Specific interaction of the unmodified bacteriocin Lactococcin 972 with the cell wall precursor lipid II.

Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits septum biosynthesis in Lactococcus lactis rather than forming pores in the cytoplasmic membrane. In this study, a deeper analysis of the molecular basis of the mode of action of Lcn972 was performed. Of several lipid cell wall precursors, only lipid II antagonized Lcn972 inhibitory activity in vivo. Likewise, Lcn972 only coprecipitated with lipid II micelles. This bacteriocin inhibited the in vitro polymerization of lipid II by the recombinant S. aureus PBP2 and the addition to lipid II of the first glycine catalyzed by FemX. These experiments demonstrate that Lcn972 specifically interacts with lipid II, the substrate of both enzymes. In the presence of Lcn972, nisin pore formation was partially hindered in whole cells. However, binding of Lcn972 to lipid II could not compete with nisin in lipid II-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, possibly indicating a distinct binding site. The existence of a putative cotarget for Lcn972 activity is discussed in the context of its narrow inhibitory spectrum and the localized action at the division septum. To our knowledge, this is the first unmodified bacteriocin that binds to the cell wall precursor lipid II.

The mode of action of the lantibiotic lacticin 3147--a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II.

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.

Lipid II-based antimicrobial activity of the lantibiotic plantaricin C.

We analyzed the mode of action of the lantibiotic plantaricin C (PlnC), produced by Lactobacillus plantarum LL441. Compared to the well-characterized type A lantibiotic nisin and type B lantibiotic mersacidin, which are both able to interact with the cell wall precursor lipid II, PlnC displays structural features of both prototypes. In this regard, we found that lipid II plays a key role in the antimicrobial activity of PlnC besides that of pore formation. The pore forming activity of PlnC in whole cells was prevented by shielding lipid II on the cell surface. However, in contrast to nisin, PlnC was not able to permeabilize Lactococcus lactis cells or to form pores in 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes supplemented with 0.1 mol% purified lipid II. This emphasized the different requirements of these lantibiotics for pore formation. Using cell wall synthesis assays, we identified PlnC as a potent inhibitor of (i) lipid II synthesis and (ii) the FemX reaction, i.e., the addition of the first Gly to the pentapeptide side chain of lipid II. As revealed by thin-layer chromatography, both reactions were clearly blocked by the formation of a PlnC-lipid I and/or PlnC-lipid II complex. On the basis of the in vivo and in vitro activities of PlnC shown in this study and the structural lipid II binding motifs described for other lantibiotics, the specific interaction of PlnC with lipid II is discussed.