The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G

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-α and Lcn-β) constituting lactococcin G. These regions include the N-terminal end of Lcn-α (residues 1 to 13) and the C-terminal part of Lcn-β (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.Lactic acid bacteria (LAB) produce ribosomally synthesized antimicrobial peptides, generally referred to as bacteriocins. There are two main classes of these bacteriocins (8, 22): the class I bacteriocins (often referred to as lantibiotics) that contain the modified amino acid residues lanthionine and/or β-methyllanthionine and the class II bacteriocins that lack modified residues (8). The class II bacteriocins are further divided into four subclasses, IIa, IIb, IIc, and IId (8). Class IIa contains the pediocin-like bacteriocins, which have very similar amino acid sequences, class IIc consists of the cyclic bacteriocins, and the one-peptide, noncyclic bacteriocins that show no sequence similarity to the pediocin-like bacteriocins are placed in class IId (8). The unmodified two-peptide bacteriocins are placed in class IIb. They are unique in that they consist of two different peptides, both of which must be present, in about equal amounts, to obtain optimal antimicrobial activity (25). More than 10 two-peptide bacteriocins have been isolated and characterized (see reference 25 for original references) since the first isolation of such a bacteriocin (lactococcin G) in 1992 (21). For the two-peptide bacteriocins that have been genetically characterized, the genes encoding the two bacteriocin peptides are always found next to each other in the same operon, along with the gene encoding the immunity protein that protects the bacteriocin producer from being killed by its own bacteriocin.Lactococcin G is perhaps the best-characterized two-peptide bacteriocin (12, 18, 19, 21, 24, 26, 27). It consists of the 39-residue α peptide (termed Lcn-α) and the 35-residue β peptide (termed Lcn-β) (Fig. ​(Fig.1A).1A). Like all two-peptide bacteriocins whose mode of action has been studied, lactococcin G causes cell death by rendering the membranes of target cells permeable to various ions (18, 19). Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have revealed that the two lactococcin G peptides adopt mainly α-helical structures when they are individually exposed to membrane-like entities (12, 27). Based on the NMR structures and findings from site-directed mutagenesis studies, a structural model of lactococcin G has recently been proposed (23, 26, 27). In this model, the two complementary peptides form parallel helices that span the target cell membrane. The 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). The model also proposes that the cationic C-terminal end (residues 35 to 39, R-K-K-K-H) of Lcn-α is unstructured and forced through the target cell membrane by the membrane potential, thereby positioning the C termini of the two peptides inside the target-cell (Fig. ​(Fig.2).2). The tryptophan-rich N-terminal end of Lcn-β is also proposed to be relatively unstructured and to position itself in the outer membrane interface, thus forcing the N termini of the two peptides to remain on the outer side of the target cell membrane and the helix-helix segment to transverse the membrane (Fig. ​(Fig.2).2). This proposed structure is presumably also valid for the two-peptide bacteriocins enterocin 1071 (4, 5, 11), enterocin C (17), and lactococcin Q (32), since their sequence similarities to lactococcin G (lactococcin G has about 88 and 57% sequence identity to lactococcin Q and enterocin 1071, respectively; enterocin 1071 and enterocin C are identical except for one residue) indicate that these four bacteriocins have similar three-dimensional structures.Open in a separate windowFIG. 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 24 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.Open in a separate windowFIG. 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 26 with permission of the publisher. Copyright 2008 American Chemical Society.)The sequence of the lactococcin G operon (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ938036","term_id":"238480353","term_text":"FJ938036"}}FJ938036) has been determined, and a gene (lagC) encoding the putative lactococcin G immunity protein has been identified downstream of the two genes encoding the two lactococcin G peptides. Downstream of the two genes encoding the two enterocin 1071 peptides, a gene (entI) encoding the enterocin 1071 immunity protein has been identified (4, 11). The putative lactococcin G immunity protein shows 38% amino acid sequence identity to the enterocin 1071 immunity protein (the sequence of which was obtained from Franz et al. [11]), and both proteins consist of 110 amino acid residues (Fig. ​(Fig.1A).1A). The aim of this study was to identify which peptides or which parts of the peptides of the two-peptide bacteriocins lactococcin G and enterocin 1071 are recognized by these immunity proteins. To achieve this, combinations of wild-type lactococcin G peptides, wild-type enterocin 1071 peptides, and hybrid lactococcin-enterocin peptides were assayed against sensitive strains that were transformed with an expression plasmid carrying either the lactococcin G or the enterocin 1071 immunity gene.