Cultivation and genomic characterization of novel and ubiquitous marine nitrite-oxidizing bacteria from the Nitrospirales

Abstract

Nitrospirales, including the genus Nitrospira, are environmentally widespread chemolithoautotrophic nitrite-oxidizing bacteria. These mostly uncultured microorganisms gain energy through nitrite oxidation, fix CO₂, and thus play vital roles in nitrogen and carbon cycling. Over the last decade, our understanding of their physiology has advanced through several new discoveries, such as alternative energy metabolisms and complete ammonia oxidizers (comammox Nitrospira). These findings mainly resulted from studies of terrestrial species, whereas less attention has been given to marine Nitrospirales. In this study, we cultured three new marine Nitrospirales enrichments and one isolate. Three of these four NOB represent new Nitrospira species while the fourth represents a novel genus. This fourth organism, tentatively named "Ca. Nitronereus thalassa", represents the first cultured member of a Nitrospirales lineage that encompasses both free-living and sponge-associated nitrite oxidizers, is highly abundant in the environment, and shows distinct habitat distribution patterns compared to the marine Nitrospira species. Partially explaining this, "Ca. Nitronereus thalassa" harbors a unique combination of genes involved in carbon fixation and respiration, suggesting differential adaptations to fluctuating oxygen concentrations. Furthermore, "Ca. Nitronereus thalassa" appears to have a more narrow substrate range compared to many other marine nitrite oxidizers, as it lacks the genomic potential to utilize formate, cyanate, and urea. Lastly, we show that the presumed marine Nitrospirales lineages are not restricted to oceanic and saline environments, as previously assumed.

Fig. 1. Nitrite-oxidizing activity of the newly cultured Nitrospirales members.

Nitrite-oxidizing activity of a the Maine enrichment, b the Elba enrichment, c the Vancouver enrichment, and d the Denmark isolate during 8 to 14 days of incubation. The rates of nitrite consumption cannot be compared across cultures, because the total biomass and NOB densities differed. The full lines show nitrite and dashed lines nitrate concentrations. Values are means ± standard deviations (error bars) for biological quadruplicates.

Fig. 2. Phylogeny of lineage IV Nitrospirales based on 16S rRNA genes.

Maximum likelihood tree based on the 16S rRNA gene sequences of Nitrospirales in lineage IV. The newly cultured Nitrospirales described in this study are highlighted in red. Isolated or enriched organisms are indicated by full and open circles, respectively, and SAGs from Pachiadaki et al. [47] are marked with an asterisk. Please refer to Materials and Methods for details of the sequence dataset used to calculate this tree. Nitrospira moscoviensis (lineage II) was used as outgroup. Numbers on the branches indicate ultra-fast bootstrap support (n = 1000). Sample sources are shown in parentheses. Lineages IVa and IVb are marked as described elsewhere [44]. The scale bar shows 0.02 estimated substitutions per nucleotide.

Fig. 3. Phylogeny of lineage IV Nitrospirales based on 120 conserved proteins.

Maximum likelihood tree based on a concatenated alignment of 120 conserved bacterial proteins from representative lineage IV Nitrospirales genomes and MAGs. The newly cultured marine Nitrospirales described in this study are highlighted in red. Isolated or enriched organisms are indicated by full and open circles, respectively. Please refer to Materials and Methods and Table S2 for details of the sequence dataset used to calculate this tree. The tree is excerpt from a more encompassing tree of the Nitrospirales (Fig. S3). The Nitrospirales outside of lineage IV are used as outgroup. Numbers on the branches indicate ultra-fast bootstrap support (n = 1000). Asterisks indicate a bootstrap value of 100. Sample sources are shown in parentheses. The scale bar shows 0.1 estimated substitutions per amino acid.

Fig. 4. Environmental distribution of lineage IV Nitrospirales and Nitrospinales.

Relative abundances of 16S rRNA genes grouped at 97% identity level from cultured and uncultured lineage IV Nitrospirales and Nitrospinales taken from publicly available amplicon sequencing datasets. The 16S rRNA gene-based abundance values were obtained from IMNGS. Each data point represents an environmental sample where the respective clade or strain, or closely related organisms, was detected with a minimal relative abundance of 0.1%. Colors represent different source environments.

Fig. 5. Selected key metabolic pathways in the newly cultured marine Nitrospirales and N. marina-Nb-295.

Schematic illustration of the genetic inventory for the membrane-bound electron transport chain, the oxidative and reductive TCA cycles, and other selected pathways in the newly cultivated Nitrospirales and N. marina Nb-295. The colored squares indicate the presence of the genes in the respective genomes, with numbers indicating gene copy numbers if more than one copy is present. Please refer to Table S6 for a complete list of gene abbreviations and details of the gene annotations. Q quinone, C c-type cytochrome, AC alternative complex, CcoNOP cyt. cbb₃ terminal oxidase, o/r TCA cycle oxidative and reductive tricarboxylic acid cycle. Roman numbers indicate the respiratory chain complexes.