Research article summary (published 30 Dec 1989):

Localization of penicillin-binding protein 1b in Escherichia coli: immunoelectron microscopy and immunotransfer studies.

Full Abstract

We report the localization of penicillin-binding protein 1b (PBP 1b) in Escherichia coli KN126 and in an overproducing construct containing plasmid pHK231. We used PBP 1b-specific antiserum for the immunoelectron microscopy of ultrathin sections of whole cells and for immunoelectrophoresis of cytoplasm and isolated membrane fractions. We studied ultrathin sections of both glutaraldehyde-fixed cells that had been embedded after progressively lowering the temperature and cryofixed cells that had been freeze-substituted in Lowicryl K4M and HM20. Most of the PBP 1b-specific label was observed in the inner membrane (IM) and the adjacent cytoplasm, much less was observed in the outer membrane (OM); appreciable amounts were also seen in the bulk cytoplasm. Distribution and intensity of label were both temperature dependent: temperature shift-up to 37 degrees C, causing PBP 1b overproduction in the construct, showed a statistically highly significant increase in label of the IM, including a cytoplasmic zone (of at least 30 nm in depth) adjacent to the IM, a zone we termed the membrane-associated area. Concomitant with the temperature shift-up, a decrease in label density was observed in the bulk cytoplasm. Increased label was also found in IM-OM contact areas (zones of membrane adhesion). The periplasm did not show significant label. Western blotting (immunoblotting) revealed PBP 1b in most of the isolated membrane fractions; however, the highest label density was found in membrane fractions of intermediate density, supporting the suggestion of an increased concentration of PBP 1b in the membrane adhesion zones. In summarizing, we propose that PBP 1b is present in the membrane-associated area of the cytoplasm, from where proteins (such as PBP 1b or thioredoxin) gain access to their specific insertion sites in the envelope. The use of several methods of immunoelectron microscopy provided the first unequivocal evidence for localization of PBP 1b at membrane adhesion sites. Since such sites are specifically labeled with anti-PBP 1b serum, we hypothesize that they contain parts of the machinery for assembly and growth of the murein layer.

Author information

Author/s: Bayer, M H (MH); Keck, W (W); Bayer, M E (ME);

Affiliation: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

Grants: AI-10414 (Agency:NIAID NIH HHS) ; CA-06927 (Agency:NCI NIH HHS) ; RR-05539 (Agency:NCRR NIH HHS)

Journal and publication information

Publication Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.

Journal: Journal of bacteriology (J Bacteriol), published in UNITED STATES. (Language: eng)

Reference: 1990-Jan; vol 172 (issue 1) : pp 125-35

Dates: Created 1990/02/08; Completed 1990/02/08; Revised 2008/11/20;

PMID: 2403537, status: MEDLINE (last retrieval date: 2/18/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Acyltransferases - analysis Bacterial Proteins Blotting, Western Carrier Proteins Cell Membrane - analysis Cytoplasm - analysis Electrophoresis, Polyacrylamide Gel Escherichia coli - analysis

Hexosyltransferases - analysis Immunoelectrophoresis Microscopy, Electron Multienzyme Complexes - analysis Muramoylpentapeptide Carboxypeptidase Penicillin G - metabolism Penicillin-Binding Proteins Peptidyl Transferases - analysis

Associated Chemicals: Bacterial Proteins (0) ; Carrier Proteins (0) ; Multienzyme Complexes (0) ; Penicillin-Binding Proteins (0) ; Penicillin G (61-33-6) ; Acyltransferases (EC 2.3.-) ; Peptidyl Transferases (EC 2.3.2.12) ; Hexosyltransferases (EC 2.4.1.-) ; Muramoylpentapeptide Carboxypeptidase (EC 3.4.17.8)

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