Summary Although metabolic pathways and associated enzymes of anaerobic ammonium oxidation (anammox) of ‘Ca. Kuenenia stuttgartiensis’ have been studied, those of other anammox bacteria are still poorly understood. NO2− reduction to NO is considered to be the first step in the anammox metabolism of ‘Ca. K. stuttgartiensis’, however, ‘Ca. Brocadia’ lacks the genes that encode canonical NO‐forming nitrite reductases (NirS or NirK) in its genome, which is different from ‘Ca. K. stuttgartiensis’. Here, we studied the anammox metabolism of ‘Ca. Brocadia sinica’. 15N‐tracer experiments demonstrated that ‘Ca. B. sinica’ cells could reduce NO2− to NH2OH, instead of NO, with as yet unidentified nitrite reductase(s). Furthermore, N2H4 synthesis, downstream reaction of NO2− reduction, was investigated using a purified ‘Ca. B. sinica' hydrazine synthase (Hzs) and intact cells. Both the ‘Ca. B. sinica’ Hzs and cells utilized NH2OH and NH4+, but not NO and NH4+, for N2H4 synthesis and further oxidized N2H4 to N2 gas. Taken together, the metabolic pathway of ‘Ca. B. sinica’ is NH2OH‐dependent and different from the one of ‘Ca. K. stuttgartiensis’, indicating metabolic diversity of anammox bacteria.

A draft genome sequence of an anaerobic ammonium-oxidizing (anammox) bacterium, “Candidatus Brocadia sinica,” was determined by pyrosequencing and by screening a fosmid library. A 4.07-Mb genome sequence comprising 3 contigs was assembled, in which 3,912 gene-coding regions, 47 tRNAs, and a single rrn operon were annotated.