Extreme Halophiles Are Models for Astrobiology
Tolerance of extremes in salinity, radiation, and temperature may permit halophiles to survive elsewhere in the universe
Shiladitya DasSarma is a Professor at the University of Maryland Biotechnology Institute, Center of Marine Biotechnology, Baltimore.
Author profile--DasSarma Focuses on Halobacteria, Hails Their Versatility
* Studying extremophiles such as halophiles on Earth may provide insights helpful in our search for life elsewhere in the universe.
* A common characteristic among halophiles is that they contain a preponderance of acidic proteins, better enabling them to dissolve and to function in high-salt environments.
* A model halophile, Halobacterium sp. NRC-1, is being studied for its ability to withstand many environmental insults, including damaging radiation, extremes of temperature, low oxygen, and exposure to heavy metals.
* Retinal-based phototrophy, first discovered in halophiles and now known to be prevalent in common marine microbes, may be one of the oldest metabolic capabilities on Earth.
Studying extremophiles on Earth may provide helpful metrics in our search for life elsewhere in the universe. Species that thrive in hypersaline environments are among the most fascinating models for studying mechanisms of survival away from our planet. The genomic sequences of six such extreme halophiles and post-genomic studies of these microorganisms are providing insights about what it takes to live in environments that were once thought uninhabitable.
A unifying requirement for life is liquid water. However, evaporative forces dissipate liquid water in arid environments on Earth, on dry planets such as Mars, and in space. In brines on Earth, evaporating water concentrates dissolved salts and increases osmotic stress for all life forms. This process, which is common in natural and artificial solar salterns, leads calcium sulfate (gypsum) and sodium chloride (halite) to precipitate from seawater, leaving mainly potassium and magnesium salts in solution. During this process, dense microbial photosynthetic mats initially form but later decline, leaving only a few extremely halophilic archaea and bacteria (Fig. 1). The saturation point of sodium chloride (5.3 M), is about nine times the salinity of seawater and marks the upper limit of resistance of cultivated biological forms.
The Diversity of Hypersaline Environments and Halophiles on Earth
Two of the largest and best-studied modern hypersaline environments are Great Salt Lake in Utah and the Dead Sea in the Middle East. Of the two, Great Salt Lake at 3,900 km2 is larger and shallower, about 10 m deep, and contains salts that closely match seawater in their relative proportions. The Dead Sea covers 800 km2, has an average depth of 340 m, and contains salts of nonmarine proportions, with a high concentration of divalent magnesium ions. Both bodies of water contain total dissolved salts close to saturation.
Extremely halophilic microorganisms thrive in these extreme environments, turning the Great Salt Lake into a deep red color that is visible from space (Fig. 2). The archaeon Halobacterium sp. NRC-1, the first halophile to have its genome sequence determined, is very similar to a dominant species present in Great Salt Lake. Two related archaea, Haloarcula marismortui and Haloferax volcanii, whose genomic sequences have also been determined, are from the Dead Sea.
Permanently cold hypersaline evaporation ponds are found in dry regions of Antarctica, including Deep Lake, Organic Lake, and Lake Suribati. The psychrophile Halorubrum lacusprofundi, whose genome sequence is being analyzed, was isolated from Deep Lake, which despite surface temperatures of -20 degrees C in winter, does not freeze because of its nearly saturating salinity. Natural lakes that consist of alkaline hypersaline soda brines include Lake Magadi in Kenya, the Wadi Natrun lakes of Egypt, and the Great Basin lakes of the western United States (such as Mono Lake in California). Soda brines lack the divalent cations magnesium and calcium because of their low solubility at alkaline pH. They support the growth of halophilic alkaliphiles, such as Natronomonas pharaonis, that can tolerate these two extremes. Halophilic microorganisms are also found in many small evaporation ponds or sabkhas near coastal areas, where seawater penetrates through seepage or via narrow inlets from the sea. Smaller hypersaline pools are especially dynamic environments in which size, salinity, and temperature fluctuate with the seasons.
Hypersaline environments are also found within the Earth subsurface, in deep-sea basins associated with undersea salt domes and in subterranean halite deposits from evaporated ancient seas. Deep-sea brines are relatively stable because of their high densities. They are found in the Red Sea, the Gulf of Mexico, and the Mediterranean Sea--and may also occur under ice crusts on the Jovian moon Europa. On Earth, intense microbial activity is associated with these deep-sea hypersaline basins. Subsurface salt deposits that are more than 10 million years old have yielded bacterial and archaeal DNA as well as culturable microorganisms. Despite skepticism about whether these recovered species are truly ancient, their similarity to modern halophiles reinforces the view that model halophiles will help us to better understand the fundamental limits to life.
Characteristics of Halophile Genomes and Acidic Proteomes
The detailed information about the sequences, for the genome sequence of Halobacterium sp. NRC-1, a typical halophilic archaeon widely distributed in hypersaline environments, became available in 2000. Since then, the complete genomes of two halophilic archaea, Haloarcula marismortui and Natronomonas pharaonis, and a bacterium, Salinibacter ruber, were published, and draft sequences of two other archaeal halophiles--Haloferax volcanii and Halorubrum lacusprofundi, have been determined.
One common characteristic among these halophiles is that their predicted proteomes have dramatically low isoelectric points. The Halobacterium sp. NRC-1 proteome, for example, contains proteins with an average pI of only ~4.9, and similar values are found for other extreme halophile genomes (Fig. 3A). By contrast, pIs of nearly all nonhalophilic proteomes are close to neutral, usually with a bimodal distribution of basic (pI ~10) and acidic (pI ~5) proteins.
Proteins from halophiles typically display high surface negative charges, and this pattern of increased negative charge and lowered pI appears to hold true for the vast majority of halophilic proteins (Fig. 3B). The acidic halophilic proteins readily dissolve with enhanced functions in a milieu with high salinity and markedly reduced water activity. These observations help to explain how metabolic functions can be maintained in hypersaline media where cytoplasmic proteins are bathed in saturated potassium chloride.
Halobacterium sp. NRC-1: Useful for Postgenomic Studies
Among extreme halophiles, Halobacterium sp. NRC-1 is being widely used for postgenomic studies because its genome sequence is available, it is easily cultured, and its metabolism is highly versatile, with both facultative anaerobic and phototrophic capabilities. This microbe tolerates and responds to many environmental stresses, including high and low temperatures, UV and ionizing radiation, heavy metals, fluctuating ionic concentrations, and microaerobic and anaerobic conditions. Moreover, Halobacterium sp. NRC-1 is genetically tractable, can be transformed, and has a readily used gene knockout system based on the selectable and counterselectable ura3 marker. Whole-genome DNA microarrays are being employed to interrogate patterns of gene expression, and biochemical studies are under way using cellular components that are released by hypotonic lysis.
Gene knockouts and DNA microarrays are being used to analyze Halobacterium sp. NRC-1 responses to a variety of environmental conditions, including several relevant to astrobiology. Inkjet technology has been used to synthesize 11,000 60-nucleotide long oligonucleotide probes, representing 99% of the genes, directly onto glass slides. This relatively low-cost analytic approach provides a flexible means for studying cellular responses to environmental fluctuations (Fig. 4). For instance, halophiles such as Halobacterium sp. NRC-1 are subject to and can withstand intense solar radiation--in this case, by producing a cyclopyrimidine photolyase (phr) that reverses photoproducts of UV radiation. When irradiated with UV, such cells also induce high levels of radA, which is related to both eukaryotic rad51 and bacterial recA, indicating the involvement of homologous recombination. Meanwhile, in response to high-energy gamma radiation and desiccation, which can lead to extensive double-strand DNA breaks, cells produce membrane pigments, such as bacterioruberin, to shield the genome. These cells also induce rfa3, which encodes a single-stranded DNA binding protein that may be important for protecting and repairing damaged DNA.
Toxic metals and fluctuating ionic conditions also can affect Halobacterium sp. NRC-1. For instance, cells resist the toxic effects of arsenic through the extrachromosomal gene cluster arsADRC, which encodes arsenate reductase, a transporter ATPase component, but not the arsenite pump. When a nearby gene, arsM, is knocked out, the cells show increased sensitivity to arsenite, suggesting a second detoxification system involving an arsenite(III)-methyltransferase that Halobacterium sp. NRC-1 may use to transform arsenite to a volatile form, trimethylarsine. Changing the concentrations of metal and ionic species affects gene expression among a complex set of regulators, including the arsenic regulator arsC and a metal-responsive regulator, sirR.
One way Halobacterium sp. NRC-1 responds to limiting oxygen levels is to induce gas vesicle production, enabling the cells to float to aerobic zones in water columns. Moreover, under strictly anaerobic conditions, cells induce a bifunctional reductase, which is related to NarG-type nitrate reductases. The reductase, encoded by the dmsEABCD operon, uses both dimethyl sulfoxide (DMSO) and trimethylamine N-oxide (TMAO) as terminal electron acceptors (Fig. 4A). Transcript levels of the dms operon depend on an upstream gene, dmsR, coding a transcriptional activator. Knocking out the dmsR gene significantly reduces dms operon transcription (Fig. 4B), and such cells no longer can grow on TMAO.
Another dramatic response to limiting oxygen occurs when Halobacterium sp. NRC-1 cells induce the bop gene, encoding bacterio-opsin, the protein component of the light-driven proton pump, bacteriorhodopsin, which allows for phototrophic growth (Fig. 4A). Bacterio-opsin synthesis is coordinately regulated with the chromophore retinal, and the two molecules form a complex with 1:1 stoichiometry. In addition to bop, this regulon includes crtB1 and brp, which encode the first and last committed steps of retinal synthesis, and bat, the sensor-activator gene.
The eukaryotic-like TATA-box in the bop promoter deviates significantly from the consensus version, suggesting that novel factors are involved in its transcription. Multiple tbp and tfb genes (coding for six TATA-binding Tbp proteins and seven TFIIB-like Tfb factors) are found in the Halobacterium sp. NRC-1 genome, suggesting an alternate Tbp-Tfb pair may be used in bop transcription, similar to some regulated genes in higher organisms. Mutagenesis defined an upstream bop gene regulatory site, UAS, which is also near the co-regulated brp and crtB1 genes, and where the Bat regulator binds. The Bat protein sequence contains GAF (cGMP-binding) and PAS/PAC (redox-sensing) motifs, indicating that it senses both light and redox potential. It also contains a C-terminal DNA-binding helix-turn-helix (HTH) motif like that in DmsR, which mediates binding to UAS. Similarities of the Bat regulator and UAS to analogous regions in diverse organisms, including a plant and a gamma proteobacterium, suggest an ancient origin for this archaeal regulon.
Evolutionary Origin of Retinal Proteins
One interesting evolutionary question concerning halophiles centers on the origin of retinal proteins, like bacteriorhodopsin. This family also includes halorhodopsin and sensory rhodopsins, which are found in Halobacterium sp. NRC-1 and in other halophilic archaea, and xanthorhodopsin in Salinibacter. These can act as light-driven ion pumps, allowing cells to grow phototrophically, or as photosensory receptors, conferring phototactic responses on cells.
Although prokaryotic rhodopsins were discovered in halophilic archaea and thought to be unique to this group, metagenomic studies show them to be prevalent in diverse bacteria and eukaryotes. Therefore, these proteins may have arisen before the three domains diverged, or they may have been dispersed through lateral gene transfers to the relatively few clades in which they are found, including oceanic planktonic bacteria, some fungi, and halophilic archaea. Although lateral gene transfers appear to be a major force in the evolution of extreme halophiles, phylogenetic analysis remains inconclusive for retinal proteins. However, their spectroscopic complementarity suggests possible coevolution of retinal pigments with chlorophyll-based pigments (Fig. 5). If true, retinal-based phototrophy may be one of the oldest metabolic capabilities on Earth.
Halophiles as “Exophiles”
Because halophiles tolerate so many forms of environmental stress, they are candidate “exophiles”—organisms that might survive on Mars or other planets. They might also be capable of surviving travel between planets--for instance, encased in salt crystals and thus protected from damaging radiation. When researchers working with the European Space Agency launched the halophile Haloarcula strain G, the organism survived for several weeks in deep space without protection, longer than any other vegetative cells. This finding is consistent with the observed capacity of Halobacterium sp. NRC-1 to withstand desiccation and radiation in laboratory studies. Moreover, several Martian meteorites, including the Shergotty and Nakhla meteorites, contain halite salt crystals, which could also be a vehicle for the interplanetary transport of halophiles. Finding entrapped halophiles within brine inclusions in ancient halite deposits on Earth thus is a subject of considerable interest.
Extreme halophiles are remarkably resilient microorganisms with hardy molecularcharacteristics, including an abundance of extremely acidic proteins that function in the presence of 5 M salt. The discovery of both archaeal and bacterial halophiles with similar strategies for survival, and anaerobic halophiles in subsurface environments such as submarine brine pools provides further evidence of the remarkable physiological capabilities among this family of microorganisms, whose study continues toexpand our understanding of the breadth of microbiology on Earth.
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