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Structural and functional properties of colicin M
Abstract:
Colicin M of Escherichia coli Cl139 was isolated in pure form. It consisted of a single polypeptide with a molecular weight of 27,000 +/- 2,000. Colicin M lysed sensitive cells of E. coli but had to act continuously up to the point when lysis commenced (after 20 min). Colicin M was largely resistant to hydrolysis by trypsin except when adsorbed to cells. Within 4 to 5 min after addition of colicin M, cells could be rescued by trypsin or sodium dodecyl sulfate. Later, colicin M was apparently inaccessible to these inactivating agents. Killing of cells by colicin M required Ca2+ ions. Cells could be rescued with ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetate (EGTA) immediately before the onset of lysis. Under these conditions, colicin M remained bound to the cells, and it became again sensitive to trypsin. We conclude that under the influence of EGTA colicin M is removed from its site of action and becomes again accessible to trypsin at the cell surface.
Insights
Colicin M from Escherichia coli requires continuous action and calcium ions to kill cells. Rescuing cells with EGTA before lysis makes colicin M accessible to trypsin again.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Colicins are bacteriocins produced by bacteria, including Escherichia coli, that exhibit antimicrobial activity against related strains.
- Colicin M is a specific type of colicin known to lyse sensitive E. coli cells, but its precise mechanism and inactivation have been areas of investigation.
Purpose of the Study:
- To isolate and characterize Colicin M from Escherichia coli Cl139.
- To investigate the mechanism of cell lysis induced by Colicin M, including its dependence on time, calcium ions, and inactivation by external agents.
- To determine the conditions under which Colicin M's activity can be reversed.
Main Methods:
- Isolation and purification of Colicin M.
- Determination of molecular weight using SDS-PAGE.
- Time-course experiments to assess lysis and rescue kinetics.
- Treatment with trypsin and sodium dodecyl sulfate (SDS) to evaluate colicin inactivation.
- Use of calcium ions (Ca2+) and ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetate (EGTA) to study rescue mechanisms.
Main Results:
- Colicin M was isolated as a single polypeptide with a molecular weight of 27,000 +/- 2,000.
- Continuous action of Colicin M for 20 minutes was required for cell lysis.
- Colicin M was resistant to trypsin unless adsorbed to cells, and could be inactivated by trypsin or SDS within 4-5 minutes.
- Cell killing by Colicin M necessitated Ca2+ ions.
- Cells could be rescued by EGTA immediately before lysis, rendering bound Colicin M sensitive to trypsin again.
Conclusions:
- Colicin M requires sustained interaction and Ca2+ ions for its bactericidal activity.
- The cell surface undergoes changes during colicin action, affecting colicin accessibility to inactivating agents.
- EGTA chelation of Ca2+ ions can reverse the effect of Colicin M, suggesting a reversible binding or conformational change at the cell surface.