Identifying bacterial heme sensor protein interacting partners under varying oxygen tensions using proximity labeling

Heme proteins play cellular roles in sensing and signaling, including reporting on labile heme pools and environmental gas/redox conditions. A major challenge in understanding these sensing pathways is the identification of proteins involved in the signaling cascade, as many of interactions are transient or low affinity. In addition, differences in oxygen levels or redox stress can alter cellular signaling, further complicating the analysis. Herein, a proximity labeling method (TurboID) is adapted for identifying proteins in proximity of the E. coli oxygen-sensing heme protein complex, DosC/P, which is involved in cyclic di-GMP metabolism, under aerobic and anaerobic conditions. These studies reveal oxygen-dependent differences in proteins in proximity of the DosC-DosP complex, which could result in differences in cellular behavior under aerobic and anaerobic conditions. Results identified glycine decarboxylase GcvP in proximity of DosCP and that GcvP binds c-di-GMP, revealing a potential new c-di-GMP signaling target and a novel role for the DosC-DosP complex in modulating aerobic one‑carbon metabolism. The application of proximity labeling developed in this work can be used not just for this oxygen-sensing complex, but can also be easily adapted to study the interactions of other heme proteins in bacteria.