Promotion of gas bubble formation by ingested nuclei in the ciliate, Tetrahymena pyriformis

Cells of the ciliate Tetrahymena pyriformis were suspended with carmine or graphite particles or with Halobacterium gas vesicles, all of which promote bubble formation in aqueous suspensions when tested with 10 atm and above (0.1-0.5 X 10(7) Pa) (carmine and graphite) or 25 atm and above (gas vesicles) of nitrogen supersaturations. All three particles were ingested, but only the gas vesicles promoted intracellular gas bubble formation if the cells containing them were nitrogen or methane saturated in a slow stepwise fashion prior to rapid decompression. Cell rupture did not occur until gas saturation pressures greater than 25 atm were used; this suggests that the ciliate pellicle and cytoplasm cannot resist the mechanical forces of an expanding gas phase induced by decompression from between 25 and 50 atm and thus provides an estimate of the physical strength of these cellular components. The inability of the ingested carmine, graphite, and collapsed gas vesicles to induce intracellular gas bubble formation suggests that the phagocytic process somehow altered them. This procedure may thus provide a tool for the study of early events in the digestive processes of ciliates.     

Promotion of gas bubble formation by ingested nuclei in the ciliate,Tetrahymena pyriformis

Cells of the ciliateTetrahymena pyriformis were suspended with carmine or graphite particles or with Halobacterium gas vesicles, all of which promote bubble formation in aqueous suspensions when tested with 10 atm and above (0.1−0.5×10⁷ Pa) (carmine and graphite) or 25 atm and above (gas vesicles) of nitrogen supersaturations. All three particles were ingested, but only the gas vesicles promoted intracellular gas bubble formation if the cells containing them were nitrogen or methane saturated in a slow stepwise fashion prior to rapid decompression. Cell rupture did not occur until gas saturation pressures greater than 25 atm were used; this suggests that the ciliate pellicle and cytoplasm cannot resist the mechanical forces of an expanding gas phase induced by decompression from between 25 and 50 atm and thus provides an estimate of the physical strength of these cellular components. The inability of the ingested carmine, graphite, and collapsed gas vesicles to induce intracellular gas bubble formation suggests that the phagocytic process somehow altered them. This procedure may thus provide a tool for the study of early events in the digestive processes of ciliates.     

Rupture of the cell envelope by decompression of the deep-sea methanogen Methanococcus jannaschii

The effect of decompression on the structure of Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen, was studied in a novel high-pressure, high-temperature bioreactor. The cell envelope of M. jannaschii appeared to rupture upon rapid decompression (ca. 1 s) from 260 atm of hyperbaric pressure. When decompression from 260 atm was performed over 5 min, the proportion of ruptured cells decreased significantly. In contrast to the effect produced by decompression from hyperbaric pressure, decompression from a hydrostatic pressure of 260 atm did not induce cell lysis.     

Rupture of the Cell Envelope by Decompression of the Deep-Sea Methanogen Methanococcus jannaschii

The effect of decompression on the structure of Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen, was studied in a novel high-pressure, high-temperature bioreactor. The cell envelope of M. jannaschii appeared to rupture upon rapid decompression (ca. 1 s) from 260 atm of hyperbaric pressure. When decompression from 260 atm was performed over 5 min, the proportion of ruptured cells decreased significantly. In contrast to the effect produced by decompression from hyperbaric pressure, decompression from a hydrostatic pressure of 260 atm did not induce cell lysis.     

Intracellular Bubble Formation: Differences in Gas Supersaturation Tolerances Between Tetrahymena and Euglena1

Cells of Tetrahymena pyriformis, T. thermophila, and Euglena gracilis were saturated with nitrogen gas at pressures up to 300 atm and rapidly decompressed. Damage was assessed by measuring post-decompression cell fragmentation or viability. Occurrence of intracellular bubbles was determined by cinephotomicrography performed during the decompression or by direct observations afterwards. The extreme gas supersaturations induced led to intracellular bubble formation and rupture in cells of Tetrahymena that contained food vacuoles, but only with supersaturations of 175 atm or higher; 225 atm left few cells intact. Bubbles were never observed in cells of Euglena or in Tetrahymena cells freed of food vacuoles, even when they were decompressed from substantially higher nitrogen supersaturations. Cells of Euglena were most resistant and were unaffected by supersaturations up to 250 atm.     

Tolerance of bacteria to extreme gas supersaturations.

Bacteria without (Escherichia coli and Corynebacterium xerosis) and with gas vacuoles (Microcyclus aquaticus) were saturated with Ar or N₂ gas at pressures up to 300 atm and then rapidly decompressed. The resulting intracellular gas supersaturations had no effect on the viability of the bacteria except when the gas vesicles were purposely kept intact by slow pressurization rates. Thus no gas bubbles form within the cells even at these extreme supersaturations. This contradicts earlier interpretations of the cause of the disruptive effect on various cells by gas pressurization and decompression.     

Bubble formation in crustaceans following decompression from hyperbaric gas exposures

In vivo bubble formation was studied in various crustaceans equilibrated with high gas pressures and rapidly decompressed to atmospheric pressure. The species varied widely in susceptibility to bubble formation, and adults were generally more susceptible than larval stages. Bubbles did not form in early brine shrimp larvae unless equilibration pressures of at least 175 atm argon or 350 atm helium were used; for adult brine shrimp, copepods, and the larvae of crabs and shrimps, 100-125 atm argon or 175-225 atm helium were required. In contrast, bubbles formed in the leg joints of megalopa and adult crabs following decompression from only 3-10 atm argon; stimulation of limb movements increased this bubble formation, whereas inhibition of movements decreased it. High hydrostatic compressions applied before gas equilibration or slow compressions did not affect bubble formation. We concluded that circulatory systems, musculature, and storage lipids do not necessarily render organisms susceptible to bubble formation and that bubbles do not generally originate as preformed nuclei. In some cases, tribonucleation appears to be the cause of the bubbles.     

High gas pressure effects on yeast

Dried microorganisms are particularly resistant to high hydrostatic pressure effects. However, exposure to high pressures of nitrogen proved to be effective in inactivating dried yeasts. In this study, we tried to elucidate this mechanism on Saccharomyces cerevisiae. High-pressure treatments were performed using different inert gases at 150 MPa and 25 degrees C with holding time values up to 12 months. The influence of cell hydration was also investigated. For fully hydrated cells, pressurized gases had little specific effect: cell inactivation was mainly due to compression effects. However, dried cells were sensitive to high pressure of gases. In this latter case, two inactivation kinetics were observed. For holding time up to 1 h, the inactivation rate increased to 4 log and was linked to a loss of membrane integrity and the presence of damage on the cell wall. In such case cell inactivation would be due to gas sorption and desorption phenomena which would rupture dried cells during a fast pressure release. Gas sorption would occur in cell lipid phases. For longer holding times, the inactivation rate increased more slightly due to compression effects and/or to a slower gas sorption. Water therefore played a key role in cell sensitivity to fast gas pressure release. Two hypotheses were proposed to explain this phenomenon: the rigidity of vitrified dried cells and the presence of glassy solid phases which would favor intracellular gas expansion. Our results showed that dried microorganisms can be ruptured and inactivated by a fast pressure release with gases.     

Glutaraldehyde treatment of proteinaceous gas vesicles from cyanobacterium Anabaena flos-aquae

As potential gas microcarriers, gas vesicles (GVs) were isolated from cultures of the filamentous cyanobacterium Anabaena flos-aquae and treated with glutaraldehyde. The effects of glutaraldehyde treatment on the stability of GVs, against elevated temperatures (40-121 degrees C) and protein-stripping agents such as urea and sodium dodecyl sulfate (SDS), were then examined with the pressure collapse curves generated using pressure nephelometry. The treatment was very beneficial to GVs against the exposure to SDS and urea; however, it did not make the evolution-optimized vesicle structure stronger or more temperature-resistant. In the presence of these protein-stripping agents, the treated vesicles had higher median (50%) collapse pressures (by > or =1 atm) than the untreated ones, at both room temperature and 40 degrees C. This increase has been presumably attributed to the cross-linking of the large GvpC protein to the ribbed GvpA shell, thereby resisting the stripping of GvpC that provides the primary mechanical strength to the vesicle wall. The glutaraldehyde treatment also restored the strength of GVs weakened by a 5-week storage in a refrigerator and, therefore, is expected to improve the stability of GVs for long-term storage. GVs could not be autoclaved. If necessary for the intended applications, glutaraldehyde treatment may also serve to chemically sterilize the vesicles, with the glutaraldehyde subsequently removed by dialysis.     

A theory of osmotic lysis of lipid vesicles

Osmotic lysis of vesicles is shown to begin when the membrane expansion due to osmotic pressure exceeds its critical value, delta S, at which a membrane ruptures to form a pore. The dependence of delta S on the vesicle radius and respective osmotic pressures are obtained. It is found that osmotic pressure necessary for small (100 A) vesicles to rupture should exceed 30 atm, for large (10 000 A) vesicles it being as small as 10(-3) atm. In the case of large (greater than or approximately 1000 A) vesicles the value of relative expansion of the membrane at which its rupture occurs in a reasonable time only depends slightly on the vesicle radius. For instance, for 10 000 A vesicles it amounts to 3%. The tension of membrane rupture is about 8 dyn/cm for large vesicles. Membrane tension, although it decreases considerably as a result of rupture and pore formation, does not vanish completely. It supports the residual intravesicular pressure causing the efflux of vesicle (cell) contents. Simultaneously, osmotic influx of water through the membrane occurs that results in either complete rupture of the membrane with the efflux of the whole of the contents, or its gradual washout in either of two, quasi-steady or pulse-wise regimes. In the first case a pore is steadily open, whereas in the second case it alternately opens and closes, ejecting about 5% of internal solution each time. Lysis kinetics is analyzed. Pulse-wise regime of lysis is shown to be the most likely one.     

The properties and buoyancy-providing role of gas vacuoles in Trichodesmium Ehrenberg

All three species of the marine blue-green alga Trichodesmium collected in the Sargasso and Caribbean seas were found to possess gas vacuoles. The constituent gas vesicles were much stronger than those found in any freshwater blue-green alga, the mean critical collapse pressures being 12 bars in T. erythraeum, 34 bars in T. contortum and 37 bars in T. thiebautii. This great strength is obviously an adaptation to the hydrostatic pressures at the depths to which these organisms occur in the ocean. In each case the gas vesicles are far too strong to be collapsed by rising cell turgor pressure, though gas-vacuolation could be slowly regulated by the differential growth of gas vesicles and cells. Since the vesicles are of a similar shape and size to those in other species, the vesicle wall material must be stronger. The majority of Trichodesmium colonies collected were positively buoyant, and in all cases tested the buoyancy was dependent on the presence of gas vacuoles. The buoyancy is important in increasing the residence time of these slowly growing algae in the euphotic zone and it is responsible for the surface water-blooms which they form.     

The pressure relationships of halophilic and non-halophilic prokaryotic cells determined by using gas vesicles as pressure probes

Abstract Gas vesicles can be used to measure the hydrostatic pressure (turgor pressure) in prokaryotic cells. Halophilic cyanobacteria have turgor pressures that are substantially less than those of cyanobacteria from fresh water. Turgor pressure acts so as to tend to burst cell walls and collapse hollow gas vesicles. The halophiles take advantage of their lower turgor pressures by producing cell walls that are relatively thinner and gas vesicles that are relatively wider than in the mesophilic cyanobacteria. In this way the halophilic structure encounters the same stress and saves on material. Extreme halophiles, with negligible turgor, have been able to adopt various shapes and to produce the weakest and widest gas vesicles.     

Variability of the turgor pressure of individual cells of the gram-negative heterotroph Ancylobacter aquaticus.

Cells of Ancylobacter aquaticus were observed under phase microscopy in a chamber to which a measured pressure could be applied. The initial collapse pressure (Ca), i.e., the lowest pressure needed to collapse the most pressure-sensitive gas vesicles, was measured for 69 cells. The cells were taken from cultures in low-density balanced exponential growth, and the experiments were performed quickly so that the bacteria were in a uniform physiological state at the time of measurement. The turgor pressure, Pt, is the difference between the pressure, C, that would cause collapse of vesicles when removed from the cell and Ca. In this paper we focus on the variability of Pt from cell to cell. Part of the observed variability of Ca was due to the variability of the collapse pressure of individual vesicles (standard deviation [SD] = 90 kPa), but because there were about 100 vesicles per cell and because a change in refracted light after the fifth vesicle (approximately) collapsed probably could be detected by the human eye, the pressure would only have an SD of 18.6 kPa due to this type of sampling error. The observed SD of Pt was 42 kPa, indicating that turgor pressure did vary considerably from cell to cell. However, the turgor pressure was independent of cell size. Statistical analysis showed that Pt would decrease 6.9 kPa over a cell cycle, but with too large an SD (19.9 kPa) to be significant. This implies that the observed change in Pt over the cell cycle is not statistically significant.     

Factors governing the assembly of cationic phospholipid-DNA complexes

The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength.     

High hydrostatic pressure effects in vivo: changes in cell morphology, microtubule assembly, and actin organization

We present the first study of the changes in the assembly and organization of actin filaments and microtubules that occur in epithelial cells subjected to the hydrostatic pressures of the deep sea. Interphase BSC-1 epithelial cells were pressurized at physiological temperature and fixed while under pressure. Changes in cell morphology and cytoskeletal organization were followed over a range of pressures from 1 to 610 atm. At atmospheric pressure, cells were flat and well attached. Exposure of cells to pressures of 290 atm or greater caused cell rounding and retraction from the substrate. This response became more pronounced with increased pressure, but the degree of response varied within the cell population in the pressure range of 290-400 atm. Microtubule assembly was not noticeably affected by pressures up to 290 atm, but by 320 atm, few microtubules remained. Most actin stress fibers completely disappeared by 290 atm. High pressure did not simply induce the overall depolymerization of actin filaments for, concurrent with cell rounding, the number of visible microvilli present on the cell surface increased dramatically. These effects of high pressure were reversible. Cells re-established their typical morphology, microtubule arrays appeared normal, and stress fibers reformed after approximately 1 hour at atmospheric pressure. High pressure may disrupt the normal assembly of microtubules and actin filaments by affecting the cellular regulatory mechanisms that control cytological changes during the transition from interphase into mitosis.     

Bubble formation in crabs induced by limb motions after decompression

In vivo bubble formation was studied in the megalopal stage of the crab Pachygrapsus crassipes. The animals were equilibrated with elevated argon, nitrogen, or helium pressures then rapidly decompressed to atmospheric pressure. Voluntary motions induced bubble nucleation in leg joints after exposures to as low as 2 atm nitrogen (gauge pressure). Delays of several minutes sometimes passed between decompression and bubble formation. Mechanically stimulating the animals to move their legs increased this bubble formation, whereas immobilizing the legs before gas equilibration prevented it, even in animals decompressed from 150 atm nitrogen. We conclude that preformed nuclei are not responsible for bubbles developing in the legs of this animal. Instead, tribonucleation of bubbles apparently occurs as a result of limb motions at relatively low gas supersaturations.     

A study of the dynamics of K42 uptake by sunflower plants with the aid of a scintillation count-rate meter

Summary The time sequential mechanism of K⁴² uptake by sunflower plants from an osmotic pressure series of K⁴² HCO₃ solutions was analyzed with the aid of a localizing scintillation count-rate meter. A range of osmotic pressures from 0.3 to 28.2 atm was selected.Three phases of potassium uptake could be distinguished; an initial, exponential, rapid uptake; a period of saturation; a final phase, of renewed rapid uptake. Solutions with osmotic pressures of < 1.5 atm did not cause appreciable leaf scorching but tended to reduce uptake rates. An osmotic pressure of 7.6 atm resulted in a maximum potassium accumulation in the damaged leaves. All kinetic trends were reversed at 28.2 atm, causing rapid death by dessication. Plasmolytic effects of potassium were discussed in view of uptake stimulation.This work was undertaken while the author was a Postdoctoral fellow of the National Research Council in conjunction with the U.S. Atomic Energy Commission.     

Ethanol and acetate synthesis from waste gas using batch culture of Clostridium ljungdahlii

Single inorganic carbon source was used for production of chemicals and fuels via fermentation processes. Clostridium ljungdahlii, a strictly anaerobic autotrophic bacterium, was grown on synthesis gas to produce acetate and ethanol from gaseous substrates. C. ljungdahlii was grown on a various concentrations of carbon monoxide with synthesis gas total pressures of 0.8–1.8 atm with an interval of 0.2 atm. The cell and product yields were 0.015 g cell/g CO and 0.41 g acetate/g CO, respectively. Formation of acetate was steady and the production trend was about the same for all of the gases initial pressure and at constant cell density. The ethanol concentration was enhanced by the initial presence of hydrogen and carbon dioxide in the liquid phase. There was no substrate inhibition while C. ljungdahlii was grown in the batch fermentation, even at high system pressure of 1.6 and 1.8 atm. A desired product molar ratio of ethanol:acetate (5:1) was achieved with total gas pressure of 1.6 and 1.8 atm.     

The sequence of the major gas vesicle protein,GvpA, influences the width and strength of halobacterial gas vesicles

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.     

Gas vesicle assembly in Microcyclus aquaticus.

When observed in the electron microscope intact gas vesicles appeared as transparent areas in whole cells of Microcylus aquaticus, whereas vesicles collapsed by centrifugation were not discernible. Within 5 min of suspending cells containing collapsed vesicles in growth medium, small transparent vesicles were detected. By 15 min the average number of vesicles per cell was 15. This number remained relatively constant while the size of the vesicles increased until they attained their maximum diamtere of 100 nm. At this time the vesicles, interpreted as biconical structures, began to elongate presumably due to the synthesis of the cylindrical midsection. Closely correlated with the time at which vesicles began to elongate was the initiation of smaller vesicles which resulted in a doubling of the number of vesicles per cell by 90 min. This evidence coupled with the isolation of a mutant which assembles only the conical portions of the vesicle suggests that assembly occurs in two distinct stages subject to genetic mutation. Protein and ribonucleic acid synthesis, and presumably adenosine triphosphate formation, were required for gas vesicle assembly. In addition, inhibition of protein or ribonucleic acid synthesis resulted in a loss of extant gas vesicles. Over the time course of our study, deoxyribonucleic acid synthesis was not required for gas vesicle assembly or stability.