Metagenomic Evidence for the Presence of Comammox Nitrospira-Like Bacteria in a Drinking Water System

Abstract

We report metagenomic evidence for the presence of a Nitrospira-like organism with the metabolic potential to perform the complete oxidation of ammonia to nitrate (i.e., it is a complete ammonia oxidizer [comammox]) in a drinking water system. This metagenome bin was discovered through shotgun DNA sequencing of samples from biologically active filters at the drinking water treatment plant in Ann Arbor, MI. Ribosomal proteins, 16S rRNA, and nxrA gene analyses confirmed that this genome is related to Nitrospira-like nitrite-oxidizing bacteria. The presence of the full suite of ammonia oxidation genes, including ammonia monooxygenase and hydroxylamine dehydrogenase, on a single ungapped scaffold within this metagenome bin suggests the presence of recently discovered comammox potential. Evaluations based on coverage and k-mer frequency distribution, use of two different genome-binning approaches, and nucleic acid and protein similarity analyses support the presence of this scaffold within the Nitrospira metagenome bin. The amoA gene found in this metagenome bin is divergent from those of canonical ammonia and methane oxidizers and clusters closely with the unusual amoA gene of comammox Nitrospira. This finding suggests that previously reported imbalances in abundances of nitrite- and ammonia-oxidizing bacteria/archaea may likely be explained by the capacity of Nitrospira-like organisms to completely oxidize ammonia. This finding might have significant implications for our understanding of microbially mediated nitrogen transformations in engineered and natural systems. IMPORTANCE Nitrification plays an important role in regulating the concentrations of inorganic nitrogen species in a range of environments, from drinking water and wastewater treatment plants to the oceans. Until recently, aerobic nitrification was considered to be a two-step process involving ammonia-oxidizing bacteria or archaea and nitrite-oxidizing bacteria. This process requires close cooperation between these two functional guilds for complete conversion of ammonia to nitrate, without the accumulation of nitrite or other intermediates, such as nitrous oxide, a potent greenhouse gas. The discovery of a single organism with the potential to oxidize both ammonia and nitrite adds a new dimension to the current understanding of aerobic nitrification, while presenting opportunities to rethink nitrogen management in engineered systems.

Keywords: Nitrospira; comammox; drinking water systems.

FIG 1

(A, left) Radial cladogram based on RAxML-based maximum-likelihood phylogeny (500 bootstraps, gamma distribution model, and LG+F substitution model) constructed using 16 syntenic ribosomal proteins, with prominent phylum-level affiliation of branches indicated. The reference sequence from the phylum Aquificae was used as the outgroup for this analysis. (Right) Expanded view showing the placement of the Nitrospira metagenome bin within the phylum Nitrospirae, with >90% bootstrap support indicated. The comammox Nitrospira species are in green, while strict NOB are in red. A detailed annotated tree is provided in the supplemental material, while the concatenated alignment used to perform phylogenetic analyses is available on figshare (http://dx.doi.org/10.6084/m9.figshare.1619897). (B, left) Radial cladogram based on RAxML-based maximum-likelihood phylogeny (1,000 bootstraps, gamma distribution model, GTR substitution model) constructed using 16S rRNA genes from 87 reference sequences within the genus Nitrospira and the partial 16S rRNA gene within the Nitrospira metagenome bin. The different lineages are in different colors. (Right) Expanded view of Nitrospira lineage 2, showing the placement of the 16S rRNA genome from the Nitrospira metagenome bin alongside recently published comammox Nitrospira organisms. Comammox Nitrospira bacteria are in green, while strict NOB are in red. (C) Bayesian inference phylogeny (20,000 generations, standard deviation = 0.02) nxrA genes from Nitrospirae and Planctomycetes, with the root placed on outgroup Nitrococcus mobilis (class Gammaproteobacteria). Nodes with >99% bootstrap support are indicated with black circles. The nxrA genes from the Nitrospira metagenome bin cluster within lineage 2. (D) Arrangement of genes in the region from kbp 69.1 to 92.7 of scaffold 158 with ammonia oxidation genes and those on scaffold 123 with an arrangement similar to that of comammox Nitrospira bacteria. Hypothetical proteins are colored in gray, while genes annotated as coding for hypothetical proteins but showing homology to orfM, amoD, and amoE are also marked. The solid line indicates continuity between two fragments of scaffold 158, while the dotted line indicates likely connectivity between scaffold 123 and scaffold 158.

FIG 2

(A) Tiled view of an ESOM map constructed using all 51 metagenome bins assembled from the samples collected in this study, with the white square encompassing the Nitrospira-like metagenome bin. Some metagenome bins expand over the edge of a single ESOM grid. Hence, a tiled view consisting of four copies of the ESOM grid is shown to allow for visualization of metagenome bins at the edge as contiguous clusters. This results in all metagenome bins included in the ESOM analyses appearing four times in the tiled view. (B) Enlarged view of panel A indicating three scaffold fragments that were outliers based on ESOM analyses. (C) Enlarged view of panel A showing fragments of scaffold 158 containing ammonia oxidation genes that were binned with the Nitrospira metagenome. The ESOM binning procedure and contents of the three outlier scaffolds/scaffold fragments are presented in Text S1 in the supplemental material. (D) RAxML-based maximum-likelihood tree constructed using amino acid sequences of the amoA gene in the Nitrospira metagenome bin and pmoA/amoA sequences from a range of ammonia-oxidizing bacteria/archaea and methane-oxidizing bacteria, including the Nitrospira comammox. The tree was built from a trimmed muscle alignment using the Dayhoff model for protein evolution, gamma distribution model, and 500 bootstraps using the archaeal amoA gene of Nitrosopumilus maritimus as the outgroup. Branches are colored according to phylogenetic affiliation, and node support of >70% is indicated. This placement of the amoA gene from the Nitrospira-like genome and overall tree topology were also confirmed by neighbor-joining analysis (500 bootstraps) and the unweighted pair group method with arithmetic mean (UPGMA) (500 bootstraps) in Geneious and Bayesian phylogeny inference (20,000 generations) (Fig. S3).