Infections with gram-positive pathogens exert immense pressure on health care systems worldwide. Especially (multi-)drug resistant bacteria are the cause for numerous hospitalizations and deaths. As resistances against approved antibiotics are spreading rapidly, the discovery of new antimicrobial compounds as well as research on potential cellular target structures are urgently necessary.

We compare the modes of action of two structurally similar antibiotic compounds. They were both found to target a broad spectrum of gram-positive bacteria. However, besides their structural similarities they significantly differ in antimicrobial potency. To determine differences in the effects on bacterial cells and on compound:target interaction, we applied a combination of in vitro and in vivo systems, as well as fluorescence microscopy. We obtained proof that both compounds target cell wall biosynthesis and bind the ultimate peptidoglycan precursor lipid II. However, we determined substantial differences in the interaction with additional target molecules. Combined with structural analysis describing the architecture of the compound:lipid complex by crystallization, we resolved the characteristics of target recognition.