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[23] Calcium-induced permeabilization of the outer membrane: A method for reconstitution of periplasmic binding protein-dependent transport systems in Escherichia coli and Salmonella typhimurium

Methods in Enzymology, ISSN: 0076-6879, Vol: 125, Issue: C, Page: 289-302
1986
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Article Description

This chapter illustrates that reconstitution of transport and chemotaxis in Escherichia coli and Salmonella typhimurium by introduction of exogenous binding protein into the periplasm of Ca 2+- treated cells of binding protein-deficient mutants is an easy and generally applicable method, which provides biochemical proof for the essential role of a specific binding protein in these processes. The described procedure opens the periplasm for a series of new studies as it allows the introduction of a variety of different macromolecules, including periplasmic binding proteins, which are chemically modified in vitro. This chapter helps in studying the in situ rate of diffusion of binding proteins by laser bleaching analysis after introduction of binding protein labeled with a fluorescent probe. These experiments enable to investigate in detail, the various aspects of the structure and functions of the periplasmic compartment of gram-negative bacteria.

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