Updated publication reference for PubMed record(s): 26969785. Chelocardin is an atypical tetracycline and its mechanism of action is discussed controversially in literature. Therefore, we analyzed the bacterial response of Bacillus subtilis to chelocardin and as a control for classical tetracyclines to tetracycline by proteomic profiling. The induced marker proteins mirror the damage in the cell due the antibiotic stress. Proteomic profiling was carried out by 2D-PAGE and spot identification by nUPLC-ESI-MS/MS. The proteomic profile of chelocardin demonstrates a dual mechanism of action with protein biosynthesis inhibition and membrane damage.