528 PHYSIOLOGY AND METABOLISM [56]

[56] Osmot i c A d j u s t m e n t : Organ ic Solu tes

By ROBERT H. REED

Introduction

Cyanobacteria survive and grow in environments with dissimilar salin- ity regimes, ranging from freshwater through brackish and marine condi- tions to hypersaline habitats approaching salt saturation. Several recent studies have shown that long-term osmotic balance in salt-stressed cyano- bacteria may be achieved by the synthesis of specific low-molecular- weight organic solutes. This was first demonstrated for the heteroside 2-O-t~-o-glucopyranosylglycerol (glucosylglycerol, lilioside) in the marine unicell Synechococcus RRIMP N I00) Subsequent research has identi- fied other organic osmolytes in salt-stressed cyanobacteria: these include the disaccharides sucrose and trehalose and the quaternary ammonium compounds glycine betaine (trimethylglycine) and glutamate betaine (tri- methylglutamate). 2 These metabolites share the common features of (1) a high solubility in water and (2) a lack of toxicity when assayed in vitro at physiologically relevant concentrations against a range of enzymes. In general, a single metabolite is accumulated, although certain strains may synthesize more than one organic solute, particularly in response to in- creased temperature) This chapter outlines some of the experimental procedures used to identify and quantify these solutes.

Natural Abundance laC-Nuclear Magnetic Resonance Spectroscopy

13C-NMR spectroscopy relies on the "resonance" of individual m3C nuclei in a magnetic field between (1) a low-energy state (magnetic mo- ment parallel to the applied field) and (2) a high-energy state (magnetic moment antiparallel to the applied field). Each 13C nucleus gives a charac- teristic resonance peak, revealing the functional group to which the reso- nance belongs and enabling individual organic molecules to be identified by their 13C "fingerprint." Natural abundance 13C-NMR spectra of salt- stressed cyanobacteria are dominated by resonances from the low-molec-

t L. J. Borowitzka, S. Demmerle, M. A. Mackay, and R. S. Norton, Science 210, 650 (1980).

2 R. H. Reed, L. J. Borowitzka, M. A. Mackay, J. A. Chudek, R. Foster, S. R. C. Warr, D. J. Moore, and W. D. P. Stewart, FEMS Microbiol. Rev. 39, 57 (1986).

3 S. R. C. Warr, R. H. Reed, and W. D. P. Stewart, New Phytol. 1 ~ , 285 (1985).

Copyright © 1988 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 167 All fights of reproduction in any form reserved.

[56] OSMOTIC ADJUSTMENT: ORGANIC SOLUTES 529

ular-weight organic osmolytes which are responsible, in part at least, for the generation of intracellular osmotic pressure. The technique has been used in recent years to screen a wide range of cyanobacteria for organic solutes, demonstrating that halotolerance may be linked to the class of accumulated organic solute (disaccharide, heteroside, or betaine). 2

Natural abundance 13C-NMR spectroscopy is noninvasive and non- destructive: analysis of intact cells can be used to determine unequivo- cally all of the principal low-molecular-weight organic compounds which are in solution intracellularly in osmotically significant amounts. Addi- tionally, a comparison of the 13C-NMR spectra of a sample containing intact cells and of an aqueous extract from a similar biomass will show whether all of the osmolyte is freely mobile within the cells.l Additional information on the physical state of the cytoplasm of cyanobacteria, in- cluding the rotational motion and mobility of organic osmotica, can be obtained from 13C-NMR spin-lattice relaxation time measurements of intact cells, as shown for Synechococcus RRIMP N 100. 4

The natural abundance level of 13C is approximately 1.1%, the remain- ing 12C fraction being NMR silent. Additionally, 13C is less sensitive to NMR analysis than other nuclei (e.g., 1H, 31p). These factors impose a requirement for (1) a high magnetic field strength, (2) long accumulation times, and/or (3) large amounts of biological material. Resolution can be improved in most cases by using homogeneous extracts, rather than intact cells, enabling samples containing more than I0 mg organic osmolytes to be analyzed in less than 12 hr on most commercially available lower-field spectrometers. Figure 1 shows a typical spectrum of an extract from salt- stressed Synechocystis PCC 6714, using 10 liters of exponentially growing cells centrifuged and extracted in 80% (v/v) ethanol:water (24 hr), dried down, and redissolved in 1 ml D20 (obtained with a BrOker WP60 FT spectrometer operating at 15.08 MHz)) The spectrum contains 8 reso- nances, corresponding to those of authentic glucosylglycerol, ~ and con- firms the osmotic significance of this metabolite in this strain. Higher-field NMR spectrometers would allow considerable savings in assay time for such samples, together with a further potential increase in sensitivity.

Recent studies have shown that ~3C-NMR spectroscopy can be used to investigate the biosynthesis and turnover of organic osmolytes in cyano- bacteria, using 13C-enriched bicarbonate as a source of inorganic carbon for photosynthesis. This enables the changes in cell osmolytes to be moni- tored in oivo, without recourse to the time-consuming extraction and

4 R. S. Norton, M. A. Mackay, and L. J. Borowitzka, Biochem. J. 202, 699 (1982). 5 S. R. C. Warr, R. H. Reed, J. A. Chudek, R. Foster, and W. D. P. Stewart, Planta 163,

424 (1985).

530 PHYSIOLOGY AND METABOLISM [56]

I I I I 100 8 0 60 40

chemical shift (ppm)

FIG. 1. Natural abundance 13C-NMR spectrum of an extract of the glucosylglycerol- accumulating euryhaline unicell Synechocystis PCC 6714.

separation procedures required for radiocarbon tracer experiments. Fur- ther details are contained in Refs. 6 and 7.

Low-Molecular-Weight Carbohydrates

The ~3C-NMR procedures described above can be used to quantify individual carbohydrates in cyanobacterial extracts. However, limitations on sample size and spectrometer access time may prevent analysis of large numbers of samples by this method. An alternative procedure for routine analytical work involves the separation of trimethylsilyl (TMS) ethers of carbohydrates by gas-liquid chromatography (GLC).

Extraction of cyanobacterial samples (each containing carbohydrate at 0.05-2.00 rag) is carried out by incubation in boiling 80% (v/v) ethanol : water (containing 0.2-1.0 mg arabitol as an internal standard) for 5 min, followed by reextraction of the residual material in 80% (v/v) ethanol : water at 25 ° for 18 hr. This procedure extracts over 99% of the

6 M. A. Mackay and R. S. Norton, J. Gen. Microbiol. 133, 1535 (1987). 7 E. Tel-Or, S. Spath, L. Packer, and R. J. Mehlhorn, Plant Physiol. 82, 646 (1986).

[56] OSMOTIC ADJUSTMENT: ORGANIC SOLUTES 531

low-molecular-weight carbohydrates from cyanobacterial cells. The etha- nolic extracts are then pooled, evaporated to dryness at 40 °, and stored in a vacuum desiccator for 24-48 hr. Samples are then dissolved in 0.2-1.0 ml of a suitable organic solvent, e.g., pyridine 8 or dimethyl sulfoxide. 9 TMS ethers are produced by the sequential addition of 2 volumes of hexamethyldisilazane and 1 volume of trimethylchlorosilane (0.1 ml and 0.05 ml, respectively). Samples are shaken for at least 90 sec and left for over 12 hr at room temperature to ensure that all free hydroxyl groups are converted to TMS ethers. Since all solvents containing free hydroxyl groups will react with the reagents used to produce the TMS derivatives, it is essential to exclude both ethanol and water from the later stages of sample preparation.

While pyridine has been widely used as a reaction solvent for the analysis of low-molecular-weight carbohydrates from plant tissues, 8 di- methyl sulfoxide has several advantages since (I) low-molecular-weight carbohydrates dissolve more rapidly in dimethyl sulfoxide prior to deriva- tization and (2) an upper phase of hexamethyldisiloxane is formed after reaction. TMS-carbohydrates show a high partition coefficient for this phase which provides (3) a useful additional selective procedure for the removal of interfering substances and (4) a method of increasing the con- centration of TMS derivatives in a smaller reaction volume. Furthermore, the hexamethyldisiloxane phase gives a rapidly eluted and reduced sol- vent peak compared with pyridine. This removes any requirement to dry down samples and redissolve in a more suitable solvent prior to analysis.

Samples can be analyzed by conventional GLC techniques, with flame-ionization detection, using a 2 m × 6 mm (i.d.) column containing 2% methyl phenyl silicone gum (SE 52, Pye Unicam, Cambridge, UK) with diatomite as solid support, or by capillary GLC using a 10 m × 0.53 mm silica heliflex column coated at 0.25/~m with RSL 150 polydimethylsi- loxane (Alltech Assoc., Carnforth, UK). A temperature program is re- quired to achieve optimum separation: with a temperature change from 140 to 280 ° at 20 ° min -1, holding the initial and final temperatures for 2 min and with suitable carrier gas flow rates (30 and 3 ml min -1, respectively), analysis is complete in under 10 min. Representative retention times and response factors (relative to the internal standard, arabitol) are shown in Table I, as obtained using a Varian 3700 GLC (Varian Instruments, Wal- nut Creek, CA). We have used this procedure to screen a wide range of cyanobacteria from freshwater, brackish, and marine habitats, 1° showing

8 p. M. Holligan and E. A. Drew, New Phytol. 70, 271 (1971). 9 D. J. Moore, R. H. Reed, and W. D. P. Stewart, J. Gen. Microbiol. 131, 1267 (1985). 10 R. H. Reed, D. L. Richardson, S. R. C. Warr, and W. D. P. Stewart, J. Gen. Microbiol.

130, 1 (1984).

532 PHYSIOLOGY AND METABOLISM [56]

TABLE I GLC CHARACTERISTICS OF CARBOHYDRATES FROM

CYANOBACTERIA

Stationary Carbohydrate phase and

characteristic Glucosylglycerol Sucrose Trehalose

SE 52 Retention time 1.47 1.77 1.84 Response factor 0.75 0.60 0.60

RSL 150 Retention time 2.18 2.88 3.06 Response factor 0.77 0.64 0.64

that the least halotolerant forms accumulate disaccharides in response to salt stress while the heteroside glucosylglycerol is accumulated by the more halotolerant strains, irrespective of their source of isolation. 11

Direct extraction of freeze-dried samples in dimethyl sulfoxide (at 100 ° for 18 hr) has been proposed as a means of reducing assay time and sample preparation procedure.lZ We have used this experimental proce- dure to investigate the synthesis of sucrose as a secondary osmolyte in unicellular glucosylglycerol-accumulating cyanobacteria grown at high temperature in hyposaline media, 3 showing that this method is a viable alternative to ethanolic extraction.

Quaternary Ammonium Compounds

A range of analytical procedures can be used to identify and quantify quaternary ammonium compounds. However, many of the methods are time-consuming, nonspecific, and/or insensitive, relying on spectrophoto- metric analysis of periodide/reineckate salts. Alternative procedures, in- cluding thin-layer chromatography, 13 pyrolysis-gas chromatography, TM

and high-performance liquid chromatography, 15 often require several ad- ditional ion-exchange purification steps per sample prior to analysis, in- creasing costs and assay times. We have developed a procedure using

1| D. L. Richardson, R. H. Reed, and W. D. P. Stewart, FEMS Microbiol. Lett. 18, 99 (1983).

~2 R. H. Reed and I. R. Davison, Br. Phycol. J. 19, 381 (1984). |3 G. Blunden, S. M. Gordon, W. F. H. McLean, and M. D. Guiry, Bot. Mar. 25, 563 (1982). |4 W. D. Hitz and A. D. Hanson, Phytochemistry 19, 2371 (1980). |s R. D. Guy, P. G. Warne, and D. M. Reid, Physiol. Plant. 61, 195 (1984).

[56] OSMOTIC ADJUSTMENT: ORGANIC SOLUTES 533

I I I 4 .0 3 .0 2 .0

chemical shift (ppm)

FIG. 2.1H-NMR spectrum of an extract of Synechococcus PCC 7418 with added sodium acetate (resonance 1.91 ppm) as an internal standard.

high-resolution continuous wave ~H-NMR spectroscopy to identify and quantify methylated osmolytes in algae and cyanobacteria. 16 Owing to the chemical shift equivalence of the protons in all three of the methyl groups, the dominant feature of 1H-NMR spectra of betaines is a singlet resonance of relative intensity 9. This singlet provides a sensitive and distinctive probe for quantitative purposes, by comparing the peak size with that of a known amount of an internal standard with dissimilar =H absorption peaks.

Cyanobacterial samples (containing 0.5-5.0 mg betaine) are extracted in 80% (v/v) ethanol:water, as described above, with 2-10 mg sodium acetate as internal standard. Samples are then dried, redissolved in 0.5 ml D20 and analyzed byIH-NMR spectroscopy without further purification. If glycine betaine is present, the major 9-proton resonance will be ob- served at a chemical shift (Sij) of 3.27, with an additional (CH2) resonance at 3.88, as shown in Fig. 2 for an extract of the halotolerant unicell Synechococcus PCC 7418 (Aphanothece halophytica), obtained using a Briiker HX90 continuous wave spectrometer operating at 90 MHz. Simi- lar spectra have been obtained for other halotolerant cyanobacteria, in- cluding Dactylococcopsis salina, Synechocystis DUN 52,17 and Oscillato-

t6 j. A. Chudek, R. Foster, D. J. Moore, and R. H. Reed, Br. Phycol. J. 22, 169 (1987). 17 R. H. Reed, J. A. Chudek, R. Foster, and W. D. P. Stewart, Arch. Microbiol. 138, 333

(1984).

534 PHYSIOLOGY AND METABOLISM [57]

ria limnetica, ~8 showing glycine betaine to be the major organic solute in these strains. Glutamate betaine, which has been reported for two isolates of Calothrix, ~9 may be quantified using the same procedure. Fourier trans- form ~H-NMR spectroscopy offers a further increase in sensitivity: sam- ples containing 0.01-0.50 mg glycine betaine can be analyzed in approxi- mately 60 min. ~6 However, this degree of sensitivity should not be required for those strains which contain glycine betaine or glutamate betaine in osmotically significant amounts.

Acknowledgments

Research supported by the Royal Society, Natural Environment Research Council UK, and Science and Engineering Research Council UK. RHR is a Royal Society Research Fellow.

~s R. H. Reed, A. Oren, and J. A. Chudek, unpublished observations (1986). 19 M. A. Mackay, R. S. Norton, and L. J. Borowitzka, J. Gen. MicrobioL 130, 2177 (1984).

[57] I n o r g a n i c C a r b o n U p t a k e b y C y a n o b a c t e r i a

By AARON KAPLAN, YEHOUDA MARCUS, and LEONORA REINHOLD

The ability to concentrate inorganic carbon (C~) within their cells en- ables cyanobacteria to compensate for the discrepancy between the Km(CO2) of ribulose-bisphosphate carboxylase (EC 4 : 1 : 1 : 39, Rubisco) (200/~M) and the concentration of dissolved COz in equilibrium with air (10/xM). This ability to accumulate Ci internally is light dependent ~ and develops as a function of the concentration of CO2 experienced by the cells during growth. 2 The rate of Ci uptake as well as the extent of accu- mulation are far greater in cells adapted to the air level of CO2 then in cells grown at elevated CO2 concentration, with the result that the apparent photosynthetic affinity for COz Show_la by the former cells is considerably higher. 2 The extent of Ci accumulation is far greater than would be pre- dicted on the basis of passive penetration of C~ species along their electro- chemical potential gradient with subsequent interspecies equilibration ac- cording to intracellular pH.

Two major questions have been addressed in the study of the CO2 concentrating mechanism: the nature of the C~ concentrating system and

I A. Kaplan, D. Zenvirth, Y. Marcus, T. Omata, and T. Ogawa, Plant Physiol. 84, 210 (1987).

2 A. Kaplan, M. R. Badger, and J. A. Berry, Planta 1497 219 (1980).

Copyright © 1988 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 167 All rights of reproduction in any form reserved.