKKGMDQAINVGARRSFVIPMHCGKNLNVSPGDIYLVMGMHNAHWR---- 1574
Ixodes ------Chlamys ------Homo ------Pongo ------Mus ------Gallus ------Mbur_1707 ------Drosophila ------Oncorhynchus GLLQYKYVLGEQTWIEYWPSQQECTSRDYREVCLGIDEFINQITTFGCPV 1620 Eptatretus NSDRTQYVLTSDTWFEKFPLESVCRLPSPPASCQVSENFKGCSLKG---- 1620
Organisms and proteins (with accession numbers) used in this alignment (chosen based upon NCBI BLAST analysis against Swissprot and for species diversity) were: Ixodes scapularis (black legged tick), EEC12664 uncharacterised alpha macroglobulin-like protein; Chlamys farreri (Zhikong scallop), ABP04060 Chlamys thioester-containing protein, alpha macroglobulin-like; Homo sapiens, P01023 alpha-2-macroglobulin; Pongo abelii (Orang-utan), Q5R4N8 alpha-2-macroglobulin; Mus musculus (house mouse), Q61838 alpha-2- macroglobulin; Gallus gallus (chicken), NP_990557 alpha-2- macroglobulin; Drysophila melanogaster (fruit fly), NP_524688 macroglobulin compliment related; Oncorhynchus mykiss (rainbow trout), AAB05029 C3 complement component; Eptatretus burgeri (inshore hagfish), P98094 C3 complement component. Blue text indicates Pfam alpha-2-macroglobulin N-terminal region 1, M. burtonii matched this region with scores of 4.8e-55, and 180 (trusted cut-off 25). Green text indicates Pfam alpha-2-macroglobulin N-terminal region 2, M. burtonii matched this region with scores of 1.4e-35, and 115 (trusted cut-off 19). Purple lettering indicates Pfam alpha-2- macroglobulin C domain, M. burtonii scores for this region were 1.7e-45, and 148 (trusted cut-off 18). Red lettering highlights the Pfam alpha-2-macroglobulin thiol-ester bond-forming region, with the characteristic GCGEQ region underlined. M. burtonii matched this region with scores of 6.7e-19 and 60 (trusted cut-off 28). Marone text indicates Pfam alpha-2-macroglobulin complement component, the scores for the M. burtonii alignment with this region were 9.4e-80 and 262 (trusted cut-off 18). Grey blue text indicates the Pfam alpha-2-macroglobulin receptor-binding domain, M. burtonii scores for this region were 2.5e-31 and 101 (trusted cut-off 17)
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Appendix A.10 GI numbers of proteins used in the phylogenetic analysis of A2M proteins. Organism Gene Organism Gene Anabaena variabilis GI:75908574 Marinitoga piezophila GI:214041341 Bacterium Ellin514 GI:186459129 Microscilla marina GI:124003208 Bacteroides thetaiotaomicron GI:29349455 Mus musculus GI:199086 Balanus amphitrite (SIPC) GI:71361896 Naja naja GI:399269 Bos taurus GI:124056491 Oncorhynchus mykiss GI:1352103 Chlamys farreri GI:144952812 Planctomyces maris GI:149174703 Danio rerio GI:189528666 Pongo abelii GI:197102064 Deinococcus radiodurans GI:15807953 Pseudomonas putida GI:26987308 Desulfatibacillum Ralstonia alkenivorans GI:218779432 solanacearum GI:17547749 Desulfovibrio desulfuricans GI:78355317 Rattus norvegicus GI:81872093 Drosophila melanogaster GI:17933686 Rhodopirellula baltica GI:32475766 Eptatretus burgeri GI:1168986 Sinorhizobium meliloti GI:16264550 Escherichia coli GI:16130445 Solibacter usitatus (a) GI:116621418 Fusobacterium nucleatum GI:34762629 Solibacter usitatus (b) GI:116623167 Gallus gallus GI:45382565 Sorangium cellulosum GI:162452347 Gemmata obscuriglobus GI:168699082 Stigmatella aurantiaca GI:115379148 Strongylocentrotus Helicobacter hepaticus GI:32266233 purpuratus GI:47551023 Homo sapiens GI:112911 Thermotoga maritima GI:15643744 Thermotoga Ixodes scapularis GI:215503170 neapolitana GI:222100568 Leeuwenhoekiella blandensis GI:85829953 Thermotoga petrophila GI:148270874 Leptospira interrogans GI:24214886 Thermotoga sp. RQ2 GI:170289587 Lethenteron japonicum GI:1352101 Xenopus tropicalis GI:62201351 Limulus sp. GI:2073373
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Appendix A.11 FKBP type PPIase alignment
M_Thermolithotrophicus_Fkbp ------XVDKGVKIKVDYIGKLESGDVF 22 Thermococcus_FKBP ------MKVEAGDYVLFHYVGRFEDGEVF 23 Mbur_2255_FKBP-type ------MVQEGDLISVNYIGQLEDGTIF 22 Mbur_2256_FKBP-type MRKALLLLILTGMLLVSGCASNGDQENTMVQEGDLISVNYIGQLDDGTIF 50 *: * : ..*:*:::.* :*
M_Thermolithotrophicus_Fkbp DTSIEEVAKEAGIYAPDREYEPLEFVVGEGQLIQGFEEAVLDMEVGDEKT 72 Thermococcus_FKBP DTSYEEIARENGILVEEREYGPMWVRIGVGEIIPGLDEAIIGMEAGEKKT 73 Mbur_2255_FKBP-type DTSLEDVAKEAGQHNPARGYEPLEFTVGAGQMIPGFDAGVVGMTVGEEKT 72 Mbur_2256_FKBP-type DTSFEDVAKEAGQYNPARGYEPLEFTVGASRLIAGFDAGVVGMTVGEEKT 100 *** *::*:* * * * *: . :* ..:* *:: .::.* .*::**
M_Thermolithotrophicus_Fkbp VKIPAEKAYGNRNEMLIQKIPRDAFKEADFEPEEGMVILAEGI-PATITE 121 Thermococcus_FKBP VTVPPEKAYGMPNPELVISVPREEFTKAGLEPQEGLYVMTDSG-IAKIVS 122 Mbur_2255_FKBP-type VTIPAAEAYGEYDEELVQPVPRTQLKEMGIEPEVGMPLFTQYG-QVTIVR 121 Mbur_2256_FKBP-type VVIPAAEAYGEYDEERVQPVPISQLKEKDIEPEVGMLLNTISGQQVPIVR 150 * :*. :*** : : :* :.: .:**: *: : : . *.
M_Thermolithotrophicus_Fkbp VTDNEVTLDFNHELAGKDLVFTIKIIEVVE------151 Thermococcus_FKBP VGESEVSLDFNHPLAGKTLVFEVEVIEVKKAEEDSEA 159 Mbur_2255_FKBP-type IDETNAYVDYNHHLAGETLTFKITLVSIGKE------152 Mbur_2256_FKBP-type MDETNAYVDNNYPLAGETLTFQITLVSIGQD------181 : :.:. :* *: ***: *.* : ::.: :
The IF region, which imparts a chaperone-like function to these proteins, is highlighted red
Appendix A.12 SuDH protein alignments
SudA
Mbur1328 ----MSIRPKMPQQAPEDRITNFDEVALGYTEEQAILEAERCLEC--KNPKCVEGCPVNI 54 SudA_P.furiosus MPRLIKDRVPTPERSVGERVRDFGEVNLGYSWELALREAERCLQCPVEYAPCIKGCPVHI 60 :. * *::: :*: :*.** ***: * *: ******:* : . *::****:*
Mbur1328 DIPGFISKIKEG------DFQEAINIIKATNILPAVCGRVCPQEEQCEKLCILGKKSEPI 108 SudA_P.furiosus NIPGFIKALRENRDNPSKAVREALRIIWRDNTLPAITGRVCPQEEQCEGACVVGKVGDPI 120 :*****. ::*. .:**:.** * ***: *********** *::** .:**
Mbur1328 AIGRLERICADIERKVGVKDPVVAQST------GKTVAIVGSGPAGLTAASDLAKAGHSV 162 SudA_P.furiosus NIGKLERFVADYAREHGIDDELLLEEIKGIKRNGKKVAIIGAGPAGLTCAADLAKMGYEV 180 **:***: ** *: *:.* :: :. **.***:*:******.*:**** *:.*
Mbur1328 TIFEALHEAGGVLVYGIPEFRLPKAIVKEEVDHIRKLGADIKMDYVIGRILTLDQLTDRF 222 SudA_P.furiosus TIYEALHQPGGVLIYGIPEFRLPKEIVKKELENLRRLGVKIETNVLVGKTITFEELREEY 240 **:****:.****:********** ***:*::::*:**..*: : ::*: :*:::* :.:
Mbur1328 DAVFLGTGAGLPKFMGIEGENLNGVYSANEFLTRVNLMKAYNN-DFDTPIKRGSNVIVVG 281 SudA_P.furiosus DAIFIGTGAGTPRIYPWPGVNLNGIYSANEFLTRINLMKAYKFPEYDTPIKVGKRVAVIG 300 **:*:***** *:: * ****:*********:******: ::***** *..* *:*
Mbur1328 GGNVAMDAARSALRLGADEVSIVYRRSDEELPARREEIEHAKEEGIVFRLLTNPVRIIEG 341 SudA_P.furiosus GGNTAMDAARSALRLGA-EVWILYRRTRKEMTAREEEIKHAEEEGVKFMFLVTPKRFIGD 359 ***.************* ** *:***: :*:.**.***:**:***: * :*..* *:* .
Mbur1328 ENMSVNGVECIKMELGEPDDSGRRRPEVVEGSEHIVDADIVIMAIGTSPNPIIFDGSKGL 401 SudA_P.furiosus ENGNLKAIELEKMKLGEPDESGRRRP-IPTGETFIMEFDTAIIAIGQTPNKTFLETVPGL 418 ** .::.:* **:*****:****** : *. .*:: * .*:*** :** ::: **
Mbur1328 EVTPWGTIVVDESGMSSIEGVCAGGDAVTGAATVISAMGAGKLAADTINNYLSK-- 455 SudA_P.furiosus KVDEWGRIVVDENLMTSIPGVFAGGDAIRGEATVILAMGDGRKAAKAIHQYLSKEK 474 :* ** *****. *:** ** *****: * **** *** *: **.:*::****
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SudB
Mbur_1329 MAYKITEKRQLVPDVHLMNIQAPDVAAAANAGQFIILRIDEAGERIPLTIADYNADEGSV 60 SudB_P.furiosus -MFKILRKERLAPGINLFEIESPRIAKHAKPGQFVMIRLHEKGERIPLTIADVDISKGSI 59 :** .*.:*.*.::*::*::* :* *:.***:::*:.* ********** : .:**:
Mbur_1329 TIIFQEMGKTTKQLAKMSAGEDLLDFVGPLGKESEIEKLGTVVLVGGGVGIAPIYPQAKE 120 SudB_P.furiosus TIVAQEVGKTTRELGTYEAGDYILDVLGPLGKPSHIDYFGTVVMIGGGVGVAEIYPVAKA 119 **: **:****::*.. .**: :**.:***** *.*: :****::*****:* *** **
Mbur_1329 YRAAGNKVISIIGARNKDMLILEDEMIAASDEVLVATDDGSKGHHGFVTDLVKQLLDGDE 180 SudB_P.furiosus MKEKGNYVISILGFRTKDLVFWEDKLRSVSDEVIVTTNDGSYGMKGFTTHALQKLIEEGR 179 : ** ****:* *.**::: **:: :.****:*:*:*** * :**.*. :::*:: ..
Mbur_1329 HIDRIVAIGPPIMMRAVAGVTASYDVETLVSLNPIMVDGTGMCGGCRVNVGGEVKFACVD 240 SudB_P.furiosus KIDLVHAVGPAIMMKAVAELTKPYGIKTVASLNPIMVDGTGMCGACRVTVGGEVKFACVD 239 :** : *:**.***:*** :* .*.::*:.**************.***.***********
Mbur_1329 GPEFNAKDVDFNLLMSRLSFYRKEEADAITKSQKGCGDDCKCQ 283 SudB_P.furiosus GPEFDAHLVDWDQLMNRLAYYRDLEKISLEKWER----ERRMV 278 ****:*: **:: **.**::**. * :: * :: : :
Appendix A.13 YidC protein alignments
Mbur_0026 ------M.mazei_GI_21228254 ------T.volcanium_GI_13541181 ------T.acidophilum_GI_10640592 ------Halobacterium_NRC-1_GI_1058118 MARTAEKVQSLVREDPEMAT-ILEDLLDHDGDLQWQDVSDDVSSGQWGRL 49 Halobacterium_NRC-1_GI_1058040 MPDTQLASLLQTAAMREAASAVLEESDARTGDIQWDDVSDAVSSSQWGRL 50 M.thermoautotrophicum_GI_74466 ------M.jannaschii_GI_2495998 ------P.horikoshii_GI_14591509 ------P.abyssi_GI_14520532 ------A.fulgidus_GI_11499485 ------
Mbur_0026 ------MADSNFKKTAEKFVLAVGI 19 M.mazei_GI_21228254 ------MVTETLKKQIDRFLLAFGF 19 T.volcanium_GI_13541181 ------MTETYNKEQQEAMKKMMSF 19 T.acidophilum_GI_10640592 ------MQLTEQYPKEQQEAMKKMMTF 21 Halobacterium_NRC-1_GI_1058118 LEKDILVEGDTGFEVGDPDGVEEGLAPDDDGD----DDSP--EGTSWSTY 93 Halobacterium_NRC-1_GI_1058040 ISAGVLTSTDTGFVVADPDELAAELAAHTDAESTEHTDAPAIESASWTIY 100 M.thermoautotrophicum_GI_74466 ------M.jannaschii_GI_2495998 ------P.horikoshii_GI_14591509 ------P.abyssi_GI_14520532 ------A.fulgidus_GI_11499485 ------MKAVIS 6
Mbur_0026 SLMIGIVVLG------QEGRESIGAIMGVFMDPISMMVGEGNFHIMLFI 62 M.mazei_GI_21228254 SLMFGIMLLG------QEFRQAVGEAVGIFMDPVLMLVGEQNFHLILLV 62 T.volcanium_GI_13541181 QMLYMLIMLGSLFIVITPSSRDAVGNLLNVVLIPT-IGFGYRYPLLSIIL 68 T.acidophilum_GI_10640592 QMIYMVVMLGSLFLVITPSSRDAVGRLLNFALIPS-IGFGYRYPVLTIIL 70 Halobacterium_NRC-1_GI_1058118 DKLAGVGAVGAMIGYSYSPIKNAIGGTINVVLGPL---DSIMPFYVVILV 140 Halobacterium_NRC-1_GI_1058040 DKAAAVGAIGLFAGYWNSSIQTTIASVDNQLLAPV---MNLLPFHIVVLV 147 M.thermoautotrophicum_GI_74466 ------MVLEIVYGALNAVFGPFIAMD---PNPQNPILTVFL 33 M.jannaschii_GI_2495998 ------MFGSIFDIYYKTLDAIFMPIIKV------LHPALAILI 32 P.horikoshii_GI_14591509 ------MLEGIYLALDKMFGPMLRN------THPMWVVTV 28 P.abyssi_GI_14520532 ------MLEGIYLALDELFGPMLKS------THPMWVVTV 28 A.fulgidus_GI_11499485 RFIMVLGALFFIGILFSREFRLELGQIVESVLFFL----SPYPFHVVILI 52 : : : :
Mbur_0026 MAAITALYASLIQKYTIDWDLMRNTQERMKSFQKEFREAQLSQNTYMVKK 112 M.mazei_GI_21228254 MAAITAIYASLIQKYTMDWDLMRNTQERMKVFQKEFREAQLSQNTYMLKK 112 T.volcanium_GI_13541181 TGVIIGVILSIPRYFFTDWVKMGKMQNTMKAFNEAIRAAYRERDMKKINK 118 T.acidophilum_GI_10640592 TGVIIGIILSIPRYFFTDWVKMGKMQNTMKAFNDAIREAYRERDMKKINK 120 Halobacterium_NRC-1_GI_1058118 LAVLTGLYTTILQANLTDMEKMSKYQEQAQELQDKMSAAKERGDDAAVER 190 Halobacterium_NRC-1_GI_1058040 LAVTTGVYSTYLQSRLMDHETLQAYKDRMAALKERREAAKERGDDEALER 197 M.thermoautotrophicum_GI_74466 ISAIVAFVITLANKLLVDQERLEELKVEMQEFQQEMMEARKKNDMAALEE 83 M.jannaschii_GI_2495998 IAIIVSLIINIATKLLVDQKRVAELKKEIQEFQVKFKKMSKNPEM--MEK 80 P.horikoshii_GI_14591509 SGVILGAFFVLLNYFLVDQEKMKRLQKMAKELQEEFKKARESGDEKKLRK 78 P.abyssi_GI_14520532 AGIILGAFFVFLNYVFVDQEKMKRLQKMAREFQEEFKKARESGDEKKLKK 78 A.fulgidus_GI_11499485 MSILTGIYSNLIQKFTIDYKRMKEIQKILREYQKEYIEATKQNNKFKLKQ 102 . . * : : : : :..
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Mbur_0026 LEEQRADMMGDQMEMSKQQ-FKPMAYISIISLPLFMWAYHY------152 M.mazei_GI_21228254 LEDQRKEMMEDQMKMSKQQ-FKPMAYISIISLPLFMWAYYF------152 T.volcanium_GI_13541181 LNSMRMQMSMDQYQLSMNT-MKPLLVISVLTILFYAWLFVF------158 T.acidophilum_GI_10640592 LNSMRMQMSMDQYQLSMNT-MKPLMVISVVTILFYAWLFVF------160 Halobacterium_NRC-1_GI_1058118 LREEQMEAFGDQASMMKEQ-FRPMVWIMLLTIPVFLWMYWK------230 Halobacterium_NRC-1_GI_1058040 IQSEQMDAAGDQLGLFKLQ-FRPMVWIMLLTIPVFLWLRWE------237 M.thermoautotrophicum_GI_74466 IQKKQMEFMDKQREMMTMS-FKPMIVTFIPIILVFYWMGQ------122 M.jannaschii_GI_2495998 LQEEQQRIMQLNAELMKMS-FRPMIYTWVPIILIFIYLRHVYGFGGVYQE 129 P.horikoshii_GI_14591509 LQQKQLELLRLQNELMKDAFLKPMLITWPIIIIFWGWLKR------118 P.abyssi_GI_14520532 LQQKQLEILRMQNELMKDAFFKPMLISWPIIIIFWGWLRR------118 A.fulgidus_GI_11499485 LEQKKNEIQQIQSEMMSMQ-FKPMFYTFVVTIPIFAWLWEKASASYELVT 151 :.. : : : ::*: : .: :
Mbur_0026 ------ISGHPGASLEFPFWGTQILVETVIGP------IQYWIFW 185 M.mazei_GI_21228254 ------ISGHEAATMVFPFWGEQLLTTSAIGP------FQHWIYW 185 T.volcanium_GI_13541181 ------VGKIPYNYIAFPWDFNINISTAHFWI------MPYWIFM 191 T.acidophilum_GI_10640592 ------VGNLPYNYVAFPWDYNINITTAHFWI------MPYWIFM 193 Halobacterium_NRC-1_GI_1058118 ----LGTGTIPQSELNMVLPLLGEIDLNGGRVLV------FPAWILW 267 Halobacterium_NRC-1_GI_1058040 ----VRGGHLGASEHGMVVPLAGHVAWQHALLGP------MATWIVW 274 M.thermoautotrophicum_GI_74466 -EPHIVNTLVILPQIAYYVLLVPLWHMFYGMPPN-----APSYAIGWLGW 166 M.jannaschii_GI_2495998 LNPGWNGVVVYLPIILSKILFIDFWHWLGSIFYKGGFKIVSNTALGWLGW 179 P.horikoshii_GI_14591509 ----WYFETAIVKYPFNFFLFDIFHKMYHSALKP------DELGYFGW 156 P.abyssi_GI_14520532 ----WYFEVAIVKYPFNFFLFDIFHKMYHSALRP------DELGYFGW 156 A.fulgidus_GI_11499485 GATNYGNLTSELPEVFLQTLKPELYTVVAPFAGQIHVVTPVLIFPWWLFW 201 ::
Mbur_0026 YFITSLAISQVVRKALNIGGV------206 M.mazei_GI_21228254 YFISSLGVSQLVRKALNIGGV------206 T.volcanium_GI_13541181 YFLTEIVVSYFITMIMKYIDFTLRLRRISRETSS---- 225 T.acidophilum_GI_10640592 YFLTELVVSYFVTMVMKYIDFSFKLRKLSRMSVSKATD 231 Halobacterium_NRC-1_GI_1058118 YMMCSFSFSNIIRKALNIQTTPT------290 Halobacterium_NRC-1_GI_1058040 YFVCSMASRQFLQKTFNIQAST------296 M.thermoautotrophicum_GI_74466 YILCSFAMSQIFRKFMGLKGGM------188 M.jannaschii_GI_2495998 YILCSFATSTVLRKILGIK------198 P.horikoshii_GI_14591509 YFLTSYVVGTILRKVLDMA------175 P.abyssi_GI_14520532 YFLTSYVVGSVLRKILDMA------175 A.fulgidus_GI_11499485 YLLCSITFGQIIKKVLRVGV------221 *:: . .. :
Putative YidC-like proteins display low homology
Appendix A.14 YidC phylogenetic tree
Proposed YidC homolgues in the archaea. The sequences are divergent from the bacteria. Mbur_0026 displays high homology to proteins from the methanosarcinaceae (M. mazei is shown as an example).
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Appendix A.15 Hydrophobicity profile of Mbur_2063
As fractions become more hydrophobic (left to right) a slight trend is seen towards more peptide identifications. 23 C samples are shown on the left while 4 C samples are shown on the right
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Appendix B. Chapter 4 Appendices
Appendix B.1 iTRAQ experimental design (thermal adaptation experiments)
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Appendix B.2 iTRAQ labelling protocol (substrate experiments)
Appendix B.3 Formulas used for calculating weighted mean and standard deviation
Formula a) weighted mean:
Where w = 1/log10(Error factor), and χ = log10 protein ratio
Formula b) weighted standard deviation: SD / b0.5
Where: SD = unweighted standard deviation
b =
and w = 1/log10(Error factor)
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Appendix B.4 Methyltransfer operon organisation in M. burtonii
(From Williams, et al., submitted – a)
0835 0836 0837 0838 0839 0840 0841 0842 0843 0844 0845 0846 0847
MtmB-1 MtmC-1 MtmB-2 MtmC-1
Appendix B.4.1 Gene arrangement of methylamine locus 1. Colour scheme: purple = increased in
abundance in methylamine media; white = not identified . Pertinent protein designations are listed
underneath gene loci. Locus tags represent (mbur_): 0835, Superoxide reductase/desulfoferrodoxin-like
protein (ER4); 0836, Hypothetical protein (ER5); 837, Hypothetical protein (ER5); 0838, Amine
permease (ER4) – possible methylamine permease; 0839, Monomethylamine methyltransferase (MtmB-
1)(ER2); 0840, Monomethylamine corrinoid protein (MtmC-1) (ER2); 0841, Protein with response
regulator receiver domain (ER4); 0842, Multisensor signal transduction histidine kinase (ER4); 0843,
Methanogenesis operon transcriptional regulator (ER2); 0844, Methanogenesis operon transcriptional
regulator (ER2); 0845, Hypothetical protein (ER5); 846, Monomethylamine methyltransferase (MtmB-
2) (ER2); 847, Monomethylamine corrinoid protein (MtmC-2) (ER2). Protein designations are listed
underneath gene loci
1363 1364 1365 1366 1367 1368 1369 1370 1371
MtbB-1 MtbC-1 MttQ-1 MttP-1 MttC-1 MttB-1 RamA
Appendix B.4.2 Gene arrangement of methylamine locus 2. Colour scheme: purple = increased in
abundance in methylamine media; green = no difference in abundance; white = not identified. Pertinent
protein designations are listed underneath gene loci. Locus tags represent: 1363, Sodium/hydrogen
antiporter (ER4); 1364, Dimethylamine methyltransferase (MtbB-1) (ER2); 1365, Dimethylamine
corrinoid protein (MtbC-1) (ER2); 1366, MttQ (function unknown) (ER4) (MttQ-1); 1367, TMA
permease MttP (ER4) (MttP-1); 1368, Trimethylamine corrinoid protein (MttC-1) (ER2); 1369,
trimethylamine methyltransferase (MttB-1) (ER2); 1370, RamA (reductive activation of methyltransfer,
amines) (ER2); 1371, Hypothetical protein (ER5).
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2082 2083 2084 2085 2086
MtbA PylD PylC PylB Pyrrolysyl-tRNA synthetase Appendix B.4.3 Gene arrangement of methylamine locus 3. Colour scheme: purple = increased in abundance in methylamine media; grey = no difference in abundance. Pertinent protein designations are listed underneath gene loci. Locus tags represent (mbur_): 2082, Methylamine-specific methylcobamide:CoM methyltransferase (mtbA) (ER2); 2083, Protein with NAD binding domain, pyrrolysine biosynthesis (pylD) (ER4); 2084, ATP-binding protein with DUF201 domain, pyrrolysine biosynthesis (pylC) (ER4); 2085, Radical SAM family protein, pyrrolysine biosynthesis (pylB) (ER4);
2086, Pyrrolysyl-tRNA synthetase (EC 6.1.1.-) (ER2)
2288 2291/2290 2292
Tn MtbC-2 MtbB-2
2285 2286 2287
Appendix B.4.4 Gene arrangement of methylamine locus 4. Blue indicates interrupting transposase.
Following locus tags: Tn = transposase. Colour scheme: white = not identified . Pertinent protein designations are listed underneath gene loci. Locus tags represent (mbur_): 2285, Protein with response regulator receiver domain (ER4); 2286, Multisensor signal transduction histidine kinase (ER4);
2287, Methanogenesis operon transcriptional regulator (ER2); 2288, Dimethylamine corrinoid protein
(MtbC-2) (ER2); 2290, transposase (ER4); 2291, Dimethylamine methyltransferase (MtbB-2) with inserted transposase (Mbur_2290) (ER4); 2292, Amino acid/polyamine transporter I (ER4).
350 D. Burg UNSW
2308/2309 2310 2311 2312 2313
MttB-2 MttC-2 MttP-2 MttQ-2 MttB-3
Appendix B.4.5 Gene arrangement of methylamine locus 5. Colour scheme: purple = increased in abundance in methylamine media; grey = no difference in abundance; white = not identified. Pertinent protein designations are listed underneath gene loci. Locus tags represent (mbur_): 2308/2309,
Trimethylamine methyltransferase (MttB-2) (ER2); 2310, Trimethylamine corrinoid protein (MttC-2)
(ER2); 2311, TMA permease (MttP-2) (ER4); 2312, MttQ (function unknown) (MttQ-2) (ER4); 2313,
TMA methyltransferase (MttB-3) (ER2).
0808 0809 0810 0811 0812 0813 0814
MtaA RamM MtaC-2 MtaB-2 MtaC-1 MtaB-1
Appendix B.4.6 Gene arrangement of methanol locus. Colour scheme: green = increased in abundance in methanol media; white = not identified. Pertinent protein designations are listed underneath gene loci. Locus tags represent (mbur_): 0808, methanol-specific methylcobalamin:CoM methyltransferase
(MtaA) (ER2); 0809, RamM (reductive activation of methyltransfer, methanol) (ER2); 0810, Multi-PAS sensor (ER4); 0811, Methanol corrinoid protein (MtaC-2) (ER2); 0812, Methanol--corrinoid methyltransferase (MtaB-2) (ER2); 0813, Methanol corrinoid protein (MtaC-1) (ER2); 0814, Methanol-- corrinoid methyltransferase (MtaB-1) (ER2).
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