Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Blackwell Science, 20024Review ArticleH. Santos and M. S. da CostaCompatible solutes of (hyper)thermophiles Environmental Microbiology (2002) 4(9), 501–509 Minireview Compatible solutes of organisms that live in hot saline environments Helena Santos1 and Milton S. da Costa2 organisms belong to the Bacteria and to the Archaea, 1Instituto de Tecnologia Química e Biológica, although the latter domain includes a larger variety of Universidade Nova de Lisboa, Apartado 127, 2780–156 organisms living at higher temperatures than the former. Oeiras, Portugal. The term thermophile is often used to define organisms 2Departamento de Bioquímica, Universidade de Coimbra, that have optimum growth temperatures between 65°C 3001–401 Coimbra, Portugal. and 80°C, while hyperthermophilic organisms are those with optimum growth temperatures above 80°C. (Blöchl et al., 1995). Summary Most thermophilic organisms have been isolated from The accumulation of organic solutes is a prerequisite continental geothermal or artificial thermal environments, for osmotic adjustment of all microorganisms. Ther- but some thermophiles have been isolated from marine mophilic and hyperthermophilic organisms generally hydrothermal environments, the best known of which accumulate very unusual compatible solutes namely, are Rhodothermus marinus and Thermus thermophilus di-myo-inositol-phosphate, di-mannosyl-di-myo- (Alfredsson et al., 1988; da Costa et al., 2001). The water inositol-phosphate, di-glycerol-phosphate, manno- venting from continental hot springs is generally low in sylglycerate and mannosylglyceramide, which have sodium and the isolates, being fresh water organisms, not been identified in bacteria or archaea that grow at rarely grow in media containing more than 1.0% NaCl low and moderate temperatures. There is also a grow- (w/v). Some organisms isolated from continental fresh ing awareness that some of these compatible solutes water hot springs are, however, halotolerant with optimum may have a role in the protection of cell components growth in media without added NaCl but, like the strains against thermal denaturation. Mannosylglycerate and of Thermus thermophilus, are able to grow in media con- di-glycerol-phosphate have been shown to protect taining 4.0–6.0% NaCl (da Costa et al., 2001). enzymes and proteins from thermal denaturation in On the other hand, hyperthermophilic organisms, with vitro as well, or better, than compatible solutes from a few notable exceptions such as the species of the Order mesophiles. The pathways leading to the synthesis of Sulfolobales, Pyrobaculum islandicum and Thermococ- some of these compatible solutes from thermophiles cus zilligii, originate from shallow or abyssal marine geo- and hyperthermophiles have been elucidated. How- thermal areas (Blöchl et al., 1995). The water in shallow ever, large numbers of questions remain unanswered. marine or abyssal environments is generally saline but, Fundamental and applied interest in compatible varies from low salinity to that of seawater. The organisms solutes and osmotic adjustment in these organisms, that originate from these environments are, as would be drives research that, will, in the near future, allow us expected, generally slightly halophilic, requiring sodium to understand the role of compatible solutes in for growth. Optimum growth occurs in media containing osmotic protection and thermoprotection of some of 0.5–2.0% NaCl. Few organisms, if any, grow at salinities the most fascinating organisms known on Earth. higher than 6.0–8.0% NaCl. Moderate halophiles are defined as organisms having higher growth rates in media containing between 0.5 M Slightly halophilic thermophiles and 2.5 M NaCl, while extreme halophiles have higher and hyperthermophiles growth rates in media containing over 2.5 M NaCl Thermophiles and hyperthermophiles have been isolated (Ventosa et al., 1998). Moderate or extremely halophilic from a large variety of thermal environments. These organisms simultaneously capable of growth at very high temperatures have not yet been identified. The reason for our inability to isolate such organisms possibly stems from Received 11 March, 2002; accepted 23 July, 2002. *For correspon- dence. E-mail [email protected]; Tel. +351 239 824024; Fax +351 239 the fact that appropriate environments may be very rare 826798. or may not even exist on Earth. There are continental © 2002 Blackwell Science Ltd 502 H. Santos and M. S. da Costa springs with salinities higher than seawater but, the water isms, ranging from archaea, bacteria, yeast, filamentous temperature is usually fairly low. Deep, hot brines in the fungi and algae, rely exclusively on the accumulation of Red Sea reach temperatures of about 60°C, although compatible solutes for osmoadaptation, indicating that this higher temperatures have been estimated but not actually strategy is very successful. Compatible solutes can also measured (Hartmann et al., 1998). The latter environ- be taken up from the environment if present or, they can ments could, in the future, be the source of truly halophilic be synthesized de novo. The most common compatible hyperthermophiles. For the time being the most thermo- solutes of microorganisms are neutral or zwitterionic and, philic and halophilic organisms known include Halother- include amino acids and amino acid derivatives, sugars, mothrix orenii isolated from sediments of a salt lake, with sugar derivatives (heterosides) and polyols, betaines and an optimum growth temperature of 60°C and a salt range the ectoines (da Costa et al., 1998). Some are widespread for growth of 4–20% (Cayol et al., 1994), and Thermoha- in microorganisms, namely trehalose, glycine betaine and lobacter berrensis isolated from solar salterns, with an α-glutamate, while others are restricted to a few organ- optimum growth temperature of 65°C and a salt range isms. Polyols, for example, are widespread among fungi between 2 and 15% (Cayol et al., 2000). Scientists inter- and algae but are very rare in bacteria and unknown in ested in osmotic adjustment in (hyper)thermophiles are, archaea. Ectoine and hydroxyectoine are examples of for now, only able to examine halotolerant and slightly compatible solutes found only in bacteria. halophilic organisms. Many slightly and moderately halophilic methanogens possess a mixed type of osmoadaptation where K+ accu- mulates to high levels along with neutral and negatively Roles of compatible solutes charged organic solutes (Martin et al., 1999). Hyperthermophiles are, like all organisms living in aque- A. D. Brown originally defined compatible solutes as ous environments, faced with alterations in the water small organic compounds used for osmotic adjustment activity due to fluctuations in the levels of dissolved salts that do not interfere with cell function (Brown, 1976). It is or sugars. To adjust to lower water activities of the envi- also implicit by this definition that compatible solutes ronment and the resulting decrease in cytoplasmic water, protect proteins and other cell components from osmotic- microorganisms must accumulate intracellular ions or induced dehydration. However, the role of compatible organic solutes to re-establish the cell turgor pressure solutes goes beyond osmotic adjustment alone, to the and/or cell volume and, at the same time, preserve protection of cells and cell components from freezing, enzyme activity (Brown, 1990). desiccation, high temperature and oxygen radicals Microorganisms have developed two main strategies for (da Costa et al., 1998; Argüelles, 2000; Welsh, 2000; osmotic adjustment. One strategy relies on the selective Benaroudj et al., 2001; Santos and da Costa, 2001). In influx of K+ from the environment to, sometimes extremely, view of these properties, many compatible solutes can, in high levels and is known as the ‘salt-in-the-cytoplasm’ type fact, be regarded as stress protectants. The protective role of osmotic adaptation (Galinski, 1995; da Costa et al., of trehalose against several stress conditions has been 1998; Roeßler and Müller, 2001). This type of osmotic amply demonstrated (Singer and Lindquist, 1998; Simola adjustment occurs in the extremely halophilic archaea of et al., 2000), and the accumulation of glycerol, the the family Halobacteriaceae, the anaerobic moderately canonical osmolyte of yeast, has also been correlated halophilic bacteria of the Order Halanaerobiales (Oren, with the acquisition of thermotolerance (Siderius et al., 1999) and the extremely halophilic bacterium Salinibacter 2000). Some hyperthermophiles accumulate very high ruber (Anton et al., 2002; Oren and Mana, 2002). The levels of di-myo-inositol-1,1′-phosphate (DIP) primarily presence of the saline type of osmotic adaptation in three during growth at supraoptimum temperatures, which may distinct lineages of organisms implies independent devel- have a role in thermoprotection of these organisms opment of this strategy, it being difficult to envision as an (Martins and Santos, 1995; Martins et al., 1997; L. ancient characteristic retained in a few scattered groups Gonçalves, R. Huber, M. S. da Costa and H. Santos, of organisms or a characteristic occurring through mas- unpublished results). sive lateral gene transfer. The majority of microorganisms have not, however, Compatible solutes of thermophiles undergone extensive genetic alterations as a prerequisite and hyperthermophiles for adaptation to a saline environment and, the intracellu- lar macromolecules are generally