The outer membrane of Proteus mirabilis II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes

Rottem, Shlomo; Hasin, Miriam; Razin, Shmuel

doi:10.1016/0005-2736(75)90355-7

Cited by 40 publications

(15 citation statements)

References 21 publications

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“…We believe that this more fluid domain corresponds to the external leaflet of the cell wall, as defined in our model (see Discussion). When spin-labeled fatty acids are added to structures containing domains of different degrees of fluidity-for example, the outer membrane of Gram-negative bacteria (14)-they become inserted nearly exclusively into the most fluid domain, as can be seen from the observation that the fluidity reported by such probes (15,16) is fairly close to that of the fluid phospholipid domain (13,16) and very far away from that of the much less fluid lipopolysaccharide domain (13,16). We have shown, furthermore, that 5-SASL added to intact M. chelonae cells produced ESR spectra of a similar 2T11 value as that added to the isolated cell wall (Fig.…”

Section: Resultsmentioning

confidence: 95%

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Fluidity of the lipid domain of cell wall from Mycobacterium chelonae.

Liu

Rosenberg²,

Nikaido³

1995

Proc. Natl. Acad. Sci. U.S.A.

134

118

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The mycobacterial cell wall contains large amounts of unusual lipids, including mycolic acids that are covalently linked to the underlying arabinogalactanpeptidoglycan complex. Hydrocarbon chains of much of these lipids have been shown to be packed in a direction perpendicular to the plane of the cell surface. In this study we examined the dynamic properties of the organized lipid domains in the cell wall isolated from Mycobacterium chelonae grown at 30°C. Differential scanning calorimetry showed that much of the lipids underwent major thermal transitions between 30°C and 65°C, that is at temperatures above the growth temperature, a result suggesting that a significant portion of the lipids existed in a structure of extremely low fluidity in the growing cells. Spin-labeled fatty acid probes were successfully inserted into the more fluid part of the cell wall. Our model of the cell wall suggests that this domain corresponds to the outermost leaflet, a conclusion reinforced by the observation that labeling of intact cells produced electron spin resonance spectra similar to those of the isolated cell wall. Use of stearate labeled at different positions showed that the fluidity within the outer leaflet increased only slightly as the nitroxide group was placed farther away from the surface. These results are consistent with the model of mycobacterial cell wall containing an asymmetric lipid bilayer, with an internal, less fluid mycolic acid leaflet and an external, more fluid leaflet composed of lipids containing shorter chain fatty acids. The presence of the low-fluidity layer will lower the permeability of the cell wall to lipophilic antibiotics and chemotherapeutic agents and may contribute to the well-known intrinsic resistance of mycobacteria to such compounds.Mycobacterium tuberculosis causes tuberculosis, a disease that kills 3 million people a year mostly in developing countries. In industrialized countries, the appearance of multiple-drugresistant strains of M tuberculosis has made it once again a major public health problem (1). Furthermore, Mycobacterium leprae causes leprosy in >10 million people worldwide, and several Mycobacterium species, including M. avium complex, M. kansasii, M. fortuitum, and M. chelonae, cause opportunistic infections in immunologically compromised patients, such as AIDS patients. The treatment of mycobacterial infection is often difficult because these bacteria are intrinsically resistant to most common antibiotics and chemotherapeutic agents (2). This general resistance is thought to be related to the unusual structure of mycobacterial cell wall, which is located outside the cytoplasmic membrane yet contains large amounts of lipids, many of them with unusual structure. The major fraction of the cell wall is occupied by unusually long-chain fatty acids containing 70-90 carbons, the mycolic acids (3). The peptidoglycan, which contains N-glycolylmuramic acid instead of the usual N-acetylmuramic acid, is linked to arabinogalactan via a phosphodiester bridge. About 10% of the...

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Section: Resultsmentioning

confidence: 95%

“…Stearic acid spin labels carrying doxyl groups at positions 5, 7, 9, 12, or 16 [5-, 7-, 9-, 12-, or 16-doxylstearic acid spin label (SASL)] were from Molecular Probes.…”

Section: Methodsmentioning

confidence: 99%

Fluidity of the lipid domain of cell wall from Mycobacterium chelonae.

Liu

Rosenberg²,

Nikaido³

1995

Proc. Natl. Acad. Sci. U.S.A.

134

118

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“…Possibly the same mechanism of "crosslinking" is responsible for the (20,21). Second, the outer membrane is less fluid, by-several criteria, than the inner membrane or membranes of most other cells (10)(11)(12); when the lipopolysaccharide of this membrane contains a long-chain polysaccharide, this polysaccharide causes the greater rigidity because the mobility of spin-labeled fatty acids is restricted in membranes with long-chain, rather than short-chain, lipopolysaccharide (13). Short-chain, lipopolysaccharides apparently have much less or no effect on mobility (13,22).…”

Section: Discussionmentioning

confidence: 99%

“…A total of 37% of the fragments (as measured by [3H]glycerol) was found in fractions 1-20. When galactose was present only during the late part of growth, no membrane fragments were found at the position of OM1 and most of the outer membrane fragments were low in density (like OM2). Only 16% of the fragments were found in fractions [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] (Fig. 3B).…”

Section: Methodsmentioning

confidence: 99%

“…Lipopolysaccharide molecules, which are made in the inner membrane and inserted into special loci in the outer membrane, are known to diffuse from these loci to cover the entire surface, indicating that they are capable of lateral diffusion within this membrane (5)(6)(7)(8)(9). However, the motion, or diffusion, of lipids or lipid probes within the bilayer appears to be more restricted in the outer membrane than in other membranes (10)(11)(12) and, at least when it has a long polysaccharide side chain, lipopolysaccharide participates in this restriction (13). These studies raise the question of whether lipopolysaccharide motion is itself restricted within the membrane and if so, how.…”

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confidence: 99%

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Domains involving nonrandom distribution of lipopolysaccharide in the outer membrane of Escherichia coli.

Leive¹

1977

Proc. Natl. Acad. Sci. U.S.A.

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The present data demonstrate that the outer membrane of Escherichia coli contains domains of lipopolysaccharide that do not intermix freely with each other. A strain of E. coli lacking galactose epimerase was grown with galactose, for varying periods of time, which permits formation of a long polysaccharide, and without galactose, which results in a short polysaccharide. Such cultures yielded outer membrane fragments that were heterogeneous in lipopolysaccharide composition, some containing more long-than short-chain lipopolysaccharide, and vice versa. The kinetics of formation of these fragments suggest that lipopolysaccharide initially enters the membrane at points from which it can diffuse but ultimately is organized into domains that do not mix with each other, at least when lipopolysaccharides of different composition are present in the same organism.The outer membrane of Gram-negative cells has a relatively simple composition in which three or four polypeptides comprise 60-80% of the total protein (1, 2), most of the phospholipid is phosphatidylethanolamine (3, 4), and lipopolysaccharide is the primary polysaccharide-containing component (4). However, the organization of these components, and their rearrangement during growth, is unknown. Lipopolysaccharide molecules, which are made in the inner membrane and inserted into special loci in the outer membrane, are known to diffuse from these loci to cover the entire surface, indicating that they are capable of lateral diffusion within this membrane (5-9). However, the motion, or diffusion, of lipids or lipid probes within the bilayer appears to be more restricted in the outer membrane than in other membranes (10)(11)(12) and, at least when it has a long polysaccharide side chain, lipopolysaccharide participates in this restriction (13). These studies raise the question of whether lipopolysaccharide motion is itself restricted within the membrane and if so, how.The current experiments show that older lipopolysaccharide, which has already diffused away from its point of insertion, is organized into domains or areas that do not intermix freely with newer lipopolysaccharide of a different composition. MATERIALS AND METHODSStrains, Media, and Isotopes. Escherichia colt J5, a mutant of E. coil 0111:B4 lacking galactose epimerase, was used throughout. Cells were grown at 370 with vigorous aeration on proteose peptone beef extract medium (4) that had been tested for absence of galactose (9).

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Die Permeabilität der äußeren Bakterienmembran

Nikaido

1979

Angewandte Chemie

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Ein beliebtes Objekt für das Studium von Struktur/Funktions‐Beziehungen bei biologischen Membranen ist die äußere Membran Gram‐negativer Bakterien. Diese Membran hat u.a. die Aufgabe, den Einstrom von Nahrungsstoffen und den Ausstrom von Abfallprodukten zu ermöglichen. Untersuchungen unter Verwendung von Mutanten zeigten, daß es mindestens zwei allgemeine Wege für die Diffusion kleiner Moleküle durch die äußere Membran gibt: einen für hydrophobe und einen für hydrophile Verbindungen. Beim „hydrophoben Weg”︁ löst sich die hydrophobe Verbindung im Inneren der Membran und verläßt es entsprechend dem Verteilungskoeffizienten. Bei Wildtypformen ist dieser Weg nicht gangbar – vermutlich wegen des Fehlens von Bereichen mit Phospholipid‐Doppelschichten. Kleine hydrophile Moleküle durchdringen die Membran dagegen beim Wildtyp und bei Mutanten durch wassergefüllte Poren.

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The outer membrane of Proteus mirabilis II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes

Cited by 40 publications

References 21 publications

Fluidity of the lipid domain of cell wall from Mycobacterium chelonae.

Fluidity of the lipid domain of cell wall from Mycobacterium chelonae.

Domains involving nonrandom distribution of lipopolysaccharide in the outer membrane of Escherichia coli.

Die Permeabilität der äußeren Bakterienmembran

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