Mode of action of LciA, the lactococcin A immunity protein.
Monoclonal antibodies were raised against a fusion between the Escherichia coli maltose-binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A. One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L. lactis producer cell. LciA was present in the cytosolic, the membrane-associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A. The monoclonal antibody specifically reacted with right-side-out vesicles obtained from a strain producing the immunity protein. It did not react with inside-out vesicles of the same strain, or with right-side-out vesicles obtained from a strain producing both LciA and lactococcin A. Also, externally added lactococcin A blocked the interaction between the antibody and right-side-out vesicles obtained from a strain producing only LciA. The epitope in LciA was localized between amino acid residues 60 and 80. As the epitope could be removed from right-side-out vesicles by proteinase K, it is located at the outside of the cell. The immunity protein contains a putative alpha-amphiphilic helix from residue 29 to 47. A model is proposed in which this helix is thought to traverse the membrane in such a way that the C-terminal part of the protein, containing the epitope, is on the outside of the cell. Vesicle-fusion studies together with leucine-uptake experiments suggest that the immunity protein interacts with the putative receptor for lactococcin A, thus preventing pore formation by the bacteriocin.
Venema
K
K
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Centre, University of Groningen, Haren, The Netherlands.
Haverkort
R E
RE
Abee
T
T
Haandrikman
A J
AJ
Leenhouts
K J
KJ
de Leij
L
L
Venema
G
G
Kok
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D001426:Bacterial Proteins; D001430:Bacteriocins / Q000276:immunology*; D001483:Base Sequence; D002462:Cell Membrane / Q000502:physiology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D019286:Endopeptidase K; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D005798:Genes, Bacterial; D013294:Lactococcus lactis / Q000235:genetics / Q000276:immunology; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology; D012697:Serine Endopeptidases
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001430:Bacteriocins; D017931:DNA Primers; D004269:DNA, Bacterial; D000939:Epitopes; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C083085:lactococcin A immunity factor, bacteria; C069325:lactococcin A; D012697:Serine Endopeptidases; D019286:Endopeptidase K