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Extremophiles are organisms that can grow and thrive in harsh conditions, e.g., extremes of temperature, pH, salinity, radiation, pressure and oxygen tension. Thermophilic, halophilic and radiation-resistant organisms are all microbes, some of which are able to withstand multiple extremes. Psychrophiles, or cold-loving organisms, include not only microbes, but fish that live in polar waters and animals that can withstand freezing. Extremophiles are structurally adapted at a molecular level to withstand these conditions. Thermophiles have particularly stable proteins and cell membranes, psychrophiles have flexible cellular proteins and membranes and/or antifreeze proteins, salt-resistant halophiles contain compatible solutes or high concentrations of inorganic ions, and acidophiles and alkaliphiles are able to pump ions to keep their internal pH close to neutrality. Their interest to veterinary medicine resides in their capacity to be pathogenic, and as sources of enzymes and other molecules for diagnostic and pharmaceutical purposes. In particular, thermostable DNA polymerases are a mainstay of PCR-based diagnostics.

Extremophiles are micro-organisms adapted to survive in ecological niches defined as ‘extreme’ for humans and characterized by the presence of adverse environmental conditions, such as high or low temperatures, extreme values of pH, high salt concentrations or high pressure. Biomolecules isolated from extremophiles possess extraordinary properties and, in particular, proteins isolated from extremophiles represent unique biomolecules that function under severe conditions, comparable to those prevailing in various industrial processes.

In this article, we will review some examples of recent applications of thermophilic proteins for the development of a new class of fluorescence non-consuming substrate biosensors for monitoring the levels of two analytes of high social interest, such as glucose and sodium.

Background

Among extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity.

Methods and Findings

A lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C β-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80°C and the pH optimum determined at 25°C was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested.

Conclusions

In this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs.

The proteome of the radiation- and desiccation-resistant bacterium D. radiodurans features a group of proteins that contain significant intrinsically disordered regions that are not present in non-extremophile homologues. Interestingly, this group includes a number of housekeeping and repair proteins such as DNA polymerase III, nudix hydrolase and rotamase. Here, we focus on a member of the nudix hydrolase family from D. radiodurans possessing low-complexity N- and C-terminal tails, which exhibit sequence signatures of intrinsic disorder and have unknown function. The enzyme catalyzes the hydrolysis of oxidatively damaged and mutagenic nucleotides, and it is thought to play an important role in D. radiodurans during the recovery phase after exposure to ionizing radiation or desiccation. We use molecular dynamics simulations to study the dynamics of the protein, and study its hydration free energy using the GB/SA formalism. We show that the presence of disordered tails significantly decreases the hydration free energy of the whole protein. We hypothesize that the tails increase the chances of the protein to be located in the remaining water patches in the desiccated cell, where it is protected from the desiccation effects and can function normally. We extrapolate this to other intrinsically disordered regions in proteins, and propose a novel function for them: intrinsically disordered regions increase the “surface-properties” of the folded domains they are attached to, making them on the whole more hydrophilic and potentially influencing, in this way, their localization and cellular activity.

Author Summary

Intrinsically disordered proteins and protein segments carry out a wide range of important biological functions despite their lack of permanent tertiary structure. Using advanced computational methods we study the biophysical properties of the intrinsically disordered regions in the enzyme nudix hydrolase from the desiccation- and radiation-resistant bacterium D. radiodurans. Interestingly, these regions are absent in homologue proteins in non-extremophile bacteria, suggesting that they might be involved in helping the key rescue-and-repair proteins in D. radiodurans, such as nudix hydrolase, adapt to the extreme absence of water. We show that the disordered regions in nudix hydrolase enlarge the overall surface of the enzyme, and most importantly, increase its overall affinity for water (i.e. its hydrophilicity). We suggest a novel hypothesis that this, indeed, may be the principal function of disordered regions in some cases: they increase the chances of the protein to be located in the remaining water patches in the desiccated cell, where it is protected from the desiccation effects and can function normally.

OLE (Ornate, Large, Extremophilic) RNAs represent a recently discovered non-coding RNA class found in extremophilic anaerobic bacteria, including certain human pathogens. OLE RNAs exhibit several unusual characteristics that indicate a potentially novel function, including exceptionally high expression and localization to cell membranes via interaction with a protein partner called OLE-associated protein (OAP). In the current study, new genetic and phenotypic characteristics of OLE RNA from Bacillus halodurans C-125 were established. OLE RNA is transcribed at high levels from its own promoter under normal growth conditions and the transcript is exceptionally stable compared to most other RNAs. Expression is increased by ∼7-fold when cells are exposed to near lethal concentrations of short-chain alcohols such as ethanol or methanol. Strains wherein the genes for OLE and/or OAP are deleted are more susceptible to growth inhibition by alcohol and also become more sensitive to cold. Normal growth characteristics can be restored by expressing the genes for OLE and OAP from plasmids or from elsewhere on the chromosome. Our findings confirm a functional link between OLE and OAP and reveal the importance of a large non-coding RNA in the response to alcohol-induced stress.

Environments that are hostile to life are characterized by reduced microbial activity which results in poor soil- and plant-health, low biomass and biodiversity, and feeble ecosystem development. Whereas the functional biosphere may primarily be constrained by water activity (aw) the mechanism(s) by which this occurs have not been fully elucidated. Remarkably we found that, for diverse species of xerophilic fungi at aw values of ≤ 0.72, water activity per se did not limit cellular function. We provide evidence that chaotropic activity determined their biotic window, and obtained mycelial growth at water activities as low as 0.647 (below that recorded for any microbial species) by addition of compounds that reduced the net chaotropicity. Unexpectedly we found that some fungi grew optimally under chaotropic conditions, providing evidence for a previously uncharacterized class of extremophilic microbes. Further studies to elucidate the way in which solute activities interact to determine the limits of life may lead to enhanced biotechnological processes, and increased productivity of agricultural and natural ecosystems in arid and semiarid regions.

Reproductive isolation among locally adapted populations may arise when immigrants from foreign habitats are selected against via natural or (inter-)sexual selection (female mate choice). We asked whether also intrasexual selection through male-male competition could promote reproductive isolation among populations of poeciliid fishes that are locally adapted to extreme environmental conditions [i.e., darkness in caves and/or toxic hydrogen sulphide (H2S)]. We found strongly reduced aggressiveness in extremophile P. oecilia mexicana, and darkness was the best predictor for the evolutionary reduction of aggressiveness, especially when combined with presence of H2S. We demonstrate that reduced aggression directly translates into migrant males being inferior when paired with males from non-sulphidic surface habitats. By contrast, the phylogenetically old sulphur endemic P. sulphuraria from another sulphide spring area showed no overall reduced aggressiveness, possibly indicating evolved mechanisms to better cope with H2S.

Background

Halophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored.

Methodology/Principal Findings

A halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data.

Conclusions/Significance

The solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that all the solutions studied were structurally inhomogeneous, it is important for future work to understand how the LipC's solution aggregation affected its activity.

We studied the immobilization of a recombinant thermostable lipase (Pf2001Δ60) from the hyperthermophilic archaeon Pyrococcus furiosus on supports with different degrees of hydrophobicity: butyl Sepabeads and octadecyl Sepabeads. The enzyme was strongly adsorbed in both supports. When it was adsorbed on these supports, the enzyme showed 140 and 237% hyperactivation, respectively. The assessment of storage stability showed that the octadecyl Sepabeads immobilized enzyme showed 100% of residual activity after 30 days of storage. However, the greatest stability at 70°C was obtained in butyl Sepabeads immobilized enzyme, which retained 77% activity after 1 hour incubation. The maximum activity of the immobilized preparations was obtained with the pH between 6 and 7, at 70°C. Thus, this study achieved a new extremophilic biocatalyst with greater stability, for use in several biotechnological processes.

The biosynthesis of Ag and Au nanoparticles (NPs) was investigated using an extremophilic yeast strain isolated from acid mine drainage in Portugal. Three distinct studies were performed, namely, the growth of yeast strain in presence of metal ions, the use of yeast biomass for the metal nanoparticles synthesis, and of the supernatant obtained after 24-hour incubation of yeast biomass in water. The extremophilic strain under study was able to grow up to an Ag ion concentration of 1.5 mM whereas an increase of Au ion concentration over 0.09 mM caused a strong inhibitory effect. A successful route for the metal NPs synthesis was obtained using the yeast biomass. When the washed yeast cells were in contact with Ag or Au solutions, AgNPs smaller than 20 nm were produced, as for the AuNPs diameter ranged from 30 to 100 nm, as determined through transmission electron microscopy and confirmed by energy-dispersive X-ray spectra. The supernatant-based strategy provided evidence that proteins were released to the medium by the yeasts, which could be responsible for the formation and stabilisation of the Ag NPs, although the involvement of the cell wall seems fundamental for AuNPs synthesis.

Natronomonas pharaonis is an archaeon adapted to two extreme conditions: high salt concentration and alkaline pH. It has become one of the model organisms for the study of extremophilic life. Here, we present a genome-scale, manually curated metabolic reconstruction for the microorganism. The reconstruction itself represents a knowledge base of the haloalkaliphile's metabolism and, as such, would greatly assist further investigations on archaeal pathways. In addition, we experimentally determined several parameters relevant to growth, including a characterization of the biomass composition and a quantification of carbon and oxygen consumption. Using the metabolic reconstruction and the experimental data, we formulated a constraints-based model which we used to analyze the behavior of the archaeon when grown on a single carbon source. Results of the analysis include the finding that Natronomonas pharaonis, when grown aerobically on acetate, uses a carbon to oxygen consumption ratio that is theoretically near-optimal with respect to growth and energy production. This supports the hypothesis that, under simple conditions, the microorganism optimizes its metabolism with respect to the two objectives. We also found that the archaeon has a very low carbon efficiency of only about 35%. This inefficiency is probably due to a very low P/O ratio as well as to the other difficulties posed by its extreme environment.

Author Summary

Extremophiles are organisms that thrive in physically or geochemically extreme conditions that are detrimental, even lethal, to the majority of life on Earth. Natronomonas pharaonis is one that has been able to adapt to both high salt concentration and an alkaline pH. In this study, we investigate the chemical reactions that occur within the microorganism, collectively referred to as its metabolic network, that allow it to convert the nutrients in its environment to biomass and energy. Specifically, we reconstructed the network by collecting evidence for the existence of reactions from the literature, and then supplemented them with computational approaches, for example by searching the genome of Natronomonas pharaonis for genes that could potentially encode analogs of known enzymes from other organisms. Finally, with the network in hand, we developed a computational model which we used to simulate growth. Among other results, we found indications that Natronomonas pharaonis regulates its metabolism such that energy production and growth are maximized. Despite this however, we also found that Natronomonas pharaonis is only able to incorporate a very small fraction of the total carbon that it consumes (approximately 35%), likely in no small part due to the difficulties posed by its environment.

Graphical abstract

Highlights

► Detailed molecular evolution of metalloenzymes that catalyse the dismutation of hydrogen peroxide. ► Three protein families of differing structure, catalytic mechanism, distribution and evolutionary age. ► Catalatic enzymes in pathogenic organisms are promising targets for drug design. ► Occurrence of biotechnological interesting representatives in extremophiles.

For efficient removal of intra- and/or extracellular hydrogen peroxide by dismutation to harmless dioxygen and water (2H2O2 → O2 + 2H2O), nature designed three metalloenzyme families that differ in oligomeric organization, monomer architecture as well as active site geometry and catalytic residues. Here we report on the updated reconstruction of the molecular phylogeny of these three gene families. Ubiquitous typical (monofunctional) heme catalases are found in all domains of life showing a high structural conservation. Their evolution was directed from large subunit towards small subunit proteins and further to fused proteins where the catalase fold was retained but lost its original functionality. Bifunctional catalase–peroxidases were at the origin of one of the two main heme peroxidase superfamilies (i.e. peroxidase–catalase superfamily) and constitute a protein family predominantly present among eubacteria and archaea, but two evolutionary branches are also found in the eukaryotic world. Non-heme manganese catalases are a relatively small protein family with very old roots only present among bacteria and archaea. Phylogenetic analyses of the three protein families reveal features typical (i) for the evolution of whole genomes as well as (ii) for specific evolutionary events including horizontal gene transfer, paralog formation and gene fusion. As catalases have reached a striking diversity among prokaryotic and eukaryotic pathogens, understanding their phylogenetic and molecular relationship and function will contribute to drug design for prevention of diseases of humans, animals and plants.

Background

The compatible solute trehalose is a non-reducing disaccharide, which accumulates upon heat, cold or osmotic stress. It was commonly accepted that trehalose is only present in extremophiles or cryptobiotic organisms. However, in recent years it has been shown that although higher plants do not accumulate trehalose at significant levels they have actively transcribed genes encoding the corresponding biosynthetic enzymes.

Results

In this study we show that trehalose biosynthesis ability is present in eubacteria, archaea, plants, fungi and animals. In bacteria there are five different biosynthetic routes, whereas in fungi, plants and animals there is only one. We present phylogenetic analyses of the trehalose-6-phosphate synthase (TPS) and trehalose-phosphatase (TPP) domains and show that there is a close evolutionary relationship between these domains in proteins from diverse organisms. In bacteria TPS and TPP genes are clustered, whereas in eukaryotes these domains are fused in a single protein.

Conclusion

We have demonstrated that trehalose biosynthesis pathways are widely distributed in nature. Interestingly, several eubacterial species have multiple pathways, while eukaryotes have only the TPS/TPP pathway. Vertebrates lack trehalose biosynthetic capacity but can catabolise it. TPS and TPP domains have evolved mainly in parallel and it is likely that they have experienced several instances of gene duplication and lateral gene transfer.

In particular niches of the marine environment, such as abyssal trenches, icy waters and hot vents, the base of the food web is composed of bacteria and archaea that have developed strategies to survive and thrive under the most extreme conditions. Some of these organisms are considered “extremophiles” and modulate the fatty acid composition of their phospholipids to maintain the adequate fluidity of the cellular membrane under cold/hot temperatures, elevated pressure, high/low salinity and pH. Bacterial cells are even able to produce polyunsaturated fatty acids, contrarily to what was considered until the 1990s, helping the regulation of the membrane fluidity triggered by temperature and pressure and providing protection from oxidative stress. In marine ecosystems, bacteria may either act as a sink of carbon, contribute to nutrient recycling to photo-autotrophs or bacterial organic matter may be transferred to other trophic links in aquatic food webs. The present work aims to provide a comprehensive review on lipid production in bacteria and archaea and to discuss how their lipids, of both heterotrophic and chemoautotrophic origin, contribute to marine food webs.

Coccomyxa acidophila is an extremophile eukaryotic microalga isolated from the Tinto River mining area in Huelva, Spain. Coccomyxa acidophila accumulates relevant amounts of β-carotene and lutein, well-known carotenoids with many biotechnological applications, especially in food and health-related industries. The acidic culture medium (pH < 2.5) that prevents outdoor cultivation from non-desired microorganism growth is one of the main advantages of acidophile microalgae production. Conversely, acidophile microalgae growth rates are usually very low compared to common microalgae growth rates. In this work, we show that mixotrophic cultivation on urea efficiently enhances growth and productivity of an acidophile microalga up to typical values for common microalgae, therefore approaching acidophile algal production towards suitable conditions for feasible outdoor production. Algal productivity and potential for carotenoid accumulation were analyzed as a function of the nitrogen source supplied. Several nitrogen conditions were assayed: nitrogen starvation, nitrate and/or nitrite, ammonia and urea. Among them, urea clearly led to the best cell growth (~4 × 108 cells/mL at the end of log phase). Ammonium led to the maximum chlorophyll and carotenoid content per volume unit (220 μg·mL·1 and 35 μg·mL·1, respectively). Interestingly, no significant differences in growth rates were found in cultures grown on urea as C and N source, with respect to those cultures grown on nitrate and CO2 as nitrogen and carbon sources (control cultures). Lutein accumulated up to 3.55 mg·g·1 in the mixotrophic cultures grown on urea. In addition, algal growth in a shaded culture revealed the first evidence for an active xanthophylls cycle operative in acidophile microalgae.

Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.

Background

Thellungiella has been proposed as an extremophile alternative to Arabidopsis to investigate environmental stress tolerance. However, Arabidopsis accessions show large natural variation in their freezing tolerance and here the tolerance ranges of collections of accessions in the two species were compared.

Results

Leaf freezing tolerance of 16 Thellungiella accessions was assessed with an electrolyte leakage assay before and after 14 days of cold acclimation at 4°C. Soluble sugars (glucose, fructose, sucrose, raffinose) and free polyamines (putrescine, spermidine, spermine) were quantified by HPLC, proline photometrically. The ranges in nonacclimated freezing tolerance completely overlapped between Arabidopsis and Thellungiella. After cold acclimation, some Thellungiella accessions were more freezing tolerant than any Arabidopsis accessions. Acclimated freezing tolerance was correlated with sucrose levels in both species, but raffinose accumulation was lower in Thellungiella and only correlated with freezing tolerance in Arabidopsis. The reverse was true for leaf proline contents. Polyamine levels were generally similar between the species. Only spermine content was higher in nonacclimated Thellungiella plants, but decreased during acclimation and was negatively correlated with freezing tolerance.

Conclusion

Thellungiella is not an extremophile with regard to freezing tolerance, but some accessions significantly expand the range present in Arabidopsis. The metabolite data indicate different metabolic adaptation strategies between the species.

The crystal structure of adenylate kinase from the psychrophile M. marinus has been determined at 2.0 Å resolution and the kinetic parameters of this cold-adapted enzyme have been examined.

Adenylate kinases (AKs; EC 2.7.4.3) are essential members of the NMP kinase family that maintain cellular homeostasis by the interconversion of AMP, ADP and ATP. AKs play a critical role in adenylate homeostasis across all domains of life and have been used extensively as prototypes for the study of protein adaptation and the relationship of protein dynamics and stability to function. To date, kinetic studies of psychrophilic AKs have not been performed. In order to broaden understanding of extremophilic adaptation, the kinetic parameters of adenylate kinase from the psychrophile Marinibacillus marinus were examined and the crystal structure of this cold-adapted enzyme was determined at 2.0 Å resolution. As expected, the overall structure and topology of the psychrophilic M. marinus AK are similar to those of mesophilic and thermophilic AKs. The thermal denaturation midpoint of M. marinus AK (321.1 K) is much closer to that of the mesophile Bacillus subtilis (320.7 K) than the more closely related psychrophile B. globisporus (316.4 K). In addition, the enzymatic properties of M. marinus AK are quite close to those of the mesophilic AK and suggests that M. marinus experiences temperature ranges in which excellent enzyme function over a broad temperature range (293–313 K) has been retained for the success of the organism. Even transient loss of AK function is lethal and as a consequence AK must be robust and be well adapted to the environment of the host organism.

Background

The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass.

Results

Here we show that a cellulase gene (sso1354) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade α-cellulose or even complex cell wall preparations under those pretreatment conditions.

Conclusion

The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.

Extreme environments, generally characterized by atypical temperatures, pH, pressure, salinity, toxicity, and radiation levels, are inhabited by various microorganisms specifically adapted to these particular conditions, called extremophiles. Among these, the microorganisms belonging to the Archaea domain are of significant biotechnological importance as their biopolymers possess unique properties that offer insights into their biology and evolution. Particular attention has been devoted to two main types of biopolymers produced by such peculiar microorganisms, that is, the extracellular polysaccharides (EPSs), considered as a protection against desiccation and predation, and the endocellular polyhydroxyalkanoates (PHAs) that provide an internal reserve of carbon and energy. Here, we report the composition, biosynthesis, and production of EPSs and PHAs by different archaeal species.

Background

Generally, extremophiles have been deemed to survive in the extreme environments to which they had adapted to grow. Recently many extremophiles have been isolated from places where they are not expected to grow. Alkaliphilic microorganisms have been isolated from acidic soil samples with pH 4.0, and thermophiles have been isolated from samples of low temperature. Numerous moderately halophilic microorganisms, defined as those that grow optimally in media containing 0.5–2.5 Molar (3–15%) NaCl, and halotolerant microorganisms that are able to grow in media without added NaCl and in the presence of high NaCl have been isolated from saline environments such as salterns, salt lakes and sea sands. It has tacitly been believed that habitats of halophiles able to grow in media containing more than 20% (3.4 M) are restricted to saline environments, and no reports have been published on the isolation of halophiles from ordinary garden soil samples.

Results

We demonstrated that many halophilic bacteria that are able to grow in the presence of 20% NaCl are inhabiting in non-saline environments such as ordinary garden soils, yards, fields and roadways in an area surrounding Tokyo, Japan. Analyses of partial 16S rRNA gene sequences of 176 isolates suggested that they were halophiles belonging to genera of the family Bacillaceae, Bacillus (11 isolates), Filobacillus (19 isolates), Gracilibacillus (6 isolates), Halobacillus (102 isolates), Lentibacillus (1 isolate), Paraliobacillus (5 isolates) and Virgibacillus (17 isolates). Sequences of 15 isolates showed similarities less than 92%, suggesting that they may represent novel taxa within the family Bacillaceae.

Conclusion

The numbers of total bacteria of inland soil samples were in a range from 1.4 × 107/g to 1.1 × 106/g. One tenth of the total bacteria was occupied by endospore-forming bacteria. Only very few of the endospore-forming bacteria, roughly 1 out of 20,000, are halophilic bacteria. Most of the halophilic bacteria were surviving as endospores in the soil samples, in a range of less than 1 to about 500/g soil. Samples collected from seashore in a city confronting Tokyo Bay gave the total numbers of bacteria and endospores roughly 1000 time smaller than those of inland soil samples. Numbers of halophilic bacteria per gram, however, were almost the same as those of inland soil samples. A possible source of the halophilic endospore originating from Asian dust storms is discussed.

Bacterial microorganisms that grow optimally at Na+ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review focuses on the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance of extremely halophilic eubacteria; the focus is on those with alkaline pH optima, above 8.5, and elevated temperature optima, above 50°C. If all three conditions are required for optimal growth, these microorganisms are termed "poly-extremophiles". However, only a very few extreme halophiles able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated so far. Therefore the question is: do the combined extreme growth conditions of the recently isolated poly-extremophiles, i.e., anaerobic halophilic alkalithermophiles, approach a physico-chemical boundary for life? These poly-extremophiles are of interest, as their adaptive mechanisms give insight into organisms' abilities to survive in environments which were previously considered prohibitive to life, as well as to possible properties of early evolutionary and extraterrestrial life forms.

Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi – Coniosporium perforans, Exophiala jeanselmei – and of the extremophilic fungus – Friedmanniomyces endolithicus – were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum.

Highlights

► The paper is the first investigation of protein patterns in rock inhabiting fungi. ► We show the first results of temperature response in black fungi. ► Protein patterns of black fungi from different ecological niches are compared. ► Adaptation to similar conditions led to a convergent response in black fungi. ► Ecological strategies of MCF are different from Penicillium chrysogenum ones.

Natranaerobius thermophilus is an unusual extremophile because it is halophilic, alkaliphilic and thermophilic, growing optimally at 3.5 M Na+, pH55°C 9.5 and 53°C. Mechanisms enabling this tripartite lifestyle are essential for understanding how microorganisms grow under inhospitable conditions, but remain unknown, particularly in extremophiles growing under multiple extremes. We report on the response of N. thermophilus to external pH at high salt and elevated temperature and identify mechanisms responsible for this adaptation. N. thermophilus exhibited cytoplasm acidification, maintaining an unanticipated transmembrane pH gradient of 1 unit over the entire extracellular pH range for growth. N. thermophilus uses two distinct mechanisms for cytoplasm acidification. At extracellular pH values at and below the optimum, N. thermophilus utilizes at least eight electrogenic Na+(K+)/H+ antiporters for cytoplasm acidification. Characterization of these antiporters in antiporter-deficient Escherichia coli KNabc showed overlapping pH profiles (pH 7.8–10.0) and Na+ concentrations for activity (K0.5 values 1.0–4.4 mM), properties that correlate with intracellular conditions of N. thermophilus. As the extracellular pH increases beyond the optimum, electrogenic antiport activity ceases, and cytoplasm acidification is achieved by energy-independent physiochemical effects (cytoplasmic buffering) potentially mediated by an acidic proteome. The combination of these strategies allows N. thermophilus to grow over a range of extracellular pH and Na+ concentrations and protect biomolecules under multiple extreme conditions.

Extremophilic archaea were stained with the LIVE/DEAD BacLight kit under conditions of high ionic strength and over a pH range of 2.0 to 9.3. The reliability of the kit was tested with haloarchaea following permeabilization of the cells. Microorganisms in hypersaline environmental samples were detectable with the kit, which suggests its potential application to future extraterrestrial halites.