The lactococcin G immunity protein recognizes specific regions in both peptides constituting the two-peptide bacteriocin lactococcin G

Abstract

Lactococcin G and enterocin 1071 are two homologous two-peptide bacteriocins. Expression vectors containing the gene encoding the putative lactococcin G immunity protein (lagC) or the gene encoding the enterocin 1071 immunity protein (entI) were constructed and introduced into strains sensitive to one or both of the bacteriocins. Strains that were sensitive to lactococcin G became immune to lactococcin G when expressing the putative lactococcin G immunity protein, indicating that the lagC gene in fact encodes a protein involved in lactococcin G immunity. To determine which peptide or parts of the peptide(s) of each bacteriocin that are recognized by the cognate immunity protein, combinations of wild-type peptides and hybrid peptides from the two bacteriocins were assayed against strains expressing either of the two immunity proteins. The lactococcin G immunity protein rendered the enterococcus strain but not the lactococcus strains resistant to enterocin 1071, indicating that the functionality of the immunity protein depends on a cellular component. Moreover, regions important for recognition by the immunity protein were identified in both peptides (Lcn-alpha and Lcn-beta) constituting lactococcin G. These regions include the N-terminal end of Lcn-alpha (residues 1 to 13) and the C-terminal part of Lcn-beta (residues 14 to 24). According to a previously proposed structural model of lactococcin G, these regions will be positioned adjacent to each other in the transmembrane helix-helix structure, and the model thus accommodates the present results.

FIG. 1.

(A) Amino acid sequence alignment of enterocin 1071 (peptides Ent1071A and Ent1071B) and lactococcin G (peptides Lcn-α and Lcn-β) and the cognate immunity proteins (Ent1071im and LcnGim, respectively). Ent1071A and Lcn-α show 59% sequence identity, whereas Ent1071B and Lcn-β show 54% sequence identity. The immunity proteins consist of 110 amino acid residues each and show 38% sequence identity. Identical amino acid residues are colored in red. (B) Amino acid sequences of the two hybrid peptides. The Lcn-α-Ent1071A hybrid peptide (α-hybrid) is termed α[1-16]/A[14-39] in this study (it is designated α2-4 in reference 24). Residues are numbered according to the corresponding amino acid positions in Lcn-α (Fig. 1A). Residues in orange are derived from Lcn-α, and residues in blue are derived from Ent1071A. The overlapping region (i.e., residues 14 to 16) is marked in red, and this region consists of residues that are identical in Lcn-α and Ent1071A. The α-hybrid peptide contains an additional lysine residue in the C-terminal end derived from Lcn-α (see reference for the construction of the hybrid peptide). The Lcn-β-Ent1071B hybrid peptide (β-hybrid) is termed β[1-13]/B[11-35] in this study (it is designated β1-6 in reference 24). Residues in orange are derived from Lcn-β, and residues in blue are derived from Ent1071B. The overlapping region (i.e., residues 11 to 13) is marked in red, and residues in this region are identical in Lcn-β and Ent1071B.

FIG. 2.

Proposed structural model of lactococcin G. The two peptides (Lcn-α and Lcn-β) form a transmembrane helix-helix structure, with the flexible tryptophan-rich N-terminal end of Lcn-β positioned in the outer membrane interface and the unstructured, highly cationic C-terminal end of Lcn-α inside the target cell membrane. The transmembrane helix-helix segment consists of the N-terminal region of Lcn-α (from about Trp-3 to Gly-22) and the C-terminal region of Lcn-β (from about Tyr-13 to Trp-32). (Adapted from reference with permission of the publisher. Copyright 2008 American Chemical Society.)