Implications for nitrogen and sulfur cycles: phylogeny and niche-range of Nitrospirota in terrestrial aquifers

Increasing evidence suggests Nitrospirota are important contributors to aquatic and subsurface nitrogen and sulfur-cycles. Here we determined the phylogenetic and ecological niche associations of Nitrospirota colonising terrestrial aquifers. Nitrospirota compositions were determined across 59 groundwater wells using 16S rRNA genes. Analyses showed Nitrospirota distributions were strongly influenced by oxygen availability in groundwater, marked by a trade-off between aerobic (Nitrospira, Leptospirillum) and anaerobic (Thermodesulfovibrionia, unclassified) lineages. Seven Nitrospirota metagenome-assembled genomes (MAGs) from a subset of wells included three from the recently designated class '9FT-COMBO-42-15'. Most were relatively more abundant, and transcriptionally active, in dysoxic groundwater. These MAGs were analysed with 743 other Nitrospirota genomes. Results illustrate the predominance of certain lineages in aquifers (e.g., non-nitrifying Nitrospiria, classes 9FT-COMBO-42-15 and UBA9217, Thermodesulfovibrionales family UBA1546). These lineages are characterised by mechanisms for nitrate reduction and sulfur-cycling, and, excluding Nitrospiria, the Wood-Ljungdahl pathway, consistent with carbon-limited, low-oxygen and sulfur-rich aquifer conditions. Class 9FT-COMBO-42-15 is a sister clade of Nitrospiria, and comprises two families spanning a transition in carbon fixation approaches: f_HDB-SIOIB13 encodes rTCA (like Nitrospiria) and f_9FT-COMBO-42-15 encodes Wood-Ljungdahl CO dehydrogenase (like Thermodesulfovibrionia and UBA9217). The 9FT-COMBO-42-15 family is further differentiated by the capacity for sulfur oxidation (via DsrABEFH and SoxXAYZB) and dissimilatory nitrate reduction to ammonium. Gene transcription by a f_9FT-COMBO-42-15 MAG, nzgw271, indicated active coupling of nitrogen and sulfur cycles in dysoxic groundwater. Results indicate that Nitrospirota are widely distributed in groundwater, and that oxygen availability drives the spatial differentiation of lineages with ecologically distinct roles related to nitrogen and sulfur metabolism.