؊1 for the wildtype peptide, and the minimum concentration for pore formation increased from the 1 nM to the 50 nM range. In contrast, peptides mutated in the flexible hinge region, e.g. [⌬N20/⌬M21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their in vivo activity. We found the remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit its incorporation into the peptidoglycan network. Therefore, through interaction with the membrane-bound cell wall precursor lipid II, nisin inhibits peptidoglycan synthesis and forms highly specific pores. The combination of two killing mechanisms in one molecule potentiates antibiotic activity and results in nanomolar MIC values, a strategy that may well be worth considering for the construction of novel antibiotics.The antimicrobial peptide nisin is produced by numerous strains of Lactococcus lactis and inhibits a broad range of Gram-positive bacteria (1, 2). It belongs to the lantibiotics, a group of antimicrobial peptides that is characterized by the presence of intramolecular rings formed by the thioether amino acids lanthionine and 3-methyllanthionine (3, 4). Nisin has had a long history as a potent and safe food preservative, although recent insight into the molecular mechanism of its bactericidal activity also make it interesting for biomedical applications (5, 6). Generally, the nisin-type subgroup of lantibiotics comprises elongated cationic peptides that have the capacity to adopt amphiphilic structures. Such peptides are assumed to kill microbes by disturbing the integrity of the energy-transducing membrane. Indeed, early experiments demonstrated that nisin or related lantibiotics induced rapid efflux of ions or cytoplasmic solutes such as amino acids and nucleotides. The concomitant depolarization of the cytoplasmic membrane resulted in an instant termination of all biosynthetic processes (7,8). Structural analysis in the presence of micelles indicated that the hydrophilic groups of the peptide interact with the phospholipid headgroups, and the hydrophobic side chains are immersed in the hydrophobic core of the membrane (9, 10). The wedge model as proposed by Driessen et al. (11) takes into account such structural data and proposes that the peptides insert into the membrane without losing contact with the membrane surface, resulting in the formation of a short-lived pore.Whereas the wedge model may illustrate results obtained with model membranes, a number of effects observed with intact living cells remain unexplained; in particular, the fact that nisin acts on model membranes at micromolar concentrations whereas in vivo minimal inhibitory concentration (MIC) 1 values are in the nanomolar range. The discrepancies were explained by the finding that nisin and epidermin use lipid II, the bactoprenol-bound precursor of the bacterial cell wall as a docking molecule for subsequent pore formation (12). The specificity of the nisin-lipid II interaction a...

Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing the plasma membrane. These peptides generally do not act via specific receptors and are active in the micromolar range. Here it is shown that vancomycin and the antibacterial peptide nisin Z use the same target: the membrane-anchored cell wall precursor Lipid II. Nisin combines high affinity for Lipid II with its pore-forming ability, thus causing the peptide to be highly active (in the nanomolar range).

Lipid II is a membrane-anchored cell-wall precursor that is essential for bacterial cell-wall biosynthesis. The effectiveness of targeting Lipid II as an antibacterial strategy is highlighted by the fact that it is the target for at least four different classes of antibiotic, including the clinically important glycopeptide antibiotic vancomycin. However, the growing problem of bacterial resistance to many current drugs, including vancomycin, has led to increasing interest in the therapeutic potential of other classes of compound that target Lipid II. Here, we review progress in understanding of the antibacterial activities of these compounds, which include lantibiotics, mannopeptimycins and ramoplanin, and consider factors that will be important in exploiting their potential as new treatments for bacterial infections.

The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.

SummaryGrowth of the meshlike peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function respectively of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/ LpoB and their PBP docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth.

Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II-targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.

The peptidoglycan layers surrounding bacterial membranes are essential for bacterial cell survival and provide an important target for antibiotics. Many antibiotics have mechanisms of action that involve binding to Lipid II, the prenyl chain-linked donor of the peptidoglycan building blocks. One of these antibiotics, the pore-forming peptide nisin uses Lipid II as a receptor molecule to increase its antimicrobial efficacy dramatically. Nisin is the first example of a targeted membranepermeabilizing peptide antibiotic. However, it was not known whether Lipid II functions only as a receptor to recruit nisin to bacterial membranes, thus increasing its specificity for bacterial cells, or whether it also plays a role in pore formation. We have developed a new method to produce large amounts of Lipid II and variants thereof so that we can address the role of the lipidlinked disaccharide in the activity of nisin. We show here that Lipid II is not only the receptor for nisin but an intrinsic component of the pore formed by nisin, and we present a new model for the pore complex that includes Lipid II.The cell wall is an essential structure of a bacterium, providing its shape and protecting it from bursting because of the high osmotic pressures of the cytoplasm. This wall is a threedimensional network built of identical subunits consisting of two amino sugars, N-acetylglucosamine (GlcNAc) and N-acetylmu-is attached to the carboxyl group of MurNAc. These subunits are assembled in the cytosol of bacteria using UDP-activated precursors on a special lipid carrier, undecaprenyl phosphate (for a review see Ref. 1). The integral membrane protein MraY and the peripherally membraneassociated MurG that synthesize the precursors Lipid I and II, respectively, are the key enzymes in the last two cytoplasmic steps in the formation of the subunits (Fig. 1). Subsequently, Lipid II is transported across the plasma membrane via an as of yet unknown mechanism. Thereafter, the subunits are polymerized and inserted into the pre-existing cell wall by means of the penicillin-binding proteins (for review see Ref.2). Numerous antibiotics target the cell wall synthesis, including a diverse group of antibiotics that bind to Lipid II. Perhaps the best known of these antibiotics is vancomycin, the antibiotic of last resort to treat MRSA infections (3). However, there are many others, including the polypeptide nisin, that kill cells by permeabilizing bacterial membranes. Efficient membrane permeabilization by nisin requires an interaction with Lipid II (4, 5). This designates nisin as the first example of a targeted poreforming peptide antibiotic.Two recent studies have shed light on the structural requirements within nisin for the interaction with Lipid II. A genetic approach indicated that the N terminus of nisin is involved in the interaction with Lipid II (6), and more recently we could map the binding interface toward specific residues in the N terminus using 15 N-labeled nisin (7). It is not clear yet what events lead to pore formation after the...

Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membraneThis study identifies FtsW as the flippase that translocates lipid-linked peptidoglycan precursors across the cell membrane during bacterial cell wall synthesis.