Arch Microbiol (1991) 156:350--355

030289339100143B Archives of

Microbiology �9 Springer-Verlag 1991

Intracellular salt and solute concentrations in Ectothiorhodospira marismortui: glycine betaine and N -carbamoyl glutamineamide as osmotic solutes Aharon Oren 1, Gilat Simon 1, and Erwin A. Galinski 2

1 Division of Microbial and Molecular Ecology, Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel 2 Institut ftir Mikrobiologie und Biotechnologie, Rheinische Friedrich-Wilhelms-Universit/it, Meckenheimer Allee 168, W-5300 Bonn 1, Federal Republic of Germany

Received November 30, 1990/Accepted April 29, 1991

Abstract. Ectothiorhodospira marismortui, a moderately halophilic purple sulfur bacterium from a hypersaline sulfur spring, contains glycine betaine and N~-carbamoyl glutamineamide (CGA) as the main intracellular osmotic solutes, with sucrose as a minor component. The concen- tration of glycine betaine was found to increase with increasing salt concentration of the medium, from 0.47 M to 1.29 M in cells grown from 0.85 to 2.56 M NaC1, while the estimated CGA concentration rose from about 0.2 M to 0.5 M. The concentration of sucrose remained con- stant at a value of around 0.05 M. Intracellular sodium and potassium concentrations were relatively low (around 0.5 and 0.3 M, respectively, at an external NaC1 concentration of 1.8 M). The concentration of the novel compound N~-carbamoyl glutamineamide was enhanced when L-glutamine was added to the growth medium, suggesting that glutamine served as a precursor for the synthesis of the compound.

Key words: Ectothiorhodospira - Halophilic - Compat- ible solutes - Glycine betaine - N~-carbamoyl gluta- mineamide - Sucrose

Microorganisms adapted to life in high salt concen- trations have developed different strategies to cope with the high osmotic pressure exerted by their surrounding medium. The extremely halophilic archaebacteria (family Halobacteriaceae) contain very high intracelullar po- tassium, sodium and chloride concentrations, and their enzymatic machinery is adapted to function in their pres- ence. On the other hand, intracellular enzymes of most halophilic eubacteria are sensitive to high salt concen- trations. These organisms contain high intracellular con- centrations of organic compounds, serving as "compat-

Offprint requests to: A. Oren

Abbreviations: CGA, Nc~-carbamoyl glutamineamide

ible solutes", enabling osmotic balance, while supporting enzymatic activity (Brown 1976; Kushner 1978; Trfiper and Galinski 1986).

Glycine betaiue was found to be widespread as com- patible solute in halophilic photosynthetic prokaryotes (Galinski and Triiper 1982; Mackay et al. 1984); in ad- dition it is accumulated by many moderately halophilic eubacteria, which are unable of de novo synthesis of the compound (Imhoff and Rodriguez-Valera 1984; Wohlfarth et al. 1990). In addition, ectoine (1,4,5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), a compound discovered for the first time in halophilic photosynthetic bacteria of the genus Ectothiorhodospira (Galinski et al. 1985), seems to occur widely in halophilic and halotolerant microorganisms (Wohlfarth et al. 1990).

We recently described a novel obligately anaerobic, moderately halophilic purple sulfur bacterium from a hypersaline sulfur spring on the shore of the Dead Sea (Oren 1989; Oren et al. 1989). The strain, described as Ectothiorhodospira marismortui, grows optimally at NaC1 concentrations between 2 and 10%, and up to 20%. As part of the characterization of the species we measured its internal salt concentrations and tested for the presence of different organic osmotic solutes. We show here that glycine betaine is the main osmotic solute in this organ- ism. In addition, a hitherto unknown compound, iden- tified as N~-carbamoyl glutamineamide (CGA) (Galinski and Oren 1991), was present in high concentrations, while ectoine was absent. Sucrose (a substance not earlier re- ported to serve as an osmotic solute in the genus Ectothiorhodospira) was found as a minor component. In the present work we estimated the intracellular concen- trations of the three osmotic solutes as a function of external salt concentrations and growth conditions.

Materials and methods

Bacterial strain and culture conditions

Ectothiorhodospira marismortui EG-I (DSM 4180) was grown an- aerobically in the light at 35~ using illumination by white fluor-

escent tubes, incident light intensity 5 x 103 erg/cm 2 �9 s. For the preparation of I 1 growth medium an autoclaved solution of 50, 100, or 150 g NaC1, as indicated, was prepared, with the addition of 1 g KC1, 0.5 g Na2SO4 and 0.1 g Bacto yeast extract, in a final volume of 900 ml. The following sterile solutions were added just before inoculation, and in the order indicated: (1) 50 ml of a solution containing 6.6 g/1 of each KH2PO4, NH4C1 and MgC12 �9 6 HzO; (2) 10 ml of trace element solution (Pfennig and Lippert 1966); (3) 5ml of a solution of 100g/1 Na-acetate. 3H20 or Na- succinate - 6 HzO, as indicated; (4) 15 ml of a solution of 100 g/1 NazCO3; (5) 10-11 ml of I M HC1, to a final pH of 6 .5- 6.8; (6) 10 ml of a solution of 33 g/1 CaCI2 �9 2 H20. Immediately before use of the medium 2.5ml of an autoclaved solution of 50g Na2S �9 9 H20/1 was added, and the medium was dispensed into 300- ml culture bottles, which were filled completely. Different modifi- cations of the above standard growth conditions were used, as indicated in the experiments. Unless stated otherwise, mid- to late exponential growth phase cells were used (2 to 3 days old cultures, depending on the salinity of the medium used).

Measurement of internal Na + and K + concentrations

Cells in the exponential growth phase were collected by centrifuga- tion (5 min, 6000 • g), and the pellet was resuspended in fresh growth medium to a density of approximately 50 - 70 mg wet pellet weight per ml. Portions (i ml) of cell suspensions were added to 1.5 ml plastic centrifuge tubes. 3H20 (3.75 gCi in 6 gl) (New England Nuclear, Boston, MA, USA) was added, together with different 14C-labelled compounds serving as markers for extracellular space (6 gl of 48 mM polyethylene glycol, and 0.75 gCi of [U-14C]poly- ethylene glycol (Amersham, Buckinghamshire, UK) (mol. wt. 4000, 60 mCi/mmol) in 15 Ixl, or 0.75 gCi [carboxyl-14C]dextran carboxyl (New England Nuclear) (50 gCi/ml, 0.78 mCi/g in 15 gl) or for extracellular + periplasmic space (0.8p~Ci D-[1-14C]fructose (Amersham) (60 mCi/mmol) in 16 gl, or 2 gCi D-[1-14C]galactose (Amersham) (60 mCi/mmol) in 10 gl, and the contents of each tube were mixed. After 5 min incubation at room temperature the tubes were centrifuged in an Eppendorf (Hamburg, FRG) model 3200 centrifuge for 4 min. A portion of 50 gl of the supernatant was mixed with 0.95 ml of 10% perchloric acid, the remainder of the supernatant was removed by means of a Pasteur pipette, and the pellet was resuspended in 0.95 ml of 10% perchloric acid and incu- bated overnight in the cold, whereafter the tubes were shaken and centrifuged again. Both supernatant fractions were assayed for 3H and 14C radioactivity by counting 100-gl portions with 8 ml Instagel (Packard, Downers Grove, IL., USA) scintillation cocktail in a Packard Tri-Carb model 3255 scintillation counter. Samples of both supernatant fractions were assayed for Na § and K § ions using a flame photometer (Evans Electroselenium, Halstead, UK), after dilution with distilled water to concentrations between i and 10 rag/1 Na + or K +.

Intracellular Na + and K § concentrations were calculated ac- cording to: Cin = Cp-C~ - V~x/(1 - V J , in which Cin is the Na + or K + concentration inside the cells, and Cp and Cs their respective concentrations in the pellet and supernatant fractions, and V,x the relative extracellular volume as expressed by the fraction of the pellet volume accessible to fructose or galactose (Shindler et al. 1977). The cytoplasmic volume was determined by subtracting the fructose or galactose space from the total cellular space, and the total cellular space by subtracting the polyethylene glycol or dextran space from the total wet pellet space (Imhoff and Riedel 1989).

Identification of intracelIular osmotic solutes

Intracellular osmotic solutes were identified and quantitated by use of high performance liquid chromatography. Following a modifi- cation of the method of Bligh and Dyer (1969), wet cell pellet was extracted with a mixture of methanol/chloroform/water (10: 5:3.4

351

by volume, 100 ml/g dry pellet weight). Phase separation was achieved by addition of 27 ml chloroform and 27 ml water per 100 ml extract. The aqueous top phase containing cytoplasmic com- pounds, soluble proteins and extracellular salts was then deproteinized by adding perchloric acid to a final concentration of 5% (w/v) and incubating in the cold for 10 rain, a procedure that did not cause significant degradation of CGA (Galinski and Oren 1991). Subsequently the solution was desalted on an ion retardation column (BioRad AGll AB). The fraction of non-ionic compounds and betaines was collected by freeze-drying and redissolved in 3 ml of demineralized water (approximately 10 times the volume of the estimated cytoplasmic space). An aliquot of the sample was further diluted with twice the volume of acetonitrile (HPLC grade) and subjected to HPLC analysis, using a column of Nucleosil-5NH2 (Macherey & Nagel, Diiren, FRG) (200 x 4 mm internal diameter), eluted with 65% (by volume) acetonitrile at a rate of 1 ml/min, and a refractive index monitor (LDC/Milton Roy, Rochester, NY, USA, model 1109). To confirm the identity of glycine betaine a column of Nucleosil-5 SA (250 • mm internal diameter) was eluted with 100ram NH~HzPO4 (pH 4.9)+5% methanol (by volume), a method which specifically separates various betaines (Gorham 1984).

~3C-NMR spectra were recorded in pulsed Fourier transform mode on a Varian (Sunnyvale, Calif., USA; Model XL 300) NMR spectrometer operating at 75 MHz and 300 MHz (for the deeoupling channel). Deuterated dioxane was used as internal standard (71 ppm), and 3300 scans were accumulated at an aquisition time of 0.9 s.

The presence of sucrose was further confirmed by thin layer chromatography of cell extracts (prepared by resuspending 0.2 g cell pellet in 2 ml water, breaking the cells by sonication for 10 s, and centrifugation) on silicagel plates, eluted with methanol/ethyl acetate/acetic acid/water (10:60:15:10 by volume). Sugar spots were visualized by spraying the plates with anisaldehyde/ethanot/ sulfuric acid ( I : l 8:I by volume), followed by heating the plates at 100~ This procedure allowed a good separation of sucrose (Rf 0.25) from trehalose (Re 0.20) and glucose (Rf 0.42).

Quantitative determination of intracellular solute concentrations

Extracts of cells in perchloric acid as above were assayed for the presence of betaine by the absorption of its periodide derivative at 365 nm in acidic solution (assaying both betaine and choline) (Speed and Richardson 1968).

Quantitative estimates of the concentrations of other intracellu- lar solutes were made from the peak areas obtained during HPLC elution, using appropriate standards for comparison for the calcu- lation of the response factors, and calculating the concentrations relative to the colorimetrically determined betaine concentration.

Results

Identification of organic osmotic solutes in Ectothiorhodospira marismortui

Mid-exponen t ia l growth phase cells were harvested and a protein-free cell extract was analyzed for the presence of organic osmotic solutes by high per formance l iquid chromatography . As can be seen in Fig. 1, glycine beta ine was found to be the d o m i n a n t compat ib le solute, ac- compan ied by two m i n o r componen ts , one of which was identif ied as sucrose. The results ob ta ined by H P L C analysis were conf i rmed by N M R data (Fig. 2). The pre- d o m i n a n t N M R signals can all be al located to glycine betaine (at 57.7, 70.6, and 173.2 ppm), which confi rms its

352 801 gl ~ %

"~ ~0 c

40 I

2 4 6 8 10mln

Retention flme

F i g . 1. Identification of organic osmotic solutes in the cytoplasm of Ectothiorhodospira marismortui by high performance liquid chromatography. Cells grown in 10% NaC1 with acetate as carbon source were extracted as described in Materials and Methods, and analyzed on a Nucleosil-5NH2 column using 65% acetonitrile as a solvent, c ~ CGA; s = sucrose; gb = glycine betaine

0

i o.~ L-

-~ o~ o L.

~ 0 2

0'8 1.5 ~6 20 24 NoCI concentr 'Gflon in med ium

{M)

Fig. 3. Intracellular salt and solute concentrations in Ecto- thiorhodospira marismortui as a function of NaC1 concentration of the growth medium. Cells grown in the presence of 5, 10 or 15% NaC1, 0.1% KC1, and with succinate as carbon source were col- lected, and the intracellular Na + (A), K + (A), glycine betaine (@), CGA ([]) and sucrose concentrations (�9 were determined in cell pellets; the amount of extracellular water in the pellets was estimated using galactose or fructose as non-penetrating solute

gb

_ _ I_l 200 180 160 1~0 120 100 80 60 /+0 20 PPM 0

Fig. 2. Natural abundance t 3 C NMR spectrum of a deionized pro- tein-free cell extract of Ectothiorhodospira marismortui, with the assignment of the signals to known compatible solutes. * = dioxane (added as standard); gb = glycine betaine; s = sucrose; c = CGA

function as the major organic solute. Another group of resonances, of markedly lower intensity, can be assigned to the disaccharide sucrose (12 signals between 64.6 and 108.1 ppm). Compar ison of N M R signal intensities of cell extract (Fig. 2) and standards revealed that the con- centration of sucrose was at least one order of magnitude lower than that of glycine betaine. The identification of sucrose was confirmed by thin-layer ch romatography (not shown). Ectoine was not detected in any of the samples tested, including cells grown under a variety of growth conditions (see below).

Analysis o f cell extracts by HPLC revealed the pres- ence o f an additional compound (Fig. 1). This compound was purified and characterized, and was identified as Na- carbamoyl glutamineamide (Galinski and Oren 1991). The presence of this novel compound was also reflected in the N M R spectrum of the cell extract (Fig. 2): the few remaining signals (31.9, 35.8, 165.1,181.9 and 182.5 ppm) indicate the presence of CGA. Another signal of this compound at 57.9 p p m (Galinski and Oren 1991) was invisible as it coincides with a signal o f glycine betaine. Two further signals in the N M R spectrum could not be related to known compounds.

Determination of intracellular salt and solute concentrations in Ectothiorhodospira marismortui cells grown at different salt concentrations

Quantitative estimates ofintracellular salt and solute con- centrations depend on a correct estimation of the fraction of the cell pellet volume that is truly intracellular. We used different non-penetrat ing solutes as markers for ex- tracellular space, and the results obtained differed with different compounds tested. Using dextran or polyethyl- eneglycol we measured an "extracellular" volume of 0.43 ml/ml total water space (or 0.27 ml/g wet pellet weight or 0.72 ml/g dry pellet weight); with fructose or galactose these values were 0.74 ml/ml, 0.46 ml/g, and 1.25 ml/g, respectively. I t was shown before ( Imhoff and Riedel 1989; Galinski, unpublished results) in volumet- ric measurements performed with Ectothiorhodospira species, that the designated marker for the extracyto- plasmatic space (sucrose) occupied a significantly larger volume than dextran. Therefore it was concluded that the macromolecule did not penetrate the outer membrane. Our data also suggest that the periplasmic space of Eco- thiorhodospira is rather substantial, possibly due to the numerous membrane intrusions. Any volumetric data on the basis of dextran or polyethylene glycol penetrat ion may lead to an overestimation of the cytoplasmic volume. Our data are comparable to those obtained by Galinski (unpublished) with Ectothiorhodospira haloehloris, in which the space accessible to ~H20 was 0.65 ml/g wet weight (of firm cell pellet), to a4C-sucrose 0.4 ml/g, and to x4C-dextran 0.25 ml/g.

Using the volume inaccesible to fructose or galactose as the "true" intracellular space, we calculated intracellu- lar concentrations of N a +, K + , glycine betaine and su- crose in cells grown in 5, 10 and 15% NaCI (Fig. 3). In all cases an accumulation of K + of about 20- to 30-fold was found, with measured intracellular K + concen- trations of 0 .28 -0 .43 M, independent of the external NaC1 concentration. Calculated intracellular Na + con-

353

'-~ lOO

-6 6o

o ~ 40 LI O

er~ 0 A B C D E F G H I

Fig. 4. Relative intracellular concentraUons of Ne-carbamoyl glut- amineamide (hatched bars), glycine betaine (open bars), and sucrose (black bars) in Ectothiorhodospzra marismortui cells grown under different growth conditions. Unless stated otherwise the NaC1 con- centration in the medium was 10%, acetate was added to the growth medium, and cells were harvested in the exponential growth phase. The growth conditions compared were: standard conditions (A), 5% NaC1 (B), 15% NaC1 (C), cultures supplemented with sodium- L-glutamate or L-glutamine (0.5 g/l) (D and E, respectively), station- ary-phase cells (a 5-day-old culture (F), succinate instead of acetate as carbon source (G), nitrogen-starved cells (NH4C1 omitted from the medium, and a yeast extract concentration reduced to 0.05 g/l) (H), and an autotrophlcally grown culture with sulfide as electron donor (but in the presence of 0.05 g/1 yeast extract) (I)

centrations were between 0.49 and 0.55 M, again without a significant increase with external salinity. A great varia- bility was observed in Na + concentration values between duplicate experiments, as may be expected in the presence of high Na + concentrations in the medium. The deter- mination of intracellular sodium concentrations in halophilic bacteria is problematic, and errors may arise from the following reasons: 1, small deviations in the determination of the cytoplasmic volume cause large errors due to the high external sodium concentration, and 2, significant amounts of ions may be bound to components of the cell envelope structure. Thus, it cannot be ascertained at this stage whether the calculated high Na + concentrations are real or artifacts resulting from the limitations of the experimental procedure.

The intracellular glycine betaine concentration (as de- termined using the colorimetric assay) was found to in- crease with the external salinity: at 5% NaC1 the calcu- lated betaine concentration was 0.47 M, at 10% 1.05 M and at 15% 1.29 M. Sucrose concentrations were found to be low - around 0.05 M, and no significant changes were found in this concentration as a function of external salt concentration. Sucrose, though present, probably plays a minor role in the osmoregulation mechanism enabling this organism to grow in a wide range of salt concentrations (Fig. 3).

The intracellular concentrations of CGA were esti- mated from the HPLC data, giving the relative amounts of the three organic solutes present (Fig. 4 A - C ) ; abso- lute concentrations being calculated using the chemically determined glycine betaine concentrations as a reference (Fig. 3). Concentrations thus calculated were in the order of magnitude of 0.2 - 0.5 M, increasing with external salt concentration.

Summarizing: increased salinity of the medium was accompanied by an increase in intracellular concen- trations of both glycine betaine and CGA, while the con-

centrations of sucrose, Na + and K + were relatively low, and did not increase significantly with the salt concen- tration in the medium.

Comparison of intracellular solute concentrations in Ectothiorhodospira marismortui cells grown under different growth conditions

Cells were grown under a variety of conditions to deter- mine how the concentrations of the three osmotic solutes are regulated; the relative amounts of the solutes present was determined by HPLC (Fig. 4). As stated before, changes in medium salinity did not greatly influence the relative concentrations, nor did the exchange of acetate by succinate as carbon source for photoheterotrophic growth, or the use of autotrophic growth conditions.

The addition of L-glutamate (as a possible precursor for the synthesis of CGA) did not cause a change in the content of the novel compatible solute. However, when L-glutamine was included in the medium, the amount of CGA rose from 30.7% to 37.5% of total osmotic solutes.

In two cases a decreased content of CGA was ob- served: in stationary phase cells (Fig. 4F), and in cells grown in a medium low in nitrogen. However, even under nitrogen starvation a significant concentration of the compound was present, and no nitrogen-free osmotic solutes (such as e.g. trehalose) appeared.

Discussion

Three organic osmotic solutes were identified in the cyto- plasm of E. marismortui: glycine betaine, a novel com- pound, identified as N~-carbamoyl glutamineamide, and sucrose. Ectoine, until now found in all Ectothiorhodospira species tested (Galinski et al. 1985), was never detected in our analyses of E. marismortui. Ectoine has also been found in a halophilic Microcoecus (together with glycine betaine) (Irnhoff 1986), and is ap- parently widespread as an osmotic solute in different bacterial groups (Wohlfarth et al. 1990).

The finding of glycine betaine as the major organic osmotic solute could be expected, as this compound was found to be present in large concentrations in photosynthetic prokaryotes capable of growth at high salinities, including photosynthetic purple bacteria of the genus Ectothiorhodospira (Galinski and Triiper 1982). The presence of glycine betaine as the dominant compat- ible solute in halophilic heterotrophic eubacteria reported previously (Imhoff 1986; Imhoff and Rodriguez-Valera 1984) was shown to be the result of accumulation from the growth medium rather than de novo synthesis (Wohlfarth et al. 1990).

The novel compound CGA was estimated to be pres- ent in concentrations between 0.2-0.5 M, which is lower than the glycine betaine concentrations found, but clearly the new compound contributes significantly (about 30%) to the intracellular concentration of organic osmotic sol- utes. Our estimates of the concentration of this com- pound were based on HPLC data; a chemical assay

354

(based on a reaction with amide bonds) may be feasable (Galinski and Oren 1991), but was not yet included in this study.

The presence of a low intracellular concentration of sucrose seems to be a unique feature in our organism when compared to other Ectothiorhodospira species. Su- crose was found in concentrations much lower than those of glycine betaine and Na-carbamoyl glutamineamide. Among the eubacteria sucrose was hitherto identified in the moderately halophilic purple sulfur bacteria Chromatium purpuratum, grown in 7.5% NaC1 [Galinski (1986) Salzadaptation durch kompatible Solute bei halo- philen phototrophen Bakterien, Ph.D. Thesis, University of Bonn], and in Chromatium salexigens (Galinski, un- published results). The only other group in which the role of sucrose in osmoregulation was previously established is that of the cyanobaeteria: as a reaction to osmotic stress the least halophilic cyanobacteria accumulate oligosaccharides, among which sucrose (Blumwald and Tel-Or 1982; Mackay et al. 1984; Reed et al. 1984; Reed et al. 1986). In some cases sucrose was found in combination with trehalose and/or glucose or fructose, and often sucrose was found as a secondary osmoticum in cyanobacteria that accumulate glucosylglycerol (Reed et al. 1986). In those cyanobacteria that accumulate sucrose its intracellular concentration ranges between 200 and 275 mM; in the marine Anabaena strain CA an increase in NaC1 concentration from 50 mM to 500 mM caused an increase in sucrose concentration from 20 to 120 mmol/kg (Reed et al. 1986).

Though an exact determination of intracellular salt concentrations is difficult, both as a result of the presence of high external concentrations, and by uncertainties in the calculation of the true intracellular water space, it can be assumed that salts (sodium, potassium) are present in E. marismortui in relatively low concentrations, compar- able to those reported from other moderately halophilic photosynthetic bacteria (estimated concentrations were around 0.5 M Na +, and 0.3 M K + in cells grown in 1.81 M NaC1, and the values did not increase greatly with extracellular salt concentration) (Fig. 3). Measured K + values were similar to the values of0.30-0.39 M internal potassium reported in Rhodospirillum salinarium, grown in 0.5 and 3 M NaC1, respectively-. In last-named strain an internal sodium concentration of 0.41 + 0.35 M was reported in cells grown in 3 M NaC1 (Nissen and Dundas 1984), and also here it was claimed that the high external sodium concentration precluded the accurate determi- nation of its internal concentration. In Ectothiorhodospira mobilis chloride was found to be excluded from the cyto- plasm, and decreasing ratios of cytoplasmic to external chloride concentration were reported with increasing ex- ternal chloride concentration, and an intracellular C1- concentration of 71 mM was measured in cells grown in 0.54 M extracellular C1- (Imhoff and Riedel 1989). Chloride is also excluded from Ectothiorhodospira halochloris (Galinski, unpublished results).

As stated before, all calculations of internal salt and solute concentrations were based on the estimated cyto- plasmic volumes, as measured using galactose or fructose as non-penetrating solutes, accessible to the periplasmic

space. In the case of Ectothiorhodospira a considerable part of the total cell volume is occupied by the periplasmic space - in E. mobilis about 40 -50% of the total cell water space (Imhoff and Riedel 1989), as compared to approximately 38% for Vibrio costieola (Shindler et al. 1977). As electron micrographs of E. marismortui show (Oren 1989; Oren et al. 1989), a significant portion of the cell is taken up by intracellular stacks of thylakoids, resembling those of other Ectothiorhodospira species. In Ectothiorhodospira it was shown that these thylakoids are open to the outside, and their interior is thus not continuous with the cytoplasm (Remsen et al. 1968; Wanner et al. 1986). As discussed by Galinski and Triiper (1982) this peculiar architecture of the photosynthetic apparatus may have a profound influence on the aqueous phase structure of the cell. As a relatively small amount of cytoplasm is in contact with the large polar surface of the thylakoid membranes, only part of the cytoplasmic water will be in a free state, and thus in equilibrium with the environment. Calculations of solute concentrations on the basis of total cytoplasmic water might not, there- fore, necessarily reflect the actual proportions.

When estimating the osmotic pressure exerted by the sum of the intracellular betaine, CGA, sucrose, Na +, and K + concentrations, it becomes clear that the sum of the concentrations may be sufficiently high to balance the extracellular NaC1 concentration at all salt concen- trations tested (based on the assumption that the mea- sured Na + and K + concentrations are balanced by equivalent concentrations of anions). An increase in salt concentration in the medium is mainly balanced by an increase in the concentrations of both glycine betaine and CGA.

Variations in the growth conditions of E. marismortui did not greatly affect the relative amounts of glycine betaine, CGA and sucrose present (Fig. 4), with the pos- sible exception of the addition of glutamine (see below). The relative concentrations were not significantly differ- ent in cells grown at different salt concentrations, and in autotrophically versus photoheterotrophically grown cells with different carbon sources. A slight decrease was found in stationary cultures (signifying that minor differ- ences in the relative amounts of the osmotic solutes may be due to differences in the state of the cultures during harvest). In nitrogen-starved cultures the concentration of CGA was only slightly decreased. As the compound contains 4 tool of nitrogen per mol, a decrease in its synthesis could be expected when the nitrogen supply is restricted. Thus, in Ectothiorhodospira halochloris ectoine is replaced by the disaccharide trehalose under nitrogen limitation, while the synthesis of betaine continues (Galinski and Herzog 1990).

The biochemical pathway leading to the formation of the novel compound CGA is unknown. Our finding that its intracellular concentration was enhanced when L-glu- tamine (but not L-glutamate) was added to the medium suggests its use as a precursor for its synthesis. Synthesis of CGA from glutamine requires amidation of the ~- carboxyl group, and the addition of the carbamoyl group (possibly derived from carbamoylphosphate) to the ~- amino group. The sequence of these reactions is presently

355

unde r s tudy, as is the fate o f the c o m p o u n d when the externa l sal t concen t r a t i on is lowered.

The isolate used in this w o r k was descr ibed as an Ectothiorhodospira species on accoun t o f the use o f sulfide as e lec t ron d o n o r wi th excre t ion o f e lementa l sulfur, its 16S r i b o s o m a l R N A o l igonuc leo t ide ca ta log , its ha lophi l i c na tu re , a n d the u l t r a s t ruc tu re o f the pho- tosyn the t i c m e m b r a n e s (Oren et al. 1989). However , the s t ra in differs f rom the h i ther to descr ibed species o f the genus Ectothiorhodospira in a t least three p rope r t i e s : the l ipid c o m p o s i t i o n (no t ab ly the absence o f card io l ip in) , its neu t roph i l i c r a the r t han a lka l iphi l ic na ture , and , as d o c u m e n t e d in the p resen t work , the absence o f ectoine as osmot ic solute, a n d the presence o f osmot ic solutes no t f o u n d in the o the r represen ta t ives o f the genus. There- fore a t a x o n o m i c revis ion o f the genus m a y be in place, in which E. marismortui m a y deserve c lass i f ica t ion in a new genus, poss ib ly toge the r wi th the s trains i so la ted by Ven tu ra et al. (1988).

Acknowledgements. We thank Brigit Amendt for valuable technical assistance, and Hans G. Trfiper for stimulating discussions. This work was supported by a grant from the G. I. F., the German-Israeli Foundation for Scientific Research and Development.

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