HALOPHILIC MICROORGANISMS and THEIR ENVIRONMENTS Cellular Origin and Life in Extreme Habitats

HALOPHILIC MICROORGANISMS AND THEIR ENVIRONMENTS Cellular Origin and Life in Extreme Habitats

Volume 5

Series Editor: Joseph Seckbach Hebrew University of Jerusalem, Israel Halophilic Microorganisms and their Environments

by Aharon Oren The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, Israel

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Visit Kluwer Online at: http://kluweronline.com and Kluwer's eBookstore at: http://ebooks.kluweronline.com DEDICATION

This book is dedicated to the memory of Donn J. Kushner, one of the founding fathers of halophile microbiology, who passed away on September 15, 2001, at the age of 74. My first meeting with Donn took place at the 1983 ASM meeting in New Orleans. At the time I was a newcomer in the field of halophile science, and I clearly remember how thrilled I was to meet one of the 'big' names in the field. It quickly appeared that we shared not only an interest in science, but in music as well. Donn was an accomplished player of the violin and the viola. We have since performed chamber music together at scientific meetings in Jerusalem (1987), in Alicante (1989, together with Morris Kates playing the violin and Masamichi Kohiyama on 'cello), in Williamsburg (1992, again with Morris Kates' violin and Larry Hochstein's clarinet), and once more in Jerusalem (1997, again joined by Larry Hochstein). I will always remember these informal concerts with colleague halophile scientists as highlights in my career. I hope that the following pages will form a fitting tribute to Donn’s many contributions to our understanding of halophilic microorganisms.

Aharon Oren

v TABLE OF CONTENTS

DEDICATION v

TABLE OF CONTENTS vii

FOREWORD BY JOSEPH SECKBACH xiii

PREFACE xv

COMMENTS ON PROKARYOTE NOMENCLATURE AND xix SALT CONCENTRATION UNITS

SECTION 1. AN HISTORICAL SURVEY 1

CHAPTER 1. HALOPHILIC MICROORGANISMS IN THEIR NATURAL ENVIRONMENT 3 AND IN CULTURE -AN HISTORICAL INTRODUCTION 1.1. Red brines - early observations and explanations 3 1.2. The first records of the isolation of halophilic 5 Archaea and Bacteria 1.3. Dunaliella and other halophilic algae 8 1.4. Ecological studies: the Great Salt Lake 9 1.5. Alkaline soda lakes 10 1.6. The Dead Sea 11 1.7. The study of biogeochemical processes in hypersaline environments 11 1.8. The beginning of the modern era of halophile research 13 1.9. References 14

SECTION 2.HALOPHILIC MICROORGANISMS AND THEIR PROPERTIES 19

INTRODUCTION 21

CHAPTER 2. TAXONOMY OF HALOPHILIC MICROORGANISMS: ARCHAEA, 23 BACTERIA AND EUCARYA 2.1. The place of halophiles within the microbial world 23 2.2. The halophilic Archaea 25 2.3. The halophilic and halotolerant Bacteria 36 2.4. The halophilic and halotolerant Eucarya 56 2.5. References 57

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CHAPTER 3. THE CELLULAR STRUCTURE OF HALOPHILIC 69 MICROORGANISMS 3.1. Cellular structures of halophilic Archaea 69 3.2. Cellular structures of halophilic Bacteria 99 3.3. Cellular structrues of halophilic Eucarya 110 3.4. References 111

CHAPTER 4. CELLULAR METABOLISM AND PHYSIOLOGY OF HALOPHILIC 125 MICROORGANISMS 4.1. Physiology of halophilic Archaea 125 4.2. Physiology of halophilic Bacteria 146 4.3. Physiology of halophilic Eucarya 152 4.4. Metabolic diversity among the halophiles: a bioenergetic approach 153 4.5. References 157

CHAPTER 5. PIGMENTS OF HALOPHILIC MICROORGANISMS 173

5.1. Algal carotenoids 174 5.2. Pigments of oxygenic and anoxygenic photosynthetic Bacteria 178 5.3. Carotenoids of aerobic heterotrophic Archaea and Bacteria 179 5.4. The retinal pigments: bacteriorhodopsin, halorhodopsin, and sensory rhodopsins 183 5.5. The photoactive yellow protein of Halorhodospira and other halophilic purple bacteria 196 5.6. References 198

CHAPTER 6. INTRACELLULAR SALT CONCENTRATIONS AND ION METABOLISM 207 IN HALOPHILIC MICROORGANISMS 6.1. Introduction 207 6.2. Methods used to estimate intracellular ionic concentrations in halophilic microorganisms 208 6.3. Ion metabolism in the Halobacteriaceae 210 6.4. Ion metabolism in aerobic halophilic Bacteria 214 6.5. Ion metabolism in the Halanaerobiales 223 6.6. Ion metabolism in Dunaliella 223 6.7. References 227

CHAPTER 7. PROPERTIES OF HALOPHILIC PROTEINS 233 7.1. Introduction 233 7.2. Halophilic behavior of enzymes from halophilic Archaea 235 7.3. Purification of halophilic proteins 240 7.4. Salt relationships of selected proteins from halophilic Archaea 249 ix

7.5. Halophilic behavior of enzymes from the aerobic halophilic Bacteria 253 7.6. Halophilic behavior of enzymes from the anaerobic halophilic Bacteria 264 7.7. Halophilic behavior of enzymes from the halophilic Eucarya 264 7.8. References 267

CHAPTER 8. ORGANIC COMPATIBLE SOLUTES 279 8.1. Organic osmotic solutes and their distribution 279 8.2. Compatible solutes in the domain Archaea 284 8.3. Compatible solutes in the domain Bacteria 286 8.4. Compatible solutes in the domain Eucarya 294 8.5. The mode of action of compatible solutes 297 8.6. References 299

CHAPTER 9. HALOPHILIC BACTERIOPHAGES AND HALOCINS 307 9.1. Bacteriophages of halophilic microorganisms 307 9.2. Halocins 316 9.3. References 319

CHAPTER 10. GENETICS AND GENOMICS OF HALOPHILIC ARCHAEA AND BACTERIA 323 10.1. Genetics of halophilic microorganisms - an historical survey 323 10.2. Genetics of halophilic Archaea 324 10.3. Genetics of halophilic Bacteria 343 10.4. References 348

CHAPTER 11. BIOTECHNOLOGICAL APPLICATIONS AND POTENTIALS OF 357 HALOPHILIC MICROORGANISMS 11.1. Introduction 357 11.2. Applications of halophlic Archaea 359 11.3. Applications of halophlic Bacteria 367 11.4. Applications of halophlic Eucarya 375 11.5. References 380

SECTION 3. HYPERSALINE ENVIRONMENTS AND THEIR BIOTA 391

INTRODUCTION 393

CHAPTER 12. GREAT SALT LAKE, UTAH 395 12.1. The lake and its setting 395 12.2. The microbial communities of the Great Salt Lake 400 12.3. Microbial isolates and their properties 407 12.4. Biogeochemical processes 409 12.5. References 415 x

CHAPTER 13. THE DEAD SEA 419 13.1. The lake and its setting 419 13.2. Early studies on the biota of the Dead Sea 423 13.3. Dynamics of microbial blooms in the Dead Sea 424 13.4. Microbial isolates and their properties 431 13.5. Adaptations of the Dead Sea biota to the ionic composition of the lake 433 13.6. The nature of the species dominant in the archaeal blooms 434 13.7. Anaerobic processes in the Dead Sea sediments 434 13.8. Biology of the Dead Sea - expected future developments 435 13.9. References 436

CHAPTER 14. SOLAR SALTERNS 441 14.1. The saltern environment and its biota 441 14.2. Benthic microbial mats in the evaporation ponds 444 14.3. Microbial isolates and their properties 448 14.4. Approaches toward the identification of the dominant Archaea in saltern crystallizer ponds 451 14.5. Salinibacter and other halophilic Bacteria in saltern ponds 453 14.6. The red pigments in salten crystallizer ponds 454 14.7. Dynamics of archaeal and bacterial communities in saltern ponds 455 14.8. The importance of the saltern biota in the production of solar salt 459 14.9. References 462

CHAPTER 15. ALKALINE HYPERSALINE LAKES IN AFRICA AND ASIA 471 15.1. The Wadi Natrun lakes 472 15.2. Lake Magadi and other East-African soda lakes 478 15.3. Hypersaline soda lakes in Asia - chemical and biological characteristics 489 15.4. References 491

CHAPTER 16. MONO LAKE, CALIFORNIA, AND BIG SODA LAKE, NEVADA 495 16.1. Hypersaline lakes in the Great Basin of North America 495 16.2. Mono Lake, California 496 16.3. Big Soda Lake, Nevada 508 16.4. References 512 xi

CHAPTER 17. MISCELLANEOUS HABITATS OF HALOPHILIC MICROORGANISMS – 517 FROM ANTARCTIC LAKES TO HYDROTHERMAL VENTS 17.1. Cold and hypersaline: Antarctic hypersaline lakes 517 17.2. Hot and hypersaline: Solar Lake (Sinai) and other warm brines 519 17.3. Seawater, deep sea brines and hydrothermal vents 525 17.4. Halophilic microorganisms in oil field brines 528 17.5. Halophiles in salt marshes, hypersaline lagoons and miscellaneous lakes 528 17.6. Hypersaline springs 531 17.7. Hypersaline soils 532 17.8. Wall paintings 532 17.9. Desert plants and animals 533 17.10. References 533

SECTION 4. EPILOGUE 541

CHAPTER 18. EPILOGUE:EVOLUTION OF HALOPHILES AND SURVIVAL 543 OF HALOPHILES ON EARTH AND IN SPACE 18.1. The evolutionary origin of halophiles 543 18.2. Long-term survival of halophiles in ancient salt crystals 545 18.3. Halophiles in space? 548 18.4. References 549

SECTION 5. SUPPLEMENT 553

METHODS FOR CULTIVATION AND HANDLING OF HALOPHILIC ARCHAEA AND 555 BACTERIA

GLOSSARY OF LIMNOLOGICAL TERMS 559

ABOUT THE AUTHOR 561

ORGANISM INDEX 563

GEOGRAPHICAL INDEX 567

SUBJECT INDEX 571 FOREWORD

"This water" he told me, "runs out to the eastern region, and flows into the Arabah; and when it comes into the sea, into the sea of foul waters [i.e., the Dead Sea], the water will become wholesome. Every living creature that swarms will be able to live wherever this stream goes; the fish will be very abundant once these waters have reached there. It will be wholesome, and everything will live wherever this stream goes. Fishermen shall stand beside it all the way from En-gedi to En-eglaim; it shall be a place for drying nets; and the fish will be of various kinds [and] most plentiful, like the fish of the Great Sea."

Ezekiel’s prophecy (Ezekiel 47: 8-10) for revival and purification of the Dead Sea waters

This new book on "Halophilic Microorganisms and their Environments" is the fifth volume in the COLE series (Cellular Origin and Life in Extreme Habitats (see: http://www.wkap.nl/prod/s/COLE). In the previous books we covered aspects of enigmatic microorganisms, microbial diversity, astrobiology, and symbiosis, so this book on halophilic microbes adds a fitting link to the rest of series' books. Since ancient times hypersaline habitats have been considered extreme environments, and some were thought not to sustain life at all. Yet, every organism requires salt for its existence. Salty places have been compared to an environment of extinction (e.g., the Dead Sea). In the Bible, Lot's wife was converted into a pillar of salt as a punishment for disobeying the request to not glance backward (Genesis 19:26). Moses warned the people that: "all its soil devastated by sulfur and salt, beyond sowing and producing, no grass growing in it, just like the upheaval of Sodom and Gomorrah...which the Lord overthrew in His fierce anger" (Deuteronomy 29:22). Abimelech razed the town of Shechem and as an act of desolation he sowed it with salt in order to punish that rebellious city (Judges 9:45). But, in the Bible too, salt also has some curative power, as the prophet Elisha cures bad water by pouring salt into it (Kings II 2:19-22). The prophet Ezekiel recognizes the universal need for salt and promises that some areas (following the purification of the Dead Sea – see above) will still remain saline (Ezekiel 47:11). Hypersaline habitats like the Dead Sea have been considered a "dead" environment devoid of life. Over a half century ago, however, Ben Volcani (1936) demonstrated that the Dead Sea is rather alive and sustains active life of microorganisms. Since then, our knowledge of the biota of the Dead Sea and of other hypersaline environments has increased tremendously. This current volume enriches our information about various aspects of the hypersaline world.

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This volume describes the highly diverse groups of microorganisms growing in hypersaline environments all over the earth. Topics covered include taxonomy, physiology, ecology, osmoregulation, as well as other properties of halophiles. Special chapters are devoted to the hypersaline lakes such as the Dead Sea (between Israel and Jordan) and the Great Salt Lake (Utah, USA), both as halo-extremophilic environments. This book is addressed to researchers and students in the fields of microbiology, marine biology, ecological biology, workers in the field of extreme environments, and to every "knowledge-thirsty" reader interested in biology. The author, Aharon Oren, a well-known expert in the area of halophilic microorganisms, took up the challenge to review the halophiles. He is an active board member of this series. On this broad subject he has published numerous journal articles and contributed several chapters to books. Dr. Oren organized an international research workshop on "Microbiology and Biogeochemistry of Hypersaline Environments" (1997) and edited its proceedings (1999). Other volumes in this series "Cellular Origin and Life in Extreme Habitats" contain additional chapters on the halophiles and the Dead Sea, first in Enigmatic Microorganisms and Life in Extreme Environments (http://www.wkap. nl/prod/b/0-7923- 5492-3) and then in Journey to Diverse Microbial Worlds (http://www.wkap.nl/prod/b/0-7923-6020-6). In these lines, I wish to thank the author, Professor Oren, for his swift completion of the project of writing this book, and his full cooperation with the editor. Much appreciation is due to the Kluwer Academic Publishers team and specifically to Mrs. Claire van Heukelom and Dr. Frans van Dunne for their constant interest, collaboration and assistance in the "making" of this new volume. Last but not least, I am grateful to my wife, Fern Seckbach for encouragement to proceed with this book series.

The Hebrew University of Jerusalem Joseph Seckbach [email protected] Chief Editor of COLE Book Series April 2002 PREFACE

How surprising it is that any creatures should be able to exist in a fluid, saturated with brine, and that they should be crawling among crystals of sulphate of soda and lime!

(Charles Darwin, 1839).

Halophilic microorganisms - organisms that prefer to take their habitat "cum grano salis", with a grain of salt - inhabit hypersaline environments with salt concentrations up to NaCl saturation. Halophiles are found in all three domains of life: Archaea, Bacteria and Eucarya. They may occur in large numbers in hypersaline lakes and in other habitats characterized by salt concentrations approaching saturation, and they often have a strong impact on the ecosystems in which they thrive. Halophilic microorganisms also present the biochemist/physiologist with interesting questions on how organisms cope with the high osmotic pressures exerted by the highly salty medium. The study of the halophiles has shown that the microbial world has developed a variety of adaptive strategies to enable growth in brines that are hostile to other forms of life. I was first introduced to the fascinating world of the halophiles in 1980, when the late Prof. Moshe Shilo invited me to join a study of the biology of the Dead Sea. In fact I had worked with halophilic microorganisms before: my Ph.D. thesis work dealt with physiological and biochemical aspects of a cyanobacterium named Oscillatoria limnetica, isolated from the hypersaline Solar Lake, Sinai (see also Section 17.2). I used to grow this organism at a salt concentration twice that of seawater, but it tolerates much higher salinities. At the time, however, I did not devote much thought to aspects of its salt tolerance, salt requirement and salt adaptation. The variety of halophiles encountered in high numbers in the Dead Sea in 1980, a year in which unusually dense blooms of the alga Dunaliella and of red halophilic Archaea developed in the lake (see Chapter 13), aroused my interest in these forms of life and in the ways they cope with the challenges presented by their environment. The biology of halophilic microorganisms has been the subject of a number of earlier monographs. These include the two-volume "Halophilic Bacteria" edited by Francisco Rodríguez-Valera (CRC Press, 1988), and "The Biology of Halophilic Bacteria", edited by Russ Vreeland and Larry Hochstein (CRC Press, 1993). There are also a number of books published as meeting proceedings: "Energetics and Structure of Halophilic Microorganisms", edited by Roy Caplan and Margaret Ginzburg (Elsevier, 1978), General and Applied Aspects of Halophilic Microorganisms (edited by Francisco Rodríguez-Valera, Plenum Press, 1991), and "Microbiology and Biogeochemistry of Hypersaline Environments", a book I edited as the proceedings of a symposium on

xv xvi halophilic microorganisms I had the pleasure of organizing in 1997 (CRC Press, 1999). These volumes all give a good overview of many aspects of the life of halophilic microorganisms. A number of excellent review articles and chapters have also been written in the course of the years. The comprehensive overview of all aspects of the ecology, physiology and biochemistry of halophiles presented by Donn Kushner in 1978 in a 52-page chapter in the book he edited on "Microbial Life in Extreme Environments" (Academic Press), a chapter that beautifully summarized the knowledge available at the time, is still relevant today. The writing of this book was inspired to a large extent by the exemplary monograph written by Barbara Javor on "Hypersaline Environments - Microbiology and Biogeochemistry" (Brock/Springer, 1989). That book covered the state of the art of halophile microbiology at the time, with a strong emphasis on the biogeochemical aspects, but without neglecting in-depth information on the taxonomy, physiology and biochemistry of those organisms that inhabit hypersaline environments. I can only hope that the present volume will provide an overview of the current state of the science as thorough and as well-balanced as Javor's book. Tremendous progress has been made in the last decades. The greatest breakthrough in the past few years came of course from the field of genomics. The first complete genome sequence of a halophile was published in 2000; more such sequences are expected to become available in the near future. New questions will be asked and deeper insights will be obtained than thought possible until even a few years ago. To some extent this volume presents an overview of halophilic science in the "pre- genomic" era, while providing glimpses of the first exciting results obtained from whole genome analyses. With all the advances in the understanding of the properties of individual model organisms we should not forget the place of the halophilic microorganisms in their natural habitats. Also here significant breakthroughs have been made in recent years, especially thanks to the application of methods derived from molecular biology, including culture-independent techniques that allow us to obtain information on the structure of the microbial community. This book contains four sections. It opens with a historical overview, showing how the ideas about the nature of the halophilic microorganisms have evolved over the centuries. The second section provides an overview of our understanding of the halophilic microorganisms themselves, including aspects of their taxonomy, physiology, genetics, and biotechnological applications. The third section is ecosystem-centered. A number of representative hypersaline environments are discussed and information is reviewed on their community structure and on the activities of the different halophilic microorganisms. The volume concludes with some thoughts about the evolution of halophiles and their longevity, addressing questions about the possibility of survival of halophilic prokaryotes within salt crystals for hundreds of millions of years, and the possible existence of halophiles elsewhere in the Universe. References to methods used for the cultivation and handling of the halophiles, as well as a glossary of limnological terms are given in the supplement. xvii

While preparing the chapters for this book the author was confronted with the question how to define a halophile and a hypersaline environment: how salty should an environment be and how much salt should be required or tolerated by a microorganism to be included in this volume? As microorganisms present us with a continuum of adaptations, from freshwater to salt saturation, and as many organisms easily adapt to a wide range of often rapidly changing salt concentrations, there is no obvious answer to such a question. Any attempt to classify the halophiles into categories according to their salt requirement and salt tolerance is to a large extent artificial, and many species defy classification within such schemes. I arbitrarily chose the value of salt as the boundary. I have attempted to include those environments in which the salt concentration exceeds salt and those microorganisms that are able to grow well above that value, even if their salinity optimum may sometimes be in the lower range. Mono Lake (California) and Big Soda Lake (Nevada) (Chapter 16) are borderline cases: with salt concentrations up to 98 and respectively, these lakes have salinities just below the above-defined boundary. However, both these lakes have many interesting properties, and their biota have been the subject of many in-depth studies. A discussion of their properties therefore appears warranted. Each chapter contains a large number of references. These cover not only the most recent articles relevant to the field, but I intentionally included citations of old papers, often going back to the first pioneering studies that opened up the field. In the course of the time I have been involved with the halophiles, I have come across many forgotten publications that demonstrate that earlier generations of scientists knew much more than we are likely to admit nowadays. Hopefully this book will therewith become a valuable source of references, including papers that have been published many decades or even more than a century ago. The large number of literature citations may give a false impression of completeness. Even lists of references twice as long as those presented here cannot cover the literature in full. This is especially true in the case of bacteriorhodopsin and the other retinal pigments of halophilic Archaea. A search for articles on bacteriorhodopsin in the ISI Web of Science, covering the period from 1988 to 2001, yielded over 3,500 entries. In such cases a rigid selection was needed. Special emphasis has then been placed on coverage of those papers published in the early days, during which the function of the pigment has become clear, and on the major breakthroughs achieved in recent years that have led to an exact picture of the functioning of the retinal proteins. I thus hope that the book provides a fair coverage of all relevant aspects of the biology of halophilic microorganisms and our current understanding of what halophilic microorganisms are and how they function in nature. I have attempted to include all relevant material that was published before the end of January 2002. A book of this scope and size will undoubtedly contain omissions, inaccuracies and errors. Comments are welcome at all times. I have learned much while working on this book and I hope to learn even more from the comments by my colleagues. xviii

I want to thank Joseph Seckbach for his invitation to contribute a volume to his book series on "Cellular Origin and Life in Extreme Habitats". Thanks also to Claire van Heukelom and her colleagues at Kluwer Academic Publishers who helped me to produce this volume within a relatively short time. I finally would like to thank those colleagues who have reviewed the different chapters of this book and have suggested many corrections and improvements: Mike Dyall-Smith (Melbourne. Australia), Erwin Galinski (Bonn, Germany), Bill Grant (Leicester, UK), Nina Gunde- Cimerman (Ljubljana, Slovenia), Tim Hollibaugh (Athens, GA, USA), Kjeld Ingvorsen (Aarhus, Denmark), Bob Jellison (Mammoth Lakes, CA, USA), Martin Kessel (Bethesda, MD, USA), Carol Litchfield (Manassas, VA, USA), Dominique Madern (Grenoble, France), Volker Müller (München, Germany), Dieter Oesterhelt (Martinsried, Germany), Ron Oremland (Menlo Park, CA, USA), Francisco Rodríguez- Valera (Alicante, Spain), and Russ Vreeland (West Chester, PA, USA).

Jerusalem, April 2002

Aharon Oren

The Institute of Life Sciences, and the Moshe Shilo Minerva Center for Marine Biogeochemistry The Hebrew University of Jerusalem

E-mail address: [email protected] COMMENTS ON PROKARYOTE NOMENCLATURE AND SALT CONCENTRATION UNITS

Taxonomy is a living science, and the nomenclature of microorganisms has seen many changes as our views on microbial taxonomy have developed. The increasing understanding of the phylogenetic relationships between different microorganisms, as enabled by 16S or 18S rRNA sequence analysis, has revolutionized microbial taxonomy in general, and the taxonomy of prokaryotes in particular. As a result, many names of halophilic microorganisms, Archaea as well as Bacteria, have changed over the years. To give a few examples: some Archaea formerly classified in the genus Halobacterium have later been transferred to genera such as Haloferax, Haloarcula, and Halorubrum. Moreover, the former species Halobacterium salinarium, Halobacterium halobium, and Halobacterium cutirubrum have been unified into a single species, Halobacterium salinarum. In the bacterial domain, Vibrio costicola became Salinivibrio costicola. The Dead Sea isolate Ba1, an organism that has been the subject of many physiological studies, was first named Halomonas israelensis, and has recently been renamed Chromohalobacter israelensis. To further complicate the matter, an isolate (DSM 3043 = ATCC 33174) formerly classified as a strain of Halomonas elongata has now been removed from the genus Halomonas and renamed Chromohalobacter salexigens. This particular strain as been used in a large number of genetic studies, and so has the type strain of Halomonas elongata. A considerable extent of confusion may result from such nomenclatural changes. This book has attempted to use the currently approved nomenclature, as given in the lists that periodically appear in the International Journal of Systematic and Evolutionary Microbiology ("Notification that new names and new combinations have appeared in volume …, part …, of the IJSEM", and "Validation of publication of new names and new combinations previously effectively published outside the IJSEM"), even when this may lead to some confusion. The prokaryote nomenclature web site ("List of bacterial names with standing in nomenclature") maintained by Jean Euzéby (www.bacterio.cict.fr) is also a reliable source of updated information. Nomenclatural information, including former species designations (basonyms), has been included in the description of the different halophilic microorganisms in Chapter 2. When performing experiments with different species of Bacteria or Archaea, the importance of the proper designation of the strain used and the desirability of using the designated type strain whenever possible cannot be sufficiently stressed. Communication between physiologists, molecular biologists and geneticists at one side and taxonomists on the other side is often poor or altogether absent. There are still too many articles, even those published in recent years, in which the source of the strain used was not clarified beyond statements such as: "Halobacterium halobium was a gift from colleague xyz". The case of Halobacterium salinarum - Halobacterium salinarium - Halobacterium halobium - Halobacterium cutirubrum is a particularly

xix xx interesting one. Complete genome sequences of two strains (Halobacterium strain NRC-1 and Halobacterium salinarum [halobium] strain R1) are now available – at the time of writing the only genomes of halophilic Archaea sequenced – and none of these is the type strain of the species. In fact, the source of strain NRC-1 is obscure. The authors of the publication describing the genome (Ng et al., 2000) state: "The precise relationships among [H. halobium. H. cutirubrum, H. salinarium, and H. salinarum] and Halobacterium sp. strain NRC-1 are not entirely clear. Strain NRC-1 was a gift from W.F. Doolittle, Dalhousie University, Halifax, Canada. The strain has been deposited with the American Type Culture Collection, Manassas, VA (reference no. ATCC 700922)." Halobacterium strain R1 is a mutant laboratory strain often used in physiological experiments. Tindall (1992) has presented an excellent overview of the source and history of many of the old Halobacterium isolates that circulate in culture collections and laboratories. In this book all isolates designated Halobacterium salinarium, Halobacterium halobium or Halobacterium cutirubrum, including the strains NRC-1 and R1, have been named Halobacterium salinarum. Details of the particular strain used or its culture collection accession number (if available at all) have not been specified in most cases. For additional details on the identity and the source of the strain used readers should therefore refer to the original publications as cited. There are probably even cases in the literature in which strains designated as "Halobacterium sp." may not actually belong to that genus. Caution should therefore be exerted when comparing data published on "Halobacterium salinarum". In the case of the cyanobacteria matters of nomenclature are also exceedingly confusing. There are many cases in which the same organism has become known under two, three and more different names in the literature. More information on the nomenclatural problems with halophilic and halotolerant cyanobacteria can be found in Chapter 2 and in a book chapter on cyanobacteria in hypersaline environments (Oren, 2000). Taxonomic issues dealing with the halophilic Archaea of the family Halobacteriaceae are discussed by the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halobacteriaceae. Such a subcommittee also exists for the photosynthetic prokaryotes, a group that also contains a considerable number of halophiles. A subcommittee that will deal with the taxonomy of the Halomonadaceae is expected to become established in the near future. The minutes of the meetings of these subcommittees, as published in the International Journal of Systematic and Evolutionary Microbiology (see e.g. Oren and Ventosa, 2002) contain much relevant information on the taxonomy and nomenclature of halophiles. In this book the word "Bacteria" (with a capital B) is reserved for describing organisms in a phylogenetic sense, i.e. as members of the domain Bacteria. In contrast, the word "bacteria" is used to refer to prokaryotes in general, both Bacteria and Archaea.

Salt concentrations can be expressed in a variety of units. These include molar units , molal units , weight per volume - as % salt = g per 100 ml solution or or on a weight per weight basis - as salinity (g per kg). While oceanographers and limnologists generally prefer the salinity unit to describe the salt xxi concentrations of saline and hypersaline water bodies, the composition of growth media is nearly always given in units of gram or mol salt per liter. While reviewing the literature during the preparation of this book it became clear that the term "salinity" is often (mis)used to describe salt concentrations in terms of grams of salt per liter rather than grams of salt per kg. Especially at high salt concentrations these terms are not equivalent. Rather than attempting to elucidate what units had been used in each article reviewed, I have assumed that the term salinity when expressed as % or referred to salt concentrations in units of gram per 100 ml or gram per liter, respectively. Errors may possibly have been introduced while doing so. To achieve consistency throughout the book I have converted salt concentration data to units of as much as possible. Only in Chapters 6-8, where the concentrations and the effects of different ions are compared, have I used molar units, and occasionally also molal units.

REFERENCES

Ng, W.V., Kennedy, S.P., Mahairas, G.G., Berquist, B., Pan, M., Shukla, H.D., Lasky, S.R., Baliga, N.S., Thorsson, V., Sbrogna, J., Swartzell, S., Weir, D., Hall, J., Dahl, T.A., Welti, R., Goo, Y.A., Leithauser, B., Keller, K., Cruz, R., Danson, M.J., Hough, D.W., Maddocks, D.G., Jablonski, P.E., Krebs, M.P., Augevine, CM., Dale, H., Isenberger, T.A., Peck, R.F., Pohlschroder, M., Spudich, J.L., Jong, K.-H., Alam, M., Freitas, T., Hou, S., Daniels, C.J., Dennis, P.P., Omer, A.D., Ebhardt, H., Lowe, T.M., Liang, P., Riley, M., Hood, L., and DasSarma, S. 2000. Genome sequence of Halobacterium species NRC-1 Proc. Natl. Acad. Sci. USA97: 12176-12181. Tindall, B.J. 1992. The family Halobacteriaceae, pp. 768-808 In: Balows, A., Trüper, H.G., Dworkin, M., Harder, W., and Schleifer, K.-H. (Eds.), The prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. Vol. I. Springer-Verlag, New York. Oren, A. 2000. Salts and brines, pp. 281-306 In: Whitton, B.A., and Potts, M. (Eds.), Ecology of cyanobacteria: their diversity in time and space. Kluwer Academic Publishers, Dordrecht. Oren, A., and Ventosa, A. 2002. International Committee on Systematics of Prokaryotes. Subcommittee on the Taxonomy of Halobacteriaceae. Minutes of the Meetings, 24 September 2001, Sevilla, Spain. Int. J. Syst. Evol. Microbiol. 52: 289-290.