Effects of Temperature, Salinity, and Medium Composition on Compatible Solute Accumulation by Thermococcus Spp

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by CiteSeerX

Effects of Temperature, Salinity, and Medium Composition on Compatible Solute Accumulation by Thermococcus spp.

1 1 2 1 PEDRO LAMOSA, LI´GIA O. MARTINS, MILTON S. DA COSTA, AND HELENA SANTOS * Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, 2780 Oeiras,1 and Departamento de Bioquı´mica, Universidade de Coimbra, 3000 Coimbra,2 Portugal

Received 29 December 1997/Accepted 23 July 1998

The effects of salinity and growth temperature on the accumulation of intracellular organic solutes were examined by nuclear magnetic resonance spectroscopy (NMR) in Thermococcus litoralis, Thermococcus celer, Thermococcus stetteri, and Thermococcus zilligii (strain AN1). In addition, the effects of growth stage and composition of the medium were studied in T. litoralis. A novel compound identiﬁed as ␤-galactopyranosyl-5- hydroxylysine was detected in T. litoralis grown on peptone-containing medium. Besides this newly discovered -compound, T. litoralis accumulated mannosylglycerate, aspartate, ␣-glutamate, di-myo-inositol-1,1؅(3,3؅)-phos phate, hydroxyproline, and trehalose. The hydroxyproline and ␤-galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the other Thermococcus strains examined. Di-myo-inositol-1,1؅(3,3؅)-phosphate, aspartate, and mannosylglycerate were detected in T. celer and T. stetteri, and the latter organism also accumulated ␣-glutamate. The only nonmarine species studied, T. zilligii, accumulated very low levels of ␣-glutamate and aspartate. The levels of mannosylglycerate and aspartate increased in T. litoralis, T. celer, and T. stetteri in response to salt stress, while di-myo-inositol-1,1؅(3,3؅)-phosphate was the major intracellular solute at supraoptimal growth temperatures. The phase of growth had a strong inﬂuence on the types and levels of compatible solutes in T. litoralis; mannosylglycerate and aspartate were the major solutes during exponential growth, while di-myo- inositol-1,1؅(3,3؅)-phosphate was the predominant organic solute during the stationary phase of growth. This work revealed an unexpected ability of T. litoralis to scavenge suitable components from the medium and to use them as compatible solutes.

Most microorganisms subjected to water stress accumulate ation; for example, DIP, cyclic-2,3-bisphosphoglycerate, and organic solutes to control their internal water activity, maintain MG have been shown to stabilize enzymes (15, 27, 31). These the appropriate cell volume and turgor pressure, and protect results suggest that these solutes play an important role in intracellular macromolecules (14). The compatible solutes, protecting cellular components in vivo that may account, in also called osmolytes, include sugars, amino acids and their part, for the thermophilic nature of the organisms. Other sol- derivatives, polyols and their derivatives, betaines, and ecto- utes, such as di-myo-inositol-1,3Ј-phosphate and 2-O-␤-di- ines (11, 12, 14). The term compatible solute is generally used mannosyl-di-myo-inositol-1,1Ј(3,3Ј)-phosphate, whose levels for low-molecular-weight organic compounds that accumulate increase in response to high temperatures in some hyperther- to high intracellular levels under osmotic stress and that are mophilic organisms, may also have similar properties (21, 22). compatible with the metabolism of the cell. Compatible solutes Therefore, in this paper, the term compatible solute is used for can be synthesized de novo or, if present in the medium, can be major intracellular organic solutes that accumulate in response taken up by the organisms. The latter strategy is preferred to osmotic or temperature stress. when appropriate substances are present in the environment or Recently, we examined several thermophiles and hyperther- in the growth medium (14). Glycine betaine, for example, is a very common compatible solute in mesophilic bacteria and mophiles for the accumulation of organic solutes (20–22, 26); archaea that is not synthesized by most microorganisms but is in particular, we observed that Pyrococcus furiosus accumulates taken up from the medium and used for osmoadaptation (11, only the following two compatible solutes: MG, whose concen- 14). tration increases as the salinity of the medium increases; and Recent investigations of water and temperature stress in DIP, whose concentration increases sharply at supraoptimum thermophilic and hyperthermophilic microorganisms led to the growth temperatures (20). Moreover, the same solutes were identiﬁcation of several new compatible solutes, some of which also observed in Pyrococcus woesei (31). Given the close phy- (mannosylglycerate [MG], di-glycerol-phosphate, and ␤-gluta- logenetic relationship between the genera Pyrococcus and mate) accumulate primarily in response to salt stress. Others, Thermococcus (30), we decided to compare the compatible such as di-myo-inositol-1,1Ј(3,3Ј)-phosphate (DIP) and cyclic- solutes and strategies of thermoadaptation and osmoadapta- 2,3-bisphosphoglycerate, accumulate primarily in response to tion in species of these two genera. In this work, we examined growth at supraoptimal temperatures (9, 15, 20–22, 26, 31). A the effects of salinity and growth temperature on the intracel- growing body of evidence also suggests that some solutes may lular solute pools of four Thermococcus species. A new solute, stabilize macromolecules facing potential thermal denatur- ␤-galactopyranosyl-5-hydroxylysine (GalHL), was identiﬁed in Thermococcus litoralis. Furthermore, we found that the composition of the medium had a major effect on the type of * Corresponding author. Mailing address: Instituto de Tecnologia organic solutes that accumulated during salt stress in this spe- Quı´mica e Biolo´gica, Apartado 127, 2780 Oeiras, Portugal. Phone: 351 cies and that the proportions of several solutes changed signif- 1 4469828. Fax: 351 1 4428766. E-mail: [email protected]. icantly with the growth stage.

3591 3592 LAMOSA ET AL. APPL.ENVIRON.MICROBIOL.

MATERIALS AND METHODS droxyproline, and aspartate (Fig. 1). Resonances were assigned Strains and culture conditions. T. litoralis DSM 5474T, Thermococcus celer by comparison with the carbon chemical shift values reported DSM 2470T, Thermococcus stetteri DSM 5262T, and Thermococcus sp. strain previously (7, 26, 31). The identities of the compounds were AN1T (ϭ DSM 2770T), recently classified as Thermococcus zilligii (30), were confirmed by 1H NMR, and assignment of the resonances due obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen to DIP was further confirmed by spiking a T. litoralis extract GmbH, Braunschweig, Germany. Unless indicated otherwise, T. litoralis, T. celer, with an aliquot of a P. woesei extract, in which DIP was initially and T. stetteri were grown in Bacto Marine Broth (Difco). For growth of T. celer 13 and T. stetteri this medium was supplemented with sodium sulfide (final concen- identified (31). Initially, the remaining resonances in the C- Ϫ tration, 2 g ⅐ liter 1). T. zilligii was grown in the medium described by Lanzotti et NMR spectrum of T. litoralis (176.0, 102.5, 75.5, 75.5, 72.8, al. (18) supplemented with S0. 71.2, 68.9, 61.4, 55.0, 43.4, 28.4, and 27.1 ppm) could not be Cultures of T. litoralis were routinely grown in a 2-liter fermentation vessel assigned to a known compound, but identification was signifi- with continuous gassing with pure N2 and stirring at 100 rpm. T. stetteri was grown under the same conditions except that the gas mixture contained N2 and cantly facilitated by partial purification of the extract. The CO2 (80:20). T. zilligii and T. celer were grown in static 2-liter flasks that were resonances at 176.0 and 102.5 ppm were assigned to a carbox- flushed with N2 for 1 h before the medium was autoclaved. Cell growth was ylic group and an anomeric CH group, respectively. The monitored by measuring turbidity at 600 nm. Most experimental cultures were grown until the late exponential phase, harvested by centrifugation (8000 ϫ g, HMQC spectrum showed that the latter resonance was con- 30°C, 10 min), and washed once with a solution lacking organic nutrients but nected to a proton resonance at 4.49 ppm (Fig. 2). The reso- otherwise identical to the medium in which the cells were grown. The effect of nances at 27.1, 28.4, 43.4, and 61.4 ppm were due to methylene salinity on the levels of intracellular solutes was examined by changing the NaCl groups, while the remaining resonances were due to methyne concentration while the same concentrations of the other medium components were maintained. The effect of growth phase on the accumulation of solutes was groups. The final structure of the molecule was established by examined in T. litoralis by growing the cultures in a 5-liter fermentation vessel using the results of a set of COSY, NOESY, TOCSY, and with medium containing 4% NaCl. Samples (500 ml) were collected at appro- HMBC experiments. The COSY and TOCSY spectra showed priate culture times and treated as described above. that the anomeric carbon at 102.5 ppm belonged to a network The effects of organic components on the accumulation of compatible solutes Ϫ by T. litoralis were examined by replacing peptone (5.0 g ⅐ liter 1) with Casitone of a six-carbon monosaccharide (resonances at 102.5, 75.5, Ϫ Ϫ (5.0 g ⅐ liter 1) or tryptone (5.0 g ⅐ liter 1) or by replacing yeast extract (1.0 g ⅐ 72.8, 71.2, 68.9, and 61.4 ppm). The HMBC experiments Ϫ Ϫ liter 1) with maltose (2.0 g ⅐ liter 1) in a medium containing 3% NaCl and showed that there was a connection between the anomeric having the same basal salts composition as Bacto Marine Broth. carbon of the hexose and the methyne group of the nonsugar Extraction of intracellular solutes, cell protein determination, partial purification and hydrolysis of ␤-galactosyl-hydroxylysine, and quantification of or- moiety at 76.5 ppm. Further analysis of the COSY and TOCSY ganic solutes. Cells were extracted twice with boiling 80% ethanol by the method spectra led to identification of this moiety as 5-hydroxylysine. of Reed et al. (28), modified as previously described by Martins and Santos (20). The hexose and the amino acid fragments were firmly identi- Freeze-dried extracts were dissolved in D2O for nuclear magnetic resonance fied as galactose and 5-hydroxylysine after hydrolysis of the (NMR) analysis. Quantification was performed by 1H NMR by using formate as an internal concentration standard. Glutamate content was determined by the intact compound and spiking of the hydrolysate with pure enzymatic assay of Lund (19), and hydroxyproline was determined by using a galactose and 5-hydroxylysine. The carbon chemical shift val- Pico-Tag amino acid analysis system (Waters, Milford, Mass.). ues of the original compound indicated that the galactose The protein content of cells was determined by the Bradford assay (6) after moiety was in the pyranose configuration rather than the treatment of the cells with 1 M NaOH (100°C, 10 min) and neutralization with 1 M HCl. furanose configuration (5). The presence of connectivities be- The novel compound GalHL was purified by ion-exchange chromatography on tween H1 and H2 and between H3 and H5 but not between H1 an SP-Sepharose C-25 (Pharmacia, Uppsala, Sweden) column (19 by 2.6 cm). and H4 in the NOESY spectra led to the conclusion that The extract was eluted with a sodium phosphate buffer (pH 7.0) gradient ranging galactose was in the ␤-pyranosyl configuration. This conclusion from 5 mM to 1 M. Fractions were eluted and examined by NMR for the presence of the compound. Fractions containing the compound were pooled and was supported by the low chemical shift value for galactose H1 freeze-dried. The residue was dissolved in a small volume of water, salt was (4.49 ppm) and the magnitude of the coupling constants 3 ϭ 1 ϭ removed by passage through a Sephadex G-25 column (type PD-10; Pharmacia), ( JH1,H2 7.7 Hz, JC1,H1 160 Hz), which unambiguously and the eluted sample was concentrated by freeze-drying. The residue was indicated that the configuration was a ␤-pyranosyl configura- dissolved in D2O for NMR analysis. Part of the sample was hydrolyzed with 3.0 tion (2, 4, 23). There was also a clear connectivity between H1 M HCl at 100°C for 3 h under an N2 atmosphere in a sealed ampoule to liberate the sugar moiety from the amino acid. of galactose and H5 of 5-hydroxylysine in the NOESY spec- NMR spectroscopy. All spectra were obtained with a Bruker model AMX500 trum, which showed that the location of the linkage was be- spectrometer. 13C NMR spectra were obtained at 125.77 MHz by using a 5-mm tween C-1 of galactose and the hydroxyl group of the amino carbon selective probe head. Typically, spectra were acquired with a repetition delay of 1.5 s and a pulse width of 7 ␮s corresponding to a 90° flip angle. Proton acid. The position of this linkage was also confirmed by the decoupling was applied during the acquisition time only by using the wide-band HMBC spectrum. On the basis of these results, the unknown alternating-phase low-power technique for zero-residue splitting sequence. compound was identified as GalHL (Fig. 2, inset). The corre- Chemical shifts were referenced to the resonance of external methanol at 49.3 sponding carbon and proton chemical shift values are shown in ppm. 1H NMR spectra were acquired with water presaturation, a 6-␮s pulse width Table 1. corresponding to a 60° flip angle, and a repetition delay of 15 s. Chemical shifts Effects of temperature and salt stress on the accumulation were determined relative to 3-(trimethylsilyl)propanesulfonic acid (sodium salt). of compatible solutes by Thermococcus spp. The optimum Formate was added as an internal concentration standard. Two-dimensional growth temperatures of T. litoralis, T. celer, T. stetteri, and T. spectra were obtained by using standard Bruker pulse programs. Phase-sensitive nuclear Overhauser effect spectroscopy (NOESY), proton-homonuclear shift zilligii are 85, 88, 88, and 75°C, respectively. All of these or- correlation spectroscopy (COSY), and total-correlation spectroscopy (TOCSY) ganisms except T. zilligii, which is not halophilic, have optimum ϫ 1 13 were performed by collecting 4,096 (t2) 512 (t1) data points; in H- C het- salinities for growth of about 2.0 to 2.5% NaCl (17, 24, 25, 37). eronuclear multiple quantum coherence (HMQC) spectra (3), a delay of 3.5 ms 1 Under optimal growth conditions in Bacto Marine Broth, the was used for evolution of JCH. The heteronuclear multiple bond connectivity (HMBC) spectrum was obtained by collecting 4,096 (t ) ϫ 256 (t ) data points; total solute pools of these organisms were small; the highest 2 1 levels of organic solutes were found in T. celer, in which they a delay of 73.5 ms was used for evolution of long-range couplings. Ϫ did not exceed 0.6 ␮mol ⅐ mg of protein 1 (Table 2). As the growth temperature or the salinity of the medium was in- RESULTS creased above the optimal level for growth, the total solute Identification of organic solutes in Thermococcus spp. The pool also increased due to differential accumulation of some 13C-NMR spectra of ethanol extracts of the thermococci grown solutes (Table 2, and Fig. 3 and 4). There was, for example, a in Bacto Marine Broth contained several sets of resonances positive correlation in T. litoralis between growth in medium that were assigned to DIP, MG, trehalose, ␣-glutamate, hy- containing higher concentrations of NaCl and pronounced in- VOL. 64, 1998 COMPATIBLE SOLUTES IN THERMOCOCCUS SPP. 3593

FIG. 1. Proton decoupled 13C-NMR spectrum of an ethanol extract of T. litoralis grown in Bacto Marine Broth containing 4% NaCl at 85°C. Resonances due to aspartate (peaks a), MG (peaks b), DIP (peaks d), glutamate (peaks g), GalHL (peaks h), and trehalose (peaks t) are indicated.

creases in the levels of aspartate, MG, and (to a lesser extent) species examined in this study, and, unlike the other species of GalHL. On the other hand, the accumulation of compatible the genus Thermococcus, T. zilligii had very low intracellular solutes was less pronounced when the temperature was raised solute concentrations (Table 2). No solutes other than aspar- above the optimum temperature for growth. However, DIP tate and glutamate were detected in this organism, and no became the major solute during growth at supraoptimum tem- clear correlation between the levels of these solutes and a high peratures. growth temperature was evident. Trehalose, GalHL, and hydroxyproline were not detected in Effect of the growth phase on the accumulation of solutes in T. celer or T. stetteri, which at elevated salinities accumulated T. litoralis. Alterations in the levels of the organic solutes signiﬁcant levels of MG and aspartate. The levels of MG also during the growth cycle of T. litoralis were determined in Bacto increased at supraoptimum temperatures, but the most pro- Marine Broth containing 4% NaCl at 85°C (Fig. 5). During the nounced alterations of the organic solute pools were due to exponential phase of growth, aspartate was the most abundant increases in the DIP concentrations from about 0.4 to 1.2 solute, but trehalose, MG, DIP, glutamate, and GalHL were Ϫ ␮mol ⅐ mg of protein 1 in T. celer and from 0.1 to 0.4 ␮mol ⅐ mg also detected in moderate amounts, while hydroxyproline was Ϫ of protein 1 in T. stetteri. present at very low levels. At the onset of the stationary phase, T. zilligii was the only nonhalophilic and nonhalotolerant increases in the concentrations of DIP and GalHL were ob- 3594 LAMOSA ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 2. 13C-1H correlation spectrum through one bond coupling (heteronuclear multiple quantum correlation) of the novel compound GalHL. (Inset) Schematic representation of GalHL.

served concomitant with slight decreases in the concentrations during the mid-exponential phase to vestigial during the early of all other solutes except glutamate, whose level decreased stationary phase. On the other hand, the levels of DIP re- notably. The total solute pool decreased signiﬁcantly after pro- mained almost constant. These results show that it is critical to longed incubation of the culture; only DIP, MG, and GalHL control the cell growth stage if meaningful compatible solute were detected, and DIP was the predominant organic solute accumulation data are going to be obtained. during this stage of growth. Effect of the growth medium on the accumulation of organic Dramatic alterations in the relative amounts of the compat- solutes in T. litoralis. Due to the uniqueness of GalLH and the ible solutes with the growth stage were observed in cells grown rarity of hydroxyproline in prokaryotes, we though that it was at 85°C in Bacto Marine Broth containing 3% NaCl when important to ascertain whether these solutes were synthesized peptone was replaced by tryptone. In particular, the levels of de novo or taken up from medium containing peptone or yeast MG changed drastically; the concentrations of MG ranged extract. For this reason, T. litoralis was grown in media that Ϫ from 0.37 ␮mol ⅐ mg of protein 1 when cells were harvested were identical to Bacto Marine Broth except that they con- VOL. 64, 1998 COMPATIBLE SOLUTES IN THERMOCOCCUS SPP. 3595

TABLE 1. NMR parameters of the novel compound GalHL

13C 1H 13C 1H Galactosyl moiety Hydroxylysine moiety ␦ 1 ␦ 3 ␦ 1 ␦ (ppm) JCH (Hz) (ppm) JHH (Hz) (ppm) JCH (Hz) (ppm)

C1 102.5 160 4.49 7.7 C1 176.0 C2 71.2 148 3.55 9.8 C2 55.0 146 3.60 C3 72.8 142 3.65 3.3 C3 27.1 126 2.00, 1.90 a C4 68.9 145 3.95 ND C4 28.4 122 1.75, 1.60 C5 75.5 139 3.75 ND C5 76.5 150 4.05 C6 61.4 144 3.80 ND C6 43.4 144 3.00, 3.20 a ND, not determined. tained Casitone or tryptone instead of peptone and in media in effect on the accumulation of compatible solutes, primarily the which maltose replaced yeast extract. T. litoralis accumulated levels of DIP, while there were only slight increases in the DIP, MG, trehalose, and glutamate in medium containing concentrations of solutes as the salinity of the medium was yeast extract plus Casitone or tryptone, but, unlike growth in increased (Fig. 3 and 4). In P. furiosus, on the other hand, both medium containing peptone, aspartate, GalHL, and hydroxy- temperature and salinity had very pronounced effects on the proline were not detected (Fig. 6). In these media there were accumulation of compatible solutes (20). Accumulation of MG also pronounced increases in the levels of DIP and MG com- and DIP in the species of the genus Thermococcus was ex- pared to the levels accumulated by the organism in peptone- pected because these organisms are closely related to Pyrococ- containing medium. Substitution of yeast extract for maltose in cus species, in which these solutes are also encountered (20). medium containing peptone, Casitone, or tryptone led to an However, we did not expect to ﬁnd that aspartate was a major absence of trehalose. compatible solute in the halophilic Thermococcus species grown in peptone-containing medium, while glutamate played a lesser DISCUSSION role in the osmoadaptation of these organisms. Glutamate has been shown to be a common compatible solute during low- The results presented in this paper show that T. celer, T. stet- level osmoadaptation in many bacteria and archaea (11, 12, teri, and T. litoralis accumulate different solutes in response to 14), while aspartate is generally found at very low intracellular two different types of stress. The levels of MG, aspartate, and concentrations and contributes only slightly to the compatible GalHL increased in response to salt stress, while the levels of solute pool (34). Aspartate accumulates to higher levels in DIP responded primarily to temperature stress. In T. litoralis, Methanococcus thermolithotrophicus and Methanosarcina ther- however, the effect of the salinity of the medium on the accu- mophila, but there is only a slight positive correlation between mulation of compatible solutes was much more pronounced the level of aspartate and salinity and this amino acid is not than the effect of the growth temperature. The reverse was true thought to behave like a compatible solute (29, 33). However, in T. celer; in this organism the growth temperature had a clear under salt stress conditions aspartate is an important compat-

TABLE 2. Quantiﬁcation of organic solutes as determined by 1H NMR in species of the genus Thermococcusa

Conditions Solute concn (␮mol/mg of protein) Species Temp (°C) NaCl concn (%) DIP MG Glutamateb Aspartate Trehalose GalHL Hydroxyprolinec T. litoralis 85 2.0 NDd ND 0.088 (19)e 0.21 (47) 0.02 (4) ND 0.148 (33) 85 3.0 0.11 (24) 0.04 (9) 0.105 (24) 0.33 (73) 0.10 (22) 0.07 (16) 0.093 (21) 85 4.0 0.20 (44) 0.31 (69) 0.260 (58) 0.75 (167) 0.23 (51) 0.14 (31) 0.052 (12) 85 5.0 0.34 (76) 0.66 (147) 0.307 (68) 0.65 (144) 0.37 (82) 0.28 (62) 0.050 (11) 90 2.0 0.18 (40) 0.07 (16) 0.070 (15) 0.13 (29) 0.06 (13) ND 0.058 (13) 95 2.0 0.37 (82) 0.09 (20) 0.030 (7) 0.11 (24) ND ND ND T. celer 88 2.0 0.38 (84) 0.02 (4) 0.006 (1) 0.15 (33) ND ND ND 88 3.0 0.35 (78) 0.08 (18) 0.007 (2) 0.27 (60) ND ND ND 88 4.0 0.31 (69) 0.25 (56) 0.017 (4) 0.27 (60) ND ND ND 91 2.0 0.95 (211) 0.09 (20) 0.004 (1) 0.22 (49) ND ND ND 94 2.0 1.20 (267) 0.11 (24) 0.009 (2) 0.37 (82) ND ND ND T. stetteri 88 2.5 0.09 (20) 0.12 (27) 0.136 (30) 0.07 (16) ND ND ND 88 3.5 0.35 (78) 0.31 (69) 0.078 (17) 0.36 (80) ND ND ND 88 4.5 0.39 (87) 0.34 (76) 0.080 (18) 0.41 (91) ND ND ND 93 2.5 0.45 (100) 0.09 (20) 0.090 (20) 0.10 (22) ND ND ND T. zilligii 75 0.2 ND ND 0.004 (1) 0.02 (4) ND ND ND 80 0.2 ND ND 0.006 (1) 0.03 (7) ND ND ND 84 0.2 ND ND 0.013 (4) 0.05 (11) ND ND ND

a The majority of the values are the means from two to four replicate determinations, but in some cases determinations were performed only once. The values obtained in replicate determinations never varied more than 15%. b Glutamate concentrations were determined by an enzymatic assay. c Hydroxyproline concentrations were determined by a Pico-Tag amino acid analysis. d ND, not detected by 1H NMR. Ϫ e The values in parentheses are crude estimates of the intracellular concentrations of solutes (millimolar) based on a cell volume of 4.5 ␮l ⅐ mg of protein 1 for P. furiosus (20). 3596 LAMOSA ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 3. Effect of the growth temperature, at the optimum salinity, on the accumulation of the following compatible solutes by T. litoralis (A), T. celer (B), and T.

123 u o 123 ᮀ s 2 stetteri (C): DIP ( ), MG ( ), glutamate123 ( ), aspartate ( ), trehalose( ), GalHL ( ), and hydroxyproline ( ).

123 ible solute of halophilic thermococci grown in medium con- were detected only in T. litoralis cells grown in peptone-containing peptone, in which it reaches levels comparable to those taining media leads us to believe that these solutes are derived of MG. from peptone. It is, however, difficult to explain why aspartate The newly identified solute GalHL was detected only in was not accumulated from Casitone or tryptone by this organ- peptone-derived T. litoralis cells, in which it accumulated in ism, since this amino acid is present in these casein-based response to increasing levels of salinity. Aside from choline preparations. It is also not known why GalHL and hydroxypro- and acetylcholine, which are taken up from the medium by line were not detected in the other Thermococcus species or in Lactobacillus plantarum (16), no other known compatible sol- the closely related species P. furiosus grown in media that also ute has a net positive charge (12, 14). However, two neutral contain peptone (22). lysine derivatives are known to serve as compatible solutes; The intracellular levels of compatible solutes in the thermo- ε N -acetyl-␤-lysine accumulates in several methanogens, such as cocci never balanced the levels of osmotically active substances ϩ Methanosarcina thermophila and Methanogenium cariaci (33), in the medium. This indicates that inorganic ions (namely, K ε Ϫ and N -acetyl-lysine behaves like an osmolyte in a few moder- and Cl ) may significantly contribute to the osmolyte pools of ately halophilic bacteria (13). It is also important to note the these organisms. Large contributions by inorganic ions to the resemblance between GalHL and the ␣-glucosyl-␤-galactosyl- osmolyte pools have been found in some methanogens and 5-hydroxylysine moiety found in mammalian collagen. In fact, have been inferred for other hyperthermophilic archaea (9, 20, GalHL is probably derived from collagen, since this protein is 21), and such contributions may also occur in the thermococci. present in peptone (13a); it may be formed from the diglycosyl- However, it should be pointed out that the values obtained hydroxylysine component after removal of the terminal glucose were calculated by assuming that the cell volume was indepen- residue by the microorganisms or during the production of dent of the salinity of the medium, which is unlikely. peptone. It should also be noted that hydroxyproline is a major Trehalose is a common disaccharide in bacteria and archaea amino acid in collagen and that low levels of this amino acid and may play a role in osmotic adaptation in several organisms also accumulate in T. litoralis. Accumulation of hydroxyproline (11, 14). Yeast extract, in which the trehalose level is about was detected previously in Listeria monocytogenes under water 11% (wt/wt) (36), is a common source of this disaccharide for stress conditions in peptone-containing media or when hy- many organisms. Our results show that trehalose accumulated droxyproline or prolyl-hydroxyproline was added to the me- in T. litoralis only when yeast extract was added to the medium; dium (1). The observation that GalHL and hydroxyproline therefore, we concluded that this disaccharide was not synthe-

FIG. 4. Effect of the NaCl concentration of the medium, at the optimum growth temperature, on the accumulation of the following compatible solutes by T. litoralis u o 12 ᮀ s 2 (A), T. celer (B), and T. stetteri (C): DIP ( ), MG ( ), glutamate (12 ), aspartate ( ), trehalose ( ), GalHL (12 ), and hydroxyproline ( ). VOL. 64, 1998 COMPATIBLE SOLUTES IN THERMOCOCCUS SPP. 3597

The differential accumulation of osmolytes observed during the growth cycle of T. litoralis corroborates previous results obtained with hyperthermophilic archaea, mesophilic bacteria, and yeasts (8, 20, 32, 35). For example, it is often observed that the total solute pool decreases during the stationary phase, although no explanation for this observation has been given. Perhaps some compatible solutes replace others that accumulate during the exponential phase because they are not as easily lost to the external environment. Some compatible solutes that accumulate during the exponential phase of growth may also be preferentially consumed when the concentrations of nutrients in the medium decrease below critical levels. Unlike the other organisms examined here, T. zilligii AN1T was isolated from a low-salinity continental hot spring and does not tolerate the levels of NaCl required by the other species of the genus Thermococcus for growth (17, 30). This organism does not accumulate appreciable levels of compatible solutes at the optimum growth temperature, nor does it FIG. 5. Correlation between growth phase and accumulation of organic sol- accumulate compatible solutes as the growth temperature is utes by T. litoralis. The culture was grown in medium containing 4.0% NaCl at raised. Other nonhalophilic thermophilic and hyperthermo- 85°C (■). The intracellular concentrations of the following solutes were deter- philic organisms, such as Thermotoga thermarum, Pyrobaculum u o 123 mined at different phases of growth: DIP ( ), MG ( ), glutamate123 ( ), aspartate (ᮀ), trehalose (s), GalHL123 ( ), and hydroxyproline (2). islandicum, and Fervidobacterium islandicum, unlike slightly 123 halophilic members of the same genera, do not accumulate compatible solutes at the optimum growth temperature (21, 22). The observation that only halophilic strains accumulate sized de novo under the conditions examined in this study. compatible solutes at supraoptimum growth temperatures sug- Rather, trehalose was taken up from yeast extract by the re- gests that the role played by these solutes may be something cently described high-affinity maltose-trehalose transport sys- other than a role in thermoadaptation. However, it is possible tem, which was shown to be induced by trehalose (36). Our that solutes such as DIP, cyclic-2,3-bisphosphoglycerate, and results reinforce the importance of solute uptake by organisms under water stress conditions and extend previous results (to some extent) MG may play a role as thermoprotectants showing that unusual compatible solutes, such as carnitine, primarily when two stresses (namely, water stress and temper- dimethylsulfoniopropionate, 3-morpholino-1-propanesulfonic ature stress) are applied simultaneously. In fact, it is difficult to acid (MOPS), and arsenobetaine (10, 14, 16, 32) are taken up. explain the consistent accumulation of specific solutes by most This work revealed the an unexpected ability of T. litoralis to hyperthermophilic organisms at supraoptimal temperatures scavenge suitable components from the medium and to use unless a role in thermoprotection is envisioned. Intrinsic prop- them as compatible solutes, thus bypassing de novo synthesis erties of cell components are very important for growth at high and saving energy. It is noteworthy that neither the other temperatures, but with the present state of knowledge we can- Thermococcus species examined nor the closely related Pyro- not discount the contribution of extrinsic factors, such as the coccus species were able to derive such a variety of solutes thermostabilizing attributes of some compatible solutes, to the from the medium. growth of microorganisms at high temperatures.

FIG. 6. Effect of medium composition on the accumulation of organic solutes by T. litoralis grown at 85°C with 3.0% NaCl. The media contained peptone plus yeast extract (bar A), peptone plus maltose (bar B), Casitone plus yeast extract (bar C), Casitone plus maltose (bar D), tryptone plus yeast extract (bar E), and tryptone plus

u o 123 ᮀ s

maltose (bar F). The intracellular concentrations of the following solutes were determined: DIP ( ), MG ( ), glutamate123 ( ), aspartate ( ), trehalose ( ), 2

GalHL (12 ), and hydroxyproline ( ). 3598 LAMOSA ET AL. APPL.ENVIRON.MICROBIOL.

ACKNOWLEDGMENTS 19. Lund, P. 1987. UV method with glutaminase and glutamate dehydrogenase, p. 357–363. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis, 3rd This work was supported by grant BIO4-CT96-0488 from the Euro- ed., vol. VIII. VCH, Weinheim, Germany. pean Community Biotech Programme (Extremophiles as Cell Facto- 20. Martins, L. O., and H. Santos. 1995. Accumulation of mannosylglycerate ries) and by Praxis XXI and FEDER Programme grants PRAXIS and di-myo-inositol-phosphate by Pyrococcus furiosus in response to salinity 2/2.1/BIO/20/94 (to H.S. and M.S.D.) and PRAXIS/2/2.1/BIO/1109/95 and temperature. Appl. Environ. Microbiol. 61:3299–3303. (to H.S.). 21. Martins, L. O., L. S. Carreto, M. S. da Costa, and H. Santos. 1996. Novel compatible solutes related to di-myo-inositol-phosphate in the order Ther- motogales. J. Bacteriol. 178:5644–5651. REFERENCES 22. Martins, L. O., R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. 1. Amezaga, M.-R., I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Santos. 1997. Organic solutes in hyperthermophilic archaea. Appl. Environ. Booth. 1995. The role of peptide metabolism in the growth of Listeria mono- Microbiol. 63:896–902. cytogenes ATCC 23074 at high osmolarity. Microbiology 141:41–49. 23. Mehlert, A., J. M. Richardson, and M. A. J. Ferguson. 1998. Structure of the 2. Baumann, H., A. O. Tzianabos, J.-R. Brisson, D. L. Kasper, and H. J. glycosylphosphatidylinositol membrane anchor glycan of a class-2 variant Jennings. 1992. Structural elucidation of two capsular polysaccharides from surface glycoprotein from Trypanossoma brucei. J. Mol. Biol. 277:379–392. one strain of Bacteroides fragilis using high-resolution NMR spectroscopy. 24. Miroshnichenko, M. L., E. A. Bonch-Osmolovskaya, A. Neuner, N. A. Kos- Biochemistry 31:4081–4089. trikina, N. A. Chernych, and V. A. Alekseev. 1989. Thermococcus stetteri sp. 3. Bax, A., and M. F. Summers. 1986. 1H and 13C assignments from sensitivity- nov., a new extremely thermophilic marine sulphur-metabolizing archaebac- enhanced detection of heteronuclear multiple-bond connectivity by 2D mul- terium. Syst. Appl. Microbiol. 12:257–262. tiple quantum NMR. J. Am. Chem. Soc. 108:2093–2094. 25. Neuner, A., H. W. Jannasch, S. Belkin, and K. O. Stetter. 1990. Thermococ- 4. Bock, K., and C. Pedersen. 1974. A study of 13CH coupling constants in cus litoralis sp. nov.: a new species of extremely thermophilic marine archae- hexopyranoses. J. Chem. Soc. Perkin Trans. II 1974:293–297. bacteria. Arch. Microbiol. 153:205–207. 5. Bock, K., and C. Pedersen. 1983. Carbon-13 nuclear magnetic resonance 26. Nunes, O. C., C. M. Manaia, M. S. da Costa, and H. Santos. 1995. Com- spectroscopy of monosaccharides. Adv. Carbohydr. Chem. Biochem. 41:27– patible solutes in the thermophilic bacteria Rhodothermus marinus and 66. “Thermus thermophilus.” Appl. Environ. Microbiol. 61:2351–2357. 6. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of 27. Ramos, A., N. D. H. Raven, R. J. Sharp, S. Bartolucci, M. Rossi, R. Cannio, microgram quantities of protein utilizing the principle of protein-dye bind- J. Lebbink, J. van der Oost, W. M. de Vos, and H. Santos. 1997. Stabilization ing. Anal. Biochem. 72:248–254. of enzymes against thermal stress and freeze-drying by mannosylglycerate. 7. Breitmaier, E., and W. Voelter. 1990. Carbon-13 NMR spectroscopy. VCH Appl. Environ. Microbiol. 63:4020–4025. Verlagsgesellschaft mbH, New York, N.Y. 28. Reed, R. H., D. L. Richardson, S. R. C. Warr, and W. D. P. Stewart. 1984. 8. Brown, A. D. 1978. Compatible solutes and extreme water stress in eukary- Carbohydrate accumulation and osmotic stress in cyanobacteria. J. Gen. otic micro-organisms. Adv. Microb. Physiol. 17:181–242. Microbiol. 130:1–4. 9. Ciulla, R. A., S. Burggraf, K. O. Stetter, and M. F. Roberts. 1994. Occur- 29. Robertson, D. E., M. F. Roberts, N. Belay, K. O. Stetter, and D. R. Boone. rence and role of di-myo-inositol-1,1Ј-phosphate in Methanococcus igneus. 1990. Occurrence of ␤-glutamate, a novel osmolyte, in marine methanogenic Appl. Environ. Microbiol. 60:3660–3664. bacteria. Appl. Environ. Microbiol. 56:1504–1508. 10. Ciulla, R. A., M. R. Diaz, B. F. Taylor, and M. F. Roberts. 1997. Organic 30. Ronimus, R. S., A. L. Reysenbach, D. R. Musgrave, and H. W. Morgan. 1997. osmolytes in aerobic bacteria from Mono Lake, an alkaline, moderately The phylogenetic position of the Thermococcus isolate AN1 based on 16S hypersaline environment. Appl. Environ. Microbiol. 63:220–226. rRNA gene sequence analysis: a proposal that AN1 represents a new species, 11. Csonka, L. N. 1989. Physiological and genetic responses of bacteria to os- Thermococcus zilligii sp. nov. Arch. Microbiol. 168:245–248. motic stress. Microbiol. Rev. 53:121–147. 31. Scholz, S., J. Sonnenbichler, W. Scha¨fer, and R. Hensel. 1992. Di-myo- 12. da Costa, M. S., H. Santos, and E. A. Galinski. 1998. An overview of the role inositol-1,1Ј-phosphate: a new inositol phosphate isolated from Pyrococcus and diversity of compatible solutes in Bacteria and Archaea. Adv. Biochem. woesei. FEBS Lett. 306:239–242. Eng. Biotechnol. 61:117–153. 32. Smith, L. T. 1996. Role of osmolytes in adaptation of osmotically stressed 13. del Moral, A., J. Severin, A. Ramos-Cormenzana, H. G. Tru¨per, and E. A. and chill-stressed Listeria monocytogenes grown in liquid media and on pro- Galinski. 1994. Compatible solutes in new moderately halophilic isolates. cessed meat surfaces. Appl. Environ. Microbiol. 62:3088–3093. FEMS Microbiol. Lett. 122:165–172. 33. Sowers, K. R., D. E. Robertson, D. Noll, R. P. Gunsalus, and M. F. Roberts. ε 13a.Difco Laboratories. Personal communication. 1990. N -acetyl-␤-lysine: an osmolyte synthesized by methanogenic archae- 14. Galinski, E. A. 1995. Osmoadaptation in bacteria. Adv. Microb. Physiol. bacteria. Proc. Natl. Acad. Sci. USA 87:9083–9087. 37:273–328. 34. Tempest, D. W., J. L. Meers, and C. M. Brown. 1970. Influence of environ- 15. Hensel, R., and H. Ko¨nig. 1988. Thermoadaptation of methanogenic bacteria ment on the content and composition of microbial free amino acid pools. by intracellular ion concentration. FEMS Microbiol. Lett. 49:75–79. J. Gen. Microbiol. 64:171–185. 16. Kets, E. P. W., E. A. Galinski, and J. A. M. de Bont. 1994. Carnitine: a novel 35. Whatmore, A. M., J. A. Chudek, and R. H. Reed. 1990. The effects of osmotic compatible solute in Lactobacillus plantarum. Arch. Microbiol. 162:243–248. upshock on the intracellular solute pools of Bacillus subtilis. J. Gen. Micro- 17. Klages, K. U., and H. W. Morgan. 1994. Characterisation of an extremely biol. 136:2527–2535. thermophilic sulphur-metabolizing archaebacterium belonging to the Ther- 36. Xavier, K. B., L. O. Martins, R. Peist, M. Kossmann, W. Boos, and H. mococcales. Arch. Microbiol. 162:261–266. Santos. 1996. High-affinity maltose/trehalose transport system in the hyper- 18. Lanzotti, V., A. Trincone, B. Nicolaus, W. Zillig, M. de Rosa, and A. Gam- thermophilic archaeon Thermococcus litoralis. J. Bacteriol. 178:4773–4777. bacorta. 1989. Complex lipids of Pyrococcus and AN1, thermophilic mem- 37. Zillig, W., I. Holtz, D. Janekovic, W. Scha¨fer, and W. D. Reiter. 1983. The bers of archaebacteria belonging to Thermococcales. Biochim. Biophys. Acta archaebacterium Thermococcus celer represents a novel genus within the 1004:44–48. thermophilic branch of the archaebacteria. Syst. Appl. Microbiol. 4:88–94.