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

Abstract

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 °C) and low affinity for nitrite (K(s)=1 mM), 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 CO(2), 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 CO(2) 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.

Figure 1

Morphology and in situ detection of N. hollandicus. (a) Phase contrast micrograph of an autotrophically grown pure culture. (b) Transmission electron micrograph of cells from the same culture as in panel a. (c) In situ detection of N. hollandicus and AOB in activated sludge from the bioreactor that was the source of the isolated N. hollandicus strain. The populations were detected by FISH using the probes Ntlc804 (specific for N. hollandicus), Cluster6a192 (specific for AOB of the N. oligotropha lineage) and the EUB338 probe mix targeting most Bacteria. Cells of N. hollandicus appear magenta, AOB green-blue and other bacteria dark blue. (d) Thin section electron micrograph of a cell from a culture that was grown mixotrophically in medium containing NO₂⁻, HCO₃/CO₂ and formate. The layered cell envelope is clearly visible. N, nucleoide; CW-EL, external layer of the cell wall; CW-PG, peptidoglycane; CM, cytoplasmic membrane.

Figure 2

Growth of N. hollandicus in mineral medium containing NO₂⁻ as the sole energy source. NO₂⁻ was stoichiometrically oxidized to NO₃⁻ while biomass protein increased. Results of two replicate experiments are shown. Nitrate was measured only in one experiment.

Figure 3

Phylogenetic affiliation of N. hollandicus. 16S rRNA-based Bayesian inference tree (s.d.=0.009202) showing the affiliation of N. hollandicus (boldface) with the class Thermomicrobia (gray box) of the phylum Chloroflexi. Pie charts indicate statistical support of nodes based on bootstrap analysis or Bayesian inference, respectively. Conflicting topologies among the different methods are also indicated. Representatives of the phylum Actinobacteria were used as outgroup. The scale bar represents 10% estimated sequence divergence. MB, Bayesian inference; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbor joining.

Figure 4

Cell metabolic cartoon constructed from the annotation of the N. hollandicus genome. Enzyme complexes of the electron transport chain are labeled by Roman numerals. CLD, chlorite dismutase; FDH, formate dehydrogenase; FmdA, formamide amidohydrolase; TCA-cycle, tricarboxylic acid cycle; CRISPR, clustered, regularly interspaced short palindromic repeats.

Figure 5

Phylogeny of NXR and related enzymes, based on the α subunit. Maximum likelihood tree showing the phylogenetic positioning of selected type II enzymes of the dimethyl sulfoxide reductase family, based on the catalytic (α) subunits. For the analysis 1102 amino acid positions were considered. Names of validated enzymes are indicated (Nxr, boldface; 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. Pie charts indicate statistical support of nodes based on bootstrap analysis or Bayesian inference, respectively. More distantly related molybdoenzymes were used as outgroup.