Genomic and kinetic analysis of novel Nitrospinae enriched by cell sorting

Anna J Mueller,Craig W Herbold,Cameron R Strachan,Rasmus H Kirkegaard,Michael Wagner,Holger Daims,Man-Young Jung

doi:10.1038/s41396-020-00809-6

Abstract

Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) are key players in global nitrogen and carbon cycling. Members of the phylum Nitrospinae are the most abundant, known NOB in the oceans. To date, only two closely affiliated Nitrospinae species have been isolated, which are only distantly related to the environmentally abundant uncultured Nitrospinae clades. Here, we applied live cell sorting, activity screening, and subcultivation on marine nitrite-oxidizing enrichments to obtain novel marine Nitrospinae. Two binary cultures were obtained, each containing one Nitrospinae strain and one alphaproteobacterial heterotroph. The Nitrospinae strains represent two new genera, and one strain is more closely related to environmentally abundant Nitrospinae than previously cultured NOB. With an apparent half-saturation constant of 8.7 ± 2.5 µM, this strain has the highest affinity for nitrite among characterized marine NOB, while the other strain (16.2 ± 1.6 µM) and Nitrospina gracilis (20.1 ± 2.1 µM) displayed slightly lower nitrite affinities. The new strains and N. gracilis share core metabolic pathways for nitrite oxidation and CO2 fixation but differ remarkably in their genomic repertoires of terminal oxidases, use of organic N sources, alternative energy metabolisms, osmotic stress and phage defense. The new strains, tentatively named “Candidatus Nitrohelix vancouverensis” and “Candidatus Nitronauta litoralis”, shed light on the niche differentiation and potential ecological roles of Nitrospinae.

Highlights

Supplementary information The online version of this article contains supplementary material, which is available to authorized users.Bioavailable nitrogen is essential to all life on earth and the growth limiting factor in many ecosystems
To expedite the purification of Nitrospinae strains from our initial enrichments, which was previously a laborious and lengthy process [24], we developed a method for the physical separation, activitybased identification of nitrite-oxidizing bacteria (NOB), and subcultivation in 96-well microtiter plates
It differs from a previously reported fluorescence activated single cell sorting (FACS) isolation approach for Nitrospira NOB from activated sludge, where the NOB were targeted based on their known cell cluster size and shape, which had been determined by Nitrospira-specific rRNAtargeted FISH analysis before FACS was performed [43]

Summary

Introduction

Nitrite oxidoreductase (NXR), the key enzyme for nitrite oxidation, was found to be highly abundant in metaproteomic studies of OMZs [11, 12] and high in situ nitrite oxidation rates were measured, nitrite oxidation is considered to be an aerobic process and OMZs are strongly oxygen limited [5, 18, 19] In addition to their importance in the nitrogen cycle, the Nitrospinae have been suggested to play a major role in dark ocean carbon fixation by contributing up to 15–45% of the fixed inorganic carbon in some environments [20]. The contribution of Nitrospinae to CO2 fixation is, still under debate [21, 22], partially due to a lack of cultured representative organisms

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Conclusion