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Appl Environ Microbiol. Nov 2010; 76(21): 6971–6981.
Published online Sep 3, 2010. doi:  10.1128/AEM.01868-10
PMCID: PMC2976238

Halophiles 2010: Life in Saline Environments [down-pointing small open triangle]

The world of halophilic microorganisms is highly diverse. Microbes adapted to life at high salt concentrations are found in all three domains of life: Archaea, Bacteria, and Eucarya. In some ecosystems salt-loving microorganisms live in such large numbers that their presence can be recognized without the need for a microscope. The brines of saltern crystallizer ponds worldwide are colored pink-red by Archaea (Haloquadratum and other representatives of the Halobacteriales), Bacteria (Salinibacter), and Eucarya (Dunaliella salina).

Hypersaline environments such as saltern pond brines and natural salt lakes present the ecologist with relatively simple ecosystems with low diversity and high community densities. In such systems fundamental questions of biodiversity, selection, biogeography, and evolution in the microbial world can be investigated much more conveniently than in the far more complex freshwater and marine systems. The sediments of such water bodies, however, are often inhabited by extremely diverse, still incompletely explored microbial communities. Different types of halophiles have solved the problem how to cope with salt stress (and often with other forms of stress as well) in different ways, so that the study of microbial life at high salt concentrations can answer many basic questions on the adaptation of microorganisms to their environments. Most known halophiles are relatively easy to grow, and genera such as Halobacterium, Haloferax, and Haloarcula have become popular models for studies of the archaeal domain as they are much simpler to handle than methanogenic and hyperthermophilic Archaea. Some halophilic and halotolerant microorganisms have found interesting biotechnological applications as well, as shown in the last section of this report.

The 9th International Conference on Halophilic Microorganisms, held from 29 June 2010 to 3 July 2010 in Beijing, China, brought together 166 participants from 25 countries. The 50 lectures and 112 posters presented provided an excellent overview of the current state of our understanding of all aspects of microbiology at high salt concentrations. The meeting was hosted by the Institute of Microbiology, the Chinese Academy of Sciences, the Chinese Society of Biotechnology, and the Chinese Society for Microbiology. Conference chair was Yanhe Ma. The series of symposia on halophiles started in Rehovot, Israel, in 1978 with a meeting devoted mainly to the properties of bacteriorhodopsin, the retinal-containing protein of Halobacterium that was discovered just a few years earlier. The delegates noted and applauded the presence of Janos Lanyi in the audience, one of the attendees at the first meeting. This initial event was followed by meetings held in 1985 (Obermarchtal, Germany), 1989 (Alicante, Spain), 1992 (Williamsburg, VA), 1997 (Jerusalem, Israel), 2001 (Seville, Spain), 2004 (Ljubljana, Slovenia), and 2007 (Colchester, United Kingdom). The proceedings of the 1978, 1989, 1997, 2001, and 2004 symposia were published as books (20, 31, 56, 68, 88); selected papers from the 1985, 2002, and 2007 symposia appeared in dedicated special volumes of journals (FEMS Microbiology Reviews, Experientia, and Saline Systems).

This review intends to capture emerging themes and to report key interesting new findings presented at the Halophiles 2010 symposium in Beijing. The following topics were the focus of attention and discussion.

DIVERSITY OF HALOPHILES—CULTURED AND UNCULTURED

“Everything is everywhere: but, the environment selects” (“Alles is overal: maar, het milieu selecteert”). This famous quotation from Lourens Baas Becking's 1934 book Geobiologie of inleiding tot the milieukunde (8) can be taken as the basis for our understanding of the distribution of halophilic microorganisms worldwide. In fact, Baas Becking (1895 to 1963) had visited many salt lakes and studied many different halophilic microorganisms. His book and his publications from the early 1930s contain a wealth of information, largely forgotten today, on the properties of the halophiles. Some phenomena described by Baas Becking at the time, including the acidic nature of the cell envelope of Dunaliella and the interrelationship of salt requirement/tolerance and temperature in halophilic prokaryotes, were “rediscovered” in the 1970s, as documented by Aharon Oren (Jerusalem, Israel) in his keynote lecture at the opening session.

To explore to what extent in the halophilic world “everything” is indeed “everywhere” and what degree of variation may be found among different high-salt environments, microbial diversity studies have been performed in a great variety of environments. These include saltern ponds worldwide, Great Salt Lake, the Dead Sea (16), saline lakes in Inner Mongolia (60), African soda lakes, deep-sea brines (85), and many others. These studies included culture-dependent approaches, leading to the isolation and characterization of many novel types of halophiles and new information on the abundance and geographic distribution of the known types, as well as culture-independent studies based on sequencing of DNA recovered from the environment. Many posters at the meeting related to culture-independent analyses of hypersaline environments from around the world, including Xinjiang salt lakes; Chinese salt mines; salterns in Goa India, Turkey, Spain, and Israel; south Siberian hypersaline lakes; the Dead Sea; and Great Salt Lake. Thane Papke (Storrs, CT) examined Halorubrum strains in Spain and Algeria, and one of his conclusions was that “migration routes are slower than mutation rates,” allowing endemism in Halorubrum strains to develop. Shaun Heaphy (Leicester, United Kingdom) provided a culture-independent microbial characterization of several Inner Mongolian salt and soda lakes and used statistical techniques to correlate the findings with physico-chemical parameters of brines and geographical location. He broadly agreed that microbial populations diverged as distance between the lakes increased, although this was only statistically significant for the Bacteria on an intercontinental scale (a hypersaline lake in Argentina was included in the analysis). Factors such as pH, temperature, and Na+ concentration were particularly correlated with the microbial community composition. Thus, everything may not be everywhere. More and more cases are being reported of the isolation of halophilic microorganisms from low salinity environments. Thus, after almost 80 years, Baas Becking's quotation still inspires experiment and debate.

Application of culture-dependent methods led to the isolation of a novel halophilic archaeon from seawater (at a salinity that does not support growth of Halobacteriaceae and causes lysis of most known representatives of the group). The properties of this new organism, to be described as a new genus and species, Halomarina oriensis, were presented by Kentaro Inoue (Chiba, Japan) (37). Poster presentations included a new fungal isolate from a Turkish salt mine; novel haloarchaea from a Chinese saltern, Inner Mongolian lakes, and Iranian salt lakes; and novel bacterial isolates from Chinese salt lakes, Inner Mongolian Lakes, Xinjiang salt lakes, Quidam Basin Quaternary sediments, brine wells in southwestern China, the Yellow Sea, The South China Sea, a Korean salt flat, Iranian salt lakes, Mexican soda environments, and salted hides. There were additional reports on the isolation of new actinomycetes from saline environments in China and on different bacterial halophiles from nonsaline sites such as soils.

HALOPHILES IN UNUSUAL ENVIRONMENTS AND HALOPHILES EXPOSED TO MULTIPLE FORMS OF STRESS

Most habitats explored for the presence of halophiles are thalassohaline environments that originated by evaporation from seawater, reflect the ionic composition of seawater, and have a nearly neutral to slightly alkaline pH.

Deep-sea brines, found on the bottom of the Red Sea, the Mediterranean Sea, and the Gulf of Mexico, are interesting environments to search for novel microbes. Apart from their increased high salinity, they are anaerobic and form characteristically sharp brine-seawater interfaces, with some of the brines displaying significant increases in temperature and metal concentration. The ionic composition of the brines generally differs from that of seawater; they are anaerobic, and in some cases the temperature can be elevated as well. The microbiology of Shaban Deep and other deep-sea brines in the Red Sea was discussed by André Antunes (Thuwal, Saudi Arabia). These sites, considered sterile in the past, have yielded a number of interesting microorganisms, including Salinisphaera shabanensis (a facultative anaerobe growing in a very large range of salt concentrations, from 1 to 28%) (5), Halorhabdus tiamatea (a nonpigmented representative of the Halobacteriales that prefers an anaerobic life style) (7), Flexistipes sinusarabici (an anaerobe tolerating between 3 to 18% NaCl) (28), and Haloplasma contractile (a contractile bacterium, phylogenetically equidistant to the Firmicutes and the Mollicutes) (6). The sites will be revisited in the near future for further microbiological exploration.

In many athalassohaline environments, life at the extremes of high salt is combined with the need to thrive at alkaline pH and elevated temperatures, and organisms growing there do so at the physico-chemical boundary for life (18). Jürgen Wiegel (Athens, GA) summarized his studies of the anaerobic halophilic, alkaliphilic, thermophilic bacteria isolated from the Wadi an-Natrun, Egypt. Natranaerobius thermophilus accumulates both glycine betaine and K+ for osmotic adaptation and has multiple Na+/H+ antiporters (49) and a Na+-extruding ATPase, which was characterized in-depth by Noha Mesbah (Alexandria, Egypt). Two new species, designated “Natranaerobius jonesii” and “Natranaerobius grantii” are currently being characterized. Natranaerobius jonesii has an extremely high requirement for chloride ions as it does not grow at less than 1.4 M Cl. Other alkaline saline environments subjected to intensive studies in recent years are the soda lakes of the Kulunda Steppe (Altai, Russia). Dimitry Sorokin (Moscow, Russia) summarized the wealth of information obtained from these studies at the level of the characterization of cultures of novel organisms, especially those participating in the reductive part of the sulfur cycle, and from culture-independent studies using molecular markers, as well as measurements of the rates of microbial sulfidogenesis. In general, sulfide production was active even in saturated soda brines, but far more sulfide was produced in these environments from elemental sulfur and from thiosulfate than from sulfate. Dismutation of thiosulfate and sulfite was a major trend in soda lake isolates (78, 79).

The Dead Sea is a rare example of a low-Na+, high-Mg2+, and high-Ca2+ chloride brine with a slightly acidic pH. Metagenomic studies are now providing information on the microbial diversity in the lake, both at the time of a bloom of microorganisms following dilution of the upper water layers by rain floods in 1992 and during the current drying out of the lake, causing a continuously decreasing ratio of monovalent/divalent cations, making conditions too extreme for even the best salt-adapted microorganisms (16).

GENOMICS, EVOLUTION, AND TAXONOMY OF HALOPHILIC ARCHAEA AND BACTERIA

Genomics of the Halobacteriaceae has come of age. Shiladitya DasSarma (Baltimore, MD) gave the closing keynote lecture, highlighting the haloarchaeal genomes from different genera which have been determined since the genome sequence of Halobacterium NRC-1 was first published (24, 53) 10 years ago. More recently completed genomes highlighted included Haloarcula marismortui (11, 14), Natronomonas pharaonis (26), Haloquadratum walsbyi (17), Halorubrum lacusprofundi (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=23834, 2009), Halomicrobium mukohataei (82), Halorhabdus utahensis (10), Halogeometricum borinquense (46), Haloterrigena turkmenica (73), and Haloferax volcanii (34). The list includes significant ecological diversity, e.g., a haloalkaliphilic species, a cold-adapted species, species adapted to life in low-Na+-high-Mg2+ environments, and isolates showing interesting cell morphologies. The sizes of these genomes range between 2.6 and 5.4 Mb. The sequencing and analysis of the genomes of Haloarcula hispanica and Haloferax mediterranei were announced by Hua Xiang and colleagues (Beijing, China). DasSarma showed that there has been an exponential increase in the sequencing of haloarchaeal genomes over the past 10 years, and, with next-generation sequencing methods now available, expects that within a few years the number of published genomes of species of Halobacteriaceae will grow even faster. Some of the conserved properties of haloarchaeal genomes were discussed, including the presence of large megaplasmids and minichromosomes (24) and the occurrence of core acidic proteomes (23). The data analysis also yielded the prediction of an expanding haloarchaeal pan-genome with increasing numbers of novel genes which may have applications in biotechnology.

In two presentations from the DasSarma group, additional postgenomic work was presented. James Coker (soon to move to Birmingham, AL) reported on studies on the expanded TATA-binding protein and transcription factor B protein families of haloarchaea, showing their importance for gene expression and stress regulation (21, 77). Satyajit DasSarma (the youngest presenter at age 13) reported on the expansion of the HaloWeb, the haloarchaeal genome database (http://halo4.umbi.umd.edu), which now provides access to all the public haloarchaeal genomes and as well as a suite of tools for data retrieval and analysis.

Environmental genomics studies increasingly show that the genome of individual strains may be only a small fraction of the pan-genome of the species in nature. Haloquadratum walsbyi has become an excellent example to illustrate this, as shown by Francisco Rodríguez-Valera (Alicante, Spain) and Mike Dyall-Smith (Martinsried, Germany). Comparisons have been made of the genome diversity within Haloquadratum populations in a single saltern crystallizer pond as well as comparisons between populations in similar environments at different geographic locations. The pan-genome of Haloquadratum walsbyi is at least 40 times the size of the genome of the type strain, and genomic microdiversity within an extremely simple and relatively constant environment is very high (17, 22, 40, 55). An interesting study of experimental evolution was presented by Jizhong Zhou (Norman, OK), using Desulfovibrio vulgaris as a model organism and monitoring genetic changes after exposure of this nonhalophilic bacterium to 0.25 M NaCl for 1,000 generations. Salt-specific mutations and deletions were detected in the salt-resistant phenotype, which used different amino acids as osmoprotectants (35).

Genome sequencing of new isolates is getting simpler and cheaper and will probably soon become routine. Undoubtedly, this development will have profound implications on the taxonomy of the halophiles. Until taxonomy can be based on comparison of complete genome sequences, multilocus sequence analysis (MLSA) is gaining popularity for the comparison of strains for taxonomic and evolutionary studies. Thane Papke (Storrs, CT) presented his extensive MLSA data on Halorubrum isolates from Spain and Algeria. Analysis of the data indicates very frequent occurrence of homologous recombination, to the extent that alleles were randomly associated, as typical of sexually reproducing species. Natural competence and conjugation (like the mating mechanism in Haloferax) (3) may be the possible mechanisms for lateral gene transfer (61). Emma White (Storrs, CT) and Hiroaki Minegishi (Saitama, Japan) showed how analysis of the RNA polymerase subunit B′(rpoB′) gene can help in reconstructing the phylogeny of the Halobacteriaceae (51). Also for the Halomonadaceae, MLSA is becoming a valuable tool for taxonomic studies, as shown by Antonio Ventosa (Seville, Spain). For both groups, sets of genes and primers have been defined that give good results consistent with other genotypic and phenotypic traits.

The list of sequenced genomes of halophilic and halotolerant Bacteria is as yet short. It does not yet even include Halomonas elongata, the organism that, since it was described 30 years ago (89), has become one of the most popular model organisms and has also found biotechnological applications (30, 57). Its genome sequence will soon be published. Genome sequence information is available for the anoxygenic halophilic phototroph Halorhodospira halophila, for an extremely salt-tolerant alkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio, for the thermophilic anaerobic halophile Halothermothrix orenii, and for the aerobic heterotrophic Chromohalobacter salexigens (58) and Salinibacter ruber.

Extensive environmental genomics data have been collected for Salinibacter. Josefa Antón (Alicante, Spain) showed a high degree of genomic variation within Salinibacter populations. Comparative analyses indicate that Salinibacter ruber genomes present a mosaic structure with conserved and hypervariable regions. Overall, 10% of the genes encoded in the genome of the Salinibacter M8 genome are absent from the type strain Salinibacter M31. Metabolomic profiles also differed in these two isolates (62).

HALOPHILIC VIRUSES

The Halophiles 2001 and 2004 symposia in Seville and Ljubljana will be remembered as the events where the importance of fungi in hypersaline ecosystems became clear. Halophiles 2010 can then be described as the congress presenting the importance of viruses. Phages attacking extremely halophilic Archaea were first described already in 1974 (83), but the role of viruses in hypersaline ecosystems remained largely unexplored.

Elina Roine (Helsinki, Finland) and her colleagues have discovered novel types of viruses attacking halophilic Archaea. The isolation and characterization of pleomorphic viruses possessing a lipid envelope, containing either a single-stranded or double-stranded DNA genome, show that viral diversity in hypersaline environments (63, 64, 69) is much larger than previously assumed. Shaun Heaphy (Leicester, United Kingdom) presented two novel lytic head/tailed viruses (virus BJ1 of the Siphoviridae and virus BJ2 of the Myoviridae), infecting Halorubrum kocurii, isolated from a salt lake in Inner Mongolia (59). Few archaeal virus genomes have been sequenced, and the complete sequence of virus BJ1 (EMBL accession number AM419438) is therefore a welcome addition.

First results of a comprehensive study of viral distribution and diversity in Great Salt Lake, UT, were presented by Bonnie Baxter (Salt Lake City, UT). Saltern crystallizer ponds are also ideal environments to study virus diversity and dynamics, as protozoa and other predators are absent, and numbers of prokaryotes and virus-like particles are extremely high, typically in the order of >107/ml and >108 to 109/ml, respectively. Forest Rohwer (San Diego, CA) showed his studies of virus dynamics in such salt-saturated ponds. At first sight, the salterns present predictable and stable communities of both Archaea and viruses, apparently different from the “kill-the-winner” behavior, with rapid cycling of microbial taxa and their viral predators that may be expected in such an environment (67). Metagenomic analysis of the viruses in the salterns near San Diego showed that the distribution of microbial taxa and viral taxa remained stable over time but with strong dynamic fluctuations of the prevalence of microbial strains and viral genotypes. Thus, at the fine level, the populations of both individual strains and viral genotypes fluctuate in a kill-the-winner fashion (67).

Activity of viruses also may have profound implications on the distribution of the extremely halophilic bacterium Salinibacter (Bacteroidetes). Josefa Antón (Alicante, Spain) studied the metagenome of viral assemblages of saltern pond in which Salinibacter accounts for around 15% of the prokaryotic community. Based on bioinformatic analysis (G+C content and dinucleotide frequency analysis), about 24% of the retrieved viral sequences could correspond to Salinibacter phages (70). It seems that phages infecting Salinibacter are more active in the environment than phages infecting Haloquadratum, and this may possibly explain why Haloquadratum outnumbers Salinibacter in every environment that supports growth of these organisms.

HALOPHILIC FUNGI

The importance of halophilic fungi, long neglected as members of hypersaline ecosystems, became recognized only in the past decade. Nina Gunde-Cimerman (Ljubljana, Slovenia) gave an overview of the biology of the most widespread and most halophilic or halotolerant fungi and yeasts. These include the black yeasts Hortaea werneckii which grows up to 5 M NaCl, the true halophile Wallemia ichthyophaga that requires at least 1.5 M NaCl and grows up to saturation, and Aureobasidium pullulans that grows up to 3 M NaCl. All of these are commonly found in hypersaline lakes and in a great variety of other, often unexpected, environments: domestic dishwashers, polar ice, and possibly even on spider webs in desert caves (32).

The halophilic and halotolerant fungi use polyols such as glycerol, erythritol, arabitol, and mannitol as osmotic solutes and retain low salt concentrations in their cytoplasm. Molecular studies on osmotic adaptation of Hortaea werneckii and Wallemia ichthyophaga were presented by Ana Plemenitaš and Janja Zajc (Ljubljana, Slovenia). Identification and structural features of Na+-sensitive 3′-phosphoadenosine-5′-phosphatase HwHal2, one of the putative determinants of halotolerance in H. werneckii and a promising transgene to improve halotolerance in crops, was presented (87). An in-depth understanding has been obtained of the HOG (high osmolarity glycerol) pathway, and this understanding may be applied in the future to the development of improved salt-resistant crops. Glycerol-3-phosphate dehydrogenase is involved in glycerol synthesis by both Wallemia and Hortaea, and heterologous expression of the gene encoding the enzyme can restore halotolerance in Saccharomyces cerevisiae deficient in glycerol production.

LONG-TERM SURVIVAL OF HALOPHILES

When brines dry out and halite crystals are formed, small fluid inclusions remain entrapped within the crystals. Microorganisms that inhabited the brine may get entrapped in these inclusions (9). Since the first controlled studies showed that such microorganisms may retain their viability for long periods (54), the question of the longevity of different types of halophiles within salt crystals has become a popular topic, relevant to disciplines including geology, biogeography, evolution, and even space exploration (48).

Terry McGenity (Colchester, United Kingdom) presented field studies and laboratory simulations of entombment of different types of microbes inside salt crystals. Salinibacter alone survives poorly within halite crystals, but when it was trapped inside a crystal together with Haloquadratum, longevity was much enhanced. Thus, simple food chains and mutual interactions occur between microorganisms in fluid inclusions in salt.

Examination of halite cores from Saline Valley, CA, representing salt deposited up to 150 thousand years ago, showed remnants of algae within fluid inclusions entrapped in the salt crystals. Morphological features as well as sequences of the internal transcribed spacer between the 18S and 5.8S rRNA genes led to the identification of Dunaliella, Ulothrix, and Nephroselmis, as shown by Krithivasan Sankaranarayanan (Binghamton, NY), who won first prize for his presentation by a young scientist. Presence of entrapped algae, with their high content of organic compatible solutes, may provide carbon and energy sources enabling halophilic heterotrophic microorganisms to survive for prolonged times (75).

OSMOTIC ADAPTATION, COMPATIBLE SOLUTES, AND ADAPTATION OF INTRACELLULAR PROTEINS TO SALT

There are basically two strategies that enable halophilic and halotolerant microorganisms to live in high salt concentrations. The “high-salt-in” strategy (used by the Halobacteriaceae, Salinibacter, and the anaerobic Halanaerobiales) requires all intracellular proteins to be stable and active in the presence of molar concentrations of KCl and other salts. The “low-salt, organic-solutes-in” strategy is based on the biosynthesis and/or accumulation of organic solutes that do not interfere greatly with the activity of normal enzymes. But even such organisms need to have salt-adapted proteins in the membrane exposed to the saline medium. It is remarkable that already in the early 1930s Baas Becking concluded that Dunaliella must have a highly acidic surface, based on the insensitivity of the alga to certain otherwise toxic anions (8).

Over the years, Haloarcula marismortui has been the most popular model organism for the study of the behavior of proteins active in a high-salt environment. These include the Haloarcula ribosome, whose structure elucidation by Ada Yonath was awarded the Nobel Prize for Chemistry in 2009. Christine Ebel (Grenoble, France) presented an overview of molecular adaptations of halophilic proteins, based on her studies of the Haloarcula marismortui malate dehydrogenase and other enzymes. Particularly, the very acidic surface of the macromolecule allows protein-salt interactions that avoid water or salt enrichment at the surface of the protein at high salt and preserve its solubility (25, 44).

Volker Müller (Frankfurt, Germany) uses Halobacillus halophilus as a model to understand the mechanisms of osmotic adaptation by a bacterium that accumulates organic-compatible solutes. Using techniques of biochemistry, genomics, DNA microarrays, etc., his group studies the way the organism senses its environment. Halobacillus is the first chloride-dependent bacterium reported, and several cellular functions depend on Cl for maximal activities, the most important being the activation of solute accumulation. Halobacillus switches its osmolyte strategy with the salinity in its environment by the production of different compatible solutes. Glutamate and glutamine dominate at intermediate salinities, and proline and ectoine dominate at high salinities. Chloride stimulates expression of the glutamine synthetase and activates the enzyme. The product glutamate then turns on the biosynthesis of proline by inducing the expression of the proline biosynthetic genes (71, 72). Halobacillus dabanensis is used by Su-Sheng Yang and his colleagues (Beijing, China) as a model organism to study the genes involved in halotolerance, including genes encoding Na+/H+ antiporters, enzymes involved in osmotic solute metabolism, and stress proteins (27, 90). Studies of a mutant of Halomonas elongata deficient in ectoine synthesis by Elisabeth Witt (Bonn, Germany) showed the production of a new cyclic compatible solute, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC). It is made by a side reaction of ectoine synthase (EctC) that forms ADPC by cyclic condensation of glutamine. She also demonstrated that ectoine synthase is a reversible enzyme, which has its equilibrium (in case of ectoine synthesis) completely on the side of the cyclic condensation product.

Ectoine and hydroxyectoine biosynthesis is widely found in halophilic and halotolerant microorganisms, and the expression of the ect structural genes is induced by salt stress. But the solutes provide protection not only against salt stress but also against temperature stress in Bacillus subtilis and other salt-tolerant bacilli, as shown by Erhard Bremer (Marburg, Germany). Quantification of the intracellular ectoine concentration in Virgibacillus pantothenticus revealed that its production is triggered either by an increase in external salinity or by a reduction in growth temperature. Transcription of the ectoine biosynthetic operon (ectABC) was enhanced under both environmental conditions (38). The crystal structure of Virgibacillus salexigens EctD, the enzyme responsible for conversion of ectoine to hydroxyectoine, is now known in detail (66).

LIPIDS, MEMBRANE-BOUND PIGMENTS, AND MEMBRANE-LINKED PROCESSES

The cytoplasmic membranes of halophilic Archaea of the family Halobacteriaceae contain interesting ether lipids and often have retinal proteins (bacteriorhodopsin, halorhodopsin, and sensory rhodopsins). Interesting lipids and retinal proteins have also been found in Salinibacter.

Heiko Patzelt (Muscat, Oman) showed that unsaturated ether lipids are far more common in the halophilic Archaea than generally assumed. Such unsaturated diether lipids were earlier reported from the psychrotolerant haloarchaeon Halorubrum lacusprofundi (29). Isolates of Haloarcula spp. and Haloferax sp. obtained from a potash mine crystallization pond in north Germany had unsaturated ether lipids up to 37% of the total membrane lipid content. Only the phospholipids were unsaturated, and these contained mostly four or six double bonds in the archaeol chain.

Novel types of acylhalocapnines were described by Angela Corcelli (Bari, Italy). Salisaeta longa, an organism that requires lower salt concentrations than the related Salinibacter (Bacteroidetes), contains the hydroxyl fatty acid ester of 2-carboxy-2-amino-3,4-hydroxy-17-methyloctadec-5-ene-1-sulfonic acid, a sulfonate sphingoid base for which the common name of halocapnine is suggested (12). Salinibacter contains similar acylhalocapnine lipids in its membrane, as well as a retinal protein named xanthorhodopsin and an unusual ketocarotenoid named salinixanthin, found in a 1:1 molar ratio with the retinal pigment. Janos Lanyi (Irvine, CA) showed how the two chromophores interact and how the carotenoid acts as an antenna, transferring the absorbed light energy to the xanthorhodopsin proton pump. Such an energy transfer phenomenon appears to be unique for the clade that includes xanthorhodopsin as it is not found between the carotenoid bacterioruberin and bacteriorhodopsin in Halobacterium and related genera. The efficiency of the energy transfer is about 50%. The three-dimensional structure of the xanthorhodopsin-salinixanthin system has been determined from X-ray diffraction of xanthorhodopsin crystals, showing how the carotenoid interacts with the retinal protein (42).

The Haloquadratum walsbyi genome encodes two different bacteriorhodopsins. Both are expressed in the cells. Angela Corcelli (Bari, Italy) reported the isolation of the two forms of bacteriorhodopsin from Haloquadratum cultures using a biochemical approach. Bacteriorhodopsin was also recovered from biomass collected from the saltern crystallizer ponds of the Margarita di Savoia saltern.

Mecky Pohlschröder (Philadelphia, PA) presented recent results pertaining to mechanisms of protein transport across haloarchaeal cytoplasmic membranes. In haloarchaea, although the Sec pathway transports important substrates, including subunits of type IV pilus-like structures, the Tat pathway is used extensively and transports a wide range of secreted proteins, the majority of which appear to be anchored to the haloarchaeal membrane via a lipid anchor. In silico analyses suggest that prominent use of the Tat pathway as well as extensive anchoring of Tat substrates via a lipid anchor is unique to halophilic Archaea (the latest views on the membranal mechanisms of protein secretion in Haloferax volcanii). The Sec pathway remains an essential mode of protein transport in halophilic Archaea (80, 84). Novel programs allowing more accurate predictions of protein subcellular location (publicly available at SignalFind.org) were also presented.

DNA REPLICATION, TRANSCRIPTION, TRANSLATION, AND POSTTRANSLATIONAL MODIFICATION IN HALOPHILIC ARCHAEA

Relatively few talks dealt with the molecular biology of halophiles and the basic properties of the DNA replication, transcription, and translation machinery in different groups of halophiles.

Stuart MacNeill (St. Andrews, United Kingdom) presented novel information on the structure of the replication fork of Haloferax. Haloferax volcanii encodes a single minichromosome maintenance protein. Its N-terminal domain has a putative DNA-binding β-hairpin, a Cys4 zinc ribbon, and a β-hairpin with a role in interdomain communication. Genetic analysis of different mutants enabled the elucidation of the roles of the different proteins associated with the replication fork (43).

The biosynthesis and assembly of gas vesicles in halophilic Archaea have been used as a model for the study of transcription and other molecular processes in the Halobacteriaceae for nearly 3 decades now. Felicitas Pfeifer (Darmstadt, Germany) presented the latest information how the Halobacterium salinarum gas vesicle, when expressed in Haloferax volcanii, can be used as a convenient model system for the study of gene expression, transcription, and translation. Gas vesicles are composed of two structural proteins: the hydrophobic GvpA that forms the core of the cylinders and the hydrophilic GvpC that cross-links the GvpA subunits at the outside and provides strength to the vesicles. GvpC is now also known to be involved in the determination of the shape of the vesicles. Anaerobiosis inhibits gas vesicle formation. Fourteen gvp genes are required for gas vesicle formation, and these are arranged in two oppositely organized clusters. The function of the different promoters and transcriptional activators is becoming increasingly clear (13, 36, 81).

Updates about the molecular mechanisms of translational control in Halobacterium salinarum and Haloferax volcanii were given by Jörg Soppa (Frankfurt, Germany). Different mechanisms for translation initiation are known: (i) interaction between 16S rRNA and a Shine-Dalgarno sequence, (ii) the eukaryotic mechanism of linear scanning of the small ribosomal subunit from the 5′-cap to the start codon, (iii) an alternative eukaryotic mechanism using internal ribosomal entry sites, and (iv) leaderless transcripts that require an undissociated ribosome and the initiator tRNA (a mechanism encountered in all three domains of life). Characterization of the 5′ and 3′ ends of haloarchaeal transcripts showed that the majority of the transcripts are leaderless, that Shine-Dalgarno sequences are very rare, and that about a third do not fall in any of these four classes and must use a novel, yet uncharacterized method of translation initiation (19).

Posttranslational modification is studied in the laboratory of Jerry Eichler (Beer-Sheva, Israel), centering on the biosynthesis of the glycoproteins so abundantly found in the cell envelope of the Halobacteriaceae. Asn-modified glycoproteins are common in Archaea, and their production was studied using Haloferax volcanii as a model. A series of agl (archaeal glycosylation) genes was defined, encoding proteins involved in the assembly and attachment of a novel pentasaccharide to Asn residues of the S-layer glycoprotein. The functions of several Agl proteins are now known (1, 2, 45, 91).

GENETIC SYSTEMS FOR HALOPHILIC PROKARYOTES

Two interesting systems for genetic manipulation of halophiles were highlighted at the meeting. Already in the original species description of Haloferax volcanii the formation of intercellular bridges was noted (52). Moshe Mevarech (Tel Aviv, Israel) presented a survey of the genetic manipulation of Haloferax volcanii, developed since genetic transfer based on cell mating was first described 25 years ago (50). The mating system of Haloferax volcanii resembles eukaryotic sexual mating rather than bacterial lateral gene transfer. Large amounts of genetic material can be transferred this way (3). The successful mating of Haloferax volcanii and Haloferax mediterranei, yielding hybrid progeny, was announced.

Saskia Köcher (Frankfurt, Germany) presented a (prize-winning) poster describing the establishment of a genetic system for the manipulation of Halobacillus halophilus, based on protoplast fusion and markerless gene disruption. Cells can be transformed by protoplast transformation, resulting in integration of a nonreplicating plasmid via single homologous recombination. The method was used to generate proline biosynthesis mutants. This genetic manipulation strategy will now open the way to study many more properties of Halobacillus at the genetic level.

BIOTECHNOLOGICAL APPLICATIONS OF HALOPHILES

In comparison to other groups of extremophilic microorganisms such as the thermophiles and the alkaliphiles, the halophiles of all three domains have been relatively little exploited in biotechnological processes, with notable exceptions of β-carotene from Dunaliella, bacteriorhodopsin from Halobacterium, and ectoine from Halomonas (57). The biotechnology section of the meeting focused on production/modification techniques of compatible solutes, bioplastics, and halophilic enzymes. In addition, attention was drawn toward secondary metabolites from halophiles as well as bioremediation and biofuel production.

One success story of halophile biotechnology is the production and application of the compatible solute ectoine, currently produced at large scale by bitop AG in Germany using “bacterial milking” of Halomonas elongata. Ectoine is the active ingredient of many cosmetics and skin care products and increasingly becomes important in medicinal preparations (30). In addition, ectoine (and/or suitable derivatives) is used as a protectant for biomolecules and enhancer in molecular biology applications such as PCR and DNA microarray techniques (47, 74). Erwin Galinski (Bonn, Germany) presented a survey of the industrial production processes of ectoine and, in particular, a critical analysis of the maximal specific production rates obtainable with H. elongata as the production strain (50 mg g−1 of dry weight h−1 at 5 to 7.5% NaCl). Different strategies have been applied in attempts to increase production, including heterologous expression of the ectoine gene cluster in Escherichia coli and concomitant overexpression of genes that increase the supply of limiting precursors for ectoine biosynthesis, thus bypassing “metabolic bottlenecks“ (15, 76). Heterologous expression of the ectoine gene cluster in E. coli is at present not a suitable alternative to ectoine production in H. elongata. With respect to the hydroxylated derivative (S,S-β-hydroxyectoine) the situation is, however, different. As this compound is always produced in a mixture with ectoine in H. elongata, a costly chromatographic separation is required. By overexpressing the ectD gene (encoding ectoine hydroxylase) in E. coli, an efficient whole-cell biotransformation system for ectoine has been established in which the product (hydroxyectoine) leaked out of the cells and accumulated in the medium (E. A. Galinski, M. Stein, A. Ures, and T. Schwarz, World patent application WO 2009/059783 A1). Novel developments concern use of genetically engineered H. elongata for production of rare and thus far inaccessible compatible solutes. The potential of this approach for the development of new production processes was demonstrated using the compatible solutes mannosylglycerate (gene cluster from the thermophilic Rhodothermus marinus) and N-acetyl-glutaminyl glutamine-1-amide (gene cluster from Pseudomonas putida) as examples.

Whereas in past meetings the production of extracellular halophilic polymers with interesting rheological properties had claimed attention, the emphasis of this year's meeting (as regards polymers) has clearly been on intracellular polyesters. Production of poly-β-hydroxyalkanoates—biodegradable polymers with plastic-like properties—although not restricted to halophilic prokaryotes, was the topic of no less than four talks and a number of posters. Some halophilic or halotolerant Bacteria were shown to be excellent producers of such bioplastics. One of these is Halomonas boliviensis, as argued by Jorge Quillaguamán (Cochabamba, Bolivia), who presented strategies to optimize the biosynthesis of such bioplastics coupled with production of the high-value products ectoine and hydroxyectoine (65, 86). Archaea of the genus Haloferax are also known as poly-β-hydroxyalkanoate producers and the biosynthetic pathway leading to their production were elucidated by Hua Xiang and colleagues (Beijing, China) (33, 41). Unfortunately none of the presenters compared the potential of halophilic producers with the current productivity of industrial strains as used, for example, by Metabolix/ADM (Cambridge, MA) for their bioplastic product Tirel.

Many alkaliphiles are halophilic as well, and many useful enzymes applied in the detergent industry (washing powders), the textile industry, and other processes were derived from bacteria growing in saline alkaline lakes. Brian Jones (Leiden, Netherlands) explained how the saline alkaline lakes in Kenya and Inner Mongolia have been a rich source of organisms and/or genes from metagenomic libraries, and some of these are already explored as starting material for the production of commercially valuable enzymes, in particular, proteases and amylases.

Halophilic enzymes (typical for Archaea and Salinibacter but also for exoenzymes of any halophile) are characterized by an excess of acidic amino acids and subsequent negative surface charge. This peculiarity allows effective competition for hydration water and enables function in solutions of low water activity, including organic solvent/water mixtures. The immediate advantages for enzyme technology are as follows: increased salt and heat tolerance, a catalytic environment which enables use of less polar educts, and potential reversal of hydrolytic reactions, all of which make them strong candidates for industrial biocatalysts.

An increasingly important industrial application of enzymes is the environmentally friendly production of stereo-specific building blocks for pharmaceuticals in “white biotechnology.” One such example, the stereo-specific production of alcohols from ketones was presented by Leanne Timpson and her colleagues Ann-Kathrin Liliensiek and Francesca Paradisi (Dublin, Ireland). Halobacterial alcohol dehydrogenases were overexpressed in Haloferax volcanii, using novel expression systems (4, 39). A number of poster presentations outlined the search for useful enzymes such as proteases, cellulases, lipases, amylases, and mannanases from halophiles, including isolates from Chinese and Iranian lakes, and a prize-winning presentation by Yasuhiro Shimane and colleagues (Saitama, Japan) on enzymes derived from haloarchaea isolated from domestic and commercial salt samples.

Nayla Munawar and Paul Engel (Dublin, Ireland) approached protein engineering of substrate specificity in a halophilic enzyme by site directed mutagenesis in the absence of a crystal structure of the enzyme. Using the crystal structure and previous mutagenesis of a mesophilic counterpart (Clostridium symbiosum glutamate dehydrogenase) as a guide, they selected corresponding residues in Halobacterium salinarum glutamate dehydrogenase (GDH) for site-directed mutagenesis and created a novel halophilic dehydrogenase which accepts l-methionine, l-norleucine, and l-norvaline as substrates instead of glutamate.

Secondary metabolites and, in particular, the untapped potential of halophilic actinomycetes as a source for novel antibiotics are increasingly becoming important, as explained by Wen-Jun Li (Kunmin, China). The abundance of culturable yet unknown types was beyond expectation, with predominance of Nocardiopsis, Saccharomonospora, and Streptomonospora. In the context of new drug discoveries, Xiukun Lin (Qingdao, China) reported on novel compounds from actinomycetes isolated from salterns and their cytotoxic effect against a range of cancer cell lines.

The worldwide problem of petroleum contamination and potential application of halophiles for bioremediation were addressed by Mohammad Amoozegar (Tehran, Iran), who described a novel isolate, similar to Alcanivorax dieselolei, able to grow on crude oil, diesel fuel, and pure aliphatic hydrocarbons but unable to degrade aromatic compounds. Its use in saline soils was investigated. A consortium of at least six culturable strains (including Marinobacter and Halomonas sp.) was able to degrade various polyaromatic hydrocarbons over a salinity range from 1 to 17% NaCl. Thus, the degrading potential of halophiles has just started to come to light and will become increasingly important in the future.

Another process in which halophiles may contribute in the future is the production of biofuel. Melanie Mormile (Rolla, MO) explained how halophilic/haloalkaliphilic and halotolerant bacteria could be used to break down biomass material and form biofuel products. Lignocellulosic biomass as a source for fermentative production of biofuel products, such as ethanol and hydrogen, may become a commercially interesting option, provided lignin components can be removed. The required alkaline pretreatment (to remove lignin) and subsequent partial neutralization will create an environment for halophilic or haloalkaliphilic fermentative bacteria in cellulose-converting processes. The general trend toward use of algae for biofuel (biodiesel) production is problematic because of the high consumption of fresh water. The use of halophilic algae may overcome such hurdles by means of efficient nonpotable water recycling and open up a bright future for halophile technology.

It is thus possible that in the future the biotechnological application of halophiles, or of genes derived from them, will extend to many more members of this extremely diverse group of microbes. Possible areas of exploitation may stretch from production of valuable compounds and remediation of contaminated waters and soils to future solutions of the world's liquid fuel crisis.

Biography

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Object name is zam9991014500001.jpgYanhe Ma is a Professor of Microbiology and the Vice-Director of State Key Laboratory of Microbial Resources in the Institute of Microbiology, Chinese Academy of Sciences (CAS). He is also the Vice-Director of the newly founded Tianjin Industrial Biotechnology R&D Center, CAS. He is the Deputy Secretary-General of the Council of Chinese Society of Biotechnology and the Vice-President of the Beijing Society for Microbiology. He is also a member of the International Committee on Systematics of Prokaryotes (ICSP) Subcommittee on Taxonomy of Halobacteriaceae. In addition, he is Associate Editor of Saline Systems and of the Chinese Journal of Bioprocess Engineering. He won the Invention Award of the Chinese Academy of Sciences in 1999 and the National Award of Advanced Science and Technology in 2000. His research interests are mainly in the biodiversity, physiology, and application of extremophiles.

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Object name is zam9991014500002.jpgErwin A. Galinski studied biology, chemistry, and biochemistry at Bonn University and at the University of St. Andrews (Scotland) as a scholar of the German National Merit Foundation. He received his Dr. rer. nat. degree in microbiology (1986) and his university lecturer qualification in microbiology and biotechnology (1993) from Bonn University. After a period as tenured Professor of Biochemistry/Biotechnology at Münster University (1997 to 2001), he returned to the Rheinische Friedrich-Wilhelms University in Bonn as full Professor of Microbiology. He has held the positions of Head of the Examination Committee, Member of the Faculty Council of Natural Sciences, and Chairman of the Biology Section and the Student Fees Financial Board. He is currently Managing Director of the Department of Microbiology and Biotechnology. His main interests are in osmoprotective mechanisms of halophilic bacteria, in particular, production and application of compatible solutes such as ectoines (ingredient of skin care products), genetic engineering of salt tolerance, and principles of anhydrobiosis.

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Object name is zam9991014500003.jpgWilliam D. Grant was born in Scotland in 1942. He received his B.Sc. (1964) and Ph.D. (1968) from the University of Edinburgh. After postdoctoral studies at the University of Wisconsin, Madison, WI, he returned to the United Kingdom as a research fellow at the University of Leicester, subsequently becoming Professor of Environmental Microbiology, latterly Emeritus Professor of Environmental Microbiology. Since the late 1970s Dr. Grant has been interested in microbial biodiversity in extreme environments, particularly in East African saline and soda lakes. He has also worked on the diversity of microbes in salt mines, ancient salt deposits, and low-level nuclear waste. His current interests are mainly in the molecular analyses of microbes and microbial signatures in hypersaline environments and accessing microbial genetic resources without the need for culture.

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Object name is zam9991014500004.jpgAharon Oren (born 1952 in Zwolle, Netherlands) received his M.Sc. (1974) from the University of Groningen and his Ph.D. (1978) from the Hebrew University of Jerusalem, Israel, where he has been a full professor since 1996. He serves as editor of International Journal of Systematic and Environmental Microbiology, Extremophiles, FEMS Microbiology Letters, and Saline Systems. He is Executive Secretary/Treasurer of the International Committee on Systematics of Prokaryotes and member of the ICSP Subcommittees on the Taxonomy of Halobacteriaceae, Photosynthetic Prokaryotes and Halomonadaceae, President of the International Society for Salt Lake Research, and board member of the Israel Society for Microbiology. He received the Moshe Shilo Prize (1993) and the Ulitzki Prize (2004) of the Israel Society for Microbiology and is a Fellow of the American Academy of Microbiology (2000). In 2010 he was awarded an honorary doctorate from the University of Osnabrück. His research focuses on the ecology, physiology, and taxonomy of halophilic microorganisms.

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Object name is zam9991014500005.jpgAntonio Ventosa is Professor and Head of the Department of Microbiology and Parasitology of the University of Seville, Spain. He has been Vice-Dean (1993 to 1997) and Dean (1997 to 2001) of the Faculty of Pharmacy and Vice-Rector of Postgraduate Studies (2003 to 2006) of his university. He is associate editor of the International Journal of Systematic and Evolutionary Microbiology and editorial board member of Systematic and Applied Microbiology, Extremophiles, International Microbiology, and Archaea. He is a member of the International Committee on Systematics of Prokaryotes (ICSP) and Chairman of the ICSP Subcommittees on the Taxonomy of Halobacteriaceae and Halomonadaceae. He won the Jaime Ferran Award (the Spanish Society of Microbiology, 1991) and the FAMA Research Prize (University of Seville, 2008). He is a Fellow of the American Academy of Microbiology (2004) and the European Academy of Microbiology (2009). His research focuses on extremophilic microorganisms, microbial diversity of hypersaline environments, taxonomy and phylogeny, and biotechnological applications of halophiles.

Footnotes

[down-pointing small open triangle]Published ahead of print on 3 September 2010.

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