Nitric oxide‐forming nitrite reductases in the anaerobic ammonium oxidizer <i>Kuenenia stuttgartiensis</i>

Anaerobic ammonium-oxidizing (anammox) bacteria contribute to the global nitrogen cycle by removing fixed nitrogen from the environment. They do so via the anaerobic oxidation of ammonium to dinitrogen gas, with nitrite as terminal electron acceptor. The first step in this so-called anammox reaction is the proposed conversion of nitrite to nitric oxide by a nitrite reductase. There is an unusual diversity and redundancy in anammox nitrite reductases and to gain more insight into the puzzling redundancy and diversity, we investigated the putative reductases in the model anammox species 'Candidatus Kuenenia stuttgartiensis'. The genome of this model anammox species encodes for three putative nitrite reductases and we investigated which of these is or are active in K. stuttgartiensis strain MBR1. Active nitric oxide-producing nitrite reductases were enriched from K. stuttgartiensis cells via fast protein liquid chromatography. Nitric oxide production by the enriched nitrite reductases was followed with membrane inlet mass spectrometry. Combining the activity assays with proteomics analysis indicated that the soluble nitrite reductases NirS and HAOr most strongly correlated with enzyme activity. This indicates that K. stuttgartiensis strain MBR1 employs two distinct nitrite reductases to keep its nitric oxide pool replenished. Containing two different nitrite reductases could improve the adaptability of K. stuttgartiensis to changes in environmental nitrite concentrations.