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|Title:||Interaction of Colicin Ia With Escherichia Coli: I. Mechanisms of Colicin Insensitivity. Ii. Inhibition of Colicin Binding by Phenolic Uncouplers|
|Author(s):||Weaver, Craig Allen|
|Department / Program:||Microbiology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The mechanisms involved in two types of colicin Ia insensitivity in Escherichia coli were examined. Although whole cells of TonB strains are insensitive to colicin Ia, cytoplasmic membrane vesicles prepared from a TonB strain are sensitive to colicin-induced membrane depolarization. This finding shows that the TonB function is not required for channel formation and maintenance within the cytoplasmic membrane, but, instead, is probably required for translocation of the colicin protein to the inner membrane.
Whole cells of TonB strains are unable to transport the siderophore ferrichrome. However, spheroplasts prepared from TonB strains are able to accumulate this substrate. Presumably, disruption of the outer membrane permeability barrier allows ferrichrome direct access to the cytoplasmic membrane resulting in TonB-independent transport. It is concluded that the TonB protein in some way functions in the translocation of substrates from the cells exterior to the cytoplasmic membrane, perhaps by forming regions of apposition between outer membrane receptors and appropriate inner membrane sites.
Colicinogenic strains of E. coli are immune (insensitive) to their homologous colicin. For example, cells that are immune to colicin Ia are fully sensitive to the closely related colicin Ib which is known to utilize the same outer membrane receptor as colicin Ia. In order to study specific colicin Ia immunity, a detailed knowledge of the mode of action of colicin Ib was required. It is found that in whole cells and in membrane vesicles colicin Ib causes collapse of the transmembrane electrical potential ((DELTA)(psi)) without collapse of the proton chemical potential ((DELTA)pH). Studies performed by other workers show that colicin Ib forms channels in artificial phospholipid bilayers. Thus, colicin Ib, like colicin Ia, functions by decreasing the proton-motive force in sensitive cells due to the formation of ion-permeable channels in the cytoplasmic membrane. It follows that colicins Ia and Ib share mode of action and receptor-binding functional specificities and differ only in immunity specificity.
Using cytoplasmic membrane vesicles, it is found that specific colicin Ia immunity is expressed at the level of the cytoplasmic membrane. That is, vesicles prepared from colicin Ia-immune strains are significantly less sensitive to depolarization by colicin Ia relative to vesicles prepared from a non-immune strain, but maintain full sensitivity to colicins Ib and El. In addition, using recombinant DNA techniques, transposon mutagenesis, and analysis of plasmid-encoded polypeptides by both in vitro and in vivo methods, it is found that the product of the colicin Ia immunity gene is a 14-14500 dalton inner membrane protein. It is proposed that the mechanism of colicin Ia immunity involves the non-covalent interaction between colicin Ia and immunity protein within the cytoplasmic membrane in such a way as to prevent colicin Ia-induced membrane depolarization.
An investigation into the observed inhibition of colicin Ia binding to whole cells by the uncouplers 2,4-dinitrophenol and pentachlorophenol is presented. It is found that this inhibition is a result of interaction between the uncouplers and the iodinated form of colicin Ia that is used in binding assays. Such interaction apparently leads to a physical change in the colicin molecule which results in decreased binding to outer membrane receptors. A preliminary examination of pentachlorophenol-resistant mutants is also included. These mutants may possess an altered cell envelope which presumably leads to decreased sensitivity to uncoupler as well as to several colicins.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1981.
|Date Available in IDEALS:||2014-12-16|