A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria

Abstract

Nitrospira are barely studied and mostly uncultured nitrite-oxidizing bacteria, which are, according to molecular data, among the most diverse and widespread nitrifiers in natural ecosystems and biological wastewater treatment. Here, environmental genomics was used to reconstruct the complete genome of "Candidatus Nitrospira defluvii" from an activated sludge enrichment culture. On the basis of this first-deciphered Nitrospira genome and of experimental data, we show that Ca. N. defluvii differs dramatically from other known nitrite oxidizers in the key enzyme nitrite oxidoreductase (NXR), in the composition of the respiratory chain, and in the pathway used for autotrophic carbon fixation, suggesting multiple independent evolution of chemolithoautotrophic nitrite oxidation. Adaptations of Ca. N. defluvii to substrate-limited conditions include an unusual periplasmic NXR, which is constitutively expressed, and pathways for the transport, oxidation, and assimilation of simple organic compounds that allow a mixotrophic lifestyle. The reverse tricarboxylic acid cycle as the pathway for CO2 fixation and the lack of most classical defense mechanisms against oxidative stress suggest that Nitrospira evolved from microaerophilic or even anaerobic ancestors. Unexpectedly, comparative genomic analyses indicate functionally significant lateral gene-transfer events between the genus Nitrospira and anaerobic ammonium-oxidizing planctomycetes, which share highly similar forms of NXR and other proteins reflecting that two key processes of the nitrogen cycle are evolutionarily connected.

Fig. 1.

Genome-based model of energy metabolism in Ca. N. defluvii. Orange arrows indicate electron flow in the oxidative branches of the electron transport chain; green arrows indicate reverse electron transport from NO₂⁻ to NAD⁺. Dashed black lines point out that the membrane-integral subunit of NXR is uncertain. Dashed orange arrows show hypothetical possibilities for electron flow from NXR to the putative cyt. c-oxidase. nH⁺ indicates that the number of translocated protons is unknown because the H⁺/e⁻ ratio of the respective complexes has not been determined for Nitrospira. FRD, fumarate reductase; SDH, succinate dehydrogenase. See Table S2 for a list of the involved proteins.

Fig. 2.

Maximum-likelihood tree showing the phylogenetic positioning of selected type II enzymes of the DMSO reductase family. For phylogenetic analysis of the catalytic (α) subunits, 1,308 amino acid positions were considered. Names of validated enzymes are indicated: Nxr, nitrite oxidoreductase; Nar, membrane-bound respiratory nitrate reductase; Pcr, perchlorate reductase; Ebd, ethylbenzene dehydrogenase; Ddh, dimethylsulfide dehydrogenase; Clr, chlorate reductase; Ser, selenate reductase. Parentheses contain the number of sequences within a group or the accession number, respectively.

Fig. 3.

Schematic of the genomic regions in Ca. K. stuttgartiensis and Ca. N. defluvii, which contain shared genes coding for NXR, putative cyt. bd-like oxidases and electron carriers, and proteins of unknown function. Solid lines connect genes that are the closest homologs on the basis of protein phylogeny. Their predicted functions are in boldface. Dashed lines connect similar genes that are not the closest relatives in the respective phylogenetic protein trees. Predicted CDS and connecting lines are colored according to functional classes. CDS and intergenic regions are drawn to scale.