ADAPTATION TO LIFE AT HIGH SALT CONCENTRATIONSIN ARCHAEA, BACTERIA, AND EUKARYA

Volume 9

Series Editor:

Joseph Seckbach

Cellular Origin, Life in Extreme Habitats and Astrobiology

The Hebrew University of Jerusalem, Israel

Bacteria, and Eukarya

Adaptation to Life at High SaltConcentrations in Archaea,

Edited by

Nina Gunde-CimermanBiotechnical Faculty, Department of Biology,University of Ljubljana,Slovenia

Aharon Oren

The Hebrew University of Jerusalem,Israel

and

Ana PlemenitašInstitute of Biochemistry,University of Ljubljana,Slovenia

for Marine Biogeochemistry,The Institute of Life Sciences and the Moshe Minerva Center

A C.I.P. Catalogue record for this book is available from the Library of Congress.

Published by Springer,P.O. Box 17, 3300 AA Dordrecht, The Netherlands.

Printed on acid-free paper

All Rights Reserved© 2005 SpringerNo part of this work may be reproduced, stored in a retrieval system, or transmittedin any form or by any means, electronic, mechanical, photocopying, microfilming, recordingor otherwise, without written permission from the Publisher, with the exceptionof any material supplied specifically for the purpose of being enteredand executed on a computer system, for exclusive use by the purchaser of the work.

Printed in the Netherlands.

ISBN-10 1-4020-3633-7 (e-book)ISBN-13 978-1-4020-3632-3 (HB)

ISBN-10 1-4020-3632-9 (HB)

ISBN-13 978-1-4020-3633-0 (e-book)

www.springeronline.com

TABLE OF CONTENTS

Foreword Joseph Seckbach xi

Introduction Nina Gunde-Cimerman, Aharon Oren and Ana Plemenitaš 1

Section I. The environments and their diversity 7

Microbial diversity of Great Salt Lake Bonnie K. Baxter, Carol D. Litchfield, Kevin Sowers,

Jack D. Griffith, Priya Arora DasSarma and Shiladitya DasSarma 9

Microbial communities in the Dead Sea – past, present and future Aharon Oren, Ittai Gavrieli, Jonah Gavrieli, Marco Kohen, Joseph Lati and Mordehay Aharoni 27

Microscopic examination of microbial communities along a salinity gradient in saltern evaporation ponds: a ‘halophilic safari’ Aharon Oren 41

The microbial diversity of a solar saltern on San Francisco Bay Carol D. Litchfield, Masoumeh Sikaroodi and Patrick M. Gillivet 59

Diversity of microbial communities: the case of solar salterns Carlos Pedrós-Alió 71

Isolation of viable haloarchaea from ancient salt deposits and application of fluorescent stains for in situ detection of halophiles in hypersaline environmental samples and model fluid inclusions

Stefan Leuko, Andrea Legat, Sergiu Fendrihan, Heidi Wieland, Christian Radax, Claudia Gruber, Marion Pfaffenhuemer, Gerhard Weidler and Helga Stan-Lotter 91

vi

Hydrocarbon degradation under hypersaline conditions. Some facts, some experiments and many open questions Heiko Patzelt 105

The relevance of halophiles and other extremophiles to Martian and extraterrestrial environments Joseph Seckbach 123

Halophiles: a terrestrial analog for life in brines on Mars – Halophiles on Mars

Rocco L. Mancinelli 137

Section II. Archaea 149

Comparative genomic survey of information transfer systems in two diverse extremely halophilic Archaea, Halobacterium sp. strain NRC-1 and Haloarcula marismortui Brian R. Berquist, Jeetendra Soneja and Shiladitya DasSarma 151

Walsby’s square archaeon; it’s hip to be square but even more hip to be culturable Henk Bolhuis 185

Gene regulation and the initiation of translation in halophilic Archaea Felicitas Pfeifer, Peter Zimmermann, Sandra Scheuch and Simone Sartorius-Neef 201

Protein translation, targeting and translocation in Haloferax volcanii Jerry Eichler, Gabriela Ring, Vered Irihimovitch, Tovit Lichi, Irit Tozik and Zvia Konrad 217

Enzymes of halophilic Archaea. Recent findings on ureases and nucleoside diphosphate kinases Toru Mizuki, Ron Usami, Masayuki Kamo, Masaru Tanokura

and Masahiro Kamekura 227

Osmoadaptation in methanogenic Archaea: recent insights from a genomic perspective Katharina Pflüger, Heidi Wieland and Volker Müller 239

Section III. Bacteria 253

Salinibacter ruber: genomics and biogeography Josefa Antón, Arantxa Peña, Maria Valens, Fernando Santos, Frank-Oliver Glöckner, Margarete Bauer, Joaquín Dopazo, Javier Herrero, Ramon Rosselló-Mora and Rudolf Amann 255

What we can deduce about metabolism in the moderate halophile Chromohalobacter salexigens from its genomic sequence

Laszlo N. Csonka, Kathleen O’Connor, Frank Larimer, Paul Richardson, Alla Lapidus, Adam D. Ewing, Bradley W. Goodner and Aharon Oren 267

K+ transport and its role for osmoregulation in a halophilic member of the Bacteria domain: characterization of the K+ uptake systems from Halomonas elongata Hans-Jörg Kunte 287

The chloride regulon of Halobacillus halophilus: a novel regulatory network for salt perception and signal transduction in bacteria Volker Müller and Stephan H. Saum 301

Biosynthesis of the compatible solute mannosylglycerate from hyperthermophiles to mesophiles. Genes, enzymes and evolutionary perspectives Milton S. da Costa and Nuno Empadinhas 311

Genes and enzymes of ectoine biosynthesis in the haloalkaliphilic obligate methanotroph “Methylomicrobium alcaliphilum 20Z”

Alexander S. Reshetnikov, Valentina N. Khmelenina, Ildar I. Mustakhimov, Yana V. Ryzhmanova and Yuri A. Trotsenko 327

vii

viii

Halophilic Archaea and Bacteria as a source of extracellular hydrolytic enzymes

Antonio Ventosa, Cristina Sánchez-Porro, Sara Martín and Encarnación Mellado 337

Biopolyester production: halophilic microorganisms as an attractive source Jorge Quillaguamán, Bo Mattiasson and Rajni Hatti-Kaul 355

Section IV. Fungi 369

Relation of halotolerance to human pathogenicity in the fungal tree of life: an overview of ecology and evolution under stress

G. Sybren de Hoog, Polona Zalar, Bert Gerrits van den Ende and Nina Gunde-Cimerman 371

Halotolerant and halophilic fungi from coastal environments in the Arctics Nina Gunde-Cimerman, Lorena Butinar, Silva Sonjak, Martina Turk, Viktor Urši , Polona Zalar and Ana Plemenitaš 397

Halotolerant and halophilic fungi and their extrolite production Jens C. Frisvad 425

Introducing Debaryomyces hansenii, a salt-loving yeast José Ramos 441

Cellular responses in the halophilic black yeast Hortaea werneckii to high environmental salinity

Ana Plemenitaš and Nina Gunde-Cimerman 453

Halotolerance and lichen symbioses Martin Grube and Juliane Blaha 471

Section V. Algae 489

A century of Dunaliella research: 1905-2005 Aharon Oren 491

Molecular determinants of protein halotolerance: structural and functional

ix

studies of the extremely salt tolerant carbonic anhydrases from Dunaliella salina

Lakshmane Premkumar, Michal Volkovitsky, Irena Gokhman, Joel L. Sussman and Ada Zamir 503

Section VI. Protozoa 517

Heterotrophic protozoa from hypersaline environments Gwen Hauer and Andrew Rogerson 519

Heterotrophic flagellates in hypersaline waters Byung C. Cho 541

Section VII. Viruses 551

Haloviruses and their hosts Mike L. Dyall-Smith, David G. Burns, Helen M. Camakaris,

Peter H. Janssen, Brendan E. Russ and Kate Porter 553

Subject Index 565

Organism Index

Author Index

569

575

FOREWORD

Salt is an essential requirement of life. Already from ancient times (e.g., see the books of the Bible) its importance in human life has been known. For example, salt symbolizes destruction (as in Sodom and Gomorra), but on the other hand it has been an ingredient of every sacrifice during the Holy Temple periods.

Microbial life in concentrated salt solutions has fascinated scientists since its discovery. Recently there have been several international meetings and books devoted entirely to halophiles. This book includes the proceedings of the “Halophiles 2004” conference held in Ljubljana, Slovenia, in September 2004 (www.uni-lj.si/~bfbhaloph/index.html). This meeting was attended by 120 participants from 25 countries. The editors have selected presentations given at the meeting for this volume, and have also invited a number of contributions from experts who had not been present in Ljubljana. This book complements “Halophilic Microorganisms”, edited by A. Ventosa and published by Springer-Verlag (2004), “Halophilic Microorganism and their Environments” by A. Oren (2002), published by Kluwer Academic Publishers as volume 5 of “Cellular Origins, Life in Extreme Habitats and Astrobiology” (COLE), and “Microbiology and Biogeochemistry of Hypersaline Environments” edited by A. Oren, and published by CRC Press, Boca Raton (1999).

Salt-loving (halophilic) microorganisms grow in salt solutions above seawater salinity (~3.5% salt) up to saturation ranges (i.e., around 35% salt). High concentrations of salt occur in natural environments (e.g., in the Dead Sea and the Great Salt Lake, Utah) and in man-made hypersaline environments such as solar salterns. The research of halophiles covers all three domains of life, i.e., Archaea, Bacteria, and Eukarya.

This multi-author review has been edited by Professors Nina Gunde-Cimerman, Aharon Oren and Ana Plemenitaš. It is volume 9 of the COLE book series [“Cellular Origins, Life in Extreme Habitats and Astrobiology”, edited by J. Seckbach, now published by Springer] (www.springeronline.com). It covers most recent research on halophiles in chapters contributed by experts in this field. I wish to thank the editors for suggesting the publication of this volume in the framework of the COLE series and for editing all the chapters of this book.

January 2005

Joseph Seckbach Chief Editor of the COLE book series The Hebrew University of Jerusalem

xi

E-mail: seckbach@huji.ac.il

Bacteria, and Eukarya, 1-6.© 2005 Springer. Printed in the Netherlands.

1

INTRODUCTION

NINA GUNDE-CIMERMAN1, AHARON OREN2 and ANA PLEMENITAŠ3

1Biology Department, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia; 2 The Institute of Life Sciences, and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel; 3Institute of Biochemistry, Medical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia

This book, entitled “Adaptations to life at high salt concentrations in Archaea, Bacteria and Eukarya”, intends to provide an overview of current research in the field of halophilic microorganisms, with special emphasis on the diversity of life forms adapted to tolerate high salt concentrations and accordingly low water activities. Most of the chapters are based on lectures given by the participants of the international symposium “Halophiles 2004”, held in Ljubljana, Slovenia, from 4-9 September 2004. To ensure a broader scope of the book we have invited a number of chapters from colleagues who had not been present at the Ljubljana symposium.

The first multi-author review written about halophilic microorganisms was, to the best of our knowledge, written in 1978 (Caplan and Ginzburg, 1978). Looking at the contents of that book, one discovers that no less than thirty-eight chapters were devoted solely or primarily to Halobacterium salinarum (= H. halobium, H. cutirubrum), four chapters dealt with other halophilic Archaea of the family Halobacteriaceae, three were on Dunaliella, and only eight chapters had as their topic other types of halophilic or halotolerant microorganisms, including one chapter that dealt with fungi. We now realize that the world of halophilic microorganisms is very diverse, and that life at high salt concentrations cannot be understood from the study of a single organism or group of organisms alone. The halophiles are as diverse as the environments they live in (see also Oren, 2002).

How large the diversity of halophilic microorganisms really is, is quickly recognized when one examines samples of water and sediment from saltern evaporation ponds in the microscope. The “halophilic safari” presented by Oren in this volume, which explores just one example of a hypersaline environment, shows the tremendous morphological variations found – variations so extensive that one sometimes wonders whether presence of high salt concentrations is indeed so stressful to the microbial world as is often assumed. Indeed, we find halophiles all over the phylogenetic tree, in each of the three domains of life, Archaea, Bacteria and Eukarya, and in different branches of each domain. Many of these still await isolation and characterization.

While the world of prokaryotic halophiles, Archaea as well as Bacteria, has been explored quite extensively in the past, halophilic and halotolerant Eukarya are still a relatively neglected group. A notable exception is the unicellular green alga Dunaliella,

N. Gunde-Cimerman et al. (eds.), Adaptation to Life at High Salt Concentrations in Archaea,

NINA GUNDE-CIMERMAN ET AL.2

which is present worldwide in hypersaline lakes and is responsible for most of the primary production in aquatic high-salt environments, thereby supporting varied communities of heterotrophs. This book is published exactly hundred years after Dunaliella was described as a new species of algae (Teodoresco, 1905), and this fact is memorized in a chapter by Oren in this volume. The chapter by Premkumar and coworkers presents an overview of the most recent advances in the study of this intriguing organism.

The fungi form another group of eukaryotes that can grow at high salt concentrations. While it has been known for long that many fungi can tolerate low water activities, the realization that fungi are an integral part of saline and hypersaline ecosystems came only recently. One group, the black yeasts, even appears to have high salt aquatic systems as its primary habitat (Gunde-Cimerman et al., 2000). Following comprehensive surveys by de Hoog et al. and Gunde-Cimerman et al. of halophilic fungal life in temperate and cold environments, more in-depth explorations of fungal life at high salt are presented in the chapters by Ramos and by Plemenitaš and Gunde-Cimerman. The potential of the fungi to produce secondary metabolites, as described by Frisvad in this volume, makes the group especially interesting, also from the point of view of applied science. We hope that the contributions in this book will give the fungi, long neglected by the halophilic scientific community, the place they deserve. Another example of a neglected group of salt-loving or salt-tolerant organisms is the lichens, as convincingly shown in the chapter by Grube and Blaha.

Another group often ignored in studies of microbial diversity at high salt concentrations is the protozoa. Although their presence is easily revealed by microscopic examination (see e.g. the “halophilic safari” chapter by Oren in this volume), only very few studies have been devoted to the function that ciliate, flagellate and amoeboid protozoa have in hypersaline environments. That such protozoa may also play a quantitatively important role even in the most salty ecosystems was nicely shown in a study of saltern crystallizer ponds by Park et al. (2003). The chapters by Hauer and Rogerson and by Cho provide an overview of our current knowledge of these organisms. Viruses are also an integral part of the biota of the hypersaline environments. The chapter by Dyall-Smith et al. summarizes our present understanding of their diversity and their importance.

We are now in the era of genomics, and approaches of genomics and proteomics have not bypassed the halophiles. The first complete genome of a halophilic archaeon, Halobacterium strain NRC-1 (recently described as a strain of Halobacterium salinarum;Gruber et al., 2004) was published already five years ago (Ng et al., 2000), and the second genome sequence, that of the Dead Sea isolate Haloarcula marismortui, was released just a few weeks before this introduction was written (Baliga et al., 2004). To what extent the approach of comparative genomics can add to our understanding of halophilic microorganisms is clearly shown in the chapter by Berquist et al. Although no complete descriptions of genomes of halophilic members of the Bacteria have been published yet, intriguing previews of what we may expect in the future can already be found in the chapters on the genomes of the extremely halophilic Salinibacter ruber by Antón et al. and on the moderately halophilic versatile Chromohalobacter salexigens by Csonka et al.

However important the study of microbial genomes may be, it can only complement, but never replace physiological studies of the organisms themselves. In this respect,

INTRODUCTION 3

those who study life at the highest salt concentrations are in a much better situation than many colleagues who work on “conventional” ecosystems. We now have a quite thorough understanding of the nature of the organisms growing there, and – most important – these organisms are now available in pure culture thanks to a few breakthroughs in recent years. Salinibacter, an extremely halophilic representative of the domain Bacteria, numerically probably the second-most important organism in saltern crystallizer ponds worldwide, was isolated and described by Antón et al. (2002). The number-one organism in this environment is the famous flat square archaeon first described by Walsby from a brine pool (Walsby, 1980). This intriguing microbe long resisted all attempts towards its isolation, but now two groups have independently succeeded the task (Burns et al., 2004a; Bolhuis et al., 2004). We are pleased that the present volume contains contributions from both groups (the chapters by Bolhuis and Dyall-Smith et al.). It was convincingly shown that also most other inhabitants of these salt-saturated environments can be cultivated when using the proper techniques as well as much patience (Burns et al., 2004b), so it may be expected that within a short time the combination of physiological studies, environmental genomics and genomic and proteomic studies of representative isolates will yield an excellent picture of the microbial communities present and the processes they perform.

Considerable progress is being made in the understanding of the physiology of halophilic microorganisms, in particular those processes that enable these organisms to cope with the high salt concentration in their surrounding medium. Careful regulation of the intracellular ionic concentrations is a prerequisite for the ability to live at high salt. Some of the mechanisms involved are presented in the chapters by Kunte and by Müller and Saum. A widespread strategy is to balance the high ionic concentration in the medium by de novo biosynthesis of by uptake of organic osmotic, “compatible” solutes. Recent advances in our understanding of the metabolism of these compounds, in part guided by information gained using genomic approaches, can be found in the contributions by Reshetnikov et al., Pflüger et al., and Da Costa and Empadinhas, each dealing with a different group of microorganisms. Halophiles are also convenient model organisms to study other aspects of microbial physiology such as gene regulation (Pfeifer et al. in this volume), the properties of salt-dependent enzymes (the chapter by Mizuki et al.), and protein translocation through membranes (Eichler et al. in this volume) – all chapters dealing with extreme halophiles belonging to the archaeal domain. Some halophiles also produce chemicals of considerable economic value, as described in the chapter by Quillaguamán and coworkers, and their exploitation may well lead to novel biotechnological processes. Halophiles have also considerable potential to degrade a variety of organic chemicals, including toxic ones, as documented on the level of the genome in the chapter by Csonka et al., culture and enzyme studies (presented by Ventosa et al., and polluted hypersaline environments (Patzelt in this volume).

Most studies that involve sampling outdoors come from the man-made environment of solar saltern evaporation and crystallizer ponds. The chapter by Pedrós-Alió gives a beautiful example of an in-depth multidisciplinary study of such a saltern environment. The study of halophilic microorganisms in natural hypersaline lakes is a relatively neglected aspect. This is understandable as salterns form a more convenient environment for sampling, and the salinity gradients present are relatively constant and to a large extent predicable and reproducible. The properties of natural lakes are often far from constant, and the impact of short term and long term changes in their salt concentration