Cells of Enterobacter cloacae (Clo DF13) produce a bacteriocin which is characterized by its very effective killing activity against sensitive bacteria. Purification and characterization of the excreted bacteriocin has revealed that this bacteriocin consists of an equimolar complex of two plasmidspecific gene products: the cloacin and its inhibitor the immunity protein. Dissociation of the complex by treatment with sodium dodecylsulfate induces the endonucleolytic activity of the cloacin but strongly reduces the killing activity. The purified complex possesses no activity in vitro. Both cloacin and immunity protein isolated from the complex were functionally identical to cloacin and immunity protein purified from the bacteriocinogenic cells by other methods. Reconstitution of the complex results in a partial restoration of killing activity.

The interaction of a complex of ['4C]cloacin and its immunity protein (cloacin complex) and of ['4C]cloacin itself with the outer and cytoplasmic membranes of sensitive cells has been studied to elucidate the difference in specific killing activity of both bacteriocin preparations [Eur. J . Biochem.2. No significant difference exists between cloacin complex and cloacin with respect to the specificity of adsorption, the adsorption characteristics and the kinetics of binding to the outer membrane receptors of sensitive cells grown in brain/heart infusion (about 425 receptors per cell).3. The adsorption of the bacteriocin onto the receptors shows a temperature optimum between 35 "C and 40 "C, but is not affected by variation in pH between 5.0 and 8.0 or extracellular potassium chloride up to a concentration of 0.5 M. An apparent association constant of 0.6 x lo8 M-' is calculated at 37 "C and pH 7.0.4. At 37 "C and pH 7.0 virtually all the cloacin molecules adsorbed by sensitive cells can be digested by trypsin during at least 3 h after adsorption, in contrast to the adsorbed molecules of cloacin complex which become gradually less accessible to trypsin.5. No significant difference exists between the affinity of cloacin complex and cloacin for cytoplasmic membrane vesicles isolated from sensitive cells. At 37 "C and pH 7.0 about lo5 bacteriocin molecules can bind per vesicle with an apparent association constant of about 8 x lo8 M-l.6. These results indicate that most probably the removal of immunity protein from the cloacin complex in vitro results in a strong reduction of the transfer of biologically active cloacin molecules from the cell surface receptors to the cytoplasmic membrane in vivo.7. This conclusion is confirmed by the observation that, in vivo, cloacin itself is less effective in the induction of a leakage of potassium ions across the cytoplasmic membrane compared to the cloacin complex. Concomitant with the reduction of the leakage of potassium ions a decrease in killing activity is observed. Increasing concentrations of extracellular potassium chloride gradually reduce the killing activity of cloacin complex to 3.1 % and of cloacin to 45.5 % in the presence of 0.2 M potassium chloride. 73,107-114(1977)].Cells of Enterobacter cloacae harbouring the bacteriocinogenic plasmid Clo DF13 excrete a bacteriocin which is a stoichiometric complex of two Clo-DF13-specific gene products, cloacin and immunity protein [l]. After adsorption of this cloacin complex onto the specific receptor sites located in the outer membrane of sensitive bacteria the cells are killed as the result of two events: (a) leakage of potasEnzymes. Lysozyme (EC 3.2.1.17); trypsin (EC 3.4.21.4). Note. Throughout this paper cloacin complex refers to the complex between cloacin and its immunity protein.sium ions across the cytoplasmic membrane and (b) enzymic inactivation of the ribosomes by a specific endoribonucleolytic cleavage of the 16-S ribosomal RNA. The cloacin complex possesses no significant activity on isolated ribosomes in vitro...

The production of the K99 antigen by enterotoxigenic Escherichia coli strains with various 0 antigens was investigated by means of slide agglutination tests, enzyme-linked immunosorbent assays, and hydrophobic interaction chromatography. The extent of K99 production appeared to be dependent on the nutrient medium, as well as on the incubation temperature. Minimal salt medium with glucose and semisynthetic Minca medium were the most suitable for K99 production. In complex media the production of K99 antigen was strongly reduced.

Extraction of the crude cell envelope fraction of cloacin DF13-susceptible Enterobacter cloacae strain 02 with Triton X-100 and ethylenediaminetetraacetate solubilized an outer membrane fraction which neutralized the lethal activity of cloacin DF13. A similar fraction could not be isolated from strains known to be lacking functional cloacin DF13 receptors. On this basis the isolated outer membrane fraction was assumed to contain the specific cloacin DF13 receptor. The receptor was purified to homogeneity by acetone precipitation and affinity chromatography, using cloacin DF13 as a ligand. The purified receptor was identified as a protein which consisted of a single polypeptide chain with an apparent molecular weight of 90,000 and a preponderance of acidic amino acids (pI = 5.0). The interaction of equimolar amounts of purified receptor and cloacin DF13 in vitro resulted in a complete, irreversible neutralization of the lethal activity of the bacteriocin. This interaction showed a temperature optimum at 430C but was only slightly affected by variation of the pH beween 5.0 and 8.5 or by increasing the ionic strength of the incubation buffer. The receptor had no neutralizing activity towards other bacteriocins, such as colicin El or colicin E3.Cloacin DF13 is a bacteriocin produced by bacteriocinogenic strains of Enterobacter cloacae(CloDF13) (16,47). It is characterized by its ability to kill cells of susceptible strains of Enterobacter and Klebsiella species (16). The bacteriocin is excreted as an equimolar complex of two CloDF13-determined gene products, designated cloacin and immunity protein (13). The cloacin inactivates bacterial ribosomes by endoribonucleolytic cleavage of 16S rRNA and induces a leakage of potassium ions from the susceptible cells (12,14,15). The immunity protein serves as an inhibitor of the endoribonucleolytic activity of the cloacin through a direct and specific stoichiometric interaction with the cloacin molecules (34).The initial event in the killing of susceptible bacteria by cloacin DF13 involves recognition of and binding to specific receptors localized on the surface of the cells (16,35). Recently, it has been reported that whole cells of cloacin DF13-susceptible E. cloacae strain 02 grown in brain heart infusion each adsorb about 425 molecules of cloacin DF13 (35). This adsorption has a temperature optimum between 35 and 400C, but it is not affected by variation in pH between 5.0 and 8.0 or by extracellular potassium chloride up to a concentration of 0.5 M. However, little is known about the nature of the interaction of this bacteriocin and its receptor or about the sequence of events that, after adsorption, leads to the biochemical effects observed.All receptors for bacteriocins affecting gramnegative bateria studied so far have been localized in the outer membrane fraction of the cell envelope (6,7,30,39,45,50). Furthermore, it has been shown that some of these receptors also serve as the attachment sites for certain bacteriophages and that they facilitate the passage of low-molec...

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