<i>In vivo</i> biosynthesis on murein—lipoprotein of the outer membrane of <i>E. coli</i>

Braun, Volkmar; Bosch, V.

doi:10.1016/0014-5793(73)80817-8

Cited by 30 publications

(11 citation statements)

References 13 publications

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“…With up to about 7.2 × 10 5 copies per cell, the lipoprotein Lpp, or Braun's lipoprotein (15), belongs to the most abundant CE proteins in exponentially growing E. coli cells. Lpp is found in both "free" and "bound" forms, the latter being covalently attached to the PG network (16,17).…”

Section: Resultsmentioning

confidence: 99%

Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

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

180

220

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Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a wellestablished imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution. cellular envelope | Escherichia coli | lipoprotein | PagL | magic angle spinning P hysiological processes rely on the concerted action of molecular entities in and across different cellular compartments. Whereas advancements in molecular imaging have provided unprecedented insights into the macromolecular organization in the subnanometer range (1), studying atomic structure and motion in situ has been challenging for structural biology. NMR has provided insight into cellular processes (2-4) and can determine entire 3D molecular structures inside living cells (5) provided that molecular entities tumble rapidly in a cellular setting. In principle, solid-state NMR (ssNMR) spectroscopy offers a complementary spectroscopic tool to monitor molecular structure and dynamics at atomic resolution in a complex setting (see ref. 6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).Here, we introduce a general approach to investigate structure and dynamics of an arbitrary molecular target and its potential molecular partners in a cellular setting. Our studies focuses on the Gram-negative bacterial cell that is characterized by a molecularly complex but architecturally unique envelope, consisting of two lipid bilayers, the inner and outer membrane (IM, OM), separated by the periplasm containing the peptidoglycan (PG) layer (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical proteins, whereas the OM is asymmetrical and consists of phospholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrelfold integral membrane proteins. LPS forms the outermost layer of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell its shape and rigidity.Using uniformly 13 C, 15 N-labeled cellular preparations of Escherichia coli, we characterized the structure and dynamics of a recombinant integral membrane protein (PagL) and other major endogenous molecular components of the cell envelope in...

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Section: Resultsmentioning

confidence: 99%

Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

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

180

220

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“…Diaminopimelate-labeled E. coli W7 cells were used to identify the chromatographic position of the released lipoprotein (21). After lysozyme treatment, the only diaminopimelate remaining is that attached to lipoprotein (21).…”

Section: Methodsmentioning

confidence: 99%

Very stable prokaryotic messenger RNA in chromosomeless Escherichia coli minicells.

Levy¹

1975

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

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E. coli minicells lack DNA, yet they make protein, the synthesis of which is sensitive to chloramphenicol but insensitive to rifamycin. This protein is coded for by very stable cellular mRNA with an estimated half-life of 40-80 min. In an R factor-containing minicell, two very different species of mRNA are observed: (i) R factor-specific mRNA with a short half-life whose synthesis is rifamycinsensitive and (if) cellular mRNA with a long half-life whose synthesis is rifamycin-insensitive. These In prokaryotic cells, gene transcription and subsequent translation to protein appear to occur coordinately (1, 2). Electron microscopic visualization has confirmed the coupling of these processes, showing protein synthesis occurring on polyribosomes attached to messenger RNA (mRNA) transcripts which are still in association with the template DNA (3). Biochemical studies have shown that prokaryotic mRNA is labile, with an average half-life of less than 3 min (4-8).More stable species of mRNAs have been described, e.g., for certain T7 phage proteins (9), for penicillinase production (10) and sporulation (11) in Bacillus cereus. Recently a group of outer membrane proteins in Escherichia coli has been identified which appear synthesized from mRNA species with half-lives of 5.5-11.5 min (12,13 (18). Sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis was performed in 10% gels for analysis of proteins (19) and in 5% gels for analysis of RNA (20).E. coli lipoprotein was determined by chromatography on Whatman 3 paper in a solvent system consisting of isobutyric acid/i M ammonium hydroxide (5:3) (21). Samples of radioactively labeled cells and minicells were boiled for 15 min to release the soluble material. After several washes in distilled water, the samples were spotted onto paper and chromatographed for 15 hr.This initial chromatography was used to remove free lipoprotein (22) Osborn, personal communication). After centrifugation at 46,000 rpm in a SW 50.1 rotor (Beckman) for 12 hr, the fractions were collected and radioactivity was determined on trichloroacetic acid-treated paper discs (25). Extraction of membranes with sodium lauryl sarcosinate (Sarkosyl) was as described (19,26).

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“…In the light of negative experiences in the past with the activity of purified colicins, especially with colicin E3 (1), we take our negative results with great caution. But interference with synthesis of murein is equally a likely possibility which we are at present investigating with an approach previously described (3).…”

Section: Resultsmentioning

confidence: 98%

Isolation, Characterization, and Action of Colicin M

Braun

Schaller

Wabl

1974

Antimicrob Agents Chemother

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Colicin M was isolated from Escherichia coli K-12 32T 19F/T1. The purified, biologically active protein had a molecular weight of 27,000. It contained phosphatidyl ethanolamine. The molecular weight found for the polypeptide chain by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 18,000. Colicin M was found to be firmly integrated in the membrane of the producing strain. The action of the colicin seems to be on the membrane, since cells of the susceptible strain E. coli K-12 ROW/V/22.1 lyse rapidly. Using the phase contrast microscope, lysis was followed by decrease in turbidity of the cell culture and release of protein into the medium. Lysis started at about 15 min after addition of colicin M and was completed after 40 to 60 min. At this time, one-third of the protein had been released from the cells. The number of viable cells dropped within 10 min to 0.01%. Colicin M induced formation of spheroplasts in the presence of 16% sucrose. The electron microscope examination revealed that at first bulges in the cell envelope appear, most frequently occurring equatorially but also occurring at sites all over the cell. In the process of spheroplast formation, the cytoplasmic membrane often retreats from one-half of the outer membrane so that the cytoplasm is confined to one hemisphere. Sucrose did not prevent cells from dying unless cells were pregrown in a sucrose containing medium for several generations before colicin M was added. With cells pregrown in the presence of sucrose, the number of survivors was 100 times higher than in the absence of sucrose.Receptors on the cell surface are of interest with regard to conjugation of bacteria, phage infection, and killing of cells by colicins. We isolated the receptor for phage T5 from Escherichia coli B and showed that colicin M binds to the same receptor (4). The receptor activity resides in a protein with a molecular weight of 85,000, which is apparently a single polypeptide chain localized in the outer membrane (5). Colicin M was originally defined by P. Fredericq on the basis that M-producing strains are consistently resistant to Ti and T5 (7). In the course of our studies on the interaction among the receptor, phage T5, and colicin M, we noticed that susceptible cells lyse rapidly in liquid culture when incubated with colicin M. Induction of rapid lysis seemed to be unique for this colicin. Cell lysis caused by other colicins studied so far is the ultimate consequence of primary damages occurring much earlier (15). In the case of colicin M, lysis is an early event. We therefore started to investigate the mode of '

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In vivo biosynthesis on murein—lipoprotein of the outer membrane of E. coli

Cited by 30 publications

References 13 publications

Cellular solid-state nuclear magnetic resonance spectroscopy

Cellular solid-state nuclear magnetic resonance spectroscopy

Very stable prokaryotic messenger RNA in chromosomeless Escherichia coli minicells.

Isolation, Characterization, and Action of Colicin M

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