Ultrastructure of intracytoplasmic membranes of Methanomonas margaritae cells grown under different conditions

The development of intracytoplasmic membranes of Methanomonas margaritae cells grown under different culture conditions was studied. Growth on methane was strongly accelerated by the addition of copper ions. Acceleration by copper, however, was not observed in the case of growth on methanol. Cells grown on methane with copper possessed intracytoplasmic membranes along the cell periphery. When the organism was grown in a medium lacking copper, intracytoplasmic membranes appeared as large vesicles surrounded by a unit membrane at the periphery of the cell. The vesicles originated from paired membranes due to the absence of copper in the medium. Cells grown on methanol with or without copper possessed a number of vesicles of different sizes arranged in a chain along the cell periphery. The possible relationship between membrane arrangement and methane oxidation is discussed.     

Methyl sterol and cyclopropane fatty acid composition of Methylococcus capsulatus grown at low oxygen tensions.

Methylococcus capsulatus contained extensive intracytoplasmic membranes when grown in fed-batch cultures over a wide range of oxygen tensions (0.1 to 10.6%, vol/vol) and at a constant methane level. Although the biomass decreased as oxygen levels were lowered, consistently high amounts of phospholipid and methyl sterol were synthesized. The greatest amounts of sterol and phospholipid were found in cells grown between 0.5 and 1.1% oxygen (7.2 and 203 mumol/g [dry weight], respectively). While sterol was still synthesized in significant amounts in cells grown at 0.1% oxygen, the major sterol product was the dimethyl form. Analysis by capillary gas chromatography-mass spectrophotometry showed that the phospholipid esterified fatty acids were predominantly 16:0 and 16:1 and that the hexadecenoates consisted of cis delta 9, delta 10, and delta 11 isomers. At low oxygen tensions, the presence of large amounts (25%) of cyclopropane fatty acids (cy 17:0) with the methylene groups at the delta 9, delta 10, and delta 11 positions was detected. Although the delta 9 monoenoic isomer was predominant, growth at low oxygen levels enhanced the synthesis of the delta 10 isomers of 16:1 and cy 17:0. As the oxygen level was increased, the amount of cyclopropanes decreased, such that only a trace of cy 17:0 could be detected in cells grown at 10.6% oxygen. Although M. capsulatus grew at very low oxygen tensions, this growth was accompanied by changes in the membrane lipids.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

Photopigments in Rhodopseudomonas capsulata cells grown anaerobically in darkness.

The phototrophic bacterium Rhodopseudomonas capsulata can obtain energy for dark anaerobic growth from sugar fermentations dependent on accessory oxidants such as trimethylamine-N-oxide or dimethyl sulfoxide. Cells grown for one to two subcultures in this fashion, with fructose as the energy source, showed approximately a twofold increase in bacteriochlorophyll content (per milligram of cell protein) and developed extensive intracytoplasmic membranes in comparison with cells grown photosynthetically at saturating light intensity. Cells harvested from successive anaerobic dark subcultures, however, showed progressively lower pigment contents. After ca. 20 transfers, bacteriochlorophyll and carotenoids were barely detectable, and the amount of intracytoplasmic membrane diminished considerably. Spontaneous mutants incapable of producing normal levels of photosynthetic pigments arose during prolonged anaerobic dark growth. Certain mutants of this kind appear to have a selective advantage over wild-type cells under fermentative growth conditions. Of four pigment mutants characterized (two being completely unable to produce bacteriochlorophyll), only one retained the capacity to grow photosynthetically.     

Nitrogen metabolism in a new obligate methanotroph, 'Methylosinus' strain 6

A new obligate methanotroph was isolated and characterized. It was classified as a 'Methylosinus' species and named 'Methylosinus' sp. strain 6. Nitrogen metabolism in 'Methylosinus' 6 was found to be similar to other Type II methanotrophs, including the assimilation of nitrogen exclusively by the glutamine synthetase/glutamate synthase system. However, unlike other Type II methanotrophs, it appeared that glutamine synthetase activity was regulated by adenylylation in this organism. 'Methylosinus' 6 was grown in continuous culture with either dinitrogen or nitrate as sole nitrogen source under various dissolved oxygen tensions. Higher rates of methane oxidation and a more developed intracytoplasmic membrane system were found at lower oxygen tensions with nitrate as the nitrogen source but at higher oxygen tensions with dinitrogen as the nitrogen source. This suggested that carbon metabolism was influenced by nitrogen metabolism in this organism.     

The use of the fluorescent probe alpha-parinaric acid to determine the physical state of the intracytoplasmic membranes of the photosynthetic bacterium, Rhodopseudomonas sphaeroides.

alpha-Parinaric acid has been used to determine the degree of ordering of the hydrocarbon region of purified intracytoplasmic membranes of Rhodopseudomonas sphaeroides. The usefulness of alpha-parinaric acid as a probe of membrane fluidity was established by comparison of its fluorescent properties in phosphatidylcholine vesicles with those of the more commonly used fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Both fluorescent probes were shown to monitor similar environments in the phosphatidylcholine vesicles when the phospholipids were maintained at temperatures above their phase transition temperature. The rotational mobility of alpha-parinaric acid in the intracytoplasmic membranes was determined from 0 to 50 degrees C, a region where no phase transitions were detectable. The rotational mobility of alpha-parinaric acid dissolved in vesicles formed from total extracted intracytoplasmic membrane phospholipids, was 2--3-fold greater than that measured in the intact intracytoplasmic membranes; demonstrating that the presence of protein greatly reduces the mobility of the phospholipid acyl chains of the intracytoplasmic membranes. Due to the high protein content of these membranes, the perturbing effect of protein on acyl chain mobility may extend to virtually all the intracytoplasmic membrane phospholipid.     

Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration

Plants of Ramonda serbica were dehydrated to 3.6% relative water content (RWC) by withholding water for 3 weeks, afterwards the plants were rehydrated for 1 week to 93.8% RWC. Plasma membranes were isolated from leaves using a two-phase aqueous polymer partition system. Compared with well-hydrated (control) leaves, dehydrated leaves suffered a reduction of about 75% in their plasma membrane lipid content, which returned to the control level following rewatering. Also the lipid to protein ratio decreased after dehydration, almost regaining the initial value after rehydration. Lipids extracted from the plasma membrane of fully-hydrated leaves were characterized by a high level of free sterols and a much lower level of phospholipids. Smaller amounts of cerebrosides, acylated steryl glycosides and steryl glycosides were also detected. The main phospholipids of control leaves were phosphatidylcholine and phosphatidylethanolamine, whereas sitosterol was the free sterol present in the highest amount. Following dehydration, leaf plasma membrane lipids showed a constant level of free sterols and a reduction in phospholipids compared with the well-hydrated leaves. Both phosphatidylcholine and phosphatidylethanolamine decreased following dehydration, their molar ratio remaining unchanged. Among free sterols, the remarkably high cholesterol level present in the control leaves (about 14 mol%) increased 2-fold as a result of dehydration. Dehydration caused a general decrease in the unsaturation level of individual phospholipids and total lipids as well. Upon rehydration the lipid composition of leaf plasma membranes restored very quickly approaching the levels of well-hydrated leaves.     

Isolation and Characterization of Bacteria That Grow on Methane and Organic Compounds as Sole Sources of Carbon and Energy

Bacteria capable of growth on methane and a variety of complex organic substrates as sole sources of carbon and energy have been isolated. Conditions used to rigorously establish the purity of the cultures are described. One facultative methylotroph has been studied in detail. This organism has peripherally arranged pairs of intracytoplasmic membranes characteristic of obligate methylotrophs. This isolate apparently utilizes the serine pathway of formaldehyde fixation. The location of methane oxidizers in a dimictic lake indicates that these organisms prefer less than saturating levels of dissolved oxygen. Laboratory experiments confirmed the preference of these organisms for atmospheres containing less oxygen than air.     

Fluorescence-based analysis of the intracytoplasmic membranes of type I methanotrophs

Most methanotrophic bacteria maintain intracytoplasmic membranes which house the methane-oxidizing enzyme, particulate methane monooxygenase. Previous studies have primarily used transmission electron microscopy or cryo-electron microscopy to look at the structure of these membranes or lipid extraction methods to determine the per cent of cell dry weight composed of lipids. We show an alternative approach using lipophilic membrane probes and other fluorescent dyes to assess the extent of intracytoplasmic membrane formation in living cells. This fluorescence method is sensitive enough to show not only the characteristic shift in intracytoplasmic membrane formation that is present when methanotrophs are grown with or without copper, but also differences in intracytoplasmic membrane levels at intermediate copper concentrations. This technique can also be employed to monitor dynamic intracytoplasmic membrane changes in the same cell in real time under changing growth conditions. We anticipate that this approach will be of use to researchers wishing to visualize intracytoplasmic membranes who may not have access to electron microscopes. It will also have the capability to relate membrane changes in individual living cells to other measurements by fluorescence labelling or other single-cell analysis methods.     

Lipid Composition of Plasma Membranes and Tonoplasts Isolated from Etiolated Seedlings of Mung Bean (Vigna radiata L.)

The lipid composition of plasma membranes and tonoplasts from etiolated mung bean hypocotyls was examined in detail. Phospholipids, sterols, and ceramide monohexoside(s) were the major lipid classes in both membranes. The content of phospholipids on a protein basis was higher in the tonoplast, but the content of total sterols was similar in both membranes. Accordingly, the sterol to phospholipid molar ratio in the plasma membrane was higher than that of the tonoplast. Phosphatidylethanolamine and phosphatidylcholine comprised the major phospholipids in both membranes. Phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol were identified as minor phospholipid components. The content of phosphatidylinositol and phosphatidylglycerol was relatively high in the tonoplast, comprising 11 and 5% of the total phospholipids, respectively. Although special care was taken against the degradative action of phospholipase D and phosphatidic acid phosphatase during the isolation of these membranes, by adding EDTA, EGTA, KF, choline, and ethanolamine to the homogenizing medium, significant amounts of phosphatidic acid, about 15% of the total phospholipids, were detected in the plasma membrane. On the other hand, the content of phosphatidic acid in tonoplasts and other membrane fractions was very low. This fact may indicate that high levels of phosphatidic acid occur naturally in plasma membranes. Phosphatidylglycerol in both membranes and phosphatidylinositol in the tonoplast contained high levels of palmitic acid, which comprised more than 50% of the total fatty acids. Significant differences were observed in the sterol compositions of plasma membranes and tonoplasts. More than 90% of the sterols in the plasma membrane were unesterified, while the tonoplast was enriched in glycosylated sterols, especially acylated sterylglycosides. Ceramide monohexoside was found to be specifically located in these membranes, in particular, in the tonoplast, in which it comprised nearly 17% of the total lipids.     

Plasma membrane lipid alterations induced by NaCl in winter wheat roots

A highly enriched plasma membrane traction was isolated by two phase partitioning from wheat roots ( Triticum aestivum L. cv. Vivant) grown with and without 100 m M NaCl. The lipids of the plasma membrane fraction were extracted and characterised. Phosphatidylcholine and phosphatidylethanolamine were the major phospholipids with lesser amounts of phosphandylinositol, phosphatidylglycerol, diphosphalidylglycerol, phosphatidic acid and phosphatidylseriae. NaCl decreased the total phospholipids and the phosphatidylcholine portion of the plasma membranes. Salt treatment had no effect on total sterols and glycolipids. but the relative abundance of the tree sterols was altered: cholesterol, stigma sterol and brassicasterol were significantly increased. Salt treatment resulted in an increase of the more planar/less planar ratio of the free sterols and in introduction of a double bond in the C₂₂ position in the side chain of stigma sterol and brassicasterol. The degree of fatty acid saturation of total phospholipids, phospha-tidylcholine and phosphatidylethanolamine was increased after salt treatment. These lipid changes are discussed in relation to the salt tolerance mechanism.     

The use of the fluorescent probe α-parinaric acid to determine the physical state of the intracytoplasmic membranes of the photosynthetic bacterium, Rhodopseudomonas sphaeroides

α-Parinaric acid has been used to determine the degree of ordering of the hydrocarbon region of purified intracytoplasmic membranes of Rhodopseudomonas sphaeroides. The usefulness of α-parinaric acid as a probe of membrane fluidity was established by comparison of its fluorescent properties in phosphatidylcholine vesicles with those of the more commonly used fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Both fluorescent probes were shown to monitor similar environments in the phosphatidylcholine vesicles when the phospholipids were maintained at temperatures above their phase transition temperature.The rotational mobility of α-parinaric acid in the intracytoplasmic membranes was determined from 0 to 50°C, a region where no phase transitions were detectable. The rotational mobility of α-parinaric acid dissolved in vesicles formed from total extracted intracytoplasmic membrane phospholipids, was 2–3-fold greater than that measured in the intact intracytoplasmic membranes; demonstrating that the presence of protein greatly reduces the mobility of the phospholipid acyl chains of the intracytoplasmic membranes. Due to the high protein content of these membranes, the perturbing effect of protein on acyl chain mobility may extend to virtually all the intracytoplasmic membrane phospholipid.     

Variants of the Obligate Methanotroph Isolate 761M Capable of Growth on Glucose in the Absence of Methane

Isolate 761M is an unusual type I methanotroph that possesses a complete tricarboxylic acid cycle. Variants of this methanotroph that were capable of growth with methanol (isolate 761AR) or glucose (isolate 761H) have been isolated. Cultures of isolate 761H grown with glucose and casein hydrolysate as the sole carbon and energy sources retained the ability to grow on methane, contained methane monooxygenase and 3-hexulose phosphate synthase, and possessed intracytoplasmic membranes similar to those found in thin sections of isolate 761M grown on methane. Methane monooxygenase was also present in cultures of isolate 761AR grown on methanol and casein hydrolysate.     

Isolate 761M: a New Type I Methanotroph That Possesses a Complete Tricarboxylic Acid Cycle

A methanotrophic bacterium, isolate 761M, grows slowly with methane as the sole carbon and energy source. Growth was stimulated by peptone, casein hydrolysate, glucose, and acetate plus malate. Sugars other than glucose did not stimulate growth. Growth yields, based on the amount of methane consumed, increased when other carbon sources were present, and less methane carbon was assimilated under these conditions. Methane was obligately required for growth of isolate 761M. This bacterium does not grow on rich media. Isolate 761M was found to possess hexulose phosphate synthase and intracytoplasmic membranes characteristic of other type I methanotrophs. Unlike other type I methanotrophs, this bacterium possessed alpha-ketoglutarate dehydrogenase and oxidized [2-¹⁴C]acetate to carbon dioxide.     

The fine structure of Methylococcus capsulatus

The fine structure of Methylococcus capsulatus is described. Particular emphasis is focused on the intracytoplasmic membrane system which is organized as a stacked array of flattened saccules. Each saccule is limited by a 75 A unit membrane and lies in close apposition to adjacent saccules. Methylococcus capsulatus is an obligate methylotroph whose sole source of carbon and energy is methane (or methanol). In this study methane oxidation is demonstrated for the first time in a cell-free system. Work is in progress to determine the cellular organelles which constitute the particulate fraction responsible for methane oxidation. The possible role of the intracytoplasmic membranes in energy transfer is considered in relation to the functions of stacked membrane arrays in other animal, plant and bacterial systems.     

Lipid composition of plasma membranes isolated from sunflower seedlings grown under water-stress

Plasma membranes were isolated by aqueous two-phase-partitioning from sunflower ( Helianthus annuus cv. Isabel) seedlings grown both under field irrigation and dryland conditions. Water-stressed plants showed a decrease in the leaf water potential and in the osmotic potential at full turgor, with the turgor pressure remaining at positive values. Dryland conditions also induced a reduction in the bulk modulus of elasticity. Plasma membranes of irrigated plants were characterized by high contents of phospholipids (68% of total lipids), free sterols (15. 7%) and glycolipids (9. 1%), mainly glycosphingolipids and steryl glycosides. Diacylglycerols, triacylglycerols and free fatty acids were also present. The major phospholipids were phosphatidylcholine and phosphatidylethanolamine with smaller amounts of phosphatidylinositol and phosphatidylglycerol. Following water stress, the plasma membranes showed a reduction of about 24 and 31% in total lipids and phospholipids, respectively. Also the amounts of glycolipids and diacylglycerols decreased significantly upon water stress. There was no change in free fatty acids, however, and triacylglycerols and free sterols increased. As a consequence, the free sterol to phospholipid molar ratio increased from 0. 4 to 0. 7 under water deficit conditions. The ratio of phosphatidylcholine to phosphatidylethanolamine increased from 1. 1 (control plants) to 1. 6 (water-stressed plants), while phosphatidic acid rose to 4% of total phospholipids. Dehydration did not result in any substantial change in the unsaturation level of the individual lipid classes, however. The results show that dryland conditions resulted in a marked alteration in the lipid composition of the sunflower leaf plasma membrane     

Characterization of two new facultative methanotrophs

Two new facultative methane-oxidizing bacteria have been isolated from lake water enrichments. The organisms have been characterized in terms of colony types, growth characteristics, the guanine plus cytosine content of their deoxyribonucleic acid, thin sections, oxidation rates, and carbon assimilation pathways. Methane-grown cells of both organisms contained intracytoplasmic membranes similar to those described as type II in other methanotrophic bacteria. Neither organism had such membranes when grown heterotrophically. Both organisms assimilated methane by way of the isocitrate lyase-negative serine pathway for formaldehyde incorporation. The enzymes of this pathway were high in specific activity in cells grown on methane and were at low levels in cells grown either on heterotrophic substrates or on heterotrophic substrates plus methane. It is proposed that both organisms be classified in the genus Methylobacterium as two new species, Methylobacterium ethanolicum and Methylobacterium hypolimneticum.     

Microsomal Membrane Changes during the Ripening of Apple Fruit

The changes in leakage and viscosity of microsomal membranes from apples (Malus sylvestris cv Calville de San Sauveur) at different stages of ripening were examined. These changes were correlated with those in the lipid composition of the membranes, sterols, phospholipids, and fatty acids of the phospholipids. The greatest changes in membrane properties occurred as the fruit reached its climacteric and this corresponded with a change in the sterol:phospholipid ratio in the membranes. Changes were also found in fatty acid unsaturation level, but primarily in the postclimacteric stage of ripening.     

Intracellular localization of the particulate methane monooxygenase and methanol dehydrogenase in Methylomicrobium album BG8

The methanotrophic bacterium Methylomicrobium album BG8 uses methane as a sole source of carbon and energy. This bacterium forms an extensive intracytoplasmic membrane. The first enzymes of the methane oxidation pathway are the membrane-bound particulate methane monooxygenase and the periplasmic methanol dehydrogenase. Immunoelectron microscopy with specific antibodies was used to localize these enzymes to the intracytoplasmic membrane.     

Role of sterols in the interaction of polyene antibiotics with lipid membranes

The problem whether the membrane sterols are indirect acceptors of polyenic antibiotics or they play the role of substances providing conditions (at the expense of putting in order the membrane phospholipids) for formation of conductive complexes (ionic canals) from the antibiotic molecules is discussed. The comparative study on the ability of sterols of various structure (ergosterol, 7-dehydrocholesterol, cholesterol, 5 alpha-cholestan-3 beta-ol) to interact with the membrane phospholipids and to increase the sensitivity of such membranes to amphotericin B showed no correlation between the levels of these properties. The value of the changes in the cross elasticity module (E) of artificial bilayer lipid membranes from egg lecithin on introduction of the above sterols into their composition was used as the criterion for the interaction level. The absence of correlation between the above properties of the sterols indicated that the role of the sterols in interaction of polyenic antibiotics with the membranes could not be considered as the only effect of the sterols on putting in order the phospholipids, which confirmed the hypothesis on the acceptor function of the sterols with respect to polyenic antibiotics. The study of the effect of amphotericin B on the elastic properties of the cholesterol-containing bilayer membranes isolated from egg lecithin showed tha the values of the longitudinal and cross elasticity modules of the membranes did not change during introduction into the membranes of the ionic canals.