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Quantification of group A colicin import sites
1Laboratorie d'Ingénierie et de Dynamique des Systèmes Membranaires, Centre National de la Recherche Scientifique, UPR 9027, Marseille, France.
Abstract:
Pore-forming colicins are soluble bacteriocins which form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, these colicins first bind to a receptor located on the outer membrane and then are translocated through the envelope. Colicins are subdivided into two groups according to the envelope proteins involved in their translocation: group A colicins use the Tol proteins; group B colicins use the proteins TonB, ExbB, and ExbD. We have previously shown that a double-cysteine colicin A mutant which possesses a disulfide bond in its pore-forming domain is translocated through the envelope but is unable to form a channel in the inner membrane (D. Duché, D. Baty, M. Chartier, and L. Letellier, J. Biol. Chem. 269:24820-24825, 1994). Measurements of colicin-induced K+ efflux reveal that preincubation of the cells with the double-cysteine mutant prevents binding of colicins of group A but not of group B. Moreover, we show that the mutant is still in contact with its receptor and import machinery when it interacts with the inner membrane. From these competition experiments, we conclude that each Escherichia coli cell contains approximately 400 and 1,000 colicin A receptors and translocation sites, respectively.
Insights
A pore-forming colicin A mutant, unable to form channels, still binds receptors. This finding helps estimate the number of colicin A receptors and translocation sites on Escherichia coli cells.
Area of Science:
- Microbiology
- Molecular Biology
- Biophysics
Background:
- Pore-forming colicins are bacteriocins that create ion channels in the Escherichia coli inner membrane.
- Colicin translocation involves outer membrane receptors and translocation machinery, with two groups (A and B) utilizing different protein pathways (Tol or TonB/ExbB/ExbD).
Purpose of the Study:
- To investigate the role of the pore-forming domain in colicin translocation and receptor interaction.
- To quantify the number of colicin A receptors and translocation sites on Escherichia coli.
Main Methods:
- Utilized a double-cysteine colicin A mutant with a disulfide bond in its pore-forming domain.
- Measured colicin-induced K+ efflux and performed competition experiments with wild-type colicins and the mutant.
Main Results:
- The colicin A mutant was translocated through the envelope but could not form an inner membrane channel.
- Preincubation with the mutant blocked group A colicin binding but not group B, indicating specific interactions.
- The mutant remained associated with the receptor and import machinery during inner membrane interaction.
Conclusions:
- Escherichia coli cells possess approximately 400 colicin A receptors.
- Each Escherichia coli cell has approximately 1,000 colicin A translocation sites.