Ancient electrogenic survival metabolism of β-proteobacterial ammonia oxidizers for oxygen deficiency

Oxygen availability is critical for microbes as some are obligatorily dependent on oxygen for energy conservation. However, aerobic microbes that live in environments with varying oxygen concentrations experience pressures over evolutionary time, selecting alternative energy metabolisms that relax the dependence on oxygen. One such capacity is extracellular electron transfer (or EET), which is the ability to transfer electrons from central metabolism to extracellular oxidants such as iron and manganese oxides. We posit that the {beta}-proteobacterial ammonia-oxidizing bacteria, highly specialized lineages heretofore recognized as strict aerobes, can be capable of EET as they have been constantly observed in oxygen-limiting and depleted environments. Here, we show that a strictly aerobic ammonia-oxidizer, Nitrosomonas communis, utilized a poised electrode to maintain metabolic activity in anoxic conditions. The presence and activity of multi-heme cytochromes suggested that direct electron transfer is the mechanism underlying EET. Molecular clock models suggest that the ancestors of {beta}-proteobacterial ammonia oxidizers appeared after the oxygenation of Earth when the oxygen levels were >10-4 pO2 (PAL), suggesting their aerobic origins. Phylogenetic reconciliations of gene and species trees show that the multi-heme c-type EET proteins in Nitrosomonas and Nitrosospira were acquired by gene transfer from {beta}-proteobacteria during oxygen scarcity. The preservation of EET metabolism over billions of years under fluctuating oxygen levels and aspects of EET physiology in {beta}-proteobacterial ammonia oxidizers might explain how they have been coped with oxygen stress and survived under oxygen deprivation. SIGNIFICANCEMetabolic versatility can permit typically aerobic microbes to survive in anaerobic conditions when oxygen is deficient as a terminal electron acceptor. This article demonstrates a previously unidentified anaerobic extracellular electron transfer metabolism that operates in aerobic {beta}-proteobacterial ammonia oxidizers and reconstructs the evolutionary history of this metabolism, linking it to the early history of Earths oxygenation. Our approach contributes to the understanding of metabolisms in the N-cycle and their evolution on Earth, as well as how aerobic microbes manage to retain energy generation under oxygen-limiting or depleted conditions. AUTHOR CONTRIBUTIONSAG designed the physiological research with PRG and the phylogenetic research with GF; AG performed the research, AG analyzed the data with PRG and GF. AG wrote the paper, and all authors edited and approved the manuscript.