Nitrification expanded: discovery, physiology, and genomics of a nitrite-oxidizing bacterium from the phylum Chloroflexii

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, a major process of the
biogeochemical nitrogen cycle, but the recognized diversity of this guild is surprisingly low and only
two bacterial phyla contain known NOB. Here, we report on the discovery of a chemolithoautotrophic
nitrite oxidizer that belongs to the widespread phylum Chloroflexi not previously known to
contain any nitrifying organism. This organism, named Nitrolancetus hollandicus, was isolated from
a nitrifying reactor. Its tolerance to a broad temperature range (25-63 1C) and low affinity for nitrite
(Ks¼1mM), a complex layered cell envelope that stains Gram positive, and uncommon membrane
lipids composed of 1,2-diols distinguish N. hollandicus from all other known nitrite oxidizers.
N. hollandicus grows on nitrite and CO2, and is able to use formate as a source of energy and
carbon. Genome sequencing and analysis of N. hollandicus revealed the presence of all genes
required for CO2 fixation by the Calvin cycle and a nitrite oxidoreductase (NXR) similar to the NXR
forms of the proteobacterial nitrite oxidizers, Nitrobacter and Nitrococcus. Comparative genomic
analysis of the nxr loci unexpectedly indicated functionally important lateral gene transfer events
between Nitrolancetus and other NOB carrying a cytoplasmic NXR, suggesting that horizontal
transfer of the NXR module was a major driver for the spread of the capability to gain energy from
nitrite oxidation during bacterial evolution. The surprising discovery of N. hollandicus significantly
extends the known diversity of nitrifying organisms and likely will have implications for future
research on nitrification in natural and engineered ecosystems.