Fluorescent protein-based Zn sensors reveal distinct responses of aerobic and anaerobic Escherichia coli cultures to excess Zn.

Zinc ions are required by all known organisms. Maintaining zinc homeostasis by preventing toxic overload while ensuring sufficient acquisition for cellular functions is crucial for survival and growth of bacteria. Bacteria, however, frequently encounter and must survive in various environments. During infection in host animals, for example, bacteria are exposed to acidic conditions in the stomach and anaerobic conditions in the intestines, but the effects of oxygen on zinc homeostasis in Escherichia coli have not been well-studied. Previously, we reported a flavin-binding fluorescent protein-based zinc sensor, CreiLOV, which can respond to changes in labile Zn levels in bacteria under both aerobic and anaerobic conditions. Here, we combined the use of CreiLOV with established oxygen-dependent fluorescent protein-based sensors, inductively coupled plasma-mass spectrometry, and growth curves to evaluate how oxygen levels affect zinc uptake in E. coli. Inductively coupled plasma-mass spectrometry results showed that cells grown aerobically with added zinc acquired more zinc, but no additional zinc was accumulated when cells were grown anaerobically. Using oxygen-independent CreiLOV and the oxygen-dependent ZapCY series of sensors, intracellular labile zinc was detected in E. coli grown with varied zinc under varied conditions. Although little to no endogenous zinc was detected by any sensor in E. coli cells grown with up to 2 mM added zinc, CreiLOV revealed that when Zn was added and detected by cells in real-time, anaerobic cells required more Zn to similarly saturate the sensor. Overall, this work reveals that zinc uptake in E. coli is impacted by oxygen levels during cell growth.