Genetic Foundations of Direct Ammonia Oxidation (Dirammox) to N 2 and MocR-like Transcriptional Regulator DnfR in Alcaligenes faecalis JQ135

Ammonia oxidation is an important process of both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox ( dir ect amm onia ox idation, NH 3 →NH 2 OH→N 2 ) has been identified in Alcaligenes ammonioxydans . However, whether Dirammox is present in other microbes and its genetic regulation remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N 2 via NH 2 OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N 2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N 2 O and trace amounts of N 2 with nitrite as sole nitrogen source. Deletion of genes nirK and nosZ that are essential for denitrification did not impair the capability of JQ135 to oxidize ammonia to N 2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5′-TGGTCTGT-3′ in the promotor region. Bioinformatic survey showed that homologs to dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that besides A. a mmonioxydans , Dirammox also occurs in other bacteria, and is regulated by the MocR-family transcriptional regulator DnfR. Importance Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previous known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation (Anammox), and complete ammonia oxidation (Comammox)) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N 2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. Bioinformatic survey suggests that homologs to dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N 2 .