Effect of a mutation preventing lipid modification on localization of the pCloDF13-encoded bacteriocin release protein and on release of cloacin DF13.
The pCloDF13-encoded bacteriocin release protein (BRP; Mr 2,871) is essential for the translocation of cloacin DF13 across the cell envelope of producing Escherichia coli cells. Overproduction of this BRP provokes lysis (quasilysis) of cells. Construction and analysis of a hybrid BRP-beta-lactamase protein (BRP-Bla) demonstrated that the BRP contains a lipid modified cysteine residue at its amino terminus and is mainly located in the outer membrane. The significance of lipid modification for the localization and functioning of the BRP was investigated. Site-directed mutagenesis was used to substitute the cysteine residue for a glycine residue in the lipobox of the BRP and the BRP-Bla protein. The mutated BRP was unable to bring about the release of cloacin DF13 and could not provide the lysis (quasilysis) of host cells. However, the mutated BRP strongly inhibited the colony-forming ability of the cells, indicating that induction of the mutated protein still affected cell viability. In contrast to the wild-type BRP-Bla protein, the mutated BRP-Bla protein was mainly located in the cytoplasmic membrane, indicating that the mutation prevented the proper localization of the protein. The results indicated that lipid modification of the BRP is required for its localization and release of cloacin DF13, but not for its lethality to host cells.
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001430:Bacteriocins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002989:Cloacin / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D005988:Glyceric Acids / Q000378:metabolism*; D007118:Immunoassay; D050356:Lipid Metabolism; D009154:Mutation; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis; D001618:beta-Lactamases / Q000235:genetics
D001426:Bacterial Proteins; D001430:Bacteriocins; D029968:Escherichia coli Proteins; D005988:Glyceric Acids; D011993:Recombinant Fusion Proteins; C052368:cloacin DF13-encoded bacteriocin release protein, E coli; D002989:Cloacin; D001618:beta-Lactamases