Intracellular Glycerol Content Research Articles

Loss of intracellular glycerol from Dunaliella by electroporation at constant osmotic pressure: subsequent restoration of glycerol content and associated volume changes

ABSTRACTThe effect of electroporation on Dunaliella tertiolecta at constant osmotic pressure (or water activity) was investigated. The following metabolic and physiological parameters were determined: extracellular and intracellular glycerol content, soluble protein content, photosynthetic oxygen evolution, mitochondrial oxygen uptake, cell volume and cell density. Electroporation conditions are described that released about 10% of intracellular glycerol to the external medium with minimal apparent effects on metabolism. Glycerol release originated from most cells rather than by total rupture of a small proportion of cells. Cell volume, measured on motile cells by video microscopy, reduced by 23% immediately after electroporation. Cell density did not increase. The uptake of mannitol, the major solute in the electroporation medium, was less than 20% of glycerol release. Following electroporation, the intracellular glycerol content and the cell volume both returned to pre‐electroporation values after about 30min. Because the cells were maintained at constant external osmotic pressure throughout the procedure, it is concluded that the regulatory mechanism responsible for setting the intracellular glycerol content does not sense external osmotic pressure per se. These findings are consistent with a mechanism that senses a parameter linked directly to cell volume to set the intracellular glycerol content.

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus S. Courtney Frasch

The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus S. Courtney Frasch.

The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.

Glycerol uptake by zooxanthellae of the temperate hard coral, Plesiastrea versipora (Lamarck)

The symbiotic algae of the coral Plesiastrea versipora have previously been shown to release photosynthate to the host, much of it in the form of glycerol. Glycerol uptake was investigated as part of studies of the mechanisms of translocation of organic carbon between zooxanthellae and their coral hosts. Experiments were done on isolated zooxanthellae suspended in coral host homogenate or sea water. Uptake of [$^{14}$C]glycerol was linear with time in the light and dark. The Photosystem II inhibitor, DCMU, had no effect on glycerol uptake rates in the light or dark, but glycerol uptake was strongly inhibited by KCN. [$^{14}$C]glycerol taken up by the zooxanthellae was rapidly metabolized and the intracellular glycerol concentration was near zero. Uptake curves were curvilinear and resolvable into a saturable Michaelis-Menten component and a linear component. Glycerol is apparently taken up both by a transport mechanism and by simple diffusion. Glycerol-3-phosphate, propan-1-ol, propan-2-ol, ethanediol, 1,2 propanediol and erythritol do not inhibit glycerol uptake, but DL-glyceraldehyde is a non-competitive inhibitor. The permeability of the zooxanthellae to glycerol is similar to that of other eukaryotic cells (p $\approx $ 0.65 nm s$^{-1}$). Host homogenate decreases the net rate of uptake of glycerol into zooxanthellae by diffusion in both the light and the dark. The apparent permeability of glycerol is slightly higher in the dark than in the light. Host homogenate does not make the plasma membrane `leaky to glycerol. Models of the mechanism of action of host homogenate are discussed in the light of these results.

Physiological responses of Zygosaccharomyces rouxii to osmotic stress

When cell suspensions of Zygosaccharomyces rouxii were subjected to osmotic shock with NaCl, the cell volume decreased sharply and plasmolysis was observed. The cell subsequently recovered and volumes similar to those of cells growing at the respective water activity (aw) values were found. Cycloheximide prevented cell recovery, indicating the involvement of protein synthesis in the recovery process. The intracellular glycerol concentration of Z. rouxii incubated in the presence of [14C]glycerol increased from 13 to 96 mmol/l during the initial 20 min after an upshock from 0.998 aw to 0.96 aw. All the intracellular glycerol was labelled and therefore derived from the medium. Labelled glycerol was subsequently utilized and replaced by unlabelled glycerol produced by the cell within 90 min. The initial increase in glycerol concentration following the upshock was confirmed by 13C-nuclear magnetic resonance (NMR) spectroscopic studies of cell extracts. The combined dihydroxyacetone and dihydroxyacetone phosphate concentrations fluctuated during this period, whereas glycerol-3-phosphate initially increased and then remained constant. This indicates that the production of glycerol is regulated. Decreases in ATP and polyphosphate levels were observed following osmotic upshock and may reflect a greater demand for ATP during the period of adjustment to decreased aw. The changes in cell volume and in ATP concentration following osmotic upshock may serve as osmoregulatory signals in Z. rouxii, as suggested previously for other microorganisms.

Energy flux and osmoregulation of Saccharomyces cerevisiae grown in chemostats under NaCl stress

The energetics and accumulation of solutes in Saccharomyces cerevisiae were investigated for cells grown aerobically in a chemostat under NaCl stress and glucose limitation. Changed energy requirements in relation to external salinity were examined by energy balance determinations performed by substrate and product analyses, with the latter including heat measurements by microcalorimetry. In both 0 and 0.9 M NaCl cultures, the catabolism was entirely respiratory at the lowest dilution rates tested but shifted to a mixed respiratory-fermentative metabolism at higher dilution rates. This shift occurred at a considerably lower dilution rate for salt-grown cells. The intracellular solute concentrations, as calculated on the basis of intracellular soluble space determinations, showed that the internal Na+ concentration increased from about 0.02 molal in basal medium to about 0.18 molal in 0.9 M NaCl medium, while intracellular K+ was maintained around 0.29 molal despite the variation in external salinity. The intracellular glycerol concentration increased from below 0.05 molal at low salinity to about 1.2 molal at 0.9 M NaCl. The concentrations of the internal solutes, however, changed insignificantly with growth rate and energy metabolism. The additional maintenance energy expenditure for growth at 0.9 M NaCl was, depending on the growth rate, 14 to 31% of the total energy requirement for growth at 0 M NaCl. Including the energy conserved in glycerol, the total additional energy demand for growth at 0.9 M NaCl corresponded to 28 to 51% of the energy required for growth at 0 M NaCl.

Regulation of glycerol metabolism in Zygosaccharomyces rouxii in response to osmotic stress

Enzyme analyses indicated that the metabolism of glycerol by Zygosaccharomyces rouxii occurred via either glycerol-3-phosphate (G3P) or dihydroxyacetone (DHA). The route via DHA is significant in osmoregulation. The specific activities of glycerol dehydrogenase (GDHG) and DHA kinase, which metabolize glycerol via DHA, increased nine- and fourfold respectively during osmotic stress [0.960 water activity (aw) adjusted with NaCl] when compared to non-stressed conditions (0.998 aw). Both pathways are under metabolic regulation. Glycerol kinase, mitochondrial G3P dehydrogenase and DHA kinase are induced by glycerol while the latter is also repressed by glucose. Cells treated with cycloheximide prior to osmotic upshock showed significantly lower DHA kinase and GDHG levels and lower intracellular glycerol concentrations when compared to untreated control cells. Thus protein synthesis is essential for osmotic adaptation.

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (aw) to 0.960. When the aw was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 aw (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 aw. Compared to steady-state cultivation at 0.998 aw, the yeast retained at 0.960 aw (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 aw (PEG 400). The intracellular glycerol concentration was insufficient to balance the aw across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active.

Determination of Volume ofDunaliellaCells by Lithium Dilution Measurements and Derivation of Internal Solute Concentrations

A method has been developed to measure the cell volume of the unicellular green alga Dunaliella parva 19/9 using Li + measurements only. Concentrations of internal solutes can also be calculated if they are assayed in the same samples as Li + . We found that D. parva cells grown in 0-4 kmol m~3 NaCl have an average aqueous cell volume of 65-1 +2-9 /nm3, a K + concentration of 126 + 6 mol m-3, a Na+ concentration of 9+ 11 mol m~3 and a glycerol concentration of 615 + 27 mol m-3 (n= 12). Algae grown in 1-5 kmol m~3 NaCl have an average aqueous cell volume of 131+7-5 ??m3, a K+ concentration of 109 + 4 mol m-3, a Na+ concentration of 10 + 39 mol m3 and a glycerol concentration of 1 425 + 59 mol m-3 (n= 12). These results indicate that D. parva cells adapted to high salinities have larger cell volumes than those adapted to lower salinities. However, there is no evidence for a significant difference in internal Na+ concentration, despite the almost 4-fold difference in the concentration of external NaCl. The intracellular glycerol concentration alone accounts for 65% and 54%, respectively, of the osmotic balance in low and high salt grown cells.

Intracellular levels of glycerol necessary for initiation of growth under salt-stressed conditions in a salt-tolerant yeast,Zygosaccharomyces rouxii

The relationship between the intracellular concentration of glycerol and the initiation of growth under salt-stressed conditions in the salt-tolerant yeast Zygosaccharomyces rouxii was studied. The results demonstrated that the accumulation of a definite intracellular concentration of glycerol is required prior to the initiation of growth under NaCl-stressed conditions. The initiation of growth in 3 M and 3.5 M NaCl media started at low intracellular concentrations such as 0.51 and 1.17 mol/l cell volume. Similar results were obtained under KCl- and MgCl2-stressed conditions. However, Z. rouxii was unable to grow under LiCl-stressed conditions, though it accumulated glycerol to the level required for the initiation of growth.

The accumulation of polyols by the yeast Debaryomyces hansenii in response to water stress

Growth of the yeast Debaryomyces hansenii in 1% (w/v) glucose medium containing NaCl or KCl resulted in the accumulation of glycerol during the exponential phase of growth and arabinitol during the stationary phase. Similar results were obtained during growth of the yeast in 12.5% (w/v) glucose, 25% glucose, 25% fructose, and 1% glucose plus 25% polyethylene glycol M.W.200 (PEG 200). The results indicate that the intracellular glycerol concentration increases concomitantly with the solute concentration of the growth medium, while the accumulation of arabinitol is less pronounced. Growth of the yeast in media containing meso-erythritol or D-mannitol as carbon sources without or with 1 M NaCl resulted in the pronounced inhibition of arabinitol accumulation because of the intracellular accumulation of the exogenous polyols. Mannitol accumulated primarily during the stationary phase, while erythritol accumulation occurred primarily during the exponential phase. The erythritol concentration attained very high levels during growth in media containing 1 M NaCl and effectively inhibited the accumulation of glycerol.

Glycerol fomation inDunaliella cells under non-growing conditions with hypertonic lithium or magnesium media

Glycerol formation ofDunaliella cells in non-growing media was investigated.Dunaliella tertiolecta andD. bioculata grew well in a NaCl medium but not at all in a LiCl or a MgCl2 medium. When the cells originally suspended in a medium containing 0.5 M NaCl were transferred to media which contained one of 1 M NaCl, 1 M LiCl or 0.7 M MgCl2, the intracellular glycerol content increased.D. tertiolecta cultured in either a 1 M LiCl or a 0.7 M MgCl2 medium did not multiply, but maintained abilities to evolve O2 in the light and absorb O2 in thedark even after about a 5 day culture. From these results, it can be concluded that the halotolerance ofDunaliella to different kinds of salts is not directly related to osmoregulation by the glycerol formation.

Polyol and water in the salt-tolerant yeastDebaryomyces hanseniias studied by1H nuclear magnetic resonance

When cultured in media containing high concentrations of solutes, xerotolerant yeasts respond by increased accumulation of polyhydroxy alcohols (polyols) [1-4]. During growth in saline media the predominant intracellular solute is glycerol [1,2]. At high salinities the intracellular glycerol concentration may attain molar levels. Any solute that is accumulated intraceUularly to such high concentrations must be compatible with metabolic functioning and the innocuous nature of glycerol is well documented [1,4-6]. Schobert [7] proposed that polyols by means of their hydroxyl groups may participate in the intracellular structure thereby preserving the hydration of the cellular biopolymers at the reduced cellular water activities prevailing in concentrated environments. The properties of water in biological systems have frequently been investigated by NMR techniques and the general observations are linewidth broadening of the water spectrum and reduced relaxation times for the water protons. Most investigators have interpreted these data as indicating that a significant fraction of the intracellular water exhibits a reduced mobility due to interaction with cellular macromolecules [8-10]. Similarly, interactions between macromolecular surfaces and polyols may sufficiently alter the physical state of the polyols to be visualized by NMR spectroscopy. The present work is a first step in a study of the NMR properties of cell water and polyol in a xerotolerant yeast grown under salt stress. A substantial increase of linewidth and reduction in spin-lattice relaxation times (T I) were observed for intracellular water and polyol. The intensity of the polyol signal corresponded, however, only to a minor fraction of the total polyol content.

Permeation of Chinese hamster ovary cells by glycerol: mechanism and kinetics.

When monolayer cultures of Chinese hamster ovary cells were exposed to 3H-glycerol ranging in concentration from 0.1 microM to 200 mM, glycerol in flux was found to increase in direct proportion to the extracellular concentration of glycerol. Other experiments indicated that the same relationship existed at concentrations in excess of 1.0 M. Similarly, glycerol efflux was found to vary in direct proportion to the intracellular concentration of glycerol. In neither case could influx or efflux be saturated. Glycerol influx was not affected by depletion of ATP, alkylation by parachloromercuribenzene sulfonic acid, or exposure to persantine. Altering the pH or temperature also had little effect. Attempts at demonstrating countertransport of glycerol were negative. These data indicate that glycerol probably passes through the membrane by a non-mediated process. For cells in monolayer, the kinetics of influx and efflux are biphasic. Similar biphasic kinetics are observed with cells in suspension culture. A close fit to the data may be obtained by adding together two first-order curves. The pair of curves for influx are clearly different from the pair for efflux. The fit provided by the two pairs of first-order functions suggested that glycerol might diffuse into and out of two intracellular compartments. However, the experimental data do not agree with the predicted behavior of a two-compartment system. As a result, the exact nature of the diffusion limiting steps which are described by the first-order equations remains undefined.

EFFECT OF LIGHT QUALITY AND INTENSITY ON GLYCEROL CONTENT INDUNALIELLA TERTIOLECTA(CHLOROPHYCEAE) AND THE RELATIONSHIP TO CELL GROWTH/OSMOREGULATION1

ABSTRACTDunaliella tertiolectaButcher was grown at two intensities (33, 150μEin · m−2· s−1) of blue light and white light at 0.25, 0.50 and 1.00 M NaCl. Growth rates were used as an indication of the relative osmoregulatory ability of cells in the various treatments. There was no significant effect on growth rate due to various NaCl molarities. No significant difference in growth rate was found between blue‐ and white‐light cultures at the high intensity, the average growth constant being 2.07 divisions/day. However, at the low intensity illumination, blue light produced a significant increase in growth rate; 1.42 vs. 0.93 divisions/day for blue light and white light grown cells respectively. The average glycerol content of exponentially dividing cells grown at 0.25, 0.50 and 1.00 M NaCl was 0.12, 0.41 and 1.12 mg/108cells, respectively, as measured by gas chromatography. The intracellular glycerol content was significantly reduced by blue light at both light intensities and at each NaCl molarity. However, high light intensity reduced cellular glycerol content more than the reduction effected by blue light. Glycerol accumulated in the medium throughout culture growth. Intracellular glycerol content also increased with cellular aging reaching 2.72 mg/108cells in stationary phase, low intensity 1.00 M NaCl cultures. A negative correlation between glycerol content and growth rate was found. Total inhibition of glycerol production could not be obtained by treatment with blue light. However, this negative correlation possibly indicates thatD. tertiolectaexpends energy producing an excess amount of glycerol over that required for osmoregulation, leading to a reduction in the growth rate for the organism.

The ATP pool in relation to the production of glycerol and heat during growth of the halotolerant yeast Debaryomyces hansenii

In a study of the halotolerant yeast Debarymyces hansenii cultured in 4 mM and 2.7 M NaCl the intracellular ATP pool, the heat production, the oxygen uptake, and, in the high culture salinity also, the intracellular glycerol concentration were found to be correlated. The intracellular ATP in the 2.7 M NaCl culture had a constant concentration of 3.5 mM ATP during the second half of the lag phase, while in 4 mM NaCl it rose to a maximum of 3.1 mM during the late log phase. The intracellular glycerol concentration in 2.7 M NaCl was about 1.3M during the entire exponential growth phase. Sine the glycerol concentration of the medium was not more than 0.23 mM, glycerol must contribute to the osmotic balance of the cells in high salinity. The corresponding maximum values for the 4 mM NaCl culture were 0.16 M and 0.08 mM. The experimental enthalpy changes were approximately the same for the two salinities, viz. about-1200 kJ per mole consumed glucose. The Ym-values for the 4 mM and 2.7 M NaCl cultures were 91 and 59, respectively, the difference being a consequence of the decreased efficiency of growth in high salinity.

The microbiology and biogeochemistry of the Dead Sea

The Dead Sea is a hypersaline water body. Its total dissolved salts content is on the average 322.6 gm/liter. The dominant cation is Mg (40.7 gm/liter), followed by Na (39.2 gm/liter), Ca (17 gm/liter) and K (7 gm/liter). The major anion is Cl (212 gm/liter), followed by Br (5 gm/liter); SO4 and HCO3, are very minor. The lake contains a limited variety of microorganisms and no higher organisms. The number of recorded species is very low, but the total biomass is reasonably high (about 10(5) bacteria/ml and 10(4) algal cells/ml). The indigenous flora is comprised mainly of obligate halophylic bacteria, such as the pink, pleomorphicHalobacterium sp., aSarcina-like coccus, and the facultative halophilic green alga,Dunaliella. Sulfate reducers can be isolated from bottom sediments. Recently a unique obligate magnesiophile bacteria was isolated from Dead Sea sediment. Several of the Dead Sea organisms possess unusual properties. TheHalobacterium sp. has extremely high intercellular K(+) concentration (up to 4.8M) and extraordinary specificity for K(+) over Na. TheDunaliella has very high intracellular concentration of glycerol (up to 2.1M). The microorganisms exert marked influence on some biogeochemical processes occurring in the lake, such as the control of the sulfur cycle and the formation and diagenesis of organic matter in the sediments. The Dead Sea is an excellent example of the development of two different mechanisms for adjusting to a hostile environment. The algae adjust to the high salinity by developing a mechanism for the exclusion of salts from the intracellular fluid and using glycerol for osmotic regulation. On the other hand, the bacteria adapt to the environment by adjusting their internal inorganic ionic strength, but not composition, to that of the medium. The problem of population dynamics and limiting factors for algal and bacterial productivity are discussed in view of the total absence of zooplankton and other consumers other than bacteria.