Modular bacteriocins represent a major group of secreted protein toxins with a narrow spectrum of activity, involved in interference competition between Gram-negative bacteria. These antibacterial proteins include a domain for binding to the target cell and a toxin module at the carboxy terminus. Self-inhibition of producers is provided by coexpression of linked immunity genes that transiently ...

The effects on planar lipid bilayer membranes of carboxyl-terminal fragments derived from the bacteriocin colicin El by either proteolysis or CNBr cleavage are indistinguishable from those of the voltage-dependent parent colicin molecule. An upper limit to the length of the COOH-terminal peptide required for channel formation is 152 amino acid residues from the COOHterminal end, as indicated by...

The genetic determinant of the lethal antibiotic colicin E3 was cloned under the control of a tightly regulated promoter in the absence of the gene for its cognate inhibitor. Combination of this killing cassette with a stringent regulatory element provided a substrate-dependent conditional suicide system that was exploited for the biological containment of a Pseudomonas putida strain. The letha...

The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA). This was inserted into the Escherichia coli inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai). We constructed a series of fusion proteins in which various numbers of sp-pfColA alpha-helices were fused to alkaline phosphatase (AP). We show...

Microcin E492 is a 5,000- to 7,000-molecular-weight peptide antibiotic which depolarizes the cytoplasmic membrane of sensitive Escherichia coli K-12 cells. The microcin has many features in common with colicin V but is distinguished from it by the absence of microcin E472 immunity in ColV+ strains and by the fact that colicin V-insensitive Cir mutants of E. coli K-12 are microcin sensitive. Nei...

Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an z 68-residue segment translocat...

A survey of colicins in the ECOR reference collection of Escherichia coli is presented. Twenty-five of the 72 ECOR strains exhibited a phenotype consistent with colicin production and E. coli isolated from human hosts were more likely to be colicinogenic than those from animal hosts. Multiple representatives of two Col plasmids, low-molecular-mass ColE1 plasmids and high-molecular-mass, conjuga...

Colicin E4 was produced in very large amounts when Escherichia coli K12 strain W3110 pColE4-CT9 was grown in the presence of 0.5 microgram mitomycin C ml-1. The colicin was discharged from the producing cells when they lysed and was then degraded by a protease located on the surface of the producing cells. Colicins and proteolytic fragments derived from them could be concentrated from spent cul...

We describe a protocol, DNA sampling, for the rapid isolation of specific segments of DNA, together with bound proteins, from Escherichia coli K-12. The DNA to be sampled is generated as a discrete fragment within cells by the yeast I-SceI meganuclease, and is purified using FLAG-tagged LacI repressor and beads carrying anti-FLAG antibody. We illustrate the method by investigating the proteins ...

We report on the application of site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy to study possible oligomerization of the bacterial toxin colicin A (ColA) upon membrane insertion in vitro and in vivo. We applied SDSL-EPR protocols and optimized experimental conditions to perform continuous wave EPR experiments and double electron-electron resonance dista...