Structural heterogeneity of the cytoplasmic and outer membranes of Escherichia coli

Leij, Lfmh de; Witholt, Bernard

doi:10.1016/0005-2736(77)90396-0

Cited by 46 publications

(33 citation statements)

References 29 publications

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“…The distributions of PBPs 4, 6, 7, and 8 (A), PBPs la, lb, and 5 (B), PBPs 2 and 3 (C), NADH oxidase and lactate dehydrogenase (LDH) (D), and total protein (E) are shown. vesicles, each associated with a unique set of proteins (10,13,15,16). This heterogeneity may reflect the existence of specific inner membrane regions having biologically discrete functions.…”

Section: Resultsmentioning

confidence: 99%

“…Distinct membrane regions or domains appear to exist which may be specially involved with cell elongation, division, or maintenance of shape (2,5,6,10,13,16). For example, electron microscopic observations suggest two circumscribed areas within the inner membrane to which proteins might be specifically localized: zones of adhesion, points where the inner and outer membranes seem to be contiguous (4), and the periseptal annulus, predicted to be the forerunner of septal division sites (reviewed in reference 31).…”

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confidence: 99%

“…Inner and outer membranes from gram-negative bacterial envelopes have been separated from one another on the basis of density by sucrose gradient ultracentrifugation (20,24,33,34). By using this general approach, inner membrane fragments from Escherichia coli exhibited asymmetries in protein and lipid composition (10,13,15). For example, Ishidate et al used a two-step sucrose separation protocol with which they were able to isolate four or five vesicle populations with different protein profiles (13).…”

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confidence: 99%

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Separation of Escherichia coli penicillin-binding proteins into different membrane vesicles by agarose electrophoresis and sizing chromatography

et al. 1989

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Membrane vesicles from the envelope of Escherichia coli were separated by electrophoresis through dilute agarose and by sizing chromatography through Sephacryl S-1000. These techniques revealed that proteins were associated with different subsets of vesicles. In particular, dilute agarose electrophoresis clearly separated the inner membrane penicillin-binding proteins (PBPs) into different vesicle groups. Vesicles containing PBPs 4, 6, 7, and 8 migrated rapidly through agarose; vesicles with PBPs 1a, 1b, 2, 3, and 5 eluted later. With the exception of PBP 4, which migrated with PBPs 1 through 5, chromatography through Sephacryl S-1000 was able to distinguish the same two vesicle sets, though the extent of separation was poorer than with agarose. The existence of these associations among vesicles and proteins suggests that there is an organization to the inner membrane of E. coli which is not observed when membrane vesicles are separated solely on the basis of density in sucrose gradients.

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Section: Resultsmentioning

confidence: 99%

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Separation of Escherichia coli penicillin-binding proteins into different membrane vesicles by agarose electrophoresis and sizing chromatography

et al. 1989

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“…Spheroplasts were disrupted by sonication as described by De Ley and Witholt (10). Inner membrane vesicles were separated from outer membrane vesicles by equilibrium centrifugation in a discontinuous sucrose gradient (30 to 70%) in a Spinco SW65 rotor (115,000 x g; 4°C for 50 h).…”

Section: Methodsmentioning

confidence: 99%

Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX)

1991

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We have described a gene coding for an Enterobacter cloacae protein, provisionally called OmpX (J. Stoorvogel, M. J. A. W. M. van Bussel, J. Tommassen, and J. A. M. van de Klundert, J. Bacteriol. 173:156-160, 1991). In the work reported here, OmpX was localized in the cell envelope by means of sucrose gradient fractionation of membrane vesicles. Overproduction of OmpX in Escherichia coli from a multicopy plasmid resulted in a reduction in the amount of OmpF. No accumulation of OmpF, of its uncleft precursor, or of its degradation products could be detected in various cell fractions by Western immunoblot analysis using monoclonal antibodies produced in response to OmpF. A decrease in the rate of synthesis of ompF mRNA was indicated by a beta-galactosidase assay in an ompF-lacZ fusion strain containing the cloned ompX gene and by Northern (RNA) blot analysis. These results indicate that the inhibition is at the level of transcription. Colony hybridization, using an internal ompX fragment as a probe, showed a widespread distribution of the ompX gene among clinical isolates of members of the family Enterobacteriaceae. To study the function of the OmpX protein and its role in the regulation of porin protein synthesis, the ompX gene was deleted from the Enterobacter cloacae chromosome and replaced by the aphA gene. The absence of the ompX gene had no apparent effect on cell growth or on the regulation of the porin proteins.

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“…They will be referred to as the polyclonal antiserum against PBP lb because they showed no difference in specificity. Goat anti-mouse immunoglobulin G (,y) (9). Thin sections of the membranes from sonicated cells showed large envelope vesicles, very small membrane vesicles, coiled envelopes, and outer membranes (results not shown).…”

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confidence: 93%

Topology of penicillin-binding protein 1b of Escherichia coli and topography of four antigenic determinants studied by immunocolabeling electron microscopy

Blaauwen

Nanninga

1990

J Bacteriol

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A method has been developed to study the orientation of proteins in the cytoplasmic membrane of Escherichia coli. Vesicles from sonicated cells were incubated in droplets on electron microscope support grids in sequence with a monoclonal antibody (MAb) against a protein with an unknown orientation (PBP lb) followed by a MAb against a periplasmic component (peptidoglycan). The different MAbs were made visible with 5-and 10-nm gold-conjugated secondary antibodies, respectively. PBP lb appeared to colabel with peptidoglycan. The labeling of PBP lb in membrane vesicles with MAbs against four different epitopes was further used to estimate the number of PBP lb molecules per cell. Approximately 1,400 PBP lb molecules per cell grown in broth were labeled. The spatial distribution of the epitopes of the MAbs was studied by inmunocolabeling of pairs of MAbs and by competitive antibody-binding inhibition. It could be tentatively concluded that the four epitopes form a cluster of antigenic determinants which occupy less than half of the surface of PBP lb.

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Structural heterogeneity of the cytoplasmic and outer membranes of Escherichia coli

Cited by 46 publications

References 29 publications

Separation of Escherichia coli penicillin-binding proteins into different membrane vesicles by agarose electrophoresis and sizing chromatography

Separation of Escherichia coli penicillin-binding proteins into different membrane vesicles by agarose electrophoresis and sizing chromatography

Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX)

Topology of penicillin-binding protein 1b of Escherichia coli and topography of four antigenic determinants studied by immunocolabeling electron microscopy

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