Anammox bacteria originally used nitric oxide as electron accepter

<strong>Anaerobic ammonium oxidation (anammox) bacteria has been known to oxidize ammonium with nitrite as the terminal electron acceptor. Recently, however, nitric oxide (NO)-dependent anammox has been shown in <em>Candidatus</em> (<em>Ca.</em>) Kuenenia stuttgartiensis with first complete genome and it is hypothesized that NO-dependent anammox bacteria existed on early Earth. Here we report second complete genome of anammox bacteria, <em>Ca.</em> Brocadia pituitae, from metagenome of anammox bacterial community (ABC). Comparative genomics of <em>B. pituitae</em>, <em>K. stuttgartiensis</em> and 6 other anammox bacteria with nearly complete genomes revealed that their core genome structure comprises syntenic 1,152 orthologous groups. Both copper-containing (NirK) and <em>cd</em>₁ (NirS) nitrite reductases which are absent in <em>B. pitutitae</em> were not included in the core although <em>Ca.</em> Brocadia sp. UTMX2 and <em>Ca.</em> B. caroliniensis possess <em>nirK</em> gene. Indeed, IS element is inserted adjacent to the <em>nirK</em> gene in <em>Ca.</em> Jettenia caeni genome and also phylogenetic analysis revealed that the origin of nitrite reductases is derived from multiple lineages. Thus, it is clear that the <em>nirK</em> and <em>nirS </em>genes<em> </em>of anammox bacteria within family <em>Brocadiaceae</em> were acquired by horizontal gene transfer. ABC fed with nitrite and ammonium shows anammox activity but it disappears when the biomass granules are dispersed by agitation. Also, because ABC can oxidize ammonium with NO like <em>K. stuttgartiensis</em>, other nitrite reductase holders in ABC are deemed to supply NO to <em>B. pituitae</em>. Our results support the previous hypothesis that anammox bacteria originally used NO as electron accepter.</strong>