Chemical Characterization, Spatial Distribution and Function of a Lipoprotein (Murein-Lipoprotein) of the E. coli Cell Wall. The Specific Effect of Trypsin on the Membrane Structure

Braun, Volkmar; Rehn, Kurt

doi:10.1111/j.1432-1033.1969.tb00707.x

Cited by 521 publications

(340 citation statements)

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“…They studied Lpp in E. coli. It had been known for some time that Lpp existed in two distinct forms -one covalently bound to the peptidoglycan, and a 'free' population that did not interact with peptidoglycan (Braun & Rehn, 1969;Braun & Bosch, 1972). The novel observation made by Cowles et al (2011) was the discovery that the 'free' population of Lpp spans the outer membrane, with one end of its trimeric structure exposed to the extracellular space.…”

Section: Introductionmentioning

confidence: 99%

Dual orientation of the outer membrane lipoprotein Pal in Escherichia coli

et al. 2015

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Peptidoglycan associated lipoprotein (Pal) of Escherichia coli (E. coli) is a characteristic bacterial lipoprotein, with an N-terminal lipid moiety anchoring it to the outer membrane. Since its discovery over three decades ago, Pal has been well studied for its participation in the TolPal complex which spans the periplasm and has been proposed to play important roles in bacterial survival, pathogenesis and virulence. Previous studies of Pal place the lipoprotein in the periplasm of E. coli, allowing it to interact with Tol proteins and the peptidoglycan layer. Here, we describe for the first time, a subpopulation of Pal which is present on the cell surface of E. coli. Flow cytometry and confocal microscopy detect anti-Pal antibodies on the surface of intact E. coli cells. Interestingly, Pal is surface exposed in an 'all or nothing' manner, such that most of the cells contain only internal Pal, with fewer cells (,20 %) exhibiting surface Pal.

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Section: Introductionmentioning

confidence: 99%

Dual orientation of the outer membrane lipoprotein Pal in Escherichia coli

et al. 2015

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“…After heating to 100 "C for 15 rnin and centrifugation (48000g, 15 "C, 1 h), the extraction was repeated with the sediment (the supernatant was discarded). The final sediment ('rigid layer') was washed with distilled water for a complete removal of SDS (Braun & Rehn, 1969; Jurgens et al, 1983). Protein was removed from the rigid layer by incubation with pronase [from Streptomycesgriseus, specific activity 8 units (mg protein)-' ; 40 units per mg rigid layer protein were used] while stirring at 37 "C for 24 h. After centrifugation at 48000g (15 "C, 20 min), the sediment (peptidoglycan-polysaccharide complex) was boiled in 4% (w/v) SDS for 15 min and subsequently freed from SDS by washing with distilled water.…”

Section: -mentioning

confidence: 99%

Isolation and Characterization of Cell Wall Components of the Unicellular Cyanobacterium Synechococcus sp. PCC 6307

1988

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Cell walls and outer membranes, free of thylakoids and cytoplasmic membranes, were isolated from the unicellular cyanobacterium Synechococcus sp. PCC 6307. Electron microscopy revealed C-shaped cell wall fragments, which were partially converted to outer membrane vesicles after removal of the peptidoglycan by lysozyme digestion. The major constituents of the outer membrane were proteins and lipopolysaccharide, while lipids and carotenoids were minor components. The polypeptide patterns of the outer membranes were dominated by two major proteins ( M , 52000 and 54000). Five strongly polar lipids (unidentified), free fatty acids and small amounts of sulpholipid were detected in extracts of partially purified cell wall fractions, but monogalactosyldiglyceride and digalactosyldiglyceride were not found. The peptidoglycan layer (10 nm thick) was also isolated. Its chemical composition indicated an Aly-type structure. The degree of cross-linkage was 57%. A polysaccharide, consisting of fucose, mannose, galactose and glucose, was bound to the peptidoglycan, most likely via muramic acid 6-phosphate. I N T R O D U C T I O NCell walls of cyanobacteria are composed of a peptidoglycan layer and an outer membrane. Outer membrane constituents include proteins, lipids and carotenoids (Drews & Weckesser, 1982;Resch & Gibson, 1983;Omata & Murata, 1984;Benz & Bohme, 1985). Lipopolysaccharides have been found in some, but not all, cyanobacteria studied (Weckesser et al., 1979; Schmidt et al., 1980a, b ; Raziuddin et al., 1983). Proteins of apparent M , 50000 to 70000 dominate the polypeptide patterns of cell walls from Synechocystis sp. PCC 6714 (Omata & Murata, 1984; Jiirgens et al., 1985), Anacystis nidulans (Golecki, 1977; Murata e t a / . , , 1983, 1985). This paper describes the isolation and chemical characterization of the cell wall, the outer membrane and the peptidoglycan of the cyanobacterium Synechococcus sp. PCC 6307, the reference strain for the high GC group (Rippka et a/., 1979). ~~Abbreviations : A,pm, diaminopimelic acid ; GlcNAc, N-acetylglucosamine ; GlcN-6-P, glucosamine 6-phosphate; ManNAc, N-acetylmannosamine ; MurNAc, N-acetylmuramic acid ; MurN-6-P, muramic acid 6-phosphate; Tyv, Tyvelose. Isolation of cell walls. Cells were broken in Tris-buffer by means of a cooled (4 "C) vibrogen shaker (type Vi 2; E. Buhler) for 20 min at full speed using a ratio of cells to glass beads (0.17-0.18 mm in diameter) of 1 : 2 (w/w). The cell homogenate was passed through a glass filter (type G-1 ; Schott) to remove glass beads. Whole cells were separated by low-speed centrifugation (300 g, 10 min). Cell envelopes were obtained from the supernatant after centrifugation at 12000 g for 30 min. They were washed with Tris-buffer until the final supernatant was colourless. The cell envelope fraction (5 ml suspension, 2 mg protein ml-l) was loaded onto a discontinuous sucrose gradient [ 10 ml each of 55, 50 and 40% (w/w) sucrose and 5 ml of 30% (w/w) sucrose in Tris-buffer] and centrifuged in a swing-out bucket rotor (AS 4.13, Ko...

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“…This uniquely bacterial modification was first observed in 1969 in a major outer membrane protein of Escherichia coli called Braun's lipoprotein and later detected in every bacterial organism studied (2). Adhesion proteins and substrate-binding subunits of ATPbinding cassette (ABC) 2 transporters are known to be attached to the membrane in this manner. Thousands of proteins predicted to be lipoproteins "by similarity" according to the presence of consensus sequences of Sec Type II or Tat Type II signal peptides are also thought to be acylated (3).…”

mentioning

confidence: 99%

“…All bacteria rely on acylation of N-terminal Cys residues of lipoproteins to allow tight anchorage on the membrane surface (1). This uniquely bacterial modification was first observed in 1969 in a major outer membrane protein of Escherichia coli called Braun's lipoprotein and later detected in every bacterial organism studied (2). Adhesion proteins and substrate-binding subunits of ATPbinding cassette (ABC) 2 transporters are known to be attached to the membrane in this manner.…”

mentioning

confidence: 99%

The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii

Serebryakova

Demina

Galyamina

et al. 2011

Journal of Biological Chemistry

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Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In Gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in Gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of Gram-negative bacteria are also present in other mollicutes and Gram-positive bacteria.Essential cellular activities such as transport, sensing, signal transduction, growth, adhesion, and pathogenesis require proteins localized on the cell surface. One of the strategies to anchor a protein at the lipid membrane is covalent modification of proteins by fatty acids or other lipids. All bacteria rely on acylation of N-terminal Cys residues of lipoproteins to allow tight anchorage on the membrane surface (1). This uniquely bacterial modification was first observed in 1969 in a major outer membrane protein of Escherichia coli called Braun's lipoprotein and later detected in every bacterial organism studied (2). Adhesion proteins and substrate-binding subunits of ATPbinding cassette (ABC) 2 transporters are known to be attached to the membrane in this manner. Thousands of proteins predicted to be lipoproteins "by similarity" according to the presence of consensus sequences of Sec Type II or Tat Type II signal peptides are also thought to be acylated (3).Bacteria of the Mollicutes class are widespread in nature and are characterized by the lack of a rigid cell wall and drastically reduced genome sizes. In fact, representatives of this class are the simplest self-replicating organisms able to independently subsist, generate energy, and adapt to changing environments (4 and references therein). Most mollicutes depend on cholesterol and are partially or totally incapable of fatty acid synthesis and therefore depend on the host or the culture medium for a constant supply of these substances (5). Acholeplasma laidlawii is a convenient model mollicute because of its relatively simple nutritional requirements and fast growth rate. A. laidlawii can synthesize limit...

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Chemical Characterization, Spatial Distribution and Function of a Lipoprotein (Murein-Lipoprotein) of the E. coli Cell Wall. The Specific Effect of Trypsin on the Membrane Structure

Cited by 521 publications

References 34 publications

Dual orientation of the outer membrane lipoprotein Pal in Escherichia coli

Dual orientation of the outer membrane lipoprotein Pal in Escherichia coli

Isolation and Characterization of Cell Wall Components of the Unicellular Cyanobacterium Synechococcus sp. PCC 6307

The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii

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