Membrane depolarization kills dormant Bacillus subtilis cells by generating a lethal dose of ROS

The bactericidal activity of several antibiotics partially relies on the production of reactive oxygen species (ROS), which is generally linked to enhanced respiration and requires the Fenton reaction. Bacterial persister cells, an important cause of recurring infections, are tolerant to these antibiotics because they are in a dormant state. Here, we use Bacillus subtilis cells in stationary phase, as a model system of dormant cells, to show that pharmacological induction of membrane depolarization enhances the antibiotics' bactericidal activity and also leads to ROS production. However, in contrast to previous studies, this results primarily in production of superoxide radicals and does not require the Fenton reaction. Genetic analyzes indicate that Rieske factor QcrA, the iron-sulfur subunit of respiratory complex III, seems to be a primary source of superoxide radicals. Interestingly, the membrane distribution of QcrA changes upon membrane depolarization, suggesting a dissociation of complex III. Thus, our data reveal an alternative mechanism by which antibiotics can cause lethal ROS levels, and may partially explain why membrane-targeting antibiotics are effective in eliminating persisters.