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

Abstract

Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (<u>dir</u>ect <u>amm</u>onia <u>ox</u>idation, NH₃→NH₂OH→N₂) has been identified in <i>Alcaligenes ammonioxydans</i>. However, whether Dirammox is present in other microbes, as well as 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 N₂ via NH₂OH under aerobic conditions. Genetic deletion and complementation results suggest that <i>dnfABC</i> is responsible for the ammonia oxidation to N₂ in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N₂O and trace amounts of N₂, with nitrite as the sole nitrogen source. Deletion of the <i>nirK</i> and <i>nosZ</i> genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N₂ (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that <i>pod</i> (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that <i>AFA_16745</i> (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 <i>dnfABC</i> operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of <i>dnf</i> genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. <b>IMPORTANCE</b> Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously 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 N₂ (Dirammox), has been identified in the heterotrophic bacterium <i>Alcaligenes ammonioxydans</i> HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by <i>dnf</i> genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of <i>dnf</i> genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N₂.