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1 Meeting Review 1 2 Halophiles 2010: Life in Saline Environments 3 4 Yanhe Ma, 1 Erwin A. Galinski, 2 William D. Grant, 3 Aharon Oren 4* and Antonio Ventosa 5 5 6 Institute of Microbiology, Chinese Academy of Sciences, Tianjin Institute of Industrial 7 Biotechnology, 1 Beichen West Road, Beijing 100101, China, 1 Institute of Microbiology & 8 Biotechnology, Meckenheimer Allee 168, 53115 Bonn, Germany, 2 Department of Infection, 9 Immunity and Inflammation, University of Leicester, Maurice Shock Medical Sciences 10 Building, Leicester LE1 9HN, UK, 3 Department of Plant and Environmental Sciences, The 11 Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel, 4 and 12 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 13 41012 Sevilla, Spain 5 14 15 * Corresponding author. Mailing address: Department of Plant and Environmental Sciences, 16 The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. 17 Phone: (972) 2-6584951. Fax: (972) 2-6584425. E-mail: [email protected]. 18 19 20 The world of halophilic microorganisms is highly diverse. Microbes adapted to life at high 21 salt concentrations are found in all three domains of life: Archaea, Bacteria, and Eucarya. In 22 some ecosystems salt loving microorganisms live in such large numbers that their presence 23 can be recognized without the need for a microscope. The brines of saltern crystallizer ponds 24 worldwide are colored pink-red by Archaea (Haloquadratum and other representatives of the 25 Halobacteriales), Bacteria (Salinibacter) and Eucarya (Dunaliella salina). 26 Hypersaline environments such as saltern pond brines and natural salt lakes present 27 the ecologist with relatively simple ecosystems with low diversity and high community 28 densities. In such systems fundamental questions of biodiversity, selection, biogeography and 29 evolution in the microbial world can be investigated much more conveniently than in the far 30 more complex freshwater and marine systems. The sediments of such water bodies, however, 31 are often inhabited by extremely diverse, still incompletely explored microbial communities. 32 Different types of halophiles have solved the problem how to cope with salt stress (and often 33 with other forms of stress as well) in different ways, so that the study of microbial life at high 34 Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Appl. Environ. Microbiol. doi:10.1128/AEM.01868-10 AEM Accepts, published online ahead of print on 3 September 2010 on April 21, 2020 by guest http://aem.asm.org/ Downloaded from
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Meeting Review 1
2
Halophiles 2010: Life in Saline Environments 3
4
Yanhe Ma,1 Erwin A. Galinski,
2 William D. Grant,
3 Aharon Oren
4* and Antonio Ventosa
5 5
6
Institute of Microbiology, Chinese Academy of Sciences, Tianjin Institute of Industrial 7
Biotechnology, 1 Beichen West Road, Beijing 100101, China,1 Institute of Microbiology & 8
Biotechnology, Meckenheimer Allee 168, 53115 Bonn, Germany,2 Department of Infection, 9
Immunity and Inflammation, University of Leicester, Maurice Shock Medical Sciences 10
Building, Leicester LE1 9HN, UK,3
Department of Plant and Environmental Sciences, The 11
Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel,4 and 12
Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 13
41012 Sevilla, Spain5 14
15
* Corresponding author. Mailing address: Department of Plant and Environmental Sciences, 16
The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. 17
Phone: (972) 2-6584951. Fax: (972) 2-6584425. E-mail: [email protected]. 18
19
20
The world of halophilic microorganisms is highly diverse. Microbes adapted to life at high 21
salt concentrations are found in all three domains of life: Archaea, Bacteria, and Eucarya. In 22
some ecosystems salt loving microorganisms live in such large numbers that their presence 23
can be recognized without the need for a microscope. The brines of saltern crystallizer ponds 24
worldwide are colored pink-red by Archaea (Haloquadratum and other representatives of the 25
Halobacteriales), Bacteria (Salinibacter) and Eucarya (Dunaliella salina). 26
Hypersaline environments such as saltern pond brines and natural salt lakes present 27
the ecologist with relatively simple ecosystems with low diversity and high community 28
densities. In such systems fundamental questions of biodiversity, selection, biogeography and 29
evolution in the microbial world can be investigated much more conveniently than in the far 30
more complex freshwater and marine systems. The sediments of such water bodies, however, 31
are often inhabited by extremely diverse, still incompletely explored microbial communities. 32
Different types of halophiles have solved the problem how to cope with salt stress (and often 33
with other forms of stress as well) in different ways, so that the study of microbial life at high 34
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01868-10 AEM Accepts, published online ahead of print on 3 September 2010
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salt concentrations can answer many basic questions on the adaptation of microorganisms to 35
their environment. Most known halophiles are relatively easy to grow, and genera such as 36
Halobacterium, Haloferax and Haloarcula have become popular models for studies of the 37
archaeal domain as they are much simpler to handle than methanogenic and 38
hyperthermophilic Archaea. Some halophilic and halotolerant microorganisms have found 39
interesting biotechnological applications as well, as shown in the last section of this report. 40
The 9th
International Conference on Halophilic Microorganisms, held from June 29 – 41
July 3, 2010 in Beijing, China, brought together 166 participants from 25 countries. The 50 42
lectures and 112 posters presented provided an excellent overview of the current state of our 43
understanding of all aspects of microbiology at high salt concentrations. The meeting was 44
hosted by the Institute of Microbiology, the Chinese Academy of Sciences, the Chinese 45
Society of Biotechnology and the Chinese Society for Microbiology. Conference chair was 46
Prof. Yanhe Ma. The series of symposia on halophiles started in Rehovot, Israel in 1978 with 47
a meeting devoted mainly to the properties of bacteriorhodopsin, the retinal-containing 48
protein of Halobacterium that was discovered just a few years earlier. The delegates noted 49
and applauded the presence of Janos Lanyi in the audience, one of the attendees at the first 50
meeting. This initial event was followed by meetings held in 1985 (Obermarchtal, Germany), 51
1989 (Alicante, Spain), 1992 (Williamsburg, VA, USA), 1997 (Jerusalem, Israel), 2001 52
(Sevilla, Spain), 2004 (Ljubljana, Slovenia), and 2007 (Colchester, UK). The proceedings of 53
the 1978, 1989, 1997, 2001, and 2004 symposia were published as books (20, 32, 57, 69, 89); 54
selected papers from the 1985, 2002 and 2007 symposia appeared in dedicated special 55
volumes of journals (FEMS Microbiology Reviews, Experientia, and Saline Systems). 56
This review intends to capture emerging themes and to report key interesting new 57
findings presented at the Halophiles 2010 symposium in Beijing. The following topics were 58
the focus of attention and discussion. 59
60
61
DIVERSITY OF HALOPHILES – CULTURED AND UNCULTURED 62
63
“Everything is everywhere: but, the environment selects” (“Alles is overal: maar, het milieu 64
selecteert”. This famous quotation from Lourens Baas Becking’s 1934 book “Geobiologie of 65
inleiding tot the milieukunde” (8) can be taken as the basis for our understanding of the 66
distribution of halophilic microorganisms worldwide. In fact, Baas Becking (1895-1963) had 67
visited many salt lakes and studied many different halophilic microorganisms. His book and 68
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his publications from the early 1930s contain a wealth of information, largely forgotten today, 69
on the properties of the halophiles. Some phenomena described by Baas Becking at the time, 70
including the acidic nature of the cell envelope of Dunaliella and the interrelationship of salt 71
requirement/tolerance and temperature in halophilic prokaryotes were “rediscovered” in the 72
1970s, as documented by Aharon Oren (Jerusalem, Israel) in his keynote lecture at the 73
opening session. 74
To explore to what extent in the halophilic world “everything” is indeed “everywhere” 75
and what degree of variation may be found among different high-salt environments, microbial 76
diversity studies have been performed in a great variety of environments. These include 77
saltern ponds world-wide, Great Salt Lake, the Dead Sea (16), saline lakes in Inner Mongolia 78
(61), African soda lakes, deep-sea brines (86), and many others. These studies included 79
culture-dependent approaches, leading to the isolation and characterization of many novel 80
types of halophiles and new information on the abundance and geographic distribution of the 81
known types, as well as culture-independent studies based on sequencing of DNA recovered 82
from the environment. Many posters at the meeting related to culture-independent analyses of 83
hypersaline environments from around the world including Xinjiang salt lakes, Chinese salt 84
mines, salterns in Goa-India, Turkey, Spain, and Israel, south Siberian hypersaline lakes, the 85
Dead Sea, and Great Salt Lake. Thane Papke (Storrs, CT, USA) examined Halorubrum 86
strains in Spain and Algeria, and one of his conclusions was that ‘migration routes are slower 87
than mutation rates’, allowing endemism in Halorubrum strains to develop. Shaun Heaphy 88
(Leicester, UK) provided a culture-independent microbial characterization of several Inner 89
Mongolian salt and soda lakes and used statistical techniques to correlate the findings with 90
physico/chemical parameters of brines and geographical location. He broadly agreed that 91
microbial populations diverged as distance between the lakes increased, although this was 92
only statistically significant for the Bacteria on an intercontinental scale – a hypersaline lake 93
in Argentina was included in the analysis. Factors such as pH, temperature, and Na+ 94
concentration were particularly correlated with the microbial community composition. Thus, 95
“everything may not be everywhere”. More and more cases are being reported of the isolation 96
of halophilic microorganisms from low salinity environments. Thus after almost 80 years, 97
Lourens Baas Becking’s quotation still inspires experiment and debate. 98
Application of culture-dependent methods led to the isolation of a novel halophilic 99
archaeon from seawater (at a salinity that does not support growth of Halobacteriaceae and 100
causes lysis of most known representatives of the group). The properties of this new 101
organism, to be described as a new genus and species, Halomarina oriensis, were presented 102
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by Kentaro Inoue (Chiba, Japan) (38). Poster presentations included a new fungal isolate from 103
a Turkish salt mine, novel haloarchaea from a Chinese saltern, Inner Mongolian lakes, and 104
Iranian salt lakes, and novel bacterial isolates from Chinese salt lakes, Inner Mongolian 105
Lakes, Xinjiang salt lakes, Quidam Basin Quaternary sediments, brine wells in southwestern 106
China, the Yellow Sea, The South China Sea, a Korean salt flat, Iranian salt lakes, Mexican 107
soda environments, and salted hides. There were additional reports on the isolation of new 108
actinomycetes from saline environments in China, and different bacterial halophiles from 109
nonsaline sites such as soils. 110
111
112
HALOPHILES IN UNUSUAL ENVIRONMENTS AND HALOPHILES EXPOSED TO 113
MULTIPLE FORMS OF STRESS 114
115
Most habitats explored for the presence of halophiles are “thalassohaline” environments that 116
originated by evaporation from seawater, reflect the ionic composition of seawater, and have a 117
near-neutral to slightly alkaline pH. 118
Deep-sea brines, found on the bottom of the Red Sea, the Mediterranean Sea, and the 119
Gulf of Mexico are interesting environments to search for novel microbes. Apart from their 120
increased high salinity, they are anaerobic, and form characteristically sharp brine-seawater 121
interfaces, with some of the brines displaying significant increases in temperature and metal 122
concentration. The ionic composition of the brines generally differs from that of seawater, 123
they are anaerobic and in some cases the temperature can be elevated as well. The 124
microbiology of Shaban deep and other deep sea brines in the Red Sea was discussed by 125
André Antunes (Thuwal, Saudi Arabia). These sites, considered sterile in the past, have 126
yielded a number of interesting microorganisms, including Salinisphaera shabanensis (a 127
facultative anaerobe growing in a very large range of salt concentrations, from 1-28%) (5), 128
Halorhabdus tiamatea (a non-pigmented representative of the Halobacteriales that prefers an 129
anaerobic life style) (7), Flexistipes sinusarabici (an anaerobe tolerating between 3-18% 130
NaCl) (28), and Haloplasma contractile (a contractile bacterium, phylogenetically equidistant 131
to the Firmicutes and the Mollicutes) (6). The sites will be revisited in the near future for 132
further microbiological exploration. 133
In many “athalassohaline” environments life at the extremes of high salt is combined 134
with the need to thrive at alkaline pH and elevated temperatures, and organisms growing there 135
do so at the physico-chemical boundary for life (18). Jürgen Wiegel (Athens, GA, USA) 136
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summarized his studies of the anaerobic halophilic, alkaliphilic, thermophilic bacteria isolated 137
from the Wadi An-Natrun, Egypt. Natranaerobius thermophilus accumulates both glycine 138
betaine and K+ for osmotic adaptation, and has multiple Na
+/H
+ antiporters (50) and a Na
+-139
extruding ATPase which was characterized in-depth by Noha Mesbah (Alexandria, Egypt). 140
Two new species, designated ‘Natranaerobius jonesii’ and ‘Natranaerobius grantii’ are 141
currently being characterized. ‘Natranaerobius jonesii’ has an extremely high requirement for 142
chloride ions, as it does not grow at less than 1.4 M Cl-. Other alkaline saline environments 143
subjected to intensive studies in recent years are the soda lakes of the Kulunda Steppe (Altai, 144
Russia). Dimitry Sorokin (Moscow, Russia) summarized the wealth of information obtained 145
from these studies, both at the level of the characterization of cultures of novel organisms, 146
especially those participating in the reductive part of the sulfur cycle, culture-independent 147
studies using molecular markers, as well as measurements of the rates of microbial 148
sulfidogenesis. In general, sulfide production was active even in saturated soda brines, but far 149
more sulfide was produced in these environments from elemental sulfur and from thiosulfate 150
than from sulfate. Dismutation of thiosulfate and sulfite was a major trend in soda lake 151
isolates (79, 80). 152
The Dead Sea is a rare example of a low-Na+, high-Mg
2+ and high-Ca
2+ chloride brine 153
with a slightly acidic pH. Metagenomic studies are now providing information on the 154
microbial diversity in the lake, both at the time of a bloom of microorganisms following 155
dilution of the upper water layers by rain floods in 1992, and during the current drying-out of 156
the lake, causing a continuously decreasing ratio of monovalent/divalent cations, making 157
conditions too extreme for even the best salt-adapted microorganisms (16). 158
159
160
GENOMICS, EVOLUTION, AND TAXONOMY OF HALOPHILIC ARCHAEA AND 161
BACTERIA 162
163
Genomics of the Halobacteriaceae has come of age. Shiladitya DasSarma (Baltimore, MD, 164
USA) gave the closing keynote lecture, highlighting the haloarchaeal genomes from different 165
genera which have been determined since the genome sequence of Halobacterium NRC-1 166
was first published (24, 54) ten years ago. More recently completed genomes highlighted 167
included Haloarcula marismortui (11, 14), Natronomonas pharaonis (26), Haloquadratum 168
walsbyi (17), Halorubrum lacusprofundi 169
(http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSea170
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rch=23834, 2009), Halomicrobium mukohataei (83), Halorhabdus utahensis (10), 171
Halogeometricum borinquense (47), Haloterrigena turkmenica (74), and Haloferax volcanii 172
(35). The list includes significant ecological diversity, e.g. a haloalkaliphilic species, a cold-173
adapted species, species adapted to life in low Na+-high Mg
2+ environments, and isolates 174
showing interesting cell morphologies. The sizes of these genomes range between 2.6 and 5.4 175
Mb. The sequencing and analysis of the genomes of Haloarcula hispanica and Haloferax 176
mediterranei was announced by Hua Xiang and colleagues (Beijing, China). DasSarma 177
showed that there has been an exponential increase in the sequencing of haloarchaeal 178
genomes over the past ten years, and with next-gen sequencing methods now available, 179
expects that within a few years the number of published genomes of species of 180
Halobacteriaceae will grow even faster. Some of the conserved properties of haloarchaeal 181
genomes were discussed, including the presence of large megaplasmids and 182
minichromosomes (24) and the occurrence of core acidic proteomes (23). The data analysis 183
also yielded the prediction of an expanding haloarchaeal “pan-genome” with increasing 184
numbers of novel genes which may have applications in biotechnology. 185
In two presentations from the DasSarma group, additional post-genomic work was 186
presented. James Coker (soon to move to Birmingham, AL, USA) reported on studies on the 187
expanded TATA-binding protein and transcription factor B protein families of haloarchaea 188
showing their importance for gene expression and stress regulation (21, 78). Satyajit 189
DasSarma (the youngest presenter at age 13) reported on the expansion of the HaloWeb, the 190
Haloarchaeal genome database (http://halo4.umbi.umd.edu), which now provides access to all 191
the public haloarchaeal genomes and as well as a suite of tools for data retrieval and analysis. 192
Environmental genomics studies increasingly show that the genome of individual 193
strains may be only a small fraction of the “pan-genome” of the species in nature. 194
Haloquadratum walsbyi has become an excellent example to illustrate this, as shown by 195
Francisco Rodríguez-Valera (Alicante, Spain) and Mike Dyall-Smith (Martinsried, Germany). 196
Comparisons have been made of the genome diversity within Haloquadratum populations in a 197
single saltern crystallizer pond as well as comparisons between populations in similar 198
environments at different geographic locations. The pan-genome of Haloquadratum walsbyi 199
is at least 40 times the size of the genome of the type strain, and genomic microdiversity 200
within an extremely simple and relatively constant environment is very high (17, 22, 41, 56). 201
An interesting experiment in “experimental evolution” was presented by Jizhong Zhou 202
(Norman, OK, USA), using Desulfovibrio vulgaris as model organism and monitoring genetic 203
changes after exposure of this non-halophilic bacterium to 0.25 M NaCl for 1000 generations. 204
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Salt-specific mutations and deletions were detected in the salt-resistant phenotype, which used 205
different amino acids as osmoprotectants (36). 206
Genome sequencing of new isolates is getting simpler and cheaper, and will probably 207
soon become routine. Undoubtedly this development will have profound implications on the 208
taxonomy of the halophiles. Until taxonomy can be based on comparison of complete genome 209
sequences, multi locus sequence analysis (MLSA) is gaining popularity for the comparison of 210
strains for taxonomic and evolutionary studies. Thane Papke (Storrs, CT, USA) presented his 211
extensive MLSA data Halorubrum isolates from Spain and Algeria. Analysis of the data 212
indicates very frequent occurrence of homologous recombination, to the extent that alleles 213
were randomly associated, as typical of sexually reproducing species. Natural competence and 214
conjugation (like the ‘mating’ mechanism in Haloferax) (3) may be the possible mechanisms 215
for lateral gene transfer (62). Emma White (Storrs, CT, USA) and Hiroaki Minegishi 216
(Saitama, Japan) showed how analysis of the RNA polymerase subunit B' (rpoB
') gene can 217
help reconstructing the phylogeny of the Halobacteriaceae (52). Also for the 218
Halomonadaceae, MLSA is becoming a valuable tool for taxonomic studies, as shown by 219
Antonio Ventosa (Sevilla, Spain). For both groups sets of genes and primers have been 220
defined that give good results consistent with other genotypic and phenotypic traits. 221
The list of sequenced genomes of halophilic and halotolerant Bacteria is as yet short. It 222
even does not yet include Halomonas elongata, the organism that, since it was described 223
thirty years ago (90) has become one of the most popular model organisms, and has also 224
found biotechnological applications (31,58). Its genome sequence will soon be published. 225
Genome sequence information is available for the anoxygenic halophilic phototroph 226
Halorhodospira halophila, for an extremely salt-tolerant alkaliphilic sulfur-oxidizing 227
bacterium of the genus Thioalkalivibrio, for the thermophilic anaerobic halophile 228
Halothermothrix orenii, and for the aerobic heterotrophic Chromohalobacter salexigens (59) 229
and Salinibacter ruber. 230
Extensive environmental genomics data have been collected for Salinibacter. Josefa 231
Antón (Alicante, Spain) showed a high degree of genomic variation within Salinibacter 232
populations. Comparative analyses indicate that Salinibacter ruber genomes present a mosaic 233
structure with conserved and hypervariable regions. Overall, 10% of the genes encoded in the 234
genome of Salinibacter M8 genome are absent from the type strain Salinibacter M31. 235
Metabolomic profiles also differed in these two isolates (63). 236
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HALOPHILIC VIRUSES 239
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The Halophiles 2001 and 2004 symposia in Sevilla and Ljubljana will be remembered as the 241
events where the importance of fungi in hypersaline ecosystems became clear. Halophiles 242
2010 can then be described as the congress presenting the importance of viruses. Phages 243
attacking extremely halophilic Archaea were first described already in 1974 (84), but the role 244
of viruses in hypersaline ecosystems remained largely unexplored. 245
Elina Roine (Helsinki, Finland) and her colleagues have discovered novel types of 246
viruses attacking halophilic Archaea. The isolation and characterization of pleomorphic 247
viruses possessing a lipid envelope, containing either a single stranded or double stranded 248
DNA genome, shows that viral diversity in hypersaline environments (64, 65, 70) is much 249
larger than previously assumed. Shaun Heaphy (Leicester, UK) presented two novel lytic 250
head/tailed viruses (virus BJ1 of the Siphoviridae and virus BJ2 of the Myoviridae), infecting 251
Halorubrum kocurii, isolated from a salt lake in Inner Mongolia (60). Few archaeal virus 252
genomes have been sequenced and the complete sequence of virus BJ1 (EMBL accession no. 253
AM419438) is therefore a welcome addition. 254
First results of a comprehensive study of viral distribution and diversity in Great Salt 255
Lake, Utah, were presented by Bonnie Baxter (Salt Lake City, UT, USA). Saltern crystallizer 256
ponds are also ideal environments to study virus diversity and dynamics, as protozoa and 257
other predators are absent and numbers of prokaryotes and virus-like particles are extremely 258
high – typically in the order of > 107/ml and >10
8-10
9/ml, respectively. Forest Rohwer (San 259
Diego, CA, USA) showed his studies of virus dynamics in such salt-saturated ponds. At first 260
sight, the salterns present predictable and stable communities of both Archaea and viruses, 261
apparently different from the “Kill-the-Winner” behavior with rapid cycling of microbial taxa 262
and their viral predators that may be expected in such an environment. Metagenomic analysis 263
of the viruses in the salterns near San Diego showed that the distribution of microbial taxa and 264
viral taxa remained stable over time, but with strong dynamic fluctuations of the prevalence of 265
microbial strains and viral genotypes. Thus, at the fine level the populations of individual 266
strains and viral genotypes both fluctuate in a “Kill-the-Winner” fashion (68). 267
Activity of viruses also may have profound implications on the distribution of the 268
extremely halophilic bacterium Salinibacter (Bacteroidetes). Josefa Antón (Alicante, Spain) 269
studied the metagenome of viral assemblages of saltern pond in which Salinibacter accounts 270
for around 15% of the prokaryotic community. Based on bioinformatic analysis (G+C 271
content, dinucleotide frequency analysis) about 24% of the retrieved viral sequences could 272
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correspond to Salinibacter phages (71). It seems that phages infecting Salinibacter are more 273
active in the environment than phages infecting Haloquadratum, and this may possibly 274
explain why Haloquadratum outnumbers Salinibacter in every environment that supports 275
growth of these organisms. 276
277
278
HALOPHILIC FUNGI 279
280
The importance of halophilic fungi, long neglected as members of hypersaline ecosystems, 281
became recognized only in the past decade. Nina Gunde-Cimerman (Ljubljana, Slovenia) 282
gave an overview of the biology of the most widespread and most halophilic or halotolerant 283
fungi and yeasts. These include the black yeasts Hortaea werneckii which grows up to 5 M 284
NaCl, the true halophile Wallemia ichthyophaga that requires at least 1.5 M NaCl and grows 285
up to saturation, and Aureobasidium pullulans that grows up to 3 M NaCl. These all are 286
commonly found in hypersaline lakes and in a great variety of other, often unexpected, 287
environments: domestic dishwashers, polar ice, and possible even on spider webs in desert 288
caves (33). 289
The halophilic and halotolerant fungi use polyols such as glycerol, erythritol, arabitol 290
and mannitol as osmotic solutes, and retain low salt concentrations in their cytoplasm. 291
Molecular studies on osmotic adaptation of Hortaea werneckii and Wallemia ichthyophaga 292
were presented by Ana Plemenitaš and Janja Zajc (Ljubljana, Slovenia). Identification and 293
structural features of Na+-sensitive 3’-phosphoadenosine-5’-phosphatase HwHal2, one of the 294
putative determinants of halotolerance in H. werneckii and promising transgene to improve 295
halotolerance in crops, was presented (88). An in-depth understanding has been obtained of 296
the HOG (High Osmolarity Glycerol) pathway, and this understanding may be applied in the 297
future to the development of improved salt-resistant crops. Glycerol-3-phosphate 298
dehydrogenase is involved in glycerol synthesis by both Wallemia and Hortaea, and 299
heterologous expression of the gene encoding the enzyme can restore halotolerance in 300
Saccharomyces cerevisiae deficient in glycerol production. 301
302
303
LONG-TERM SURVIVAL OF HALOPHILES 304
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When brines dry out and halite crystals are formed, small fluid inclusions remain entrapped 306
within the crystals. Microorganisms that inhabited the brine may get entrapped in these 307
inclusions (9). Since the first controlled studies showed that such microorganisms may retain 308
their viability for long periods (55), the question of the longevity of different types of 309
halophiles within salt crystals has become a popular topic, relevant to disciplines including 310
geology, biogeography, evolution, and even space exploration (49). 311
Terry McGenity (Colchester, UK) presented field studies and laboratory simulations of 312
entombment of different types of microbes inside salt crystals. Salinibacter alone survives 313
poorly within halite crystals, but when trapped inside a crystal together with Haloquadratum, 314
longevity was much enhanced. Thus, simple food chains and mutual interactions occur 315
between microorganisms in fluid inclusions in salt. 316
Examination of halite cores from Saline Valley, CA, representing salt deposited up to 317
150 thousand years ago, showed remnants of algae within fluid inclusions entrapped in the 318
salt crystals. Morphological features as well as sequences of the internal transcribed spacer 319
between the 18S and 5.8S rRNA genes led to the identification of Dunaliella, Ulothrix, and 320
Nephroselmis, as shown by Krithivasan Sankaranarayanan (Binghamton, NY, USA) who won 321
first prize for his presentation by a young scientist. Presence of entrapped algae, with their 322
high content of organic compatible solutes, may provide a carbon and energy source enabling 323
halophilic heterotrophic microorganisms to survive for prolonged times (76). 324
325
326
OSMOTIC ADAPTATION, COMPATIBLE SOLUTES, AND ADAPTATION OF 327
INTRACELLULAR PROTEINS TO SALT 328
329
There are basically two strategies that enable halophilic and halotolerant microorganisms to 330
live in high salt concentration. The “high-salt-in” strategy (used by the Halobacteriaceae, 331
Salinibacter, and the anaerobic Halanaerobiales) requires all intracellular proteins to be 332
stable and active in the presence of molar concentrations of KCl and other salts. The “low-333
salt, organic solutes in” strategy is based on the biosynthesis and/or accumulation of organic 334
solutes that do not interfere greatly with the activity of “normal” enzymes. But even such 335
organisms need to have salt-adapted proteins in the membrane exposed to the saline medium. 336
It is remarkable that already in the early 1930s Baas Becking concluded that Dunaliella must 337
have a highly acidic surface, based on the insensitivity of the alga to certain otherwise toxic 338
anions (8). 339
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Over the years, Haloarcula marismortui has been the most popular model organism 340
for the study of the behavior of proteins active in a high salt environment. These include the 341
Haloarcula ribosome, whose structure elucidation by Ada Yonath was awarded the Nobel 342
Prize for Chemistry in 2009. Christine Ebel (Grenoble, France) presented an overview of 343
molecular adaptations of halophilic proteins, based on her studies of the Haloarcula 344
marismortui malate dehydrogenase and other enzymes. Particularly the very acidic surface of 345
the macromolecule allows protein–salt interactions that avoid water or salt enrichment at the 346
surface of the protein at high salt and preserve its solubility (25, 45). 347
Volker Müller (Frankfurt, Germany) uses Halobacillus halophilus as a model to 348
understand the mechanisms of osmotic adaptation by a bacterium that accumulates organic 349
compatible solutes. Using techniques of biochemistry, genomics, DNA microarrays, etc. his 350
group studies the way the organism senses its environment. Halobacillus is the first chloride-351
dependent bacterium reported and several cellular functions depend on Cl- for maximal 352
activities, the most important being the activation of solute accumulation. Halobacillus 353
switches its osmolyte strategy with the salinity in its environment by the production of 354
different compatible solutes. Glutamate and glutamine dominate at intermediate salinities, and 355
proline and ectoine at high salinities. Chloride stimulates expression of the glutamine 356
synthetase and activates the enzyme. The product glutamate then turns on the biosynthesis of 357
proline by inducing the expression of the proline biosynthetic genes (72, 73). Halobacillus 358
dabanensis is used by Su-Sheng Yang and his colleagues (Beijing, China) as a model 359
organism to study the genes involved in halotolerance, including genes encoding Na+/H
+ 360
antiporters, enzymes involved in osmotic solute metabolism, and stress proteins (27, 91). 361
Studies of a mutant of Halomonas elongata deficient in ectoine synthesis by Elisabeth Witt 362
(Bonn, Germany) showed the production of a new cyclic compatible solute, 5-amino-3,4-363
dihydro-2H-pyrrole-2-carboxylate (ADPC). It is made by a side reaction of ectoine synthase 364
(EctC) that forms ADPC by cyclic condensation of glutamine. She also demonstrated that 365
ectoine synthase is a reversible enzyme, which has its equilibrium (in case of ectoine 366
synthesis) completely on the side of the cyclic condensation product. 367
Ectoine and hydroxyectoine biosynthesis is widely found in halophilic and halotolerant 368
microorganisms, and the expression of the ect structural genes is induced by salt stress. But 369
the solutes not only provide protection against salt stress, but also against temperature stress 370
in Bacillus subtilis and other salt-tolerant bacilli, as shown by Erhard Bremer (Marburg, 371
Germany). Quantification of the intracellular ectoine concentration in Virgibacillus 372
pantothenticus revealed that its production is triggered either by an increase in external 373
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salinity or by a reduction in growth temperature. Transcription of the ectoine biosynthetic 374
operon (ectABC) was enhanced under both environmental conditions (39). The crystal 375
structure of Virgibacillus salexigens EctD, the enzyme responsible for conversion of ectoine 376
to hydroxyectoine, is now known in detail (67). 377
378
379
LIPIDS, MEMBRANE-BOUND PIGMENTS, AND MEMBRANE-LINKED 380
PROCESSES 381
382
The cytoplasmic membranes of halophilic Archaea of the family Halobacteriaceae 383
contain interesting ether lipids and often have retinal proteins (bacteriorhodopsin, 384
halorhodopsin, sensory rhodopsins). Interesting lipids and retinal proteins have also been 385
found in Salinibacter. 386
Heiko Patzelt (Muscat, Oman) showed that unsaturated ether lipids are far more 387
common in the halophilic Archaea than generally assumed. Such unsaturated diether lipids 388
were earlier reported from the psychrotolerant haloarchaeon Halorubrum lacusprofundi (30). 389
Isolates of Haloarcula spp. and Haloferax sp. obtained from a potash mine crystallization 390
pond in North Germany had unsaturated ether lipids up to 37% of the total membrane lipid 391
content. Only the phospholipids were unsaturated, and these contained mostly 4 or 6 double 392
bonds in the archaeol chain. 393
Novel types of acylhalocapnines were described by Angela Corcelli (Bari, Italy). 394
Salisaeta longa, an organism that requires lower salt concentrations than the related 395
Salinibacter (Bacteroidetes), contains the hydroxyl fatty acid ester of 2-carboxy-2-amino-3,4-396
hydroxy-17-methyloctadec-5-ene-1-sulfonic acid, a sulfonate sphingoid base for which the 397
common name of halocapnine is suggested (12). Salinibacter contains similar acylhalocapnin 398
lipids in its membrane, as well as a retinal protein named xanthorhodopsin and an unusual 399
ketocarotenoid named salinixanthin, found in a 1:1 molar ratio with the retinal pigment. Janos 400
Lanyi (Irvine, CA, USA) showed how the two chromophores interact and how the carotenoid 401
acts as an antenna, transferring the absorbed light energy to the xanthorhodopsin proton 402
pump. Such an energy transfer phenomenon appears to be unique for the clade that includes 403
xanthorhodopsin, as it is not found between the carotenoid bacterioruberin and 404
bacteriorhodopsin in Halobacterium and related genera. The efficiency of the energy transfer 405
is about 50%. The three-dimensional structure of the xanthorhodopsin-salinixanthin system 406
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has been determined from X-ray diffraction of xanthorhodopsin crystals, showing how the 407
carotenoid interacts with the retinal protein (43). 408
The Haloquadratum walsbyi genome encodes for two different bacteriorhodopsins. 409
Both are expressed in the cells. Angela Corcelli (Bari, Italy) reported the isolation of the two 410
forms of bacteriorhodopsin from Haloquadratum cultures using a biochemical approach. 411
Bacteriorhodopsin was also recovered from biomass collected from the saltern crystallizer 412
ponds of the Margarita di Savoia saltern. 413
Mecky Pohlschröder (Philadelphia, PA, USA) presented recent results pertaining to 414
mechanisms of protein transport across haloarchaeal cytoplasmic membranes. In haloarchaea, 415
although the Sec pathway transports important substrates, including subunits of type IV pilus-416
like structures, the Tat pathway is used extensively and transports a wide range of secreted 417
proteins, the majority of which appear to be anchored to the haloarchaeal membrane via a 418
lipid anchor. In silico analyses suggest that prominent use of the Tat pathway as well as 419
extensive anchoring of Tat substrates via a lipid anchor is unique to halophilic Archaea (the 420
latest views on the membranal mechanisms of protein secretion in Haloferax volcanii). The 421
Sec pathway remains an essential mode of protein transport in halophilic Archaea (81, 85). 422
Novel programs allowing more accurate predictions of protein subcellular location – publicly 423
available at SignalFind.org – were also presented. 424
425
426
DNA REPLICATION, TRANSCRIPTION, TRANSLATION, AND POST-427
TRANSLATIONAL MODIFICATION IN HALOPHILIC ARCHAEA 428
429
Relatively few talks dealt with the molecular biology of halophiles and the basic properties of 430
the DNA replication, transcription and translation machinery in different groups of halophiles. 431
Stuart MacNeill (St. Andrews, UK) presented novel information on the structure of the 432
replication fork of Haloferax. Haloferax volcanii encodes a single mini-chromosome 433
maintenance protein. Its N-terminal domain has a putative DNA-binding β-hairpin, a Cys4 434
zinc ribbon and a β-hairpin with a role in interdomain communication. Genetic analysis of 435
different mutants enabled the elucidation of the roles of the different proteins associated with 436
the replication fork (44). 437
The biosynthesis and assembly of gas vesicles in halophilic Archaea has been used as 438
a model for the study of transcription and other molecular processes in the Halobacteriaceae 439
for nearly three decades now. Felicitas Pfeifer (Darmstadt, Germany) presented the latest 440
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information how the Halobacterium salinarum gas vesicle, when expressed in Haloferax 441
volcanii, can be used as a convenient model system for the study of gene expression, 442
transcription and translation. Gas vesicles are composed of two structural proteins: the 443
hydrophobic GvpA the core of the cylinders and the hydrophilic GvpA that cross-links the 444
GvpA subunits at the outside and provide strength to the vesicles. GvpC is now also known to 445
be involved in the determination of the shape of the vesicles. Anaerobiosis inhibits gas vesicle 446
formation. Fourteen gvp genes are required for gas vesicle formation, and these are arranged 447
in two oppositely organized clusters. The function of the different promoters and 448
transcriptional activators is becoming increasingly clear (13, 37, 82). 449
Updates about the molecular mechanisms of translational control in Halobacterium 450
salinarum and Haloferax volcanii were given by Jörg Soppa (Frankfurt, Germany). Different 451
mechanisms for translation initiation are known: 1, interaction between 16S rRNA and a 452
“Shine-Dalgarno sequence”; 2, the eukaryotic mechanism of linear scanning of the small 453
ribosomal subunit from the 5’-cap to the start codon; 3, an alternative eukaryotic mechanism 454
using “internal ribosomal entry sites”; and 4, leaderless transcripts that require an 455
undissociated ribosome and the initiator tRNA (a mechanism encountered in all three domains 456
of life). Characterization of the 5’- and 3’-ends of haloarchaeal transcripts showed that the 457
majority of the transcripts are leaderless, that “Shine-Dalgarno sequences” are very rare, and 458
that about a third does not fall in any of these four classes and must use a novel, yet 459
uncharacterized method of translation initiation (19). 460
Post-translational modification is studied in the laboratory of Jerry Eichler (Beer-461
Sheva, Israel), centering on the biosynthesis of the glycoproteins so abundantly found in the 462
cell envelope of the Halobacteriaceae. Asn-modified glycoproteins are common in Archaea, 463
and their production was studied using Haloferax volcanii as a model. A series of agl 464
(archaeal glycosylation) genes was defined, encoding proteins involved in the assembly and 465
attachment of a novel pentasaccharide to Asn residues of the S-layer glycoprotein. The 466
functions of several Agl proteins are now known (1, 2, 46, 92). 467
468
469
GENETIC SYSTEMS FOR HALOPHILIC PROKARYOTES 470
471
Two interesting systems for genetic manipulation of halophiles were highlighted at the 472
meeting. Already in the original species description of Haloferax volcanii the formation of 473
intercellular bridges was noted (53). Moshe Mevarech (Tel Aviv, Israel) presented a survey of 474
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the genetic manipulation of Haloferax volcanii, developed since genetic transfer based on cell 475
mating was first described twenty-five years ago (51). The mating system of Haloferax 476
volcanii resembles eukaryotic sexual mating rather than bacterial lateral gene transfer. Large 477
amounts of genetic material can be transferred this way (3). The successful mating of 478
Haloferax volcanii and Haloferax mediterranei, yielding hybrid progeny, was announced. 479
Saskia Köcher (Frankfurt, Germany) presented a (prize-winning) poster describing the 480
establishment of a genetic system for the manipulation of Halobacillus halophilus, based on 481
protoplast fusion and markerless gene disruption. Cells can be transformed by protoplast 482
transformation, resulting in integration of a non-replicating plasmid via single homologous 483
recombination. The method was used to generate proline biosynthesis mutants. This genetic 484
manipulation strategy will now open the way to study many more properties of Halobacillus 485
at the genetic level. 486
487
488
BIOTECHNOLOGICAL APPLICATIONS OF HALOPHILES 489
490
In comparison to other groups of extremophilic microorganisms such as the thermophiles and 491
the alkaliphiles, the halophiles of all three domains have been relatively little exploited in 492
biotechnological processes with notable exceptions of β-carotene from Dunaliella, 493
bacteriorhodopsin from Halobacterium and ectoine from Halomonas (58). The biotechnology 494
section of the meeting focused on production/modification techniques of compatible solutes, 495
bioplastics and halophilic enzymes. In addition, attention was drawn towards secondary 496
metabolites from halophiles as well as bioremediation and bio-fuel production. 497
One success story of halophile biotechnology is the production and application of the 498
compatible solute ectoine, currently produced at large scale by Bitop AG in Germany using 499
“bacterial milking” of Halomonas elongata. Ectoine is the active ingredient of many 500
cosmetics and skin-care products, and increasingly becomes important in medicinal 501
preparations (31). In addition, ectoine (and/or suitable derivatives) are used as protectants for 502
biomolecules and enhancers in molecular biology applications such as PCR and DNA 503
microarray techniques (48, 75). Erwin Galinski (Bonn, Germany) presented a survey of the 504
industrial production processes of ectoine and, in particular, a critical analysis of the maximal 505
specific production rates obtainable with H. elongata as the production strain (50 mg g-1
dry 506
weight h-1
at 5-7.5% NaCl). Different strategies have been applied in attempts to increase 507
production, including heterologous expression of the ectoine gene cluster in Escherichia coli 508
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and concomitant overexpression of genes that increase supply of limiting precursors for 509
ectoine biosynthesis, thus bypassing “metabolic bottle-necks" (15, 77). Heterologous 510
expression of the ectoine gene cluster in E. coli is at present not a suitable alternative to 511
ectoine production in H. elongata. With respect to the hydroxylated derivative (S,S-β-512
hydroxyectoine) the situation is however different. As this compound is always produced in a 513
mixture with ectoine in H. elongata, a costly chromatographic separation is required. By 514
overexpressing the ectD gene (encoding ectoine hydroxylase) in E. coli, an efficient whole 515
cell biotransformation system for ectoine has been established, in which the product 516
(hydroxectoine) leaked out of the cells and accumulated in the medium (29). Novel 517
developments concern use of genetically engineered H. elongata for production of rare and so 518
far inaccessible compatible solutes. The potential of this approach for the development of new 519
production processes was demonstrated, using the compatible solutes mannosylglycerate 520
(gene cluster from the thermophilic Rhodothermus marinus) and N-acetyl-glutaminyl 521
glutamine-1-amide (gene cluster from Pseudomonas putida) as examples. 522
Whereas in past meetings the production of extracellular halophilic polymers with 523
interesting rheological properties had claimed attention, the emphasis of this years meeting (as 524
regards polymers) has clearly been on intracellular polyesters. Production of poly-β-525
hydroxyalkanoates – biodegradable polymers with plastic-like properties, although not 526
restricted to halophilic prokaryotes, was the topic of no less than four talks and a number of 527
posters. Some halophilic or halotolerant Bacteria were shown to be excellent producers of 528
such bioplastics. One of these is Halomonas boliviensis, as argued by Jorge Quillaguamán 529
(Cochabamba, Bolivia), who presented strategies to optimize the biosynthesis of such 530
bioplastics coupled with production of the high-value products ectoine and hydroxyectoine 531
(66, 87). Archaea of the genus Haloferax are also known as poly-β-hydroxyalkanoate 532
producers, and the biosynthetic pathway leading to their production was elucidated by Hua 533
Xiang and colleagues (Beijing, China) (34, 42). Unfortunately none of the presenters 534
compared the potential of halophilic producers with the current productivity of industrial 535
strains as for example used by Metabolix/ADM (USA) for their bioplastic product Tirel. 536
Many alkaliphiles are halophilic as well, and many useful enzymes applied in the 537
detergent industry (washing powders), the textile industry, and other processes, were derived 538
from bacteria growing in saline alkaline lakes. Brian Jones (Leiden, The Netherlands) 539
explained how the saline alkaline lakes in Kenya and Inner Mongolia have been a rich source 540
of organisms and/or genes from metagenomic libraries, and some of these are already 541
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explored as starting material for the production of commercially valuable enzymes, in 542
particular proteases and amylases. 543
Halophilic enzymes (typical for Archaea and Salinibacter, but also for exoenzymes of 544
any halophile) are characterized by an excess of acidic amino acids and subsequent negative 545
surface charge. This peculiarity allows effective competition for hydration water and enables 546
function in solutions of low water activity, including organic solvent/water mixtures. The 547
immediate advantages for enzyme technology are as follows: increased salt- and heat 548
tolerance, a catalytic environment which enables use of less polar educts and potential 549
reversal of hydrolytic reactions, all of which make them strong candidates for industrial 550
biocatalysts. 551
An increasingly important industrial application of enzymes is the environmentally 552
friendly production of stereospecific building blocks for pharmaceuticals in “White 553
Biotechnology”. One such example, the stereospecific production of alcohols from ketones 554
was presented by Leanne Timpson and her colleagues Ann-Kathrin Liliensiek and Francesca 555
Paradisi (Dublin, Ireland). Halobacterial alcohol dehydrogenases were overexpressed in 556
Haloferax volcanii, using novel expression systems (4, 40). A number of poster presentations 557
outlined the search for useful enzymes such as proteases, cellulases, lipases, amylases and 558
mannanases from halophiles, including isolates from Chinese and Iranian lakes, and a prize-559
winning presentation by Yasuhiro Shimane and colleagues (Saitama, Japan) on enzymes 560
derived from haloarchaea isolated from domestic and commercial salt samples. 561
Nayla Munawar and Paul Engel (Dublin, Ireland) approached protein engineering of 562
substrate specificity in a halophilic enzyme by site directed mutagenesis in the absence of a 563
crystal structure of the enzyme. Using the crystal structure and previous mutagenesis of a 564
mesophilic counterpart (Clostridium symbiosum glutamate dehydrogenase) as a guide, they 565
selected corresponding residues in Halobacterium salinarum GDH for site-directed 566
mutagenesis and created a novel halophilic dehydrogenase which accepts L-methionine, L-567
norleucine and L-norvaline as substrates instead of glutamate. 568
Secondary metabolites, and in particular the untapped potential of halophilic 569
actinomycetes as a source for novel antibiotics, are increasingly becoming important, as 570
explained by Wen-Jun Li (Kunmin, China). The abundance of culturable yet unknown types 571
was beyond expectation, with predominance of Nocardiopsis, Saccharomonospora and 572
Streptomonospora. In the context of new drug discoveries, Xiukun Lin (Qingdao, China) 573
reported on novel compounds from actinomycetes isolated from salterns and their cytotoxic 574
effect against a range of cancer cell lines. 575
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The world-wide problem of petroleum contamination and potential application of 576
halophiles for bioremediation was addressed by Mohammad Amoozegar (Tehran, Iran), who 577
described a novel isolate, similar to Alcanivorax dieselolei, able to grow on crude oil, diesel 578
fuel and pure aliphatic hydrocarbons, but unable to degrade aromatic compounds. Its use in 579
saline soils was investigated. A consortium of at least six culturable strains (including 580
Marinobacter and Halomonas sp.) was able to degrade various polyaromatic hydrocarbons 581
over a salinity range from 1-17% NaCl. Thus, the degrading potential of halophiles has just 582
started to come to light and will become increasingly important in the future. 583
Another process in which halophiles may contribute in the future is the production of 584
bio-fuel. Melanie Mormile (Rolla, MO, USA) explained how halophilic/haloalkaliphilic and 585
halotolerant bacteria could be used to break down biomass material and form bio-fuel 586
products. Lignocellulosic biomass as a source for fermentative production of bio-fuel 587
products such as ethanol and hydrogen, may become a commercially interesting option, 588
provided lignin components can be removed. The required alkaline pre-treatment (to remove 589
lignin) and subsequent partial neutralization will create an environment for halophilic or 590
haloalkaliphilic fermentative bacteria in cellulose-converting processes. The general trend 591
towards use of algae for biofuel (biodiesel) production is problematic because of the high 592
consumption of fresh water. The use of halophilic algae may overcome such hurdles by means 593
of efficient non-potable water recycling and open up a bright future for halophile technology. 594
It is thus possible that in the future the biotechnological application of halophiles, or 595
genes derived from them, will extend to many more members of this extremely diverse group 596
of microbes. Possible areas of exploitation may stretch from production of valuable 597
compounds and remediation of contaminated waters and soils to future solutions of the 598
world’s liquid fuel crisis. 599
600
601
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Biographical sketches of the authors: 900
901
902
903
Yanhe Ma is a Professor of Microbiology and the vice director of State Key Laboratory of 904
Microbial Resources in the Institute of Microbiology, Chinese Academy of Sciences (CAS). 905
He is also the Vice-Director of the newly founded Tianjin Industrial Biotechnology R&D 906
Center, CAS. He is the Deputy Secretary-General of the Council of Chinese Society of 907
Biotechnology and the Vice-President of the Beijing Society for Microbiology. He is also a 908
member of the International Committee on Systematics of Prokaryotes (ICSP) Subcommittee 909
on Taxonomy of Halobacteriaceae. In addition, he is Associate Editor of Saline Systems and 910
of the Chinese Journal of Bioprocess Engineering. He won the Invention Award of the 911
Chinese Academy of Sciences in 1999 and the National Award of Advanced Science and 912
Technology in 2000. His research interests are mainly in the biodiversity, physiology and 913
application of extremophiles. 914
915
916
917
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Erwin A. Galinski studied biology, chemistry and biochemistry at Bonn University and the 918
University of St. Andrews (Scotland) as a scholar of the German National Merit Foundation. 919
He received his Dr. rer. nat. in microbiology (1986) and his university lecturer qualification in 920
microbiology and biotechnology (1993) from Bonn University. After a period as tenured 921
Professor of Biochemistry/Biotechnology at Münster University (1997-2001) he returned to 922
the Rheinische Friedrich-Wilhelms University in Bonn as full Professor of Microbiology. He 923
has held the positions of Head of the Examination Committee, Member of the Faculty 924
Council of Natural Sciences, Chairman of the Biology Section and the Student Fees Financial 925
Board. He is currently Managing Director of the Department of Microbiology & 926
Biotechnology. His main interests are in osmoprotective mechanisms of halophilic bacteria, in 927
particular production and application of compatible solutes such as ectoines (ingredient of 928
skin-care products), genetic engineering of salt-tolerance and principles of anhydrobiosis. 929
930
931
William D. Grant was born in Scotland in 1942. He received his BSc (1964) and PhD (1968) 932
from the University of Edinburgh. After post-doctoral studies at the University of Wisconsin, 933
Madison, in the USA, he returned to the UK as a research fellow at the University of 934
Leicester, subsequently becoming Professor of Environmental Microbiology, latterly 935
Emeritus Professor of Environmental Microbiology. Since the late 1970s Emeritus Professor 936
Grant has been interested in microbial biodiversity in extreme environments, particularly in 937
East African saline and soda lakes. He has also worked on the diversity of microbes in salt 938
mines, ancient salt deposits and low level nuclear waste. His current interests are mainly in 939
the molecular analyses of microbes and microbial signatures in hypersaline environments, and 940
accessing microbial genetic resources without the need for culture. 941
942
943
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944
945
Aharon Oren (1952, Zwolle, the Netherlands) received his MSc (1974) from the University of 946
Groningen and his PhD (1978) from the Hebrew University of Jerusalem, Israel, where he is 947
full professor since 1996. He serves as editor of International Journal of Systematic and 948
Environmmental Microbiology, Extremophiles, FEMS Microbiology Letters, and Saline 949
Systems. He is Executive Secretary/Treasurer of the International Committee on Systematics 950
of Prokaryotes and member of the ICSP Subcommittees on the Taxonomy of 951
Halobacteriaceae, Photosynthetic Prokaryotes and Halomonadaceae, President of the 952
International Society for Salt Lake Research and board member of the Israel Society for 953
Microbiology. He received the Moshe Shilo Prize (1993) and the Ulitzki Prize (2004) of the 954
Israel Society for Microbiology, and is Fellow of the American Academy of Microbiology 955
(2000). In 2010 he was awarded an honorary doctorate from the University of Osnabrück. His 956
research focuses on the ecology, physiology and taxonomy of halophilic microorganisms. 957
958
959
960
961
Antonio Ventosa is Professor and Head, Department of Microbiology and Parasitology of the 962
University of Sevilla, Spain. He has been Vice–Dean (1993-1997) and Dean (1997-2001) of 963
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the Faculty of Pharmacy, and Vice-Rector of Postgraduate Studies (2003-2006) of his 964
university. He is associate editor of the International Journal of Systematic and Evolutionary 965
Microbiology and editorial board member of Systematic and Applied Microbiology, 966
Extremophiles, International Microbiology, and Archaea. He is a member of the International 967
Committee on Systematics of Prokaryotes (ICSP) and Chairman of the ICSP Subcommittees 968
on the Taxonomy of Halobacteriaceae and Halomonadaceae. He won the Jaime Ferran Award 969
(the Spanish Society of Microbiolopy, 1991) and the FAMA Research Prize (University of 970
Sevilla, 2008). He is Fellow of the American Academy of Microbiology (2004) and the 971
European Academy of Microbiology (2009). His research focuses on extremophilic 972
microorganisms, microbial diversity of hypersaline environments, taxonomy and phylogeny 973
and biotechnological applications of halophiles. 974
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