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Applied and Environmental Microbiology, December 2005, p. 8091-8098, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8091-8098.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Compatible Solutes of the Hyperthermophile Palaeococcus ferrophilus: Osmoadaptation and Thermoadaptation in the Order Thermococcales

Clélia Neves,1* Milton S. da Costa,2 and Helena Santos1*

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal,1 Departamento de Bioquímica and Centro de Neurociências de Coimbra e Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal2

Received 26 July 2005/ Accepted 12 September 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effect of salinity and growth temperature on the accumulation of intracellular organic solutes was examined in the hyperthermophilic archaeon Palaeococcus ferrophilus. The genus Palaeococcus represents a deep-branching lineage of the order Thermococcales, which diverged before Thermococcus and Pyrococcus. Palaeococcus ferrophilus accumulated mannosylglycerate, glutamate, and aspartate as major compatible solutes. Unlike members of the genera Pyrococcus and Thermococcus, Palaeococcus ferrophilus did not accumulate di-myo-inositol phosphate, a canonical solute of hyperthermophiles. The level of mannosylglycerate increased in response to both heat and salt stress; glutamate increased at supraoptimal growth temperatures, whereas aspartate increased at supraoptimal salt concentration. Proline, alanine, and trehalose were also found in lesser amounts, but their levels did not respond significantly to any of the stresses imposed. Additionally, the genes involved in the synthesis of mannosylglycerate in Palaeococcus ferrophilus and Thermococcus litoralis were identified. In both organisms the synthesis proceeds via the two-step pathway comprising mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The mpgS and mpgP genes of Palaeococcus ferrophilus were expressed in Escherichia coli and the proteins were characterized. MPGS had maximal activity at 90°C and pH near 7.0, and was strictly dependent on Mg2+. MPGP had optimal activity at 90°C and pH 6.0 and was barely dependent on Mg2+. The half-life values for inactivation of MPGS and MPGP at 83°C were 18 and 25 min, respectively. A comparative discussion of the osmo- and thermoadaptation strategies in these three genera of the Thermococcales is presented.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The three genera of the order Thermococcales, Thermococcus, Pyrococcus, and Palaeococcus, constitute a large and diverse group of organisms widely used as model systems for biochemical and physiological studies of hyperthermophilic archaea (1). Strains of the genera Pyrococcus and Thermococcus have been the subject of a large number of studies, in contrast with the paucity of information on the genus Palaeococcus, a deep-branching lineage of the Thermococcales, diverging before the species of the genera Thermococcus and Pyrococcus. The genomes of three Pyrococcus species (Pyrococcus horikoshii, Pyrococcus abyssi, and Pyrococcus furiosus) have been completely sequenced and recently the genome sequence of Thermococcus kodakaraensis KOD1 has also been disclosed (15).

In general, the members of the Thermococcales are anaerobic heterotrophs which, depending on the species, have optimal growth temperatures in the range of 80°C to 100°C. The type strains of Pyrococcus furiosus and Thermococcus litoralis and the recently described Palaeococcus helgesonii were isolated from shallow-water hydrothermal springs on the island of Vulcano, Italy, and Palaeococcus ferrophilus, Pyrococcus horikoshii, and Pyrococcus abyssi type strains were isolated from hydrothermal vents of the Pacific Ocean floor (2, 13, 14, 19, 32, 44).

Hyperthermophiles isolated from marine environments, like other moderate halophilic organisms, adapt to the salinity of the milieu by de novo synthesis or uptake of low-molecular-mass organic compounds, designated compatible solutes, that allow them to control the internal water activity and protect intracellular macromolecules against desiccation (7, 40). The compatible solutes of hyperthermophiles, however, are generally different from those found in mesophiles: typically they are negatively charged compounds and are never or rarely encountered in mesophiles (31, 41).

Mannosylglycerate (MG), diglycerol phosphate, di-myo-inositol phosphate (DIP) and derivatives are archetypical solutes of archaea and bacteria that thrive in hot environments (31). MG and DIP are widespread among (hyper)thermophiles, whereas diglycerol phosphate has been found, thus far, only in strains of the genus Archaeoglobus (18).

Mannosylglycerate is widely distributed among thermophilic bacteria and hyperthermophilic archaea, including euryarchaeotes of the genera Pyrococcus and Thermococcus, and also in Archaeoglobus veneficus and Archaeoglobus profundus (10, 18, 26, 28). The crenarchaeotes Aeropyrum pernix and Stetteria hydrogenophila also accumulate MG (our unpublished data). Among thermophilic bacteria, MG has been identified in Thermus thermophilus, Rhodothermus marinus, and Rubrobacter xylanophilus (33, 41). DIP accompanies MG in all the above-mentioned archaea and also occurs in the extreme hyperthermophilic archaeon Pyrolobus fumarii (17). (Hyper)thermophilic bacteria belonging to the genera Thermotoga, Aquifex, and Rubrobacter also accumulate DIP (9, 29, 40). At least in vitro, MG was better than other solutes to protect proteins against heat damage and its potential application as stabilizer of biomaterials was often reported (4, 31, 35).

Given the close phylogenetic relationship between the genera Pyrococcus, Thermococcus, and Palaeococcus, we deemed it interesting to compare the strategy of thermoadaptation and osmoadaptation in the Thermococcales. The heat and osmotic stress responses in Pyrococcus and Thermococcus spp. have been characterized but, in this respect, information is not available for any member of the third genus. The genus Palaeococcus comprises two species, Palaeococcus ferrophilus (44) and Palaeococcus helgesonii (2). Palaeococcus ferrophilus is a barophilic hyperthermophilic archaeon displaying an absolute requirement for either elemental sulfur or ferrous iron for growth. It grows between 60 and 88°C and the optimal temperature for growth is 83°C (44). Herein we report the osmotic and heat stress responses of Palaeococcus ferrophilus in regard to compatible solute accumulation; furthermore, the genes for the synthesis of MG were identified and the recombinant proteins were characterized.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains, plasmids, and culture conditions.
Palaeococcus ferrophilus (DSM 13482T) and Thermococcus litoralis (DSM 5474T) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. Palaeococcus ferrophilus was grown in MJ(-N) synthetic seawater described by Takai et al. (44), containing 2 g yeast extract, 5 g tryptone, 0.5 g Na2S · 9H2O and 1.7 g NaNO3 per liter. Thermococcus litoralis was grown in Bacto Marine Broth (Difco). In both media the final pH was adjusted to 6.5. Sterile elemental sulfur (S0) was added (3.0 g/liter) after cooling to 83°C.

For the quantification of compatible solutes in Palaeococcus ferrophilus the cells were grown in 5-liter fermentation vessels with continuous gassing with argon and stirring at 120 rpm. Cultures were grown at 83°C, 87°C, and 90°C in medium containing 3% NaCl. To study the effect of osmotic stress on the synthesis of intracellular solutes, the salinity of the growth medium was varied between 3.0 and 5.0% NaCl. Cells were grown until late-exponential growth phase, harvested, and washed twice with a solution lacking organic nutrients but otherwise identical to the medium in which the cells were grown. For genomic DNA extraction both species were grown in 100 ml serum bottles with rubber stoppers, at 83°C, without agitation.

Escherichia coli strains DH5{alpha} and BL21 (Amersham Pharmacia Biotech) were used as hosts for the cloning vectors pGEM-T Easy (Promega) and pGEX-4T-2 (Amersham Pharmacia Biotech), respectively. E. coli was grown in LB broth and ampicillin (100 µg/ml) was used for selection of plasmids. IPTG (isopropyl-ß-D-thiogalactopyranoside) and X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) were obtained from Roche Molecular Biochemicals (Germany) and added to final concentrations of 0.25 mM and 80 µg/ml, respectively.

Ethanol extraction, solute quantification, and protein determination.
Extraction of compatible solutes was performed with boiling 80% ethanol by the method of Reed et al. (37), modified as previously described by Silva et al. (43). The final aqueous phase was lyophilized and the residue was dissolved in 2H2O for nuclear magnetic resonance analyses. Mannosylglycerate and trehalose were quantified by 1H-nuclear magnetic resonance. Amino acids were determined in a Pico-Tag amino acid analysis system (Waters, Milford, Mass.). Protein contents of the cells were determined by the bicinchoninic acid method (Pierce) after hydrolysis of the samples with 0.1 M NaOH overnight at 37°C.

Analysis of mannosylglycerate formation by thin-layer chromatography.
Thin-layer chromatograms were performed on Silica Gel 60 plates (Merck) with a solvent system composed of n-propanol and ammonia (1:1, vol/vol). MG and other sugar derivatives were visualized by spraying with {alpha}-naphtholsulfuric acid solution, followed by charring at 120°C. Authentic standards of MG, mannosyl-3-phosphoglycerate (MPG), mannose, and GDP-mannose were used for comparative purposes.

DNA methodology, cloning, and analysis.
DNA manipulations followed standard molecular techniques and procedures (39). Chromosomal DNA was isolated from Palaeococcus ferrophilus and Thermococcus litoralis as described by Marmur (27). Based on the known sequences of the mpgS and mpgP genes from Pyrococcus furiosus, Pyrococcus abyssi, and Pyrococcus horikoshii, sets of degenerate sense and antisense primers were designed and used to amplify the homologous genes from genomic DNA of Palaeococcus ferrophilus and Thermococcus litoralis. PCR amplification products were purified from agarose gels and ligated to the pGEM-T Easy vector (Promega). Nucleotide sequences of the inserts were determined in both directions by AGOWA (Berlin, Germany) using vector-specific and insert-specific oligodeoxynucleotide primers. Nucleic acid and protein sequence analysis were conducted with programs in the European Bioinformatics Institute. Genome sequence databases were screened for homologies using the (T)FASTA and (T)BLAST algorithms.

Based on the complete gene sequence, mpgS was amplified by the forward primer (5'-GCGGGATCCATGCTTCTAGAGGCTCCTGTATAC-3') and adding a recognition sequence for BamHI (underlined) immediately upstream of the start codon ATG. The reverse primer (5'-GCGGAATTCTCACACCTCGAAGTGGAAGAG-3') was constructed by adding an EcoRI recognition sequence (underlined) directly behind the stop codon. A similar procedure was followed to amplify mpgP with the forward primer (5'-GCGGGATCCATGAGGGTGATATTCCTCGAC-3') and the reverse primer (5'-GCGGAATTCTCATAACACCACCTCCAGCAA-3'). PCR products were digested, purified and ligated into the glutathione-S-transferase (GST) fusion vector pGEX-4T-2 (Amersham Pharmacia Biotech). E. coli BL21 cells (Amersham Pharmacia Biotech), bearing the constructs, were grown at 37°C in LB medium supplemented with ampicillin to an A600 of 0.6 and induced with IPTG for 3 h.

Purification of the recombinant mannosyl-3-phosphoglycerate synthase (MPGS) and mannosyl-3-phosphoglycerate phosphatase (MPGP).
GST fusion proteins were purified by fast protein liquid chromatography (Amersham Pharmacia Biotech). The cell extracts were disrupted in a French press and the cleared supernatant was applied onto a GSTPrep FF16/10 column prepacked with glutathione Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) and equilibrated with phosphate-buffered saline buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3). Elution was carried out with 50mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. Fractions corresponding to the eluted peak were pooled, concentrated and equilibrated with 20 mM Tris-HCl (pH 7.6). The GST/MPGS and GST/MPGP fusion proteins were cleaved with thrombin at 22°C overnight and incubated for 10 min at 60°C to denature host proteins.

Characterization of MPGS.
To determine the activity of the recombinant MPGS in cell extracts, the reaction mixtures, containing 5 mM GDP-mannose (Sigma) plus 5 mM D-3-phosphoglycerate (sodium salt, Sigma) in 20 mM Tris-HCl (pH 7.6) with 10 mM MgCl2, were incubated at 83°C for 30 min and MPG formation was visualized by thin-layer chromatography. As glycosyl donors in the reaction catalyzed by the MPGS we tested ADP-mannose, GDP-mannose, UDP-mannose, ADP-glucose, GDP-glucose, UDP-glucose, mannose, mannose-1-phosphate and mannose-6-phosphate. Glycerol, glycerate, D-3-phosphoglycerate, D-2-phosphoglycerate, and phosphoenolpyruvate were tested as putative glycosyl acceptors. The temperature profile for the activity of pure MPGS was determined between 30°C and 104°C, in reaction mixtures containing 5 mM GDP-mannose and 5 mM D-3-phosphoglycerate in 50 mM BisTris/propane (pH 7.6) with 10 mM MgCl2.

The effect of pH on MPGS activity was determined at 83°C in 50 mM BisTris/propane buffer (pH 5.0 to 9.0) and 50 mM cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO, pH 8.0 to 11.0). All pH values were measured at room temperature (25°C); pH values at 83°C were calculated using the conversion factor {Delta}pKa/{Delta}T °C = –0.015 for BisTris/propane and –0.018 for CAPSO. In all experiments the samples were preheated for 3 min and the reactions were initialized by the addition of the recombinant enzyme. The reaction mixtures were incubated at 83°C during 1, 2, or 3 min and immediately frozen in liquid nitrogen.

The MPG synthesized was quantified by 1H-nuclear magnetic resonance after freeze-drying and dissolution in 2H2O. Formate was used as an internal concentration standard. This protocol was used to examine also the effect of MgCl2 concentration and thermal stability of the recombinant MPGS. Enzyme thermostability was determined at 83°C by incubating enzyme solutions (0.5 mg/ml) in 20 mM Tris-HCl (pH 7.6). At appropriate times, samples were withdrawn and immediately examined for residual activity at 83°C. All experiments were performed in duplicate.

Characterization of MPGP.
The activity of the purified recombinant MPGP was measured in 50 mM BisTris/propane (pH 7.6) containing 10 mM MgCl2 and 2 mM MPG. Besides MPG, several sugar phosphates were examined as possible substrates for MPGP, mannose-1-phosphate, mannose-6-phosphate, glucose-1-phosphate, glucose-6-phosphate, fructose-1-phosphate, fructose-6-phosphate, trehalose-6-phosphate, and ribose-5-phosphate, as well as GTP, GDP, and GMP. Of these, only MPG was dephosphorylated (results not shown). The reactions were followed by thin-layer chromatography.

The temperature profile for the activity of MPGP was determined between 30°C and 104°C, in the same conditions. The effect of pH on MPGP activity was determined at 83°C in 50 mM acetate buffer (pH 3.5 to 5.5), 50 mM BisTris/propane buffer (pH 5.0 to 9.0) and 50 mM CAPSO (pH 8.0 to 11.0). In all the experiments samples were preheated for 1 min and the reactions initialized by the addition of the recombinant enzyme. The reaction mixtures were incubated at 83°C during 30, 45, or 60 seconds and immediately frozen in liquid nitrogen.

The activity of MPGP was quantified by monitoring the release of inorganic phosphate using the method described by Ames (3). This protocol was used to examine also the effect of MgCl2 concentration and thermal stability. Enzyme thermostability was determined at 83°C by incubating enzyme solutions (0.5mg/ml) in 20 mM Tris-HCl (pH 7.6). At appropriate times, samples were withdrawn and immediately examined for residual activity at 83°C. All experiments were performed in triplicate. MPG was obtained from a reaction catalyzed by pure recombinant MPGS with 15 mM GDP-mannose and 15 mM D-3-phosphoglycerate as substrates in 50 mM BisTris/propane (pH 7.6) and 10 mM MgCl2 at 83°C for 2 h. Quantification of MPG was carried out by 1H-nuclear magnetic resonance.

Nucleotide sequence accession numbers.
The nucleotide sequences of mannosyl-3-phosphoglycerate synthase and mannosyl-3-phosphoglycerate phosphatase from Palaeococcus ferrophilus have been deposited in GenBank under accession number AY998560. The nucleotide sequences of mannosyl-3-phosphoglycerate synthase and mannosyl-3-phosphoglycerate phosphatase from Thermococcus litoralis were deposited in GenBank under accession number AY998561.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of temperature and salt concentration on growth and solute accumulation by Palaeococcus ferrophilus.
The 13C- and 1H-nuclear magnetic resonance spectra of ethanol extracts contained several sets of major resonances that were assigned to MG, trehalose, glutamate, and aspartate by comparison with the chemical shifts reported previously (26). The major organic solutes were MG, aspartate, and glutamate, but minor amounts of alanine, proline, and trehalose were also detected. The total amount of internal solutes increased remarkably at temperatures and salinity above the optimum values for growth (Fig. 1). However, the nature of the solute accumulation changed according to the type of stress imposed.



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  		FIG. 1. Effect of NaCl concentration (A) and temperature (B) on the accumulation of solutes by Palaeococcus ferrophilus. Solid circles indicate growth rates. Cells were grown at the optimal temperature, 83°C (A), or optimal NaCl concentration, 3% (B), and samples were collected in the late exponential phase of growth. Aspartate (checkered), glutamate (white dotted), alanine (white), proline (black dotted), trehalose (gray), and mannosylglycerate (stippled) were determined.

 
Palaeococcus ferrophilus had a slightly halophilic behavior: the range of NaCl concentrations for growth was 1.5 to 6.0% (wt/vol) NaCl with an optimum at about 3% NaCl (results not shown). Cells grown at higher salt concentrations were enlarged compared to cells grown at optimum conditions. At the optimum salinity the total solute pool was low, MG being the major compatible solute present (0.28 µmol/mg protein). An increase of the NaCl concentration of the medium to 4% resulted in a small increase of MG concentration (0.31 µmol/mg protein), but a notable increase in aspartate (0.62 µmol/mg protein) and glutamate levels (0.63 µmol/mg protein). At the highest salinity examined (5% NaCl) the level of MG reached 0.84 µmol/mg protein, representing 44% of the total organic solutes present and showing a threefold increase over those of the optimal growth conditions. Glutamate and aspartate were also found in reasonably high amounts, representing 16% and 28% of the organic solute pool, respectively. Trehalose was detected at low levels in cells grown at 5% NaCl.

In cell cultures subjected to heat stress, MG was the predominant compatible solute (0.75 µmol/mg protein), but glutamate also seemed to have an important role, reaching values of 0.6 µmol/mg protein in cells grown at 90°C. In comparison with the optimal cell growth conditions (83°C) there was a 2.7-fold increase in MG and a 4.5-fold increase in glutamate. DIP was not detected under any of the growth conditions examined.

Identification of mpgS and mpgP genes.
The genes encoding MPGS and MPGP of Palaeococcus ferrophilus were identified and the respective functions were confirmed by functional overexpression in E. coli. The alignment of the amino acid sequences of known MPGS and MPGP from Thermococcales indicated a high amino acid identity among the species of this order (Fig. 2). The degree of sequence identity is high within the Thermococcales and significantly lower compared to the bacterium Rhodothermus marinus or the crenarchaeote Aeropyrum pernix. Immediately downstream of mpgP in Palaeococcus ferrophilus and Thermococcus litoralis there is a sequence encoding a putative bifunctional mannose-1-phosphate guanylyltransferase/phosphomannose isomerase (M1P-GT/PMI), which indicates a putative operon structure similar to that found in Pyrococcus strains (10).



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  		FIG. 2. Amino acid identity of mannosyl-3-phosphoglycerate synthase (gray background) and mannosyl-3-phosphoglycerate phosphatase (white background) from Palaeococcus ferrophilus and Thermococcus litoralis with other known MPGSs and MPGPs. Pfer, Palaeococcus ferrophilus; Phor, Pyrococcus horikoshii (GenPept BAA30023 and BAA30022); Paby, Pyrococcus abyssi (GenPept CAB50138 and CAB50139); Pfur, Pyrococcus furiosus (GenPept AAL80715 and AAL80714); Tlit, Thermococcus litoralis; Rmar, Rhodothermus marinus (GenPept AAP74552 and AAP74553); Tthe, Thermus thermophilus (GenPept AAO43097 and AAO43098); Aper, Aeropyrum pernix (GenPept BAA79872 and BAA79870).

 
Cloning, overexpression, and purification of recombinant MPGS and MPGP.
Activity assays carried out with the purified recombinant proteins revealed MPG production by the MPGS clones and MPG dephosphorylation by MPGP clones. We estimate yields of 2 to 3 mg of protein per liter of culture. The final preparations of recombinant enzymes yielded single bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses determined for MPGS and MPGP were in excellent agreement with the sizes of 44.3 and 27.1 kDa predicted from the respective gene sequences.

Catalytic properties of MPGS and MPGP.
Of the sugar donors and acceptors examined as possible substrates for MPGS (see list in Materials and Methods) only GDP-mannose and D-3-phosphoglycerate led to the formation of MPG, demon-strating the high specificity of the transferase for these substrates. Of the phosphorylated compounds examined as putative substrates of the MPGP, only MPG was dephosphorylated. MPGS showed absolute requirement for Mg2+; in contrast, the activity of MPGP in the absence of Mg2+ reached 90% of that in the presence of this divalent cation. At 20°C the activity of both MPGS and MPGP was undetectable and maximal activity was reached at around 90°C (Fig. 3). Within the pH range examined (4.0 to 9.0) the activity of MPGS at 83°C was maximal between pH 6.5 and 7.4 and the activity of MPGP was maximal between 5.7 and 7.0 (Fig. 3). The Vmax for MPGS and MPGP, determined at 90°C and pH 7.0 and 6.5, respectively, was 331 and 101 mmol/min/mg protein, respectively. At the optimal temperature for growth of Palaeococcus ferrophilus, 83°C, the half-life for inactivation of MPGS and MPGP was 18 min and 25 min, respectively.



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  		FIG. 3. Temperature profile (A) and pH dependence (B) of the activity of recombinant mannosyl-3-phosphoglycerate synthase (solid symbols) and recombinant mannosyl-3-phosphoglycerate phosphatase (open symbols) of Palaeococcus ferrophilus. The enzyme activities were determined between 20°C and 104°C and between pH 3.7 and 9.0 in acetate buffer ({diamond}), BisTris/propane ({blacksquare} and {square}) and CAPSO (• and {circ}) at 83°C. Error bars indicate standard deviation values.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To gain insights into the physiological role of compatible solutes and the evolutionary relationships underlying their distribution in (hyper)thermophiles, we compared the temperature and osmotic stress responses with respect to compatible solute accumulation in members of the three genera of the order Thermococcales. The genes and enzymes required for the synthesis of mannosylglycerate, a solute widespread in (hyper)thermophiles, were also considered for this comparison.

As in Pyrococcus furiosus (28) the organic solute pool of Palaeococcus ferrophilus increased notably in response to either temperature (2.5-fold) or salinity (2.8-fold). Mannosyl-glycerate, aspartate, and glutamate were the major solutes of Palaeococcus ferrophilus. In particular, the level of MG increased considerably in response to either supraoptimal temperature or NaCl concentration. Glutamate and aspartate, on the other hand, appear to play different roles: the level of glutamate responded primarily to heat stress, whereas aspartate accumulated in response to salt stress. Aspartate is not a common compatible solute, but it also plays a role in adaptation to supraoptimal NaCl concentrations in Thermococcus spp. (26). Glutamate is used in many mesophilic bacteria and archaea as a compatible solute during low-level osmoadaptation (7, 16), but in Thermococcus litoralis and Archaeoglobus fulgidus this amino acid is clearly part of the salt stress response (18, 26). Curiously, in Palaeococcus ferrophilus, glutamate, along with MG, accumulated in response to supraoptimal temperature.

DIP occurs in all strains of Thermococcus and Pyrococcus examined to date (10, 26, 28, 42) but was not present in Palaeococcus ferrophilus. Furthermore, the DIP level increases consistently in response to heat stress in all the organisms examined so far. Thus, the absence of DIP in Palaeococcus ferrophilus was unexpected. The specialized role of DIP in thermoadaptation was replaced in Palaeococcus ferrophilus by glutamate and MG, which appear to render the required degree of protection at the growth temperature of this organism, which has one of the lowest optimum growth temperatures among the Thermococcales.

The preferential involvement of MG and DIP in the osmotic and heat stress responses, respectively, has been observed in several cases and is particularly evident in Pyrococcus furiosus, in which MG responds mainly to increased NaCl concentration of the medium, and DIP is the only solute accumulating at supraoptimal growth temperatures (28). Interestingly, in Palaeococcus ferrophilus, MG increased in response to both salt stress and heat stress, behavior resembling that observed in Rhodothermus marinus (43).

Thermococcus litoralis shows a remarkable capacity to scavenge components from the medium and use them as compatible solutes (26); on the other extreme, members of the genus Pyrococcus rely mainly on de novo synthesis for osmotic adjustment and accumulate a restricted number of solutes. Palaeococcus ferrophilus appears to have an intermediate ability to take up solutes from the medium, at least if we assume that trehalose, aspartate, proline, and glutamate are amassed in this way. Obviously, the capacity to scavenge components from the medium is related to the presence of suitable transporters. ABC-transporters for trehalose have been described in Thermococcus litoralis and Pyrococcus furiosus and highly homologous sequences are present in the genomes of Pyrococcus horokoshii and Pyrococcus abyssi (8, 47); however, as judged from the genome sequence, Thermococcus kodakaraensis does not possess a similar transport system for this ubiquitous solute.

A comparison of the distribution of compatible solutes in members of the Thermococcales (Table 1) shows a great predominance of negatively charged solutes, the only exception being trehalose, which generally occurs in low levels. The superior efficacy of negatively charged solutes (phosphodiester compounds or carboxylic acids) to enhance protein thermostability has been demonstrated in several studies from our and other groups (4, 6, 23, 35). Therefore, it is interesting that hyperthermophiles have adapted their cellular components through evolution to be able to take advantage of the most effective protectors available against heat.


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  		TABLE 1. Accumulation of compatible solutes in species of the order Thermococcales in response to high salinity of the growth medium

 
The use of charged solutes as stabilizers of cell components implies the intracellular accumulation of positive counterions, such as potassium; high salt concentrations are known to be toxic for most mesophilic bacteria, but archaea, regardless of their temperature requirement for growth, use preferentially charged solutes for osmotic adjustment (38). Interestingly, hyperthermophilic and extreme thermophilic bacteria, such as Thermotoga maritima, Aquifex pyrophilus, Thermus thermophilus, and Rhodothermus marinus, also show a strong preference for charged compatible solutes whose level responds notably not only to osmotic stress but also to heat stress. It is therefore tempting to speculate that during evolution some (hyper)thermophilic bacteria acquired this common archaeal attribute, which seems important to endow them with the level of extrinsic stabilization needed for adjustment to supraoptimal temperatures in hot environments.

The biosynthetic pathways for MG have been characterized at the genetic and enzyme levels in Pyrococcus horikoshii, Thermusthermophilus, and Rhodothermus marinus (10, 11, 30). All the microorganisms examined to date use the two-step pathway exclusively with the exception of Rhodothermus marinus, in which the single-step pathway is also functional (5). According to our results, in Palaeococcus ferrophilus and Thermococcus litoralis, the synthesis of MG proceeds exclusively via the two-step pathway involving the phosphorylated intermediate mannosyl-3-phosphoglycerate, since the activity of mannosylglycerate synthase, the enzyme catalyzing the single-step pathway, was not detected in cell extracts of these organisms (results not shown).

In Palaeococcus ferrophilus and Thermococcus litoralis the genes encoding MPGS and MPGP are adjacent and putative bifunctional mannose-1-phosphate guanylyltransferase/phosphomannose isomerase (M1P-GT/PMI) and phosphomannomutase sequences appear immediately downstream the mpgP gene. Apparently this gene cluster, initially identified in Pyrococcus horikoshii (10), is a feature common to the three genera of the Thermococcales. Interestingly, Thermococcus kodakaraensis has the genes for M1P-GT/PMI immediately upstream of the phosphomannomutase gene but lacks the mpgS/mpgP cluster (36). In other archaea and bacteria examined, mpgS and mpgP have an adjacent location but the genes encoding M1P-GT/PMI and phosphomannomutase appear elsewhere in the genome (5, 11, 12).

The MPGS of Palaeococcus ferrophilus shares many properties with its counterparts from other MG-accumulating organisms (Table 2). In particular, they show very high substrate specificity for GDP-mannose and 3-phosphoglycerate, at least at the temperature examined (83°C); moreover, the temperature for maximal activity follows the optimal temperature for growth of the organism. Like most MPGSs characterized to date, the enzyme from Palaeococcus ferrophilus shows an absolute requirement for Mg2+. Pyrococcus horikoshii MPGS is different in this respect, retaining about half of maximal activity in the absence of this divalent cation.


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  		TABLE 2. Biochemical properties of recombinant mannosylphosphoglycerate synthases from Archaea and Bacteria

 
The MPGS and MPGP from Palaeococcus ferrophilus, Thermococcus litoralis, and Pyrococcus spp. show a high degree of identity at the amino acid level (at least 69% and 55%, respectively), indicating high conservation in line with the close phylogenetic relationship among the members of the Thermococcales. In contrast, the homologues found in the bacteria Rhodothermus marinus and Thermus thermophilus or in the archaeon Aeropyrum pernix show considerable less identity (Fig. 2). The amino acid identity is higher for MPGS than for MPGP, indicating that the phosphatase is more flexible in evolutionary terms than the synthase. The unrooted phylogenetic tree constructed on the basis of the alignment of the amino acid sequences of known MPGSs predicts the existence of three different clusters in agreement with the phylogenetic organization of the organisms based on 16S rRNA sequences (Fig. 4). The bacterial MPGSs from Thermus thermophilus and Rhodothermus marinus are grouped together in a separate cluster; the MPGS from Aeropyrum pernix, the only crenarchaeote represented, forms a distinct branch between the bacterial and the euryarchaeote MPGSs; the representatives of the Thermococcales form a very tight cluster, in which the most divergent MPGS belongs to the genus Palaeococcus, which had an early separation from the common ancestor of Thermococcus and Pyrococcus.



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  		FIG. 4. Unrooted phylogenetic tree based on known sequences of mannosyl-3-phosphoglycerate synthase genes. The ClustalX and TreeView programs (34, 45) were used for sequence alignment and to generate the phylogenetic tree. The significance of the branching order was evaluated by bootstrap analysis of 1,000 computer-generated trees. The bootstrap values are indicated. Bar, 0.1 change per site. Abbreviations: Aper, Aeropyrum pernix; Rmar, Rhodothermus marinus; Tthe, Thermus thermophilus; Pfer, Palaeococcus ferrophilus; Pfur, Pyrococcus furiosus; Paby, Pyrococcus abyssi; Phor, Pyrococcus horikoshii; and Tlit, Thermococcus litoralis.

 
The accumulation of MG is a common trait in (hyper)thermophiles, but recently, putative mpgS genes have been reported in samples of uncultured mesophilic archaea originating from soil and river sediments (20, 46) as well as in fungi (9), but the function of these genes remains to be proven. The mesophilic bacterium "Dehalococcoides ethenogenes" has genes with high sequence homology to known mpgS and mpgP and in this case the recombinant products have been shown to be functional in the heterologous synthesis of MG (12). Also, it is relevant to recall that MG was originally found in mesophilic red algae (21, 22, 24, 25). We have shown, however, that in the alga Caloglossa leprieurii, the synthesis of MG proceeds in a single-step catalyzed by mannosylglycerate synthase (C. Neves and H. Santos, unpublished data), an alternative biosynthetic pathway, otherwise found only in Rhodothermus marinus; therefore, the synthesis of MG in red algae probably constitutes a particular line that evolved in a restricted number of organisms.

The scattered distribution of the genes responsible for the synthesis of MG in different lineages raises interesting questions on the origin and evolution of the synthesis of MG. In respect to the members of the order Thermococcales, we conclude that the osmo/thermoregulation strategies adopted by this group of organisms are more diverse than anticipated on the basis of their close phylogenetic relationships. While members of the genus Pyrococcus use DIP and MG almost exclusively for osmo- and thermoadaptation, a variety of solutes, namely amino acids, are used in the genera Thermococcus and Palaeococcus. Moreover, the absence of DIP in Palaeococcus ferrophilus and loss of the genes for the synthesis of MG in Thermococcus kodakaraensis are eloquent illustrations of how genomes are shaped in the course of evolution.


   ACKNOWLEDGMENTS
 
This work was funded by European Commission Contracts QLK3-CT-2000-00640 and COOP-CT-2003-508644 and Fundação para a Ciência e a Tecnologia and FEDER, Portugal, POCTI/35715/BIO/2000 and POCTI/42331/BIO/2001. C. Neves acknowledges a postdoctoral grant from PRAXIS XXI (SFRH/BPD/7119/2001).

We thank Paula Chicau (Laboratório de Análise de Amino Ácidos e Sequenciação de Proteínas, ITQB-UNL, Oeiras, Portugal) for performing the amino acid analysis.


   FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal. Phone: 351-214469800. Fax: 351-214428766. E-mail: santos{at}itqb.unl.pt. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Adams, M. W. 1993. Enzymes and proteins from organisms that grow near and above 100°C. Annu. Rev. Microbiol. 47:627-658.[Medline]
  2. Amend, J. P., D. R. Meyer-Dombard, S. N. Sheth, N. Zolotova, A. C. Amend. 2003. Palaeococcus helgesonii sp. nov., a facultatively anaerobic, hyperthermophilic archaeon from a geothermal well on Vulcano Island, Italy. Arch. Microbiol. 179:394-401.[CrossRef][Medline]
  3. Ames, B. N. 1966. Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol. 8:115-118.
  4. Borges, N., A. Ramos, N. D. H. Raven, J. S. Sharp, and H. Santos. 2002. Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes. Extremophiles 6:209-216.[CrossRef][Medline]
  5. Borges, N., J. D. Marugg, N. Empadinhas, M. S. da Costa, and H. Santos. 2004. Specialized roles of the two pathways for the synthesis of mannosylglycerate in osmoadaptation and thermoadaptation of Rhodothermus marinus. J. Biol. Chem. 279:9892-9898.[Abstract/Free Full Text]
  6. Busby, T. F., and K. C. Ingham. 1984. Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects of nonenzymatic glycosylation. Biochim. Biophys. Acta 799:80-89.[Medline]
  7. da Costa, M. S., H. Santos, and E. A. Galinski. 1998. An overview of the role and diversity of compatible solutes in Bacteria and Archaea. Adv. Biochem. Eng. Biotechnol. 61:117-153.[Medline]
  8. DiRuggiero, J., D. Dunn, D. L. Maeder, R. Holley-Shanks, J. Chatard, R. Horlacher, F. T. Robb, W. Boos, and R. B. Weiss. 2000. Evidence of lateral gene transfer among hyperthermophilic archaea. Mol. Microbiol. 38: 684-693.[CrossRef][Medline]
  9. Empadinhas, N. 2004. Ph.D. thesis. University of Coimbra, Coimbra, Portugal.
  10. Empadinhas, N., J. D. Marugg, N. Borges, H. Santos, and M. S. da Costa. 2001. Pathway for the synthesis of mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii. J. Biol. Chem. 276:43580-43588.[Abstract/Free Full Text]
  11. Empadinhas, N., L. Albuquerque, A. Henne, H. Santos, and M. S. da Costa. 2003. The bacterium Thermus thermophilus, like hyperthermophilic archaea, uses a two-step pathway for the synthesis of mannosylglycerate. Appl. Environ. Microbiol. 69:3272-3279.[Abstract/Free Full Text]
  12. Empadinhas, N., L. Albuquerque, J. Costa, S. H. Zinder, M. A. Santos, H. Santos, and M. S. da Costa. 2004. A gene from the mesophilic bacterium Dehalococcoides ethenogenes encodes a novel mannosylglycerate synthase. J. Bacteriol. 186:4075-4084.[Abstract/Free Full Text]
  13. Erauso, G., A. L. Reysenbach, A. Godfroy, J. R. Meunier, B. Crump, F. Partensky, J. A. Baross, V. Marteinsson, G. Barbier, N. R. Pace, and D. Prieur. 1993. Pyrococcus abyssi sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Arch. Microbiol. 160:338-349.
  14. Fiala, G., and K. O. Stetter. 1986. Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C. Arch. Microbiol. 145:56-61.[CrossRef]
  15. Fukui, T., H. Atomi, T. Kanai, R. Matsumi, S. Fujiwara, and T. Imanaka. 2005. Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genes. Genome Res. 15:352-363.[Abstract/Free Full Text]
  16. Galinski, E. A. 1995. Osmoadaptation in bacteria. Adv. Microb. Physiol. 37:273-328.
  17. Gonçalves, L. G., P. Lamosa, R. Huber, and H. Santos. 2004. New phosphorylated compounds accumulating in the extreme hyperthermophile Pyrolobus fumarii. Proceedings of the 5th International Conference on Extremophiles, Cambridge, Maryland.
  18. Gonçalves, L. G., R. Huber, M. S. da Costa, and H. Santos. 2003. A variant of the hyperthermophile Archaeoglobus fulgidus adapted to grow at high salinity. FEMS Microbiol. Lett. 218:239-244.[CrossRef][Medline]
  19. González, J. M., Y. Masuchi, F. T. Robb, J. W. Ammerman, D. l. Maeder, M. Yanagibayashi, J. Tamaoka, and C. Kato. 1998. Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough. Extremophiles 2:123-130.[CrossRef][Medline]
  20. Hallam, S. J., N. Putnam, C. M. Preston, J. C. Detter, D. Rokhsar, P. M. Richardson, and E. F. DeLong. 2004. Reverse methanogenesis: testing the hypothesis with environmental genomics. Science 305:1457-1462.[Abstract/Free Full Text]
  21. Karsten, U., J. A. West, G. C. Zuccarello, R. Engbrodt, A. Yokoyama, Y. Hara, and J. Brodie. 2003. Low molecular weight carbohydrates of the Bangiophycidae (Rhodophyta) J. Phycol. 39:584-589.[CrossRef]
  22. Karsten, U., K. D. Barrow, A. S. Mostaert, R. J. King, and J. A. West. 1994. 13C- and 1H-NMR studies on digeneaside in the red alga Caloglossa leprieurii. A re-evaluation of its osmotic significance. Plant Physiol. Biochem. 32:669-676.
  23. Kaushik, J. K., and R. Bhat. 1999. A mechanistic analysis of the increase in the thermal stability of proteins in aqueous carboxylic acid salt solutions. Protein Sci. 8:222-233.[Abstract]
  24. Kirst, G. O. 1980. Low MW carbohydrates and ions in Rhodophyceae: quantitative measurement of floridoside and degeneaside. Phytochemistry 19:1107-1110.[CrossRef]
  25. Kremer, B. P., and R. Vogl. 1975. Zur chemotaxonomischen Bedeutun des (14C)-Markierungsmusters bei Rhodophyceen. Phytochemistry 14:1309-1314.[CrossRef]
  26. Lamosa, P., L. O. Martins, M. S. da Costa, and H. Santos. 1998. Effects of temperature, salinity and medium composition on compatible solute accumulation by Thermococcus spp. Appl. Environ. Microbiol. 64:3591-3598.[Abstract/Free Full Text]
  27. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms J. Mol. Biol. 3:208-218.
  28. Martins, L. O., and H. Santos. 1995. Accumulation of mannosylglycerate and di-myo-inositol phosphate by Pyrococcus furiosus in response to the salinity and temperature. Appl. Environ. Microbiol. 61:3299-3303.[Abstract]
  29. Martins, L. O., L. S. Carreto, M. S. da Costa, and H. Santos. 1996. New compatible solutes related to di-myo-inositol-phosphate in members of the order Thermotogales. J. Bacteriol. 178:5644-5651.[Abstract/Free Full Text]
  30. Martins, L. O., N. Empadinhas, J. D. Marugg, C. Miguel, C. Ferreira, M. S. da Costa, and H. Santos. 1999. Biosynthesis of mannosylglycerate in the thermophilic bacterium Rhodothermus marinus. J. Biol. Chem. 274:35407-35414.[Abstract/Free Full Text]
  31. Martins, L. O., R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos. 1997. Organic solutes in hyperthermophilic Archaea. Appl. Environ. Microbiol. 63:896-902.[Abstract]
  32. Neuner, A., H. W. Jannasch, S. Belkin, and K. O. Stetter. 1990. Thermococcus litoralis sp. nov.: a new species of extremely thermophilic marine archaebacteria. Arch. Microbiol. 153:205-207.[CrossRef]
  33. Nunes, O. C., C. M. Manaia, M. S. da Costa, and H. Santos. 1995. Compatible solutes in the thermophilic bacteria Rhodothermus marinus and Thermus thermophilus. Appl. Environ. Microbiol. 61:2351-2357.[Abstract]
  34. Page, R. D. M. 1996. Treeview: An application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357-358.[Free Full Text]
  35. Ramos, A., N. D. H. Raven, R. J. Sharp, S. Bartolucci, M. Rossi, R. Cannio, J. Lebbink, J. van der Oost, W. M. de Vos, and H. Santos. 1997. Stabilization of enzymes against thermal stress and freeze-drying of mannosylglycerate. Appl. Environ. Microbiol. 63:4020-4025.[Abstract]
  36. Rashid, N., T. Kanai, H. Atomi, and T. Imanaka. 2004. Among multiple phosphomannomutase gene orthologues, only one gene encodes a protein with phosphoglucomutase and phosphomannomutase activities in Thermococcus kodakaraensis. J. Bacteriol. 186:6070-6076.[Abstract/Free Full Text]
  37. Reed, R. H., D. L. Richardson, S. R. C. Warr, and W. D. P. Stewart. 1984. Carbohydrate accumulation and osmotic stress in cyanobacteria. J. Gen. Microbiol. 130:1-4.
  38. Roberts, M. F. 2004. Osmoadaptation and osmoregulation in archaea: update 2004. Front Biosci. 9:1999-2019.[Medline]
  39. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  40. Santos, H., and M. S. da Costa. 2001. Organic solutes from thermophiles and hyperthermophiles. Methods Enzymol. 334:302-315.[Medline]
  41. Santos, H., and M. S. da Costa. 2002. Compatible solutes of organisms that live in hot saline environments. Environ. Microbiol. 4:501-509.[CrossRef][Medline]
  42. Scholz, S., J. Sonnenbichler, W. Schäfer, and R. Hensel. 1992. Di-myo-inositol-1,1'-phosphate: a new inositol phosphate isolate from Pyrococcus woesei. FEBS Lett. 306:239-242.[CrossRef][Medline]
  43. Silva, Z., N. Borges, L. O. Martins, R. Wait, M. S. da Costa, and H. Santos. 1999. Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis. Extremophiles 3:163-172.[CrossRef][Medline]
  44. Takai, K., A. Sugai, T. Itoh, and K. Horikoshi. 2000. Palaeococcus ferrophilus gen. nov., sp. nov., a barophilic, hyperthermophilic arcaeon from a deep sea hydrothermal vent chimney. Int. J. Syst. Evol. Microbiol. 50:489-500.[Abstract]
  45. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882.
  46. Treush, A. H., A. Kletzin, G. Raddatz, T. Ochsenreiter, A. Quaiser, G. Meurer, S. C. Schuster, and C. Schleper. 2004. Characterization of large-insert DNA libraries from soil for environmental genomic studies of Archaea. Environ. Microbiol. 6:970-980.[CrossRef][Medline]
  47. Xavier, K. B., L. O. Martins, R. Peist, M. Kossmann, W. Boos, and H. Santos. 1996. High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis. J. Bacteriol. 178:4773-4777.[Abstract/Free Full Text]

Applied and Environmental Microbiology, December 2005, p. 8091-8098, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8091-8098.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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