Intracytoplasmic membrane, phospholipid, and sterol content of Methylobacterium organophilum cells grown under different conditions.

Intracytoplasmic membranes were present in Methylobacterium organophilum when cells were grown with methane, but not methanol or glucose, as the sole carbon and energy source. Cells grown with methane as the carbon and energy source and low levels of dissolved oxygen had the greatest amount of intracytoplasmic membrane. Cells grown with increased levels of dissolved oxygen had less intracytoplasmic membrane. The amount of total lipid correlated with the amount of membrane material observed in thin sections. The individual phospholipids varied in amount, but the same four were present in M. organophilum grown with different substrates and oxygen levels. Phosphatidyl choline was present as a major component of the phospholipids. Sterols were present, and they differed from those in the type I methylotroph Methylococcus capsulatus. The relative amounts of different sterols and squalene changed with the substrate provided for growth. The greatest amounts of sterols were found in methane-grown cells grown at low levels of dissolved oxygen. None of the unusual or usual membrane components assayed was uniquely present in the intracytoplasmic membranes.     

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.     

Disappearence of the intracytoplasmic membranes inRhodopseudomonas sphaeroides

The photosynthetic bacterium,Rhodopseudomonas sphaeroides, can be grown phototrophically (light, anaerobiosis), of chemotrophically (dark, aerobiosis). In the first case, it contains intracytoplasmic membranes with photosynthetic pigments. When shifted from phototrophy to chemotrophy these membranes disappear in an unknown fashion. In the present experiment, samples were taken for electron microscopy, cell density and bacteriochlorophyll determinations after shift from phototrophy to chemotrophy. The density of intracytoplasmic vesicles was measured on micrographs. During the first 2h growth is very slow and the ultrastructure remains unaltered. As growth resumes, the vesicles disappear at a rate which implies that they are not incorportated into the cytoplasmic membrane, nor actively digested, but remain intact and become increasingly diluted in the cytoplasm as the culture grows. The size of the vesicles was estimated to about 500 Å. The number of vesicles in phototrophically grown cells was calculated to about 575 per cell, and after 6h chemotrophic growth to about 100. The areas of the cytoplasmic and intracytoplasmic membranes are roughly calculated.Abbreviations Bchl bacteriochlorophyll - CM cytoplasmic membranes - ICM intracytoplasmic membranes     

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.     

Ultrastruct of Methylosinus trichosporium as revealed by freeze etching.

The methane-oxidizing bacterium Methylosinus trichosporium forms extensive intracytoplasmic membranes that lie near the cell periphery and paralled to it. These membranes enclose cavities within the cytoplasm and exist as flattened, balloon-like vesicles. The internal membranes are passed along to both cells during budding. The bacteria accumulate poly-beta-hydroxybutyrate granules that lie in the center of the cells, neither within the internal membrane vesicles nor attached to them. Intercellular bridges result in the formation of chains of bacteria two to four cells in length.     

Morphological and ultrastructural variations inAzotobacter vinelandii growing in oxygen-controlled continous culture

Membrane development as a response to growth at different oxygen tensions (from about 1% to 100% saturation of the medium with air) was determined inAzotobacter vinelandii strain OP. The organisms were grown in a carbonlimited chemostat either on atmospheric nitrogen or on ammonium as nitrogen sources. Both types of cultures increased not only the number of intracytoplasmic membrane vesicles per cell but also the cell volume with aeration. As the ratio of length per width stayed largely constant increases of volume resulted in decreases of the cell surface area, representing the surface area of the peripheral cytoplasmic membrane, per cell volume. While in nitrogen-fixing cells the proportion of intracytoplasmic membrane surface area per cytoplasmic membrane surface area increased from 1:2 to 3:1 the ratio stayed almost constant in ammonium-assimilating cells. The data suggest that oxygen controls changes in the ratio of intracytoplasmic to cytoplasmic membrane surface areas only under conditions of nitrogen fixation.Abbreviations CM Cytoplasmic membrane - ICM intracytoplasmic membrane     

Isolation of Intracytoplasmic Membrane from the Methanotrophic Bacterium Methylomicrobium album BG8

Methane-oxidizing bacteria, including Methylomicrobium album BG8, form an intracytoplasmic membrane in addition to the cytoplasmic and outer membranes of the cell envelope. Techniques to isolate the intracytoplasmic membrane of M. album BG8 were developed. An intracytoplasmic membrane fraction was separated from a cell envelope fraction on the basis of sedimentation velocity in sucrose density gradients. Proteins associated with the particulate methane monooxygenase were found in both membrane fractions. Received: 27 July 1999 / Accepted: 30 August 1999     

Growth yields of methanotrophs

Summary The growth yield ofMethylococcus capsulatus (Bath) on methane was dependent on the availability of copper in the growth medium. In nitrate mineral salts medium the carbon conversion efficiency increased by 38%, concomitant with the transition from soluble to particulate methane monooxygenase, after transfer from low to high copper medium. An increase in growth efficiency was also observed with ammonia as nitrogen source but not when methanol replaced methane as carbon source. The high growth efficiency is attributed to a reduced NADH requirement for methane oxidation. This could only arise if methanol dehydrogenase was capable of electron transfer, either directly or indirectly to the particulate methane monooxygenase (MMO). The carbon conversion efficiency from methanol with nitrate as nitrogen source was as high as theoretically predicted. It is suggested that the previously low yields of methanotrophs grown on methanol resulted from the use, as nitrogen source, of ammonia which was oxidised by the MMO still present under these growth conditions. The term ‘methanotroph’ is used throughout to distinguish those organisms capable of growth on methane from ‘methylotrophs’ capable of growth on reduced C, compounds other than methane     

Cytochemistry of cytochrome oxidase in the cytoplasmic and intracytoplasmic membranes of Azotobacter vinelandii

Vegetative cells of Azotobacter vinelandii contain a system of intracytoplasmic membranes in the form of numerous internal vesicles. The three-dimensional morphology of these internal vesicles was established by an examination of stereopair electron micrographs of negatively stained cells. The vesicles assumed a variety of forms ranging from nearly spherical units to short, curved tubules. These structures were found at the periphery of the cytoplasm, subjacent to the cytoplasmic membrane. Large flattened cisternae were also present in some cells. The amount of intracytoplasmic membrane varied widely even among individual cells from the same culture. The total surface area of the intracytoplasmic membranes was greater than that of the cytoplasmic membrane in many cells. To assess the possible association of cytochrome oxidase activity with the intracytoplasmic membranes, enzyme localization experiments were conducted with the cytochemical substrate 3,3'-diaminobenzidine. The results showed that a cyanide-sensitive cytochrome oxidase activity is located at the intracytoplasmic membrane. The quantity of cytochrome oxidase activity present in the internal membranes is probably less than that present in the cytoplasmic membrane.     

Ultrastructure of carotenoid mutant strain R-26 of Rhodopseudomonas sphaeroides

Stained thin-sections and freeze-fractured preparations of the carotenoid-less mutant strain R-26 of Rhodopseudomonas sphaeroides grown photosynthetically revealed 2 morphological kinds of intracellular membrane systems- spherical vesicles distributed throughout the cytoplasm and lamellae confined to the periphery of the cell. The lamellar membranes appeared to be large, flattened vesicles.Non-Standard Abbreviations R Rhodopseudomonas - E exoplasmic-half - P protoplasmic-half     

Structure of Methylosinus trichosporium exospores

Methylosinus trichosporium exospores did not display a well-defined cortex or an exosporium. A thick, electron-dense exospore wall was characteristic of the exospores. Located on the exterior of the exospore wall was a cell wall to which a well-defined capsule was attached. An extensive lamellar intracytoplasmic membrane system characteristic of the kind in vegetative cells of this bacterium was present along the interior periphery of the exospore wall. Upon germination of M. trichosporium exospores, the thick exospore wall gradually disappeared and a germ tube formed. The intracytoplasmic membranes of the exospores extended into the germ tube which did not possess the extensive fibrillar capsule observed on the dormant exospore. Cup-shaped exospores which have an ultrastructure similar to that of mature exospores except that they are invaginated also germinated upon exposure to methane.     

Effect of Copper on Methylomonas albus BG8

Addition of copper to the medium for Methylomonas albus BG8 increased cell yield and methane monooxygenase activity. Intracytoplasmic membrane was formed only in cells grown with copper supplementation. Additionally, the abundances of two major membrane proteins were affected by copper in the growth medium. These findings indicate that effects of copper on the physiology of methanotrophic bacteria are not limited to those on types II and X.     

ONTOGENY AND CYTOCHEMISTRY OF ALKALOIDAL VESICLES IN LATICIFERS OF PAPAVER SOMNIFERUM L. (PAPAVERACEAE)

Development of alkaloidal vesicles in laticifers of opium poppy, Papaver somniferum L., was investigated at the ultrastructural level. Laticifer initials possessed abundant endoplasmic reticulum throughout their dense cytoplasm. During differentiation the endoplasmic reticulum organized into long, folded sheets that were parallel to the longitudinal walls along the periphery of the cell. Vesicles appeared to be derived from dilation of endoplasmic reticulum. This relationship was confirmed through cytochemical data obtained with zinc iodide-osmium tetroxide and osmium tetroxide impregnation. Alkaloidal vesicles had electron-dense regions or caps that occurred early in laticifer differentiation, but these caps became less conspicuous in mature cells. Caps appeared to be derived from small particles which condensed along the inner surface of the vesicle membrane and subsequently accumulated at one or two positions along the membrane of the vesicle.     

Die Fraktionierung des Membransystems von Rhodopseudomonas capsulata und seine Morphogenese

Zusammenfassung Das Membransystem von Rps. capsulata setzt sich aus Cytoplasmamembran und intracytoplasmatischen Membranen zusammen. In anaeroben Lichtzellen und in Dunkelzellen unter geringen Sauerstoffpartialdrucken bestehen die intracytoplasmatischen Membranen aus Vesikeln, bei Anzucht unter hohen Sauerstoffspannungen sind sie tubulär. Auch nach einer 8stündigen Kultur bei 400 mm (Hg) Sauerstoffpartialdruck, d.h. unter Bedingungen, die eine BChl-Synthese vollständig hemmen, enthalten die Zellen noch intracytoplasmatische Tubuli an einem Zellpol.Nach Zellaufschluß mit der French pressure cell gelang es durch anschließende fraktionierte Zentrifugation und Reinigung der Partialfraktionen über Ficoll-Gradienten 3 membranhaltige Banden zu isolieren. Die leichte Bande besteht vorwiegend aus Membranfragmenten der Cytoplasmamembran. Die mittlere Bande enthält die intracytoplasmatischen Tubuli aerob angezogener Zellen. Die schwere Bande, die den höchsten Reinheitsgrad aufweist, setzt sich aus den intracytoplasmatischen Vesikeln der Licht-bzw. der semiaeroben Dunkelzellen zusammen. ¹⁴C-Markierungsexperimente und elektronenmikroskopische Beobachtungen sprechen für morphologische und morphogenetische Zusammenhänge zwischen den Membranfraktionen und stützen damit die Hypothese, daß alle Membrantypen in einer Zelle Teile eines zusammenhängenden Membransystems sind. Die einzelnen Membrantypen können reversibel ineinander überführt werden.

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The fractionation and morphogenesis of the membrane system of Rhodopseudomonas capsulata

Summary The membrane system of Rhodopseudomonas capsulata, strain 37b4 is investigated in cells cultivated anaerobically in the light and in the dark, respectively, at different oxygen partial pressures (pO₂). The intracytoplasmic membrane vesicles of anaerobically light grown and semiaerobically [5 mm (Hg) pO₂] dark grown cells show similar diameters (30–50 nm). Growing aerobically in darkness the cells contain tubular intracytoplasmic membranes with comparable diameters. An increase of the pO₂ up to 400 mm (Hg) results in a slightly decreased growth rate and in a complete inhibition of bacteriochlorophyll synthesis and intracytoplasmic membrane formation. After 8 h of cultivation under these conditions tubular membranes are still found. However, they are restricted to one cell pole only.In order to isolate the membranes, cells were broken by means of a French pressure cell. The crude, membrane fractions (sedimented at 104000-314000xg, 60 min) are purified by centrifugation on a Ficoll density gradient. This results in the formation of three membrane fractions. The light fraction consists of small vesicular particles derived from the cytoplasmic membrane. Crude membrane fractions of all cells sedimented at 314 000xg contain a relative high percentage of these particles. Intracytoplasmic membranes of aerobically grown cells are found in a middle band. Their tubular structure remains unaffected if the cells are treated by lower pressures during homogenization. The heavy band contains the intracytoplasmic membrane vesicles from light grown and from semiaerobically dark grown cells in a highly purified form.After treatment with butanol or NaCl and subsequently with lysozyme plus EDTA cells release flat membrane fragments which still exhibit invaginations. This shows once more that the membranes are connected to each other.Pulse chase experiments with (2-¹⁴C)-acetate support the hypothesis that the cytoplasmic membrane and intracytoplasmic membranes are transformable into each other. So they should be looked at as a morphogenetical unit.

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Abkürzungen BChl Bacteriochlorophyll - CM Cytoplasmamembran - ICM intracytoplasmatische Membran - pO₂ Sauerstoffpartialdruck     

Particulate methane monooxygenase from Methylosinus trichosporium is a copper-containing enzyme

Particulate methane monooxygenase (pMMO) has been exfoliated and isolated from membranes of the Methylosinus trichosporium IMV 3011. It appears that the stability of pMMO in the exfoliation process is increased with increasing copper concentration in the growth medium, but extensive intracytoplasmic membrane formed under higher copper concentration may inhibit the exfoliation of active pMMO from membrane. The highest total activity of purified pMMO is obtained with an initial concentration of 6 microM Cu in the growth medium. The purified MMO contains only copper and does not utilize NADH as electron donor. Treatment of purified pMMO with EDTA resulted in little change in copper level, suggesting that the copper in the pMMO is tightly bound with pMMO.     

Methane and trichloroethylene oxidation by an estuarine methanotroph, Methylobacter sp. strain BB5.1.

An estuarine methanotroph was isolated from sediment enrichments and designated Methylobacter sp. strain BB5.1. In cells grown on medium with added copper, oxidation of methane and trichloroethylene occurred with similar Ks values, but the Vmax for trichloroethylene oxidation was only 0.1% of the methane oxidation Vmax. Cells grown on low-copper medium did not oxidize trichloroethylene and showed a variable rate of methane oxidation.     

Intracytoplasmic membranes in oxygen-limited chemostat cultures of Methylosinus trichosporium OB3b: Biocatalytic implications of physiologically balanced growth

Summary The continuous culture growth conditions for induction of intracytoplasmic membranes in Methylosinus trichosporium OB3b are described. During oxygen-limited, nitrate-excess chemostat culture, organisms have an extensive intracytoplasmic membrane system and particulate, cell-free methane mono-oxygenase (MMO). Under methane limitation fewer intracytoplasmic membranes are seen, while under all other conditions tested, membranes are absent and cell-free MMO is entirely soluble. These findings may be important in relation to the development of oxidative biotransformation processes using this bacterium.     

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.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

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.