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

Abstract Increasing evidence suggests Nitrospirota are important contributors to aquatic and subsurface nitrogen and sulphur cycles. We determined the phylogenetic and ecological niche associations of Nitrospirota colonizing terrestrial aquifers. Nitrospirota compositions were determined across 59 groundwater wells. 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), or populations, were recovered from a subset of wells, including 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, and Thermodesulfovibrionales family UBA1546). These lineages are characterized by mechanisms for nitrate reduction and sulphur cycling, and, excluding Nitrospiria, the Wood–Ljungdahl pathway, consistent with carbon-limited, low-oxygen, and sulphur-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 its capacity for sulphur oxidation (via DsrABEFH and SoxXAYZB) and dissimilatory nitrate reduction to ammonium, and gene transcription indicated active coupling of nitrogen and sulphur cycles by f_9FT-COMBO-42-15 in dysoxic groundwater. Overall, 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 sulphur metabolism.


Introduction
The microbial N and S cycles are catalyzed by diverse microorganisms in terrestrial aquifers [1][2][3].Their intersection occurs in bacteria that couple sulphur oxidation with nitrogen reduction [4][5][6], which may be favourable in characteristically organic carbon-poor groundwater [6,7].Nitrogen species are usually present in low concentrations in groundwater, particularly nitrite and ammonia, which are unstable and easily convert to nitrate through microbial nitrification [8].Excess nitrogen concentrations stem primarily from anthropogenic sources [9].Denitrification is the primary process for nitrate attenuation in groundwater [10].The rate of N loss is usually dependent on the availability of dissolved organic carbon for heterotrophic denitrification [11].However, given limited dissolved organic carbon [6], autotrophic denitrification is likely an ecologically important process in groundwater.
Autotrophic denitrifiers use inorganic electron donors, such as reduced sulphur, Fe(II), and H 2 in groundwater [5].Sulphur can be plentiful in aquifers and is typically present as sulphate, bisulphide, or hydrogen sulphide [12], with sulphate concentrations ranging from 0 to 230 mg/L [13].Reduced forms (e.g.hydrogen sulphide, iron sulphide precipitate) or partially reduced forms (elemental sulphur, sulphite, and thiosulphate) can derive from the activity of sulphate-reducing ± sulphur-oxidizing bacteria [12,14].Sulphur oxidation is among the most energetically favourable processes for chemoautotrophs (transferring up to eight electrons per sulphur atom) [4] and releases almost twice as much kJ per mol electron donor than iron oxidation [15].Recent research suggests the functional capacity for autotrophic sulphur (and nitrogen) oxidation in aquifers can be substantial [7].Nonetheless, sulphur oxidation remains understudied across the large differences in oxygen, nitrate, and dissolved organic carbon (DOC) concentrations found in groundwater [3].
Here, we determined the distribution of Nitrospirota lineages associated with oxic to anoxic terrestrial groundwater ecosystems.Groundwater samples, encompassing large differences in nitrate, oxygen, and DOC concentrations, were collected for 16S rRNA gene amplicon analysis to determine the diversity and distribution of Nitrospirota.Metagenome-assembled genomes (MAGs), reconstructed from eight groundwater wells, included seven Nitrospirota MAGs paired with transcriptional data.These seven and 743 other Nitrospirota and Nitrospirota_A genomes were compared to determine the relationship between groundwater lineages and carbon, nitrogen, and sulphur metabolisms.Analyses show that certain lineages of Nitrospirota are common to aquifers, occupying distinct niches governed by oxygen availability, and illustrate metabolic divergences among groundwaterderived lineages.

Samples for amplicon and omic analyses
Amplicon and omic data, as well as accompanying geochemical profiles, were generated from groundwater samples as described previously [3,29].In brief, 80 filtered groundwater samples (biomass ≥0.22 μm) were collected from 59 wells across 10 aquifers (Auckland, Waikato, Wellington, and Canterbury regions, New Zealand).All were used for amplicon analyses.A subset of 16 samples from eight wells across two alluvial aquifers in Canterbury were used for metagenomics (samples gwj01-gwj16) and six for metatranscriptomics (samples gwj09, gwj11, and gwj13-gwj16 with RNA integrity numbers ≥6% or 30% of fragments >200 nucleotides [3]).The two samples per well were collected sequentially: first, filtered groundwater, and, second, biofilm-enriched filtered groundwater following low-frequency sonication [29].

Average nucleotide identities and phylogenetic core gene trees
Comparative analyses used all 750 available Nitrospirota genomes with < 5% estimated contamination in the Genome Taxonomy Database (GTDB, release 214), including Nitrospirota_A and Nitro-spirota_B, collectively referred to as Nitrospirota below, and seven Nitrospirota MAGs we reconstructed (Table S1).The total available pool of Nitrospirota genomes available in the 214 release before filtering was 802 (also see Table S1).Pairwise ANIs were determined using fastANI v1.33 [36].Core gene alignments were undertaken using GTDB-Tk v2.1.1.Concatenated alignments (based on 120 bacteria marker genes and 5036 amino acids, Table S2) were used to construct a maximum-likelihood phylogenomic tree in IQ-TREE v2.2.2.2 [37] using the ModelFinder [38] best-fit non-taxa-specific model LG + F + I+ with 1000 ultrafast bootstrap replicates (see Fig. S1 for tree with bootstrap values).Tree construction was repeated as above using 1000 standard bootstrap replicates, and a core gene alignment based on a subset of 115 Nitrospirota genomes representing all seven classes and 19 orders (with 75 representatives from the 30 families with genomes from terrestrial aquifers) (Fig. S1).Consensus trees were rooted to representatives of 11 different phyla (Table S1).

Metabolic predictions
First, metabolic predictions based on protein-coding gene sequences were carried out on eight Nitrospirota MAGs we reconstructed from groundwater samples, as summarized here and described previously [29].Protein-coding gene sequences for MAGs were predicted using Prodigal v2.6.3 [39].Predicted proteincoding sequences were annotated using USEARCH v9.02132 [40] with the usearch_global command (−id 0.5 -e-value 0.001maxhits 10), UniRef100 [41], UniProt [42], and KEGG databases [43].Additional hidden Markov model (HMM) searches were also carried out on MAGs and reference genomes using HMMER v3.3 [44] against Pfam [45], TIGRfam [46] databases, and databases with HMM cutoffs provided by Anantharaman et al. [1].Additionally, protein sequences were searched against the eggNOG v5.0 database [47] using eggNOG-mapper v2 [48] with default parameters.Second, for comparative analyses with other publicly available Nitrospirota genomes, metabolic predictions of all 750 Nitrospirota genomes used for the phylogenomic tree (including MAGs derived from this study) were undertaken with DRAM v1.3.5 with default settings [49].The functional assignments of key metabolic genes identified by these two sets of annotations were interrogated further and validated, as described below.
Nitrospirota genomes derived from terrestrial aquifers were found across most taxonomic classes (six out of seven in GTDB).Representatives of Leptospirillia were lacking, although 16S rRNA gene data indicate the presence of this lineage in oxic groundwater (Fig. 2A).Instead, groundwater genomes included those from canonical nitrifiers and comammox bacteria [3], and most were derived from poorly studied and populated classes.Of these, certain taxonomic groups were almost entirely represented by MAGs recovered from aquifers: deep-branching Nitrospiria (earlier branching orders than Nitrospirales), class 9FT-COMBO-42-15, class UBA9217, four genomes comprising class RBG-16-64-22, and clusters of Thermodesulfovibrionia spanning multiple families in both the major orders Thermodesulfovibrionales (notably families UBA1546, SM23-35, and UBA6898) and UBA6902 (grey-shading in Fig. 1A, Table S1).The broad geographic distribution of aquifers that these over-represented taxonomic groups were reproducibly found across (e.g.different U.S. states, Germany, New Zealand) suggests these taxa are particularly well-adapted to aquifer conditions.For example, class 9FT-COMBO-42-15 almost exclusively comprises uncultivated taxa recently sampled from aquifers in the USA (Colorado [1] and California [60]), Germany (Thuringia [21]), and New Zealand (our sampling, Canterbury [3]) (18 out of 19 genomes, Fig. 1B, Table S1).

Spatial differentiation of Nitrospirota lineages in groundwater based on dissolved oxygen content
To explore the spatial distribution of Nitrospirota in aquifers, and its association with water chemistry, we analysed 16S rRNA gene amplicon sequences classified as Nitrospirota from 59 geographically widespread wells.Results showed proportionally more unclassified Thermodesulfovibrionia and genus 4-29-1 (SILVA taxonomy) at low oxygen sites, and more Nitrospira, and, to a lesser extent, Leptospirillum, at high oxygen sites (Fig. 2A).The relationship between 4-29-1 and GTDB-designated taxonomies is unclear, but 16S rRNA gene based sequence similarities are shared between the 4-29-1 class and GTDB classes 9FT-COMBO-42-15, UBA9217, and Thermodesulfovibrionia (Table S1).The presence of 4-29-1 could potentially indicate the presence of any of these lineages (e.g.9FT-COMBO-42-15, which was sampled via metagenomics from some of the same samples; see discussion below).The spatial distribution of Nitrospirota taxa was significantly associated with changes in dissolved oxygen and ORP (Fig. 2B), whether aquifers were confined or unconfined, aquifer location, sample type (groundwater, biomass-enriched groundwater), sulphate, and pH, which collectively explained 9% (R 2 adjusted) of variation in Nitrospirota community composition (db-RDA permutation-test, P < 0.05, permutations = 999, Table S5).Nitrospirota are therefore present in diverse aquifers, with distinct environmental niches, such as those governed by oxygen availability, occupied by different lineages.
Broadly similar results were obtained based on the eight MAGs recovered from a subset of sampled aquifers.Comammox Nitrospira nzgw279 was proportionally most abundant in oxic groundwater, whereas members of the 9FT-COMBO-42-15 class (MAGs nzgw271-273), and most earlier-branching and poorly characterized Nitrospiria (MAGs nzgw274, nzgw276, and nzgw278) were more abundant under dysoxic conditions (excluding Candidatus Manganitrophus nzgw269, Fig. 3A).As indicated above, class 9FT-COMBO-42-15 represents one of the Nitrospirota lineages overrepresented in terrestrial aquifers (Fig. 1A).Representatives of both families in the class (9FT-COMBO-42-15 and HDB-SIOI813) were relatively much more abundant and exhibited higher relative gene expression in dysoxic versus oxic groundwater (MAG Figure 1.Maximum likelihood phylogenomic tree of 750 Nitrospirota and their genomically inferred capacity for carbon metabolism, nitrogen and sulphur cycling; (A) full tree with branches coloured by class.Grey shading denotes parts of the tree largely comprising genomes derived from terrestrial aquifers; (B) tree highlighting the two orders of class 9FT-COMBO-42-15 (upper clade = order 9FT-COMBO-42-15; lower clade = order HDB-SIOI1813) with other classes collapsed; branch labels give GTDB species designations, source country and NCBI strain identifiers; in (A) and (B), red font and arrows indicate MAGs from this study.Genes present are indicated by coloured squares to the right: genes associated with the TCA/rTCA cycles (dark red = CO 2 to oxaloacetate; red = second C oxidation pathway; orange = first), Wood-Ljungdahl pathway CO dehydrogenase (yellow), comammox/nitrification (green), DNRA (dark blue), denitrification (different blue shades denote genes associated nitrate reductases, nitrite reductases, nitric oxide reductase, and nitrous oxide reductase), sulphate reduction or oxidation (different purple shades denotes genes associated with ATP-sulfurylase and adenylyl-sulphate reductases, dissimilatory sulphite reductase, reverse dissimilatory reductase, the sulphur oxidation pathway, quinone-modifying oxidoreductase, and sulphide-quinone oxidoreductase); only TCA/rTCA genes annotated in at least one genome are shown, and numbers prefixing gene names indicate steps (0-8) from CO 2 to oxaloacetate to malate, etc., scale bars represent number of substitutions per site; coloured squares with black outlines in (B) denote lactonase domain encoding nosZ like genes; bootstrap support is shown in Fig. S1; acronym: Re-citrate syn, Re-citrate synthase.S1).Acronyms: G, genus; c, class; p, phylum.(B) Distance-based redundancy analysis of the 16S amplicon data based on a Bray-Curtis dissimilarity matrix of ASV relative abundances.Each point in the ordination represents the Nitrospirota and Nitrospirota_A community composition in each sample, and is shaded dark to light blue based on increasing groundwater DO content.Vectors show environmental variables that significantly (P < 0.05) constrain the variability in community composition.nzgw271, genus 9FT-COMBO-42-15; HDB-SIOI813 MAGs nzgw272 and nzgw273, genus JAHFEQ01 and JACRGV01; Fig. 3A-C).Both families also share the capacity for nitrate reduction (via NarGH) and nitrite reduction to NO (via NirK or NirS) (Fig. 1A) [62], and hence anaerobic metabolism.Similarly, another member of the 9FT-COMBO-42-15 family (MAG H51-bin250-1, genus JACPZJ01) was found to dominate suboxic-anoxic groundwater elsewhere (Hainich Critical Zone Exploratory, Germany) [21].Results therefore indicate members of class 9FT-COMBO-42-15 are adapted to low-oxygen groundwater habitats.

Metabolic trait differences among Nitrospirota in terrestrial aquifers
Collectively, Nitrospirota lineages, including the diversity of lineages found in aquifers, are associated with aerobic, microaerobic, and anaerobic lifestyles, and the transformation of nitrogen, sulphur, iron, and manganese species for energy gain.Wellcharacterized genera, Nitrospira (class Nitrospiria) and Leptospirillum (class Leptospirillia), are capable of autotrophic and aerobic nitrite oxidation, comammox, or microaerobic-anaerobic iron oxidation [24,26], and dominated Nitrospirota in oxic groundwaters in  S1) is included [3].Groundwater and biomass-enriched groundwater (i.e.groundwater collected post sonication to detach aquifer biofilms and particles) were obtained sequentially from the same groundwater wells.Geochemical parameters per site/sample are given below the bar plot (abbreviations: ORP, oxidation-reduction potential; DO, dissolved oxygen; DOC, dissolved organic carbon; DRP, dissolved reactive phosphorus).(B) Functional categories (eggNOG) of all transcribed genes predicted from MAG nzgw271.(C) Abundance of nitrogen and sulphur cycling transcripts per million (TPM) across each sample.Asterisks indicate presence of transcripts (including where expression was relatively low).
this study ( Fig. 2A).Candidatus Manganitrophus (Nitrospiria), which couples manganese oxidation with aerobic respiration [25], was also observed in groundwater (MAG nzgw269, oxic groundwater in this study, and two locations in the USA, Table S1).However, most genomes derived from aquifers to date are characterized by genes associated with nitrate reduction and sulphur metabolism (Fig. 1A) and are suggestive of anaerobic (or microaerobic) lifestyles (e.g.members of the 9FT-COMBO-42-15 class).In addition, magnetotactic Thermodesulfovibrionia, such as Candidatus Magnetoovum (from a partially oxic sample, Fig. 2A), are thought to oxidize sulphur as aerotolerant anaerobes or microaerobes [63].Moreover, Thermodesulfovibrio (reported from a couple of groundwater sites) are heterotrophic sulphate reducers [64,65], and hence anaerobic, or are suggested to couple sulphate reduction to C1 metabolism [20].
To further illustrate the metabolic traits characteristic of Nitrospirota lineages alongside those over-represented in terrestrial aquifers, we searched for genes associated with carbon fixation and nitrogen and sulphur cycling in the 750 genomes.Results highlight the distinct metabolic strategies encoded by laterbranching Nitrospiria (i.e.aerobic nitrification and rTCA-based carbon fixation [23,66]) versus most other Nitrospirota lineages (i.e.anaerobic/microaerobic sulphur and nitrogen metabolism, and Wood-Ljungdahl (WL) CO dehydrogenase) (Fig. 1A), consistent with a recent phylogenomic analysis of the phylum [20].Genomes derived from groundwater to date are largely associated with WL-dependent lineages (i.e.classes UBA9217, 9FT-COMBO-42-15, and Thermodesulfovibrionia).Although those associated with the intermediate class, 9FT-COMBO-42-15, span both WLdependent and WL-independent groups.In addition, the four available RBG-16-64-22 genomes lack the WL pathway, and some very early branching non-nitrifying Nitrospiria orders that lack the WL pathway are also largely comprised of aquifer-derived genomes (grey-shading, Fig. 1A).Overwhelmingly, lineages overrepresented in terrestrial aquifers (including early branching Nitrospiria) are characterized by genes encoding for sulphate reduction (dissimilatory sulphite reductase, DsrAB), sulphur oxidation (rDsrAB or the sulphur oxidation pathway, Sox), dissimilatory nitrate reduction to ammonium (DNRA), and/or the denitrification pathway.The metabolic properties of these overrepresented lineages [20] highlight the tendency for aquifers to be oxygen-depleted and sulphur-rich (Fig. 3A) [3], and consequently rich in sulphur-cycling taxa [1].

Metabolic transitions within class 9FT-COMBO-42-15
Results show a transition in carbon fixation mechanisms within the class 9FT-COMBO-42-15, from WL CO dehydrogenase in the 9FT-COMBO-42-15 family to rTCA in family HDB-SIOIB13 (the only families of their respective orders, Fig. 1B).The same transition is repeated within the clade encompassing classes UBA9217 and RBG-16-64-22, although the latter is poorly represented by available genomes.These transitions ref lect the rTCA dependence in later-evolved Nitrospiria, and the phylogenetic relatedness of class 9FT-COMBO-42-15 to Nitrospiria (Fig. 1A).Nitrospiria, HDB-SIOIB13, and RBG-16-64-22 are characterized by the presence of aclAB (ATP citrate lyase) genes lacking from the 9FT-COMBO-42-15 family and the UBA9217 and Thermodesulfovibrionia classes, which encode incomplete rTCA cycles (Fig. 1A).These other lineages (excluding Leptospirillia) instead encode WL acsABCDE CO dehydrogenase genes.The 9FT-COMBO-42-15 and HDB-SIOI813 families are also distinguished by their capacities for anaerobic nitrogen metabolism and sulphur metabolism.Notably, 9FT-COMBO-42-15 harbours an expanded repertoire of genes for sulphur oxidation, as discussed further below (Fig. 1B).
Although rTCA carbon fixation is employed by sulphideoxidizers in anoxic groundwater [14], the incomplete rTCA cycle encoded by members of the 9FT-COMBO-42-15 family likely functions to biosynthesize intermediates and replenish the oxaloacetate pool that condenses with acetyl-CoA (Fig. 1, Supplementary Information).Incomplete TCA and rTCA cycles can still convert pyruvate to necessary biosynthetic intermediates under anaerobic or microaerophilic conditions.They are suggested to be from an intermediate stage in the evolution of the oxidative catabolic TCA cycle that was primarily for amino acid biosynthesis, and comprise a non-cyclic "horseshoe" pathway lacking the oxidative enzymatic step to form succinate (catalyzed by succinyl-CoA synthetase, SucCD, in the rTCA cycle) (Fig. 1B, Supplementary Information) [71].
Ammonia-forming dissimilatory nitrite reductase genes, nrfAH, were present in MAG nzgw271, and two other species in the genus 9FT-COMBO-42-15, demonstrating the capacity to generate energy via the six-electron reduction of nitrite to ammonium [76].DNRA prevails over denitrification under nitrate-limiting conditions [77], under more reducing conditions where electron acceptors are scarcer due to a greater consumption of electrons [78], and where carbon/nitrogen ratios are high [77].In comparison, groundwater where nzgw271 was most abundant and transcriptionally active (sample gwj15) had mixed redox conditions (DO 0.37 mg/L) and high nitrate concentrations (4.4 g/m 3 ) compared to nitrite (0.031 g/m 3 ), which suggests a lack of resource limitation for denitrification (Fig. 3A).Although DOC was relatively high for groundwater (10.1 g/m 3 ) [6], the C/N ratio (2:3) was much lower than previously shown to favour DNRA [79].Accordingly, analysis of transcripts indicates nzgw271 was more likely engaged in denitrification at the time of sampling (Fig. 3C).Transcripts for the key step in DNRA (nrfAH) were not detected, unlike those for nitrate reduction (narGH) and steps specific to the denitrification pathway (nirK, nirS, norC, nosZ-like).Analysis of the overall prokaryotic community likewise indicated that gene transcription associated with denitrification was one-to-two orders of magnitude greater than DNRA [ 3].

Role in groundwater denitrification: capacity for steps from nitrate reduction to nitrous oxide generation
The most transcriptionally active prokaryotes associated with denitrification in the sampled groundwater were from phyla, such as Pseudomonadota, Methylomirabilota, and Nitrospinota, based on summed nir, nor, and nos transcripts (TPM, Fig. 4).Nonetheless, denitrification tends to be a community effort [3,62], and Nitrospirota from class 9FT-COMBO-4215 and Nitrospiraceae were among the more transcriptionally active contributors overall (five MAGs).This included nzgw271 (family 9FT-COMBO-4215) and nzgw272 (family HDB-SIOI813), which ranked 17th and 12th out of 60 taxa, respectively.Notably, all these Nitrospirota expressed the nir genes for converting nitrite into nitric oxide, particularly in dysoxic groundwater, and made the biggest transcriptional contribution to this step in the pathway (Fig. 4).
Nir comprises two structurally different, but functionally equivalent enzymes, cytochrome cd 1 -type nitrite reductase NirS, and copper-containing nitrite reductase NirK [62].Most Nitrospiria and Leptospirilla contain nirK.Other classes contain an equal mix (9FT-COMBO-42-15), a few of which are mostly nirS (Thermodesulfovibrionia), or neither (UBA9217) (Fig. 1A and B).9FT-COMBO-42-15 bacterium nzgw271 possesses both genes (Fig. 1A) and expresses both concomitantly in dysoxic groundwater (Fig. 3C).Oligotrophic strains of Bradyrhizobia also encode both nitrite reductases, and use NirS to facilitate swimming motility in the presence of nitrate [80].Swimming motility would be advantageous for nutrient acquisition [81] in oligotrophic groundwater and in unconfined aquifers with f luctuating oxygen levels [82].Members of the 9FT-COMBO-42-15 family largely lack genes for f lagellar synthesis (an exception being the biosynthesis protein gene f lgG), but most members of the HDB-SIOI813 family encode numerous synthesis genes, and likely undertake swimming motility (28-40 genes annotated in each of nine genomes, Table S8).Of these, members of genus HDB-SIOI813 encode f lagellar (all five) and NirS (four of five) (Fig. 1B, Table S8), potentially providing a secondary role for NirS.
Nitric oxide is reduced to nitrous oxide by NorB (a hemerich subunit containing the active site) and NorC (cytochrome c) [83].NorBC genes are prevalent in Thermodesulfovibrionia and UBA9217, and norBCDQ was identified in one member of the HDB-SIOI813 genus.In contrast, nzgw271 and other members of the 9FT-COMBO-42-15 family are devoid of norB, although norD and norQ genes, essential for the activation of NorBC [83], are universally present [1].The norC gene of nzgw271 was, nonetheless, transcribed in dysoxic groundwater alongside genes encoding nitrate and nitrite reductases, and a putative nitrous oxide reductase ( Fig. 3C).Members of the 9FT-COMBO-42-15 family could therefore use a novel mechanism for NO reduction.Alternatively, other bacteria could complete this step [62], as illustrated by diverse taxa transcribing norBC in dysoxic groundwater (Fig. 4).
Proteins annotated as Sec-type NosZ are almost exclusively encoded by bacteria in class 9FT-COMBO-42-15 (Table S9).These proteins (including one from a Nitrospiria in the SBBL0 order) are phylogenetically distinct from canonical clade II Sectypes (Fig. S3).They form a distantly related clade shared by sequences from a diverse collection of other taxa-Nitrospinota bacterium "DRJW01 sp011052055" (GCA_011052055.1,derived from a hydrothermal vent) [87], Sporomusa acidovorans DSM 3132 (which also has genes for DNRA) [88], and a Burkholderiales species (GCA_905339285.1).N 2 O reductases are characterized by a 7bladed β-propeller domain and cupredoxin domain [89].The novel clade of NosZ-like proteins contains a cupredoxin domain.The 7-bladed beta-propeller domain is homologous to a lactonase (IPR019405, Fig. S4, Table S9) rather than the usual Nos β-propeller, explaining the sequence divergence.Inspection of MAG nzgw271 showed the presence of the novel nosZ-like gene in addition to nosDFLYX genes (Fig. 5), which are found in canonical nos clusters [84].The nosZ-like gene was expressed in dysoxic groundwater along with other denitrification genes (Figs 3A and 4; Table S6).However, experimentation is needed to confirm if this protein performs nitrous oxide reduction or some other function.
The capacity for sulphur oxidation strongly differentiated the two 9FT-COMBO-42-15 families.Little evidence was found for sulphur oxidation (except via Sqr or potentially Hdr/Qmo) in the HDB-SIOIB13 family, whereas all members of the 9FT-COMBO-42-15 family possessed genes associated with reverse Dsr and the sulphur oxidation pathway (Sox), which were most complete for genus 9FT-COMBO-42-15 (Fig. 1A).Strikingly, one member of the 9FT-COMBO-42-15 genus (MAG nzgw271) possessed a full complement (soxXAYZB and rdsrABCEFHL), excluding soxCD, in addition to Sqr and Hdr/Qmo genes (Fig. 1B; Table S6), which suggests it can oxidize a variety of sulphur species (sulphide, elemental sulphur, sulphite, thiosulphate) to sulphate (Fig. 5), and that sulphur represents an important energy source.The sulphide:quinone oxidoreductase (Sqr) predicted is involved in poly-S formation and catalyzes the oxidation of sulphide to elementary sulphur for respiration and potentially also detoxification [91].Heterodisulphide reductase genes (hdrABC/qmoABC), present in nzgw271, could be used to oxidize sulphur compounds to sulphite [22].Sulphite could then be oxidized to sulphate by genes encoding adenosine 5 -phosphosulphate reductase (aprAB) and adenosine 5 -triphosphate (ATP) sulfurylase (sat) (Figs 1B and 5) [22].Alternatively, elementary sulphur could be oxidized to sulphite by the dissimilatory sulphite reductase encoded by rdsrABCEFHL genes [22].
Thiosulphate is a product of sulphur-reducing bacteria or is formed through the chemical oxidation of hydrogen sulphide [92].Thiosulphate oxidation could be catalyzed by the sulphur oxidation multienzyme complex (soxXAYZB) [93].MAG nzgw271 was devoid of genes encoding sulphur dehydrogenase SoxCD, and they were likewise entirely missing from all other early branching/basal clade genomes, including early branching Nitrospiria (Fig. 1A) [20].This is consistent with soxCD absence in other bacteria encoding rdsrAB [92].In Paracoccus pantotrophus (the model organism for Sox), SoxCD facilitates growth on thiosulphate by oxidizing protein-bound sulphur (sulfane to sulfone) and increasing the electron yield from two to eight [93].However, rDsr is proposed to substitute for SoxCD in green sulphur bacteria to enable complete oxidation of thiosulphate to sulphate [92,94], and may be likewise utilized by nzgw271 and other similarly equipped Nitrospirota.Sox systems devoid of SoxCD are found in a broad range of sulphur-oxidizers.Genes encoding SoxCD are missing from Thiobacillus denitrificans and Thioalkalivibrio HL-EbGR7, which are capable of sulphur-driven autotrophic denitrification [4], and subsurface-derived Sulfuricurvum species [95,96].Sulfuricurvum kujiense oxidizes various sulphur species aerobically/anaerobically, including coupling nitrate reduction with thiosulphate-oxidation [95].The Sox pathway sulphur dehydrogenase genes were instead exclusively found among the Nitrospirales (Nitrospiria) (Fig. 1A).Although SoxCD in P. pantotrophus was shown to have no catalytic activity in the absence of other sox proteins [93], soxCD genes are prevalent among Nitrospirales [20] (Fig. 1A).Nitrospirales are characterized by genes for nitrification (nxrAB + amoABC) and overwhelmingly lack other sox genes (Fig. 1A).They possibly use reduced forms of sulphur (via SoxCD or Sqr) as an alternative electron sink or for detoxification [91].

Coupled sulphur and nitrogen metabolism
Reduced sulphur compounds, such as sulphide, elemental sulphur, and thiosulphate, can act as electron donors when coupled to the reduction of nitrate, leading to partial or complete denitrification and the formation of oxidized sulphur species, such as sulphate [95].As nzgw271 encodes nitrate reductase and multiple nitrite reductases (NirS, NirK, NrfA) and mechanisms for sulphur oxidation (rDsr, Sox, Sqr, Hdr/Qmo), it could f lexibly couple either of two respiratory nitrate/nitrite reduction pathways (denitrification or DNRA) with sulphur oxidation (Fig. 5).Co-expression of denitrification genes (narGH, nirS/nirK, norC, nosDLZ) with dsrA and qmoC genes additionally suggests nzgw271 may have actively coupled sulphur oxidation to denitrification at the time of sampling (Fig. 1C).Simultaneous expression of hdr/qmo genes with those for denitrification has been observed in Thiobacillus and Thauera [97], indicating a role for hdr/qmo in sulphur-based denitrification.Similar N and S cycling mechanisms in the 9FT-COMBO-42-15 family (e.g.nzgw271) and class RBG_16_64_22 suggest that phylogenetically disparate Nitrospirota in aquifers are capable of sulphur-driven denitrification (Fig. 1, Supplementary Information).

Conclusion
Results show that phylogenetically and metabolically diverse Nitrospirota genera are present in aquifers, spanning all seven known classes.However, certain lineages are almost entirely comprised of members from aquifers (three clusters of Thermodesulfovibrionia, classes UBA9217, RBG-16-64-22, and 9FT-COMBO-42-15, and very early branching Nitropiria).In aquifers, Nitrospirota community fractions are structured based on groundwater oxygen contents ranging from oxic (Nitrospira-dominated with Leptospirillum) to anoxic (Thermodesulfovibrionia-dominated with other early branching clades, including Nitrospiria).This distribution ref lects the distinct metabolic attributes of these lineages, in particular those characterized by aerobic nitrite oxidation versus nitrate reduction and sulphur oxidation or reduction.Most Nitrospirota genomes recovered from aquifers to date exhibit features associated with anaerobic nitrogen-and sulphur-based metabolisms and utilization of the WL pathway, rather than the rTCA cycles encoded by Nitrospiria and Leptospirillum.Genomic analysis of the aquifer-associated and recently established 9FT-COMBO-42-15 class indicates a transition among the two constituent families in their mechanisms for both sulphur metabolism (extensive in the 9FT-COMBO-42-15 family) and carbon acquisition (WL versus rTCA mediated).The transition ref lects the intermediate phylogenetic placement of this class, and attributes consistent with basal clades or the later-evolved Nitrospiria class, respectively.Analysis of the 9FT-COMBO-42-15 family demonstrates its capacity for versatile autotrophic sulphur and nitrogen-based metabolisms, including DNRA and the denitrification pathway coupled to sulphur oxidation, which likely confers a broad niche breadth to this group of Nitrospirota in aquifers.The predicted coupling of nitrogen and sulphur metabolisms is supported by associated transcriptional activity in dysoxic groundwater indicative of sulphide oxidation and denitrification (alongside carbon fixation), although the capacity to undertake the complete denitrification pathway remains to be determined.Overall, results illustrate that metabolic adaptations among Nitrospirota, associated with the capacity to undertake oxidative or reductive transformations of nitrogen and sulphur species, enable members of this phylum to colonize geochemically distinct groundwaters and that these adaptations contribute to the spatial differentiation of lineages in terrestrial aquifers.

Figure 2 .
Figure 2. Stacked bar graph and ordination plot showing the environmental variables and factors inf luencing the structure of the Nitrospirota (and Nitrospirota_A) groundwater community based on 16S rRNA gene amplicon data.(A) Composition of the Nitrospirota community across 80 samples, ordered by dissolved oxygen (DO) concentrations.Black points within plot = DO content.Bars represent different genera.Black font = SILVA classifications (genus level or next rank with a classification).Based on GTDB small subunit rRNA gene BLAST bast comparisons, SILVA classes shown correspond to the same GTDB classes, except for: c_661256, c_BMS9AB35, and c_HDB-SIOI1093 (GTDB classes unknown), and c_4-29-1 (GTDB classes 9FT-COMBO-42-15, UBA9217, and Thermodesulfovibrionia) (TableS1).Acronyms: G, genus; c, class; p, phylum.(B) Distance-based redundancy analysis of the 16S amplicon data based on a Bray-Curtis dissimilarity matrix of ASV relative abundances.Each point in the ordination represents the Nitrospirota and Nitrospirota_A community composition in each sample, and is shaded dark to light blue based on increasing groundwater DO content.Vectors show environmental variables that significantly (P < 0.05) constrain the variability in community composition.

Figure 3 .
Figure 3. Relative abundance and gene expression of Nitrospiria MAG nzgw271 across different geochemical conditions at sites A-D.(A) Bar plots showing the relative abundance of Nitrospirota MAGs based on genome coverages normalized to library size across each site (samples right to left: gwj01-gwj16) and sample type (groundwater and biomass-enriched groundwater).An eighth MAG (comammox Nitrospira nzgw279, 5.45% contamination, TableS1) is included[3].Groundwater and biomass-enriched groundwater (i.e.groundwater collected post sonication to detach aquifer biofilms and particles) were obtained sequentially from the same groundwater wells.Geochemical parameters per site/sample are given below the bar plot (abbreviations: ORP, oxidation-reduction potential; DO, dissolved oxygen; DOC, dissolved organic carbon; DRP, dissolved reactive phosphorus).(B) Functional categories (eggNOG) of all transcribed genes predicted from MAG nzgw271.(C) Abundance of nitrogen and sulphur cycling transcripts per million (TPM) across each sample.Asterisks indicate presence of transcripts (including where expression was relatively low).

Figure 4 .
Figure 4. Activity of the denitrifying microbial community (TPM) across sites C (gwj09, gwj11) and D (gwj13-gwj16).Gene transcripts are shown for nitrite reductases (nirK and nirS), nitric oxide reductase (norBC), and nitrous oxide reductase (nosZ) for each MAG in which these genes were detected.MAG identifiers are shown along the x-axis.Bolded font and dashed grey lines indicate Nitrospiria MAGs.

Figure 5 .
Figure 5. Schematic of autotrophic sulphur-dependant denitrification pathways genomically inferred from MAG nzgw271.Orange font indicate substrates and products involved in S metabolism; blue indicates denitrification.Dashed lines = electron f low.Solid lines = substrate transformations.Red font denotes uncertain protein function.Pale grey font denotes missing genes.Shapes: Orange = proteins involved in sulphur and nitrogen reactions, grey = ATP synthesis pathways, brown = quinone pool, green = substrate transporters.