Extremophiles-Basic Concepts

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EXTREMOPHILES

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©Encyclopedia of Life Support Systems (EOLSS) EXTREMOPHILES

CONTENTS

VOLUME I

Extremophiles: Basic Concepts 1 Charles Gerday, Laboratory of Biochemistry, University of Liège, Belgium

1. Introduction 2. Effects of Extreme Conditions on Cellular Components 2.1. Membrane Structure 2.2. Nucleic Acids 2.2.1. Introduction 2.2.2. Desoxyribonucleic Acids 2.2.3. Ribonucleic Acids 2.3. Proteins 2.3.1. Introduction 2.3.2. Thermophilic Proteins 2.3.2.1. Enthalpically Driven Stabilization Factors: 2.3.2.2. Entropically Driven Stabilization Factors: 2.3.3. Psychrophilic Proteins 2.3.4. Halophilic Proteins 2.3.5. Piezophilic Proteins 2.3.5.1. Interaction with Other Proteins and Ligands: 2.3.5.2. Substrate Binding and Catalytic Efficiency: 2.3.6. Alkaliphilic Proteins 2.3.7. Acidophilic Proteins 3. Conclusions

Extremophiles: Overview of the Biotopes 43 Michael Gross, University of London, London, UK

1. Introduction 2. Extreme Temperatures 2.1. Terrestrial Hot Springs 2.2. Hot Springs on the Ocean Floor and Black Smokers 2.3. Life at Low Temperatures 3. High Pressure 3.1. The Deep Sea 3.2. The Deep Subsurface 4. Chemical Stress Factors 4.1. High-Salinity Biotopes 4.2. Life at Extreme pH 4.3. Hydrophobic Environments 5. Other Extremes 5.1. Resistance to Drought and Radiation 5.2. Yet to be Discovered 6. Extremophiles and the Evolution of Life 6.1. Primeval Earth as an Extreme Habitat 6.2. Archaea: A New, Very Old Domain of Life 6.3. Does Life Come out of the Heat?

Phylogeny of Extremophiles 71 Nicolas Glansdorff, Vrije Universiteit Brussel, Brussels, Belgium Ying Xu, Vrije Universiteit Brussel, Brussels, Belgium

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1. Introduction 2. The Structure of the Tree of Life 2.1. The Last Common Ancestor (LCA) and the Three Domains 2.2. The Root of the Tree of Life 3. Was the LCA a Hyperthermophile? 4. Was the LCA a Prokaryote? 5. The Origin of Prokaryotes and of Hyperthermophily 6. Thermophilic Prokaryotes 7. Piezophilic Prokaryotes 8. Psychrophilic Prokaryotes 9. Extreme Halophiles 10. Concluding Remarks

Survival Strategies and Membrane Properties of Extremophiles 98 Wil N. Konings, University of Groningen, The Netherlands Sonja-Verena Albers, University of Groningen, The Netherlands Sonja M. Koning, University of Groningen, The Netherlands Arnold J.M. Driessen, University of Groningen, The Netherlands

1. Introduction 2. Composition of the Membrane 3. Bioenergetics 4. Bioenergetic Problems of Extremophiles 4.1. Temperature 4.2. Salt 4.3. pH 5. Transport of Solutes in Extremophiles 5.1. Secondary Transporters 5.2. ABC Transporters 5.2.1. Solute-Binding Proteins 5.2.2. ATP-Binding Domains 5.3. Distribution of Transporters 6. Conclusions

Thermophily 117 Gudmundur O. Hreggvidsson, University of Iceland and Prokaria Ltd, Reykjavik, Iceland Jakob K. Kristjansson, University of Iceland and Prokaria Ltd, Reykjavik, Iceland

1. Introduction 1.1. Thermophily and Geochemical History 1.2. Definitions and Terminology 2. Habitats and Ecology 2.1. Diversity of Thermal Environments 2.2. Energy Sources and Physiology 2.3. Development of Isolation Methods 2.4. Culture-Independent Studies of Thermophilic Communities 3. Thermophile Diversity and Population Structure 3.1. Phylogeny and Species 3.2. Main Thermophilic Bacterial Phyla 3.2.1. Thermophilic Cyanobacteria 3.2.2. The Genus Thermus 3.2.3. The Order Aquificales 4. Archaea 5. Distribution and Speciation of Thermophiles 5.1. Global Distribution of Thermophiles 5.2. Dispersal of Thermophiles

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5.3. Evolution and Speciation of Thermophiles 5.4. Lateral Gene Transfer in Thermophiles 6. Conclusion

Hyperthermophilic Microorganisms 158 Karl O. Stetter, Universität Regensburg, Lehrstuhl für Mikrobiologie, Universitätsstraße 31, D-93053 Regensburg, Germany

1. Introduction 2. Biotopes of Hyperthermophiles 2.1. Terrestrial Biotopes 2.2. Marine Biotopes 3. Phylogeny of Hyperthermophiles 4. Taxonomy of Hyperthermophiles 5. Sampling and Isolation of Hyperthermophiles 6. Strategies of Life and Environmental Adaptations of Hyperthermophiles 6.1. General Metabolic Potentialities 6.2. Physiological Properties of the Different Groups of Hyperthermophiles 6.2.1. Terrestrial Hyperthermophiles 6.2.2. Marine Hyperthermophiles 7. Distribution of Species and Complexity in Hyperthermophilic Ecosystems 8. Basis of Heat Stability and the Upper Temperature Limit for Life 9. Conclusions: hyperthermophiles in the history of life

Strategies of Hyperthermophiles in Nucleic Acids Adaptation to High Temperature 189 Patrick Forterre, Universite de Paris-Sud, France

1. Introduction 2. The General Problem of Nucleic Acid Stability at High Temperature 3. Thermoprotection of RNA in Hyperthermophiles 3.1. Protection of RNA against Thermodenaturation 3.2. The Problem of RNA Thermodegradation 3.3. Hyperthermophiles and the Possibility of a Very Hot RNA World 4. Thermoprotection of DNA in Hyperthermophiles 4.1. Effect of Temperature on the DNA Secondary and Tertiary Structures 4.2. Effect of Temperature on the DNA Chemical Integrity 4.2.1. Depurination 4.2.2. DNA—Cytosine Deamination 4.3. DNA Repair in Hyperthermophiles 4.4. Reverse Gyrase 5. Conclusion

Thermostability and Thermoactivity of Extremozymes 209 Michael John Danson, University of Bath, UK David W. Hough, University of Bath, UK

1. Introduction 2. Enzyme Stability 2.1. Thermodynamic Stability 2.2. Kinetic Stability 2.3. Thermostability 3. The Structural Basis of Thermostability 3.1. Conformational Flexibility and Loop Regions 3.2. Increased Hydrophobicity 3.3. Ionic Interactions and Networks

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3.4. Packing Density and Other Electrostatic Interactions 3.5. Other Structural Features 3.6. Oligomeric Enzymes and Subunit Interactions 3.7. Thermolabile Amino Acids 4. Observations from Nature 4.1. Systematic Structural Comparisons 4.2. Specific Enzyme Studies 4.3. Citrate Synthase: an Homologous Series Spanning the Biological Temperature Range 5. Lessons From Directed Evolution 5.1. Thermal Adaptation of Enzyme Stability 5.2. Adaptation of Catalytic Activity 5.3. Flexibility and Stability 6. Intrinsic versus Extrinsic Factors in Enzyme Thermostability 7. Thermoactivity 7.1. Catalytic Activity at Physiological Temperatures 7.2. The Temperature Optimum of an Enzyme 8. Concluding Remarks

Unique Aspects of the Hyperthermophile Proteome 239 James F. Holden, University of Georgia, Athens, Georgia, USA Michael W. W. Adams, University of Georgia, Athens, Georgia, USA

1. Introduction 2. Systematics of the Order Thermococcales 2.1. Phylogeny 2.2. Growth Characteristics 2.3. Habitats 3. Characterized Enzymes and Proteins 3.1. Oxidoreductases (EC 1.-.-.-) 3.2. Transferases (EC 2.-.-.-) 3.3. Hydrolases (EC 3.-.-.-) 3.3.1. Esterases (EC 3.1.-.-) 3.3.2. Glycosylases (EC 3.2.-.-) 3.3.3. Peptidases (EC 3.4.-.-) 3.3.4. Other Hydrolases 3.4. Lyases (EC 4.-.-.-) 3.5. Isomerases (EC 5.-.-.-) 3.6. Ligases (EC 6.-.-.-) 3.7. Other Proteins 4. Starch Catabolism 5. Functional and Structural Genomics 6. Conclusions

Compatible Solutes in Microorganisms that Grow at High Temperature 265 Milton S. da Costa, Universidade de Coimbra, Coimbra, Portugal Helena Santos, Universidade Nova de Lisboa, Oeiras, Portugal

1. Introduction 2. Strategies for Osmotic Adaptation in Microorganisms 3. Compatible Solutes of Organisms that Live at High Temperatures 4. The Distribution of Compatible Solutes Within the Tree of Life 5. Reflections on the Physiological Role of Compatible Solutes in Thermoadaptation 6. The Effect of Hypersolutes on Protein Stability 6.1. The Effect of Mannosylglycerate on the Stabilization of Enzymes 6.2. The Effect of Di-myo-Inositol-Phosphate on the Stabilization of Enzymes 6.3. Stabilization of Proteins and Enzymes by Diglycerol Phosphate

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6.4. Stabilization of Enzymes by Cyclic 2,3-Bisphosphoglycerate 7. Pathways for the Synthesis of Compatible Solutes in Thermophiles and Hyperthermophiles 8. Concluding Remarks

Heat-Shock Response in Thermophilic Microorganisms 282 Mose Rossi, Istituto di Biochimica delle Proteine ed Enzimologia, Napoli, Italy and Università “Federico II”, Napoli, Italy Annamaria Guagliardi, Università “Federico II”, Napoli, Italy

1. Introduction 1.1. The Concept of Molecular Chaperone 2. Even Extreme Thermophiles Display Heat-Shock Response 3. Archaeal Chaperonins 3.1. Chaperonin-Assisted In Vitro Folding of Denatured Proteins 3.2. Chaperonin-Mediated Prevention of Native Protein Denaturation 3.3. Chaperonin-Dependent Formation of Filaments 4. Archaeal Chaperonins Are Biotechnological Tools 5. Perspectives

Thermoactive Enzymes in Biotechnological Applications 294 Costanzo Bertoldo, Technical University Hamburg-Harburg, Hamburg, Germany Garabed Antranikian, Technical University Hamburg-Harburg, Hamburg, Germany

1. Introduction 2. Extreme Environments as a Source of Novel Thermoactive Enzymes 2.1. Biology at the Boiling Point of Water 2.2. Microbial Life at High Temperatures and at Extremes of pH 3. Cultivation of Extremophilic Microorganisms 4. Screening Strategies for Thermoactive Enzymes 5. Starch-Processing Enzymes 5.1. Heat-Stable a-Amylases, Glucoamylases, and a -Glucosidases 5.2. Thermoactive Pullulanase and CGTase 6. Cellulases 7. Thermoactive Xylanases 8. Pectin-Degrading Enzymes 9. Chitinases 10. Proteolytic Enzymes 11. Glucose Isomerases, Alcohol Dehydrogenases, and Esterases 12. Polymerase Chain Reaction (PCR)

Index 317

About EOLSS 323

VOLUME II

Psychrophily and Resistance to Low Temperature 1 Nicholas J. Russell, Imperial College London, Wye Campus, UK

1. Introduction 2. Cold-Adapted Microorganisms 2.1. Terminology 2.2. Habitats

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2.3. Biotypes 3. Cold-Adaptation Mechanisms 3.1. Growth 3.2. Membrane Lipids 3.3. Proteins (Enzymes) 4. Cold Shock and Cold Acclimation 5. Cold Resistance and Cold Sensitivity 5.1. Antifreeze Protection 5.2. Cold-Resistant and Cold-Sensitive Mutants

Ice Ecosystems and Biodiversity 33 Roland Psenner, University of Innsbruck, Austria Anton Wille, University of Innsbruck, Austria Birgit Sattler, University of Innsbruck, Austria John C. Priscu, Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana, USA Marisol Felip, University of Barcelona, Department of Ecology, Diagonal Barcelona, Spain Dietmar Wagenbach, University of Heidelberg, Institute of Environmental Physics, Im Neuenheimer Feld, Heidelberg, Germany

1. Introduction 1.1. Definitions and dimensions 1.2. Physico-chemical constraints 1.3. Effects on microorganisms 2. Known and unknown ice ecosystems 2.1. Ice Covers Over Liquid Water 2.1.1. Sea Ice 2.1.2. Seasonal Ice Covers on Alpine Lakes 2.1.3. Permanent Ice Covers on Antarctic Lakes 2.2. Snow Cover 2.3. Glaciers 2.4. Supercooled Cloud Droplets 3. Open questions 3.1. Cold = Oligotrophic? 3.2. Barrier versus Connectivity—– Are Ice Ecosystems Ecotones? 3.3. Origin of Organisms 3.4. Applications 3.5. Perspectives under a Global Warming Scenario 3.6. Can Ice Ecosystems Exist without Solar Radiation? 3.7. Implications for the Origin of Life 4. Conclusion

Membrane Adaptation and Solute Uptake Systems 67 Nicholas J. Russell, Imperial College London (Wye Campus), UK

1. Introduction 2. Membrane Structure and Lipid Organization 3. Structure of Transport Proteins 4. Lipid Adaptation to the Cold 5. Transport of Small Molecules 5.1. General Mechanisms 5.2. Bacterial Porins 5.3. Binding-Protein-Dependent ABC Transporters 5.4. Lactose Permease 5.5. Phosphotransferase Systems 6. Effects of Lipid Composition on Transport

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7. Solute Uptake and Microbial Ecology in the Cold

Catalysis and Low Temperature: Molecular Adaptations 88 Georges Feller, University of Liege, Belgium Charles Gerday, University of Liege, Belgium

1. The Psychrophilic Context 2. Kinetic Optimization of Cold-Active Enzymes 2.1. Cold-Active Enzymes 2.2. Improving the Turnover Number kcat : A Thermodynamic Challenge 2.3. Alterations and Optimization of Km in Cold-Active Enzymes 2.3.1. Adaptive Drift of Km with Flexibility 2.3.2. Adaptive Optimization of the Km Parameter 2.4. The Active Site of Psychrophilic Enzymes in Crystal Structures 2.4.1. Conservation of Catalytic Residues 2.4.2. Conservation of the Active Site Architecture 2.4.3. Better Accessibility and Larger Active Site 2.4.4. Electrostatic Potentials 3. Stability of Psychrophilic Enzymes 3.1. Heat-Lability and Weak Structural Stability 3.2. Stability Analyzed by Microcalorimetry 3.3. Structural Determinants of Cold Adaptation in a Large Protein 3.4. Thermodynamic Stability 3.5. Limits of Enzyme Adaptation to Cold 3.6. Three Types of Conformational Stability 3.7. Local Stability 3.8. Irreversible Unfolding 3.9. Structural Factors Involved in the Weak Stability 4. Activity-Flexibility-Stability Relationships 4.1. Basic Aspects 4.2. Experimental Approaches 4.3. Motions and Flexibility in Proteins

Cold-Shock Response in Microorganisms 122 Ricardo Cavicchioli, The University of New South Wales, Sydney, Australia Neil Saunders, The University of New South Wales, Sydney, Australia Torsten Thomas, The University of New South Wales, Sydney, Australia

1. Introduction 2. Membranes 3. Transport Systems 4. Metabolic Processes 5. Antifreeze Compounds and Intracellular Effectors 6. Nucleic Acids and Nucleic Acid-Binding Proteins 6.1. The Role of Cold-shock Domain (CSD), Nucleic Acid Binding Proteins 7. Protein Synthesis 7.1. Protein Folding 8. Cold-Sensing Mechanisms 9. Cold Shock and Other Stress Responses 10. Conclusion

Heterologous Gene Expression in Cold-Adapted Micro-Organisms 148 A. Duilio, Universita di Napoli Federico II, Italy Maria Luisa Tutino, Universita di Napoli Federico II, Italy Gennaro Marino, Universita di Napoli Federico II, Italy

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1. Introduction 2. Heterologous Protein Production in Bacteria other than Escherichia coli 2.1. Use of Gram-Positive Bacteria 2.2. Use of Cold-Adapted Bacteria 3. Cold-Adapted Bacteria Transformation 3.1. Use of BHR Plasmids 3.2. Use of Naturally Cold-Adapted Plasmids 3.2.1. Screening for Cold-Adapted Replicons 3.2.2. Psychrobacter sp. TA144 and its Plasmids 3.2.3. Pseudoalteromonas haloplanktis TAC 125 and pMtBL Plasmid 4. Construction of Cold Genetic Systems and their Cold Host Profile 5. The α -Amylase Example of Heterologous Protein Production in Cold-Adapted Bacteria 6. Conclusions and Future Perspectives

Cold-Active Enzymes as New Tools in Biotechnology 164 Rosa Margesin, Institute of Microbiology, Leopold Franzens University, Innsbruck, Austria

1. Introduction 2. Advantages of Cold-Active Enzymes in Biotechnology 3. Improvement of Enzyme Yield and Thermostability 4. Biotechnological Potential of Cold-Active Enzymes 4.1. Detergent Industry 4.1.1. Proteases 4.1.2. Lipases, Amylases, and Cellulases 4.2. Food Industry 4.2.1. Fermentation Products 4.2.2. Baking 4.2.3. Lactose Hydrolysis 4.2.4. Food Processing 4.2.5. Food Supplements 4.2.6. Food Preservation 4.3. Organic Synthesis 4.4. New Tools in Molecular Biology 4.5. Pharmaceuticals from Antarctic Krill 4.6. Textile Industry 4.7. Biosensors 4.8. Bioremediation of Organic Contaminants in Cold Environments 4.8.1. Oil Spills 4.8.2. Aromatic Compounds 4.8.3. Chlorinated Compounds 4.8.4. Other Compounds 4.9. Waste Treatment 4.9.1. Anaerobic Wastewater Treatment 4.9.2. Anaerobic Digestion 4.9.3. Composting 4.10. Agriculture

Freeze Tolerance 184 Kenneth B. Storey, Carleton University, Ottawa, Ontario, Canada Janet M. Storey, Carleton University, Ottawa, Ontario, Canada

1. Introduction 1.1. Low temperature and freezing injury 2. Strategies for Survival at Subzero Temperatures 2.1. Anhydrobiosis 2.2. Vitrification

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2.3. Freeze Avoidance 2.3.1. Nucleator Control 2.3.2. Antifreeze Proteins 2.3.3. Carbohydrate Antifreezes 3. Freeze Tolerance 3.1. Adaptations for Freeze Tolerance 3.2. Ice Nucleators 3.3. Ice Management and AFPs 3.4. Cryoprotectants 3.5. Membrane Protection 3.6. Gene and Protein Adaptations 3.6.1. Animals 3.6.2. Plants 3.6.3. Bacterial Cold-Shock Proteins 3.7. Ischemia Resistance, Metabolic Rate Depression, and Antioxidant Defenses

Freezing Avoidance in Polar Fishes 215 Chi-Hing Christina Cheng, University of Illinois, Urbana-Champaign, Illinois, USA

1. Introduction 2. Diversity of Fish Antifreeze Proteins 2.1. Antifreeze Glycoprotein 2.2. Type I, II, III, and IV Antifreeze Peptides (AFPs) 3. Antifreeze Property and Function 3.1. Adsorption-Inhibition Mechanism of Antifreeze Action 3.2. Antifreeze Protein in Freezing Avoidance in Fish 4. Evolutionary Origins and Pathways of Antifreeze Proteins 4.1. Type II and IV AFP—Evolution by Gene or Domain Duplication 4.2. Notothenioid AFGP—Evolution by Partial Gene Recruitment and De Novo Amplification 4.3. Evolution of Arctic Cod AFGP by Protein Sequence Convergence 5. Environmental Driving Force for Antifreeze Evolution 6. Conclusion

Halophily (Halophilism and Halophilic Microorganisms) 233 Antonio Ventosa, Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Spain David R. Arahal, Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Spain

1. Introduction 2. Halophilism: Concept and Classifications 3. Phylogeny and Taxonomy 3.1. Archaea 3.2. Bacteria 3.3. Eukarya 4. Ecology and Diversity 5. Physiology 6. Genetics and Genomics 7. Biotechnological Applications

Physico-Chemical Characteristics of Hypersaline Environments and Their Biodiversity 247 Antonio Ventosa, University of Sevilla, Spain David R. Arahal, University of Sevilla, Spain

1. Introduction

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2. Thalassohaline Environments 2.1. Solar Salterns and Coastal Lagoons 2.2. Soils 2.3. Great Salt Lake 2.4. Salt Mines 3. Athalassohaline Environments 3.1. Dead Sea 3.2. Soils 3.3. Alkaline Lakes 3.4. Unusual Habitats 4. Biodiversity 4.1. Studies Based on Cultivation 4.2. Molecular Ecology 5. Future Trends

Osmoregulation in Halophilic Bacteria 263 Hans Jorg Kunte, Institute for Microbiology & Biotechnology, Rheinische Friedrich-Wilhelms- Universität, Bonn, Germany

1. Introduction 2. Mechanisms of Osmoadaptation in Prokaryotes 2.1. Thermodynamic Principles Underlying Osmotic Stress 2.2. Salt-in-Cytoplasm Mechanism 2.3. Organic-Osmolyte Mechanism 2.3.1. Stress Protection by Compatible Solutes 2.3.2. Compatible Solutes of Halophiles and Their Synthetic Pathways 2.3.3. Compatible Solute Transport and Osmosensing 3. Primary Response of the Halophilic Cell to Fluctuation in External Salinity 3.1. Response to Sudden Decrease in External Salinity (Osmotic Downshock) 3.2. Primary Response to Sudden Increase in Salinity (Osmotic Upshock) 3.2.1. Osmoregulatory Response of Nonhalophiles to Osmotic Upshock 3.2.2. Osmoregulatory Response of Halophilic Bacteria to Osmotic Upshocks

Molecular Adaptation of Halophilic Proteins 278 Christine Ebel, Institut de Biologie Structurale, Grenoble, France Dominique Madern, Institut de Biologie Structurale, Grenoble, France Giuseppe Zaccai, Institut de Biologie Structurale, Grenoble, France

1. Introduction 1.1. Halophilic Organisms, Halophilic Proteins 1.2. Evolution 1.3. Molecular Adaptation 2. Three-Dimensional Structures of Soluble Halophilic Proteins 2.1. Overall Structures as seen by Crystallography 2.2. Protein-Solvent as seen by Crystallography 2.3. Modeling 3. Site-Directed Mutagenesis 3.1. Mutation of Acidic Amino Acids 3.2. Salt-Bridge Clusters 4. Halophilic Enzyme Activity 4.1. Effects of Salt Concentration 4.2. Effects of Salt Type 4.3. In Vivo Activity 4.4. Detailed Description of Enzymatic Activity 5. Halophilic Protein Solubility 5.1. As a Function of the Solvent

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5.2. For Purification 5.3. For Crystallization 6. Halophilic Protein Stability 6.1. Stability Measurements 6.2. Systematic Studies of Salt Type 6.3. Temperature Dependence 6.4. Isotopic Effects of Heavy Water 7. Solvent Interactions for Halophilic Proteins 8. Conclusion 8.1. Cellular Adaptation 8.2. In Vitro Studies for the Study of Solvent Interactions 8.3. Specific Binding Sites 8.4. Halophilic Proteins are Dynamic Protein-Salt-Water Complexes

Ectoines: A New Type of Compatible Solutes with Great Commercial Potential 298 Georg Melmer, Bitop Aktiengesellschaft für Biotechnische Optimierung, Witten, Germany Thomas Schwarz, Bitop Aktiengesellschaft für Biotechnische Optimierung, Witten, Germany

1. Introduction 2. Ectoine—A Compatible Solute in Halophilic Microorganisms 3. Industrial Production of Ectoines 4. Effects of Ectoines in Stabilization 5. Ectoine—A New Cosmetic Ingredient 5.1. Stabilization of Membranes 5.2. Ectoine and Immune Protection 5.3. Ectoine Reduces Cell Damage 5.4. Ectoine Accelerates the Formation of Heat-Shock Proteins 6. Stabilization of Pharmaceuticals 7. Concluding Remarks

Index 315

About EOLSS 323

VOLUME III

Alkaliphily 1 Terry Ann Krulwich, Mount Sinai School of Medicine, New York, USA

1. Introduction 2. The Place of Alkaliphiles Among Extremophilic Bacteria 2.1. Ecological Niches 2.2. Diversity of Organisms 2.3. Multiple Challenges 3. Genomics, Proteomics, and Adaptations to Alkaliphily 3.1. An Alkaliphile Genome 3.2. Initial Proteomics—Extreme Alkaliphiles are Hardwired for Alkaliphily 3.3. The pH Homeostasis Problem 3.4. Energization of Motility, Solute Transport, and Oxidative Phosphorylation 4. Applications 4.1. Production of Natural Products of Interest 4.2. Use as Assay Vehicles for Natural Products of Interest 4.3. Use in Bioremediation 5. Future Perspectives

Alkaline Environments and Biodiversity 21 Wiliam D. Grant, University of Leicester, UK

1. Introduction 2. Genesis of Soda Lakes. 3. Microbial Diversity 3.1. Primary Production in Soda Lakes 3.2. Chemo-Organotrophs in Soda Lakes 3.3. Methanogenesis in Soda Lakes 3.4. Sulfur in Soda Lakes 3.5. Nitrogen in Soda Lakes 4. Conclusion

Adaptation Processes in Alkaliphiles When Cell Wall Acidity is Elevated 39 Rikizo Aono, Tokyo Institute of Technology, Japan

1. Introduction 2. Growth pH Ranges of Alkaliphilic Microorganisms 2.1. Diversity of Physiological Properties of Alkaliphilic Strains of Bacillus spp. 2.2. Growth pH Ranges of Alkaliphilic Strains of Bacillus spp. and Their Alkaline pH-Sensitive Mutants 2.3. Growth pH Ranges of Protoplasts Prepared From Alkaliphilic Strains of Bacillus spp. 3. Cell Surface Structure of Alkaliphilic Strains of Bacillus spp. 3.1. Structural Cell Wall Components of Bacillus spp. 3.2. Peptidoglycan Compositions of Alkaliphilic Strains of Bacillus spp. 3.3. Nonpeptidoglycan Components of the Cell Walls of Alkaliphilic Strains of Bacillus spp. 3.4. Chemical Natures of Acidic Polymers in the Cell Walls of Strain C-125 3.5. Wide Distribution of Teichuronopeptide in the Cell Walls of the Group 2 Alkaliphilic Strains of Bacillus spp. 4. Alkaline pH Sensitivity of Cell Wall-Defective Mutants of Strain C-125 4.1. Isolation of Mutants Defective in Synthesis of Cell Wall Components 4.2. Growth of Mutants Defective in Cell Wall Components in Alkaline Environments 4.3. Culture pH-Dependent Increase in Anionic Charges in the Cell Walls of Strain C-125 4.4. Gene tupA Restores Synthesis of Teichuronopeptide in the Mutant C-125-90 5. Intracellular pH Homeostasis of Strain C-125 and its Cell Wall-Defective Mutants in Alkaline Environments 5.1. Culture pH-Dependent Development of Intracellular pH Homeostasis of Strain C-125 5.2. Intracellular pH Homeostasis in Mutants Defective in Cell Wall Components 5.3. Improvement of pH Homeostasis in C-125-90 By Introduction of the tupA Gene 6. Conclusion

Metallophiles and Acidophiles in Metal-Rich Environments 65 Max Mergeay, Laboratory for Microbiology, Center of Studies for Nuclear Energy, Mol, Belgium

1. Introduction: Industrial Biotopes as a Reservoirs for Extremophiles 2. Bacteria and the Periodic Table 3. Bacteria and Metals 4. How to Track Metal-Resistant Bacteria? 5. Metal-Rich Biotopes as Sources of Metal-Resistant Bacteria 5.1. Natural Metal-Rich Soil Biotopes 5.2. Anthropogenic Metal-Rich Soil Biotopes 6. Metal Resistance: A Role for Mobile Genetic Elements 7. The Organization of Metal-Resistance Genes in Ralstonia metallidurans 7.1. A Preliminary View at the Genomic Level

7.2. Regulation Modules in Metal-Resistance Operons 8. Other Bacteria Involved in Biogeochemical Processes Involving Metals 9. Environmental Applications of Bacteria Adapted to Heavy Metals or Able to Process Heavy Metals 10. Conclusions

Ecology and Biodiversity of Extremely Acidophilic Microorganisms 89 Douglas Eric Rawlings, University of Stellenbosch, South Africa D. Barrie Johnson, University of Wales, Bangor, UK

1. Definition of Extreme Acidophily 2. Low pH Environments 2.1. Geothermal and Volcanic Areas 2.2. As a Result of Human Activity 2.2.1. Acid Mine Drainage 2.2.2. Biooxidation Heaps and Dumps 2.2.3. Biooxidation Tanks 3. Carbon and Energy Sources of Extreme Acidophiles 3.1. Carbon Dioxide Fixation by Acidophilic Autotrophs 3.2. Electron Donors for Autotrophic Acidophiles 3.2.1. Reduced Inorganic Sulfur Compounds (RISCs) 3.2.2. Iron 3.2.3. Alternative Electron Donors 3.3. Electron Donors and Sources of Carbon for Heterotrophic Acidophiles 3.4. Electron Acceptors 4. Biodiversity of Extremely Acidophilic Bacteria 4.1. Iron-Oxidizing Autotrophs 4.1.1. Leptospirillum ferrooxidans 4.2. Sulfur-Oxidizing Autotrophs 4.2.1. Acidithiobacillus thiooxidans 4.2.2. Acidithiobacillus caldus 4.2.3. Hydrogenomonas acidophilus 4.3. Iron- and Sulfur-Oxidizing Autotrophs 4.3.1. Acidithiobacillus ferrooxidans 4.3.2. Thiobacillus prosperus 4.4. Iron-Oxidizing Mixotrophs and Heterotrophs 4.4.1. Acidimicrobium ferrooxidans 4.4.2. Ferrimicrobium acidophilum 4.5. Sulfur-Oxidizing Mixotrophs 4.5.1. Acidiphilium acidophilum 4.5.2. Thiomonas cuprina 4.5.3. Sulfobacillus disulfidooxidans 4.6. Iron- and Sulfur-Oxidizing Mixotrophs 4.6.1. Sulfobacillus spp. 4.7. Iron-Reducing Heterotrophs 4.7.1. Acidiphilium spp. 4.7.2. Alicyclobacillus-Like Bacteria 4.8. Other Acidophilic Heterotrophic Bacteria 4.8.1. Acidocella spp. 4.8.2. Acidobacterium capsulatum 4.8.3. Acidomonas methanolica 4.8.4. Alicyclobacillus spp. 5. Acidophilic Archaea 5.1. Crenarchaeot 5.1.1. Sulfolobus spp. 5.1.2. Acidianus spp. 5.1.3. Metallosphaera spp. 5.1.4. Stygiolobus azoricus

5.1.5. Other Thermo-Acidophilic Crenarchaeota 5.2. Euryarchaeota 5.2.1. Thermoplasma spp. 5.2.2. Picrophilus spp. 5.2.3. Ferroplasma spp. 6. Eukaryotic Acidophiles 6.1. Fungi and Yeasts 6.2. Protozoa 6.3. Microalgae 6.4. Rotifera 7. Relationships Between Acidophilic Microorganisms 7.1. Competition 7.2. Mutualism 7.3. Synergism 7.4. Ammensalism 7.5. Predation 8. Conclusions

Ion Transport in Acidophiles 119 A.C. Matin, Stanford University, California, USA

1. Introduction 2. Acid Resistance Mechanisms in Neutrophiles 3. Mechanism of Growth Under Extreme Acid Conditions 3.1. Proton Motive Force 3.2. Effect of Protonophores 3.3. H+ Circulation 3.4. H+ Impermeability Acidophilic Microorganisms 3.5. Buffering Capacity 3.6. Nature of the Membrane Potential 3.6.1. H+ Diffusion Potential 3.6.2. Donnan Potential 3.7. Maintenance of ΔpH and Cell Viability 3.8. Active Transport of Ions Other than H+ 3.8.1. Electrogenic K+ Transport 3.8.2. Cl- Transport 3.9. Interaction of Different Forms of ∆Ψ , and Active Ionic Circulation in the Maintenance of ∆pH 4. Acid Resistance of H. pylori 4.1. Is H. pylori a True Acidophile? 4.2. Role of Intracellular Urease 5. Conclusion

Efflux Systems in Metallophiles 143 Christopher Rensing, University of Arizona, Tucson, Arizona, USA Gregor Grass, University of Arizona, Tucson, Arizona, USA

1. Introduction 2. Metal Efflux Systems in Bacteria 3. Soft Metal Translocating P-type ATPases 4. CDF proteins 5. RND Type Efflux Complexes 6. MFS Transporters 7. Selfish Operons 8. Conclusion

Piezophily: Prokaryotes Exposed to Elevated Hydrostatic Pressure 157 Daniel Prieur, Université de Bretagne occidentale, Plouzané, France.

1. Introduction 2. Deep-Sea Microbiology 2.1. A Brief History 2.2. Deep-Sea Psychrophiles 2.2.1. General Features 2.2.2. Adaptations to Elevated Hydrostatic Pressure 2.3. Deep-Sea Hydrothermal Vents 2.3.1. Deep-Sea Hyperthermophiles 2.3.2. Responses to Hydrostatic Pressure 3. Other Natural Environments Exposed to Hydrostatic Pressure 3.1. Deep Marine Sediments 3.2. Deep Oil Reservoirs 3.3. Deep Rocks and Aquifers 3.4. Sub-Antarctic Lakes 4. Other Worlds 4.1. Mars 4.2. Europa 5. Conclusions

Characteristics of Deep-Sea Environments and Biodiversity of Piezophilic Organisms 174 Chiaki Kato, Department of Marine Ecosystems Research, Japan Marine Science and Technology Center, Japan Koki Horikoshi, Department of Engineering, Toyo University, Japan

1. Investigation of Life in a High-Pressure Environment 2. JAMSTEC Exploration of the Deep-Sea High-Pressure Environment 3. Taxonomic Identification of Piezophilic Bacteria 3.1. Isolation of Piezophiles and their Growth Properties 3.2. Taxonomic Characterization and Phylogenetic Relations 4. Biodiversity of Piezophiles in the Ocean Environment 4.1. Microbial Diversity of the Deep-Sea Environment at Different Depths 4.2. Changes in Microbial Diversity under High-Pressure Cultivation 4.3. Diversity of Deep-Sea Shewanella Is Related to Deep Ocean Circulation 4.3.1. Diversity, Phylogenetic Relationships, and Growth Properties of Shewanella Species Under Pressure Conditions 4.3.2. Relations between Shewanella Phylogenetic Structure and Deep Ocean Circulation 5. Molecular Mechanisms of Adaptation to the High-Pressure Environment 5.1. Mechanisms of Transcriptional Regulation under Pressure Conditions in Piezophiles 5.1.1. Pressure-Regulated Promoter of S. violacea Strain DSS12 5.1.2. Analysis of the Region Upstream From The Pressure-Regulated Genes 5.1.3. Possible Model of Molecular Mechanisms of Pressure-Regulated Transcription By The Sigma 54 Factor 5.2. Effect of Pressure on Respiratory Chain Components in Piezophiles 5.2.1. Respiratory Systems In S. violacea Strain DSS12 5.2.2. Respiratory Systems In Another Piezophile, S. benthica Strain DB172F 6. Conclusions

Pressure Effects on Biomolecules 199 Karel Heremans, Department of Chemistry, Katholieke Universiteit Leuven, Belgium

1. Introduction 2. Pressure effects compared with temperature effects 2.1. The Le Chatelier and Braun principle

2.2. Compressibility, thermal expansivity, heat capacity 2.2.1. Definitions 2.2.2. Statistical thermodynamics: Molecular fluctuations 2.3. Phase changes in single component systems: Clausius-Clapeyron 2.4. Solutions 2.4.1. Molecular interpretation of compressibility and thermal expansivity 2.4.2. Molecular intrepretation of reaction volumes: Electrostriction 3. Role of water and solvent composition 3.1. Cavities in biopolymers 3.2. Hydration of biopolymers 3.3. Water activity and osmotic pressure 3.4. Pressure effect on glass transitions 4. Modeling the pressure-temperature behavior of biomolecules 4.1. Thermodynamic modeling 4.2. Computer modeling 5. Lipids and biomembranes 6. Nucleic acids 7. Polysaccharides 8. Proteins 8.1. Ligand-binding 8.2. Protein-protein interactions 8.3. Protein unfolding and intermolecular aggregation 8.3.1. Protein unfolding: Conformational intermediates 8.3.2. Stability phase diagram and intermolecular interactions 8.4. Gel formation 8.5. Mixtures of proteins and polysaccharides 9. From molecules to cells 10. Conclusion: the specificity of pressure effects

Piezophiles: Microbial Adaptation to the Deep-Sea Environment 231 Eric E. Allen, Scripps Institution of Oceanography, University of California, San Diego–La Jolla, California, USA Douglas H. Bartlett, Scripps Institution of Oceanography, University of California, San Diego–La Jolla, California, USA

1. Introduction 2. Deep-Sea Habitats 3. Isolation and Characterization of Piezophiles 3.1. Nomenclature 3.2. Isolation and Cultivation 3.3. Taxonomy 3.4. Growth and Physiology of Piezophiles 4. High-Pressure Adaptation Mechanisms 4.1. Metabolic Responses to Pressure 4.2. Membrane Proteins and Pressure Regulated Gene Expression 4.3. Pressure Regulated Operons in Piezophilic Shewanella sp. 4.4. Enzyme Stability and Activity at High Pressure 4.5. Cell Division 4.6. Membrane Lipids 5. Future Prospects

Enzymes from Deep-Sea Microorganisms 257 Hideto Takami, Microbial Genome Research Group, Japan Marine Science and Technology Center, 2- 15 Natsushima, Yokosuka, 237-0061 Japan

1. Introduction 2. Collection of Deep-sea Mud

3. Isolation of Microorganisms from Deep-sea Mud 3.1. Bacteria From The Mariana Trench 3.2. Bacteria From Other Deep-Sea Sites Located Off Southern Japan 4. 16S rDNA Sequences of Deep-sea Isolates 5. Exploring Unique Enzyme Producers Among Deep-sea Isolates 5.1. Screening for Amylase Producers 5.2. Purification of Amylase Produced By Pseudomonas Strain MS300 5.3. Enzyme Profiles

Index 273

About EOLSS 283