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J Bacteriol. 2010 January; 192(1): 191–197.
Published online 2009 October 30. doi:  10.1128/JB.01115-09
PMCID: PMC2798264

Thermococcus kodakarensis Mutants Deficient in Di-myo-Inositol Phosphate Use Aspartate To Cope with Heat Stress[down-pointing small open triangle]§

Abstract

Many of the marine microorganisms which are adapted to grow at temperatures above 80°C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakarensis (formerly Thermococcus kodakaraensis), manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and α-glutamate. Under heat stress the level of DIP increased 20-fold compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, overexpression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85°C) and supraoptimal (93.7°C) temperatures for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakarensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptation of (hyper)thermophilic organisms.

Hyperthermophilic bacteria and archaea isolated from saline environments accumulate unusual organic solutes in response to osmotic as well as heat stress. Mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, mannosyl-di-myo-inositol phosphate (DIP), diglycerol phosphate, and glycero-phospho-myo-inositol are examples of compatible solutes highly restricted to thermophiles and hyperthermophiles (27, 31). Our team has, over several years, examined the compatible solute composition in a large number of hyperthermophiles and their accumulation under stressful conditions. The data reveal a trend toward specialization of roles in thermoadaptation and osmoadaptation. Indeed, mannosylglycerate and diglycerol phosphate typically accumulate in response to increased NaCl concentration in the growth medium, whereas the levels of DIP and derivatives consistently increase at supraoptimal growth temperatures (11, 16, 17, 27, 31).

DIP is widespread among extreme archaeal hyperthermophiles, such as Methanotorris igneus, Aeropyrum pernix, Stetteria hydrogenophila, Pyrodictium occultum, Pyrolobus fumarii, Archaeoglobus spp., and all the members of the Thermococcales examined thus far, except Palaeococcus ferrophilus (5, 7, 11, 13, 16, 18, 31). This organic solute has also been found in representatives of the two hyperthermophilic bacterial genera, Aquifex and Thermotoga (14, 17, 22).

The specific chemical nature of solutes encountered in hyperthermophiles, together with their accumulation in response to elevated temperatures, led to the hypothesis that they play a role in thermoprotection of cellular components in vivo. However, there is a lack of convincing experimental evidence, such as that obtained with suitable mutants. Progress toward understanding the physiological functions of these solutes critically depends on two conditions: the availability of genetic tools to manipulate hyperthermophilic organisms and knowledge about the genes and enzymes implicated in the synthesis of these unusual solutes.

Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) is a member of the order Thermococcales with an optimal growth temperature of 85°C and is able to grow at temperatures up to 94°C in batch cultures. The NaCl concentration for optimal growth matches that of seawater (1). T. kodakarensis is the only marine hyperthermophile for which a number of genetic tools have been developed, including Escherichia coli-T. kodakarensis shuttle vectors and a reliable gene disruption system (19, 29, 32, 34). The genome of T. kodakarensis possesses a gene encoding CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase (IPCT/DIPPS), a key enzyme in DIP synthesis (2, 25, 26). This enzyme catalyzes the synthesis of CDP-inositol from CTP and inositol-1-phosphate as well as the transfer of the inositol group from CDP-inositol to a second molecule of inositol-1-phosphate to yield a phosphorylated form of DIP (2). Therefore, we set out to investigate whether DIP was involved in thermoadaptation of T. kodakarensis. A DIP-deficient mutant was constructed by deleting the IPCT/DIPPS gene; subsequently, this strain was complemented in this activity by inserting the gene under the control of a constitutive promoter, resulting in a construct with restored ability to synthesize DIP. The effects of heat and osmotic stress on the pattern of solute accumulation and on the growth profiles of the two mutants provided evidence for the involvement of DIP in thermoprotection.

MATERIALS AND METHODS

Strains and culture conditions.

T. kodakarensis strain KUW1 (ΔpyrF ΔtrpE) was used as the host strain for genetic manipulation (34). This organism was routinely cultivated anaerobically in a nutrient-rich medium (MA-YT) supplemented with sodium pyruvate (0.5%, wt/vol) and in synthetic medium (ASW-AA) containing artificial seawater, amino acids, and elemental sulfur (0.2%, wt/vol) (32). T. kodakarensis was transformed according to described protocols (32).

The pools of intracellular organic solutes were determined in T. kodakarensis strains cultivated in medium (TK medium) containing, per liter, 19 g of NaCl, 5 g of yeast extract, 0.24 g of CaCl2·2H2O, 0.52 g of KCl, 12.6 g of MgCl2·6H2O, 3.28 g of MgSO4·7H2O, and 10 ml of a modified solution of Wolfe's trace minerals (23). The final pH was adjusted to 7.0. The medium was degassed with N2 and sterilized by autoclaving. Prior to inoculation, the medium was supplemented with sodium pyruvate (0.3%, wt/vol) and Na2S (0.01%, wt/vol). Two consecutive preinoculum cultures were performed to ensure the reproducibility of the growth curves. The organism was cultured in 0.5-liter static vessels containing 0.3 liter of medium under anaerobiosis (N2 gas phase); cell growth was assessed by measuring the optical density (OD) at 600 nm.

The effect on growth of the NaCl concentration was studied. Cells were grown at 85°C in medium containing different NaCl concentrations (0.0%, 1.9%, 3.0%, and 4.0%, wt/vol). The growth rate was maximal in media containing 1.9 to 3.0% NaCl (data not shown).

To study the growth characteristics and the effect of osmotic and heat stresses on the accumulation of solutes in T. kodakarensis KUW1 and mutant strains, cells were grown at optimal conditions (1.9% NaCl and 85°C) under osmotic stress (4.0% NaCl, 85°C) and under heat stress (1.9% NaCl, 93.7°C) in TK medium. Special attention was given to measuring the temperatures of the cultures by using a thermocouple inserted in an extra vessel containing the same volume of medium. In each experiment, the temperature fluctuation did not exceed 0.1°C. This is important under heat stress conditions, since the level of compatible solutes is very sensitive to changes in the set temperature.

DNA manipulation and sequence analysis.

Restriction and modification enzymes were purchased from Toyobo (Osaka, Japan) or Takara (Kyoto, Japan). Pfu DNA polymerase (Fermentas, Burlington, Canada) or KOD polymerase (Toyobo, Japan) was used for PCR. Plasmid DNA was isolated and purified from E. coli cells with the plasmid mini-kit from Qiagen (Hilden, Germany). A GFX PCR DNA and gel band purification kit (GE Healthcare, Little Chalfont, United Kingdom) was used to recover DNA fragments from agarose gels. DNA sequencing was performed using a BigDye Terminator cycle sequencing kit (version 3.1) and a model 3100 capillary DNA sequencer (Applied Biosystems, Foster City, CA).

Construction of vectors.

Chromosomal DNA from T. kodakarensis was isolated according to the method of Ramakrishnan and Adams (21). The disruption vector, pUMT2-Δdipps, was constructed to delete the gene encoding the enzyme CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase (IPCT/DIPPS) (Fig. (Fig.1).1). The 5′ and 3′ flanking regions (1,000 bp) of the IPCT/DIPPS gene were separately amplified from the genomic DNA by PCR. The set of primers used is presented in Table Table1.1. The 5′ and 3′ flanking regions were amplified using the primer sets UP-F/UP-R and DOWN-F/DOWN-R, respectively. Amplified fragments were cloned into the pUMT2 (33) between SphI-PstI and KpnI-EcoRI sites, respectively, for the 5′ and 3′ flanking regions.

FIG. 1.
Genetic organization of T. kodakarensis mutants after double homologous recombination using vectors pUMT2-Δdipps (DIP-deficient mutant) and pTKV1-dipps (DIP-complemented mutant). ABC, ABC-type multidrug transport system; ATP-bp, ATP-binding protein; ...
TABLE 1.
Primers used in this worka

A second vector, pTKV1-dipps, was constructed to insert a copy of the IPCT/DIPPS gene into the genome of the DIP-deficient T. kodakarensis, under the control of a strong constitutive promoter, the glutamate dehydrogenase (GDH) promoter (Fig. (Fig.1).1). pTKV1 was constructed for the insertion of the IPCT/DIPPS gene into the TKV1 genetic element loci (9) via double-crossover recombination. The 5′ and 3′ flanking regions of the TKV1 genetic element were separately amplified from the genomic DNA of T. kodakarensis with primer sets TKV1-F1/TKV1-R1 and TKV1-F2/TKV1-R2, respectively. The amplified fragments were inserted into the SmaI site (5′ flanking) or the PstI-SphI sites (3′ flanking) of pUD2, which includes the pyrF marker gene (34). To construct the IPCT/DIPPS gene cassette under the control of the GDH promoter and terminator, the coding region of the gdh gene, along with the promoter and terminator regions, was amplified by PCR from genomic DNA using primers GDH-F/GDH-R. The PCR product was inserted into pUC118 previously digested at the HincII site and phosphorylated. The promoter and terminator regions of the gdh gene and the pUC118 vector were amplified by inverse PCR using primers Inv-GDH-F/Inv-GDH-R. The IPCT/DIPPS gene was amplified by PCR from genomic DNA using the 5′-phosphorylated primers IPCT-F/DIPPS-R and ligated to the inverse PCR product. Finally, the IPCT/DIPPS gene and the promoter and terminator regions were digested with BamHI and inserted into pTKV1 at the corresponding site. DNA sequencing confirmed the correctness of the constructed vectors, pUMT2-Δdipps and pTKV1-dipps. DNA manipulation was carried out in E. coli DH5α (Invitrogen, Carlsbad, CA), by following standard protocols (28).

Transformation of strains.

T. kodakarensis strain KUW1, a tryptophan and uracil auxotrophic strain, was transformed with pUMT2-Δdipps. Selection was carried out in ASW-AA medium without tryptophan, leading to the isolation of DIP-deficient mutant strains. To construct the DIP-complemented mutant, the Δdipps strain was used as the host strain and transformed with pTKV1-dipps. Transformants were selected in ASW-AA medium without uracil. PCR analyses confirmed that the DIP-deficient mutant lacks the IPCT/DIPPS gene and that the DIP-complemented mutant possesses the IPCT/DIPPS gene fused with the gdh promoter (Pgdh) (see Fig. S1 in the supplemental material). Additionally, 2.2 kb on each flank of the recombination region was sequenced in the two mutants, revealing a single insertion of the trpE cassette and the Pgdh-ipct/dipps-pyrF cassette into the target genes of the DIP-deficient and DIP-complemented mutants, respectively.

Extraction, identification, and quantification of intracellular solutes.

Cells were harvested during the mid-exponential phase of growth by centrifugation (7,000 × g, 10 min, 4°C) and washed twice with an NaCl solution identical in concentration to that of the growth medium. Cell pellets were suspended in water and disrupted by sonication. Aliquots were removed for determination of total protein. The remaining cell extract was treated twice with boiling 80% ethanol as described previously (30). Freeze-dried extracts were dissolved in 2H2O and analyzed by nuclear magnetic resonance (30).

Preparation of crude cell extracts and detection of enzyme activity.

Cells were harvested by centrifugation as described above and washed twice with Tris-HCl buffer (20 mM, pH 7.6) containing 1.9% NaCl. The cell pellet was resuspended in the same buffer, and cells were disrupted in a French press. Cell debris was removed by centrifugation (30,000 × g, 45 min, 4°C). Low-molecular-mass compounds were removed in a PD-10 column (GE Healthcare Bio-Science AB, Uppsala, Sweden) equilibrated with Tris-HCl (20 mM, pH 7.6) containing 10 mM MgCl2. Protein content was estimated by the Bradford method (3). The IPCT/DIPPS activity in cell extracts of T. kodakarensis was determined by monitoring the 31P-NMR resonances due to the phosphodiester groups of the reaction products, DIP, di-myo-inositol-phosphate phosphate, and CDP-inositol, as described previously (26). Diglycerol phosphate (bitop AG, Witten, Germany) was added as a concentration standard.

NMR spectroscopy.

Compatible solutes accumulating in T. kodakarensis under several growth conditions were identified and quantified by 1H-NMR. Spectra were acquired on a Bruker AVANCEII500 spectrometer (Bruker, Rheinstetten, Germany) using a broadband inverse probe head and a repetition delay of 60 s. Formate was added to provide an internal concentration standard.

Determination of the melting temperature by differential scanning calorimetry.

Measurements were carried out with a MicroCal VP DSC calorimeter as previously described (8). Samples contained staphylococcal nuclease (SNase) (30 μM) in 10 mM potassium phosphate buffer, pH 7.5. Aspartate and glutamate (potassium salts) were added at a final concentration of 0.5 M. The melting temperature was calculated by a nonlinear fit of raw data to a Gaussian curve using the MicroCal software. Determinations of the melting temperature (Tm) were done in duplicate, and the standard deviation was approximately 0.3°C.

Reverse transcription-PCR (RT-PCR) experiments.

T. kodakarensis KUW1 and mutants were grown at optimal growth conditions (85°C, 1.9% NaCl) and under heat stress (93.7°C, 1.9% NaCl) as described above. Total RNA was isolated from cells harvested during the mid-exponential phase of growth using the SV total RNA isolation system (Promega). An additional incubation step with the kit DNase I (1.5 h, 24°C) was required to remove chromosomal DNA. Total RNA (0.73 μg), deoxynucleoside triphosphates (final concentration of 0.5 mM) and random oligonucleotides (12 μg ml−1) (Invitrogen) were heated at 65°C for 5 min and chilled on ice. Dithiothreitol (final concentration, 5 μM), first-strand RT buffer, and Superscript III (1/20, vol/vol) (Invitrogen) were added, and samples were incubated for 5 min at 25°C, 60 min at 50°C, and 15 min at 70°C for enzyme inactivation. A parallel sample was treated in the same way, except for the addition of enzyme. cDNA was subsequently used at a 1/4 (vol/vol) dilution as a template for standard PCRs. To test for DNA contamination in the RNA preparations, the RNA samples without reverse transcriptase were used as negative controls for all conditions examined. Chromosomal T. kodakarensis KUW1 was used as a positive control for the PCR. The primers used to amplify internal fragments of the IPCT/DIPPS gene (RT-IPCT-F/RT-DIPPS-R) and the elongation factor 1-alpha (EF-1-alpha, tk0308) gene (RT-EF-F/RT-EF-R) are shown in Table Table1.1. The T. kodakarensis EF-1-alpha gene, a housekeeping gene coding for the elongation factor required for continued translation of mRNA, was used as a control. The band intensity was quantified with Quantity One software (Bio-Rad, Hercules, CA), and the signal of the IPCT/DIPPS gene was normalized with respect to that of the EF-1-alpha gene.

RESULTS

Effect of osmotic and heat stress on the accumulation of compatible solutes in the T. kodakarensis parental strain.

The pool of compatible solutes in T. kodakarensis was determined with cells grown at optimal conditions (85°C, 1.9% NaCl) and also with cells subjected to osmotic stress (85°C, 4% NaCl) and heat stress (93.7°C, 1.9% NaCl). 1H-NMR spectra of ethanol extracts showed the presence of DIP, aspartate, and α-glutamate. The assignment was confirmed by spiking with the pure compounds. Under optimal growth conditions, aspartate (0.082 μmol/mg of protein) was the major solute, whereas α-glutamate and DIP were present in small amounts (Table (Table2).2). The total solute pool (0.104 μmol/mg of protein) increased to 0.425 and 0.342 μmol/mg of protein in cells grown under osmotic and heat stress, respectively. Aspartate was the predominant compound in cells grown under osmotic stress, accounting for 83% of the total solute pool; α-glutamate and DIP represented 14% and 3% of the total pool, respectively (Fig. (Fig.2A).2A). In response to heat stress, the levels of DIP and α-glutamate increased substantially (20- and 9.8-fold, respectively), while the level of aspartate was fairly constant.

FIG. 2.
Effect of the growth temperature and the NaCl concentration on the accumulation of α-glutamate (checkered), aspartate (gray), di-myo-inositol-phosphate (white), and myo-inositol-1-phosphate (black) in the T. kodakarensis parental strain (A), a ...
TABLE 2.
Accumulation of compatible solutes in T. kodakarensis KUW1 (parental strain), a DIP-deficient mutant, and a DIP-complemented mutant

Construction of T. kodakarensis mutants and evaluation of transcription levels and enzyme activity.

To assess the contribution of DIP in the process of thermoadaptation of T. kodakarensis, two mutants were constructed. In one of them, the IPCT/DIPPS gene encoding the key enzyme in DIP synthesis was deleted by double-crossover homologous recombination. This DIP-deficient mutant was used as the host strain to construct the second mutant, in which the IPCT/DIPPS gene was overexpressed. This gene was inserted into the TKV1 provirus region of the T. kodakarensis genome, under the control of a constitutive, strong glutamate dehydrogenase promoter.

The transcription level of the IPCT/DIPPS gene and the IPCT/DIPPS activity were determined in the DIP-deficient and the DIP-complemented mutants and compared with those of the parental strain. The strains were grown at 85°C (optimal growth temperature) and also at 93.7°C. As expected, no transcript of the IPCT/DIPPS gene was detected in the DIP-deficient mutant. The transcriptional level in the parental strain increased 1.6-fold when the growth temperature was augmented from 85 to 93.7°C. An approximately 4-fold increase in the transcription level, compared to that of the parental strain, was observed in cells of the DIP-complemented mutant, and the level was independent of the growth temperature (Fig. (Fig.3).3). Additionally, the activity of IPCT/DIPPS was determined in cell extracts of the three strains. Under optimal growth conditions, the parental strain had an activity of 8.0 nmol/min/mg of protein and a 14-fold higher activity was detected in the DIP-complemented mutant. The DIP-deficient mutant showed no activity.

FIG. 3.
Transcriptional levels of the IPCT/DIPPS gene in the T. kodakarensis parental strain (A), DIP-deficient mutant (B), and DIP-complemented mutant (C) at optimal growth conditions (85°C, 1.9% NaCl) and under heat stress (93.7°C, 1.9% ...

Effect of osmotic and heat stress on growth parameters and solute accumulation.

Growth rates and compatible solute composition of the DIP-deficient and DIP-complemented strains were studied at optimal growth conditions (85°C, 1.9%) and under heat stress (93.7°C, 1.9%) and compared with those of the parental strain. The growth rate of the parental strain at optimal growth conditions was 0.71 h−1; under heat stress, the growth rate was reduced to 0.41 h−1 (Fig. (Fig.4).4). Surprisingly, the DIP-deficient strain exhibited growth parameters identical to those of the parental strain under optimal as well as stress conditions (Fig. (Fig.4).4). The final growth yield was also fairly similar (data not shown). It seems that the absence of DIP was compensated for by an alternative strategy that endowed the mutant with equal ability to cope with heat stress.

FIG. 4.
Growth curves of T. kodakarensis parental strain (solid circles) and DIP-deficient mutant (open circles) at optimal growth conditions (A), under heat stress (B), and under osmotic stress (C). Points for two independent growths are plotted for each strain ...

With respect to solute composition, the DIP-deficient mutant under optimal growth conditions showed a pattern similar to that of the parental strain, except for the absence of DIP (Fig. (Fig.2B).2B). However, in response to a supraoptimal growth temperature, the mutant accumulated large amounts of aspartate (0.265 μmol/mg protein) corresponding to a 3.3-fold increase relative to optimal conditions, and l-myo-inositol-1-phosphate was detected at a level of 0.039 μmol/mg protein (Table (Table2).2). The level of α-glutamate increased from 0.018 μmol/mg protein (optimal conditions) to 0.032 μmol/mg protein (heat stress).

The behavior of the DIP-deficient mutant in response to osmotic stress was also analyzed. The solute pool was composed of aspartate and α-glutamate in the proportion of 5.6:1. The growth rate and the profile of solute accumulation were similar to those of the parental strain when equal growth conditions were considered (Fig. (Fig.22 and Fig. Fig.4).4). The DIP-complemented strain also showed profiles of solute accumulation very similar to those of the parental strain under all the conditions examined (Fig. (Fig.22).

Effect of aspartate on the stability of a model protein.

DIP was replaced by aspartate in the DIP-deficient strain. Therefore, we deemed it interesting to compare the abilities of these two compounds to protect proteins against thermal denaturation. For this purpose, differential scanning calorimetry was used to measure the melting temperature (Tm) of staphylococcal nuclease (SNase) in the absence and presence of aspartate. A Tm of 54.4°C was determined for SNase without the addition of solutes. We have shown that the Tm of SNase was not significantly affected by the presence of 0.5 M KCl (8). Upon addition of 0.5 M aspartate, the Tm of SNase increased to 61.6°C. The value of Tm in the presence of 0.5 M DIP is 62.3°C (8). For comparison, the Tm of SNase was also determined in the presence of 0.5 M α-glutamate, the third solute accumulating in T. kodakarensis, and a value of 62.0°C was found.

DISCUSSION

The solute pool of the hyperthermophilic archaeon T. kodakarensis comprises three negatively charged compounds: di-myo-inositol phosphate, aspartate, and α-glutamate. This observation reinforces the proposed correlation between hyperthermophily and the accumulation of negatively charged compatible solutes (31).

The levels of α-glutamate and DIP clearly increased in response to heat stress, whereas osmotic stress induced a strong increase in the concentration of aspartate, an amino acid rarely used as a compatible solute. A few members of the order Thermococcales (Palaeococcus ferrophilus and several Thermococcus species) accumulate aspartate, along with mannosylglycerate, in response to osmotic stress (13, 20); in addition, several Methanothermococcus spp. use aspartate for osmoregulation (15, 24). To our knowledge, the use of aspartate as a compatible solute is restricted to hyperthermophilic Archaea.

The absence of mannosylglycerate in the solute pool of T. kodakarensis is curious, since this compound has been detected in all the members of the Thermococcales that have been examined (7, 13, 18, 20). Moreover, three of the five Thermococcus strains with sequenced genomes available (T. gammatolerans, T. barophilus, Thermococcus sp. AM4) possess genes with high similarity to mpgS, the gene encoding mannosyl-3-phosphoglycerate synthase (7). These observations, combined with the fact that all members of the order Thermococcales cluster in a tight branch of the 16S RNA Tree of Life (http://www.arb-silva.de/), led to the speculation that the common ancestor of the Thermococcales possessed genes for mannosylglycerate synthesis which were lost during evolution toward T. kodakarensis. A parallel suggestion applies to Palaeococcus ferrophilus, the sole member of the Thermococcales lacking DIP (20).

Di-myo-inositol phosphate (DIP) is a compatible solute restricted to microorganisms with optimal growth temperatures above 60°C; moreover, DIP accumulation is induced by heat stress, suggesting that this solute plays a role during thermoadaptation of (hyper)thermophiles. In this study, a gene implicated in the synthesis of DIP in T. kodakarensis was deleted, and the resulting mutant strain was examined with respect to its ability to cope with heat stress. The mutant strain lacked DIP, showing that there is no pathway for DIP synthesis other than that involving IPCT/DIPPS (26). Intriguingly, the growth parameters of the DIP-deficient mutant under heat stress were identical to those of the parental strain, which accumulated considerable amounts of DIP. Analysis of the solute pool revealed that the absence of DIP in the mutant was offset by an increase in the content of aspartate. Both the mutant and the parental strain used aspartate for osmoregulation. Interestingly, the parental strain accumulated DIP and glutamate under heat stress conditions, while the mutant accumulated aspartate to cope with the same type of stress. Therefore, aspartate was used for both osmo- and thermoadaptation in the DIP-deficient strain. Interestingly, aspartate and DIP were equally efficient as protectors of the structure of a model enzyme against heat denaturation in vitro. The chain of events triggered by heat stress in the mutant and resulting in the switch from DIP to aspartate accumulation is elusive and deserves future investigation. However, this result is less intriguing in view of the observation that aspartate (and glutamate) accumulated in the parental strain in response to heat stress, when the organism was grown in a nutrient-rich medium (data not shown). Moreover, there are several examples of solutes from hyperthermophiles whose levels respond both to heat and salt stress (31). For instance, α-glutamate accumulates in Thermotoga maritima in response to these two types of stress (27).

Inositol-1-phosphate is a substrate for the reaction catalyzed by IPCT/DIPPS; therefore, the accumulation of this precursor of DIP synthesis in the strain deficient in this synthase is not too surprising. Likewise, accumulation of N-γ-acetyl-diaminobutyric acid, the precursor for the synthesis of the compatible solute ectoine, has been reported in a mutant of Halomonas elongata with disruption of the ectoine synthase (4).

An efficient strategy was developed in the DIP-deficient mutant to circumvent the absence of DIP. This strategy led to the replacement of the missing solute by aspartate, which apparently allowed the mutant to grow as well as the parental strain under heat stress. This is the first report of the replacement of a thermoprotector by another compound with an equivalent role. Replacement of solutes used in osmoregulation has been previously observed: in the Methanosarcina mazei mutant defective in the synthesis of Nepsilon-acetyl-β-lysine, this compatible solute was replaced by glutamate and alanine (35). This strategy allows the mutant to exhibit growth rates similar to those of the wild-type strain at low and moderate salinities, but the substitutes were less efficient than Nepsilon-acetyl-β-lysine, as growth of the mutant at high salinity was poorer.

The IPCT/DIPPS activity in the T. kodakarensis DIP-complemented mutant was 14-fold higher than that in the parental strain, but this great increase in activity did not lead to a larger DIP pool. Therefore, this enzyme does not constitute a bottleneck in DIP synthesis. Substrate availability could be a relevant factor in the control of DIP accumulation.

To our knowledge, this is the first study using mutants of (hyper)thermophilic organisms to demonstrate the involvement of compatible solutes in thermoadaptation. The role of trehalose in the acquisition of thermotolerance in trehalose-deficient mutants of the mesophiles Saccharomyces cerevisiae and Escherichia coli has been investigated (6, 12), and a similar study of the role of hydroxyectoine in Chromohalobacter salexigens has been reported (10). Unlike T. kodakarensis, alternative solutes did not accumulate in these mutant strains; furthermore, their thermotolerance was clearly impaired.

In conclusion, this work shows that DIP contributes to the thermoprotection of T. kodakarensis and that a different negatively charged compound, aspartate, was able to replace DIP with comparable efficacy.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was funded by Fundação para a Ciência e a Tecnologia (FCT), POCTI Portugal, and FEDER, Projects PTDC/BIA-MIC/71146/2006 and POCTI/BIA-MIC/59310/2004, and by a grant-in-aid for scientific research on priority areas “Applied genomics” (no. 20018013) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The NMR spectrometers at CERMAX are part of the National NMR Network and were acquired with funds from FCT (REDE/1517/RMN/2005) and FEDER.

We thank Mafalda Henriques for technical support.

Footnotes

§Supplemental material for this article may be found at http://jb.asm.org/.

[down-pointing small open triangle]Published ahead of print on 30 October 2009.

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