Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions

Abstract

The genus Nitrospira is the most widespread group of nitrite-oxidizing bacteria and thrives in diverse natural and engineered ecosystems. Nitrospira marina Nb-295^T was isolated from the ocean over 30 years ago; however, its genome has not yet been analyzed. Here, we investigated the metabolic potential of N. marina based on its complete genome sequence and performed physiological experiments to test genome-derived hypotheses. Our data confirm that N. marina benefits from additions of undefined organic carbon substrates, has adaptations to resist oxidative, osmotic, and UV light-induced stress and low dissolved pCO₂, and requires exogenous vitamin B₁₂. In addition, N. marina is able to grow chemoorganotrophically on formate, and is thus not an obligate chemolithoautotroph. We further investigated the proteomic response of N. marina to low (∼5.6 µM) O₂ concentrations. The abundance of a potentially more efficient CO₂-fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high-affinity cbb₃-type terminal oxidase increased under O₂ limitation, suggesting a role in sustaining nitrite oxidation-driven autotrophy. This putatively more O₂-sensitive POR complex might be protected from oxidative damage by Cu/Zn-binding superoxide dismutase, which also increased in abundance under low O₂ conditions. Furthermore, the upregulation of proteins involved in alternative energy metabolisms, including Group 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides insights into the metabolic diversity and niche differentiation of NOB in marine environments.

Fig. 1. Maximum likelihood phylogenetic tree of representatives of the phylum Nitrospirae.

The multiple sequence alignment consisting of 120 concatenated phylogenetic marker genes contained 95 genomes and metagenome-assemble genomes (MAGs) from the Genome Taxonomy Database (GTDB) (Release 04-RS89, 19 June 2019), the genome of N. marina, the MAG of Ca. N. alkalitolerans and two open ocean single-cell amplified genomes (SAGs), AC-738-G23 and AC-732-L14. All MAGs and SAGs were estimated to be ≥50% complete with ≤5% contamination. Nodes with UFBoot support of at least 95% are indicated as black filled circles. Cultured representatives are shown in bold. The scale bar represents 0.1 substitutions per site.

Fig. 2. The effect of undefined organic carbon substrates on nitrite oxidation and growth of N. marina Nb-295^T.

Error bars represent the range of measurements from duplicate cultures.

Fig. 3. Growth of N. marina Nb-295^T on formate (1 mM), pyruvate (1 mM) and yeast extract and tryptone (150 mg L⁻¹ each) in the absence of nitrite.

Error bars represent the range of measurements from duplicate cultures.

Fig. 4. Nitrite consumption by N. marina Nb-295^T in artificial seawater mineral medium with and without addition of vitamin B₁₂ (Table S1).

Cultures of strain Nb-295^T were repeatedly transferred into medium without vitamin B₁₂ using 2% inocula. Error bars represent the range of measurements from duplicate cultures.

Fig. 5. Nitrite consumption by N. marina Nb-295^T grown under atmospheric (filled circles) and low O₂ conditions (open circles).

Cells for proteome analysis were harvested after 12 and 27 days, respectively. Error bars represent standard deviations from measurements of triplicate cultures.

Fig. 6. Heat map of N. marina Nb-295^T proteins that were more abundant under low O₂ concentrations compared to atmospheric O₂ concentration.

Relative protein abundance values were square-root transformed and hypothetical proteins were excluded to improve readability. The complete set of untransformed values can be found in Data Set 3. Fold-changes and significance values (adj. P value ≤ 0.001, ***; ≤0.01, **; ≤0.05, *) are shown in white boxes next to the corresponding protein. Select low abundant proteins of interest with high fold-changes were included despite being not statistically significant (see “Material and Methods”). Fold-changes of proteins that were not detected under atmospheric O₂ conditions are omitted to avoid dividing by zero (not available, na). Functional categories of depicted proteins are indicated by different colors. Gene clusters are indicated by black brackets.