The renowned antimicrobial activity of copper stems in part from its ability to undergo redox cycling between Cu11/21 oxidation states. Bacteria counter copper toxicity with a network of sensors that often include two-component signaling systems to direct transcriptional responses. As in typical two-component systems, ligand binding by the extracellular domain of the membrane bound copper sensor component leads to phosphorylation and activation of the cognate response regulator transcription factor. In Listeria monocytogenes, the plasmid-borne CopRS two-component system upregulates both copper resistance and lipoprotein remodeling genes upon copper challenge, but the oxidation state of copper bound by CopS is unknown. Herein, we show CopS utilizes a triad of key residues (His-His-Phe) that are predicted to be at the dimerization interface and that are analogous with the Escherichia coli CusS copper sensor to specifically bind Cu11/Ag11 and activate CopR transcription. We demonstrate Cu21 only induces CopRS if first reduced by electron transport systems, as strains lacking menaquinone carriers were unable to respond to Cu21. The flavin-dependent extracellular electron transport system (EET) was the main mechanism for metal reduction, capable of either generating inducing ligand (Cu21 to Cu11) or removing it by precipitation (Ag11 to Ag0). We show that EET flux is directly proportional to the rate of Cu21 reduction and that since EET activity is low under oxygenated conditions when a competing respiratory chain is operating, CopRS signaling in turn is activated only under anaerobic conditions. EET metal reduction thus sensitizes cells to copper while providing resistance to silver under anaerobic growth. IMPORTANCE Two-component extracellular copper sensing from the periplasm of Gram-negative bacteria has been well studied, but copper detection at the cell surface of the Gram-positive L. monocytogenes is less understood. Collectively, our results show that EET is most active under anaerobic conditions and reduces Cu21 and Ag11 to, respectively, generate or remove the monovalent ligands that directly bind to CopS and lead to the induction of lipoprotein remodeling genes. This reducing activity regulates CopRS signaling and links the upregulation of copper resistance genes with increasing EET flux. Our studies provide insight into how a two-component copper sensing system is integrated into a model monoderm Firmicute to take cues from the electron transport chain activity.
All Science Journal Classification (ASJC) codes
- Molecular Biology