Comparative genomics sheds light on niche differentiation and the evolutionary history of comammox Nitrospira

Abstract

The description of comammox Nitrospira spp., performing complete ammonia-to-nitrate oxidation, and their co-occurrence with canonical β-proteobacterial ammonia oxidizing bacteria (β-AOB) in the environment, calls into question the metabolic potential of comammox Nitrospira and the evolutionary history of their ammonia oxidation pathway. We report four new comammox Nitrospira genomes, constituting two novel species, and the first comparative genomic analysis on comammox Nitrospira. Unlike canonical Nitrospira, comammox Nitrospira genomes lack genes for assimilatory nitrite reduction, suggesting that they have lost the potential to use external nitrite nitrogen sources. By contrast, compared to canonical Nitrospira, comammox Nitrospira harbor a higher diversity of urea transporters and copper homeostasis genes and lack cyanate hydratase genes. Additionally, the two comammox clades differ in their ammonium uptake systems. Contrary to β-AOB, comammox Nitrospira genomes have single copies of the two central ammonia oxidation pathway operons. Similar to ammonia oxidizing archaea and some oligotrophic AOB strains, they lack genes involved in nitric oxide reduction. Furthermore, comammox Nitrospira genomes encode genes that might allow efficient growth at low oxygen concentrations. Regarding the evolutionary history of comammox Nitrospira, our analyses indicate that several genes belonging to the ammonia oxidation pathway could have been laterally transferred from β-AOB to comammox Nitrospira. We postulate that the absence of comammox genes in other sublineage II Nitrospira genomes is the result of subsequent loss.

Fig. 1

Differential coverage plot of two metagenomes obtained from Islevbro waterworks at different sampling depth. Scaffolds are displayed as circles, scaled by length and colored according to phylum-level taxonomic affiliation. Only scaffolds >4 kbp are shown. A second differential coverage plot showing the CG24_E bin extraction (enclosed by the polygon) is presented

Fig. 2

Communality and uniqueness in the Nitrospira pangenome as derived from the clustering of 16 genomes based on 12,337 protein clusters (PCs). Each radial layer represents a genome, and each bar in a layer represents the occurrence of a PC (dark presence, light absence). Clade A and Clade B comammox Nitrospira genomes are denoted in green and blue, respectively

Fig. 3

Cartoon of core and specific key metabolic features in the Nitrospira pangenome, as predicted from genome annotation. AIO arsenite oxidase, CynS cyanate hydratase, FDH formate dehydrogenase, H2ase hydrogenase, MSP methionine salvage pathway, SOR sulfite dehydrogenase, TrHb2 2/2 hemoglobin group 2. Enzyme complexes of the electron transport chain are labeled by Roman numerals. * N. moscoviensis possesses an octaheme nitrite reductase (ONR) putatively involved in nitrite reduction to ammonia

Fig. 4

Relationship between average phylogenetic distance of genomes and protein sequence divergence for housekeeping proteins (left) and for ammonia-oxidation related proteins (right) for comammox Nitrospira, β-AOB and γ-AOB genomes. Boxplot are colored according to the groups to which the compared genomes belong. The y-axis shows the pairwise protein dissimilarity (fraction of differing amino-acid sites) for a set of 18 housekeeping proteins or 27 ammonia-oxidation related proteins, while the x-axis shows the corresponding pairwise dissimilarity for a set of 14 ribosomal proteins. Asterisks(* and **) indicates non-significant (P > 0.01) and significant (P < 0.01) difference between means of sequence dissimilarities of two studied groups, respectively

Fig. 5

Schematic of the ammonia oxidation pathway genomic region as well as other AOB-related genomic features in comammox Nitrospira clade A (Ca. N. nitrificans), clade B (GWW3 bin) and selected ammonia-oxidizing bacteria (Nitrosospira multiformis and Nitrosomonas europaea). Homologous genes are connected by lines. Functions of the encoded proteins are represented by color. Parallel double lines designate a break in locus organization (other genes are in between the genes of interest). Single line designates a break probably due to contig fragmentation. Position of the blocks denotes the orientation of the coding strand (upper strands for forward; down strands for reverse)

Fig. 6

Reconciliation of functional gene trees (based on amino acid sequences. Top. AmoA; bottom. HaoA) with species tree for comammox Nitrospira, β-AOB, and γ-AOB genomes. The species tree, based on 14 ribosomal proteins, is shown in gray with the gene-trees super-imposed on top in narrower black lines. Arrows and red dots denote transfer and loss events, respectively. The displayed tree is the most parsimonious tree. Dashed arrows indicate an alternative reconciliation scenario