This review focuses on the structure and mode-of-action of non-lanthionine-containing peptide bacteriocins produced by Gram-positive bacteria. These bacteriocins may be divided into four groups: (i) the anti-listerial one-peptide pediocin-like bacteriocins that have very similar amino acid sequences, (ii) the two-peptide bacteriocins that consist of two different peptides, (iii) the cyclic bacteriocins, and (iv) the linear non-pediocin-like one-peptide bacteriocins. These bacteriocins are largely cationic, contain 20 to 70 residues, and kill cells through membrane-permeabilization. The pediocin-like bacteriocins are the ones that are best characterized. Upon contact with target membranes, their cationic N-terminal half forms a beta-sheet-like structure that binds to the target cell surface, while their more hydrophobic helical-containing C-terminal half penetrates into the hydrophobic core of target-cell membranes and apparently binds to the mannose phosphotransferase permease in a manner that results in membrane leakage. Immunity proteins that protect cells from being killed by pediocin-like bacteriocins bind to the bacteriocin-permease complex and prevent bacteriocin-induced membrane-leakage. Recent structural analyses of two-peptide bacteriocins indicate that they form a helix-helix structure that penetrates into cell membranes. Also these bacteriocins may act by binding to integrated membrane proteins. It is proposed that many membrane-active peptide bacteriocins kill target-cells through basically the same mechanism; the common theme being that a membrane-penetrating part of bacteriocins bind to a membrane embedded region of an integrated membrane protein, thereby causing conformational alterations in the protein that in turn lead to membrane-leakage and cell death.

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.

The two-peptide class II bacteriocins consist of two different unmodified peptides, both of which must be present in about equal amounts in order for these bacteriocins to exert optimal antimicrobial activity. These bacteriocins render the membrane of target cells permeable to various small molecules. The genes encoding the two peptides of two-peptide bacteriocins are adjacent to each other in the same operon and they are near the genes encoding (i) the immunity protein that protects the bacteriocin-producing bacteria from being killed by their own bacteriocin, (ii) a dedicated ABC transporter that transports the bacteriocin out of the bacteriocin-producing bacteria, and (iii) an accessory protein whose specific role is not known, but which also appears to be required for secretion of the bacteriocin. The production of some two-peptide bacteriocins is transcriptionally regulated through a three-component regulatory system that consists of a membrane-interacting peptide pheromone, a membrane-associated histidine protein kinase, and response regulators. Structure analysis of three two-peptide bacteriocins (plantaricin E/F, plantaricin J/K, and lactococcin G) by CD (and in part by NMR) spectroscopy reveal that these bacteriocins contain long amphiphilic α-helical stretches and that the two complementary peptides interact and structure each other when exposed to membrane-like entities. Lactococcin G shares about 55% sequence identity with enterocin 1071, but these two bacteriocins nevertheless kill different types of bacteria. The target-cell specificity of lactococcin G-enterocin 1071 hybrid bacteriocins that have been constructed by site-directed mutagenesis suggests that the β-peptide is important for determining the target-cell specificity.

SummaryMost bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor-mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two-peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G-resistant mutants and performed whole-genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non-sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.

The membrane-permeabilizing two-peptide bacteriocin lactococcin G consists of two different peptides, LcnG-alpha and LcnG-beta. The bacteriocin contains several tryptophan and tyrosine residues and three putative helix-helix interacting GxxxG-motifs, G 7xxxG 11 and G 18xxxG 22 in LcnG-alpha and G 18xxxG 22 in LcnG-beta. The tryptophan and tyrosine residues and residues in the GxxxG-motifs were altered by site-directed mutagenesis to analyze the structure and membrane-orientation of lactococcin G. Substituting the glycine residues at position 7 or 11 in the G 7xxxG 11-motif in LcnG-alpha with large hydrophobic or hydrophilic residues was highly detrimental, whereas small residues were tolerated. Qualitatively similar results were obtained for the G 18xxxG 22-motif in LcnG-beta. In contrast, replacement of the glycine residues in the middle of these two motifs with large hydrophilic residues was tolerated. All mutations in the G 18xxxG 22-motif in LcnG-alpha were relatively well-tolerated, indicating that this motif is not involved in helix-helix interactions. The four aromatic residues in the N-terminal part of LcnG-beta could individually be replaced by other aromatic residues, a hydrophilic positive residue, and a hydrophobic residue without a marked reduced activity, indicating that this region is structurally flexible and not embedded in a strictly hydrophobic or hydrophilic environment. The results are in accordance with a structural model where the G 7xxxG 11-motif in LcnG-alpha and the G 18xxxG 22-motif in LcnG-beta interact and allow the two peptides to form a parallel transmembrane helix-helix structure, with the tryptophan-rich N-terminal part of LcnG-beta positioned in the outer membrane interface and the cationic C-terminal end of LcnG-alpha inside the cell.

Lactobacillus plantarum produces a number of antimicrobial peptides (bacteriocins) that mostly target closely related bacteria. Although bacteriocins are important for the ecology of these bacteria, very little is known about how the peptides target sensitive cells. In this work, a putative membrane protein receptor of the two‐peptide bacteriocin plantaricin JK was identified by comparing Illumina sequence reads from plantaricin JK‐resistant mutants to a crude assembly of the sensitive wild‐type Weissella viridescens genome using the polymorphism discovery tool VAAL. Ten resistant mutants harbored altogether seven independent mutations in a gene encoding an APC superfamily protein with 12 transmembrane helices. The APC superfamily transporter thus is likely to serve as a target for plantaricin JK on sensitive cells.

The two peptides (Lcn-␣ and Lcn-␤) of the two-peptide bacteriocin lactococcin G (Lcn) were changed by stepwise site-directed mutagenesis into the corresponding peptides (Ent-␣ and Ent-␤) of the two-peptide bacteriocin enterocin 1071 (Ent), and the potencies and specificities of the various hybrid constructs were determined. Both Lcn and, to a lesser extent, Ent were active against all the tested lactococcal strains, but only Ent was active against the tested enterococcal strains. The two bacteriocins thus differed in their relative potencies to various target cells, despite their sequence similarities. The hybrid combination Lcn-␣؉Ent-␤ had low potency against all strains tested, indicating that these two peptides do not interact optimally. The reciprocal hybrid combination (i.e., Ent-␣؉Lcn-␤), in contrast, was highly potent, indicating that these two peptides may form a functional antimicrobial unit. In fact, this hybrid combination (Ent-␣؉Lcn-␤) was more potent against lactococcal strains than wild-type Ent was (i.e., Ent-␣؉Ent-␤), but it was inactive against enterococcal strains (in contrast to Ent but similar to Lcn). The observation that Ent-␣ is more active against lactococci in combination with Lcn-␤ and more active against enterococci in combination with Ent-␤ suggests that the ␤ peptide is an important determinant of target cell specificity. Especially the N-terminal residues of the ␤ peptide seem to be important for specificity, since Ent-␣ combined with an Ent-␤ variant with Ent-to-Lcn mutations at positions 1 to 4, 7, 9, and 10 was >150-fold less active against enterococcal strains but one to four times more active against lactococcal strains than Ent-␣؉Ent-␤. Moreover, Ent-to-Lcn single-residue mutations in the region spanning residues 1 to 7 in Ent-␤ had a more detrimental effect on the activity against enterococci than on that against lactococcal strains. Of the single-residue mutations made in the N-terminal region of the ␣ peptide, the Ent-to-Lcn mutations N8Q and P12R in Ent-␣ influenced specificity, as follows: the N8Q mutation had no effect on activity against tested enterococcal strains but increased the activity 2-to 4-fold against the tested lactococcal strains, and the P12R mutation reduced the activity >150-fold and only ϳ2-fold against enterococcal and lactococcal strains, respectively. Changing residues in the C-terminal half/part of the Lcn peptides (residues 20 to 39 and 25 to 35 in Lcn-␣ and Lcn-␤, respectively) to those found in the corresponding Ent peptides did not have a marked effect on the activity, but there was an ϳ10-fold or greater reduction in the activity upon also introducing Lcn-to-Ent mutations in the mid-region (residues 8 to 19 and 9 to 24 in Lcn-␣ and Lcn-␤, respectively). Interestingly, the Lcn-to-Ent F19L؉G20A mutation in an Lcn-Ent-␤ hybrid peptide was more detrimental when the altered peptide was combined with Lcn-␣ (>10-fold reduction) than when it was combined with Ent-␣ (ϳ2-fold reduction), suggesting that residues 19 and 20 (which are part of a GXXXG motif)...

Plantaricin EF is a two-peptide bacteriocin that depends on the complementary action of two different peptides (PlnE and PlnF) to function. The structures of the individual peptides have previously been analyzed by nuclear magnetic resonance spectroscopy (18555030Biochim. Biophys. Acta2008178417111719), but the bacteriocin structure and how the two peptides interact have not been determined. All two-peptide bacteriocins identified so far contain GxxxG motifs. These motifs, together with GxxxG-like motifs, are known to mediate helix–helix interactions in membrane proteins. We have mutated all GxxxG and GxxxG-like motifs in PlnE and PlnF in order to determine if any of these motifs are important for antimicrobial activity and thus possibly for interactions between PlnE and PlnF. Moreover, the aromatic amino acids Tyr and Trp in PlnE and PlnF were substituted, and four fusion polypeptides were constructed in order to investigate the relative orientation of PlnE and PlnF in target cell membranes. The results obtained with the fusion polypeptides indicate that PlnE and PlnF interact in an antiparallel manner and that the C-terminus of PlnE and N-terminus of PlnF are on the outer part of target cell membranes and the N-terminus of PlnE and C-terminus of PlnF are on the inner part. The preference for an aromatic residue at position 6 in PlnE suggests a positioning of this residue in or near the membrane interface on the cells inside. Mutations in the GxxxG motifs indicate that the G5xxxG9 motif in PlnE and the S26xxxG30 motif in PlnF are involved in helix–helix interactions. Atomistic molecular dynamics simulation of a structural model consistent with the results confirmed the stability of the structure and its orientation in membranes. The simulation approved the anticipated interactions and revealed additional interactions that further increase the stability of the proposed structure.