Strategies of adaptation of microorganisms of the three domains of life to high salt concentrations

Strategies of adaptation of microorganisms of the three domains of life to high salt concentrations Abstract Hypersaline environments with salt concentrations up to NaCl saturation are inhabited by a great diversity of microorganisms belonging to the three domains of life. They all must cope with the low water activity of their environment, but different strategies exist to provide osmotic balance of the cells’ cytoplasm with the salinity of the medium. One option used by many halophilic Archaea and a few representatives of the Bacteria is to accumulate salts, mainly KCl and to adapt the entire intracellular machinery to function in the presence of molar concentrations of salts. A more widespread option is the synthesis or accumulation of organic osmotic, so-called compatible solutes. Here, we review the mechanisms of osmotic adaptation in a number of model organisms, including the KCl accumulating Halobacterium salinarum (Archaea) and Salinibacter ruber (Bacteria), Halomonas elongata as a representative of the Bacteria that synthesize organic osmotic solutes, eukaryotic microorganisms including the unicellular green alga Dunaliella salina and the black yeasts Hortaea werneckii and the basidiomycetous Wallemia ichthyophaga, which use glycerol and other compatible solutes. The strategies used by these model organisms and by additional halophilic microorganisms presented are then compared to obtain an integrative picture of the adaptations to life at high salt concentrations in the microbial world. compatible solutes, ion metabolism, halophilic, halotolerant, diversity, hypersaline ecosystems OSMOTIC ADAPTATION IN MICROORGANISMS: BASIC PRINCIPLES Microbial life at high salt concentrations is phylogenetically very diverse. Hypersaline environments with salt concentrations up to NaCl saturation are inhabited by halophilic and highly halotolerant representatives of all three domains of life: Archaea, Bacteria and Eukarya. The mechanisms used by these salt-requiring or highly salt-tolerant microorganisms to withstand the high salt concentrations, and in many cases also to adapt their physiology to changes in the salt concentrations in their environments, are diverse as well. Still, a number of general principles apply (see e.g. Brown 1976, 1990), which are as follows: Biological membranes are permeable to water. Therefore, water moves into and out of cells driven by differences in water activity between the cytoplasm and the outside medium. Active inward pumping of water lost to the medium when cells are suspended in a high-salt medium is not feasible: no such mechanisms are known, and the process would be energetically far too expensive. Therefore, the intracellular environment must be at least isosmotic with the outside medium. With the possible exception of the halophilic Archaea of the class Halobacteria (Walsby 1971), all microorganisms possess a positive turgor. Maintenance of this outward-directed pressure is essential as the driving force for cell expansion (Kempf and Bremer 1998). This means that the osmotic pressure of the cytoplasm must even exceed that of the outside medium. To maintain a high osmotic pressure inside the cells, different strategies can be used, which are as follows: The ‘salt-in’ strategy where osmotic balance is achieved by accumulating high concentrations of inorganic salts in the medium. As Na+ ions are excluded as much as possible from cells in all three domains of life, the ‘salt-in’ strategy is based on KCl rather than on NaCl as the main intracellular salt. The ‘low-salt-in’, ‘compatible solute’ strategy. The term compatible solute was first used in 1972 and was defined as a solute which, at high concentration, allows an enzyme to function effectively (Brown and Simpson 1972). This definition was later extended to allow all essential cell processes to function effectively (Brown 1990). Many organic compounds belonging to different classes have been shown to serve as compatible solutes in different groups of microorganisms, including polyols, sugars, amino acids, betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine (Galinski 1993, 1995; da Costa, Santos and Galinski 1998; Kempf and Bremer 1998; Martin, Ciulla and Roberts 1999; Roberts 2005). Compatible solutes are strong water structure formers and are probably excluded from the hydration shell of proteins, thus stabilizing the hydration shell (Galinski 1995), and they decrease water activity coefficients (Held, Neuhaus and Sadowski 2010). In many extremophiles, such low molecular weight compounds are accumulated not only in response to increased salt concentrations, but also as a response to other environmental changes such as temperature stress. Examples of organic compatible solutes in thermophiles and in psychrophiles are di-myoinositol-1,1΄-phosphate, cyclic 2,3-diphosphoglycerate, α-diglycerol phosphate, mannosylglycerate and mannosylglyceramide (Lentzen and Schwarz 2006; Cowan 2009; Casanueva et al.2010; Klähn and Hagemann 2011). Biosynthesis of organic osmotic solutes is energetically more expensive than the ‘salt-in’ strategy (Oren 1999, 2011b), and therefore microorganisms that use the ‘low-salt-in’, ‘compatible solute’ strategy (and sometimes even those that accumulate salts) will also accumulate suitable solutes if available in their medium (Kempf and Bremer 1998; Pflüger and Müller 2004). Being adapted to life at high salt concentrations is not sufficient when the properties of the environment vary; adaptability to fluctuations in salinity is also necessary. Microorganisms that use organic solutes are generally more flexible and can easier adjust to dilution stress or to sudden increases in salinity than organisms that use the ‘salt-in’ strategy. Upon salt downshock, excess of compatible solutes can be excreted via mechanosensitive channels or converted inside the cell to osmotically inactive forms. Inorganic ions can be transiently accumulated following sudden increases in salinity, to be later replaced by newly synthesized organic solutes (Wood et al.2001). This review article first presents a number of case studies, exemplifying the ways used by different groups of Bacteria, Archaea and Eukarya to adapt to life at high salt concentrations. Then follows a comparative section in which we attempt to integrate all the information to discover the general principles behind the functioning of halophiles, both alone and in the complex ecosystems in which they live. Of course, it is not possible to review here the entire literature about adaptation of microorganisms to life at high salt. Further valuable information can be found in older reviews (Kushner 1978; Csonka 1989; Roeßler and Müller 2001; Oren 2002, 2008, 2011a, 2013a; Grant 2004; Kunte 2006; Gunde-Cimerman, Ramos and Plemenitaš 2009; Gostinčar et al.2011; Plemenitaš et al.2014). CASE STUDIES Extremely halophilic Archaea of the class Halobacteria The Archaea of the class Halobacteria are the best studied extremely halophilic prokaryotes. Until 2015 the class contained a single order, the Halobacteriales, with a single family, the Halobacteriaceae. Recent phylogenomic studies led to the splitting of the group into three orders and six families. Most studies of osmotic adaptation, ion metabolism and the properties of the cellular proteins were performed on two model organisms: Halobacterium salinarum (Halobacteriaceae) and Haloarcula marismortui (Haloarculaceae). Members of the Halobacteria are characteristic inhabitants of salt lakes at or approaching halite saturation, saltern crystallizer ponds and other high salt environments. Most members of the group are obligate halophiles that require at least 150–200 g L−1 of salts and show no adaptability to lower concentrations. Except for Halococcus and a few other genera that possess a rigid cell wall, members of the Halobacteria lyse when suspended in low salt solutions as their glycoprotein cell wall requires molar concentrations of salt for structural stability. As Halobacterium and other members of a group lack a measurable turgor pressure (Walsby 1971), they do not need to maintain a higher osmotic pressure in the cell than that of the brines in which they live. Measurements of intracellular ionic concentrations in H. salinarum, Har. marismortui and related organisms performed already in the 1960s–1970s (Christian and Waltho 1962; Ginzburg, Sachs and Ginzburg 1970; Lanyi and Silverman 1972; Matheson et al.1976) showed that the cells contain molar concentrations of salts, sufficient to osmotically balance the salinity of the external medium. K+ is always present as the dominant cation, and Na+ is found at much lower concentrations. Chloride is the main anion to balance the intracellular cations. In view of the difficulty to reliably assess the intracellular Na+ concentrations in the presence of molar concentrations in the surrounding media, the true Na+ may well be lower than the apparent values reported. Probably in view of this experimental difficulty, surprisingly few later studies have tried to verify and refine the early results. One recent study used microprobe analysis in a scanning electron microscope equipped with an X-ray spectrometer to assess the intracellular ionic concentrations of H. salinarum grown in medium containing 4.28 mol NaCl and 0.036 mol K+ per kg water. The intracellular K+ concentration was 110 times that of the medium, Na+ inside the cells was about one-third of that of the medium concentration, and chloride inside the cells was 1.1 times higher than in the medium, so that the apparent cation sum exceeded the anion sum (Engel and Catchpole 2005). Measurements of intracellular ion concentrations in Har. marismortui grown in 23% salt as determined by inductively coupled mass spectrometry indicated a minimum intracellular total ion requirement of 1.13 M (Jensen et al.2015). To achieve such ion gradients across the cell membrane (accumulation of K+, exclusion of Na+, accumulation of Cl− against the inside-negative membrane potential) requires energy-dependent mechanisms. Analysis of genome sequences of model organisms such as Halobacterium sp. NRC-1 (a strain of H. salinarum) (Ng et al.2000), Har. marismortui (Baliga et al.2004), Haloquadratum walsbyi (Bolhuis et al.2006) and other isolates (Becker et al.2014) have contributed much understanding about the types of ion pumps involved. The necessary energy is derived from the proton gradient over the membrane, generated by respiratory electron transport and/or the light-dependent proton pump bacteriorhodopsin. Na+ is extruded from the cells by Na+/H+ antiporter systems. K+ can enter the cells passively through K+ channels in the membrane, as driven by the inside-negative membrane potential, but active, ATP-dependent K+ transport systems are also present. In low K+ media, H. salinarum expresses a K+-transporting KdpFABC P-type ATPase together with an additional protein annotated as Cat3 in Halobacterium sp. NRC-1 (Table 1) and as UspA protein in H. salinarum R1. K+ limitation can also lead to a lowered intracellular K+ concentration. Table 1. Major alkali metal–cation transporters in halotolerant/halophilic microorganisms. Description Transporters identified in halotolerant/halophilic microorganisms K+ efflux Tok1 : Hortaea werneckii  channel Aureobasidium sp. K+ uptake Trk1,2 : Hortaea werneckii  uniporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii DhHak1 : Debaryomyces hansenii Trk AI : Halomonas elongata TrkAH : Halobacterium salinarum NRC-1 Halococcus hamelinensis Halomonas elongata TrkA2 : Halomonas beimenensis K+(Na+) efflux Hak1 : Aureobasidium  antiporter Debaryomyces hansenii K+ efflux Acu : Wallemia ichthyophaga  P-type ATPase Aureobasidium Na+ efflux Nha1 : Hortaea.werneckii  antiporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii NhaC : Halobacterium salinarum NRC-1 Na+ efflux Ena1,2 : Hortaea werneckii  P-type Wallamia ichthyophaga  ATPase Aureobasidium sp. Debarymyces hansenii Na+ P-type ATPase : Dunaliella maritima Na+/Pi Pho89 : Hortaea werneckii  symporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii H+ exporter Pma1 : Hortaea werneckii  P-type Wallemia ichthyophaga  ATPase Aureobasidium sp. Debaryomyces hansenii Na+/H+ Nhx1 (late endosome) : Hortaea werneckii  antiporter Debaryomyces hansenii  for Na+ Vnx1 (vacuolar) : Hortaea werneckii  extrusion Wallemia ichthyophaga Na+/H+ antiporter : Dunaliella Na+/H+ antiporter : Halobacterium salinarum Salinivibrio costicola Na+/H+ antiporter NhaD : Halomonas elongata Halomonas alkaliphila Na+(K+)/H+ antiporter : Natranaerobius thermophilus Na+/H+ antiporter : Halocafeteria seosinensis K+/H+ Kha1 (Golgi apparatus)  :  Hortaea werneckii  antiporter Wallemia ichthyophaga Debaryomyces hansenii K+/H+ antiporter : Chromohalobacter israelensis ATP-driven K+ transport system KpdFABC : Halobacterium salinarum NRC-1 Description Transporters identified in halotolerant/halophilic microorganisms K+ efflux Tok1 : Hortaea werneckii  channel Aureobasidium sp. K+ uptake Trk1,2 : Hortaea werneckii  uniporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii DhHak1 : Debaryomyces hansenii Trk AI : Halomonas elongata TrkAH : Halobacterium salinarum NRC-1 Halococcus hamelinensis Halomonas elongata TrkA2 : Halomonas beimenensis K+(Na+) efflux Hak1 : Aureobasidium  antiporter Debaryomyces hansenii K+ efflux Acu : Wallemia ichthyophaga  P-type ATPase Aureobasidium Na+ efflux Nha1 : Hortaea.werneckii  antiporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii NhaC : Halobacterium salinarum NRC-1 Na+ efflux Ena1,2 : Hortaea werneckii  P-type Wallamia ichthyophaga  ATPase Aureobasidium sp. Debarymyces hansenii Na+ P-type ATPase : Dunaliella maritima Na+/Pi Pho89 : Hortaea werneckii  symporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii H+ exporter Pma1 : Hortaea werneckii  P-type Wallemia ichthyophaga  ATPase Aureobasidium sp. Debaryomyces hansenii Na+/H+ Nhx1 (late endosome) : Hortaea werneckii  antiporter Debaryomyces hansenii  for Na+ Vnx1 (vacuolar) : Hortaea werneckii  extrusion Wallemia ichthyophaga Na+/H+ antiporter : Dunaliella Na+/H+ antiporter : Halobacterium salinarum Salinivibrio costicola Na+/H+ antiporter NhaD : Halomonas elongata Halomonas alkaliphila Na+(K+)/H+ antiporter : Natranaerobius thermophilus Na+/H+ antiporter : Halocafeteria seosinensis K+/H+ Kha1 (Golgi apparatus)  :  Hortaea werneckii  antiporter Wallemia ichthyophaga Debaryomyces hansenii K+/H+ antiporter : Chromohalobacter israelensis ATP-driven K+ transport system KpdFABC : Halobacterium salinarum NRC-1 View Large Table 1. Major alkali metal–cation transporters in halotolerant/halophilic microorganisms. Description Transporters identified in halotolerant/halophilic microorganisms K+ efflux Tok1 : Hortaea werneckii  channel Aureobasidium sp. K+ uptake Trk1,2 : Hortaea werneckii  uniporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii DhHak1 : Debaryomyces hansenii Trk AI : Halomonas elongata TrkAH : Halobacterium salinarum NRC-1 Halococcus hamelinensis Halomonas elongata TrkA2 : Halomonas beimenensis K+(Na+) efflux Hak1 : Aureobasidium  antiporter Debaryomyces hansenii K+ efflux Acu : Wallemia ichthyophaga  P-type ATPase Aureobasidium Na+ efflux Nha1 : Hortaea.werneckii  antiporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii NhaC : Halobacterium salinarum NRC-1 Na+ efflux Ena1,2 : Hortaea werneckii  P-type Wallamia ichthyophaga  ATPase Aureobasidium sp. Debarymyces hansenii Na+ P-type ATPase : Dunaliella maritima Na+/Pi Pho89 : Hortaea werneckii  symporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii H+ exporter Pma1 : Hortaea werneckii  P-type Wallemia ichthyophaga  ATPase Aureobasidium sp. Debaryomyces hansenii Na+/H+ Nhx1 (late endosome) : Hortaea werneckii  antiporter Debaryomyces hansenii  for Na+ Vnx1 (vacuolar) : Hortaea werneckii  extrusion Wallemia ichthyophaga Na+/H+ antiporter : Dunaliella Na+/H+ antiporter : Halobacterium salinarum Salinivibrio costicola Na+/H+ antiporter NhaD : Halomonas elongata Halomonas alkaliphila Na+(K+)/H+ antiporter : Natranaerobius thermophilus Na+/H+ antiporter : Halocafeteria seosinensis K+/H+ Kha1 (Golgi apparatus)  :  Hortaea werneckii  antiporter Wallemia ichthyophaga Debaryomyces hansenii K+/H+ antiporter : Chromohalobacter israelensis ATP-driven K+ transport system KpdFABC : Halobacterium salinarum NRC-1 Description Transporters identified in halotolerant/halophilic microorganisms K+ efflux Tok1 : Hortaea werneckii  channel Aureobasidium sp. K+ uptake Trk1,2 : Hortaea werneckii  uniporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii DhHak1 : Debaryomyces hansenii Trk AI : Halomonas elongata TrkAH : Halobacterium salinarum NRC-1 Halococcus hamelinensis Halomonas elongata TrkA2 : Halomonas beimenensis K+(Na+) efflux Hak1 : Aureobasidium  antiporter Debaryomyces hansenii K+ efflux Acu : Wallemia ichthyophaga  P-type ATPase Aureobasidium Na+ efflux Nha1 : Hortaea.werneckii  antiporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii NhaC : Halobacterium salinarum NRC-1 Na+ efflux Ena1,2 : Hortaea werneckii  P-type Wallamia ichthyophaga  ATPase Aureobasidium sp. Debarymyces hansenii Na+ P-type ATPase : Dunaliella maritima Na+/Pi Pho89 : Hortaea werneckii  symporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii H+ exporter Pma1 : Hortaea werneckii  P-type Wallemia ichthyophaga  ATPase Aureobasidium sp. Debaryomyces hansenii Na+/H+ Nhx1 (late endosome) : Hortaea werneckii  antiporter Debaryomyces hansenii  for Na+ Vnx1 (vacuolar) : Hortaea werneckii  extrusion Wallemia ichthyophaga Na+/H+ antiporter : Dunaliella Na+/H+ antiporter : Halobacterium salinarum Salinivibrio costicola Na+/H+ antiporter NhaD : Halomonas elongata Halomonas alkaliphila Na+(K+)/H+ antiporter : Natranaerobius thermophilus Na+/H+ antiporter : Halocafeteria seosinensis K+/H+ Kha1 (Golgi apparatus)  :  Hortaea werneckii  antiporter Wallemia ichthyophaga Debaryomyces hansenii K+/H+ antiporter : Chromohalobacter israelensis ATP-driven K+ transport system KpdFABC : Halobacterium salinarum NRC-1 View Large Presence of the KdpFABC complex enables H. salinarum to grow at K+ concentrations as low as 20 μM. Deletion of the kdpFABC cat3 gene resulted in a reduced ability to grow under K+-limiting concentrations (Strahl and Greie 2008). Comparative genomics indicated a considerable diversity of potassium transport complex subunits in the halophilic archaea (Jensen et al.2015). Chloride can be accumulated into the cells by cotransport with Na+ or by the primary light-driven inward chloride pump halorhodopsin (Duschl and Wagner 1986). The light-driven chloride pump halorhodopsin, a hybrid histidine kinase, and the mechanosensitive channels MscA1 and MscA2 have been identified as putative osmosensors H. salinarum and in Haloferax volcanii (Table 2) (Le Dain et al.1998; Kolbe et al.2000; Zhang and Shi 2005). Table 2. Putative osmosensors identified in prokaryotic halophiles. Archaea Halobacterium salinarum The light-driven chloride pump halorhodopsin Kolbe et al.2000 Halobacterium salinarum NRC-1 Hybrid histidine kinase Zhang and Shi 2005 Haloferax volcanii Mechanosensitive channels MscA1, MscA2 Le Dain et al.1998 Bacteria Chromohalobacter salexigens Two-component response regulator EupR Rodríguez-Moya et al.2010 Chromohalobacter salexigens Hybrid histidine kinase Rodríguez-Moya et al.2010 Halomonas elongata Transporter for ectoine accumulation Tea Kunte, Trüper and Stan-Lotter 2012 Archaea Halobacterium salinarum The light-driven chloride pump halorhodopsin Kolbe et al.2000 Halobacterium salinarum NRC-1 Hybrid histidine kinase Zhang and Shi 2005 Haloferax volcanii Mechanosensitive channels MscA1, MscA2 Le Dain et al.1998 Bacteria Chromohalobacter salexigens Two-component response regulator EupR Rodríguez-Moya et al.2010 Chromohalobacter salexigens Hybrid histidine kinase Rodríguez-Moya et al.2010 Halomonas elongata Transporter for ectoine accumulation Tea Kunte, Trüper and Stan-Lotter 2012 View Large Table 2. Putative osmosensors identified in prokaryotic halophiles. Archaea Halobacterium salinarum The light-driven chloride pump halorhodopsin Kolbe et al.2000 Halobacterium salinarum NRC-1 Hybrid histidine kinase Zhang and Shi 2005 Haloferax volcanii Mechanosensitive channels MscA1, MscA2 Le Dain et al.1998 Bacteria Chromohalobacter salexigens Two-component response regulator EupR Rodríguez-Moya et al.2010 Chromohalobacter salexigens Hybrid histidine kinase Rodríguez-Moya et al.2010 Halomonas elongata Transporter for ectoine accumulation Tea Kunte, Trüper and Stan-Lotter 2012 Archaea Halobacterium salinarum The light-driven chloride pump halorhodopsin Kolbe et al.2000 Halobacterium salinarum NRC-1 Hybrid histidine kinase Zhang and Shi 2005 Haloferax volcanii Mechanosensitive channels MscA1, MscA2 Le Dain et al.1998 Bacteria Chromohalobacter salexigens Two-component response regulator EupR Rodríguez-Moya et al.2010 Chromohalobacter salexigens Hybrid histidine kinase Rodríguez-Moya et al.2010 Halomonas elongata Transporter for ectoine accumulation Tea Kunte, Trüper and Stan-Lotter 2012 View Large One of the most characteristic properties of the halophilic Archaea is their highly acidic proteome: their proteins have a high excess of negatively charged amino acids (aspartate, glutamate) over amino acids with a positive charge (lysine, arginine). This property is shared by other ‘salt-in’ strategists such as Salinibacter (see below). The acidic nature of the proteins of the Halobacteria was already recognized nearly half a century ago (Reistad 1970), and it was fully confirmed during genomic analyses (e.g. Dennis and Shimmin 1997; Ng et al.2000; Baliga et al.2004; Bolhuis et al.2006). Only few proteins encoded by these genomes are not acidic, the membrane-bound retinal proteins being notable exceptions. Comparative genomic analyses including halophilic Archaea, mesophiles and thermophiles showed that, compared with non-halophilic mesophiles, the protein surface of the halophiles is enriched in aspartate and is low in lysine and asparagine, the interior of the proteins being enriched in valine and low in isoleucine. Compared with thermophiles the protein surface of the halophiles is enriched in aspartate, alanine and threonine and is low in lysine, the interior of the proteins being enriched in threonine and low in isoleucine (Fukuchi et al.2003) The highly acidic enzymes and other proteins of the Halobacteria typically require molar concentrations of salt for activity and structural stability. Since the classic review of the salt-dependent properties of proteins from the extreme halophiles by Lanyi (1974), many studies have been devoted to the elucidation of the mechanisms of the salt requirement and salt tolerance of halophilic enzymes in terms of the thermodynamics of solvent–protein interactions, hydration, ion binding and unfolding kinetics. The availability of high-resolution crystal structures for selected halophilic enzymes such as the malate dehydrogenase and the 2Fe-2S ferredoxin of Har. marismortui has greatly contributed to such studies. Techniques such as analytical ultracentrifugation, small angle neutron and X-ray scattering, crystallography, and protein dynamics by energy resolved neutron scattering have been employed in the study of halophilic proteins. Such proteins are folded similar to their non-halophilic homologues. The excess in negative charge is found predominantly on the protein surfaces where extensive hydration interactions are observed. Complex salt bridges associated with solvent ion binding sites also appear as likely halo-adaptation features (Eisenberg, Mevarech and Zaccai 1992; Madern, Ebel and Zaccai 2000; Ebel and Zaccai 2004; Tehei and Zaccai 2005; Soppa 2006, and many others). The high negative surface charge makes the proteins more soluble and renders them more flexible at high salt concentrations when non-halophilic proteins tend to aggregate and become rigid. This high surface charge is neutralized mainly by tightly bound water dipoles. The requirement for a high salt concentration for the stabilization of the halophilic enzymes is due to a low-affinity binding of the salt to specific sites on the protein surface (Mevarech, Frolow and Gloss 2000). Analysis of protein–solvent interactions in the glucose dehydrogenase of Hfx. mediterranei showed that the acidic residues on the protein surface are only partially neutralized by bound potassium. A highly ordered, multilayered solvation shell is present on the surface of the protein (Britton et al.2006). Cumulative weak cation–protein interactions are expected to stabilize the folded conformations (Ortega, Diercks and Millet 2015). The acidic residues on the protein surface may also be important to prevent aggregation of the proteins (Elcock and McCammon 1998). In bacteria and nonhalophilic archaea the majority of exported proteins are Sec-dependent, while haloarchaea use primarily the Tat pathway for export, which, in contrast to Sec pathway, enables the transport of fully folded proteins. Proteins from non-halophile microorganisms would probably aggregate and precipitate at conditions of high salinity at which haloarchaea thrive, while halobacterial proteins have increased negative surface charge that prevents aggregation (Bolhuis 2002). Most members of the class Halobacteria have only a limited ability to adapt to salt concentrations below their optimum. Accordingly, relatively few studies have tried to monitor changes in the types of lipids in the cell membrane following hypoosmotic shock. Among the effects observed are the formation of cardiolipin at the expense of phosphatidylglycerol, a decrease in content of the methyl ester of phosphatidylglycerol phosphate, and an increase in the content of C25C20 lipids at the expense of C20C20 lipids. These and other changes are further documented in Table 3. Table 3. Lipid composition of selected obligately halophilic Archaea that require at least 150–200 g L−1 NaCl for growth, and changes in lipid composition following lowering of the salt concentration to values still high enough to provide structural stability. Archaeal taxa Lipid composition Changes induced by hypoosmotic shock References Class Halobacteria Branched C20 and C25 lipids (from 1–89%); great variety of polar lipids: PL, SL, GL; maintenance of a highly negative charge surface density by a high concentration of acidic lipids Neo-synthesis of BPG at the expense of PG Lopalco et al.2004, 2008 Haloarcula marismortui 86% polar lipids (GL, PG, PGP-Me, PGS and 14% non-polar lipids) (squalenes, vitamin MK-8 and bacterioruberins, β-carotene, lycopene and retinal) Increase in glycosyl cardiolipin analogues, BPG, decreased PGP-Me Evans, Kushwaha and Kates 1980 Halobacterium salinarum C20C20 DGD, abundant PGP-Me (50–80% of the polar lipids) Increased S-TGD-1-PA Russell 1989a,b; Russell et al. 1995; Tenchov et al.2006; Lobasso et al.2008 Haloferax volcanii, Hfx. mediterranei C20C20 DGD and other lipids; absence of SL Increased BPG, glycosyl cardiolipin analogues, decrease in PGP-Me Halorubrum trapanicum, Hrr. vacuolatum C20C20 DGD and other lipids Increased S-DGD-5-PA van de Vossenberg et al.1999 Haloquadratum walsbyi Neutral lipids: vitamin MK-8, squalene, bacterioruberin carotenoids and several retinal isomers. Polar lipids: PGP-Me, PGS, PG, S-DGD-1. Cardiolipins: tetra-phytanyl or dimeric phospholipids, no glycosyl-cardiolipin, trace amounts of BPG. No changes, both qualitatively and quantitatively Lobasso et al.2008 Natronobacterium gregoryi C20C20 DGD, C25C20 DGD, and other lipids; isoprenoid chains fully saturated; GL absent Increase in C25C20 lipids; increase in unsaturation of DGD Morth and Tindall 1985a,b; Tindall, Amendt and Dahl 1991 Natrialba magadii C20C20 DGD, C25C20 DGD, and other lipids Increase in C25C20 lipids; increase in unsaturation of DGD Morth and Tindall 1985a,b; Tindall, Amendt and Dahl 1991 Archaeal taxa Lipid composition Changes induced by hypoosmotic shock References Class Halobacteria Branched C20 and C25 lipids (from 1–89%); great variety of polar lipids: PL, SL, GL; maintenance of a highly negative charge surface density by a high concentration of acidic lipids Neo-synthesis of BPG at the expense of PG Lopalco et al.2004, 2008 Haloarcula marismortui 86% polar lipids (GL, PG, PGP-Me, PGS and 14% non-polar lipids) (squalenes, vitamin MK-8 and bacterioruberins, β-carotene, lycopene and retinal) Increase in glycosyl cardiolipin analogues, BPG, decreased PGP-Me Evans, Kushwaha and Kates 1980 Halobacterium salinarum C20C20 DGD, abundant PGP-Me (50–80% of the polar lipids) Increased S-TGD-1-PA Russell 1989a,b; Russell et al. 1995; Tenchov et al.2006; Lobasso et al.2008 Haloferax volcanii, Hfx. mediterranei C20C20 DGD and other lipids; absence of SL Increased BPG, glycosyl cardiolipin analogues, decrease in PGP-Me Halorubrum trapanicum, Hrr. vacuolatum C20C20 DGD and other lipids Increased S-DGD-5-PA van de Vossenberg et al.1999 Haloquadratum walsbyi Neutral lipids: vitamin MK-8, squalene, bacterioruberin carotenoids and several retinal isomers. Polar lipids: PGP-Me, PGS, PG, S-DGD-1. Cardiolipins: tetra-phytanyl or dimeric phospholipids, no glycosyl-cardiolipin, trace amounts of BPG. No changes, both qualitatively and quantitatively Lobasso et al.2008 Natronobacterium gregoryi C20C20 DGD, C25C20 DGD, and other lipids; isoprenoid chains fully saturated; GL absent Increase in C25C20 lipids; increase in unsaturation of DGD Morth and Tindall 1985a,b; Tindall, Amendt and Dahl 1991 Natrialba magadii C20C20 DGD, C25C20 DGD, and other lipids Increase in C25C20 lipids; increase in unsaturation of DGD Morth and Tindall 1985a,b; Tindall, Amendt and Dahl 1991 Dialkylglycerol diether lipids (DGD); Phospholipids (PL): phosphatidylglycerophosphate methyl ester (PGP-Me); phosphatidylglycerosulfate (PGS); phosphatidylglycerol (PG), cardiolipin = bisphosphatidylglycerol (BPG); glycosyl-substituted cardiolipins: S-TGD1-PA (Halobacterium), S-DGD-5-PA (Halorubrum); sulfolipids (SL); glycolipids (GL). View Large Table 3. Lipid composition of selected obligately halophilic Archaea that require at least 150–200 g L−1 NaCl for growth, and changes in lipid composition following lowering of the salt concentration to values still high enough to provide structural stability. Archaeal taxa Lipid composition Changes induced by hypoosmotic shock References Class Halobacteria Branched C20 and C25 lipids (from 1–89%); great variety of polar lipids: PL, SL, GL; maintenance of a highly negative charge surface density by a high concentration of acidic lipids Neo-synthesis of BPG at the expense of PG Lopalco et al.2004, 2008 Haloarcula marismortui 86% polar lipids (GL, PG, PGP-Me, PGS and 14% non-polar lipids) (squalenes, vitamin MK-8 and bacterioruberins, β-carotene, lycopene and retinal) Increase in glycosyl cardiolipin analogues, BPG, decreased PGP-Me Evans, Kushwaha and Kates 1980 Halobacterium salinarum C20C20 DGD, abundant PGP-Me (50–80% of the polar lipids) Increased S-TGD-1-PA Russell 1989a,b; Russell et al. 1995; Tenchov et al.2006; Lobasso et al.2008 Haloferax volcanii, Hfx. mediterranei C20C20 DGD and other lipids; absence of SL Increased BPG, glycosyl cardiolipin analogues, decrease in PGP-Me Halorubrum trapanicum, Hrr. vacuolatum C20C20 DGD and other lipids Increased S-DGD-5-PA van de Vossenberg et al.1999 Haloquadratum walsbyi Neutral lipids: vitamin MK-8, squalene, bacterioruberin carotenoids and several retinal isomers. Polar lipids: PGP-Me, PGS, PG, S-DGD-1. Cardiolipins: tetra-phytanyl or dimeric phospholipids, no glycosyl-cardiolipin, trace amounts of BPG. No changes, both qualitatively and quantitatively Lobasso et al.2008 Natronobacterium gregoryi C20C20 DGD, C25C20 DGD, and other lipids; isoprenoid chains fully saturated; GL absent Increase in C25C20 lipids; increase in unsaturation of DGD Morth and Tindall 1985a,b; Tindall, Amendt and Dahl 1991 Natrialba magadii C20C20 DGD, C25C20 DGD, and other lipids Increase in C25C20 lipids; increase in unsaturation of DGD Morth and Tindall 1985a,b; Tindall, Amendt and Dahl 1991 Archaeal taxa Lipid composition Changes induced by hypoosmotic shock References Class Halobacteria Branched C20 and C25 lipids (from 1–89%); great variety of polar lipids: PL, SL, GL; maintenance of a highly negative charge surface density by a high concentration of acidic lipids Neo-synthesis of BPG at the expense of PG Lopalco et al.2004, 2008 Haloarcula marismortui 86% polar lipids (GL, PG, PGP-Me, PGS and 14% non-polar lipids) (squalenes, vitamin MK-8 and bacterioruberins, β-carotene, lycopene and retinal) Increase in glycosyl cardiolipin analogues, BPG, decreased PGP-Me Evans, Kushwaha and Kates 1980 Halobacterium salinarum C20C20 DGD, abundant PGP-Me (50–80% of the polar lipids) Increased S-TGD-1-PA Russell 1989a,b; Russell et al. 1995; Tenchov et al.2006; Lobasso et al.2008 Haloferax volcanii, Hfx. mediterranei C20C20 DGD and other lipids; absence of SL Increased BPG, glycosyl cardiolipin analogues, decrease in PGP-Me Halorubrum trapanicum, Hrr. vacuolatum C20C20 DGD and other lipids Increased S-DGD-5-PA van de Vossenberg et al.1999 Haloquadratum walsbyi Neutral lipids: vitamin MK-8, squalene, bacterioruberin carotenoids and several retinal isomers. Polar lipids: PGP-Me, PGS, PG, S-DGD-1. Cardiolipins: tetra-phytanyl or dimeric phospholipids, no glycosyl-cardiolipin, trace amounts of BPG. No changes, both qualitatively and quantitatively Lobasso et al.2008 Natronobacterium gregoryi C20C20 DGD, C25C20 DGD, and other lipids; isoprenoid chains fully saturated; GL absent Increase in C25C20 lipids; increase in unsaturation of DGD Morth and Tindall 1985a,b; Tindall, Amendt and Dahl 1991 Natrialba magadii C20C20 DGD, C25C20 DGD, and other lipids Increase in C25C20 lipids; increase in unsaturation of DGD Morth and Tindall 1985a,b; Tindall, Amendt and Dahl 1991 Dialkylglycerol diether lipids (DGD); Phospholipids (PL): phosphatidylglycerophosphate methyl ester (PGP-Me); phosphatidylglycerosulfate (PGS); phosphatidylglycerol (PG), cardiolipin = bisphosphatidylglycerol (BPG); glycosyl-substituted cardiolipins: S-TGD1-PA (Halobacterium), S-DGD-5-PA (Halorubrum); sulfolipids (SL); glycolipids (GL). View Large The Halobacteria were always considered to be a coherent group of extreme halophiles that only use KCl for osmotic balance and do not employ organic osmotic solutes. This view now needs modification. Some representatives of the group can also grow at salt concentrations below 100 g L−1, and a few strains (which unfortunately were not further documented) were reported to grow slowly even at seawater salinity (Purdy et al.2004). Accumulation of an organic osmotic solute, 2-sulfotrehalose, correlated with the medium salinity, was first described in a few alkaliphilic members of the Halobacteria (Natronococcus and Natronobacterium spp.) (Desmarais et al.1997) (Table 4). Recent comparative genomic analyses showed that the potential for the biosynthesis of trehalose and/or 2-sulfotrehalose, as well as the uptake of glycine betaine, may be widespread among the Halobacteria. Out of the 83 analysed genomes, genes encoding the trehalose-6-phosphate synthase/trehalose-6-phosphatase OtsAB pathway and glycine betaine BCCT family transporters were identified in 38 and 60 genomes, respectively, and production of trehalose or sulfotrehalose was experimentally verified in 17 species. There was a general trend suggesting that trehalose-producing genera prefer habitats of intermediate and (relatively) low salinity, while genera restricted to life at the highest salt concentrations lack the trehalose production capability (Youssef et al.2014). The mechanism of osmoadaptation in Haladaptatus paucihalophilus, a low-salt adapted member of the group, was investigated in further depth. Its genome contains genes for trehalose synthesis (the OtsAB pathway) and the trehalose glycosyl-transferring synthase pathway, and for glycine betaine uptake (BCCT family of secondary transporters and QAT family of ABC transporters). When grown in defined medium, H. paucihalophilus cells synthesized and accumulated ∼2.0–3.7 μmol of trehalose per mg of protein. Exogenously supplied glycine betaine was increasingly accumulated at higher salinities (Youssef et al.2014). Another (relatively) low-salt adapted member of the Halobacteria that may use trehalose as an osmotic solute is Halococcus hamelinensis isolated from living stromatolites in Shark Bay, W. Australia. When cells were exposed to osmotic shock or to a gradual increase in salinity, no increase in intracellular K+ was measured. However, 1H-NMR spectroscopy showed accumulation of glycine betaine, trehalose and glutamate. The levels of glycine betaine increased with salinity, but at the highest NaCl concentrations intracellular trehalose levels were decreased (Goh et al.2011; Gudhka, Neilan and Burns 2015). Table 4. Compatible solutes in halotolerant/halophilic microorganisms The salt sensitive Saccharomyces cerevisiae was included for comparison. Microorganism Primary compatible solutes Secondary compatible solutes References Archaea Alkaliphilic halobacteria Natronococcus and Natronobacterium Trehalose and 2-sulfotrehalose; glycine betaine uptake Desmarais et al.1997; Goh et al.2011; Youssef et al.2014 Genera inhabiting habitats with intermediate and relatively low salinity, Haladaptatus paucihalophilus and Halococcus hamelinensis Trehalose and 2-sulfotrehalose; glycine betaine uptake Goh et al.2011 Bacteria Halomonadaceae Halomonas elongata; H. halophila; H. halmophila Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and hydroxyectoine or glycine betaine (uptake) Imhoff and Rodriguez-Valera 1984; Severin, Wohlfarth and Galinski 1992; Cánovas et al.1996, 1998 Chromohalobacter salexigens; C. marismortui Ectoine Severin, Wohlfarth and Galinski 1992 Ectothiorhodospiraceae Halorhodospira halochloris Glycine betaine (synthesis and uptake) Ectoine, trehalose Galinski and Trüper 1982; Galinski, Pfeiffer and Trüper 1985 H. halophila Glycine betaine (synthesis and uptake), Ectoine, trehalose Ectothiorhodospira marismortui glycine betaine, Nα-carbamoyl-L-glutamine 1-amide (CGA), sucrose Galinski and Trüper 1982; Galinski and Oren 1991; Oren et al., 1991; Lippert, Galinski and Trüper 1993 Bacillaceae Halobacillus halophilus Proline, glutamine and glutamate Ectoine, N-acetyl ornithine and N-acetyl lysine Saum and Müller 2008a,b; Saum et al.2013 Actinobacteria Actinopolyspora halophila Glycine betaine Severin, Wohlfarth and Galinski 1992 Nocardiopsis sp. Ectoine Hydroxyectoine Severin, Wohlfarth and Galinski 1992 Cyanobacteria Phormidium-type Sucrose and/or trehalose Oren 2012 Freshwater strains Sucrose and/or trehalose Marine strains Glycosylglycerol (O-α-D-glucopyranosyl-(1→2)-glycerol) Hagemann and Pade 2015 Coleofasciculus chthonoplastes Glycosylglycerol (O-α-D-glucopyranosyl-(1→2)-glycerol) glucosylglycerate or proline Hagemann and Pade 2015 Halophilic Aphanothece/Halothece glycine betaine, L-glutamate betaine (N-trimethyl-L-glutamate) glucosylglycerate or proline; sucrose and trehalose, ectoine Mackay, Norton and Borowitzka 1984; Klähn and Hagemann 2011; Hagemann 2013, Oren 2012 Synechococcus Glucosylglycerol Eukarya Algae Chlorophyceae : Dunaliella parva; D. salina; D. viridis Glycerol Craigie and McLachlan, 1964; Ben-Amotz and Avron, 1973; Ben-Amotz, Sussman and Avron 1983; Goyal 2007; Asteromonas Glycerol Ben-Amotz and Avron 1980 Protists Stramenopiles : Halocafeteria seosinensis Hydroxyectoine and myo-inositol (synthesis and uptake; based on genomics and gene expression studies) Harding et al.2016, 2017 Fungi Hortaea werneckii Glycerol Erythritol, arabitol and mannitol; mycosporine-glutaminol-glucoside (only at lower salinities) Petrovič, Gunde-Cimerman and Plemenitaš 2002; Kogej et al.2007 Wallemia ichthyophaga Glycerol Arabitol Zajc et al.2014 Halotolerant fungi André, Nilsson and Adler 1988; Blomberg and Adler 1992; Blomberg 2000; Hohmann 2002; Petrovič, Gunde-Cimerman and Plemenitaš 2002; Kogej et al.2007; Lenassi et al.2011 Saccharomyces cerevisiae Glycerol Gori et al.2005 Debaryomyces hansenii Glycerol Adler, Blomberg and Nilsson 1985; Gori et al.2005 Microorganism Primary compatible solutes Secondary compatible solutes References Archaea Alkaliphilic halobacteria Natronococcus and Natronobacterium Trehalose and 2-sulfotrehalose; glycine betaine uptake Desmarais et al.1997; Goh et al.2011; Youssef et al.2014 Genera inhabiting habitats with intermediate and relatively low salinity, Haladaptatus paucihalophilus and Halococcus hamelinensis Trehalose and 2-sulfotrehalose; glycine betaine uptake Goh et al.2011 Bacteria Halomonadaceae Halomonas elongata; H. halophila; H. halmophila Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and hydroxyectoine or glycine betaine (uptake) Imhoff and Rodriguez-Valera 1984; Severin, Wohlfarth and Galinski 1992; Cánovas et al.1996, 1998 Chromohalobacter salexigens; C. marismortui Ectoine Severin, Wohlfarth and Galinski 1992 Ectothiorhodospiraceae Halorhodospira halochloris Glycine betaine (synthesis and uptake) Ectoine, trehalose Galinski and Trüper 1982; Galinski, Pfeiffer and Trüper 1985 H. halophila Glycine betaine (synthesis and uptake), Ectoine, trehalose Ectothiorhodospira marismortui glycine betaine, Nα-carbamoyl-L-glutamine 1-amide (CGA), sucrose Galinski and Trüper 1982; Galinski and Oren 1991; Oren et al., 1991; Lippert, Galinski and Trüper 1993 Bacillaceae Halobacillus halophilus Proline, glutamine and glutamate Ectoine, N-acetyl ornithine and N-acetyl lysine Saum and Müller 2008a,b; Saum et al.2013 Actinobacteria Actinopolyspora halophila Glycine betaine Severin, Wohlfarth and Galinski 1992 Nocardiopsis sp. Ectoine Hydroxyectoine Severin, Wohlfarth and Galinski 1992 Cyanobacteria Phormidium-type Sucrose and/or trehalose Oren 2012 Freshwater strains Sucrose and/or trehalose Marine strains Glycosylglycerol (O-α-D-glucopyranosyl-(1→2)-glycerol) Hagemann and Pade 2015 Coleofasciculus chthonoplastes Glycosylglycerol (O-α-D-glucopyranosyl-(1→2)-glycerol) glucosylglycerate or proline Hagemann and Pade 2015 Halophilic Aphanothece/Halothece glycine betaine, L-glutamate betaine (N-trimethyl-L-glutamate) glucosylglycerate or proline; sucrose and trehalose, ectoine Mackay, Norton and Borowitzka 1984; Klähn and Hagemann 2011; Hagemann 2013, Oren 2012 Synechococcus Glucosylglycerol Eukarya Algae Chlorophyceae : Dunaliella parva; D. salina; D. viridis Glycerol Craigie and McLachlan, 1964; Ben-Amotz and Avron, 1973; Ben-Amotz, Sussman and Avron 1983; Goyal 2007; Asteromonas Glycerol Ben-Amotz and Avron 1980 Protists Stramenopiles : Halocafeteria seosinensis Hydroxyectoine and myo-inositol (synthesis and uptake; based on genomics and gene expression studies) Harding et al.2016, 2017 Fungi Hortaea werneckii Glycerol Erythritol, arabitol and mannitol; mycosporine-glutaminol-glucoside (only at lower salinities) Petrovič, Gunde-Cimerman and Plemenitaš 2002; Kogej et al.2007 Wallemia ichthyophaga Glycerol Arabitol Zajc et al.2014 Halotolerant fungi André, Nilsson and Adler 1988; Blomberg and Adler 1992; Blomberg 2000; Hohmann 2002; Petrovič, Gunde-Cimerman and Plemenitaš 2002; Kogej et al.2007; Lenassi et al.2011 Saccharomyces cerevisiae Glycerol Gori et al.2005 Debaryomyces hansenii Glycerol Adler, Blomberg and Nilsson 1985; Gori et al.2005 View Large Table 4. Compatible solutes in halotolerant/halophilic microorganisms The salt sensitive Saccharomyces cerevisiae was included for comparison. Microorganism Primary compatible solutes Secondary compatible solutes References Archaea Alkaliphilic halobacteria Natronococcus and Natronobacterium Trehalose and 2-sulfotrehalose; glycine betaine uptake Desmarais et al.1997; Goh et al.2011; Youssef et al.2014 Genera inhabiting habitats with intermediate and relatively low salinity, Haladaptatus paucihalophilus and Halococcus hamelinensis Trehalose and 2-sulfotrehalose; glycine betaine uptake Goh et al.2011 Bacteria Halomonadaceae Halomonas elongata; H. halophila; H. halmophila Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and hydroxyectoine or glycine betaine (uptake) Imhoff and Rodriguez-Valera 1984; Severin, Wohlfarth and Galinski 1992; Cánovas et al.1996, 1998 Chromohalobacter salexigens; C. marismortui Ectoine Severin, Wohlfarth and Galinski 1992 Ectothiorhodospiraceae Halorhodospira halochloris Glycine betaine (synthesis and uptake) Ectoine, trehalose Galinski and Trüper 1982; Galinski, Pfeiffer and Trüper 1985 H. halophila Glycine betaine (synthesis and uptake), Ectoine, trehalose Ectothiorhodospira marismortui glycine betaine, Nα-carbamoyl-L-glutamine 1-amide (CGA), sucrose Galinski and Trüper 1982; Galinski and Oren 1991; Oren et al., 1991; Lippert, Galinski and Trüper 1993 Bacillaceae Halobacillus halophilus Proline, glutamine and glutamate Ectoine, N-acetyl ornithine and N-acetyl lysine Saum and Müller 2008a,b; Saum et al.2013 Actinobacteria Actinopolyspora halophila Glycine betaine Severin, Wohlfarth and Galinski 1992 Nocardiopsis sp. Ectoine Hydroxyectoine Severin, Wohlfarth and Galinski 1992 Cyanobacteria Phormidium-type Sucrose and/or trehalose Oren 2012 Freshwater strains Sucrose and/or trehalose Marine strains Glycosylglycerol (O-α-D-glucopyranosyl-(1→2)-glycerol) Hagemann and Pade 2015 Coleofasciculus chthonoplastes Glycosylglycerol (O-α-D-glucopyranosyl-(1→2)-glycerol) glucosylglycerate or proline Hagemann and Pade 2015 Halophilic Aphanothece/Halothece glycine betaine, L-glutamate betaine (N-trimethyl-L-glutamate) glucosylglycerate or proline; sucrose and trehalose, ectoine Mackay, Norton and Borowitzka 1984; Klähn and Hagemann 2011; Hagemann 2013, Oren 2012 Synechococcus Glucosylglycerol Eukarya Algae Chlorophyceae : Dunaliella parva; D. salina; D. viridis Glycerol Craigie and McLachlan, 1964; Ben-Amotz and Avron, 1973; Ben-Amotz, Sussman and Avron 1983; Goyal 2007; Asteromonas Glycerol Ben-Amotz and Avron 1980 Protists Stramenopiles : Halocafeteria seosinensis Hydroxyectoine and myo-inositol (synthesis and uptake; based on genomics and gene expression studies) Harding et al.2016, 2017 Fungi Hortaea werneckii Glycerol Erythritol, arabitol and mannitol; mycosporine-glutaminol-glucoside (only at lower salinities) Petrovič, Gunde-Cimerman and Plemenitaš 2002; Kogej et al.2007 Wallemia ichthyophaga Glycerol Arabitol Zajc et al.2014 Halotolerant fungi André, Nilsson and Adler 1988; Blomberg and Adler 1992; Blomberg 2000; Hohmann 2002; Petrovič, Gunde-Cimerman and Plemenitaš 2002; Kogej et al.2007; Lenassi et al.2011 Saccharomyces cerevisiae Glycerol Gori et al.2005 Debaryomyces hansenii Glycerol Adler, Blomberg and Nilsson 1985; Gori et al.2005 Microorganism Primary compatible solutes Secondary compatible solutes References Archaea Alkaliphilic halobacteria Natronococcus and Natronobacterium Trehalose and 2-sulfotrehalose; glycine betaine uptake Desmarais et al.1997; Goh et al.2011; Youssef et al.2014 Genera inhabiting habitats with intermediate and relatively low salinity, Haladaptatus paucihalophilus and Halococcus hamelinensis Trehalose and 2-sulfotrehalose; glycine betaine uptake Goh et al.2011 Bacteria Halomonadaceae Halomonas elongata; H. halophila; H. halmophila Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and hydroxyectoine or glycine betaine (uptake) Imhoff and Rodriguez-Valera 1984; Severin, Wohlfarth and Galinski 1992; Cánovas et al.1996, 1998 Chromohalobacter salexigens; C. marismortui Ectoine Severin, Wohlfarth and Galinski 1992 Ectothiorhodospiraceae Halorhodospira halochloris Glycine betaine (synthesis and uptake) Ectoine, trehalose Galinski and Trüper 1982; Galinski, Pfeiffer and Trüper 1985 H. halophila Glycine betaine (synthesis and uptake), Ectoine, trehalose Ectothiorhodospira marismortui glycine betaine, Nα-carbamoyl-L-glutamine 1-amide (CGA), sucrose Galinski and Trüper 1982; Galinski and Oren 1991; Oren et al., 1991; Lippert, Galinski and Trüper 1993 Bacillaceae Halobacillus halophilus Proline, glutamine and glutamate Ectoine, N-acetyl ornithine and N-acetyl lysine Saum and Müller 2008a,b; Saum et al.2013 Actinobacteria Actinopolyspora halophila Glycine betaine Severin, Wohlfarth and Galinski 1992 Nocardiopsis sp. Ectoine Hydroxyectoine Severin, Wohlfarth and Galinski 1992 Cyanobacteria Phormidium-type Sucrose and/or trehalose Oren 2012 Freshwater strains Sucrose and/or trehalose Marine strains Glycosylglycerol (O-α-D-glucopyranosyl-(1→2)-glycerol) Hagemann and Pade 2015 Coleofasciculus chthonoplastes Glycosylglycerol (O-α-D-glucopyranosyl-(1→2)-glycerol) glucosylglycerate or proline Hagemann and Pade 2015 Halophilic Aphanothece/Halothece glycine betaine, L-glutamate betaine (N-trimethyl-L-glutamate) glucosylglycerate or proline; sucrose and trehalose, ectoine Mackay, Norton and Borowitzka 1984; Klähn and Hagemann 2011; Hagemann 2013, Oren 2012 Synechococcus Glucosylglycerol Eukarya Algae Chlorophyceae : Dunaliella parva; D. salina; D. viridis Glycerol Craigie and McLachlan, 1964; Ben-Amotz and Avron, 1973; Ben-Amotz, Sussman and Avron 1983; Goyal 2007; Asteromonas Glycerol Ben-Amotz and Avron 1980 Protists Stramenopiles : Halocafeteria seosinensis Hydroxyectoine and myo-inositol (synthesis and uptake; based on genomics and gene expression studies) Harding et al.2016, 2017 Fungi Hortaea werneckii Glycerol Erythritol, arabitol and mannitol; mycosporine-glutaminol-glucoside (only at lower salinities) Petrovič, Gunde-Cimerman and Plemenitaš 2002; Kogej et al.2007 Wallemia ichthyophaga Glycerol Arabitol Zajc et al.2014 Halotolerant fungi André, Nilsson and Adler 1988; Blomberg and Adler 1992; Blomberg 2000; Hohmann 2002; Petrovič, Gunde-Cimerman and Plemenitaš 2002; Kogej et al.2007; Lenassi et al.2011 Saccharomyces cerevisiae Glycerol Gori et al.2005 Debaryomyces hansenii Glycerol Adler, Blomberg and Nilsson 1985; Gori et al.2005 View Large Salinibacter ruber (Bacteroidetes) Salinibacter ruber is an extreme halophilic member of the domain Bacteria and belongs phylogenetically to the Bacteroidetes. Although it is a common inhabitant of saltern crystallizer ponds worldwide, it was recognized and isolated only in 1999 (Antón et al.2002). The bacterium is aerobic, brightly red pigmented by carotenoid pigments (salinixanthin) and retinal pigments (xanthorhodopsin and others). It grows optimally at salt concentrations between 200 and 300 g L−1, and at least 150 g L−1 salt is required for growth. Salinibacter is thus one of the most halophilic organisms known within the domain Bacteria. In spite of its affiliation with the Bacteroidetes, Salinibacter has many properties in common with the extremely halophilic Archaea of the class Halobacteria with which it shares its habitat: use of KCl for osmotic stabilization, presence of salt-requiring enzymes and a highly acidic proteome, and the ability to use photons for energy generation by a bacteriorhodopsin-like retinal pigment (Antón et al.2002; Oren 2013c). Measurements of intracellular ion concentrations showed an extremely high potassium content, the ratio K+/protein being similar to that of the Halobacteria. The presence of high intracellular K+ as well as Cl− concentrations was confirmed by X-ray microanalysis in the electron microscope (Oren et al.2002a). Moreover, measurements of intracellular concentrations of potential organic osmotic solutes, using 13C-NMR and HPLC techniques, yielded only small amounts of glutamate, glycine betaine and possibly N-α-acetyllysine in concentrations that can contribute only very little to the osmotic balance (Oren et al.2002a,b). As expected for such a ‘high-salt-in’ strategist, Salinibacter has a strongly acidic proteome because of a high abundance of acidic amino acid and low amounts of basic amino acids (Oren and Mana 2002; Mongodin et al.2005). Some of its enzymes, such as NAD-dependent isocitrate dehydrogenase and NADP-dependent isocitrate activity require 0.5–2 M salt for optimal activity and function well at salt concentrations about 3 M (Oren and Mana 2002). However, the NAD-dependent malate dehydrogenase is completely stable in absence of salt, functions best in the absence of salt, but in the presence of 3–3.5 M KCl or NaCl the activity is still 25%–30% of the optimum value (Oren and Mana 2002; Madern and Zaccai 2004). Changes in the salinity of the medium have litte effect on the lipid composition of the cell membrane (Table 5) (Corcelli et al.2004; Lattanzio et al.2009). Table 5. Salinity-induced changes of the plasma membrane in selected halophilic Bacteria. Bacteria Lipid composition and changes due to hyperosmotic shock Fluidity References Bacteroidetes Salinibacter ruber Main lipids PC, PE, PS, PG, BPG, GL and a sulfonolipid (capnoid). Increased PG; low ratio of PG/C; no significant differences in the lipid profile or the unsaturation of the lipid fatty acyl chains Corcelli et al.2004; Lattanzio et al.2009 Salisaeta longa Sulfonate sphingoids or sulfonolipids (about 10% of the total cellular lipids) Baronio et al.2010 Firmicutes Marinococcus halophilus Increase in anionic phospholipids (PG and DPG) and decrease in PE Oren 2002 Gammaproteobacteria Chromohalobacter salexigens Increased amount of negatively charged lipids; PE and PG decreased or unchanged; increase in DPG, CL, CFA Vreeland, Anderson and Murray 1984; Russell 1989a, 1993; Oren 2002; Vargas et al.2005 Chromohalobacter israelensis Decreased PE/PG ratio, increase in CL and CFA Russell 1989a, 1993; Oren 2002; Mutnuri et al.2005; Vargas et al.2005 Increased degree of fatty acid saturation up to optimal salinity Halomonas halmophila, H. halophila Major polar lipids: PG, PC and PE, additionally CL Increased Russell 1989a, 1993; Oren 2002 Increased anionic lipids (CL and moderate increase in PG), increase in neutral PC relative to phospholipids (PE) Decrease in branched-chain fatty acids. Increase in CFA, unsaturated fatty acids and GL Halomonas campisalis TBS present only in non-saline conditions Russell 1989a, 1993; Aston and Peyton 2007 Decrease in trans fatty acids, increase in CFA and monoenoic fatty acids; normal saturates initially decreased, then increased H. halophila Strong increase in PC, moderate increase in PG Russell 1989a, 1993; Oren 2002 ‘Pseudomonas halosaccharolytica’ Increased CFA, PG and CL, decrease in PE, saturated PG and monounsaturated fatty acids Increased Russell 1993; Oren 2002 Salinivibrio costicola Increased PG and decreased PE Oren 2002 Bacteria Lipid composition and changes due to hyperosmotic shock Fluidity References Bacteroidetes Salinibacter ruber Main lipids PC, PE, PS, PG, BPG, GL and a sulfonolipid (capnoid). Increased PG; low ratio of PG/C; no significant differences in the lipid profile or the unsaturation of the lipid fatty acyl chains Corcelli et al.2004; Lattanzio et al.2009 Salisaeta longa Sulfonate sphingoids or sulfonolipids (about 10% of the total cellular lipids) Baronio et al.2010 Firmicutes Marinococcus halophilus Increase in anionic phospholipids (PG and DPG) and decrease in PE Oren 2002 Gammaproteobacteria Chromohalobacter salexigens Increased amount of negatively charged lipids; PE and PG decreased or unchanged; increase in DPG, CL, CFA Vreeland, Anderson and Murray 1984; Russell 1989a, 1993; Oren 2002; Vargas et al.2005 Chromohalobacter israelensis Decreased PE/PG ratio, increase in CL and CFA Russell 1989a, 1993; Oren 2002; Mutnuri et al.2005; Vargas et al.2005 Increased degree of fatty acid saturation up to optimal salinity Halomonas halmophila, H. halophila Major polar lipids: PG, PC and PE, additionally CL Increased Russell 1989a, 1993; Oren 2002 Increased anionic lipids (CL and moderate increase in PG), increase in neutral PC relative to phospholipids (PE) Decrease in branched-chain fatty acids. Increase in CFA, unsaturated fatty acids and GL Halomonas campisalis TBS present only in non-saline conditions Russell 1989a, 1993; Aston and Peyton 2007 Decrease in trans fatty acids, increase in CFA and monoenoic fatty acids; normal saturates initially decreased, then increased H. halophila Strong increase in PC, moderate increase in PG Russell 1989a, 1993; Oren 2002 ‘Pseudomonas halosaccharolytica’ Increased CFA, PG and CL, decrease in PE, saturated PG and monounsaturated fatty acids Increased Russell 1993; Oren 2002 Salinivibrio costicola Increased PG and decreased PE Oren 2002 Glycerophospholipids (GPL): phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS); diphosphatidylglycerol (DPG) = Cardiolipin (CL). Glycolipids (GL); Terminally branched saturates (TBS); cyclopropane fatty acids (CFA). View Large Table 5. Salinity-induced changes of the plasma membrane in selected halophilic Bacteria. Bacteria Lipid composition and changes due to hyperosmotic shock Fluidity References Bacteroidetes Salinibacter ruber Main lipids PC, PE, PS, PG, BPG, GL and a sulfonolipid (capnoid). Increased PG; low ratio of PG/C; no significant differences in the lipid profile or the unsaturation of the lipid fatty acyl chains Corcelli et al.2004; Lattanzio et al.2009 Salisaeta longa Sulfonate sphingoids or sulfonolipids (about 10% of the total cellular lipids) Baronio et al.2010 Firmicutes Marinococcus halophilus Increase in anionic phospholipids (PG and DPG) and decrease in PE Oren 2002 Gammaproteobacteria Chromohalobacter salexigens Increased amount of negatively charged lipids; PE and PG decreased or unchanged; increase in DPG, CL, CFA Vreeland, Anderson and Murray 1984; Russell 1989a, 1993; Oren 2002; Vargas et al.2005 Chromohalobacter israelensis Decreased PE/PG ratio, increase in CL and CFA Russell 1989a, 1993; Oren 2002; Mutnuri et al.2005; Vargas et al.2005 Increased degree of fatty acid saturation up to optimal salinity Halomonas halmophila, H. halophila Major polar lipids: PG, PC and PE, additionally CL Increased Russell 1989a, 1993; Oren 2002 Increased anionic lipids (CL and moderate increase in PG), increase in neutral PC relative to phospholipids (PE) Decrease in branched-chain fatty acids. Increase in CFA, unsaturated fatty acids and GL Halomonas campisalis TBS present only in non-saline conditions Russell 1989a, 1993; Aston and Peyton 2007 Decrease in trans fatty acids, increase in CFA and monoenoic fatty acids; normal saturates initially decreased, then increased H. halophila Strong increase in PC, moderate increase in PG Russell 1989a, 1993; Oren 2002 ‘Pseudomonas halosaccharolytica’ Increased CFA, PG and CL, decrease in PE, saturated PG and monounsaturated fatty acids Increased Russell 1993; Oren 2002 Salinivibrio costicola Increased PG and decreased PE Oren 2002 Bacteria Lipid composition and changes due to hyperosmotic shock Fluidity References Bacteroidetes Salinibacter ruber Main lipids PC, PE, PS, PG, BPG, GL and a sulfonolipid (capnoid). Increased PG; low ratio of PG/C; no significant differences in the lipid profile or the unsaturation of the lipid fatty acyl chains Corcelli et al.2004; Lattanzio et al.2009 Salisaeta longa Sulfonate sphingoids or sulfonolipids (about 10% of the total cellular lipids) Baronio et al.2010 Firmicutes Marinococcus halophilus Increase in anionic phospholipids (PG and DPG) and decrease in PE Oren 2002 Gammaproteobacteria Chromohalobacter salexigens Increased amount of negatively charged lipids; PE and PG decreased or unchanged; increase in DPG, CL, CFA Vreeland, Anderson and Murray 1984; Russell 1989a, 1993; Oren 2002; Vargas et al.2005 Chromohalobacter israelensis Decreased PE/PG ratio, increase in CL and CFA Russell 1989a, 1993; Oren 2002; Mutnuri et al.2005; Vargas et al.2005 Increased degree of fatty acid saturation up to optimal salinity Halomonas halmophila, H. halophila Major polar lipids: PG, PC and PE, additionally CL Increased Russell 1989a, 1993; Oren 2002 Increased anionic lipids (CL and moderate increase in PG), increase in neutral PC relative to phospholipids (PE) Decrease in branched-chain fatty acids. Increase in CFA, unsaturated fatty acids and GL Halomonas campisalis TBS present only in non-saline conditions Russell 1989a, 1993; Aston and Peyton 2007 Decrease in trans fatty acids, increase in CFA and monoenoic fatty acids; normal saturates initially decreased, then increased H. halophila Strong increase in PC, moderate increase in PG Russell 1989a, 1993; Oren 2002 ‘Pseudomonas halosaccharolytica’ Increased CFA, PG and CL, decrease in PE, saturated PG and monounsaturated fatty acids Increased Russell 1993; Oren 2002 Salinivibrio costicola Increased PG and decreased PE Oren 2002 Glycerophospholipids (GPL): phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS); diphosphatidylglycerol (DPG) = Cardiolipin (CL). Glycolipids (GL); Terminally branched saturates (TBS); cyclopropane fatty acids (CFA). View Large Analysis of the genome of S. ruber confirmed a great resemblance with the haloarchaea. This may have arisen through convergence at the physiological level (different genes producing similar overall phenotype) and the molecular level (independent mutations yielding similar sequences or structures). Several genes and gene clusters may also derive by lateral transfer from, or may have been laterally transferred to, haloarchaea (Mongodin et al.2005). Halomonas elongata, Chromohalobacter salexigens and other heterotrophic halophilic and halotolerant Gammaproteobacteria Moderately halophilic bacteria of the family Halomonadaceae (Gammaproteobacteria) have become popular objects for research on osmotic adaptation in the Bacteria based on the accumulation of organic osmotic solutes (Table 4). Halomonas elongata (Vreeland et al.1980) is the first described representative of this versatile group of halophiles that typically grow in a very broad range of salt concentrations and show a high degree of adaptability to changing salinity (Ventosa, Nieto and Oren 1998). Currently, this family of halophiles consists of 12 genera and over 110 species. Halomonas strains are exploited for the industrial production of ectoine, and therefore research on this group is also relevant to biotechnology. Most studies on osmotic adaptation by the group were performed with H. elongata and with Chromohalobacter salexigens or C. marismortui. When grown in defined media with glucose as the carbon source, the members of the Halomonadaceae produce tetrahydropyrimidines as osmotic solutes. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and hydroxyectoine are accumulated by H. elongata, H. halophila and H. halmophila; in C. marismortui ectoine but no hydroxyectoine was found (Severin, Wohlfarth and Galinski 1992). The level of ectoine and hydroxyectoine are closely regulated according to the external salt levels. Genomic analysis of H. elongata led to the identification of the genes involved in the biosynthesis and the degradation of ectoine. Ectoine is synthesized from L-aspartate-β-semialdehyde by stepwise transamination (EctB), acetylation (EctA) and closure of the ring (EctC). In some organisms ectoine can be hydroxylated to hydroxyectoine by ectoine hydroxylase (EctD). Ectoine degradation proceeds via hydrolysis of ectoine (DoeA) to Nα-acetyl-L-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). Diaminobutyric acid can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation (Schwibbert et al.2011). Chromohalobacter salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and very high activities of anaplerotic pathways were shown, necessary to replenish the tricarboxylic acid cycle with intermediates withdrawn for the biosynthesis of ectoines (Pastor et al.2013). A mathematical model was developed to simulate growth and osmoregulation in H. elongata to predict the substrate and salt dependence of growth as well as ectoine synthesis and uptake of potassium and ectoine from the medium. Potassium serves as a second messenger for hyperosmotic stress. Following osmotic upshifts a transient overregulation of the intracellular solute levels occurs (Dötsch et al.2008). Metabolic reconstruction using a systems biology approach including genomic analysis and proteomic profiling was recently used to refine our insights in the osmoregulatory strategies of H. elongata (Kindzierski et al.2017). Transport of ectoine from the medium was studied in detail in C. salexigens. This bacterium can use ectoine both as an osmotic solute and as a source of carbon, nitrogen and energy for growth. When incubated in medium containing both glucose and ectoine, C. salexigens shows a biphasic growth pattern as ectoine utilization is partially downregulated by glucose. The catabolic pathways for ectoine and hydroxyectoine operate best at optimal and high salinity (Vargas et al.2006). Halomonas and its relatives are unable of de novo biosynthesis of glycine betaine, but when supplied in the medium betaine is preferentially taken up and used as compatible solute, efficiently repressing ectoine biosynthesis. The early observation that H. elongata grown in complex medium containing yeast extract contains glycine betaine as the main osmotic solute (Imhoff and Rodriguez-Valera 1984) (Table 4) can be explained by the presence of betaine in the growth medium. A high-affinity betaine transport system was characterized in C. salexigens (an isolate that requires >0.5 M NaCl for growth, a concentration higher than that needed by the type strain of the species). Betaine, choline and choline-O-sulfate enhanced growth on agar plates containing 3 M NaCl with glucose as carbon and energy source. Competition assays showed that proline betaine and ectoine are also transported by the betaine permease, but this carrier does not transport proline, choline or choline-O-sulfate (Cánovas et al.1996). When used as carbon source, betaine effectively suppresses synthesis of ectoines in C. salexigens even at high salt (Vargas et al.2006). Halomonas elongata can take up exogenous choline and transform it to betaine (Cánovas et al.1996). Accumulation of betaine synthesized from choline increased with salt concentration up to 2.5 M NaCl (Cánovas et al.1998). As Halomonas and other members of the Halomonadaceae family use organic solutes for osmotic stabilization and maintain low intracellular ionic concentrations, no special adaptation of the intracellular proteome to the presence of high salt concentration is necessary. Still, mean isoelectric points below 6.8 were predicted for the proteins encoded by the genomes of H. elongata and C. salexigens. However, a similar trend was found even in marine Gammaproteobacteria of the genera Alteromonas and Aliivibrio, genera not known to be highly halophilic or extremely halotolerant (Elevi Bardavid and Oren 2012a; Oren 2013b). Markedly, low pI values were calculated for the periplasmic binding proteins of the ABC transport systems of C. salexigens as compared to similar proteins from non-halophilic representatives of the Gammaproteobacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae). These periplasmic proteins are exposed to the salt concentration of the medium, and their acidic nature may thus enable them to function at high salt concentrations (Oren et al.2005). Following an increase in medium salinity, the percentage of negatively charged lipids such as phosphatidylcholine and cardiolipin increases at the expense of neutral lipids such as phosphatidylethanolamine. Hyperosmotic shock also leads to increased membrane fluidity. Further documentation of the effect of salinity changes in the cell membrane is given in Table 5. Different putative osmosensors have been identified in the Halomonas—Chromohalobacter group (Table 2), including a hybrid histidine kinase, the two-component response regulator EupR, and the ectoine transporter Tea (Rodríguez-Moya et al.2010; Kunte, Trüper and Stan-Lotter 2012). Halobacillus halophilus, an aerobic halophilic representative of the Firmicutes Halobacillus halophilus (basonym : Sporosarcina halophila) was isolated from salt marsh soil, can grow over a wide range of salt concentrations and is nutritionally versatile. It is obligately dependent on chloride in its growth medium, optimal growth being possible at Cl− concentrations between 0.5 and 2.0 M (Roeßler and Müller 1998). Specific functions that are chloride-dependent include endospore germination, motility and flagellar synthesis, and glycine betaine transport (Müller and Oren 2003). Halobacillus halophilus uses both inorganic ions and organic osmotic solutes for osmotic adaptation. Organic solutes identified in the cells include glutamine and glutamate, proline and ectoine. Biosynthetic routes for N-acetyl ornithine and N-acetyl lysine are also predicted from the genome sequence (Saum et al.2013). Transcription of glnA2 (encoding a glutamine synthetase) and glutamine synthetase activity are chloride-dependent. At high salinities H. halophilus switches to the production of proline as its main compatible solute. Glutamate was identified as a ‘second messenger’ essential for proline production. During the transition from the exponential to the stationary phase proline is exchanged by ectoine. Thus, the ectoine to proline ratio increased from 0.04 in exponential culture to 27.4 in the late stationary phase (Table 4). Chloride had no effect on the expression of ect genes, but it stimulated cellular EctC synthesis and ectoine production. Changes in the chloride concentration may be sensed by a yet-to-be-identified osmosensor (Saum and Müller 2008b). Thus, H. halophilus uses a hybrid strategy to cope with high salinities, combining organic solutes with molar concentrations of salt (Cl−) intracellularly (Saum and Müller 2008a,b). With respect to the acidic nature of the proteome, H. halophilus occupies an intermediate position between the ‘salt-in’ strategists and microorganisms that rely entirely on organic compatible solutes for osmotic stabilization (Saum et al.2013). The halophilic anaerobes of the order Halanaerobiales (Firmicutes) The order Halanaerobiales, families Halanaerobiaceae and Halobacteroidaceae, form a group of obligatory anaerobic halophiles most of which grow by fermentation of sugars and amino acids, typically producing acetate, ethanol, hydrogen and CO2 as fermentation products (Kivistö and Karp 2011; Oren 2013d). One of the members of the group, Halothermothrix orenii, isolated from the sediment of a Tunisian salted lake, is a thermophile that grows optimally at 60°C and does not show growth below 42°C. Its optimal NaCl concentration is 50–100 g L−1, and it tolerates NaCl concentrations up to 200 g L−1 (Cayol et al.1994). Measurements of the intracellular ionic concentrations in Halanaerobium praevalens, an isolate from the sediments of Great Salt Lake, Utah, and Halobacteroides halobius, retrieved from the sediments of the Dead Sea, showed high intracellular K+ concentrations (0.76–2.05 M, not well correlated with the external NaCl concentration) and high apparent intracellular sodium concentrations (0.28–2.6 M, increasing with increasing extracellular NaCl concentration). The sum of the intracellular K+ and Na+ concentrations were similar to the total cation concentration of the medium. Intracellular Cl− concentrations in H. praevalens were similar to the extracellular concentration (Oren 1986). Measurements of intracellular K+, Na+ and Cl− in H. praevalens cells by X-ray microanalysis in the transmission electron microscope yielded apparent intracellular cation concentrations between 1.22 and 1.91 M and chloride concentrations of 0.93–1.57 M in cells grown in 2.6 M total salts. K+ was the major cation in exponentially growing cells. In stationary phase cells, the ratio of Na+ than K+ concentrations was highly variable (Oren, Heldal and Norland 1997). In Halanaerobium acetethylicum (basonym : Halobacteroides acetoethylicus) grown at the optimal salinity of 1.7 M NaCl, the reported internal cytoplasmic Na+, K+, Mg2+ and Cl− were 0.92, 0.24, 0.02 and 1.2 M, respectively, i.e. Na+ rather than K+ being the main intracellular cation (Rengpipat, Lowe and Zeikus 1988). No significant concentrations of organic solutes such as glycine betaine, glycerol or amino acids were detected in H. praevalens and in Halobacter. halobius (Oren 1986). Intracellular enzymes of H. acetethylicum are typically salt-resistant: activities >60% of the optimal values were found for glyceraldehyde-3-phosphate dehydrogenase in 1–2 M salt; for NAD-dependent alcohol dehydrogenase at 2–4 M salt; for pyruvate dehydrogenase at 0.5–1 M salt; and for hydrogenase (methyl viologen linked) at 0.5–3 M salt (Rengpipat, Lowe and Zeikus 1988). The fatty acid synthetase complex of H. praevalens is stimulated by 1 M NaCl or KCl, and is only slightly inhibited by 3 M salt. Thus, it can be expected to be fully active at the high intracellular salt concentrations reported for this organism (Oren and Gurevich 1993). The carbon monoxide dehydrogenase of another representative of the group, the haloalkaliphilic Natroniella acetigena, showed optimal activity in the presence of 0.7 M NaCl (Detkova and Boltyanskaya 2006). Analyses of the amino acid composition of the bulk proteins of H. praevalens and H. halobius in cell pellets first suggested a high excess of acidic amino acids over basic amino acids, i.e., the typical ‘halophilic’ signature of proteins that must be stable and active at molar concentrations of salts (Oren 1986). A high excess of acidic amino acids in the bulk protein was also reported for N. acetigena (Detkova and Boltyanskaya 2006). However, the presence of an acidic proteome was not confirmed during genomic analyses. Complete genome sequences are now available for H. praevalens (Ivanova et al.2011), H. hydrogeniformans (an isolate from Soap Lake, Washington State) (Mormile 2014) and the halothermophilic H. orenii (Mijts and Patel 2001; Mavromatis et al.2009). Analysis of these genomes did not show any unusually high contents of acidic amino acids or low contents of basic amino acids of the encoded proteins. The earlier report of an apparent excess of acidic amino acids in H. praevalens (Oren 1986) can be explained by the high content of glutamine and asparagine in the proteins, which yield glutamate and aspartate upon acid hydrolysis (Elevi Bardavid and Oren 2012b). Thus, the presence of high intracellular ionic concentrations and lack of organic compatible solutes is not combined with an acidic proteome in the members of the Halanaerobiales tested. Another interesting group of halophilic anaerobes that in addition are adapted to high temperature and high pH values is represented by Natranaerobius thermophilus and related organisms isolated from the Wadi Natrun lakes in Egypt. Phylogenetically, these organisms form a separate lineage (order Natranaerobiales) affiliated with the Clostridia. Natranaerobius thermophilus grows optimally at 3.5 M Na+, pH 9.5 and 53°C. Tolerance to alkaline pH, high salt concentrations and high temperatures necessitates mechanisms for cytoplasmic pH acidification. For that purpose N. thermophilus possesses at least eight electrogenic Na+(K+)/H+ antiporters (Table 1) (Mesbah and Wiegel 2008; Mesbah, Cook and Wiegel 2009). It has a markedly acidic proteome, with an average pI of 6.27 (Elevi Bardavid and Oren 2012a). Anoxygenic phototrophic Gammaproteobacteria of the genera Ectothiorhodospira and Halorhodospira The genera Ectothiorhodospira (Gammaproteobacteria) and Halorhodospira, which was split off from the genus Ectothiorhodospira in 1997 to accommodate the most halophilic representatives of that genus, including H. halochloris and H. halophila, consist of anoxygenic phototrophs that use reduced sulfur compounds as electron donors for CO2 reduction and that excrete elemental sulfur outside the cells when grown on sulfide as the electron donor. Some members, including H. halochloris, were isolated from soda lakes and are obligate alkaliphilic, in addition to their requirement for high salt concentrations for growth. The most halophilic representatives of the genus Halorhodospira grow optimally at salt concentrations above 150–200 g L−1. The identification of glycine betaine as the main osmotic solute in H. halochloris in 1982 (Galinski and Trüper 1982) (Table 4) marked the first documentation of the use of organic ‘compatible’ solutes by a prokaryote. Its biosynthesis proceeds via stepwise methylation of glycine. Glycine betaine has since then been identified as an osmotic solute in many other groups of prokaryotes, including the most halophilic types of cyanobacteria (see below). As further documented elsewhere in this review, many bacteria that are unable of de novo biosynthesis of glycine betaine can take the compound up from the medium. Halorhodospira halochloris also has the ability of active transport of glycine betaine present in the medium, driven by the electrochemical proton gradient generated by photosynthesis (Peters, Tel-Or and Trüper 1992). Glycine betaine is not the only organic osmotic solute produced by H. halochloris and other members of the genus Halorhodospira. The cyclic amino acid derivative ectoine, now known as the compatible solute most widespread also among non-phototrophic prokaryotes (see above), was first identified in H. halochloris, and it derives its common name from the genus Ectothiorhodospira in which the species was classified at the time (Galinski, Pfeiffer and Trüper 1985). Biosynthesis of ectoine proceeds from L-aspartate β-semialdehyde through L-2,4-diaminobutyric acid and Nγ-acetyl-L-2,4-diaminobutyric acid (Peters, Galinski and Trüper 1990). Halorhodospira halochloris produces minor amounts of a third osmotic solute: trehalose. It is produced from UDP-glucose and glucose 6-phosphate by a trehalose-6-phosphate synthase (Lippert, Galinski and Trüper 1993) (Table 4). Trehalose is synthesized especially under nitrogen limitation as a nitrogen-free alternative for the nitrogen containing glycine betaine and ectoine (Galinski and Herzog 1990). When subjected to dilution stress. Halorhodospira halochloris excretes its major compatible solute, glycine betaine, to the medium. A suddenly induced dilution stress leads to an overshoot of this reaction, releasing more glycine betaine than necessary to compensate the external osmotic change. Glycine betaine can then be taken up again until a new osmotic balance is reached. Ectoine is also excreted during dilution stress. Excess trehalose is degraded intracellularly by a trehalase with a low affinity for its substrate (Km ∼0.5 M), whose activity may be regulated by the glycine betaine concentration (Herzog, Galinski and Trüper 1990; Tschichholz and Trüper 1990). In a less halophilic representative of the group, Ectothiorhodospira marismortui (considered a later heterotypic synonym of Ect. mobilis), isolated from a hypersaline sulfur spring on the shore of the Dead Sea, three organic osmotic solutes were identified: glycine betaine, a novel compound that has not been encountered yet elsewhere: Nα-carbamoyl-L-glutamine 1-amide (CGA), which accounts for approximately 30% of the cells’ compatible solutes, and a minor amount of sucrose (Galinski and Oren 1991). The concentration of glycine betaine was estimated to increase from 0.47 to 1.29 M in cells grown from 0.85 to 2.56 M NaCl, while the estimated CGA concentration rose from about 0.2 to 0.5 M, and sucrose remained constant at ∼0.05 M (Table 4). Intracellular Na+ and K+ concentrations were estimated around 0.5 and 0.3 M, respectively, in cells grown in 1.8 M NaCl (Oren, Simon and Galinski 1991). Upon hypoosmotic shock, glycine betaine is excreted from the cells to be taken up again later, while CGA and sucrose are degraded intracellularly (Fischel and Oren 1993). The concentration of the novel compound Nα-carbamoyl glutamineamide was enhanced when l-glutamine was added to the growth medium, suggesting that glutamine served as a precursor for the synthesis of the compound (Oren, Simon and Galinski 1991). Although Hlr. halophila (neutrophilic, containing bacteriochlorophyll a as photosynthetic pigment) and H. halochloris (alkaliphilic, containing bacteriochlorophyll b) are both extremely halophilic and phylogenetically closely related as based on their 16S rRNA gene sequences, their modes of osmotic adaptation may differ greatly. Halorhodospira halophila possesses a highly acidic proteome, as apparent both from its genome sequence and from isoelectric focusing gel electrophoresis experiments, and was found to contain molar concentrations of KCl as detected by X-ray microanalysis and plasma emission spectrometry. Halorhodospira halochloris does not exhibit an acidic proteome and does not accumulate K+. When grown in low salt medium, Hlr. halophila contains only low concentrations of K+ (Deole et al.2013; Oren 2013b). This proves that an organism with a highly acidic proteome can also function in the absence of high cytoplasmic salt concentrations. Halophilic and halotolerant cyanobacteria Cyanobacteria can be found over a very large range of salt concentrations, from freshwater to hypersaline brines and microbial mats at salinities of 250 g L−1 and above. Among the most salt-tolerant types are both unicellular forms (Aphanothece/Halothece) and filamentous (Phormidium-type) cyanobacteria (Oren 2012). To adapt to high salt concentrations, cyanobacteria accumulate organic osmotic solutes. The main solutes used were identified in the early 1980s based on 13C nuclear magnetic resonance spectroscopy. When subjected to salt stress, freshwater strains generally accumulate sucrose and/or trehalose. In marine strains and some mat-forming filamentous types such as Coleofasciculus chthonoplastes that also grow at salinities above that of seawater, glucosylglycerol (O-α-d-glucopyranosyl-(1→2)-glycerol) is the solute of choice. Such ‘heterosides’ are also found in many eukaryotic algae as osmotic solutes (Hagemann and Pade 2015). The most halophilic or halotolerant cyanobacteria generally accumulate glycine betaine to achieve osmotic balance. Use of l-glutamate betaine (N-trimethyl-l-glutamate) has also been reported (Mackay, Norton and Borowitzka 1984). Many strains accumulate mixtures of these compounds, including minor compounds such as glucosylglycerate or proline (Table 4). Accumulation of sucrose and trehalose also results in an increased tolerance to desiccation and high temperature stress (Klähn and Hagemann 2011). From the genome sequences, including species that are difficult to grow in the laboratory, the types of compatible solutes accumulated could be deduced for more than 60 strains (Hagemann 2013). Osmotic solute metabolism has been studied in-depth in a number of model organisms (Oren 2012). Thus, biosynthesis of glucosylglycerol was studied in the marine unicellular cyanobacterium Synechococcus N100 by 13C-NMR. Following a hyperosmotic shock, a rapid increase in the level of this heteroside was observed, most of the solute being synthesized from photosynthetically fixed CO2, but up to 10% of the newly synthesized material could be derived from intracellular organic carbon. In the dark, no increase in glucosylglycerol was observed following a hyperosmotic shock (Mackay and Norton 1987). In recent years, Synechocystis 6803, a moderately salt-tolerant species that produces glucosylglycerol, has become a popular model organism to study molecular mechanisms of osmotic adaptation in cyanobacteria based on extrusion of ions (Na+, Cl–) and uptake and/or synthesis of organic solutes, as well as the bioenergetics basis of salt adaptation (Joset, Jeanjean and Hagemann 1996; Hagemann 2011, 2013). The unicellular green alga Dunaliella Members of the halophilic or highly halotolerant algal genus Dunaliella (Chlorophyceae) have been intensively studied, both because of their interesting adaptations to life at high salt concentrations and because of their economic importance for the production of carotenoid pigments and other valuable products (Oren 2005; Ben-Amotz, Polle and Subba Rao 2009). Dunaliella species maintain low salt concentrations in their cytoplasm as well as in their chloroplast. This was realized as early as 1968 when it was observed that NaCl at concentrations far lower than present in the growth medium (3.75 m) strongly inhibited key enzymes such as pentose phosphate isomerase, ribulose bisphosphate carboxylase, glucose-6-phosphate dehydrogenase and phosphohexose isomerase in Dunaliella viridis (Johnson et al.1968). 23Na-NMR studies in D. salina yielded apparent intracellular Na+ concentrations of 88 ± 28 mM in cells grown between 0.1 and 4.0 M NaCl (Bental, Degani and Avron 1988). Assays of the intracellular ions in D. salina grown in 1–4 M NaCl and 5 mM Mg2+ yielded values of 20–40 mM Na+, 150–300 mM K+, 20 mM SO42−, 30–50 mM Cl− and 200–350 mM Mg2+. Thus, the ionic concentrations can account only for a small part of the osomotically active substances needed to balance the outside salinity. Mg2+ and K+ probably serve as the major counterions for the negative charge of polyphosphates accumulating in the cell (Karni and Avron 1988). The importance of glycerol, the first organic osmotic solute documented in the microbial world, in the metabolism of Dunaliella spp. was first suggested by Craigie and McLachlan (1964). Production of glycerol in a salt-dependent manner as a major fraction of the photosynthetically fixed carbon was described in the early 1970s (Wegmann 1971; Ben-Amotz and Avron 1973). Thus, D. parva cells grown in the presence of 1.5 M NaCl were estimated to contain 2.1 M glycerol (Table 4). Following shifts in the external salt concentration the cells, which lack a rigid cell wall, immediately react by decreasing or increasing their volume (Chen and Jiang 2009), then the intracellular glycerol concentration is rapidly adjusted to the new salinity. A new equilibrium is reached in about 90 min, restoring the normal volume of the cells. Above a salinity of 0.6 M, no leakage of glycerol to the medium was observed in most studies, also not following a salinity downshock; excess glycerol is thus metabolized within the cells. However, the study by Enhuber and Gimmler (1980) on D. parva, based on efflux studies with labelled glycerol and other techniques, suggested that its cytoplasmic membrane does not show any special low permeability for glycerol. Here significant amounts of glycerol were found to diffuse continuously into the medium, so that continuous synthesis of glycerol at a high energetic cost is required to remain adapted to high salt. In D. parva, the internal concentration of glycerol is approximately isosmotic with the salt content of the medium over a broad range of 0.6–2.1 M NaCl. Intracellular glycerol can account for >50% of the total dry weight of the cells (Ben-Amotz and Avron 1973, 1980; Ben-Amotz 1975; Ben-Amotz, Sussman and Avron 1983). Glycerol is an excellent compatible solute: different enzymatic and photosynthetic reactions tested in cell-free preparations of D. parva showed a high tolerance to high glycerol concentrations (Ben-Amotz 1975). Biosynthesis and degradation of glycerol in Dunaliella proceeds through the so-called ‘glycerol cycle’ (Chen and Jiang 2009; Oren 2017). For the production of glycerol, dihydroxyacetone phosphate is reduced to glycerolphosphate by an NAD-specific glycerol-3-phosphate dehydrogenase, and the product is then dephosphorylated to glycerol. Excess glycerol can be returned to the central metabolism by its oxidation to dihydroxyacetone by an NADP-specific glycerol dehydrogenase (dihydroxyacetone reductase), followed by ATP-dependent phosphorylation to dihydroxyacetone phosphate, which can be used e.g. for the production of starch. A study of glycerol biosynthesis in D. tertiolecta showed that the reserve starch pool is an important carbon source for glycerol biosynthesis, not only in the dark but also in the light, where carbon for glycerol production is simultaneously derived from photosynthesis and from starch (Goyal 2007). The glycerol-3-phosphate dehydrogenase activity was found to be located in the chloroplast, whereas the glycerol dehydrogenase (dihydroxyacetone reductase) was found in the cytosol. The glycerol phosphate phosphatase reaction may also occur in the chloroplast, but the distribution of the location of the dihydroxyacetone kinase is uncertain (Brown, Lilley and Marengo 1982; Gimmler and Lotter 1982; Ben-Amotz, Sussman and Avron 1983). NaCl stimulated the activities of all four enzymes in various degrees when D. salina was grown under continuous salt stress. Following hyperosmotic or hypoosmotic shock, only the activity of glycerol-3-phosphate dehydrogenase was significantly increased, and this enzyme was proposed to be responsible for the emergency response of the ‘glycerol cycle’ (Chen, Lu and Jiang 2012). A study of the transcription of the genes for glucose-6-phosphate isomerase, fructose-1,6-diphosphate aldolase, glycerol-3-phosphate dehydrogenase and dihydroxyacetone reductase following changes in the salinity of the medium showed the regulation of glycerol metabolism to be very complex. Dihydroxyacetone reductase was indicated as the key enzyme involved in the regulation of the glycerol metabolic pathway (Zhao et al.2013). Hyperosmotic shock also induces a decrease in fructose 6-phosphate and an increase in fructose-1,6-bisphosphate, indicating the activation of phosphofructokinase. Studies of mutants defective in their response to hyperosmotic shock showed that the oxidative pentose-phosphate pathway plays a major role in glycerol synthesis by maintaining the oxidation-reduction balance (Chitlaru and Pick 1991). Hyperosmotic shock in D. salina also induces rapid changes in phospholipid metabolism mainly based on a strong activation of phospholipase A: the level of phosphatidic acid dropped and that of phosphatidylinositol 4,5-bisphosphate rose sharply within 4 min, turnover of phosphatidylcholine increased and the concentration of free fatty acids increased (Einspahr, Maeda and Thompson 1988) (Table 6). Table 6. Increased salinity-induced changes in the plasma membrane composition and fluidity in selected eukaryotic halophilic and halotolerant microorganisms The salt sensitive Saccharomyces cerevisiae was included for comparison. Microorganism Fatty acids Sterols Fluidity References Algae Dunaliella salina Main lipids: GL, PG, PC, PE, PI. Different Dunaliella species contain different quantities of lipid diacylglycerol-O-(N,N,N-trimethyl)- homoserine (3%–16%) High amount of sterols Hyperosmotic shock leads to rigidification, a hypoosmotic shock to transient fluidization and increased sterol content Curtain et al.1983; Russell 1993; Azachi et al.2002; Katz et al.2007 Increase of salinity from 0.4 to 3 M NaCl resulted in increase of PG (4×), a general increase in anionic lipids (2×), increase in GL, a higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids At high salinity fatty acid chain elongation and increased overall desaturation Protists Halocafeteria seosinensis Increased PI, lowered PE; increased shorter fatty acid chains; repressed fatty acid desaturases Lowered Increased fluidity Harding et al.2017 Fungi Wallemia ichthyophaga Decreased fluidity at suboptimal NaCl concentrations, increased at optimal concentrations. Gunde-Cimerman, unpublished data Hortaea werneckii Increase in fatty acid unsaturation Almost unchanged total sterol content; significantly lower sterol-to-phospholipid ratio than in salt-sensitive fungi Lowered fluidity at suboptimal (<5%) and above-optimal (>15%) NaCl concentrations; highest fluidity at optimal salinities Turk et al.2004, 2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Phaeotheca triangularis Slight decrease in fatty acid unsaturation, decrease in PE Almost unchanged total sterol content Higher fluidity than in salt-sensitive fungi Turk et al.2004 Aureobasidium pullulans Increase in fatty acid unsaturation Almost unchanged total sterol content; significantly higher sterol-to-phospholipid ratio than in halophilic fungi Lower fluidity than in halophilic fungi; increased fluidity at salinities above the optimal range Turk et al.2004, 2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Rhodotorula mucilaginosa Increased fluidity at above-optimal salinities Turk et al.2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Debaryomyces hansenii No great effect on PC, PE or anionic GPL, a substantial increase in PG and a decrease in PI and PS; a slight decrease in fatty acid unsaturation Significant increase in ergosterol content; increased sterol to GPL ratio No significant change of fluidity Russell 1993; Turk et al.2007b; Michán et al.2012 Yarrowia lipolytica Decreased sterols Tunblad-Johansson, Andre' and Adler 1987 Saccharomyces cerevisiae Almost unchanged total sterol content; significantly higher sterol-to-phospholipid ratio than in halophilic fungi Increased fluidity at salinities that exceed the optimal range Turk et al.2004; Simonin, Beney and Gervais 2008; Turk, Plemenitaš and Gunde-Cimerman 2011 Microorganism Fatty acids Sterols Fluidity References Algae Dunaliella salina Main lipids: GL, PG, PC, PE, PI. Different Dunaliella species contain different quantities of lipid diacylglycerol-O-(N,N,N-trimethyl)- homoserine (3%–16%) High amount of sterols Hyperosmotic shock leads to rigidification, a hypoosmotic shock to transient fluidization and increased sterol content Curtain et al.1983; Russell 1993; Azachi et al.2002; Katz et al.2007 Increase of salinity from 0.4 to 3 M NaCl resulted in increase of PG (4×), a general increase in anionic lipids (2×), increase in GL, a higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids At high salinity fatty acid chain elongation and increased overall desaturation Protists Halocafeteria seosinensis Increased PI, lowered PE; increased shorter fatty acid chains; repressed fatty acid desaturases Lowered Increased fluidity Harding et al.2017 Fungi Wallemia ichthyophaga Decreased fluidity at suboptimal NaCl concentrations, increased at optimal concentrations. Gunde-Cimerman, unpublished data Hortaea werneckii Increase in fatty acid unsaturation Almost unchanged total sterol content; significantly lower sterol-to-phospholipid ratio than in salt-sensitive fungi Lowered fluidity at suboptimal (<5%) and above-optimal (>15%) NaCl concentrations; highest fluidity at optimal salinities Turk et al.2004, 2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Phaeotheca triangularis Slight decrease in fatty acid unsaturation, decrease in PE Almost unchanged total sterol content Higher fluidity than in salt-sensitive fungi Turk et al.2004 Aureobasidium pullulans Increase in fatty acid unsaturation Almost unchanged total sterol content; significantly higher sterol-to-phospholipid ratio than in halophilic fungi Lower fluidity than in halophilic fungi; increased fluidity at salinities above the optimal range Turk et al.2004, 2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Rhodotorula mucilaginosa Increased fluidity at above-optimal salinities Turk et al.2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Debaryomyces hansenii No great effect on PC, PE or anionic GPL, a substantial increase in PG and a decrease in PI and PS; a slight decrease in fatty acid unsaturation Significant increase in ergosterol content; increased sterol to GPL ratio No significant change of fluidity Russell 1993; Turk et al.2007b; Michán et al.2012 Yarrowia lipolytica Decreased sterols Tunblad-Johansson, Andre' and Adler 1987 Saccharomyces cerevisiae Almost unchanged total sterol content; significantly higher sterol-to-phospholipid ratio than in halophilic fungi Increased fluidity at salinities that exceed the optimal range Turk et al.2004; Simonin, Beney and Gervais 2008; Turk, Plemenitaš and Gunde-Cimerman 2011 Glycerophospholipids (GPL): phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS); Glycolipids (GL). View Large Table 6. Increased salinity-induced changes in the plasma membrane composition and fluidity in selected eukaryotic halophilic and halotolerant microorganisms The salt sensitive Saccharomyces cerevisiae was included for comparison. Microorganism Fatty acids Sterols Fluidity References Algae Dunaliella salina Main lipids: GL, PG, PC, PE, PI. Different Dunaliella species contain different quantities of lipid diacylglycerol-O-(N,N,N-trimethyl)- homoserine (3%–16%) High amount of sterols Hyperosmotic shock leads to rigidification, a hypoosmotic shock to transient fluidization and increased sterol content Curtain et al.1983; Russell 1993; Azachi et al.2002; Katz et al.2007 Increase of salinity from 0.4 to 3 M NaCl resulted in increase of PG (4×), a general increase in anionic lipids (2×), increase in GL, a higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids At high salinity fatty acid chain elongation and increased overall desaturation Protists Halocafeteria seosinensis Increased PI, lowered PE; increased shorter fatty acid chains; repressed fatty acid desaturases Lowered Increased fluidity Harding et al.2017 Fungi Wallemia ichthyophaga Decreased fluidity at suboptimal NaCl concentrations, increased at optimal concentrations. Gunde-Cimerman, unpublished data Hortaea werneckii Increase in fatty acid unsaturation Almost unchanged total sterol content; significantly lower sterol-to-phospholipid ratio than in salt-sensitive fungi Lowered fluidity at suboptimal (<5%) and above-optimal (>15%) NaCl concentrations; highest fluidity at optimal salinities Turk et al.2004, 2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Phaeotheca triangularis Slight decrease in fatty acid unsaturation, decrease in PE Almost unchanged total sterol content Higher fluidity than in salt-sensitive fungi Turk et al.2004 Aureobasidium pullulans Increase in fatty acid unsaturation Almost unchanged total sterol content; significantly higher sterol-to-phospholipid ratio than in halophilic fungi Lower fluidity than in halophilic fungi; increased fluidity at salinities above the optimal range Turk et al.2004, 2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Rhodotorula mucilaginosa Increased fluidity at above-optimal salinities Turk et al.2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Debaryomyces hansenii No great effect on PC, PE or anionic GPL, a substantial increase in PG and a decrease in PI and PS; a slight decrease in fatty acid unsaturation Significant increase in ergosterol content; increased sterol to GPL ratio No significant change of fluidity Russell 1993; Turk et al.2007b; Michán et al.2012 Yarrowia lipolytica Decreased sterols Tunblad-Johansson, Andre' and Adler 1987 Saccharomyces cerevisiae Almost unchanged total sterol content; significantly higher sterol-to-phospholipid ratio than in halophilic fungi Increased fluidity at salinities that exceed the optimal range Turk et al.2004; Simonin, Beney and Gervais 2008; Turk, Plemenitaš and Gunde-Cimerman 2011 Microorganism Fatty acids Sterols Fluidity References Algae Dunaliella salina Main lipids: GL, PG, PC, PE, PI. Different Dunaliella species contain different quantities of lipid diacylglycerol-O-(N,N,N-trimethyl)- homoserine (3%–16%) High amount of sterols Hyperosmotic shock leads to rigidification, a hypoosmotic shock to transient fluidization and increased sterol content Curtain et al.1983; Russell 1993; Azachi et al.2002; Katz et al.2007 Increase of salinity from 0.4 to 3 M NaCl resulted in increase of PG (4×), a general increase in anionic lipids (2×), increase in GL, a higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids At high salinity fatty acid chain elongation and increased overall desaturation Protists Halocafeteria seosinensis Increased PI, lowered PE; increased shorter fatty acid chains; repressed fatty acid desaturases Lowered Increased fluidity Harding et al.2017 Fungi Wallemia ichthyophaga Decreased fluidity at suboptimal NaCl concentrations, increased at optimal concentrations. Gunde-Cimerman, unpublished data Hortaea werneckii Increase in fatty acid unsaturation Almost unchanged total sterol content; significantly lower sterol-to-phospholipid ratio than in salt-sensitive fungi Lowered fluidity at suboptimal (<5%) and above-optimal (>15%) NaCl concentrations; highest fluidity at optimal salinities Turk et al.2004, 2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Phaeotheca triangularis Slight decrease in fatty acid unsaturation, decrease in PE Almost unchanged total sterol content Higher fluidity than in salt-sensitive fungi Turk et al.2004 Aureobasidium pullulans Increase in fatty acid unsaturation Almost unchanged total sterol content; significantly higher sterol-to-phospholipid ratio than in halophilic fungi Lower fluidity than in halophilic fungi; increased fluidity at salinities above the optimal range Turk et al.2004, 2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Rhodotorula mucilaginosa Increased fluidity at above-optimal salinities Turk et al.2007a; Turk, Plemenitaš and Gunde-Cimerman 2011 Debaryomyces hansenii No great effect on PC, PE or anionic GPL, a substantial increase in PG and a decrease in PI and PS; a slight decrease in fatty acid unsaturation Significant increase in ergosterol content; increased sterol to GPL ratio No significant change of fluidity Russell 1993; Turk et al.2007b; Michán et al.2012 Yarrowia lipolytica Decreased sterols Tunblad-Johansson, Andre' and Adler 1987 Saccharomyces cerevisiae Almost unchanged total sterol content; significantly higher sterol-to-phospholipid ratio than in halophilic fungi Increased fluidity at salinities that exceed the optimal range Turk et al.2004; Simonin, Beney and Gervais 2008; Turk, Plemenitaš and Gunde-Cimerman 2011 Glycerophospholipids (GPL): phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS); Glycolipids (GL). View Large Sensing and responding to osmotic changes and other environmental stresses in Dunaliella spp. is mediated by the high-osmolarity glycerol (HOG) signal transduction pathway, similar to that found in in yeast and other fungi (see below). The presence of this pathway has been confirmed in D. viridis and in D. tertiolecta (Table 7). In D. viridis, a Hog1-like protein was upregulated and phosphorylated under stress conditions. Treatment with a specific inhibitor of mitogen-activated protein kinases (MAPK) markedly impaired the response of Dunaliella to osmotic stress, thereby revealing its critical role in adaptation and survival under such environmental conditions (Jiménez et al.2004). In D. tertiolecta, upregulation of Hog1-like MAPK (DtMAPK) was observed under hyperosmotic conditions (Zhao et al.2016). Moreover, it was found, that this MAPK regulates the synthesis of glycerol, as suppressed transcription of the key regulatory enzyme of glycerol synthesis, glycerolphosphate dehydrogenase (GPDH), together with delayed accumulation of intracellular glycerol were observed in cells lacking the DtMAPK gene. The HOG signal transduction pathway thus probably plays a critical role in adaptation to hypersaline stress in Dunaliella, at least by turning on the expression of the osmoresponsive gene GPDH for the synthesis of glycerol. Table 7. Major identified proteins involved in sensing increased salinity and in the signal transduction pathway in eukaryotic halotolerant/halophilic microorganisms The salt sensitive Saccharomyces cerevisiae was included for comparison. HOG (high osmolarity glycerol pathway) Saccharomyces cerevisiae Hortaea werneckii Wallemia ichthyophaga Aureobasidium pullulans Debaryomyces hansenii Dunaliella viridis Dunaliella tertiolecta Transmembrane osmosensor Sho1 HwSho1A/B MAPKKK Ste11 HwSte11A/B WiSte11 Hybrid histidine kinase Sln1 HwHhk7A/B WiNik1 DhNik1 Phosphorelay response regulator Ssk1 HwSsk1A/B WiSsk1 MAPKKK Ssk2 HwSsk2A/B WiSsk2 MAPKK Pbs2 HwPbs2A/B WiPbs2 DhPbs2 MAPK Hog1 HwHog1A/B WiHog1A/B ApHog1 DhHog1 DvHog1 like MAPK DtMAPK-Hog1like function HOG (high osmolarity glycerol pathway) Saccharomyces cerevisiae Hortaea werneckii Wallemia ichthyophaga Aureobasidium pullulans Debaryomyces hansenii Dunaliella viridis Dunaliella tertiolecta Transmembrane osmosensor Sho1 HwSho1A/B MAPKKK Ste11 HwSte11A/B WiSte11 Hybrid histidine kinase Sln1 HwHhk7A/B WiNik1 DhNik1 Phosphorelay response regulator Ssk1 HwSsk1A/B WiSsk1 MAPKKK Ssk2 HwSsk2A/B WiSsk2 MAPKK Pbs2 HwPbs2A/B WiPbs2 DhPbs2 MAPK Hog1 HwHog1A/B WiHog1A/B ApHog1 DhHog1 DvHog1 like MAPK DtMAPK-Hog1like function View Large Table 7. Major identified proteins involved in sensing increased salinity and in the signal transduction pathway in eukaryotic halotolerant/halophilic microorganisms The salt sensitive Saccharomyces cerevisiae was included for comparison. HOG (high osmolarity glycerol pathway) Saccharomyces cerevisiae Hortaea werneckii Wallemia ichthyophaga Aureobasidium pullulans Debaryomyces hansenii Dunaliella viridis Dunaliella tertiolecta Transmembrane osmosensor Sho1 HwSho1A/B MAPKKK Ste11 HwSte11A/B WiSte11 Hybrid histidine kinase Sln1 HwHhk7A/B WiNik1 DhNik1 Phosphorelay response regulator Ssk1 HwSsk1A/B WiSsk1 MAPKKK Ssk2 HwSsk2A/B WiSsk2 MAPKK Pbs2 HwPbs2A/B WiPbs2 DhPbs2 MAPK Hog1 HwHog1A/B WiHog1A/B ApHog1 DhHog1 DvHog1 like MAPK DtMAPK-Hog1like function HOG (high osmolarity glycerol pathway) Saccharomyces cerevisiae Hortaea werneckii Wallemia ichthyophaga Aureobasidium pullulans Debaryomyces hansenii Dunaliella viridis Dunaliella tertiolecta Transmembrane osmosensor Sho1 HwSho1A/B MAPKKK Ste11 HwSte11A/B WiSte11 Hybrid histidine kinase Sln1 HwHhk7A/B WiNik1 DhNik1 Phosphorelay response regulator Ssk1 HwSsk1A/B WiSsk1 MAPKKK Ssk2 HwSsk2A/B WiSsk2 MAPKK Pbs2 HwPbs2A/B WiPbs2 DhPbs2 MAPK Hog1 HwHog1A/B WiHog1A/B ApHog1 DhHog1 DvHog1 like MAPK DtMAPK-Hog1like function View Large Halophilic heterotrophic protists Although heterotrophic protists can be found in salt lakes and saltern ponds up to NaCl saturation, very little is known about their strategies for osmotic adaptation. The first studies to elucidate their mode of salt adaptation were published only recently. Transcriptomic studies of the obligately halophilic protist Halocafeteria seosinensis (Stramenopiles) showed increased expression of enzymes involved in synthesis and transport of organic osmotic solutes such as hydroxyectoine and myo-inositol when grown at the highest salt concentrations, suggesting that such compounds may be used as osmolytes (Table 4). Comparative studies showed that H. seosinensis and Pharyngomonas kirbyi (Heterolobosea) had undergone fewer substitutions from hydrophilic to hydrophobic residues since divergence from their closest non-halophilic relatives, and it was concluded that the halophilic protists may have a higher intracellular salt content than marine protists. However, their proteins lack the characteristic acidic signature of microorganisms that use the salt-in strategy (Harding et al.2016). Genes whose expression was highly responsive to salinity variations included those involved in stress response (e.g. chaperones), in ion homeostasis (e.g. a Na+/H+ transporter) (Table 1), and in metabolism and transport of lipids (e.g. sterol biosynthetic genes) (Table 6) (Harding et al.2016; Harding, Roger and Simpson 2017). The black yeast Hortaea werneckii Hortaea werneckii (Horta) Nishim & Miyaji is a cosmopolitan black yeast (Capnodiales, Dothideomycetidae, Dothideomycetes, Pezizomycotina) without any known teleomorph. Hortaea werneckii was for long known only as the primary etiological agent of tinea nigra, occurring on salty human hands and feet. Although it can be isolated from different marine environments including the deep-sea, brine in eutrophic solar salterns is its primary ecological niche (Butinar et al.2005; Gunde-Cimerman and Zalar 2014). In salterns it represents up to 80% of all fungal isolates. Besides NaCl, H. werneckii also tolerates high concentrations of the chaotropic salts MgCl2 and CaCl2, and it can be isolated from Mg-rich bitterns (Zalar, unpublished data). Hortaea werneckii is the only black yeast that can grow across the whole range of NaCl concentrations, from 0% to NaCl saturation at 32%, with a broad optimum from 6% to 14% NaCl (Butinar et al.2005). Therefore, it is an important model organism for the study of halotolerance in Eukarya (Turk et al.2004; Kogej et al.2005, 2006, 2007; Lenassi et al.2011; Plemenitaš et al.2014). Extremely saline conditions induce in H. werneckii an extremophilic phenotype, characterized by enhanced melanisation, meristematic growth, and changes in size and appearance of the colonies. The molecular responses involve rigorous changes in gene expression that lead to subsequent synthesis of compatible solutes (Table 4), regulation of intracellular alkali-metal cations, changed composition of the cell membrane (Table 6) and changed cell-wall ultrastructure and morphology. Inhibition of melanine synthesis perturbs the integrity of the cell wall and cell division, and exposes cells to the harmful effects of high NaCl concentrations (Kogej et al.2006), while at lower NaCl concentrations increased porosity of the cell wall contributes to glycerol leakage (Kogej et al.2007). In yeasts, the branched MAPK signal-transduction system, HOG signaling transduction pathway is used for sensing increased osmolarity of the medium, required for cellular adaptation to hypersaline stress, as well as to oxidative, heavy-metal and temperature stress. The upstream part of this pathway consists of two functionally redundant, but structurally distinct, branches, known as the SHO1 and SLN1 branches, that converge at Pbs2 MAP2K, which is an activator of Hog1 MAPK (Hohmann 2002, 2009; Bahn 2008). Homologues of the HOG pathway components have been extensively studied in H. werneckii (Fettich et al.2011; Plemenitaš et al.2014; Kejžar et al.2015). The HOG signal transduction pathway in H. werneckii is very robust and crucial for its survival at high NaCl concentrations, but not at low and moderate NaCl concentrations. Its genome contains the key components of HOG involved in sensing increased NaCl and regulation of the transcription of osmoresponsive genes (Table 7; Fig. 1). It includes two functionally redundant MAPK homologues, HwHog1A and HwHog1B, that show osmolyte-type-dependent phosphorylation. Unlike in other fungal species, the key MAP kinase HwHog1 is fully activated by constitutive phosphorylation with the upstream kinase Hw Pbs2 only above 3 M NaCl. MAPK Hog1 is activated by both branches, SLN1 and SHO1 (Fettich et al.2011). Besides the typical membrane Sln1 histidine kinase of the SLN1 branch, H. werneckii contains an additional cytoplasmic group VII histidine kinase HwHhk7B with a putative role in osmosensing (Lenassi et al.2007). Despite constitutive phosphorylation, the activation of osmoresponsive gene transcription is transient, suggesting interplay with additional factors (Kejžar et al.2015). Hortaea werneckii can discriminate between different osmolytes: NaCl induces continuous phosphorylation of HwHog1, whereas KCl or sorbitol induce transient phosphorylation. A similar effect was observed also at the level of transcription of HOG responsive genes, with early induction of transcription as a response to NaCl stress, early or late induction with sorbitol, and without effects with KCl (Kejžar et al.2015). This osmolyte-specific response was also observed in the expression of genes related to mitochondrial function. At high concentrations of NaCl increased expression of genes involved in energy production and oxidative damage protection was observed, whereas adaptation to the non-ionic osmolytes resulted in decreased ATP synthesis and lipid peroxidation. These results were confirmed with the study of the mitochondrial proteome. At high NaCl energy metabolism-related enzymes accumulated, while non-ionic osmolytes resulted in the accumulation of protein chaperones (Vaupotič et al.2008). Figure 1. View largeDownload slide Proposed architecture of HOG pathway in halotolerant H. werneckii and halophilic W. ichthyophaga. Environmental osmolarity is detected in halotolerant H. werneckii by HwSho1A/B and HwSln1A/B sensors and hyperosmolarity signal is then transduced to MAP kinase kinase kinases (MAPKKK) HwSte11A/B and HwSsk2A/B, respectively, and further to MAP kinase (MAPKK) HwPbs2A/B1/B2, which finally doubly-phosphorylates MAP kinase (MAPK) HwHog1A/B. Activated HwHog1A/B is then transferred to the nucleus, where it regulates the expression of osmoresponsive genes. In halophilic W. ichthyophaga, the hyperosmolarity signal is transduced only via MAPKKK WiSsk2A to MAPKK WiPbs2, which phosphorylates WiHog1A/B. To get osmoadaptation in hypo- and hyperosmolarity conditions, WiHog1A/B are dephosphorylated. Figure 1. View largeDownload slide Proposed architecture of HOG pathway in halotolerant H. werneckii and halophilic W. ichthyophaga. Environmental osmolarity is detected in halotolerant H. werneckii by HwSho1A/B and HwSln1A/B sensors and hyperosmolarity signal is then transduced to MAP kinase kinase kinases (MAPKKK) HwSte11A/B and HwSsk2A/B, respectively, and further to MAP kinase (MAPKK) HwPbs2A/B1/B2, which finally doubly-phosphorylates MAP kinase (MAPK) HwHog1A/B. Activated HwHog1A/B is then transferred to the nucleus, where it regulates the expression of osmoresponsive genes. In halophilic W. ichthyophaga, the hyperosmolarity signal is transduced only via MAPKKK WiSsk2A to MAPKK WiPbs2, which phosphorylates WiHog1A/B. To get osmoadaptation in hypo- and hyperosmolarity conditions, WiHog1A/B are dephosphorylated. Global transcriptomic studies revealed many novel osmoresponsive genes, not previously connected to adaptation to increased salinity in the not highly salt-tolerant Saccharomyces cerevisiae and other fungi. At high NaCl concentrations (4.5 M NaCl), genes functionally associated with energy supply were highly represented (Petrovič, Gunde-Cimerman and Plemenitaš 2002; Vaupotič and Plemenitaš 2007; Gostinčar et al.2011). Direct interaction with MAP kinase HwHog 1 was demonstrated for more than one-third of the salt-responsive genes (Vaupotič and Plemenitaš 2007). Proteome analysis of H. werneckii cell lysates showed that the proteins are more acidic than those of the salt sensitive S. cerevisiae or the halotolerant Debaryomyces hansenii. Further analysis of subcellular proteome fractions revealed that only plasma membrane proteins in H. werneckii are highly acidic, while the cytosolic proteins did not diverge from the proteins of other investigated fungi (Gostinčar et al.2011). Hortaea werneckii uses glycerol as the primary compatible solute (Petrovič et al.2002; Kogej et al.2007). The genes that encode the key regulatory enzyme involved in glycerol biosynthesis, glycerol-3-phosphate dehydrogenase (Gpd), are present in two isoforms. Expression of both is salt-responsive and upregulated by the HOG pathway at high salt concentrations (Vaupotič and Plemenitaš 2007; Lenassi et al.2011). Hortaea werneckii also accumulates other complementary compatible solutes such as erythritol, arabitol and mannitol (Kogej et al.2007), and (only at lower salinities) a mycosporine-glutaminol-glucoside, involved otherwise in fungal sporulation and UV protection (Table 4) (Kogej et al.2006). Hortaea werneckii maintains low intracellular concentrations of sodium and potassium cations, and it is therefore considered as a Na+ excluder with an effective transport system for extrusion of Na+ cations, accompanied by prevention of their influx (Kogej et al.2005). One of the most important transporters for the extrusion of Na+ is the P-type (ENA-like) ATPase, a sodium–potassium pump. Two salt-responsive Ena pumps and their salt-dependent activities were discovered in H. werneckii (Gostinčar et al.2011), while later genome analysis uncovered an enrichment in genes coding for alkali–metal cation transporters (Table 1). They are not only duplicated but exist in multiple copies, and are unusually expanded (Lenassi et al.2013; Sinha et al.2017). Enrichment was observed in transporters which maintain high intracellular K+/Na+ ratios and are involved in the efflux of Na+ and the uptake of K+, like ENA, NHA and TRK at high NaCl concentration. Enrichment was also found in transporters involved in K+ efflux and with Na+ intake, TOK and PHO, respectively, at low NaCl concentration. This fits well with the lifestyle of H. werneckii which thrives in environments with fluctuating NaCl concentrations: maintenance of appropriate K+/Na+ ratios is not only crucial at high NaCl concentration but also when the concentration drops (Lenassi et al.2013; Plemenitaš et al.2016; Sinha et al.2017). Additional specific biochemical signatures of halophily that were recognized in H. werneckii are 3΄-phosphoadenosine-5΄-phosphatase encoded by the HAL2 gene and HMGCoA reductase, the main regulatory enzyme in the mevalonate pathway. Both are present in two isoforms. Hal2 is essential for sulfur assimilation in yeast and was found to be associated with halotolerance. In S. cerevisiae, the gene HAL2/MET22 encodes a sodium and lithium-sensitive Hal2 protein (Glaser et al.1993). Improvement of halotolerance was observed in the halotolerant yeast D. hansenii, when DHAL2 was overexpressed (Aggarwal, Bansal and Monda 2005). Hal2 proteins, which contribute significantly to the adaptation of H. werneckii to fluctuations in environmental salinity, differ from mesophilic enzymes in the specific META sequence motif with evident effect on salt tolerance in the HwHal2B isoform (Vaupotič et al.2008). Regulation of HMG-CoA reductase in H. werneckii was also salinity-dependent, both on the level of enzyme activity and protein. Salinity-dependent regulation of HMG-CoA reductase was shown also in other halotolerant fungi, like Aureobasidium pullulans, Trimmatostroma salinum and Eurotium amstelodami (Vaupotič et al.2008). Regulation of expression or activity of HMGR in response to non-optimal salinity was demonstrated for the moderately halophilic archaeon Hfx. volcanii (Bidle et al.2007). In H. werneckii, the biological consequence of HMG-CoA regulation is related to post-translational modification of proteins by prenylation and not to sterol synthesis as shown in S. cerevisiae. Maintenance of the demonstrated high levels of HMG-CoA reductase both in hyposaline and hypersaline environments may reflect physiological adaptation to metabolic demands under extreme conditions. Eukaryotes that use glycerol as compatible solute need to prevent its leaking through the membrane. The H. werneckii plasma membrane, in contrast to membranes of salt-sensitive fungi, retained fluidity over a wide range of salinities due to an increase in the content and length of unsaturated fatty acids (Table 6) (Turk et al.2004; Turk, Plemenitaš and Gunde-Cimerman 2011). In H. werneckii, changes in the expression of fatty-acid-modifying enzymes (e.g. desaturases, elongases), which also appear in two forms, can allow for precise regulation of membrane fluidity (Gostinčar et al.2011). Furthermore, the melanised cell wall of H. werneckii with a continuous layer of melanin granules in the outer part of the cell wall contributes to the glycerol retention (Kogej et al.2007). Genome sequencing (Lenassi et al.2013; Sinha et al.2017) revealed an almost 50 Mb genome. This large size is not due to repeated DNA, but to close to 16 000 genes of which 90% are in duplicates, with an average of 5% protein sequence divergence between them. Halotolerance genes, in particular, metallo-cation transporters, are all present in multiple copies. It appears that whole genome duplication was recent and is stable, since the majority of the genome's ohnologs (paralogous genes that have originated by a process of whole-genome duplication) have not yet diverged at the level of gene expression of chromatin structure, and since even after long-term growth in the presence of high concentrations of NaCl the genome was not reduced (Sinha et al.2017). The basidomycetous fungus Wallemia ichthyophaga Wallemia Johan-Olsen (Wallemiales, Wallemiomycetes) is a genus of cosmopolitan xerophilic filamentous fungi that grow as powdery, brown colonies in a wide variety of low water activity environments (Zalar et al.2005). The monotypic genus initially included only W. ichthyophaga, later renamed as W. sebi. Analysis of molecular data enabled description of seven xerophilic species, namely W. sebi, W. muriae and W. ichthyophaga (Zalar et al.2005), W. hederae (Jančič et al.2016), W. mellicola, W. canadensis and W. tropicalis (Jančič et al.2015). The phylogenetic position of the class Wallemiomycetes was unclear until genome sequencing showed that it is a sister group of the Agaricomycotina (Zajc et al.2013), recently upgraded to the subphylum Wallemiomycotina (Zhao et al.2017). Although xerotolerance is rare in the Basidiomycota, W. ichthyophaga is the most halophilic fungus known, with no known teleomorph and no discernible MAT locus (Zajc et al.2013). It needs a minimum of 10% (w/v) NaCl in the medium, is metabolically active even at 32% NaCl and has the growth optimum at 15–20% NaCl (Zajc et al.2014). To date, only 24 strains of W. ichthyophaga have been isolated, all from hypersaline waters of solar salterns, bitterns and salted meat (Zalar et al.2005; Jančič et al.2016). Wallemia ichthyophaga, like H. werneckii, detects changes in environmental salinity by the HOG signaling pathway, although with specific adaptations. All components of the HOG pathway but the final MAP kinase Hog 1 (WiHog1 A and B) are present in only one form (Table 7; Fig. 1). Only WiHog1B is functionally fully active and can interact optimally with the upstream kinase WiPbs2. Transcript levels of WiHog1 are salt-dependent (Konte and Plemenitaš 2013). The absence of orthologs of the mucins Msb2 and Hkr1 and the membrane anchor Opy2, which in yeasts are involved in the osmosensing apparatus of the SHO1 branch of the HOG pathway, and the poor interactions between the Sho1 protein and MAPKK Pbs2 suggest that the SHO1-branch is not involved in the activation of WiHog1 (Konte et al.2016). While there is no evidence for the presence of a membrane-spanning Sln1-like histidine kinase of the SLN1 branch, W. ichthyophaga contains a homologue of the cytosolic group III histidine kinase WiNik1 that can act as an osmosensor through the HAMP domain repeats (Konte et al.2016). The presence of all other components of the SLN1 branch in the W. ichthyophaga, together with the demonstrated fludioxonil sensitivity, support activation of the HOG pathway by the SLN1 branch (Konte et al.2016). In contrast to most studied fungi, the MAP kinase WiHog1 is constitutively phosphorylated under optimal osmotic conditions and is dephosphorylated in both hyposaline and hypersaline conditions (Konte and Plemenitaš 2013). This suggests the importance of the phosphatases in the unique salt-dependent modulation of the HOG signal transduction pathway (Fig. 1). In W. ichthyophaga besides the HOG pathway, HMGCoA reductase also acts as a salt-sensing protein and responds with a characteristic salt-dependent U-shape activity pattern, both at hypo- and hypersaline conditions (Vaupotič et al.2008). The main compatible solutes in W. ichthyophaga are glycerol and to a minor extent arabitol (Table 4) (Zajc et al.2014). Expression studies uncovered a relatively low level of glycerol-3-phosphate-dehydrogenase (WiGPD1) and a slow response to a hyperosmotic shock (Lenassi et al.2011). The expression of GPD1 is regulated by MAP kinase WiHog1 (Konte and Plemenitaš 2013). Efficient interaction between GPD1 promoter and WiHog1B but not WiHog1A was demonstrated. The GPD homologue WiGPD1 lacks the N-terminal peroxisomal targeting PTS2 sequence (Lenassi et al.2011) suggesting the cytosolic localization of the enzyme. In line with the strategy of compatible solutes, the intracellular levels of K+ and Na+ in W. ichthyophaga remain low at constant salinities (Zajc et al.2013). However, when under hyperosmotic shock, the levels of both cations increase significantly, indicating its poor capability to adjust to changing environments. Additionally, in cells grown at 10% and 30% NaCl, the large majority of the genes that encode metal–cation transporters (Table 1) were not differentially expressed. Furthermore, in W. ichthyophaga the expression of transporter coding genes is relatively low: of the total of 4884 genes, neither the Na+-exporting P-type ATPases nor the two Na+/H+ antiporters are among the 2000 most expressed genes at high salinity. The possible post-transcriptional regulation was not yet investigated (Zajc et al.2013). Morphological studies of W. ichthyophaga have shown that the changes at the level of cell aggregates and cell wall thickness are crucial. The thickness of the cell wall can increase up to 3-fold, while the size of cell clumps can increase even more (Zajc et al.2014). The genome of W. ichthyophaga is unusually small—only 9.6 Mb. Its compactness is reflected in its low level of repetitive sequences (1.67%) and high gene density (514 genes/Mb scaffold). Almost three quarters of the genome are covered by coding DNA sequences. The absolute number of predicted proteins in W. ichthyophaga (4884) (Zajc et al.2013) is also unusually small for a basidiomycete, where more than 10 000 proteins are not uncommon. Several protein families are significantly expanded or contracted in the genome. Some orthologs that are involved in the osmosensing apparatus of the SHO1 branch of the HOG pathway and membrane histidine kinase Sln1 are missing. However, the genome contained homologs of the cytosolic group III histidine kinase WiNik1 (Table 7) (Zajc et al.2013; Konte et al.2016). Although the genes for the enzymes known to be involved in compatible solute management were found in the genome, there were only a low number genes coding for cation transporters. Genes like SLT1 for the plasma membrane glycerol/H+ symporter and FPS1 that codes for aquaglyceroporin channel Fps1 were found in multiple copies, suggesting their involvement in the regulation of glycerol levels. Among expanded gene families were the P-type ATPase cation transporters and genes coding for cell wall proteins—hydrophobins, that were also differentially expressed. Their transcriptional response shows their important role in adaptation to salinity, either by modulation of the cell walls or by aggregating the cells into multicellular clumps. When compared to other fungi, hydrophobins in W. ichthyophaga contain a higher proportion of acidic amino acids at the protein surface, indicating adaptation to salt exposure (Zajc et al.2013). GENERAL DISCUSSION Glycerol—‘God's gift to solute-stressed eukaryotes’ Glycerol is by far the simplest organic osmotic solute: it is the smallest of all, and accordingly its synthesis is simple and requires much less energy than the biosynthesis of all other compatible solutes detected in halophilic and halotolerant microorganisms. Production of organic osmotic solutes is energetically expensive, and therefore there is a clear advantage for organisms that can reduce the energy cost of osmotic adaptation by producing smaller and simpler solutes (Oren 1999, 2011b). Still, glycerol is used as an osmotic solute only by some halophilic and halotolerant eukaryotic microorganisms: algae such as Dunaliella and Asteromonas (Ben-Amotz and Avron 1980) and many fungi and yeasts (Table 4) (André, Nilsson and Adler 1988; Blomberg and Adler 1992; Blomberg 2000; Hohmann 2002; Petrovič, Gunde-Cimerman and Plemenitaš 2002; Kogej et al.2007; Lenassi et al.2011). The statement that glycerol is ‘God's gift to solute stressed eukaryotes’ (Brown 1990) is therefore very appropriate. The question must therefore be addressed why this simple compound is not used by salt-stressed prokaryotes as well. The answer must be sought in the high permeability of most biological membranes to glycerol. One of the first conditions to be met for an osmotic solute is that it must be retained by the cytoplasmic membrane. Some leakage can possibly be tolerated, and it is generally compensated by the presence of active transport mechanisms in the membrane that can retrieve solutes lost to the medium. Such transport mechanisms are commonly found for glycine betaine, but also for some larger osmotic solutes such as glucosylglycerol in some cyanobacteria (Mikkat, Hagemannn and Schoor 1996). Indeed, the transport of solutes against a concentration gradient requires energy, but it is still much cheaper than de novo synthesis of the compounds. While an early study on Dunaliella parva did not show an especially low permeability to glycerol of the membrane, claiming that significant amounts of glycerol diffuse continuously into the medium (Enhuber and Gimmler 1980), several later studies have ascertained that the permeability of the cytoplasmic membrane of D. salina and D. parva for glycerol is several orders of magnitude lower than other membranes tested (Brown et al.1976; Gimmler and Hartung 1988). The reason for this unusually low glycerol permeability is not completely clear, but it may be connected with the high content of sterol peroxides and sterols in the membrane. The cytoplasmic membrane of D. salina contains 7-dehydroporiferasterol peroxide and ergosterol peroxide (22% of the total lipids) and free sterol (5%) (Sheffer et al.1986). Cell membranes of fungi and yeasts are also rich in sterols (Table 6) (Turk et al.2004, 2007a,b), but with no known effects on glycerol permeability. In mesophilic S. cerevisiae, the level of intracellular glycerol very much depends on the regulation of the transmembrane transport through the channel protein Fps1p, which opens to release glycerol at low NaCl concentrations and closes to retain glycerol inside the cell at high NaCl. A similar kind of channel was proposed for the halotolerant D. hansenii (Gori et al.2005). In H. werneckii on the other hand, melanization of the outer part of the cell wall seems to effectively reduce the permeability and leaking of its major compatible solute glycerol (Kogej et al.2007), while in W. ichthyophaga up to 3-fold increase of thickness of the cell wall and the hydrophobins at its surface at high salinities prevent glycerol leakage (Zajc et al.2013). Glycine betaine: an osmotic solute used by many prokaryotes, even by those that cannot synthesize it After glycerol, which can only be used as an osmotic solute by eukaryotes with a cytoplasmic membrane able to retain the solute inside the cell, glycine betaine is the smallest compatible solute. Accordingly, its biosynthesis is energetically less expensive than the production of other solutes found in the prokaryotic world (Oren 1999, 2011b). It also has a very high solubility in water. Therefore, it is not surprising that it is used by a great variety of Bacteria as well as Archaea (Table 4), either by de novo biosynthesis or following accumulation from the environment. It is generally produced by stepwise methylation of glycine but formation from choline is also possible. Among the Bacteria, biosynthesis of glycine betaine is mainly, but not exclusively, found in phototrophic species. The most halophilic or halotolerant among the cyanobacteria use betaine as their sole or main osmotic solute (Mackay, Norton and Borowitzka 1984; Oren 2012). Also in anoxygenic phototrophs glycine betaine is the main compatible solute, often combined with minor compounds such as ectoine, trehalose, and others (Galinski and Trüper 1982; Galinski 1995). Most heterotrophic bacteria cannot synthesize betaine but possess uptake systems enabling accumulation of the compound or its precursor choline when present in their environment. This explains why glycine betaine was found as the osmotic stabilizer in H. elongata and in other Gram-negative heterotrophic halophilic or halotolerant bacteria when grown in complex medium with yeast extract as a carbon source (Imhoff and Rodriguez-Valera 1984). In defined media that do not contain glycine betaine or choline, such organisms generally produce ectoine as the main osmotic solute. Glycine betaine uptake is also possible in Halobacillus and in other salt-tolerant members of the Firmicutes. Although the halophilic Archaea of the class Halobacteria generally rely on KCl for osmotic stabilization (the ‘salt-in’ strategy), glycine betaine transport proteins appear to be quite common: glycine betaine BCCT family transporters were found in 60 out of the 83 genomes analyzed (Youssef et al.2014). Halophilic methanogenic Archaea can produce glycine betaine, in addition to other organic osmotic solutes including Nε-acetyl-β-lysine and β-glutamine (Lai et al.1991; Martin, Ciulla and Roberts 1999). In Methanohalophilus portucalensis, a high level of potassium (>400 mM) was necessary for betaine formation by stepwise methylation of glycine in vitro (Lai, Yang and Chuang 1999). Ectoine—an osmotic solute for all three domains of life Ectoine was identified relatively late as an osmotic solute. Its name was derived from the anoxygenic photosynthetic gammaproteobacterium Ectothiorhodospira halochloris (now renamed Halorhodospira halochloris) (Galinski, Pfeiffer and Trüper 1985). Halorhodospira halochloris has glycine betaine as its main osmotic solute, but in addition it makes minor amounts of ectoine and trehalose. Soon after it was discovered that many heterotrophic members of the Bacteria, including Halomonas and relatives (Table 4) (Severin, Wohlfarth and Galinski 1992), produce ectoine and its 5-hydroxy derivative when grown in defined media that do not contain glycine betaine or other compounds that can be taken up to serve for osmotic stabilization. Because of its great biotechnological importance, including applications in skin protection products in the cosmetic market and in sprays against asthma, and because its industrial synthesis still depends on microbial processes based on Halomonas isolates, much effort has been invested in the elucidation of the metabolism of ectoine and the regulation of its accumulation (Schwibbert et al.2011). In the domain Bacteria, ectoine is produced not only by members of the Proteobacteria: it is found also in Firmicutes (e.g. Halobacillus) (Saum et al.2013), in the actinobacteria, and elsewhere. Both betaine and ectoine are better retained due to alterations of the membrane at increased salinity. In halotolerant and halophilic bacteria, the main adaptive changes in membrane lipids in response to a rise in salt concentration has been shown to be a rise in the anionic lipid(s), usually phosphatidylglycerol (PG) and/or cardiolipin (CL), matched with a corresponding fall in the major zwitterionic phospholipid, phosphatidylethanolamine (PE). In recent years, it has become clear that ectoine is not only found in the domain Bacteria. Analysis of the genome of the marine thaumarchaeon Nitrosopumilus maritimus, an organism that leads a chemolithotrophic lifestyle by oxidizing ammonia to nitrite and is widespread in the ocean, showed that it harbors an ectoine/hydroxyectoine gene cluster. Its ectABCD operon is osmotically induced and functional. In the archaeal domain, genes for the production of ectoine were found in some Methanothrix and Methanobacterium genomes. These genes may have been derived from horizontal gene transfer events (Widderich et al.2016). And the above-mentioned finding of enzymes involved in synthesis and transport of hydroxyectoine in the stramenopile protist H. seosinensis shows that some eukaryotes may also use ectoine and derivatives for osmotic purposes (Harding et al.2016). In spite of the intensive studies made of the properties of ectoine, its mode of action is still not completely understood. Using a combination of in vivo deuterium labeling, small angle neutron scattering, neutron membrane diffraction and inelastic scattering and neutron liquids diffraction to characterize the effects of ectoine on purple membrane of H. salinarum and on the maltose binding protein of E. coli, ectoine was shown to be excluded from the hydration layer at the membrane surface and it does not affect membrane molecular dynamics, confirming an earlier hypothesis that ectoine is excluded from a monolayer of dense hydration water around proteins (Zaccai et al.2016). Formation of strongly hydrogen-bonded water molecules around ectoine which compensate the influence of the salt on the water dynamics, as deduced from Raman spectroscopy data, shows that ectoine and NaCl have are opposing effects on proteins (Hahn et al.2016). Organic osmotic solutes as major carbon sources in hypersaline ecosystems Organic compatible solutes are important not only for the organisms that produce them so that they can withstand the high salt concentration in their environment, they can also be major factors determining the properties of hypersaline ecosystems. The fact that such solutes are accumulated in the cells’ cytoplasm in molar concentrations implies that such compounds, when leaking through the membranes of living cells or when released to the environment after the death of the cells, will become available to other components of the ecosystem, to be used either as readily available osmotic stabilizers or as carbon and energy sources. This has been well documented for two major compatible solutes: glycerol and glycine betaine. Dunaliella is the main primary producer in hypersaline lakes and saltern crystallizer ponds. Photosynthetically produced glycerol can therefore be expected to be a major compound that drives the functioning of such ecosystems. Accordingly, well-developed metabolic pathways for the utilization of glycerol are present in many halophilic Archaea and Bacteria, aerobic as well as anaerobic (Oren 2017). Among 32 haloarchaeal genomes surveyed, 27 showed a genomic potential to utilize glycerol as a source of carbon and energy (Williams et al.2017). Measurements of the uptake and metabolism of radiolabeled glycerol in the Dead Sea and in the crystallizer points of salterns in Eilat, Israel yielded showed very rapid uptake and short turnover times (Oren 1993). An especially interesting food chain based on glycerol may be functional in saltern crystallizer ponds and other salt lakes at or near NaCl saturation. Characteristic components of the biota in such environments are Dunaliella salina, Archaea including the flat square Hqr. walsbyi, and Salinibacter (Bacteroidetes). When Salinibacter is grown in the presence of glycerol, up to 20% of the substrate added is incompletely oxidized to yield dihydroxyacetone (Elevi Bardavid and Oren 2008). Genomic information suggests that Hqr. walsbyi possesses an efficient uptake system for dihydroxyacetone based on a phosphoenol pyruvate dependent phosphotransferase system (Bolhuis et al.2006). Dihydroxyacetone is indeed metabolized by Haloquadratum cultures and by the heterotrophic prokaryotic community of the saltern crystallizer ponds in Eilat dominated by Haloquadratum-like cells (Elevi Bardavid and Oren 2008; Elevi Bardavid, Khristo and Oren 2008). Glycerol derived from Dunaliella may also be the secret how halophilic Archaea may have survived for millions of years in fluid inclusions within salt crystals buried deep below the earth surface for millions of years. Living cells need a certain amount of energy for maintenance and repair of damaged DNA and other cell components. If some Dunaliella cells were trapped in a fluid inclusion at the time the salt was deposited, the archaeal cells present will find a generous supply of glycerol as energy and carbon source. It was calculated that a single Dunaliella cell of 10 μm diameter containing 5.5 M glycerol may support survival of a prokaryotic cell for 12 million years (Schubert et al.2009, 2010). In complex hypersaline ecosystems populated by glycine betaine-producing phototrophs as well as diverse heterotrophs, sufficient glycine betaine released from living or dead phototrophs can be present to provide osmotic stabilization also by the heterotrophs that are unable to synthesize the compound but can accumulate it from the medium. This explains why glycine betaine was the only osmotic solute identified in the stratified microbial community inhabiting a hypersaline evaporitic gypsum crust in a saltern evaporation pond (∼194 g L−1 total dissolved salts): presence of glycine betaine produced by the oxygenic and the anoxygenic photoautotrophic members of the community may relieve the heterotrophs from the need to synthesize other compounds at a high-energy cost (Oren et al.2013). Glycine betaine not taken up to serve as an osmotic stabilizer can also be degraded, both aerobically and anaerobically. Glycine betaine breakdown in the absence of oxygen is often performed by interesting consortia that yield methane based on methylated amines as intermediates. Thus, the homoacetogen Acetohalobium arabaticum (Halanaerobiales) can grow between 100 and 250 g L−1 salt and produces acetate and mono-, di- and trimethylamines from betaine (Zhilina and Zavarzin 1990). Degradation of trimethylamine in hypersaline environments to methane, carbon dioxide and ammonia by halophilic methanogens is well documented (Oren 1999, 2011b), so that food chains can function based on betaine-derived methylated amines (Oremland and King 1989; Oren 1990; Andrei, Banciu and Oren 2012). Recently, the first methanogen able to directly use glycine betaine, a Methanolobus strain, was isolated from an estuarine sediment (Ticak et al.2015). How widespread this type of metabolism is among the methanogenic Archaea remains to be ascertained. Interesting glycine-betaine based food chains were identified in two deep-sea hypersaline anaerobic lakes on the bottom of the Mediterranean Sea. In Lake Medee, the largest deep-sea hypersaline lake found to-date, the brine community almost exclusively consists of the members of euryarchaeal MSBL1 and bacterial KB1 candidate divisions. These organisms rely at least partially on reductive cleavage of glycine betaine (Yakimov et al.2013). A three-component microbial consortium was enriched from lake Thetis, linking anaerobic glycine betaine degradation with methanogenesis. The trophic network relies on fermentative decomposition of glycine betaine by its reductive cleavage to trimethylamine and acetate, carried out by a strain of Halobacter. lacunaris (Halanaerobiales). The trimethylamine formed is the substrate for a methylotrophic methanogen belonging to the genus Methanohalophilus. The third member of the consortium, Halanaerobium sp., can obtain energy primarily by fermenting sugars and producing hydrogen as one of the end products, thus providing necessary reducing equivalents for reductive degradation of glycine betaine. Anaerobic degradation of osmoregulatory molecules such as glycine betaine may thus play an important role in the turnover of organic carbon in anoxic hypersaline biotopes (La Cono et al.2015). Acknowledgements The authors would like to thank Dr Cene Gostinčar for computer drawing of the graphic abstract and Dr Tilen Konte for Fig. 1. FUNDING AO acknowledges support from the Israel Science Foundation Grant no. 343/13. NGC acknowledges support from the Slovenian Research Agency Grants no. J4-7246, J4 0814, J4-1019 and P1-170. Conflict of interest: None declared. REFERENCES Adler L Blomberg A Nilsson A . Glycerol metabolism and osmoregulation in salt tolerant yeast Debaryomyces hansenii . J Bacteriol 1985 ; 162 : 300 – 6 . Google Scholar PubMed Aggarwal M Bansal P Monda A . Molecular cloning and biochemical characterization of a 3’(2’).5’-biphosphate nucleotidase from Debaryomyces hansenii . Yeast 2005 ; 22 : 457 – 70 . Google Scholar CrossRef Search ADS PubMed André L Nilsson A Adler L . The role of glycerol in osmotolerance of the yeast Debaryomyces hansenii . J Gen Microbiol 1988 ; 134 : 669 – 77 . Andrei A-Ş Banciu HL Oren A . 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Strategies of adaptation of microorganisms of the three domains of life to high salt concentrations

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Oxford University Press
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© FEMS 2018.
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0168-6445
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10.1093/femsre/fuy009
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Abstract

Abstract Hypersaline environments with salt concentrations up to NaCl saturation are inhabited by a great diversity of microorganisms belonging to the three domains of life. They all must cope with the low water activity of their environment, but different strategies exist to provide osmotic balance of the cells’ cytoplasm with the salinity of the medium. One option used by many halophilic Archaea and a few representatives of the Bacteria is to accumulate salts, mainly KCl and to adapt the entire intracellular machinery to function in the presence of molar concentrations of salts. A more widespread option is the synthesis or accumulation of organic osmotic, so-called compatible solutes. Here, we review the mechanisms of osmotic adaptation in a number of model organisms, including the KCl accumulating Halobacterium salinarum (Archaea) and Salinibacter ruber (Bacteria), Halomonas elongata as a representative of the Bacteria that synthesize organic osmotic solutes, eukaryotic microorganisms including the unicellular green alga Dunaliella salina and the black yeasts Hortaea werneckii and the basidiomycetous Wallemia ichthyophaga, which use glycerol and other compatible solutes. The strategies used by these model organisms and by additional halophilic microorganisms presented are then compared to obtain an integrative picture of the adaptations to life at high salt concentrations in the microbial world. compatible solutes, ion metabolism, halophilic, halotolerant, diversity, hypersaline ecosystems OSMOTIC ADAPTATION IN MICROORGANISMS: BASIC PRINCIPLES Microbial life at high salt concentrations is phylogenetically very diverse. Hypersaline environments with salt concentrations up to NaCl saturation are inhabited by halophilic and highly halotolerant representatives of all three domains of life: Archaea, Bacteria and Eukarya. The mechanisms used by these salt-requiring or highly salt-tolerant microorganisms to withstand the high salt concentrations, and in many cases also to adapt their physiology to changes in the salt concentrations in their environments, are diverse as well. Still, a number of general principles apply (see e.g. Brown 1976, 1990), which are as follows: Biological membranes are permeable to water. Therefore, water moves into and out of cells driven by differences in water activity between the cytoplasm and the outside medium. Active inward pumping of water lost to the medium when cells are suspended in a high-salt medium is not feasible: no such mechanisms are known, and the process would be energetically far too expensive. Therefore, the intracellular environment must be at least isosmotic with the outside medium. With the possible exception of the halophilic Archaea of the class Halobacteria (Walsby 1971), all microorganisms possess a positive turgor. Maintenance of this outward-directed pressure is essential as the driving force for cell expansion (Kempf and Bremer 1998). This means that the osmotic pressure of the cytoplasm must even exceed that of the outside medium. To maintain a high osmotic pressure inside the cells, different strategies can be used, which are as follows: The ‘salt-in’ strategy where osmotic balance is achieved by accumulating high concentrations of inorganic salts in the medium. As Na+ ions are excluded as much as possible from cells in all three domains of life, the ‘salt-in’ strategy is based on KCl rather than on NaCl as the main intracellular salt. The ‘low-salt-in’, ‘compatible solute’ strategy. The term compatible solute was first used in 1972 and was defined as a solute which, at high concentration, allows an enzyme to function effectively (Brown and Simpson 1972). This definition was later extended to allow all essential cell processes to function effectively (Brown 1990). Many organic compounds belonging to different classes have been shown to serve as compatible solutes in different groups of microorganisms, including polyols, sugars, amino acids, betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine (Galinski 1993, 1995; da Costa, Santos and Galinski 1998; Kempf and Bremer 1998; Martin, Ciulla and Roberts 1999; Roberts 2005). Compatible solutes are strong water structure formers and are probably excluded from the hydration shell of proteins, thus stabilizing the hydration shell (Galinski 1995), and they decrease water activity coefficients (Held, Neuhaus and Sadowski 2010). In many extremophiles, such low molecular weight compounds are accumulated not only in response to increased salt concentrations, but also as a response to other environmental changes such as temperature stress. Examples of organic compatible solutes in thermophiles and in psychrophiles are di-myoinositol-1,1΄-phosphate, cyclic 2,3-diphosphoglycerate, α-diglycerol phosphate, mannosylglycerate and mannosylglyceramide (Lentzen and Schwarz 2006; Cowan 2009; Casanueva et al.2010; Klähn and Hagemann 2011). Biosynthesis of organic osmotic solutes is energetically more expensive than the ‘salt-in’ strategy (Oren 1999, 2011b), and therefore microorganisms that use the ‘low-salt-in’, ‘compatible solute’ strategy (and sometimes even those that accumulate salts) will also accumulate suitable solutes if available in their medium (Kempf and Bremer 1998; Pflüger and Müller 2004). Being adapted to life at high salt concentrations is not sufficient when the properties of the environment vary; adaptability to fluctuations in salinity is also necessary. Microorganisms that use organic solutes are generally more flexible and can easier adjust to dilution stress or to sudden increases in salinity than organisms that use the ‘salt-in’ strategy. Upon salt downshock, excess of compatible solutes can be excreted via mechanosensitive channels or converted inside the cell to osmotically inactive forms. Inorganic ions can be transiently accumulated following sudden increases in salinity, to be later replaced by newly synthesized organic solutes (Wood et al.2001). This review article first presents a number of case studies, exemplifying the ways used by different groups of Bacteria, Archaea and Eukarya to adapt to life at high salt concentrations. Then follows a comparative section in which we attempt to integrate all the information to discover the general principles behind the functioning of halophiles, both alone and in the complex ecosystems in which they live. Of course, it is not possible to review here the entire literature about adaptation of microorganisms to life at high salt. Further valuable information can be found in older reviews (Kushner 1978; Csonka 1989; Roeßler and Müller 2001; Oren 2002, 2008, 2011a, 2013a; Grant 2004; Kunte 2006; Gunde-Cimerman, Ramos and Plemenitaš 2009; Gostinčar et al.2011; Plemenitaš et al.2014). CASE STUDIES Extremely halophilic Archaea of the class Halobacteria The Archaea of the class Halobacteria are the best studied extremely halophilic prokaryotes. Until 2015 the class contained a single order, the Halobacteriales, with a single family, the Halobacteriaceae. Recent phylogenomic studies led to the splitting of the group into three orders and six families. Most studies of osmotic adaptation, ion metabolism and the properties of the cellular proteins were performed on two model organisms: Halobacterium salinarum (Halobacteriaceae) and Haloarcula marismortui (Haloarculaceae). Members of the Halobacteria are characteristic inhabitants of salt lakes at or approaching halite saturation, saltern crystallizer ponds and other high salt environments. Most members of the group are obligate halophiles that require at least 150–200 g L−1 of salts and show no adaptability to lower concentrations. Except for Halococcus and a few other genera that possess a rigid cell wall, members of the Halobacteria lyse when suspended in low salt solutions as their glycoprotein cell wall requires molar concentrations of salt for structural stability. As Halobacterium and other members of a group lack a measurable turgor pressure (Walsby 1971), they do not need to maintain a higher osmotic pressure in the cell than that of the brines in which they live. Measurements of intracellular ionic concentrations in H. salinarum, Har. marismortui and related organisms performed already in the 1960s–1970s (Christian and Waltho 1962; Ginzburg, Sachs and Ginzburg 1970; Lanyi and Silverman 1972; Matheson et al.1976) showed that the cells contain molar concentrations of salts, sufficient to osmotically balance the salinity of the external medium. K+ is always present as the dominant cation, and Na+ is found at much lower concentrations. Chloride is the main anion to balance the intracellular cations. In view of the difficulty to reliably assess the intracellular Na+ concentrations in the presence of molar concentrations in the surrounding media, the true Na+ may well be lower than the apparent values reported. Probably in view of this experimental difficulty, surprisingly few later studies have tried to verify and refine the early results. One recent study used microprobe analysis in a scanning electron microscope equipped with an X-ray spectrometer to assess the intracellular ionic concentrations of H. salinarum grown in medium containing 4.28 mol NaCl and 0.036 mol K+ per kg water. The intracellular K+ concentration was 110 times that of the medium, Na+ inside the cells was about one-third of that of the medium concentration, and chloride inside the cells was 1.1 times higher than in the medium, so that the apparent cation sum exceeded the anion sum (Engel and Catchpole 2005). Measurements of intracellular ion concentrations in Har. marismortui grown in 23% salt as determined by inductively coupled mass spectrometry indicated a minimum intracellular total ion requirement of 1.13 M (Jensen et al.2015). To achieve such ion gradients across the cell membrane (accumulation of K+, exclusion of Na+, accumulation of Cl− against the inside-negative membrane potential) requires energy-dependent mechanisms. Analysis of genome sequences of model organisms such as Halobacterium sp. NRC-1 (a strain of H. salinarum) (Ng et al.2000), Har. marismortui (Baliga et al.2004), Haloquadratum walsbyi (Bolhuis et al.2006) and other isolates (Becker et al.2014) have contributed much understanding about the types of ion pumps involved. The necessary energy is derived from the proton gradient over the membrane, generated by respiratory electron transport and/or the light-dependent proton pump bacteriorhodopsin. Na+ is extruded from the cells by Na+/H+ antiporter systems. K+ can enter the cells passively through K+ channels in the membrane, as driven by the inside-negative membrane potential, but active, ATP-dependent K+ transport systems are also present. In low K+ media, H. salinarum expresses a K+-transporting KdpFABC P-type ATPase together with an additional protein annotated as Cat3 in Halobacterium sp. NRC-1 (Table 1) and as UspA protein in H. salinarum R1. K+ limitation can also lead to a lowered intracellular K+ concentration. Table 1. Major alkali metal–cation transporters in halotolerant/halophilic microorganisms. Description Transporters identified in halotolerant/halophilic microorganisms K+ efflux Tok1 : Hortaea werneckii  channel Aureobasidium sp. K+ uptake Trk1,2 : Hortaea werneckii  uniporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii DhHak1 : Debaryomyces hansenii Trk AI : Halomonas elongata TrkAH : Halobacterium salinarum NRC-1 Halococcus hamelinensis Halomonas elongata TrkA2 : Halomonas beimenensis K+(Na+) efflux Hak1 : Aureobasidium  antiporter Debaryomyces hansenii K+ efflux Acu : Wallemia ichthyophaga  P-type ATPase Aureobasidium Na+ efflux Nha1 : Hortaea.werneckii  antiporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii NhaC : Halobacterium salinarum NRC-1 Na+ efflux Ena1,2 : Hortaea werneckii  P-type Wallamia ichthyophaga  ATPase Aureobasidium sp. Debarymyces hansenii Na+ P-type ATPase : Dunaliella maritima Na+/Pi Pho89 : Hortaea werneckii  symporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii H+ exporter Pma1 : Hortaea werneckii  P-type Wallemia ichthyophaga  ATPase Aureobasidium sp. Debaryomyces hansenii Na+/H+ Nhx1 (late endosome) : Hortaea werneckii  antiporter Debaryomyces hansenii  for Na+ Vnx1 (vacuolar) : Hortaea werneckii  extrusion Wallemia ichthyophaga Na+/H+ antiporter : Dunaliella Na+/H+ antiporter : Halobacterium salinarum Salinivibrio costicola Na+/H+ antiporter NhaD : Halomonas elongata Halomonas alkaliphila Na+(K+)/H+ antiporter : Natranaerobius thermophilus Na+/H+ antiporter : Halocafeteria seosinensis K+/H+ Kha1 (Golgi apparatus)  :  Hortaea werneckii  antiporter Wallemia ichthyophaga Debaryomyces hansenii K+/H+ antiporter : Chromohalobacter israelensis ATP-driven K+ transport system KpdFABC : Halobacterium salinarum NRC-1 Description Transporters identified in halotolerant/halophilic microorganisms K+ efflux Tok1 : Hortaea werneckii  channel Aureobasidium sp. K+ uptake Trk1,2 : Hortaea werneckii  uniporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii DhHak1 : Debaryomyces hansenii Trk AI : Halomonas elongata TrkAH : Halobacterium salinarum NRC-1 Halococcus hamelinensis Halomonas elongata TrkA2 : Halomonas beimenensis K+(Na+) efflux Hak1 : Aureobasidium  antiporter Debaryomyces hansenii K+ efflux Acu : Wallemia ichthyophaga  P-type ATPase Aureobasidium Na+ efflux Nha1 : Hortaea.werneckii  antiporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii NhaC : Halobacterium salinarum NRC-1 Na+ efflux Ena1,2 : Hortaea werneckii  P-type Wallamia ichthyophaga  ATPase Aureobasidium sp. Debarymyces hansenii Na+ P-type ATPase : Dunaliella maritima Na+/Pi Pho89 : Hortaea werneckii  symporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii H+ exporter Pma1 : Hortaea werneckii  P-type Wallemia ichthyophaga  ATPase Aureobasidium sp. Debaryomyces hansenii Na+/H+ Nhx1 (late endosome) : Hortaea werneckii  antiporter Debaryomyces hansenii  for Na+ Vnx1 (vacuolar) : Hortaea werneckii  extrusion Wallemia ichthyophaga Na+/H+ antiporter : Dunaliella Na+/H+ antiporter : Halobacterium salinarum Salinivibrio costicola Na+/H+ antiporter NhaD : Halomonas elongata Halomonas alkaliphila Na+(K+)/H+ antiporter : Natranaerobius thermophilus Na+/H+ antiporter : Halocafeteria seosinensis K+/H+ Kha1 (Golgi apparatus)  :  Hortaea werneckii  antiporter Wallemia ichthyophaga Debaryomyces hansenii K+/H+ antiporter : Chromohalobacter israelensis ATP-driven K+ transport system KpdFABC : Halobacterium salinarum NRC-1 View Large Table 1. Major alkali metal–cation transporters in halotolerant/halophilic microorganisms. Description Transporters identified in halotolerant/halophilic microorganisms K+ efflux Tok1 : Hortaea werneckii  channel Aureobasidium sp. K+ uptake Trk1,2 : Hortaea werneckii  uniporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii DhHak1 : Debaryomyces hansenii Trk AI : Halomonas elongata TrkAH : Halobacterium salinarum NRC-1 Halococcus hamelinensis Halomonas elongata TrkA2 : Halomonas beimenensis K+(Na+) efflux Hak1 : Aureobasidium  antiporter Debaryomyces hansenii K+ efflux Acu : Wallemia ichthyophaga  P-type ATPase Aureobasidium Na+ efflux Nha1 : Hortaea.werneckii  antiporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii NhaC : Halobacterium salinarum NRC-1 Na+ efflux Ena1,2 : Hortaea werneckii  P-type Wallamia ichthyophaga  ATPase Aureobasidium sp. Debarymyces hansenii Na+ P-type ATPase : Dunaliella maritima Na+/Pi Pho89 : Hortaea werneckii  symporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii H+ exporter Pma1 : Hortaea werneckii  P-type Wallemia ichthyophaga  ATPase Aureobasidium sp. Debaryomyces hansenii Na+/H+ Nhx1 (late endosome) : Hortaea werneckii  antiporter Debaryomyces hansenii  for Na+ Vnx1 (vacuolar) : Hortaea werneckii  extrusion Wallemia ichthyophaga Na+/H+ antiporter : Dunaliella Na+/H+ antiporter : Halobacterium salinarum Salinivibrio costicola Na+/H+ antiporter NhaD : Halomonas elongata Halomonas alkaliphila Na+(K+)/H+ antiporter : Natranaerobius thermophilus Na+/H+ antiporter : Halocafeteria seosinensis K+/H+ Kha1 (Golgi apparatus)  :  Hortaea werneckii  antiporter Wallemia ichthyophaga Debaryomyces hansenii K+/H+ antiporter : Chromohalobacter israelensis ATP-driven K+ transport system KpdFABC : Halobacterium salinarum NRC-1 Description Transporters identified in halotolerant/halophilic microorganisms K+ efflux Tok1 : Hortaea werneckii  channel Aureobasidium sp. K+ uptake Trk1,2 : Hortaea werneckii  uniporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii DhHak1 : Debaryomyces hansenii Trk AI : Halomonas elongata TrkAH : Halobacterium salinarum NRC-1 Halococcus hamelinensis Halomonas elongata TrkA2 : Halomonas beimenensis K+(Na+) efflux Hak1 : Aureobasidium  antiporter Debaryomyces hansenii K+ efflux Acu : Wallemia ichthyophaga  P-type ATPase Aureobasidium Na+ efflux Nha1 : Hortaea.werneckii  antiporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii NhaC : Halobacterium salinarum NRC-1 Na+ efflux Ena1,2 : Hortaea werneckii  P-type Wallamia ichthyophaga  ATPase Aureobasidium sp. Debarymyces hansenii Na+ P-type ATPase : Dunaliella maritima Na+/Pi Pho89 : Hortaea werneckii  symporter Wallemia ichthyophaga Aureobasidium sp. Debaryomyces hansenii H+ exporter Pma1 : Hortaea werneckii  P-type Wallemia ichthyophaga  ATPase Aureobasidium sp. Debaryomyces hansenii Na+/H+ Nhx1 (late endosome) : Hortaea werneckii  antiporter Debaryomyces hansenii  for Na+ Vnx1 (vacuolar) : Hortaea werneckii  extrusion Wallemia ichthyophaga Na+/H+ antiporter : Dunaliella Na+/H+ antiporter : Halobacterium salinarum Salinivibrio costicola Na+/H+ antiporter NhaD : Halomonas elongata Halomonas alkaliphila Na+(K+)/H+ antiporter : Natranaerobius thermophilus Na+/H+ antiporter : Halocafeteria seosinensis K+/H+ Kha1 (Golgi apparatus)  :  Hortaea werneckii  antiporter Wallemia ichthyophaga Debaryomyces hansenii K+/H+ antiporter : Chromohalobacter israelensis ATP-driven K+ transport system KpdFABC : Halobacterium salinarum NRC-1 View Large Presence of the KdpFABC complex enables H. salinarum to grow at K+ concentrations as low as 20 μM. Deletion of the kdpFABC cat3 gene resulted in a reduced ability to grow under K+-limiting concentrations (Strahl and Greie 2008). Comparative genomics indicated a considerable diversity of potassium transport complex subunits in the halophilic archaea (Jensen et al.2015). Chloride can be accumulated into the cells by cotransport with Na+ or by the primary light-driven inward chloride pump halorhodopsin (Duschl and Wagner 1986). The light-driven chloride pump halorhodopsin, a hybrid histidine kinase, and the mechanosensitive channels MscA1 and MscA2 have been identified as putative osmosensors H. salinarum and in Haloferax volcanii (Table 2) (Le Dain et al.1998; Kolbe et al.2000; Zhang and Shi 2005). Table 2. Putative osmosensors identified in prokaryotic halophiles. Archaea Halobacterium salinarum The light-driven chloride pump halorhodopsin Kolbe et al.2000 Halobacterium salinarum NRC-1 Hybrid histidine kinase Zhang and Shi 2005 Haloferax volcanii Mechanosensitive channels MscA1, MscA2 Le Dain et al.1998 Bacteria Chromohalobacter salexigens Two-component response regulator EupR Rodríguez-Moya et al.2010 Chromohalobacter salexigens Hybrid histidine kinase Rodríguez-Moya et al.2010 Halomonas elongata Transporter for ectoine accumulation Tea Kunte, Trüper and Stan-Lotter 2012 Archaea Halobacterium salinarum The light-driven chloride pump halorhodopsin Kolbe et al.2000 Halobacterium salinarum NRC-1 Hybrid histidine kinase Zhang and Shi 2005 Haloferax volcanii Mechanosensitive channels MscA1, MscA2 Le Dain et al.1998 Bacteria Chromohalobacter salexigens Two-component response regulator EupR Rodríguez-Moya et al.2010 Chromohalobacter salexigens Hybrid histidine kinase Rodríguez-Moya et al.2010 Halomonas elongata Transporter for ectoine accumulation Tea Kunte, Trüper and Stan-Lotter 2012 View Large Table 2. Putative osmosensors identified in prokaryotic halophiles. Archaea Halobacterium salinarum The light-driven chloride pump halorhodopsin Kolbe et al.2000 Halobacterium salinarum NRC-1 Hybrid histidine kinase Zhang and Shi 2005 Haloferax volcanii Mechanosensitive channels MscA1, MscA2 Le Dain et al.1998 Bacteria Chromohalobacter salexigens Two-component response regulator EupR Rodríguez-Moya et al.2010 Chromohalobacter salexigens Hybrid histidine kinase Rodríguez-Moya et al.2010 Halomonas elongata Transporter for ectoine accumulation Tea Kunte, Trüper and Stan-Lotter 2012 Archaea Halobacterium salinarum The light-driven chloride pump halorhodopsin Kolbe et al.2000 Halobacterium salinarum NRC-1 Hybrid histidine kinase Zhang and Shi 2005 Haloferax volcanii Mechanosensitive channels MscA1, MscA2 Le Dain et al.1998 Bacteria Chromohalobacter salexigens Two-component response regulator EupR Rodríguez-Moya et al.2010 Chromohalobacter salexigens Hybrid histidine kinase Rodríguez-Moya et al.2010 Halomonas elongata Transporter for ectoine accumulation Tea Kunte, Trüper and Stan-Lotter 2012 View Large One of the most characteristic properties of the halophilic Archaea is their highly acidic proteome: their proteins have a high excess of negatively charged amino acids (aspartate, glutamate) over amino acids with a positive charge (lysine, arginine). This property is shared by other ‘salt-in’ strategists such as Salinibacter (see below). The acidic nature of the proteins of the Halobacteria was already recognized nearly half a century ago (Reistad 1970), and it was fully confirmed during genomic analyses (e.g. Dennis and Shimmin 1997; Ng et al.2000; Baliga et al.2004; Bolhuis et al.2006). Only few proteins encoded by these genomes are not acidic, the membrane-bound retinal proteins being notable exceptions. Comparative genomic analyses including halophilic Archaea, mesophiles and thermophiles showed that, compared with non-halophilic mesophiles, the protein surface of the halophiles is enriched in aspartate and is low in lysine and asparagine, the interior of the proteins being enriched in valine and low in isoleucine. Compared with thermophiles the protein surface of the halophiles is enriched in aspartate, alanine and threonine and is low in lysine, the interior of the proteins being enriched in threonine and low in isoleucine (Fukuchi et al.2003) The highly acidic enzymes and other proteins of the Halobacteria typically require molar concentrations of salt for activity and structural stability. Since the classic review of the salt-dependent properties of proteins from the extreme halophiles by Lanyi (1974), many studies have been devoted to the elucidation of the mechanisms of the salt requirement and salt tolerance of halophilic enzymes in terms of the thermodynamics of solvent–protein interactions, hydration, ion binding and unfolding kinetics. The availability of high-resolution crystal structures for selected halophilic enzymes such as the malate dehydrogenase and the 2Fe-2S ferredoxin of Har. marismortui has greatly contributed to such studies. Techniques such as analytical ultracentrifugation, small angle neutron and X-ray scattering, crystallography, and protein dynamics by energy resolved neutron scattering have been employed in the study of halophilic proteins. Such proteins are folded similar to their non-halophilic homologues. The excess in negative charge is found predominantly on the protein surfaces where extensive hydration interactions are observed. Complex salt bridges associated with solvent ion binding sites also appear as likely halo-adaptation features (Eisenberg, Mevarech and Zaccai 1992; Madern, Ebel and Zaccai 2000; Ebel and Zaccai 2004; Tehei and Zaccai 2005; Soppa 2006, and many others). The high negative surface charge makes the proteins more soluble and renders them more flexible at high salt concentrations when non-halophilic proteins tend to aggregate and become rigid. This high surface charge is neutralized mainly by tightly bound water dipoles. The requirement for a high salt concentration for the stabilization of the halophilic enzymes is due to a low-affinity binding of the salt to specific sites on the protein surface (Mevarech, Frolow and Gloss 2000). Analysis of protein–solvent interactions in the glucose dehydrogenase of Hfx. mediterranei showed that the acidic residues on the protein surface are only partially neutralized by bound potassium. A highly ordered, multilayered solvation shell is present on the surface of the protein (Britton et al.2006). Cumulative weak cation–protein interactions are expected to stabilize the folded conformations (Ortega, Diercks and Millet 2015). The acidic residues on the protein surface may also be important to prevent aggregation of the proteins (Elcock and McCammon 1998). In bacteria and nonhalophilic archaea the majority of exported proteins are Sec-dependent, while haloarchaea use primarily the Tat pathway for export, which, in contrast to Sec pathway, enables the transport of fully folded proteins. Proteins from non-halophile microorganisms would probably aggregate and precipitate at conditions of high salinity at which haloarchaea thrive, while halobacterial proteins have increased negative surface charge that prevents aggregation (Bolhuis 2002). Most members of the class Halobacteria have only a limited ability to adapt to salt concentrations below their optimum. Accordingly, relatively few studies have tried to monitor changes in the types of lipids in the cell membrane following hypoosmotic shock. Among the effects observed are the formation of cardiolipin at the expense of phosphatidylglycerol, a decrease in content of the methyl ester of phosphatidylglycerol phosphate, and an increase in the content of C25C20 lipids at the expense of C20C20 lipids. These and other changes are further documented in Table 3. Table 3. Lipid composition of selected obligately halophilic Archaea that require at least 150–200 g L−1 NaCl for growth, and changes in lipid composition following lowering of the salt concentration to values still high enough to provide structural stability. Archaeal taxa Lipid composition Changes induced by hypoosmotic shock References Class Halobacteria Branched C20 and C25 lipids (from 1–89%); great variety of polar lipids: PL, SL, GL; maintenance of a highly negative charge surface density by a high concentration of acidic lipids Neo-synthesis of BPG at the expense of PG Lopalco et al.2004, 2008 Haloarcula marismortui 86% polar lipids (GL, PG, PGP-Me, PGS and 14% non-polar lipids) (squalenes, vitamin MK-8 and bacterioruberins, β-carotene, lycopene and retinal) Increase in glycosyl cardiolipin analogues, BPG, decreased PGP-Me Evans, Kushwaha and Kates 1980 Halobacterium salinarum C20C20 DGD, abundant PGP-Me (50–80% of the polar lipids) Increased S-TGD-1-PA Russell 1989a,b; Russell et al. 1995; Tenchov et al.2006; Lobasso et al.2008 Haloferax volcanii, Hfx. mediterranei C20C20 DGD and other lipids; absence of SL Increased BPG, glycosyl cardiolipin analogues, decrease in PGP-Me Halorubrum trapanicum, Hrr. vacuolatum C20C20 DGD and other lipids Increased S-DGD-5-PA van de Vossenberg et al.1999 Haloquadratum walsbyi Neutral lipids: vitamin MK-8, squalene, bacterioruberin carotenoids and several retinal isomers. Polar lipids: PGP-Me, PGS, PG, S-DGD-1. Cardiolipins: tetra-phytanyl or dimeric phospholipids, no glycosyl-cardiolipin, trace amounts of BPG. No changes, both qualitatively and quantitatively Lobasso et al.2008 Natronobacterium gregoryi C20C20 DGD, C25C20 DGD, and other lipids; isoprenoid chains fully saturated; GL absent Increase in C25C20 lipids; increase in unsaturation of DGD Morth and Tindall 1985a,b; Tindall,