Free polar amino acids in cells might be expected to play a role in osmotic balance. However, neutral amino acids are not accumulated to high concentrations, presumably because they are intermediates in protein biosynthesis. High and varying concentrations of these compounds could affect diverse cell pathways. Instead, many bacterial and archaeal cells synthesize and accumulate a few zwitterionic molecules derived from amino acids as compatible solutes. Structures of these solutes and where they are found are presented in Figure 1.

Few molecules that are polar but lack any formal charges have been identified as osmolytes in halophilic bacteria, although they are well represented in eukaryotes. For example, glycerol is prevalent as an osmolyte in marine and halophilic Dunaliella 3233. Glycerol accumulation is also a characteristic of halotolerant yeast Debaryomyces hansenii as well as the black yeast Hortea werneckii, and adaptation of this eukaryotic organism to high NaCl requires glycerol accumulation 33. Myo-inositol, another polyol, is used as an osmolyte in several eukaryotes. Neither of these polar noncharged solutes has been identified as an osmolyte in bacteria or archaea (or associated with halophiles). However, negatively charged derivatives of both glycerol and inositol are accumulated by archaea. The few uncharged solutes that are used by halotolerant bacteria and archaea include several carbohydrates and an amino acid/dipeptide modified to neutralize all charged groups (Figure 2).

Cells have a negative potential inside and often quite high intracellular K⁺. Negatively charged solutes could serve to balance high intracellular K⁺ as well as counteract osmotic pressure. Indeed, at lower external NaCl, many bacteria (including H. elongata which also synthesizes ectoine) and archaea use L-α-glutamate as an osmolyte. In methanogens, high NaCl often causes the cells to switch from anionic glutamate isomers to the zwitterionic solute Nε-acetyl-β-lysine for osmotic balance 2931. Anionic solutes used by bacteria and archaea for osmotic balance can have a carboxylate supply the negative charge (Table 3) or contain phosphate or sufate groups (Figure 4).

The high concentration of organic anions in many halophiles requires counterions such as K⁺ and/or Na⁺. However, there are halophiles that exclusively use inorganic ions for osmotic balance. Halophilic aerobic archaea have been shown to have high K⁺ and Cl^- 67, although absolute amounts appear to depend dramatically on the method of analysis and/or growth conditions. There are also bacteria with exceedingly high intracellular K⁺. In Salinibacter ruber, an obligatory aerobic chemoorganitrophic and very halophilic bacterium, K⁺ is the major intracellular component of osmotic balance with 11–15 μmol K⁺/mg protein 68. Organic solutes are relatively low in this organism; these studies used elemental analysis with EM and an X-ray spectrum to describe constituents and flame-photometric determination of K⁺. S. ruber occupies a relatively unique position in the bacterial kingdom. Its closest relative, Rhodothermus marinus, uses α-mannosylglycerate and α-mannosylglyceramide as osmolytes for osmotic balance 34.

Other microorganisms that do not appear to use organic osmolytes for balance include anaerobic fementative members of the families Halobacteroidaceae and Halanaerobiaceae 67686970. Halobacterium salinarum, an archaeon, has been reported to accumulate 12 μmol K⁺/mg protein. While some organic solutes were observed (≤50 mM), at those low concentrations they are unlikely to play a major role in osmotic balance, although they may aid in charge balance within the cells. Using a cell volume of 2.75 μl/mg proteins for H. salinarum, the estimated intracellular K⁺ is 4.4–4.8 M comparable to the Na⁺ concentration in the medium. Extracting cell pellets prior to K⁺ analyses led to considerably lower values (~1 M for S. ruber and ~3 M for H. salinarum) for K⁺, suggesting ion leakage. Intracellular Na⁺ was also high in the pellet extracts, which could suggest problems with this type of analysis. Alternatively, the difference might reflect complexed versus uncomplexed ions. Halophilic sulfate-reducing bacteria, e.g., Desulfohalobium retbaense and Desulfovibrio halophilus, like the haloarchea, appear to use inorganic salts for osmotic balance.

In contrast to the archaeal halophiles, many of the halophilic bacteria do not have exceptional high K⁺. For example, Halomonas elongata accumulates 1.1 μmol K⁺/mg protein when grow with yeast extract and 2.2 μmol/mg protein when grown in defined medium with glucose as the sole carbon source. In Halanaerobium acetethylicum grown in medium with 1.7 M NaCl, the internal cytoplasmic Na⁺ and Cl^- are 0.92 and 1.2 M, respectively, while K⁺ and Mg²⁺ concentrations in cells are 0.24 and 0.02 M, respectively 70.

Although K⁺ (and occasionally Na⁺) appears to be the major intracellular cation, there are reports that, in some cells, Mg²⁺ can reach moderately high concentrations. Heldal and coworkers 71 have found high Mg²⁺ (close to 0.9 M) in native marine bacteria under conditions of low dissolved organic carbon. The intracellular Mg²⁺ was dramatically reduced (<0.2 M) when nutrient levels increased. They suggested that high intracellular Mg²⁺ is a marker of carbon limitation.

Chloride is the most prevalent inorganic anion in halophiles that do not accumulate organic anions. Molar concentrations of chloride have been detected in several halophilic archaea. This anion is pumped into cells by halorhodopsin or cotransported with Na⁺. While this anion certainly can contribute to osmotic balance, it appears to have more critical roles in haloadaptation 72. For example, chloride has been shown to regulate betaine transport. Aside from chloride, little is known about the inorganic anion composition of halophiles. However, a recent FT-IR study of intact bacteria during growth indicates that in H. salinarum and Halococcus morrhuae, large changes occur in the concentration of sulfate ion in the cells 73. Maximum sulfate occurs during the mid-part of the exponential phase.

One of the things arising from the studies of different halotolerant and halophilic organisms is that most cells use an array of solutes, not a single one, for osmotic balance. When a single solute is detected it is often supplied by the medium and efficiently transported into the cell. However, left to its own device, the typical bacterial or archaeal cell synthesizes several molecules that together contribute to osmotic balance. Sometimes this is a combination of anions and zwitterions, but often several solutes with the same net charge. Archaea provide particularly intriguing examples of this strategy, although an explanation for the diversity of solutes in a given organism is lacking.

Methanothermococcus thermolithotrophicus accumulates the anionic α- and β-glutamate when grown in medium with less than 1 M NaCl 49. Cells adapted to higher external NaCl concentrations switch to accumulating a zwitterions, Nε-acetyl-β-lysine 314850. Since the glutamate concentration is roughly the same as intracellular K⁺, the switch to accumulating the zwitterions could be the result of an impaired K⁺ pump.

Methanohalophilus portucalensis, a halophilic methanogen, accumulates three zwitterions over its growth range: betaine, Nε-acetyl-β-lysine, and β-glutamine 8. α-Glutamate is detected, but its intracellular concentration is relatively low and does not increase with increased external NaCl. Of the three zwitterions, β-glutamine is only accumulated to large amounts at the high NaCl end of the growth range. Several conditions can affect the balance among these three zwitterions 30. The cells can grow on trimethylamine or methanol as the substrate for methanogensis, and the substrate with nitrogen promoted accumulation of the two solutes containing two nitrogen atoms (Nε-acetyl-β-lysine, and β-glutamine). Supplying precursors of these solutes (glycine, lysine, glutamate) has little effect on the distribution of the three zwitterions. Betaine is the only solute that could suppress synthesis of both Nε-acetyl-β-lysine and β-glutamine when it is present in the medium 74.

Balancing several different anionic osmolytes has also been observed in the halotolerant, hyperthermophilic Methanotorris igneus, which accumulates L-α-glutamate, β-glutamate, and DIP 10. Increased external NaCl leads to preferential increases in the intracellular β-amino acid; thermal stress causes increases in DIP levels. Multiple anionic solutes are also accumulated in other hyperthermophilic archaea. In Archaeoglobus fulgidus, glutamate, DIP and α-diglycerol phosphate are used for osmotic balance 53. In these cells, it is the α-diglycerol phosphate that was most sensitive to external NaCl while heat enhanced DIP synthesis and accumulation.

Microorganisms have two different general pathways for synthesizing betaine (Figure 5). The oxidative pathway can occur with a single soluble enzyme (choline oxidase in Gram-positive soil bacteria 75) or require two distinct soluble enzymes (choline monooxygenase and betaine-aldehyde dehydrogenase in higher plants 76), or it can occur with a membrane-associated system coded by four genes in the bet operon (in marine invertebrates and bacteria including Escherichia coli). The last system has been studied genetically 77, with genes identified for choline dehydrogenase (betA), betaine-aldehyde dehydrogenase (betB), a choline transporter (betT) and a putative regulator (betI). The choline dehydrogenase catalyzes oxidation of choline to betaine aldehyde, which is then oxidized to betaine by the betB gene product. In Pseudomonas, an electron acceptor other than O₂ is used for choline oxidation with suggestions that PQQ is the acceptor 78. In Actinopolyspora halophila, choline is oxidized to betaine aldehyde then to betaine 36. The aldehyde is produced with O₂ consumption and H₂O₂ generation. The final oxidation to betaine uses reduction of NADP⁺.

Even organisms that do not accumulate betaine in response to osmotic stress may have homologues of the genes for synthesizing this solute. Halomonas elongata does not appear to accumulate betaine. However, the organism does have a gene that codes for the oxidation of choline to betaine 79. Recently the choline dehydrogenase from that organism was expressed in E. coli and characterized. This enzyme can use O₂ if no other electron acceptors are available, although V_max decreases four-fold compared to kinetics with an acceptor such as phenazine methosulfate. Both choline and the betaine-aldehyde are converted to betaine. Although a glycine box suggestive of FAD⁺ as a cofactor was seen in the sequence, there is no experimental evidence for FAD⁺ as a cofactor. These observations prompt two questions: (1) what cofactor is used by this choline dehydrogenase, and (ii) under what conditions is this gene expressed?

Several microorganisms can also generate betaine by successively methylating glycine. GSMT (glycine sarcosine methyltransferase) and SDMT (sarcosine dimethylglycine methyltransferase) in Halorhodospira halochloris and Actinopolyspora halophila transfer the methyl group of S-adenosylmethionine to two different types of amines 183680. Betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica was also carried out by GSMT and DNMT activities 81. Only one methanogen, Methanohalophilus portucalensis, has been shown to synthesize betaine de novo 8. In these anaerobic cells, ¹³C NMR labeling experiments suggest that betaine is generated by successive methylation of glycine 20. If cells are grown with 10 mM betaine in the medium, accumulation of other osmolytes (Nε-acetyl-β-lysine, β-glutamine and glucosylglycerate) is suppressed 74. However, supplying cells with betaine precursors does not necessarily alter the distribution of osmolytes. Exogenous glycine or sarcosine had no effect on betaine accumulation. Even though the glycine (¹³C-labeled) was shown to be internalized by the cells 74, betaine synthesis and suppression of other osmolytes only occurs with N, N-dimethylglycine added. This suggests that it is the N, N-dimethylglycine intracellular concentration that regulates betaine synthesis and accumulation. Recently a 240 kDa N-methyl transferase has been isolated and partially characterized (M.-C. Lai, C.-C. Wang, M.-J. Chuang, Y.-C. Wu, and Y.-C. Lee, personal communication). The source of the methyl groups transferred to glycine is (perhaps not surprisingly) S-adenosylmethionine. While mammalian activites usually show very specific methyltransferase activity (e.g., glycine N-methyltransferase), an aggregate of similar mass proteins (but with different pI values) carries out all three methylation activities in the methanogen. Different subunits in the aggregate are optimized for different methyl transfers; the K⁺ concentration also differentially modulates the methyltransferase activities [82, M.-C. Lai and coworkers, personal communication]. The last result suggests that betaine accumulation is likely regulated by the internal K⁺ concentration in these cells.

The biosynthesis and regulation of ectoine in cells have been studied in several different bacteria, both Gram-negative and Gram-positive. Biosynthesis of ectoine in H. elongata has been studied in the greatest detail. The entry molecule into ectoine biosynthesis is aspartate semialdehyde, which is an intermediate in amino acid metabolism 83. A shown in Figure 6, the aldehyde is converted to L-2,4-diaminobutyric acid, which is then acetylated to from Nγ-acetyldiaminobutyric acid (NADA). The final step is the cyclization of this solute to form ectoine. The genes for biosynthesis of this solute were identified after the isolation of salt-sensitive mutants led to cloning of genes 84. Ectoine synthesis is carried out by the products of three genes: ectABC. The ectA gene codes for diaminobutyric acid acetyltransferase; ectB codes for the diaminobutyric acid aminotransferase; ectC codes for ectoine synthase 1985. The three recombinant H. elongata Ect enzymes have been characterized. The first enzyme (a 260 kDa complex of 44 kDa subunits) generates the diaminobutyrate by transaminating the aspartate semialdehyde with glutamate. Both pyridoxal 5'-phosphate and K⁺ are necessary for the diaminobutyrate aminotransferase activity 86. The aminotransferase of step two is activated by 0.5 M NaCl (and similarly by KCl). The last enzyme involved, ectoine synthase, is also activated by NaCl. This suggests that ectoine accumulation is partially regulated by intracellular cations.

In Chromohalobacter salexigens, the ectABC genes are regulated at the transcriptional level 87. Osmoregulated promoters with sequence homology to general stress σ factor have been identified. Since ectoine levels are modulated with betaine present, there must be additional post-transcriptional control. The effect of betaine on ectABC expression and ectoine accumulation was also shown in Marinococcus halophilus 88. In defined medium, the intracellular level of ectoine increases with NaCl and suppresses accumulation of trehalose. However, in complex medium, betaine is accumulated and ectoine synthesis is suppressed.

In most organisms, it is thought that hydroxyectoine is synthesized directly from ectoine. However, H. elongata has an alternate pathway that was observed in strains defective in EctC. These mutants that can not synthesize ectoine can still convert NADA directly to hydroxyectoine 89. Canovas et al. 89 proposed that NADA is hydroxylated to 3-hydroxyl-Nγ-acetyldiaminobutyrate, which is then cyclized to hydroxyectoine by 'hydroxyectoine synthase.' The zwitterionic precursor of ectoine can also be accumulated for osmotic balance. NADA has been detected in a salt-sensitive strain of H. elongata 89 that grows optimally with 0.75 to 1.0 M NaCl. It accounts for 80% of the organic solute pool for cells grown in 1.5 M NaCl) with ectoine (6%) and hydroxyectoine (12%) also present. NADA confers higher osmotic stability to the cells than in a H. elongata mutant where diaminobutyrate accumulates 84. Thus, this solute, but not its diaminobutyrate precursor (which would have a net positive charge) can act as a compatible solute if ectoine synthesis is blocked.

Over the past few years, genes have been identified or proteins isolated or cloned that confirm pathways initially proposed based on ¹³C isotopic labeling of these solutes. The pathway originally proposed for biosynthesis of Nε-acteyl-β-lysine has two key enzymes: (i) isomerization of α-lysine to β-lysine catalyzed by a lysine aminomutase, then (ii) acetylation of the ε-amino group 2031. Recently the genes coding for these two enzymes were identified in Methanosarcina mazei Gö1 90: ablA codes for the aminomutase while ablB codes for the β-lysine acetyltransferase. Expression of the two genes, which are organized in an operon, is salt dependent in M. mazei. Several other methanogens, including Methanococcus maripaludis, have homologous genes 90. Deletion of the abl operon in M. maripaludis generates cells incapable of growth in high salt medium. It will be interesting to characterize the methanogen lysine aminomutase and to compare it to the catabolic enzyme from bacteria that carries out the same chemistry.

Early NMR evidence ruled out a glutamate aminomutase activity as a means of generating β-glutamate 31 but did not identify precursors. As shown in Figure 7, the most likely pathway (proposed based on enzyme activities found in Methanocaldococcus jannaschii by M. Graupner, H. Xu, and R. H. White, personal communication) starts with the reduction of α-ketoglutarate to α-hydroxyglutarate, which is converted to its coenzyme A ester. Elimination of water from the α-hydroxyglutaryl-CoA generates glutaconyl-CoA, which forms β-glutamyl-CoA when ammonia is added (although the direct source of ammonia is not clear). Hydrolysis of the CoA ester generates β-glutamate. The products of the MJ0800 and MJ0400 genes have been identified as the enzymes responsible for water elimination in this pathway by R. H. White and coworkers (personal communication).

While not all the enzymes for synthesizing β-glutamate have been identified, conversion of β-glutamate to β-glutamine is done in Methanohalophilus portucalensis FDF1 by glutamine synthetase 2091. That GS has unusual properties compared to other studied GS enzymes. In particular, its activity with β-glutamate as substrate is much higher than that of other organisms 91. Regulation of the enzyme must occur in the cell, because β-glutamine is only accumulated to NMR-detectable levels in M. portucalensis when the cells are grown at higher NaCl 8. Although the in vitro K_m values for both α- and β-glutamate in this organism appear quite high, there is likely to be another mechanism responsible for regulation of the synthesis of this solute by glutamine synthetase and accumulation for osmotic balance.

Data from NMR experiments using ¹³C-labeled precursors to label DIP in Methanotorris igneus coupled with in vitro assays with postulated intermediates 92 led to a pathway for the biosynthesis of DIP that includes four steps (Figure 8): (i) conversion of D-glucose-6-phosphate to L-inositol-1-phosphate (L-I-1-P) via inositol-1-phosphate synthase (IPS); (ii) hydrolysis of the L-I-1-P by inositol monophoshatase; (iii) coupling of the L-I-1-P with CTP to form CDP-inositol; and (iv) generation of the phosphodiester linkage by condensing CDP-inositol with L-I-1-P (via a 'DIP synthase' activity for whom there is yet no candidate in genomes of organisms that accumulate DIP). In P. woesei, but not in M. igneus, DIP could also be generated from incubations of crude cell extracts with GTP and I-1-P 93. This finding can be explained by the same condensation mechanism, but assuming a multifunctional 'DIP synthase' that catalyzes not only the condensation of CDP-I and myo-inositol but the dephosphorylation of I-1-P as well (presumably without releasing the dephosphorylated product, myo-inositol).

The IPS reaction has been examined in several hyperthermophiles (Archaeoglobus fulgidus, Methanotorris igneus, Pyrococcus furiosus, P. woesei, and Thermotoga maritima) known to accumulate this solute (L. Chen and M.F. Roberts, unpublished results). IPS activities in crude extracts are ubiquitous in these organisms and fall into two classes: (i) IPS dependent on divalent cations (Mn²⁺ or Zn²⁺) is detected in A. fulgidus, while (ii) the IPS activities from the other organisms are not activated by metal ions or NH₄ ⁺ (the cofactor for all other known IPS). Although it is the first step in DIP synthesis, the IPS reaction is unlikely to be the point where DIP synthesis and accumulation are regulated since many archaea incorporate inositol into their lipids. If they incorporate L-I-1-P into lipids, then the second step, the generation of myo-inositol could be a way to regulate flow of resources into DIP.

The archaeal IMPase enzymes, easily identified by sequence homology to mammalian IMPases, have unusual properties. They exhibit similar substrate specificity to eukaryotic IMPases with one curious exception – they very specifically can dephosphorylate the phosphate on C1 of fructose bisphosphate 94. The FBPase activity identifies this class of enzymes as dual phosphatases that can process substrates in completely different pathways. FBPase activity gives it a potential role in gluconeogenesis. However, a 'true' FBPase with a homologue in all archaeal genomes was recently purified and cloned 9596, so that the IMPase/FBPase in archaea may normally function as an IMPase, (although, Methanocaldococcus jannaschii does not accumulate DIP, has no IPS sequence homologue, yet still has a gene for an IMPase/FBPase homologue which has been expressed and characterized 97). Nonetheless, it is intriguing that an enzyme that could act as either an IMPase or FBPase under the right circumstances (salt or temperature stress?) could link carbohydrate synthesis with responses to stress. At least for the IMPase from A. fulgidus, there are hints as to what could regulate this enzyme. This IMPase has two spatially close cysteine residues that can be oxidized to form a disulfide (either by vigorus bubbling with O₂ at 85°C or by adding oxidized E. coli thioredoxin 98). Formation of the intramolecular disulfide inactivates the enzyme; treatment with either a reducing agent or a reduced thioredoxin can regenerate active enzyme. Unfortunately, the lack of genetics with A. fulgidus makes it difficult to see what role this protein does play in hyperthermophiles. Although enzymes for the third and fourth steps in DIP production have not been identified, the last step has been demonstrated with cell extracts and added CDP-inositol and myo-inositol (L. Chen and M.F. Roberts, unpublished results). DIP synthesis required the presence of Mg²⁺.

The synthesis of this osmolyte has been examined in several hyperthermophiles. There appear to be two distinct pathways (Figure 9). In R. marinus, there is a direct condensation of GDP-mannose and D-glycerate to form α MG catalyzed by mannosylglycerate synthase 99. A second pathway, used by hyperthermophilic archaea, converts GDP-mannose and D-3-phosphoglycerate to mannosyl-3-phosphoglycerate via mannosyl-3-phosphoglycerate synthase, followed by phosphoglycerate phosphatase activity to remove the phosphate group 100101.

The biosynthesis of the solute cDPG diverts resources from gluconeogenesis by phosphorylation of 2-phosphoglycerate (2-PG) with ATP to form 2,3-diphosphoglycerate (DPG) via 2-phosphoglycerate kinase 102. Different transformations involving cDPG production and hydrolysis are shown in Figure 10. A novel enzyme, cyclic 2,3-diphosphoglycerate synthetase (cDPGS) 103104 then converts DPG to the cyclic form with ATP hydrolysis driving the reaction. Hydrolysis of cDPG to 3-PG would shuttle carbon and phosphate back into gluconeogenesis. In Methanothermus fervidus cDPGS is reversible, although it prefers the direction of cDPG synthesis 104. The ability to generate ATP from ADP, inorganic phosphate Pi) and cDPG under these conditions could argue for a role in energy storage in that organism. However, in Methanobacter thermoautotrophicus, the K⁺-activated cDGPS appears irreversible (and membrane-bound), suggesting it may have a different role 105. In soluble cell extracts, hydrolysis of cDPG in the presence of ADP and Pi could not generate ATP 106. However, a membrane bound hydrolase that is inhibited by K⁺ and Pi has also been identified 105. The regulation of cDPG degradation in M. thermautotrophicus is consistent with it playing a role as a carbon and phosphate storage compound, although its high concentration in cells clearly indicates it contributes to osmotic balance.

Betaine transport is common to a wide variety of halotolerant and halophilic organisms, both bacteria and archaea. There are basically two superfamilies of betaine transporters: (i) secondary transporters that use either the proton motive force or sodium motive force to drive betaine accumulation, and (ii) ATP binding cassette (ABC) transporters that couple ATP hydrolysis to uptake.

Most organisms that internalize this solute do so via a member of betaine choline carnitine transporter (BCCT) family of secondary transporters 108. The transporter can be quite specific for betaine as in the moderate halophilic lactic acid bacterium Tetragenococcus halophilus 108 or Gram-positive Marinococcus halophilus 109. Alternatively, the transporters available can internalize a wider range of solutes. For example, Listeria monocytogenes internalizes acetylcarnitine, carnitine, γ-butyrobetaine and 3-dimethylsulfoniopropionate as well as betaine, and the uptake increases the growth rate 110.

Other secondary transport systems have also been described. Corynebacterium glutamicum has the usual high affinity BetP uptake system. However, if this and the genes for three other compatible solute uptake systems are deleted, betaine can still be internalized, although the uptake is significantly reduced. The gene identified for the low capacity osmoregulated permease (lcoP) codes for a protein (LcoP) resembling a member of the BCCT-family 111. External osmolarity regulates expression and activity of LcoP.

Methanogens that can transport betaine into the cell tend to use a high affinity transporter 112 that is an ABC transporter. ABC betaine transporters have a nucleotide binding domain that hydrolyzes ATP, a membrane spanning domain, and a substrate binding domain (and/or a periplasmic or extracellular binding protein with a high affinity for betaine). The ota (

o

t

A

How does betaine finds the transporter? In hyperthermophiles, the high affinity ligand-binding protein ProX serves to bring the betaine to the transporter. Crystal structures of the A. fulgidus ProX in the absence and presence of betaine have identified cation-π interactions and non-classical hydrogen bonds between protein and ligand 114. Similar ligand binding domains have been identified in ORFs in the genomes of other archaea.

Ectoine that is provided in the medium can be internalized by some microorganisms. Growth of halotolerant Brevibacterium sp. JCM 6894 is stimulated by exogenous ectoine or hydroxyectoine 115. In H. elongata the transporter for ectoine and hydroxyectoine (TeaA, TeaB, TeaC) is similar to members of the tripartite ATP-independent periplasmic transporter family (TRAP-T) 116. The K_s(ect) is 21.7 μM, indicating a high affinity for external ectoine. The role of this transporter appears to be recovery of ectoine leaked from the cell. Marinococcus halophilus also can transport external ectoine. In this cell, the EctM gene product is a BCCT family member 108.

In the same vein, a proteomic analysis of Sinorhizobium meliloti in media that was supplemented with ectoine detected increased synthesis of ten proteins, eight of which were identified by MALDI-TOF analysis of peptides from the two-dimensional gels 117. Five of these belong to the same gene cluster (localized on the pSymB megaplasmid), whose components code for the ATP-binding cassette transporter ehu (ectoine/hydroxyectoine uptake). Another cluster of genes (eutABCDE) would produce proteins capable of ectoine catabolism. The net result of exposing S. meliloti to ectoine is to enhance the production of proteins to internalize and use any of these molecules that escape the cell.

Halobacterium salinarum has two ORFs upstream of transducer genes with significant homology to binding proteins for amino acids and compatible solutes 118. Deletion mutants indicate that the CoSB/CosT binding/transducer pair, in which the CosB is a membrane-anchored receptor, is critical for chemotaxis towards compatible solutes (in this case betaine). Whether or not the organism accumulates large amounts of betaine (which seems not to occur in Halobacterium NRC-1 119), this protein pair could function as a chemotaxis signaling pathway for organic osmolytes.

A variety of K⁺ channels have been identified in microorganisms. Structures of various K⁺ channels, initially a closed, small bacterial channel 120 and more recently a gated K⁺-channel (MthK) from Methanobacter thermoautotrophicus 121, have contributed to understanding how these proteins are arranged in membranes. However, these K⁺-channels do not respond to altered osmotic pressure. Rather, different protein complexes appear to regulate intracellular K⁺ in response to osmotic stress. H. elongata uses K⁺-glutamate as an osmolyte. Recent work has identified three genes required for K⁺ uptake: trkA, trkH, and trkI 122. The protein expressed by trkA would be analogous to the cytoplasmic NAD⁺/NADH binding protein TrkA in E. coli that is required for K⁺ uptake by the Trk system, while the H. elongata TrkH and TrkI are likely to be transmembrane proteins. Experiments with H. elongata indicate the TrkI is the main K⁺-transporter in this organism. Similar uptake systems may exist in other halophiles as well.

Cells will swell upon hypoosmotic shock as water rushes into the cell. To return to the original cell volume, cells need a rapid means of cytoplasmic solute efflux. All microorganisms have families of gated transmembrane channels that open for solute release when the lateral pressure of the membrane drops below a critical value (for review see 123).

M

s

c

V

a

c

Osmolytes in high concentrations compete with water molecules for interactions with protein surfaces. However, it has been proposed that these organic solutes are preferentially excluded from the surface of proteins 127128129130. This in turn leads to preferential hydration of the protein. The increased osmotic pressure generated by the solutes should favor compact folded proteins, which expose less surface area than denatured protein (Figure 11A). The size of internal cavities and internal water should be reduced as well 131. Differential interactions of organic solutes with folded and denatured proteins also contribute to their stabilization effects. Bolen and coworkers 132 have proposed that, compared to water, solutes have more unfavorable interactions with the peptide backbone and since unfolded protein has more available backbone, this biases the equilibrium to a folded protein (Figure 11B). This would suggest that osmolytes that impart stability actually interact with the unfolded state of the protein, shifting the equilibrium to promote the folded configuration (the 'osmophobic effect' 132). The free energy of the denatured state is higher than that of the native state, making population of this state energetically unfavorable (Figure 11B). Any interactions of osmolytes with hydrophobic residues of the unfolded protein do not overcome the osmophobic effect, nor do they interfere with the hydrophobic effect.

Ionic solutes will have a pronounced effect on water structure and these interactions will affect macromolecule stability. Neutral salts do not have the same effect on structure and solubilities of proteins. The Hofmeister series of ions reflects the ability of different ions to bind water 133134. 'Kosmotropes' (order makers) exhibit a strong interaction with water, while 'chaotropes' (disorder makers) exhibit weaker interactions with water than water has with itself. It is thought that kosmotropes bind water strongly and aid in preserving the hydration layer around the macromolecules. These solutes prefer interactions with water rather than the protein surface, hence preserve preferential hydration of the protein. Chaotropes can displace water from the protein surface and contribute to destabilization of structure by dehydrating the macromolecule. Many osmolytes are strikingly similar to the ions of the Hofmeister series: amino acids resemble ammonium acetate and the methylamines are functionally similar to quarternary ammonium ions 135. Their effects on proteins should then be similar to those in the Hofmeister series.

Collins and coworkers have shown that the Hofmeister series is actually a function of the apparent dynamic hydration number of the ion 136137, with the more hydrated an ion, the greater the stabilization of macromolecules. The calculated hydrated radius for each ion nicely coincides with the ionic strength. Collins postulated that the effect of an osmolyte on another solute (in this case the macromolecule) depends on the extent it perturbs the solvation layer of the other solute. If the osmolyte is tightly hydrated, it cannot as easily interact with the macromolecule solvation layer, an event that would destabilize the macromolecule.

Other considerations of water structure and the influence of solutes on charged regions of macromolecules have suggested that the water layer at the protein surface is more dense and reactive than bulk water 138. Patches of dense water, along with counterions, would cover charged surface regions of the protein, while inert zones of low-density water would be found around hydrophobic groups. Macromolecular crowding (see below) would also influence this. In dilute solutions, changing the density of the bulk water would be energetically unfavorable because of the large relative volume. When the solution is concentrated (a cell typically has 2 to 4 g water/g dry weight 138), the volume of surface water becomes comparable to the volume of bulk water, allowing density changes to occur.

The crowded and inhomogeneous environment of the cell also contributes to stabilization of folded proteins 139140141. The presence of solutes such as osmolytes, 'macrosolutes' such as cofactors, and other macromolecules aid in stabilizing proteins by decreasing the accessible volume, shifting the equilibrium between the folded and unfolded state of proteins to favor the more compact folded state.

This has relevance to pressure stress as well. Increasing hydrostatic pressure should promote water penetration from protein surface to the core. With osmolytes that are bigger than water, this penetration is less likely if critical water is around the protein and bulk water is diluted with the osmolyte. Studies of Photobacterium profundum solutes at 1 and 280 atm are perhaps the best evidence that small solutes can repel / inhibit water penetration at high hydrostatic pressures 52. Elevated hydrostatic pressure tends to denature proteins 142, presumably by enhancing water penetration into the protein core 143. In this deep sea organism, β-hydroxybutyrates accumulate to high intracellular levels at 280 atm. Perhaps these negatively charged solutes aid in preventing water penetration, both with an excluded volume effect but also by altering water structure in the vicinity of the proteins.

The observation of osmolyte cocktails in different types of cells likely reflects selection of solutes that cover different aspects of these effects – destabilization of the denatured state, retardation of water penetration in protein cores, and optimal modulation of water density for a particular cytoplasm. For example, one might expect thermophiles to have different solutes than mesophiles if solute exclusion is less important than other aspects of osmolyte effects (perhaps at high temperatures altering water structure is more important). The accumulation of multiple solutes may be explained by slightly different (and potentially overlapping) effects for each specific solute. For example, in a cell with multiple zwitterions, perhaps solute size or charge distribution are important in stabilizing water at some protein surfaces, while for other proteins preferential exclusion is the dominant effect. In cells with mixed zwitterions and anions, perhaps the anions are better excluded by proteins with a net negative charge, but if the organic anions are balanced by intracellular K⁺ there may be a limit on their intracellular concentration and so zwitterions are also accumulated.

Heat stress often provokes similar responses to salt stress. Organisms adapt to high external salinity by accumulating osmolytes, and the same solutes accumulated in vivo can also affect stability of microorganisms to thermal stress. E. coli cells adapted to grow in high salt contain increased betaine. Diamant et al. 144 showed that heat shock of these salt-adapted cells dramatically reduces the protein aggregation seen in non-adapted cells under the same stress. This behavior was suggested to result from osmolyte (specifically betaine, glycerol, proline or trehalose) activation of chaperones GroEL, DnaK, and ClpB. While such interactions could be shown at low osmolyte levels, at high osmolyte levels, refolding of proteins was reduced, possibly because of specific deleterious interactions of the osmolytes with chaperones.

In many cases, these small molecules assist in protein stabilization and/or refolding in vitro. The in vitro studies with purified enzymes allow one to explore any protective effects or unusual behavior of novel compatible solutes. These studies are consistent with the hypothesis that osmolytes are selected for their unfavorable interactions with peptide backbones 132.

Rabbit muscle lactate dehydrogenase has been used to test the effect of ectoine, hydroxyectroine, and their biosynthetic precursors DA and NADA on thermostability of this enzyme. At 55°C NADA enhances thermostability as measured by protection of the enzyme from thermal inactivation 89. Hydroxyectoine is more effective than ectoine and NADA at stabilizing proteins to heat stress. The one real difference with hydroxyectoine is a hydroxyl group on the ring. Perhaps this further functionalization of the (now trihydro)pyrimidine ring aids in organizing water and maintaining high surface tension at high temperatures.

Ribonuclease has been a popular target for osmolyte stabilization studies. This disulfide crosslinked enzyme can be reversibly unfolded in the absence of reducing agents. The effects of some of the more exotic osmolytes have been examined with this enzyme. 2-O-α-Mannosylglycerate, 0.5 M, increases the mid-point of the thermal denaturation curve, T_m, by 7°C as well as increases the heat capacity for the protein 145, an effect consistent with the solute destabilizing the denatured state with respect to folded protein. Other studies with ribonuclease D show that a variety of zwitterionic osmolytes dramatically increase T_m for the protein. As an example, 6 M sarcosine increases T_m by 24.6° at pH 5 146. Crystal structures of the ribonuclease fail to detect any bound osmolyte or alterations in water bound to the protein. The data support the hypothesis that osmolytes stabilize proteins by perturbing unfolded states, which biases the equilibrium to a compact, folded state.

The charge distribution of an osmolyte can be important to its biological activity as well. Solutes used by bacteria and archaea have not been examined since the pKa of any functional groups are well outside accessible pH ranges for maintaining native proteins. Trimethylamine N-oxide (TMAO), a common solute in eukaryotes, is zwitterionic above pH 6 (pKa of 4.66), and it is this state of the molecule that is critical for its stabilization of proteins 147. At low pH it no longer acts as a good thermoprotectant. While this observation may not have physiological relevance, it aids in our understanding of osmolyte properties important for their biological effects. TMAO has been shown to decrease the entropy of the unfolded state of onconase through a solvophobic effect 148. This solute clearly diminishes the unfolding rate while having little effect on the stability of the native protein. For onconase, TMAO appears to induce a local structural change that retards unfolding.

The solute exclusion theory would argue for little specificity in osmolyte effects on macromolecules. However, there are many studies that clearly show preferential stabilization by solutes. Osmolytes can counteract denaturants such as urea. Potassium D- or L-glutamate (0.25 M) counteracts the effect of urea on glutaminyl-tRNA synthetase from Escherichia coli by shifting the equilibrium between the native and molten globule and molten globule to unfolded protein to a higher urea concentration 149. However, for this protein other osmolytes (sorbitol, TMAO, inositol) cannot induce the same shift. A major conclusion of these studies is that the ability of an osmolyte to counteract urea denaturation depends on specific osmolyte-protein interactions.

As another example, two archaeal rubredoxins have been shown to be stabilized to quite different extents by α-diglycerol phosphate 150. Their structures are similar, except that one is missing a hairpin loop. There are small conformational changes induced by α-DGP (or mannosylglycerate) and evidence for solute inducing a more compact state of the protein, and the occurrence of weak, specific interactions between osmolyte and protein surface.

Osmolytes can affect protein conformation and motions of native structures as well. TMAO induces α-helix formation of alanine-base peptides 151. Compatible solutes also attenuate structural fluctuations as measured by amide hydrogen-deuterium exchange rates 152153154. Osmolytes certainly inhibit slow, large unfolding transitions, but they can also modulate fast exchange rates as well 155. Tryptophan phosphorescence has been used to probe the flexibility of the native structure of azurin and a number of mutants 131. The sugar dampens fluctuations only for loose internally hydrated macromolecules and those with thermally expanded conformations. The sucrose (and presumably other polyols) will shift the equilibrium of protein conformations to a more compact rigid form.

One of the interesting questions is whether or not solutes in halophiles stabilize proteins in the same manner as for nonhalophiles. An interesting case in point is the effect of KCl on the dihydrofolate rductase (DHFR) from Haloferax volcanii compared to that from E. coli 156. The protein from the extreme halophile is much more acidic and one might think the stabilization effects by K⁺ would differ compared to the mesophilic, non-halophilic homologue. The H. volcanii DHFR requires at least 0.5 M KCl to stay folded, while the E. coli protein is inactive above 1 M KCl. Yet the effect of salts on the stability of the proteins to urea is similar, if one compares stability at the appropriate physiological ionic strength. This work shows that salts stabilize the DHFRs by a common mechanism – preferential hydration and the Hofmeister effect of salt on the activity and entropy of the aqueous solvent. Although one could imagine hydrated salt networks occurring in the halophilic protein leading to halophile-specific stabilization, that is not the case.

Although most research into how osmolytes affect macromolecular stability has concentrated on proteins/enzymes, these solutes also affect nucleic acid stability. The addition of high concentrations of zwitterionic solutes increases the dielectric constant of the solution that, in turn, decreases ionic interactions and affects the DNA duplex. Isolated studies have explored the effect of zwitterionic solutes on nucleic acid stability. For example, betaine has been shown to eliminate the dependence of dsDNA melting on the base pair composition 157 and to enhance amplification of GC-rich templates 158 by lowering the T_m for the template. High concentrations of compatible solutes also alter accessibility of regions of the DNA to nucleases. Malin et al. 159 showed that ectoine and hydroxyectoine alter the DNA conformation such that endonucleases can no longer cleave it.

Insoluble or misfolded overexpressed proteins can often be partially denatured and refolded in the presence of osmolytes. A specific example is the use of osmolytes to enhance the yield of folded, functional cytotoxic proteins directed to the periplasm of E. coli 161. Cells grown in 4% NaCl with 0.5 M sorbitol and supplemented with 10 mM betaine can accumulate large amounts of the target protein in the periplasm (this was tried with immunotoxins). Protein is released by freeze-thaw cycles. Both high osmotic strength and added compatible solutes (in this case betaine and sorbitol) are necessary for high yields of protein.

In the same vein, ectoine, betaine, trehalose, and citrulline have been shown to inhibit insulin amyloid formation in vitro 162. This observation may provide directions for designing small molecules to inhibit myelin formation associated with neurodegenerative disorders.

Several osmolytes (notably betaine, ectoine) have been shown to be useful in PCR amplification of GC-rich (72.6% GC) DNA templates with a high T_m. In particular, ectoine was shown to outperform regular PCR enhancers; it works by reducing the DNA T_m 163. Interestingly, hydroxyectoine increases the T_m of duplex DNA. However, the optimal solute for these experiments is homoectoine (4,5,6,7-tetrahydro-2-methyl-1H-13-diazepine-4-carnoic acid), a synthetic derivative of ectoine with the ring expanded by one carbon. For betaine the effective range of solute is 0.5 to 2.0 M; for ectoine much less (0.25 to 0.5 M) is needed for the same effect. It would be intriguing to see what effect DIP type solutes have on PCR since they are synthesized by hyperthermophiles above 80°C.

Organic osmolytes have also been used as cryo-protectants. In a recent study, the ability of betaine to act as a cryo-protectant during freezing of diverse bacteria was examined. Betaine is often much better than two common cryo-protectant mixtures, serum albumin and trehalose/dextran, particularly under conditions simulating long-term storage 164. It is better than the other treatments at preserving long term viability for microorganisms like Neisseria gonorrhoeae and Streptococcus pneumoniae. Betaine is as effective as glycerol for liquid nitrogen freezing of halophilic archaea, and neutrophilic Fe-oxidizing bacteria.

The ability of osmolytes to aid in protecting cells from diverse stresses has led to the use of at least one of them, ectoine, in the cosmeceutical industry. Ectoine has been shown to protect skin from UVA-induced cell damage 65. Based on this, RonaCare™ Ectoin, produced by Merck KgaA, Darmstadt, is presently in use as a moisturizer in cosmetics and skin care products.

Osmolytes have not been developed as reagents in the pharmaceutical industry, in part because as 'compatible solutes' they interact minimally with cellular machinery. However, their ability to stabilize biomolecules may have some very specific uses. As an example, the German company Bitop in collaboration with researchers at the Cologne University Clinic is exploring the use of these solutes in certain cancer therapies where they may protect tissues against vascular leak syndrome, a severe side effect of anti-caner agents.

Insertion of genes for osmolytes into non-halotolerant organisms should increase their ability to withstand salt stress. Plants are a good target for these types of experiments since they are often exposed to drought conditions that would concentrate salt. A few reports of transgenic plants suggest that eventually this strategy might be useful. Arabidopsis thaliana transformed with a choline oxidase gene (which is needed to synthesize betaine) from Arthrobacter globiformis has a significantly improved tolerance of salt stress along with improved cold and heat tolerance 166. Transgenic tobacco with E. coli betA and bet B genes has also been constructed. This modified plant exhibits better salt and cold tolerance 167. Inserting the H. elongata ectABC genes also confers hyperosmotic tolerance on cultured tobacco cells 168. This was shown to increase the hyperosmotic tolerance of cultured cells, although only a small amount of ectoine accumulated. Other recent work to introduce genes for synthesizing osmolytes in plants 169 as a way to improve stress tolerance has, so far, not led to high accumulation of the osmolytes. Further developments await a determination of what limits osmolyte levels in plant cells.