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Membrane lipid composition, fluidity, and surface charge changes in response to growth of the fresh water cyanobacterium Synechococcus 6311 under high salinity
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OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 277, No. 2, March, pp. 263-267,199O
Membrane Lipid Composition, Fluidity, and Surface Charge Changes in Response to Growth of the Fresh Water Cyanobacterium Synechococcus 6311 under High Salinity’ Gennady
Khomutov,’
Ian V. Fry,3 Margaret
E. Huflejt,
and Lester
Packer
Membrane Bioenergetics Group, Applied Science Division, Lawrence Berkeley Laboratory, Molecular and Cell Biology, University of California, Berkeley, California 94720
and Department
of
Received July 19,1989, and in revised form October 23,1989
The effect of adaptation to saline growth of a fresh water cyanobacterium Synechococcus 63 11 on components of the cytoplasmic membranes and thylakoids was investigated. Significant changes in membrane surface charge, lipid, fatty acid, and carotenoid composition were observed upon transfer of the cells from a low salt (0.015 M NaCl) to a high salt (0.50 M NaCl) growth medium. Very similar changes in the polar lipid classes and fatty acid composition were observed in both membranes, but changes in fluidity and surface charge and a significant shift in the protein to lipid ratio were only apparent in the cytoplasmic membranes. The fluidity and surface charge data correlate well with functional studies and we can attribute the cytoplasmic membrane as the major site of interaction and adaptation to the saline environment. 8 igso Academic press. IN.
Synechococcus 6311, a freshwater unicellular cyanobacterium, exhibits the ability to adapt to and grow at high salinity (1). The adaptive process is characterized by elevated respiration (2) and glycogen content (2, 3), de nouo synthesis of cytochrome oxidase (2) located in the cytoplasmic membrane (4,5), changes in membrane particle size and distribution (3), and an increase in the Na+/H+ antiporter activity (6). Recent advances in sepi This research was supported by the Office of Basic Energy Sciences of the U.S. Department of Energy under contracts DE-AC0376SF0009S and DE-FG03-87ER13736, the NASA-CELSS Interagency agreement A-1456~ and an IREX fellowship under the United States/Soviet exchange program, Princeton, New Jersey. ’ On leave from Biophysical Department, Physical Faculty Moscow State University, Leninskie Gory Moscow, 119899 GSP MCU USSR. 3 To whom correspondence should be addressed. 0003.9861/90
Copyright All
rights
$3.00 0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
aration techniques (5,7) have allowed us to separate and purify cytoplasmic membranes from the thylakoids of cells grown at high (0.50 M) and low (15 mM) NaCl. To determine if the physical nature of the membranes contributes to the observed functional changes, biophysical and chemical analyses of surface charge, fluidity, phospholipid, fatty acid, and carotenoid composition of the cytoplasmic membrane and thylakoids were undertaken. EXPERIMENTAL Organism and culture conditions. Axenic cultures of SynechococCLLS6311 cells were grown photoautotrophically in Kratz and Myers medium C (8) at 32°C. Purity of the cellular suspensions was confirmed by plating of the culture samples on sterile nutrient broth-enriched Petri agar plates. During each experiment, the cell growth was monitored by measuring the protein and chlorophyll content and the optical density of the culture. The cells were harvested by centrifugation and washed twice in double-distilled HzO. The wet weight was obtained after washed cells were centrifuged in Sorval SS-34 rotor at 13,000 rpm for 15 min. Preparation of subcellular fractions. Cyanobacterial membranes were isolated using a flotation sucrose gradient method as described (7). The purity of cytoplasmic membrane preparations was confirmed spectrophotometrically by scanning of membrane solutions in methanol to determine contamination by chlorophyll. For analyses, only the lightest, top fraction was used. Purity of the thylakoid fraction was monitored using the following two criteria: the thylakoid membrane band appears on sucrose gradient as an extremely uniform fraction, and the two membranes have distinctively different monogalactosyl diacylglycerol to digalactosyl diacylglycerol ratios. From this we assume that the contamination of the thylakoid membrane fraction by cytoplasmic membranes is insignificant. Isolation and characterization of lipids. For fatty acids and lipid analysis, 1-2 ml of washed and concentrated membranes (2-3 mg protein/ml for cytoplasmic membrane suspension or 6-7 mg/ml for thylakoid membranes) in buffer were dried under N2, then washed with acetone for quantitative isolation of galactolipids (9). Lipids were isolated according to Van Walraven et al. (9). Dry lipids were stored under N2 at -14°C. For separation of lipid classes, the dry lipid fraction 263
264
KHOMUTOV
0
20
40
60
Mn (bound)
80
100
120
x 10m5 M
FIG. 1. Langmuir adsorption isotherm for purified thylakoids from Syrzechococcus 6311.0, From salt grown cells; 0, from control cells.
was quantitatively dissolved in dichloromethane and separated by TLC4 (silica gel 60, Merck) with the development solvent acetone/ benzene/H,0 (91:30:8, vol/vol). TLC plates were sprayed with 0.01% primuline in acetone/H,0 (4~1) and lipids were detected under long wavelength uv light. The lipid classes were identified qualitatively by comparison of their R, with Rf of lipid standards (Serdary Research, Ontario, Canada). For fatty acid analysis, silica gel containing individual lipid was recovered from plates and transmethylation was performed with boron trifluoride (14% in methanol, Alltech reagent) in llO”C/90 min, as described (lo), without prior extraction of lipids from silica gel. Fatty acid methyl esters were removed from reaction mixture by multiple washing with hexane, with the last washing in hexane/water 1:l and analyzed with a Varian Gas Chromatograph Model 3700, equipped with Supelco column No. 2330, a flame ionization detector and a CDS IIIC Varian Data System Integrator, using the following temperature program: 17O”C, 5 min; 170-23O”C, 4”C/ min; 23O”C, 5 min, with nitrogen as carrier gas. Mn’+ binding. Due to the effect of surface electrical potential, the cation concentration at the surface can be very different from the bulk concentration which was used for the determination of the apparent binding constant (11). To eliminate the effect of surface electrical potential on the binding of Mn2+ to thylakoid and cytoplasmic membranes all experiments were performed in the presence of 300 mM KCl. In buffer containing 0.3 M KC1 the dependence of the manganese (II) hexahydrate ion EPR-signal amplitude was linear with concentration, and this dependence was used as a calibration curve for calculating the free manganese in the membrane samples. Amplitude of the EPR signal due to free Mn*+ was measured at the high field or low field feature to negate any overlap of the EPR signal arising from the chlo-
ET AL. rophyll radical in the middle of the spectrum (g = 2 region). EPR spectra were recorded at room temperature on a Varian E-109 spectrometer at 1 mW power, 8 gauss modulation amplitude, with a scan rate of 250 G per minute. The concentration of MnCl, was 0.3 mM, and the volume of additions to the membrane suspensions did not exceed 2% (v/v). To record EPR spectra, 50 ~1 samples were placed in 75.~1 glass capillaries (sealed at one end) then placed in a standard 3.mm diameter quartz EPR tube in the resonance cavity. Error due to sample placement was determined for a 0.3 mM MnCl, solution which was removed and replaced into the resonance cavity 10 times. The arithmetical mean and the standard deviation were 4.4 and 0.06 (arbitrary units), respectively, representing an error of less than 2%. Thylakoid membrane concentration (control and salt grown) in all experiments was 1.91 mg chlorophyll/ml, corresponding to 10.5 and 15.1 mg protein/ml for control and salt grown thylakoid membranes, respectively. Cytoplasmic membrane concentration was between 12.5 and 14.2 mg protein/ml. The number of Mn2+ binding sites and the K' and K" binding constants were calculated from the Langmuir adsorption isotherm as described ( 12). Order parameter (S) and correlation times (7~) of the spin labels were determined as described (13,14). CATI (N,N,dimethyl-N-hexadecyle 2,2,6,6-tetramethyl-4-hydroxypiperidine-l-oxyl) was a gift from Dr. Rolf Mehlhorn, 5-doxyl stearic acid (5.DSA) and 16.doxyl stearic acid methyl ester (16-DME) were obtained from Sigma. Stock solutions of CATI were aqueous, while 5-DSA and 16-DME were in ethanol. The spin labels were diluted at least 1:lOO into the samples, giving a final ethanol concentration of less than l%, and a spin label concentration between 0.1 and 0.5 mM. Chlorophyll and protein were determined by the methods of Mackinney (15) and Lowry et al. (16), respectively.
RESULTS
Mn2+ Binding The cytoplasmic membrane and thylakoid preparations from salt and control cells exhibited Mn2+ binding similar to that observed in thylakoids from higher plants. Two distinct affinity sites were observed in both salt and control thylakoids (Fig. 1). The number of high affinity binding sites was higher than in control thylakoids, and the magnitude of the binding constant (PC’) was lower than in control samples (Fig. 1, Table I). However, the number of low affinity sites and the binding constants (K*) were very similar (Fig. 1, Table I) in both salt and control thylakoid preparations. The cytoplasmic membrane exhibited interactions with Mn*+ ions similar to the thylakoid preparations
TABLE
Manganese Binding Constants and Number of Binding Sites in Thylakoids from Control and Salt Grown
Synechococcw6311 High affinity
K' 4 Abbreviations used: TLC, thin layer chromatography; CATIG, N,N,dimethyl-N-hexadecyle 2,2,6,6-tetramethyl-4.hydroxypiperidine-1-oxyl; 5-DSA, 5-Doxyl stearic acid; 16.DME, 16-Doxyl stearic acid methyl ester.
I
Control Salt
(M-l)
5.7 x lo* 2.4 X lo4
site
Mn bound per chlorophyll 0.066
0.156
Low affinity
@ CM-‘) 2.4 X lo3 3.2 X lo3
site
Mn bound per chlorophyll 0.52-0.53 0.52-0.53
RESPONSE OF CYANOBACTERIAL
MEMBRANES
TABLE III
TABLE II Manganese Binding to Cytoplasmic Membranes and Thylakoids from Control and Salt Grown Cells
Phase Transition Temperatures of Isolated Cytoplasmic Membranes and Thylakoids from Control and Salt Grown Synechococcus 6311
Thylakoids”
Cytoplasmic membranes
Cytoplasmic membranes
Mn2+ binding, mol/mg protein 13.33 x 1o-3 21.87 X 1O-9
6.35-19.05 x 10m9 1.27 X 10m7
Control Salt
’ Thylakoid high affinity binding site.
Spin probe
Parameter measured”
16.DME 5-DSA
vz and S
CATIS
(Table II), but resolution of a K1 and K2 constant could not be made. However, relative levels of Mn2+ binding between salt and control cytoplasmic membrane preparations did show that growth under salt exposure increased the number of Mn2+ binding sites by 1 to 2 orders of magnitude (Table II). Correlation
Time and Order Parameter Analysis
Correlation times (TC) and the order parameter (S) of spin labels 16-DME, CATI,, and 5-DSA were calculated from their EPR spectra as described under Experimental. Thylakoids from salt and control cells demonstrated, using S values for the 5-DSA spin probe, breaks in the Arrhenius plot characteristic of lipid phase transitions at 27°C (Fig. 2, Table III). Arrhenius plots of 0.9
1
I
,
3
I
S
Control
Salt
Control
Salt
30 23 23
26 19
29 27 27
29 27 27
19
a Correlation time (x) and/or order parameter (S).
changes in the order parameters or w of CATI and 16DME in salt and control thylakoids showed very similar phase transition temperatures, between 27 and 30°C (Fig. 2, Table III). However, the order parameter (S) of 5-DSA in cytoplasmic membranes from control and salt grown cells exhibited a decrease in the phase transition from 23 to 19”C, respectively (Fig. 2). Analysis of Arrhenius plots for 16-DME, 5-DSA, and CATI, (using both TC and S) demonstrate consistent results for the phase transition(s) in cytoplasmic membrane and thylakoids, although 16-DME gave slightly higher results (Table III).
I
Fatty acid analysis of cytoplasmic membranes and thylakoids from control and salt grown cells showed that the protein/fatty acid ratio in thylakoid membrane was fairly constant (a 10% change), but this ratio increased by 40 to 50% in the cytoplasmic membrane from salt grown cells (Table IV). Analysis of lipid constituents showed an increase in the 18:l over the 16:l acyl chains in phospholipids and that the charged phospholipid content of the membranes also increased (Table V). There was no preferred localization of either the longer side chain containing or charged phospholipids to the cytoplasmic membrane or thylakoids. Each type of phospholipid was evenly distributed through both membrane systems (data not shown).
iz
b 0E
7C
Thylakoids
Lipid Analysis iI,
0.8
ii 5 E F x
265
TO SALT STRESS
Salt
0.7
0.6
TABLE IV Cytoplasmic Membrane
3.1
3.3
Protein to Fatty Acid Ratio (wt/wt) in Cytoplasmic Membranes and Thylakoids from Control and Salt Grown Synechococcus 6311 3.5
3.7
+ x 10-3 FIG. 2. Arrhenius plot for cytoplasmic membranes and thylakoids using the order parameter calculated for the spin probe 5-DSA.
Growth conditions
Cytoplasmic membranes
Control (0.015 M NaCl) Salt (0.50 M NaCl)
1.17 1.63
Thylakoids 5.65
5.09
266
KHOMUTOV TABLE V
Lipid Classes in Cytoplasmic Membranes Isolated from Salt and Control Grown Synechococcus 63 11 Fatty acid
Control (0.015 M NaCl) Salt (0.50 M NaCl)
Phospholipid”
16:l (% total)
(W total)
MGDG (% total)
DGDG (% total)
17.6 12.9
16.8 24.3
51.61 34.8
14.3 20.4
18:l
a MGDG, monogalactosyl diacylglycerol; DGDG, digalactosyl diacylglycerol. Upon transfer of the cells from low salt (0.015 M NaCl) to high salt (0.5 M NaCl) growth medium a rapid decrease in palmitoleic acid (C16:lA9) was accompanied by a concomitant increase in the percentage of the two oleic acid isomers (C18:lA9, C18:lAll) with the increase in the percentage of the C18:lA9 isomer being the most significant. The changes occurred within the first hour after the increase in salt concentration and progressed for up to 150 h depending on the state of growth during which the salt shock was applied. Subsequently the new ratios of fatty acids stabilized, with a decrease of about 15% of palmitoleic acid and a concomitant 13315% increase in octadecanoic acids, compared to controls. During 14 days of growth in the high salt media the total content of anionic lipids (phosphatidylglycerol and sulfoquinovosyl diacylglycerol) increased from 23.5 to 31.5% and the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol decreased from 3.5 to 1.6. The same direction of changes, but to a lesser extent was observed in aging, control (0.015 M NaCl-grown) cultures. These rearrangements in lipid environment occurred in both the thylakoid and the cytoplasmic membranes and were only partially reversible. After cells were transferred back to a low salt medium, the proportions between major fatty acids did not return to control values, even after 24 h of growth (data not shown).
Carotenoids The carotenoid fractions of cytoplasmic membranes from both control and salt grown cells exhibited absorption spectra characteristic of p-carotene (17) and control levels of 9.4 mmol/mg protein decreased to 3.5 mmol/mg protein over six days of exposure to high salinity. The carotenoids in the thylakoid membranes could not be quantified due to rapid degradation. The absorption spectrum line shape of the carotenoids in the thylakoids changed with time and was similar to that reported in (7). DISCUSSION
The effect of salinity on the membrane systems of Sy6311 is manifest at several levels, and is
nechococcus
ET AL.
most apparent in the cytoplasmic membranes rather than in the thylakoids. Although the lipid classes and fatty acids present in the cytoplasmic membranes and thylakoids both change to the same degree upon exposure to salt, the gross physical nature of the membranes, the fluidity and surface charge density, are constant in the thylakoids and only change in the cytoplasmic membrane. Protein to lipid ratios by chemical analysis show that there is a slight decrease in thylakoid membrane protein to lipid ratio, which would be expected to cause a lowering of the phase transition and a decrease in the number of Mn2+ binding sites. However, no such changes were observed, indicating that any change in the protein content of the thylakoid membrane is compensated for by changes in the fatty acid chain length and in the phospholipid classes, allowing the net fluidity and ordering of the system to remain constant. This is in agreement with the functional studies (2,5) where critical enzyme functions in the thylakoids (cytochrome oxidase, photosynthetic electron transport) were not changed after adaptation of the cells to the saline environment. The decrease in the cytoplasmic membrane phase transition may be interpreted as a more fluid membrane (due to the longer fatty acid side chains) or a decrease in the protein content (18) in response to salt. Since the chemical analysis of the membrane constituents demonstrated an increase in both the protein content and the stabilizing lipid digalactosyl diacylglycerol, which would be expected to increase the phase transition temperature, the observed fluidity increase can only be attributed to overcompensation by the elongation of the fatty acid side chains. In addition to the influence on membrane fluidity, the lipid content has been shown to critically affect the functions of some membrane proteins (19). We previously reported on the de novo synthesis of cytochrome oxidase in response to increased salinity (2). Reassessment of the data show that although the levels of the cytochrome oxidase increased by a factor of 5.42, activity of the enzyme actually increased by a factor of 11.51 (2). Such a stimulation of the enzyme activity may be directly attributable to the shift in the membrane lipid ratios as reported for other systems (19), and would certainly benefit the cell by augmenting the process of salt extrusion by the redox driven (respiratory) proton gradient (2) coupled to a Na+/H+ antiporter (6). The change in the number of Mn2+ high affinity sites and the K1 binding constant reflect the changes in the protein content and protein particle size distribution in the cytoplasmic membranes (3). In addition, the increase in the cytoplasmic membrane negative charge would certainly cause changes in the permeability of the cytoplasmic membrane to anions such as Cll and therefore alter the permeability to NaCl(20).
RESPONSE
OF CYANOBACTERIAL
The changes in the levels of carotenoid are difficult to interpret in terms of its influence on the fluidity of the membrane. Cholesterol in mammalian systems has been shown to perturb membrane structure at low concentrations and to stabilize the system when the concentration is increased (19), and carotenoids may exert a similar effect in this case by a decrease in its concentration leading to an increase in fluidity. Apart from the fluidity effects, the substantial decrease in carotenoids would certainly affect the cell’s ability to counter the process of photooxidative damage and it has been observed that cells exposed to high salinity are more sensitive to light (data not shown). It is known that there is an increase in the hydrocarbon chain motion at the terminal methyl groups in phospholipid membrane bilayers (flexibility gradient) (21), and the spectrum of spin labels are strongly dependent on the position of the -N-O’ paramagnetic group in the bilayer. From Table III the phase transition temperatures are the same for 5-DSA and CAT,,; however, the phase transition temperature reported by 16-DME (using TC) is different, and may reflect this flexibility gradient in cytoplasmic and thylakoid membranes due to the different localization of this spin label within the membranes. The results presented clearly demonstrate that there is significant modification to the surface change, phase transition, and fatty acid composition of the cytoplasmic membranes upon prolonged exposure to saline environments, while the thylakoid membranes are relatively unaffected. The data is consistent with the cytoplasmic membrane being the primary barrier to the environment, and the major site of adaptive response to the stresses imposed by increased salinity. REFERENCES 1. Blumwald, E., Mehlhorn, R. J., and Packer, L. (1983) Proc. N&l. Acad. Sci. USA 80,2599-2602.
MEMBRANES
TO SALT
STRESS
267
2. Fry, I. V., Huflejt, M., Erber, W. W. A., Peschek, G. A., and Packer, L. (1986) Arch. Biochem. Biophys. 244,689-691. 3. Lefort-Tran, M., Pouphile, Plant Physiol. 87,767-l%.
M., Spath, S., and Packer, L. (1988)
4. Fry, I. V., and Peschek, G. A. (1988) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 167 pp. 450-458, Academic Press, San Diego. 5. Peschek, G. A., Molitor, V., Trnka, M., Wastyn, M., andErber, W. (1988) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 167, pp. 437-449, Academic Press, San Diego. 6. Blumwald, E., Wolosin, J. M., and Packer, L. (1984) Biochem. Biophys. Res Commun. 122,452-459. 7. Murata, N., and Omata, T. (1988) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 167, pp. 245-251, Academic Press, San Diego. 8. Kratz, W. A., and Myers, J. (1955) Amer. J. Bot. 42,282-287. 9. Van Walraven, M. S., Kopenaal, E., Marvin, H. J. P., Magendorn, M. J. M., and Kraayenhof, R. (1984) Eur. J. Biochem. 144, 563-
564. 10. Morrison, W. R., and Smith, L. M. (1964) J. Lipid Res. 5, 600609. 11. McLohian, A. K., Zschoring, O., Kertsehen, H. P., and Ebert, B. (1984) Biochim. Biophys. Acta, 12,217-223. 12. Aveyard, R., and Haydon, D. A. (1973) An Introduction to the Principles of Surface Chemistry, Cambridge University Press, London/New York. 13. Freed, J. H. (1976) in Spin Labeling, Theory and Practice (Berlinger, L. J., Ed.), pp. 53-132, Academic Press, New York. 14. Griffith, 0. H., and Jost, P. C. (1976) in Spin Labeling, Theory and Practice (Berlinger, L. J., Ed.), pp. 454-524, Academic Press, New York. 15. Mackinney, G. (1941) J. Biol. Chem. 140,315-322. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 17. Rabinowitch, E. (1951) Photosynthesis and Related Processes, Vol. II, pp. 603-828, Interscience Pub. Inc., New York. 18. Grant, C. W. M., and McConnell, H. M. (1974) Proc. Natl. Acad. Sci. USA 71,4653-4657. 19. Yeagle, P. L. (1989) FASEB J. 3,1833-1842. 20. Blumwald, E., Mehlhorn, R. J., and Packer, L. (1983) Plant Physiol. 73,377-380. 21. McConnell, H. M. (1976) in Spin Labeling, Theory and Practice (Berlinger, L. J., Ed.), pp. 525-561, Academic Press, New York.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 277, No. 2, March, pp. 263-267,199O
Membrane Lipid Composition, Fluidity, and Surface Charge Changes in Response to Growth of the Fresh Water Cyanobacterium Synechococcus 6311 under High Salinity’ Gennady
Khomutov,’
Ian V. Fry,3 Margaret
E. Huflejt,
and Lester
Packer
Membrane Bioenergetics Group, Applied Science Division, Lawrence Berkeley Laboratory, Molecular and Cell Biology, University of California, Berkeley, California 94720
and Department
of
Received July 19,1989, and in revised form October 23,1989
The effect of adaptation to saline growth of a fresh water cyanobacterium Synechococcus 63 11 on components of the cytoplasmic membranes and thylakoids was investigated. Significant changes in membrane surface charge, lipid, fatty acid, and carotenoid composition were observed upon transfer of the cells from a low salt (0.015 M NaCl) to a high salt (0.50 M NaCl) growth medium. Very similar changes in the polar lipid classes and fatty acid composition were observed in both membranes, but changes in fluidity and surface charge and a significant shift in the protein to lipid ratio were only apparent in the cytoplasmic membranes. The fluidity and surface charge data correlate well with functional studies and we can attribute the cytoplasmic membrane as the major site of interaction and adaptation to the saline environment. 8 igso Academic press. IN.
Synechococcus 6311, a freshwater unicellular cyanobacterium, exhibits the ability to adapt to and grow at high salinity (1). The adaptive process is characterized by elevated respiration (2) and glycogen content (2, 3), de nouo synthesis of cytochrome oxidase (2) located in the cytoplasmic membrane (4,5), changes in membrane particle size and distribution (3), and an increase in the Na+/H+ antiporter activity (6). Recent advances in sepi This research was supported by the Office of Basic Energy Sciences of the U.S. Department of Energy under contracts DE-AC0376SF0009S and DE-FG03-87ER13736, the NASA-CELSS Interagency agreement A-1456~ and an IREX fellowship under the United States/Soviet exchange program, Princeton, New Jersey. ’ On leave from Biophysical Department, Physical Faculty Moscow State University, Leninskie Gory Moscow, 119899 GSP MCU USSR. 3 To whom correspondence should be addressed. 0003.9861/90
Copyright All
rights
$3.00 0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
aration techniques (5,7) have allowed us to separate and purify cytoplasmic membranes from the thylakoids of cells grown at high (0.50 M) and low (15 mM) NaCl. To determine if the physical nature of the membranes contributes to the observed functional changes, biophysical and chemical analyses of surface charge, fluidity, phospholipid, fatty acid, and carotenoid composition of the cytoplasmic membrane and thylakoids were undertaken. EXPERIMENTAL Organism and culture conditions. Axenic cultures of SynechococCLLS6311 cells were grown photoautotrophically in Kratz and Myers medium C (8) at 32°C. Purity of the cellular suspensions was confirmed by plating of the culture samples on sterile nutrient broth-enriched Petri agar plates. During each experiment, the cell growth was monitored by measuring the protein and chlorophyll content and the optical density of the culture. The cells were harvested by centrifugation and washed twice in double-distilled HzO. The wet weight was obtained after washed cells were centrifuged in Sorval SS-34 rotor at 13,000 rpm for 15 min. Preparation of subcellular fractions. Cyanobacterial membranes were isolated using a flotation sucrose gradient method as described (7). The purity of cytoplasmic membrane preparations was confirmed spectrophotometrically by scanning of membrane solutions in methanol to determine contamination by chlorophyll. For analyses, only the lightest, top fraction was used. Purity of the thylakoid fraction was monitored using the following two criteria: the thylakoid membrane band appears on sucrose gradient as an extremely uniform fraction, and the two membranes have distinctively different monogalactosyl diacylglycerol to digalactosyl diacylglycerol ratios. From this we assume that the contamination of the thylakoid membrane fraction by cytoplasmic membranes is insignificant. Isolation and characterization of lipids. For fatty acids and lipid analysis, 1-2 ml of washed and concentrated membranes (2-3 mg protein/ml for cytoplasmic membrane suspension or 6-7 mg/ml for thylakoid membranes) in buffer were dried under N2, then washed with acetone for quantitative isolation of galactolipids (9). Lipids were isolated according to Van Walraven et al. (9). Dry lipids were stored under N2 at -14°C. For separation of lipid classes, the dry lipid fraction 263
264
KHOMUTOV
0
20
40
60
Mn (bound)
80
100
120
x 10m5 M
FIG. 1. Langmuir adsorption isotherm for purified thylakoids from Syrzechococcus 6311.0, From salt grown cells; 0, from control cells.
was quantitatively dissolved in dichloromethane and separated by TLC4 (silica gel 60, Merck) with the development solvent acetone/ benzene/H,0 (91:30:8, vol/vol). TLC plates were sprayed with 0.01% primuline in acetone/H,0 (4~1) and lipids were detected under long wavelength uv light. The lipid classes were identified qualitatively by comparison of their R, with Rf of lipid standards (Serdary Research, Ontario, Canada). For fatty acid analysis, silica gel containing individual lipid was recovered from plates and transmethylation was performed with boron trifluoride (14% in methanol, Alltech reagent) in llO”C/90 min, as described (lo), without prior extraction of lipids from silica gel. Fatty acid methyl esters were removed from reaction mixture by multiple washing with hexane, with the last washing in hexane/water 1:l and analyzed with a Varian Gas Chromatograph Model 3700, equipped with Supelco column No. 2330, a flame ionization detector and a CDS IIIC Varian Data System Integrator, using the following temperature program: 17O”C, 5 min; 170-23O”C, 4”C/ min; 23O”C, 5 min, with nitrogen as carrier gas. Mn’+ binding. Due to the effect of surface electrical potential, the cation concentration at the surface can be very different from the bulk concentration which was used for the determination of the apparent binding constant (11). To eliminate the effect of surface electrical potential on the binding of Mn2+ to thylakoid and cytoplasmic membranes all experiments were performed in the presence of 300 mM KCl. In buffer containing 0.3 M KC1 the dependence of the manganese (II) hexahydrate ion EPR-signal amplitude was linear with concentration, and this dependence was used as a calibration curve for calculating the free manganese in the membrane samples. Amplitude of the EPR signal due to free Mn*+ was measured at the high field or low field feature to negate any overlap of the EPR signal arising from the chlo-
ET AL. rophyll radical in the middle of the spectrum (g = 2 region). EPR spectra were recorded at room temperature on a Varian E-109 spectrometer at 1 mW power, 8 gauss modulation amplitude, with a scan rate of 250 G per minute. The concentration of MnCl, was 0.3 mM, and the volume of additions to the membrane suspensions did not exceed 2% (v/v). To record EPR spectra, 50 ~1 samples were placed in 75.~1 glass capillaries (sealed at one end) then placed in a standard 3.mm diameter quartz EPR tube in the resonance cavity. Error due to sample placement was determined for a 0.3 mM MnCl, solution which was removed and replaced into the resonance cavity 10 times. The arithmetical mean and the standard deviation were 4.4 and 0.06 (arbitrary units), respectively, representing an error of less than 2%. Thylakoid membrane concentration (control and salt grown) in all experiments was 1.91 mg chlorophyll/ml, corresponding to 10.5 and 15.1 mg protein/ml for control and salt grown thylakoid membranes, respectively. Cytoplasmic membrane concentration was between 12.5 and 14.2 mg protein/ml. The number of Mn2+ binding sites and the K' and K" binding constants were calculated from the Langmuir adsorption isotherm as described ( 12). Order parameter (S) and correlation times (7~) of the spin labels were determined as described (13,14). CATI (N,N,dimethyl-N-hexadecyle 2,2,6,6-tetramethyl-4-hydroxypiperidine-l-oxyl) was a gift from Dr. Rolf Mehlhorn, 5-doxyl stearic acid (5.DSA) and 16.doxyl stearic acid methyl ester (16-DME) were obtained from Sigma. Stock solutions of CATI were aqueous, while 5-DSA and 16-DME were in ethanol. The spin labels were diluted at least 1:lOO into the samples, giving a final ethanol concentration of less than l%, and a spin label concentration between 0.1 and 0.5 mM. Chlorophyll and protein were determined by the methods of Mackinney (15) and Lowry et al. (16), respectively.
RESULTS
Mn2+ Binding The cytoplasmic membrane and thylakoid preparations from salt and control cells exhibited Mn2+ binding similar to that observed in thylakoids from higher plants. Two distinct affinity sites were observed in both salt and control thylakoids (Fig. 1). The number of high affinity binding sites was higher than in control thylakoids, and the magnitude of the binding constant (PC’) was lower than in control samples (Fig. 1, Table I). However, the number of low affinity sites and the binding constants (K*) were very similar (Fig. 1, Table I) in both salt and control thylakoid preparations. The cytoplasmic membrane exhibited interactions with Mn*+ ions similar to the thylakoid preparations
TABLE
Manganese Binding Constants and Number of Binding Sites in Thylakoids from Control and Salt Grown
Synechococcw6311 High affinity
K' 4 Abbreviations used: TLC, thin layer chromatography; CATIG, N,N,dimethyl-N-hexadecyle 2,2,6,6-tetramethyl-4.hydroxypiperidine-1-oxyl; 5-DSA, 5-Doxyl stearic acid; 16.DME, 16-Doxyl stearic acid methyl ester.
I
Control Salt
(M-l)
5.7 x lo* 2.4 X lo4
site
Mn bound per chlorophyll 0.066
0.156
Low affinity
@ CM-‘) 2.4 X lo3 3.2 X lo3
site
Mn bound per chlorophyll 0.52-0.53 0.52-0.53
RESPONSE OF CYANOBACTERIAL
MEMBRANES
TABLE III
TABLE II Manganese Binding to Cytoplasmic Membranes and Thylakoids from Control and Salt Grown Cells
Phase Transition Temperatures of Isolated Cytoplasmic Membranes and Thylakoids from Control and Salt Grown Synechococcus 6311
Thylakoids”
Cytoplasmic membranes
Cytoplasmic membranes
Mn2+ binding, mol/mg protein 13.33 x 1o-3 21.87 X 1O-9
6.35-19.05 x 10m9 1.27 X 10m7
Control Salt
’ Thylakoid high affinity binding site.
Spin probe
Parameter measured”
16.DME 5-DSA
vz and S
CATIS
(Table II), but resolution of a K1 and K2 constant could not be made. However, relative levels of Mn2+ binding between salt and control cytoplasmic membrane preparations did show that growth under salt exposure increased the number of Mn2+ binding sites by 1 to 2 orders of magnitude (Table II). Correlation
Time and Order Parameter Analysis
Correlation times (TC) and the order parameter (S) of spin labels 16-DME, CATI,, and 5-DSA were calculated from their EPR spectra as described under Experimental. Thylakoids from salt and control cells demonstrated, using S values for the 5-DSA spin probe, breaks in the Arrhenius plot characteristic of lipid phase transitions at 27°C (Fig. 2, Table III). Arrhenius plots of 0.9
1
I
,
3
I
S
Control
Salt
Control
Salt
30 23 23
26 19
29 27 27
29 27 27
19
a Correlation time (x) and/or order parameter (S).
changes in the order parameters or w of CATI and 16DME in salt and control thylakoids showed very similar phase transition temperatures, between 27 and 30°C (Fig. 2, Table III). However, the order parameter (S) of 5-DSA in cytoplasmic membranes from control and salt grown cells exhibited a decrease in the phase transition from 23 to 19”C, respectively (Fig. 2). Analysis of Arrhenius plots for 16-DME, 5-DSA, and CATI, (using both TC and S) demonstrate consistent results for the phase transition(s) in cytoplasmic membrane and thylakoids, although 16-DME gave slightly higher results (Table III).
I
Fatty acid analysis of cytoplasmic membranes and thylakoids from control and salt grown cells showed that the protein/fatty acid ratio in thylakoid membrane was fairly constant (a 10% change), but this ratio increased by 40 to 50% in the cytoplasmic membrane from salt grown cells (Table IV). Analysis of lipid constituents showed an increase in the 18:l over the 16:l acyl chains in phospholipids and that the charged phospholipid content of the membranes also increased (Table V). There was no preferred localization of either the longer side chain containing or charged phospholipids to the cytoplasmic membrane or thylakoids. Each type of phospholipid was evenly distributed through both membrane systems (data not shown).
iz
b 0E
7C
Thylakoids
Lipid Analysis iI,
0.8
ii 5 E F x
265
TO SALT STRESS
Salt
0.7
0.6
TABLE IV Cytoplasmic Membrane
3.1
3.3
Protein to Fatty Acid Ratio (wt/wt) in Cytoplasmic Membranes and Thylakoids from Control and Salt Grown Synechococcus 6311 3.5
3.7
+ x 10-3 FIG. 2. Arrhenius plot for cytoplasmic membranes and thylakoids using the order parameter calculated for the spin probe 5-DSA.
Growth conditions
Cytoplasmic membranes
Control (0.015 M NaCl) Salt (0.50 M NaCl)
1.17 1.63
Thylakoids 5.65
5.09
266
KHOMUTOV TABLE V
Lipid Classes in Cytoplasmic Membranes Isolated from Salt and Control Grown Synechococcus 63 11 Fatty acid
Control (0.015 M NaCl) Salt (0.50 M NaCl)
Phospholipid”
16:l (% total)
(W total)
MGDG (% total)
DGDG (% total)
17.6 12.9
16.8 24.3
51.61 34.8
14.3 20.4
18:l
a MGDG, monogalactosyl diacylglycerol; DGDG, digalactosyl diacylglycerol. Upon transfer of the cells from low salt (0.015 M NaCl) to high salt (0.5 M NaCl) growth medium a rapid decrease in palmitoleic acid (C16:lA9) was accompanied by a concomitant increase in the percentage of the two oleic acid isomers (C18:lA9, C18:lAll) with the increase in the percentage of the C18:lA9 isomer being the most significant. The changes occurred within the first hour after the increase in salt concentration and progressed for up to 150 h depending on the state of growth during which the salt shock was applied. Subsequently the new ratios of fatty acids stabilized, with a decrease of about 15% of palmitoleic acid and a concomitant 13315% increase in octadecanoic acids, compared to controls. During 14 days of growth in the high salt media the total content of anionic lipids (phosphatidylglycerol and sulfoquinovosyl diacylglycerol) increased from 23.5 to 31.5% and the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol decreased from 3.5 to 1.6. The same direction of changes, but to a lesser extent was observed in aging, control (0.015 M NaCl-grown) cultures. These rearrangements in lipid environment occurred in both the thylakoid and the cytoplasmic membranes and were only partially reversible. After cells were transferred back to a low salt medium, the proportions between major fatty acids did not return to control values, even after 24 h of growth (data not shown).
Carotenoids The carotenoid fractions of cytoplasmic membranes from both control and salt grown cells exhibited absorption spectra characteristic of p-carotene (17) and control levels of 9.4 mmol/mg protein decreased to 3.5 mmol/mg protein over six days of exposure to high salinity. The carotenoids in the thylakoid membranes could not be quantified due to rapid degradation. The absorption spectrum line shape of the carotenoids in the thylakoids changed with time and was similar to that reported in (7). DISCUSSION
The effect of salinity on the membrane systems of Sy6311 is manifest at several levels, and is
nechococcus
ET AL.
most apparent in the cytoplasmic membranes rather than in the thylakoids. Although the lipid classes and fatty acids present in the cytoplasmic membranes and thylakoids both change to the same degree upon exposure to salt, the gross physical nature of the membranes, the fluidity and surface charge density, are constant in the thylakoids and only change in the cytoplasmic membrane. Protein to lipid ratios by chemical analysis show that there is a slight decrease in thylakoid membrane protein to lipid ratio, which would be expected to cause a lowering of the phase transition and a decrease in the number of Mn2+ binding sites. However, no such changes were observed, indicating that any change in the protein content of the thylakoid membrane is compensated for by changes in the fatty acid chain length and in the phospholipid classes, allowing the net fluidity and ordering of the system to remain constant. This is in agreement with the functional studies (2,5) where critical enzyme functions in the thylakoids (cytochrome oxidase, photosynthetic electron transport) were not changed after adaptation of the cells to the saline environment. The decrease in the cytoplasmic membrane phase transition may be interpreted as a more fluid membrane (due to the longer fatty acid side chains) or a decrease in the protein content (18) in response to salt. Since the chemical analysis of the membrane constituents demonstrated an increase in both the protein content and the stabilizing lipid digalactosyl diacylglycerol, which would be expected to increase the phase transition temperature, the observed fluidity increase can only be attributed to overcompensation by the elongation of the fatty acid side chains. In addition to the influence on membrane fluidity, the lipid content has been shown to critically affect the functions of some membrane proteins (19). We previously reported on the de novo synthesis of cytochrome oxidase in response to increased salinity (2). Reassessment of the data show that although the levels of the cytochrome oxidase increased by a factor of 5.42, activity of the enzyme actually increased by a factor of 11.51 (2). Such a stimulation of the enzyme activity may be directly attributable to the shift in the membrane lipid ratios as reported for other systems (19), and would certainly benefit the cell by augmenting the process of salt extrusion by the redox driven (respiratory) proton gradient (2) coupled to a Na+/H+ antiporter (6). The change in the number of Mn2+ high affinity sites and the K1 binding constant reflect the changes in the protein content and protein particle size distribution in the cytoplasmic membranes (3). In addition, the increase in the cytoplasmic membrane negative charge would certainly cause changes in the permeability of the cytoplasmic membrane to anions such as Cll and therefore alter the permeability to NaCl(20).
RESPONSE
OF CYANOBACTERIAL
The changes in the levels of carotenoid are difficult to interpret in terms of its influence on the fluidity of the membrane. Cholesterol in mammalian systems has been shown to perturb membrane structure at low concentrations and to stabilize the system when the concentration is increased (19), and carotenoids may exert a similar effect in this case by a decrease in its concentration leading to an increase in fluidity. Apart from the fluidity effects, the substantial decrease in carotenoids would certainly affect the cell’s ability to counter the process of photooxidative damage and it has been observed that cells exposed to high salinity are more sensitive to light (data not shown). It is known that there is an increase in the hydrocarbon chain motion at the terminal methyl groups in phospholipid membrane bilayers (flexibility gradient) (21), and the spectrum of spin labels are strongly dependent on the position of the -N-O’ paramagnetic group in the bilayer. From Table III the phase transition temperatures are the same for 5-DSA and CAT,,; however, the phase transition temperature reported by 16-DME (using TC) is different, and may reflect this flexibility gradient in cytoplasmic and thylakoid membranes due to the different localization of this spin label within the membranes. The results presented clearly demonstrate that there is significant modification to the surface change, phase transition, and fatty acid composition of the cytoplasmic membranes upon prolonged exposure to saline environments, while the thylakoid membranes are relatively unaffected. The data is consistent with the cytoplasmic membrane being the primary barrier to the environment, and the major site of adaptive response to the stresses imposed by increased salinity. REFERENCES 1. Blumwald, E., Mehlhorn, R. J., and Packer, L. (1983) Proc. N&l. Acad. Sci. USA 80,2599-2602.
MEMBRANES
TO SALT
STRESS
267
2. Fry, I. V., Huflejt, M., Erber, W. W. A., Peschek, G. A., and Packer, L. (1986) Arch. Biochem. Biophys. 244,689-691. 3. Lefort-Tran, M., Pouphile, Plant Physiol. 87,767-l%.
M., Spath, S., and Packer, L. (1988)
4. Fry, I. V., and Peschek, G. A. (1988) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 167 pp. 450-458, Academic Press, San Diego. 5. Peschek, G. A., Molitor, V., Trnka, M., Wastyn, M., andErber, W. (1988) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 167, pp. 437-449, Academic Press, San Diego. 6. Blumwald, E., Wolosin, J. M., and Packer, L. (1984) Biochem. Biophys. Res Commun. 122,452-459. 7. Murata, N., and Omata, T. (1988) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 167, pp. 245-251, Academic Press, San Diego. 8. Kratz, W. A., and Myers, J. (1955) Amer. J. Bot. 42,282-287. 9. Van Walraven, M. S., Kopenaal, E., Marvin, H. J. P., Magendorn, M. J. M., and Kraayenhof, R. (1984) Eur. J. Biochem. 144, 563-
564. 10. Morrison, W. R., and Smith, L. M. (1964) J. Lipid Res. 5, 600609. 11. McLohian, A. K., Zschoring, O., Kertsehen, H. P., and Ebert, B. (1984) Biochim. Biophys. Acta, 12,217-223. 12. Aveyard, R., and Haydon, D. A. (1973) An Introduction to the Principles of Surface Chemistry, Cambridge University Press, London/New York. 13. Freed, J. H. (1976) in Spin Labeling, Theory and Practice (Berlinger, L. J., Ed.), pp. 53-132, Academic Press, New York. 14. Griffith, 0. H., and Jost, P. C. (1976) in Spin Labeling, Theory and Practice (Berlinger, L. J., Ed.), pp. 454-524, Academic Press, New York. 15. Mackinney, G. (1941) J. Biol. Chem. 140,315-322. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 17. Rabinowitch, E. (1951) Photosynthesis and Related Processes, Vol. II, pp. 603-828, Interscience Pub. Inc., New York. 18. Grant, C. W. M., and McConnell, H. M. (1974) Proc. Natl. Acad. Sci. USA 71,4653-4657. 19. Yeagle, P. L. (1989) FASEB J. 3,1833-1842. 20. Blumwald, E., Mehlhorn, R. J., and Packer, L. (1983) Plant Physiol. 73,377-380. 21. McConnell, H. M. (1976) in Spin Labeling, Theory and Practice (Berlinger, L. J., Ed.), pp. 525-561, Academic Press, New York.