The use of mutants in the study of structure-function relationships in cloacin DF13.
A bacteriocin from cells with a mutant Clo DF13 plasmid (cloacin clp03 . immunity protein complex) and a bacteriocin from cells containing the recombinant plasmic Clo DF13 :: Tn901 (cloacin pJN82) have been isolated. Both bacteriocins like wild-type cloacin DF13, are still able to inhibit in vitro protein synthesis, but their in vivo killing activity is absent. Comparison of some physicochemical characteristics of the cloacin clp03 . immunity protein complex and wild-type cloacin complex showed no significant differences. From a comparison of the binding capacity to specific receptors on sensitive cells, the translocation through the cell wall, and the interaction with cytoplasmic membranes, it could be concluded that the cloacin clp03 complex is hampered in its translocation from the outer membrane receptor site to the cytoplasmic membrane, resulting in the observed lack in killing activity. Cloacin pJN82 is shortened at the C-terminal of the molecule by approximately ten amino acid residues. Together with its loss of in vivo killing activity it has lost its capacity to bind immunity protein. Since the immunity protein probably not only provides cloacin-producing cells with "immunity" but is also involved in the translocation of the bacteriocin to the interior of sensitive cells, the absence of this protein is probably the reason for the lack of killing activity of cloacin pJN82. The implications of these findings for the topography of the cloacin molecule as suggested by de Graaf et al. (de Graaf, F.K., Stukart, M.J., Boogerd, F.C. and Metselaar, K. (1978) Biochemistry, in press) are discussed.
D001430:Bacteriocins / Q000378:metabolism; D002268:Carboxypeptidases; D002462:Cell Membrane / Q000378:metabolism; D004754:Enterobacter / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005779:Immunodiffusion; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011955:Receptors, Drug / Q000378:metabolism; D014357:Trypsin
D001430:Bacteriocins; D010446:Peptide Fragments; D011955:Receptors, Drug; D002268:Carboxypeptidases; D014357:Trypsin