Glutamine, Glutamate, and {alpha}-Glucosylglycerate Are the Major Osmotic Solutes Accumulated by Erwinia chrysanthemi Strain 3937

Erwinia chrysanthemi is a phytopathogenic soil enterobacteriumclosely related to Escherichia coli. Both species respond tohyperosmotic pressure and to external added osmoprotectantsin a similar way. Unexpectedly, the pools of endogenous osmolytesshow different compositions. Instead of the commonly accumulatedglutamate and trehalose, E. chrysanthemi strain 3937 promotesthe accumulation of glutamine and ${alpha}$ -glucosylglycerate, whichis a new osmolyte for enterobacteria, together with glutamine.The amounts of the three osmolytes increased with medium osmolarityand were reduced when betaine was provided in the growth medium.Both glutamine and glutamate showed a high rate of turnover,whereas glucosylglycerate stayed stable. In addition, the balancebetween the osmolytes depended on the osmolality of the medium.Glucosylglycerate and glutamate were the major intracellularcompounds in low salt concentrations, whereas glutamine predominatedat higher concentrations. Interestingly, the ammonium contentof the medium also influenced the pool of osmolytes. Duringbacterial growth with 1 mM ammonium in stressing conditions,more glucosylglycerate accumulated by far than the other organicsolutes. Glucosylglycerate synthesis has been described in somehalophilic archaea and bacteria but not as a dominant osmolyte,and its role as an osmolyte in Erwinia chrysanthemi 3937 showsthat nonhalophilic bacteria can also use ionic osmolytes.

INTRODUCTION

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Introduction

Erwinia chrysanthemi is the agent of soft rot disease in a widerange of plants. Various studies suggest that adaptation toosmotic stress is an important factor for the survival of thisbacterium in soil (23, 30) and for its pathogenic behavior (17,25). It is well known that bacterial behavior under hyperosmoticstress is widely influenced by the presence of osmoprotectantsin the growth medium. These compounds allow bacteria to strengthentheir natural resistance to osmotic stress. Many of them, likeglycine betaine, its precursor choline, the imino acid proline,and pipecolic acid, are abundant in plants (21, 43).

Considering that, during pathogenesis, E. chrysanthemi is subjectedto osmotic stress in a medium containing osmoprotrectants, moststudies focused on identification of the osmoprotectants ofE. chrysanthemi and their role in pathogenesis (17, 37). E.chrysanthemi is phylogenetically close to Escherichia coli,so it is not surprising that it behaves like E. coli towardsosmoprotectants (19). All osmoprotectants tested so far areactively taken up by two transporters, OusA and OusB, the orthologuesof E. coli ProP and ProU, respectively (19), and accumulatedat levels equivalent to those previously described in E. coli(18, 19). In addition, they reduce the production of enzymesinvolved in plant maceration (17).

The pathogenicity of E. chrysanthemi is also linked to its abilityto survive in soil during desiccation periods. Osmoprotectantsare generally present in small amounts in soil; they are releasedby plant exudates and the decay of various organisms. Soil microorganismscompete for the same osmoprotectants, so the availability ofthese compounds for each one remains low. In such conditions,the ability of bacteria to proliferate and survive in osmoticallychanging media depends highly on their own capacity to faceosmotic stress.

Most of the studies on osmotic stress adaptation deal with enterobacteria.A two-step response was described in E. coli and Salmonellaenterica serovar Typhimurium (9). The initial response to anosmotic upshift is to rapidly transport and accumulate K⁺ ions.To counterbalance the potassium charge, glutamate levels increasedby de novo synthesis (7, 10). This step allows a transient adaptation,but potassium glutamate enhances intracellular ionic strengthproportionally to medium osmolarity, inducing a deleteriouseffect on cellular metabolism during a prolonged stress (7).Potassium glutamate acts as an intracellular signal of osmoticstress (2, 34), inducing the uptake of osmoprotectants (39)and the synthesis of nonionic organic solutes, which do notalter intracellular macromolecules and replace potassium glutamate(13). Trehalose biosynthesis was described in E. coli and S.enterica serovar Typhimurium strains as related to the strengthof the osmotic stress (5, 24). Trehalose not only substitutesfor potassium glutamate but also displays protective propertieson macromolecules and membranes subjected to an osmotic stress(8, 46). Trehalose can also protect cells against various injurieslike desiccation (44) and high temperature (5, 6); it is alsoessential for survival at low temperatures (28).

Trehalose is the only compatible solute synthesized in enterobacteria,including E. chrysanthemi strains ECC and SR 237 (40). Therefore,trehalose was expected to be a compatible solute in other E.chrysanthemi strains. In most organisms, de novo trehalose synthesisis catalyzed by trehalose-6-phosphate synthase and trehalose-6-phosphatephosphatase, encoded by otsAB in E. coli (27). Two additionalways for trehalose synthesis were described in mycobacteria(11), Corynebacterium glutamicum (45), Sulfolobus solfataricus(31), and Rhizobium spp. (35). One way produces trehalose frommaltose by a single transglycosylation catalyzed by the trehalosesynthase TreS. The other way catalyzes the conversion of oligo-and polymaltodextrins and glycogen into trehalose in a two-stepreaction by maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalosetrehalohydrolase (TreZ). Orthologues of otsAB, treS, and treYZare absent in the E. chrysanthemi strain 3937 genome (https://asap.ahabs.wisc.edu/annotation/php/logon.php).Hence, E. chrysanthemi strain 3937 is unable to synthesize trehaloselike other known enterobacteria; however, it may use an unknownpathway. Thus, we analyzed the E. chrysanthemi 3937 endogenousosmolytes and showed that this bacterium does not synthesizetrehalose but does synthesize glutamine and ${alpha}$ -glucosylglycerate,two uncommon osmolytes in enterobacteria.

MATERIALS AND METHODS

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Materials and Methods

Culture and growth conditions.
E. chrysanthemi 3937 (32) was grown in mineral medium M63 (38)at 30°C. Glucose as a carbon and energy source was addedat 10 mM. Low-NH₄ medium was M63 containing only 1 mM ammoniumsulfate instead of 15 mM. Media of elevated osmotic strengthwere made by adding NaCl. Their osmotic pressure was determinedby measuring the freezing point with an osmometer. The osmoprotectantglycine betaine was supplied at a final concentration of 0.5mM. Bacterial growth was monitored by optical density measurementat 570 nm.

Extraction of cellular solutes.
Bacterial cells were harvested by centrifugation (8,000 x g,15 min), and the pellet was extracted twice with 80% (vol/vol)ethanol under vigorous magnetic stirring at room temperaturefor 15 min. After centrifugation, the combined supernatantswere lyophilized. The dried extract was dissolved in water beforeanalysis.

Natural-abundance ¹³C-nuclear magnetic resonance spectroscopy.
The dried cellular extract prepared from about 2 x 10¹² cells(350 mg of protein) was dissolved in 0.8 ml of deuterated water.The natural-abundance ¹³C nuclear magnetic resonance spectrawere recorded in the pulsed Fourier transform mode at an operationalfrequency of 300 MHz. The spectra of purified glucosylglycerate,hydrolyzed glucosylglycerate fractions, and synthesized glucosylglyceratewere performed as for cellular extracts.

Hydrolysis of ${alpha}$ -glucosylglycerate.
E. chrysanthemi 3937 was grown to the stationary growth phasein 0.5 M NaCl-M63 medium; bacteria were collected by centrifugationand extracted in 80% (vol/vol) ethanol. The ethanolic extractwas partially purified by passage through a cation exchangecolumn of Dowex 50Wx8 in the H⁺ form. The effluent was concentratedby evaporation and hydrolyzed by 2 N HCl for 75 min at 100°C.The hydrolysate was fractionated with an anion exchange columnIRA 400 (HCOO^–). The effluent and eluted fractions (in4 M HCOOH) were concentrated and analyzed by natural-abundance¹³C nuclear magnetic resonance spectroscopy.

Chemical synthesis of ${alpha}$ -glucosylglycerate.
Glucosylglycerate was synthesized from leucrose according toHunter et al. (26). Its structure and purity were determinedby ¹³C nuclear magnetic resonance spectroscopy and by chromatography.

Glucosylglycerate isomerization.
Infrared spectra in the attenuated total reflectance mode wereperformed with a ZnSe crystal at 45°C, and 128 scans wereadded at 2 cm^–1 spectral resolution at room temperature.Samples were dissolved in distilled water prior to acquisition.To determine the anomeric form of the molecule, D-glucose andD-trehalose were analyzed in the same conditions. D-Glucoseis present as a mixture of the ${alpha}$ and ß anomers, whereasD-trehalose exhibits an ${alpha}$ anomeric form.

Separation and quantification of osmolytes.
Ethanolic extracts of cells grown in M63 medium containing D-[U-¹⁴C]glucose(1.2 MBq mmol^–1) were analyzed by a two-dimensional systemcombining high-voltage electrophoresis (Whatman 3MM paper, 3%formic acid, electric field value of 40 V/cm) as the first dimensionand paper chromatography run in butanol-acetic acid-water (12:3:5,vol/vol) as the second dimension. Nonradiolabeled amino acidsand glucosylglycerate were added as internal standards. Afterdrying at 60°C, the chromatograms were sprayed with ninhydrin(0.4% in butanol) and heated at 60°C; then they were analyzedby autoradiography. Identified spots were cut out, and theirradioactivity was quantified by scintillation counting. A calibrationwas established between radioactivity and the amount of eachcompound, taking into account that all the carbon atoms bearan identical radioactivity. Quantification was confirmed forglutamate and glutamine by direct assay as previously described(20). All experiments were repeated three times.

Uptake and catabolism of compatible solutes.
${alpha}$ -[¹⁴C]glucosylglycerate was purified from cells grown in 0.5M NaCl-M63 medium supplemented with D-[U-¹⁴C]glucose (1.2 MBqmmol^–1). It was used for uptake experiments as previouslydescribed (20). The use of ${alpha}$ -glucosylglycerate as a growth substratewas analyzed in M63 medium containing 10 mM ${alpha}$ -glucosylglycerateas the sole carbon and energy source. Glutamate, glutamine,and betaine uptake and behavior were determined as previouslydescribed (19).

[methyl-¹⁴C]glycine betaine (2.0 GBq mmol^–1 was preparedand its uptake and accumulation were determined as described(17, 20).

RESULTS

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Results

Identification of the osmolytes accumulated by E. chrysanthemi grown in high-salt medium.
E. chrysanthemi 3937 cells grown in M63 with 0.5 M NaCl werecollected in the mid-exponential and in the stationary growthphases. Analysis of ¹³C nuclear magnetic resonance spectra showedthat both extracts contained the same signals, but in differentratios. During the exponential growth phase (Fig. 1A), the mainpeaks belonged to glutamate and glutamine; other peaks couldbe attributed collectively to a carbohydrate molecule that didnot match the expected trehalose peaks.

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FIG. 1. ¹³C nuclear magnetic resonance spectra of an ethanol extract of E. chrysanthemi 3937 grown in M63 medium with 0.5 M NaCl in the mid-exponential (A) and stationary (B) phases and ¹³C nuclear magnetic resonance spectrum control of chemically synthesized ${alpha}$ -glucosylglycerate (C). The main peaks correspond to carbons of glutamate (e), glutamine (q), and glucosylglycerate (gg).

During the stationary growth phase (Fig. 1B), the peaks of glutamateand glutamine were severely reduced and that of the supposedcarbohydrate molecule became the most significant. This compoundwas purified and hydrolyzed by HCl; the resulting moleculeswere purified by passage on an anion exchange column. The permeantand eluted fractions were characterized by ¹³C nuclear magneticresonance and compared to the nuclear magnetic resonance spectraof commercial molecules to confirm the nature of these two compounds.The permeant fraction spectrum matched ${alpha}$ and ß glucoseand the eluate fraction matched glycerate. These results suggestthat the carbohydrate is composed of a glucosyl moiety linkedto a glycerate, as in glucosylglycerate. For this molecule,the chemical shifts of carbons with directly bounded protonsoccurred at 97.6 ppm (C-1'), 72.6 ppm (C-2'), 73.4 ppm (C-3'),69.7 ppm (C-4'), 71.5 ppm (C-5'), and 60.8 ppm (C-6') for theglucosyl and at 177.5 ppm (C-1), 78.5 ppm (C-2), and 63.4 ppm(C-3) for the glycerate. Confirmation of the structure as glucosylglyceratewas provided by comparison between the ¹³C chemical shifts ofcellular extract and those of glucosylglycerate produced bychemical synthesis (Fig. 1C).

Glucosylglycerate anomerization.
The anomerization of glucosylglycerate purified from E. chrysanthemiwas analyzed by infrared spectroscopy. Figure 2 shows the infraredspectrum of the isolated glucosylglycerate along with its invertedsecond derivative (29) in the 1,400 to 900 cm^–1 frequencydomain. Peak positions were determined by analyzing the correspondingsecond derivatives (14) and spectra were band fitted. Indeed,the influence of glucose ${alpha}$ - and ß-anomerization hasbeen investigated (1). Bands at 1,339 cm^–1 and 1,320 cm^–1in distilled water were found to be associated with the ${alpha}$ - andß-anomers of glucose, respectively (36). Another studyhas shown that the bands at 1,020 cm^–1 and 1,060 cm^–1in distilled water are associated with the anomeric, C-O stretchingof the ${alpha}$ and ß forms, respectively. D-Glucose in distilledwater exhibited a major band at 1,035 cm^–1 with a shoulderat 1,066 cm^–1 whereas D-trehalose exhibited a band at1,340 cm^–1 and a shoulder at 1,023 cm^–1 which isrevealed by a second derivative maximum (data not shown). Theglucosylglycerate compound (Fig. 2) exhibited a peak at 1,335cm^–1 and a shoulder at 1,025 cm^–1 which stronglysuggest that it presents the ${alpha}$ -anomeric form. Furthermore, apeak at 1,083 cm^–1 reinforces this suggestion, since thispeak has been assigned to the ${alpha}$ -anomeric C-O stretching (36).Hence, it can be assumed that the isolated molecule is ${alpha}$ -glucosylglycerate.

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FIG. 2. Infrared spectrum of the isolated glucosylglycerate along with its inverted second derivative (29) in the 1,400 to 900 cm^–1 frequency domain.

Evolution of osmolyte content during growth in high-salt medium.
The level of E. chrysanthemi 3937 intracellular osmolytes wasanalyzed in M63 medium containing 0.5 M NaCl and D-[U-¹⁴C]glucose.Accumulated osmolytes were extracted and analyzed by chromatographythroughout growth (Fig. 3). The three osmolytes (glutamine,glutamate, and glucosylglycerate) began to accumulate immediatelyafter the osmotic upshift. Glutamate and glutamine reached theirmaximal level (700 nmol/mg of dry weight) 3 to 4 h after inoculation(Fig. 3). Then the level of glutamate decreased. Glutamine stayedat its maximal level until the late exponential growth phase.Then it decreased throughout the stationary growth phase. Glucosylglycerateappeared as the minor solute during growth; its level increasedslowly but continuously to reach a maximum of 220 nmol/mg ofdry weight during the stationary growth phase. Since glutamateand glutamine had disappeared by this time, glucosylglyceratebecame the sole osmolyte.

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FIG. 3. Evolution of osmolyte content during growth. Intracellular levels of glucosylglycerate (diamonds), glutamate (squares), and glutamine (triangles) during growth of E. chrysanthemi 3937 in 0.5 M NaCl-M63 medium with 15 mM (NH₄)₂SO₄. Growth (open circles) was monitored by optical density measurements at 570 nm.

To explain the evolution of osmotic pool components, the turnoverof osmolytes was analyzed on cells grown in M63 medium with0.4 M NaCl and containing [U-¹⁴C]glucose. After 6 h of growth,cells were collected and resuspended in the same medium withoutthe radioactive glucose. Cells were collected at regular intervals,and solutes were extracted and analyzed. Spots correspondingto glutamate, glutamine, and glucosylglycerate were excised,and their radioactivity was determined. The radioactivity ofglutamate decreased immediately, with a half-life of 23 min.This high turnover is in accordance with its central role inbiosynthetic processes. Glutamine radioactivity decreased moreslowly than that of glutamate (half-life of 30 min). The labelingof glucosylglycerate remained constant throughout the experiment.Therefore, glucosylglycerate appears to be a stable compoundand not an intermediate metabolite. This high metabolic stabilitycould explain why it did not disappear during the stationarygrowth phase.

Glutamate, glutamine, and glucosylglycerate behave as compatible solutes but are not osmoprotectants.
When a hypoosmotic shock was applied to E. chrysanthemi 3937cells grown in M63 medium containing 0.5 M NaCl and D-[U-¹⁴C]glucose,the three osmolytes were recovered in the surrounding mediumwhatever the growth phase in which the cells were collected.However, glutamate, glutamine, and glucosylglycerate were notdetected in the external medium during growth at high salinity,suggesting that they were not released during growth in hyperosmoticconditions as described for most compatible solutes (10) orthat they are efficiently reabsorbed from the medium. To testthis hypothesis, ¹⁴C-radiolabeled glutamate, glutamine, andglucosylglycerate were added to the E. chrysanthemi growth medium.Glutamate and glutamine were efficiently taken up; in contrast,[U-¹⁴C]glucosylglycerate was not taken up by E. chrysanthemi3937 cells cultivated in M63 medium without or containing 0.4M NaCl. In addition, E. chrysanthemi 3937 was unable to growin M63 medium supplemented with ${alpha}$ -glucosylglycerate as the solecarbon and energy source. Moreover, when 1 mM glutamate, glutamine,or glucosylglycerate was added to M63 medium with 0.4 M NaCl,none of them improved growth. Therefore, these three compoundsare compatible solutes but not osmoprotectants for E. chrysanthemi3937.

Influence of the medium osmolality on organic solute composition.
The intracellular osmolyte content was examined in E. chrysanthemi3937 cells cultivated to the mid-log phase of growth in M63medium in the presence of D-[U-¹⁴C]glucose and various NaClconcentrations (0 to 0.4 M) (Fig. 4). The levels of glutamate,glutamine, and glucosylglycerate increased with medium osmolality;the ratio of these molecules was dependent on the medium osmolality.Thus, glutamate and ${alpha}$ -glucosylglycerate were present in similarproportions in M63 medium deprived of salt. When the salt concentrationwas increased to 0.2 M, glutamate became the main solute. Theglutamine content was not significant at low osmolality (0 and0.1 M NaCl); it began to rise in media containing 0.2 M NaCl.Above this salt concentration, glutamine increased spectacularlyreaching more than 60% of the accumulated solutes (Fig. 4).Therefore, during E. chrysanthemi 3937 osmotic adaptation, glutamateand glucosylglycerate are the favorite solutes accumulated atlow medium osmolality, whereas glutamine is preferred at highmedium osmolality.

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FIG. 4. Influence of medium osmolarity on the ratio of internal solutes. The effect of the NaCl concentration on the intracellular glucosylglycerate (GG, grey), glutamine (Gln, black), and glutamate (Glu, white) content of E. chrysanthemi 3937 grown to exponential phase in M63 medium was determined.

Evolution of osmolyte content during growth in medium of low osmolality.
E. chrysanthemi 3937 cells were grown in M63 medium containingD-[U-¹⁴C]glucose. At regular intervals, cells were collectedand osmolytes were quantified. Glucosylglycerate and glutaminewere estimated to 30 and 10 nmol/mg of dry weight, respectively,throughout growth and did not vary in the stationary growthphase for at least 40 h (data not shown). The glutamate levelwas estimated to 50 nmol/mg of dry weight all during the exponentialgrowth phase; it decreased to 15 nmol/mg of dry weight duringthe stationary growth phase. Therefore, glutamine and glucosylglyceratecontent are not affected by growth phase in M63 medium.

Organic osmolyte ratio depends on medium composition.
Glucose, the carbon and energy source provided at 10 mM in M63,constitutes the growth-limiting nutrient rather than nitrogen.Glutamate and glutamine need both nitrogen and carbon for theirbiosynthesis, whereas glucosylglycerate is built only from carbon.Therefore, the balance between these two groups of osmolytescould depend on the C/N ratio of the growth medium. To analyzethis point, E. chrysanthemi 3937 was grown in modified M63 mediumcontaining 0.5 M NaCl, 1 mM (NH₄)₂SO₄ and D-[U-¹⁴C]glucose asthe carbon and energy source. The results presented in Fig.5 are compared to those obtained previously with cells grownin M63 medium containing 15 mM (NH₄)₂SO₄ (Fig. 3). Loweringthe level of nitrogen in the medium did not affect the growthrate but, as expected, reduced the growth yield by about 30%.During the first hours of growth, the osmolyte pool compositionwas identical in low- and high-nitrogen medium (Fig. 3 and 5).Glutamate and glutamine were the main solutes accumulated immediatelyafter the upshock. Glutamate had the same accumulation profilewhatever the medium used (Fig. 3 and 5). In contrast, glutamineaccumulation depended on the nitrogen concentration and wasonly transient in low-nitrogen media. Its level decreased simultaneouslywith that of glutamate during the early steps of exponentialgrowth (Fig. 5). The glucosylglycerate content was maintainedat the same level in high and low nitrogen throughout the glutamateand glutamine accumulation phase (Fig. 5).

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FIG. 5. Glucosylglycerate accumulation is favored under NH₄ limitation. Intracellular levels of glucosylglycerate (diamonds), glutamate (squares), and glutamine (triangles) were determined during growth of E. chrysanthemi 3937 in M63 medium with 0.5 M NaCl and containing only 1 mM (NH₄)₂SO₄. Growth (open circles) was monitored by optical density measurements at 570 nm.

In contrast, the decrease in glutamine and glutamate contentwas accompanied by a spectacular increase in the glucosylglyceratelevel. It became by far the main solute detected at the endof exponential growth, and its level was greater than that observedin high-nitrogen medium (Fig. 5). Its concentration went onincreasing after growth arrest, reaching 1,000 nmol/mg of dryweight (Fig. 5), versus 220 nmol/mg of dry weight in high-nitrogenmedium (Fig. 3).

Glycine betaine suppresses the accumulation of glutamate, glutamine, and glucosylglycerate.
In all bacteria, external compatible solutes (i.e., osmoprotectants)replace newly synthesized internal ones (9). Thus, to examinethe implication of glutamate, glutamine, and ${alpha}$ -glucosylglyceratein osmoprotection, their behavior was followed after additionof 0.5 mM glycine betaine to M63 medium containing 0.5 M NaCl.The levels of glutamate, glutamine, and glucosylglycerate wereanalyzed before and after the addition of glycine betaine duringthe exponential growth phase (Fig. 6). Glycine betaine was readilyaccumulated at a level identical to that reported previously(19). Glycine betaine accumulation abolished the accumulationof glutamate, glutamine, and glucosylglycerate. This resultconfirms that glutamate, glutamine, and glucosylglycerate areinvolved in osmoprotection and constitute the internal osmolytepool of E. chrysanthemi 3937 cells.

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FIG. 6. Suppression of endogenous solute accumulation. Intracellular levels of glucosylglycerate (diamonds), glutamate (squares), glutamine (triangles), and glycine betaine (closed circles) during growth of E. chrysanthemi 3937 in M63 medium with 0.5 M NaCl and 15 mM (NH₄)₂SO₄ was determined; 500 µM [methyl-¹⁴C]glycine betaine was added after 18 h of growth. Growth (open circles) was monitored by optical density measurements at 570 nm.

DISCUSSION

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Discussion

Bacteria subjected to a hyperosmotic stress rapidly lose intracellularwater and go on to the plasmolysis state. In response to thisstress, they must accumulate intracellular solutes, allowingmaintenance of their turgor pressure (10). However, not allbacteria produce the same compatible solutes. Halophilic bacteriaare able to accumulate salts at high levels and charged organicosmolytes (42). Nonhalophilic bacteria such as E. chrysanthemicannot support excess intracellular ionic strength (13). Therefore,they accumulate compounds that are not electrically chargedat their physiological pH (47).

The analysis of the osmolyte content of E. chrysanthemi 3937cells cultivated in high-salt media revealed the presence ofglutamate, glutamine, and ${alpha}$ -glucosylglycerate. Among these threecompounds, only glutamine is neutral at physiological pH, whichcould explain why this amino acid constitutes the main soluteaccumulated at high osmolality. Glutamine has never been describedas a compatible solute in enterobacteria; it is not a commonosmolyte in bacteria. It was described as the major endogenousosmolyte accumulated in Corynebacterium spp. (15), and its ßform was found in the compatible solute pool of halophilic methanogenicarchaea (33).

The other two solutes accumulated by E. chrysanthemi (glutamateand glucosylglycerate) are charged at physiological pH and shouldincrease the ionic strength of the cytoplasm. They representthe only accumulated solutes at low osmolarity and should respondto the entry of potassium into the cell (13), acting as a K⁺counterion to maintain electroneutrality. E. chrysanthemi 3937did not accumulate trehalose like other enterobacteria (5, 24)but glucosylglycerate mainly during stationary growth phase.This absence of trehalose accumulation is intriguing, becausethis compound was already reported for other E. chrysanthemistrains (40). Accordingly, neither the otsAB genes commonlyfound in enterobacteria nor the treS and treYZ trehalose biosyntheticgenes found in other bacteria (11, 45) were detected on theE. chrysanthemi strain 3937 genome.

E. chrysanthemi strain 3937 is totally without trehalose biosyntheticpathways. Glucosylglycerate, a solute initially identified inMethanohalophilus portucalensis strain FDF1 (41), was recentlyreported in Halomonas elongata CHR63 (4). In comparison withthese strains, the glucosylglycerate content is greater in E.chrysanthemi. Glucosylglycerate is a stable molecule in E. chrysanthemi,while it was considered a metabolic intermediate in Methanohalophilusportucalensis (41), suggesting that in E. chrysanthemi thismolecule functions solely as an osmolyte. Glucosylglyceratehas only been described in two halophilic strains and so faris not known as a compatible solute in enterobacteria. The presenceof this molecule in the endogenous osmolyte pool of E. chrysanthemistrain 3937 is surprising because this bacterium is phylogeneticallydistant from these halophilic strains. However, glucosylglycerateresponds as a classical compatible solute in that its accumulationincreased proportionally with the medium osmolality and wassuppressed by the addition of the osmoprotectant glycine betaine.

The ratio of E. chrysanthemi 3937 osmolytes varied with mediumcomposition and growth phase. Bacteria able to synthesize variousosmolytes adapt their ratio to medium composition; in Ectothiorodospiraspecies, trehalose constitutes a minor solute in high-nitrogenmedia but becomes the preferred solute under nitrogen-limitedconditions (16). Similarly, the ratio of the various osmolytesof Methanohalophilus portucalensis is highly dependent on thegrowth substrate (41). Glucosylglycerate is the major soluteof E. chrysanthemi 3937 in the stationary growth phase; an identicalsituation was reported for Halomonas elongata, which also synthesizesglucosylglycerate at this stage of growth (4). It is also thecase for trehalose, the most frequent organic solute found inbacteria (20, 22).

Glucosylglycerate was believed to be accumulated only by halophilicorganisms, while its counterpart glucosylglycerol is accumulatedby nonhalophilic ones (42). The present study shows that thisdistinction is not always true.

Compatible solutes are compounds which are restricted to a smallfamily of molecules that were selected during evolution becausethey do not affect cellular metabolism (47). While their primaryrole is to recover turgor, compatible solutes were also shownto improve resistance against other stresses, including desiccation(44) and heat treatment (5, 6); they may act as chemical chaperonesfavoring native protein folding both in vitro and in vivo (3,12). This role for glutamate has already been reported (12);such a protective effect could be expected for glutamine andglucosylglycerate and must be confirmed in the future.

ACKNOWLEDGMENTS

We thank O. Sire from L2PIC of UBS for determination of infraredglucosylglycerate spectra. We are grateful to S. Georgeault,C. Monnier, M. C. Savary, and M. Uguet for excellent technicalassistance and to C. Staines and J. P. Besnard for improvingthe English usage of the manuscript.

This work was supported by the Ministère de la Rechercheand by the CNRS.

FOOTNOTES

* Corresponding author. Mailing address: Osmorégulation chez les Bactéries, CNRS UMR 6026, Université de Rennes I, Campus de Beaulieu, Av. du Général Leclerc, 35042 Rennes, France. Phone: 33-223236140. Fax: 33-223236775. E-mail: carlos.blanco{at}univ-rennes1.fr.

REFERENCES

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