Putative novel hydrogen- and iron-oxidizing sheath-producing Zetaproteobacteria thrive at the Fåvne deep-sea hydrothermal vent field

ABSTRACT Iron-oxidizing Zetaproteobacteria are well known to colonize deep-sea hydrothermal vent fields around the world where iron-rich fluids are discharged into oxic seawater. How inter-field and intra-field differences in geochemistry influence the diversity of Zetaproteobacteria, however, remains largely unknown. Here, we characterize Zetaproteobacteria phylogenomic diversity, metabolic potential, and morphologies of the iron oxides they form, with a focus on the recently discovered Fåvne vent field. Located along the Mohns ridge in the Arctic, this vent field is a unique study site with vent fluids containing both iron and hydrogen with thick iron microbial mats (Fe mats) covering porously venting high-temperature (227–267°C) black smoker chimneys. Through genome-resolved metagenomics, we demonstrate that Zetaproteobacteria, Ghiorsea spp., likely produce tubular iron oxide sheaths dominating the Fe mats at Fåvne, as observed via microscopy. With these structures, Ghiorsea may provide a surface area for members of other abundant taxa such as Campylobacterota, Gammaproteobacteria, and Alphaproteobacteria. Furthermore, Ghiorsea likely oxidizes both iron and hydrogen present in the fluids, with several Ghiorsea populations co-existing in the same niche. Homologs of Zetaproteobacteria Ni,Fe hydrogenases and iron oxidation gene cyc2 were found in genomes of other community members, suggesting exchange of these genes could have happened in similar environments. Our study provides new insights into Zetaproteobacteria in hydrothermal vents, their diversity, energy metabolism and niche formation. IMPORTANCE Knowledge on microbial iron oxidation is important for understanding the cycling of iron, carbon, nitrogen, nutrients, and metals. The current study yields important insights into the niche sharing, diversification, and Fe(III) oxyhydroxide morphology of Ghiorsea, an iron- and hydrogen-oxidizing Zetaproteobacteria representative belonging to Zetaproteobacteria operational taxonomic unit 9. The study proposes that Ghiorsea exhibits a more extensive morphology of Fe(III) oxyhydroxide than previously observed. Overall, the results increase our knowledge on potential drivers of Zetaproteobacteria diversity in iron microbial mats and can eventually be used to develop strategies for the cultivation of sheath-forming Zetaproteobacteria.

photoferrotrophs, while there are acidophilic FeOB inhabiting low pH environments (1).Fe-oxidizers influence biogeochemical cycling of iron and other elements through transforming the soluble Fe(II) to insoluble Fe(III), which often takes the form of Fe(III) oxyhydroxides.These Fe(III) oxyhydroxides have the ability to co-precipitate and adsorb carbon, nutrients, and heavy metals (1)(2)(3)(4)(5)(6).FeOB can also play a role in corrosion (7,8) and bioremediation of metal pollution and recovery of resources (9,10).Seafloor hydrothermal fluids are typically rich in diverse electron donors such as hydrogen sulfide and methane but with variable hydrogen and Fe contents (11)(12)(13)(14).The exact chemical composition of these fluids varies widely between and often within vent fields, depending on the hydrothermal system's geological setting, and exerts a strong influence on the microbial communities present (15)(16)(17).
FeOB are found in iron microbial mats (Fe mats) around the globe, such as at the Kama'ehuakanaloa (Lō'ihi) seamount (18)(19)(20)(21)(22), Vailuluʻu seamount (23), Mid-Atlantic Ridge (24,25), the Mariana region (26)(27)(28)(29)(30)(31), Kermadec Arc (32), South Tonga Arc (33), Mid-Cayman Ridge (34), and Arctic Mid-Ocean Ridges (AMOR) (35)(36)(37).The dominant FeOB in these Fe mats are Zetaproteobacteria, first proposed as a class in 2007 (38) and collectively divided into operational taxonomic units specific to subgroups of Zetaproteobacteria (ZetaOTUs) (39).To obtain energy for CO 2 fixation, Zetaproteobac teria oxidize soluble Fe(II) under low oxygen conditions (40).Micrometer-scale struc tures composed largely of extracellular polymeric substances and precipitated Fe(III) oxyhydroxides are often formed as a result of their Fe metabolism.The morphology of Fe(III) hydroxides varies between types of Zetaproteobacteria.Some produce twisted stalks, others produce hollow tubular sheaths, bifurcating tubular structures or dreads (18,20,(41)(42)(43).It has been hypothesized that these structures prevent the cells from becoming encrusted in Fe and that they keep the cells within the gradient of oxygen and Fe required for growth (20,38).While stalk formation genes have recently been proposed (44,45), the molecular mechanisms for formation of other structures are not well studied, and not all morphologies have a known isolated representative.Fe(III) oxyhydroxide sheaths in marine environments were found associated with Zetaproteobacteria (18); however, to date, no sheath-forming Zetaproteobacteria have been isolated nor have the Zetaproteobacteria responsible for sheath formation been identified and confirmed.Since sheaths and stalks make up the majority of Fe mats, stalk-and sheath-forming Zetaproteobacteria are recognized as ecosystem engineers that produce the structure of these mats, providing a suitable environment for other species (20).
Only a few FeOB belonging to the Zetaproteobacteria have been cultured, most of which are members of the genus Mariprofundus (38,41,(46)(47)(48)(49).The cyc2 gene has been validated as the main gene involved in the Fe oxidation pathway of Zetaproteobac teria and other bacteria in near-neutral pH environments (31,(50)(51)(52).While most of Zetaproteobacteria are strict FeOB, it has been shown that Ghiorsea bivora, a ZetaOTU9 representative, can obtain energy from hydrogen oxidation by using hydrogen as either the sole electron donor or in combination with Fe(II) (53).The co-occurrence of Fe(II) and H 2 may play an important role in defining the niche of ZetaOTU9 (40).However, we have a limited understanding of the functioning of FeOB that also use H 2 and how this affects their diversity and ecology.In this paper, we contribute toward narrowing this knowledge gap.
At the recently discovered Fåvne deep-sea hydrothermal vent field located on the Mohns Ridge (54)(55)(56), dense Fe mats cover porous black smoker chimney surfaces at in situ temperatures of ~50°C (see Supplementary Material 4 at https://doi.org/10.5281/zenodo.8297777).The venting fluids at Fåvne contain both abundant dissolved H 2 and Fe(II) as energy sources, with measured concentrations (±10%) of 22 and 24 mmol/L, respectively, in the North Tower duplicate isobaric gas-tight (IGT) fluid samples (55).In contrast to lower temperature Fe mat systems where H 2 is below detection or absent (14), these levels at Fåvne instead are more characteristic of black smoker fluids at sites such as Rainbow, Logatchev, and Azhadze 1 and 2 (57).This geochemistry makes Fåvne a valuable study site to investigate FeOB that can use H 2 as an alternate electron donor.Our genome-resolved metagenomics and microscopy study characterize Fåvne Fe mats as a deep-sea hydrothermal habitat formed using abundant byproducts of novel sheath-forming Fe-oxidizing Zetaproteobacteria that potentially also utilize H 2 .The identification of sheath-producing Ghiorsea belonging to ZetaOTU9 extends previous knowledge on Fe(III) oxyhydroxide morphologies.In addition, our results suggest that hydrogen could be the main driver of diversity of Zetaproteobacteria interacting with vent fluids containing both Fe(II) and H 2 , where flexible lithotrophic energy metabolism of Ghiorsea provides an advantage.

Zetaproteobacteria produce Fe(III) oxyhydroxide tubular sheaths in Fe mats at Fåvne
The porous black smoker chimneys at Fåvne show focused flow venting at 227°C of fluids containing abundant Fe(II) and H 2 (55), and support growth of extensive Fe mats covering tall black smoker chimney spires (Fig. 1a and b).The temperature within the Fe mats close to the venting orifice on chimney exteriors was measured at ~50°C (Fig. 1b).The chimney structures appear to lack defined central conduits (54), leading to copious venting of hydrothermal fluids (55) through permeable and porous chimney walls.Analysis of the microbial community composition based on metagenome-assem bled genome (MAG) coverage (and supported by 16S sequence read abundance) revealed that Zetaproteobacteria comprised 7% of the observed community (Fe Mat, see Supplementary Material 1 Table S1 at https://doi.org/10.5281/zenodo.8297777).
To compare the taxonomy of Zetaproteobacteria in Fe mats with those present at other locations at Fåvne, we recovered MAGs from other Fåvne sampling sites (see Supplementary Material 1 Table S1 at https://doi.org/10.5281/zenodo.8297777).While black smoker Fe mats Zetaproteobacteria were all assigned to the genus Ghiorsea, low-temperature diffuse venting at Fåvne supported a higher number of other Zetaproteobacteria taxa (see Table S3 at https://doi.org/10.5281/zenodo.8297777).A total of 28 unique species-representative genomes of Zetaproteobacteria were recovered at Fåvne (based on 95% ANI cutoff and publicly available MAGs) consisting of high-and medium-quality MAGs (average completeness 81.7%, contamination 2.1% based on CheckM2; see Table S4 at https://doi.org/10.5281/zenodo.8297777).These Fåvne MAGs were associated with two families defined by GTDB and seven defined genera, with three MAGs remaining unclassified to genus level and most of taxa lacking cultured representatives (see Fig. S6 at https://doi.org/10.5281/zenodo.8297777).

Ghiorsea in Fåvne Fe mats can oxidize H 2 in addition to Fe(II)
In alignment with the presence of H 2 and Fe(II) in endmember fluids at Fåvne (55), genes encoding all subunits of a transmembrane H 2 -uptake Ni,Fe hydrogenase (Group 1d) and Cyc2 for Fe(II) oxidation were identified in Fåvne Ghiorsea genomes belonging to both Cluster A and Cluster B (Fig. 2 and 4).In addition, codon usage bias analysis predicts high expression of Fe(II) oxidation and H 2 oxidation genes of the Ghiorsea MAGs from Fåvne (see Table S5 at https://doi.org/10.5281/zenodo.8297777).A broader functional screening revealed that H 2 -based metabolism with a Group 1d hydrogenase is common to other dominant MAGs within the Fe mat belonging to the Gammaproteobacteria, Ignavibacteria, Calditrichia, KSB1, and Aquificae (see Fig. S7; Table S6 at https://doi.org/10.5281/zenodo.8297777).Ghiorsea and some Gammaproteobacteria in the Fe mat also encode genes of an Ni,Fe H 2 -sensing hydrogenase histidine kinase-linked Group 2b (hup) located in the cytosol responsible for activating hydrogenase expression (see Fig. S8 at https://doi.org/10.5281/zenodo.8297777)(70).In contrast, hydrogenases were not detected in Zetaproteobacteria MAGs not belonging to genus Ghiorsea from other locations at Fåvne.
A phylogenetic tree constructed using the large subunit of the transmembrane Ni,Fe hydrogenase (Fig. 3

Ghiorsea
of Fåvne Ghiorsea hydrogenases with hydrogenases of other Ghiorsea (53, 67) and Gammaproteobacteria.Interestingly, the closest non-Zetaproteobacteria homolog was identified as a hydrogenase from a Gammaproteobacteria MAG (encoding genes for sulfur oxidation) from the same Fe mat.The cyc2 gene has previously been identified as one of the key genes in Fe(II) oxidation, with three distinct phylogenetic clusters of functionally verified and Research Article mSystems biochemically characterized representative Fe oxidases (51,71,72).Identified Cluster 3 and Cluster 1 cyc2 genes of Ghiorsea in the Fe mat at Fåvne showed highest similarity to cyc2 in other Ghiorsea MAGs from hydrothermal vents.Cyc2 genes were also identified in MAGs belonging to Gammaproteobacteria, Alphaproteobacteria, Aquificae, Planctomy cetes, and Calditrichia (Fig. 4, see Table S7 at https://doi.org/10.5281/zenodo.8297777).
Enzymes involved in carbon fixation were identified (see Fig. S10 at https://doi.org/10.5281/zenodo.8297777)for expected CO 2 fixation pathways in representative lineages (40,63,81,82); however, key genes for the serine variant of the reductive glycine pathway were observed in a Campylobacterota MAG (83).Form I RubisCO genes were identified in the Ghiorsea MAG Faaavne_M6_B18, and in a Gammaproteobacteria and Alphaproteobacteria MAG, using LithoGenie within MagicLamp (84).In one Alphaproteo bacteria MAG, gene for Form II RubisCO was also identified.
Given that Fe(III) oxyhydroxides adsorb heavy metals ( 5), an analysis of heavy metal resistance genes within the full metagenomic assembly of the Fe mat was performed.Heavy-metal resistance genes were identified for copper, cobalt, sodium acetate, chromium, tellurium, selenium, and silver (see Table S9 at https://doi.org/10.5281/zenodo.8297777).

DISCUSSION
Fe-oxidizing Zetaproteobacteria are globally distributed, yet our knowledge on the importance of hydrogen for their distribution is still limited.Here, we phylogenetically and functionally characterized Fe(II)-and H 2 -oxidizing Zetaproteobacteria from the Fåvne vent field belonging to Ghiorsea genus, adding four novel species-representa tive genomes predicted to use H 2 .We reconstructed 28 novel species-representative genomes of diverse Zetaproteobacteria taxa, extending the known Zetaproteobacteria Aquificae ( 1) Phycisphaerae ( 1) UBA2214 ( 1) Anaerolineae ( 2) Campylobacteria ( 5) Until recently, the identity of sheath-forming Zetaproteobacteria has remained elusive.We show that at least two populations of Ghiorsea (ZetaOTU9) most likely produce Fe(III) oxyhydroxide sheaths and form dense Fe mats.

Hydrogen as a driver of Zetaproteobacteria diversification
Most Zetaproteobacteria genera are metabolic specialists only able to obtain energy from the oxidation of Fe(II).The only known cultivated exception is G. bivora, capable of using H 2 simultaneously with Fe(II), or as sole electron donor (53).It has been suggested that members of Ghiorsea (ZetaOTU9) not only occupy environments rich in Fe(II) but also combined with predicted presence of H 2 , such as at hydrothermal vents (53), in corrosion of steel (8,85), and mineral weathering (40,85,86).The presence of hydrogen in these Ghiorsea environments has mainly been based on hypotheses until the current study.Remarkably, in the Fe mat close to the venting orifice at Fåvne and in contact with fluids containing abundant H 2 , the reconstructed Zetaproteobacteria MAGs are represented by only Ghiorsea within ZetaOTU9.
In contrast, a higher diversity of Zetaproteobacteria is present in low-temperature diffuse-venting areas at around ~10°C (see Table S3; Fig. S11 and S12 at https:// doi.org/10.5281/zenodo.8297777).All these genomes, except for Ghiorsea, lack uptake hydrogenases (see Fig. S6 at https://doi.org/10.5281/zenodo.8297777).Low-temperature diffuse-venting areas may reflect a low availability of H 2 relative to Fe(II), lost by abiotic or other subsurface mixing processes and low-temperature fluid formation (87).Ghiorsea, with its hydrogen uptake capability, emerges as the sole specialist in the presence of H 2 .Members within Ghiorsea are also observed in likely H 2 -poor diffuse-flow environ ments.Here, the diversity of Zetaproteobacteria is higher, also reflected by a diver sity of Fe(III) oxyhydroxide structures (see Fig. S13 and S14 at https://doi.org/10.5281/zenodo.8297777).Hence, this diversity indicates an absence of a monopolizing niche player in H 2 -poor diffuse flow, in contrast to Ghiorsea in the black smoker Fe mat where H 2 is available.This pattern of distribution supports the hypothesis that H 2 acts as a niche-determining factor for Ghiorsea at Fe(II)-rich hydrothermal vents (40).The ability of Ghiorsea to utilize H 2 affords it a competitive advantage as H 2 is a thermodynami cally more favorable energy source than Fe(II), supporting faster cell growth (53).The competitive advantage of growing on H 2 is likely linked to evading the need for reverse electron flow to replenish the reducing agent NADH needed for CO 2 fixation (Fig. 6).
Hydrogenases restricted to Ghiorsea ZetaOTU9 at Fåvne show that potential for growth on H 2 is a trait limited to ZetaOTU9.However, through the analysis of publicly available genomes of Zetaproteobacteria, transmembrane uptake hydrogenases were detected in Zetaproteobacteria outside of Ghiorsea, beyond hydrothermal vents (see Fig. S6 and S7 at https://doi.org/10.5281/zenodo.8297777).Even so, all Ghiorsea do not necessarily share the ability to oxidize H 2 .Outside of Ghiorsea Clusters A and B, two species representatives of Ghiorsea from freshwater and a subsea tunnel do not appear to possess hydrogenases (Fig. 2).Thus, far evidence suggests that presence of hydroge nases within Ghiorsea may be unique to hydrothermal vents.
Other H 2 oxidizers besides Ghiorsea are also present in the black smoker Fe mat which possess different hydrogenases (Fig. 5).Ni,Fe hydrogenases found in Ghiorsea MAGs were most closely related to hydrogenase subunits from other Ghiorsea and a Gammap roteobacteria MAG within the same Fe mat (Fig. 3) and symbiont chemolithotrophic sulfur-oxidizing microorganisms in hydrothermal vent fauna.These observations further strengthen the possibility of horizontal gene transfer of H 2 oxidation genes between Zetaproteobacteria and lithotrophic sulfur-oxidizing Gammaproteobacteria (53).We hypothesize this may have happened at hydrothermal vents.Fe mats at Fåvne cover black smokers at temperatures of up to 50°C (the maximum measured inside of a single Fe mat), which is at the high end of the temperature spectrum where Fe mats and ZetaOTU9 have been observed (40,53).The role of temperature in distribution patterns of Zetaproteobacteria cannot be ignored; however, minor differences in predicted optimum growth temperatures (see Fig. S6 at https:// doi.org/10.5281/zenodo.8297777)indicate fluid composition plays a larger role on the Ghiorsea (ZetaOTU9) niche differentiation than temperature.It is worth noting that the Fe mat studied is a bulk sample, where different microenvironments likely exist with varying degrees of exposure to the high-temperature-reducing venting fluids.Obtaining small-scale samples and corresponding in situ measurements to capture this variability in such environments remains a challenge.

Several Ghiorsea populations at Fåvne share the same metabolic niche
Within the Fe mats at Fåvne, four novel uncultured species or populations represented by MAGs of Ghiorsea (ZetaOTU9) co-exist, belonging to two distinct phylogenetic clusters (Cluster A and Cluster B; Fig. 2).Co-existence of multiple Ghiorsea populations has also been observed in Rimicaris vent shrimp, where the difference in the presence of hydrogenase in Ghiorsea MAGs has been proposed to contribute to niche partitioning, avoiding potential competition (67).In contrast, at Fåvne, all species-representative Ghiorsea genomes seem to possess genes for common metabolic functions, including presence of uptake hydrogenases, suggesting that multiple Ghiorsea species occupy the same ecological niche at Fåvne.It remains unknown what kind of interactions arise from co-occupying the niche in Fe mats.Possible competitive relationships among closely related populations of Zetaproteobacteria could be responsible for differential distribution across physical space ultimately leading to divergence within the ZetaOTU, as hypothesized for cosmopolitan ZetaOTU2 (88).In this context, the use of genome- resolved metagenomics offers valuable information about distinct subpopulations belonging to the same ZetaOTU and their genomic makeup.

Production of Fe(III) oxyhydroxide sheaths by members of the Fe-and H 2 -oxidizing genus Ghiorsea (Zetaproteobacteria)
In contrast to stalk-forming Zetaproteobacteria (44), the identity of sheath-forming Zetaproteobacteria has not been established through either cultivation or identification of the environmental and genetic drivers for sheath formation.Fe(III) oxyhydroxide sheaths in marine environments were shown to be associated with Zetaproteobacteria (18), and previous research has suggested ZetaOTU6, ZetaOTU9, and ZetaOTU15 as candidates for sheath-forming Zetaproteobacteria (22,24,85), with ZetaOTU6 sequen ces identified from an enrichment of sheath-forming Zetaproteobacteria (85).Specific ZetaOTU could not definitively be assigned to sheath morphology; however, ZetaOTU2, ZetaOTU6, and ZetaOTU14 were present in sheath-rich Fe mats (18).Ghiorsea within ZetaOTU9 accounting for 100% of all Zetaproteobacteria present in the Fe mat and the abundant homogenous Fe(III) oxyhydroxide tubular sheaths containing Zetaproteo bacteria cells (Fig. 1d, see Fig. S11 and S14 at https://doi.org/10.5281/zenodo.8297777)strongly suggest that, at Fåvne Ghiorsea, ZetaOTU9 is uniquely forming these structures.The presence of two-size morphotypes of Fe(III) oxyhydroxide sheaths suggests that more than one Ghiorsea population is producing Fe(III) oxyhydroxide sheaths.The comparison of MAG relative abundances with abundance of the two sheath variants (Fig. 1c) indicates the large 2 µm sheaths are produced by the Faavne_M6_B18 (Cluster A) Ghiorsea population, while the 1-µm-wide sheaths are produced by the AMOR20_M1306 (Cluster B) Ghiorsea population.Variable width Fe(III) oxyhydroxide sheaths hypothesized to be created by two different unidentified Zetaproteobacteria have previously been observed in Fe mats at Beebe's vents (34).Despite these concurring observations, the possibility remains that variation in sheath width is instead a consequence of a later, secondary colonization of the same species under different conditions, resulting in variations in cell size.Due to sheathed cells themselves being relatively rare and usually observed only at the tip of the sheaths while producing these structures moving forward (18), we cannot completely exclude the possibility that sheaths could be produced by a different rare ZetaOTU that was not detected in sequencing.Nonetheless, only through cultivation and targeted FISH staining, the formation of Fe(III) oxyhydroxide sheaths by Ghiorsea can be confirmed.
Similar to stalk formation, the genetic features for dread-forming Zetaproteobacteria are associated with the presence of distant homologs of stalk-forming Zetaproteobacte ria sfz genes (44,45).However, no homologs of the sfz genes were identified in Ghiorsea MAGs from Fåvne (Sfz1-6 genes; see Fig. S6 at https://doi.org/10.5281/zenodo.8297777)or the full metagenome assembly even at low sequence identity, suggesting a different genetic mechanism for sheath formation.
Whether sheaths are uniquely formed by Ghiorsea ZetaOTU9 globally remains an open question.Previous microscopy studies of Ghiorsea did not reveal any sheath formation (53,67), with the cultured representative strains of Ghiorsea instead producing amorphous Fe(III) oxyhydroxide particulates during growth on FeCl 2 (53).This aligns with the observation that closely related species vary in their capacity to produce distinct Fe(III) oxyhydroxide structures (20).Sheath-dominated Fe mat communities have been observed at several locations (18,20,23,24,32,34).Given that ZetaOTU9 has been described as having a cosmopolitan distribution (22,35,89), member species could be producing Fe(III) oxyhydroxide sheaths in numerous environments worldwide.This emphasizes the importance of microscopy in microbial ecology as not everything can be easily observed through genetic analyses.As preserved biogenic Fe(III) oxyhydr oxide structures can help us understand the environmental conditions of early Earth through studying ancient iron oxide deposits (90)(91)(92)(93) and can also be potentially used as biosignatures (20,94), knowledge of a sheath-forming Zetaproteobacteria capable of oxidizing both Fe(II) and H 2 might prove valuable for the interpretation.

Ghiorsea (ZetaOTU9) is the architect of Fe mats with abundant H 2
Although sheath-forming Ghiorsea is not the most abundant community member (7% relative abundance), it can be considered the main community engineer with respect to the amount of produced material (Fig. 7), in agreement with Zetaproteobacteria previously characterized as ecosystem engineers and primary colonizers in Fe mats (20,95).The generation of the architectural character of the Fe mat is subsequently followed by recruitment of other community members (42).At Fåvne, the dense Fe mats close to the venting orifice show a high abundance of Campylobacterota (formerly Epsilon proteobacteria), Gammaproteobacteria, and Alphaproteobacteria; taxa commonly seen in Fe mats (27).Diversity of primary producers in Fe mats at Fåvne appears high in comparison with other hydrothermal vent mats dominated by sulfur oxidizers (59)(60)(61).This is in-line with previous findings that FeOB support higher diversity (20,27).Differences in relative abundances of common lineages in microbial mats have been observed at sites with differing chemistry in previous studies (27,88,96), emphasizing the influence of vent fluids on microbial mat communities.The mixing of oxygen-rich seawater and reduced vent fluids in the porous chimney structures at Fåvne gives rise to steep chemical gradients which are reflected in the available electron donors and acceptors utilized within the Fe mat (Fig. 5).A strong association of Fe and Mn has been shown for hydrothermal fluids elsewhere ( 73), and at Fåvne, genes likely involved in manganese oxidation or detoxification were detected in proteomics analysis (Fig. 5, see Table S8, Supplementary Material 2 Text 1 at https:// doi.org/10.5281/zenodo.8297777).Consistent with the notion that Fe(III) oxyhydroxides adsorb heavy metals (5), the presence of various heavy metal resistance genes in the Fe mat (see Table S9 at https://doi.org/10.5281/zenodo.8297777)suggests adaptation to heavy metals.

Diversity of Fe oxidation based on Cyc2 genes at Fåvne
Potential for iron oxidation at Fåvne vent field is not limited to Zetaproteobacteria based on the presence of Cyc2 genes across several phyla.The identification of cyc2 in abundant members of the Gammaproteobacteria and Alphaproteobacteria may indicate a broader taxonomic range for neutrophilic iron oxidation (Fig. 4 and 5), as seen in previous studies (31,51,52,85,97,98), including in vent fauna endosymbionts (45,50).Based on metabolic profiling, MAGs possessing cyc2 seem able to use oxygen and nitrate (Fig. 5, see Fig. S15 at https://doi.org/10.5281/zenodo.8297777).The presence of nitrate and nitrite reductase genes in Zetaproteobacteria MAGs suggests a possi bility of an advantageous metabolic plasticity in Zetaproteobacteria able to reduce nitrate and nitrite in the absence of oxygen.Such an anaerobic metabolism has not yet been observed in isolates under laboratory conditions (19,80),z and Zetaproteobac teria terminal oxidases are predicted to be highly expressed (see Table S5 at https:// doi.org/10.5281/zenodo.8297777).Similarly, in addition to the common microaerophilic Zetaproteobacteria, anaerobic iron oxidizers have been detected in deeper layers of Fe mats at Kama'ehuakanaloa (Lō'ihi) (21).It remains unknown whether the widespread occurrence of Cyc2 genes at Fåvne is involved in Fe oxidation to obtain energy for carbon fixation in several lineages or whether some of these microorganisms rather use the Fe oxidase in other processes such as detoxification (52,99).It is, however, unlikely that Fe-oxidizing Ghiorsea at Fåvne are competing with other organisms for Fe resources, as there is an abundant supply of Fe(II) in the venting fluids (55).
Most Zetaproteobacteria encode for Form II RubisCO (41,48,100,101), including Ghiorsea genomes (53,67), suggesting a preference for environments with high CO 2 and low oxygen concentrations (102,103).A few Zetaproteobacteria encode for both Form I RubisCO, adapted to higher O 2 concentrations, and Form II RubisCO (48,104,105), suggesting a certain environment flexibility.Notably, Ghiorsea MAG Faaavne_M6_B18 (98.5% complete) encoded genes for Form I RubisCO exclusively, possibly indicating a tolerance to higher oxygen concentrations than most other Zetaproteobacteria.This oxygen tolerance could be in-line with their hypothesized presence on the outer, more oxygenated side of the black smoker Fe mat.

Porous black smoker chimneys support growth of Fe mats with iron and hydrogen at Fåvne
Previous studies of Fe mats constructed by Zetaproteobacteria have generally focused on low-temperature diffuse-venting areas rather than high-temperature hydrothermal vents (21,22,35).Although Fe mats on black smoker chimneys have been observed (29), the communities and interactions of their members have not yet been characterized.Whereas Fe mats studied previously are associated with fluids depleted in H 2 (18,24,27,31), vent fluids at Fåvne contain both H 2 and Fe(II) as abundant energy sources for Fe mats growing on black smoker chimney surfaces (55).Other hydrothermal vents with similar geochemistry to Fåvne, such as Rainbow at Mid-Atlantic Ridge and Beebe's vents (Piccard) at Mid-Cayman Rise where both H 2 and Fe(II) are present, have chimneys that contain much higher-temperature mineralized conduits that are likely not as porous (106,107), which may establish a much steeper chemical gradient through the chimney walls that is unable to sustain large exterior Fe mats.In contrast, the chimneys at Fåvne appear to be highly porous, visibly allowing vent fluid to advect outward and permeate through to the chimney surface.This is evident by the relatively high measured exterior temperatures (50°C) and visible shimmering, compared with the often much lower, near bottom water temperatures typically observed on more mineralized chimney exteriors (108).We propose this high fluid flux setting creates a suitable environment for microbial life to access higher abundances of electron donors at warmer temperatures, thereby forming dense Fe mats.

Conclusion
The presence of abundant (mmolar) Fe(II) and H 2 in the hydrothermal fluids at the Fåvne vent field offers a unique opportunity to investigate the interactions and adaptations of FeOB in response to the presence of elevated H 2 , providing valuable insights into their physiology and ecological dynamics.Our study is a first look into the microbial communities of black smoker Fe mats and the first microbiological exploration of the newly discovered Fåvne hydrothermal vent field.The findings strongly suggest that Zetaproteobacteria of Fe-and H 2 -oxidizing genus Ghiorsea at Fåvne produce Fe(III) oxyhydroxide sheaths and form dense Fe mats.With these Fe(III) oxyhydroxide struc tures, Ghiorsea provide the environment for other microorganisms, ultimately maintain ing the carbon, nitrogen, sulfur, and iron cycling in the Fe mats.Exclusive presence of Fe(II)-and H 2 -oxidizing Ghiorsea in the black smoker Fe mat exposed to abundant H 2 compared with occupation by diverse Zetaproteobacteria without hydrogenases at likely low H 2 environments at Fåvne supports the notion that H 2 availability plays a crucial role in driving the niche partitioning of Zetaproteobacteria.

Sampling site
The Fåvne vent field is located at 72°45.4′N, 3°49.9′E on the Mohns Ridge section of the series of AMOR at 3,030 m below sea level (54-56) (see Fig. S16 at https://doi.org/10.5281/zenodo.8297777).Black smoker chimneys have a porous structure and are rich in iron oxide and oxyhydroxide minerals, with high cobalt concentration of hydrother mal deposits (54,56).The black smoker hydrothermal fluids there are characterized by abundant iron and hydrogen (55).

Sample collection
Iron microbial mat samples were collected using an AEgir6000 remotely operating vehicle (ROV) on board the R/V G.O. SARS in June 2019, equipped with a biosyringe (a hydraulic sampling cylinder) connected to the ROV manipulator arm (Fig. 1a).Temperature (±1°C uncertainty) in the iron microbial mat was taken in real time using a temperature probe attached to the isobaric gas-tight fluid sampler snorkel inlet (109), which was used for vent fluid sample collection (55).Iron microbial mat was collected on the exterior of the North Tower vent (coordinates 72°45.4′N, 3°50′ E), a 13-m-tall active black smoker chimney 3,025 m below sea level, 1-2 m below the orifice (Fig. 1, see Supplementary Material 4, Supplementary Material 1 Table S1 at https://doi.org/10.5281/zenodo.8297777).Additional samples of the iron microbial mat mixed with underlying chimney, chimney, and iron oxide deposits were collected (see Supplemen tary Material 1 Table S1 at https://doi.org/10.5281/zenodo.8297777).Samples retrieved were centrifuged at 6,000 rcf for 5 minutes, and the supernatant was removed.Iron microbial mat pellet for on-ship metagenome sequencing using Nanopore MinION was processed directly.Aliquots for other analyses were frozen in liquid nitrogen and stored at −80°C until processing.Samples for scanning electron microscopy were fixed in 2.5% glutaraldehyde and stored at 4°C until further processing.Samples for fluorescence microscopy were fixed in 2% formaldehyde at 4°C overnight.

Scanning electron microscopy and elemental composition analysis
Fixed samples for SEM were filtered onto 0.2 µm polycarbonate filters with subsequent incubation in a series of increasing ethanol concentrations to remove water with and without critical point drying in CO 2 .Filters were then mounted on Al stubs and sputter coated with Ir using a Gatan 682 Precision etching coating system.The sputter coater was set for intended coating thickness of 10 nm.SEM images for morphological observation were produced at 5 keV using a Zeiss SUPRA 55VP scanning electron microscope equipped with a Thermo Noran Six Energy Dispersive Spectrometer at ELMILAB (laboratory for analytical electron microscopy) at the Department of Earth Science (Faculty of Mathematics and Natural Sciences, University of Bergen).For analysis of elemental composition, energy-dispersive X-ray spectroscopy (EDS) was performed at an accelerating voltage of 15 keV and a working distance of 8 mm.Data were processed with Pathfinder X-ray Microanalysis Software v.1.2(Thermo Fisher Scientific) with default settings.Spot scanning setting was used, and Ir peaks were removed due to the Ir signal from the coating.Qualitative elemental abundances of all abundant elements were measured, with the focus on C, N, O, P, S, Fe, Mn, Cu, Ca, Mg, Al, Si, and Zn.Table of elements for EDS analysis was used to inform us whether there were elements with similar energies that could be mixed up.

Fluorescence in situ hybridization
Samples were fixed in 2% formaldehyde at 4°C overnight, rinsed three times with phosphate-buffered saline (PBS), resuspended in 1:1 PBS:ethanol solution, and stored at −20°C following a protocol for preservation of material for FISH (110).Samples were spread on microscopy slides, air dried, and embedded in 0.5% low melting point agarose.For visualizing Zetaproteobacteria, the Zeta674 probe labeled with Atto488 fluorochrome was used (18).The Zeta674 probe specificity was analyzed, and the probe was successfully hybridized in silico using the SILVA Test-Probe tool, local BLAST, and the 16S sequence of the highest-quality Ghiorsea MAG recovered (Faavne_M6_B18).FISH was performed according to a previously published protocol (111).Slides were incubated at 46°C for 1 h with 20% formamide hybridization buffer in a hybridization chamber.The probe was added, followed by hybridization for 2 h at 46°C.Slides were then incubated in a washing solution (0.1 M NaCl, 20 mM Tris-HCl [pH 8.0], 5 mM EDTA, and 0.01% SDS] at 48°C for 15 minutes, washed twice with PBS, and air dried.Vectashield antifade solution was added.Slides were visualized with fluorescence microscopy using an overlay of phase-contrast and fluorescence images.Non-EUB338 was used as a negative control (112).SYBRGreen was used to visualize all cells.

On-ship Nanopore MinION sequencing workflow
DNA extraction, sequencing, and preliminary analysis were performed on board the research vessel during the expedition.DNA was extracted from a 1-mL Fe Mat sam ple aliquot using FastDNA Spin Kit for soil (MP Biomedicals), according to the manu facturer's protocol.Metagenomic sequencing of total DNA was carried out using the rapid sequencing library (SQK-RAD004) and the Oxford Nanopore Technologies MinION 1Mk1B sequencer equipped with a FLO-MIN106 SpotON Flow cell v.R9.Sequencing and raw data acquisition were controlled with the MinKNOW software.Basecalling was performed with a local version of the guppy basecaller v.3.4.4 (https://community.nanoporetech.com).Filtering of raw reads on length and quality was performed twice using Nanofilt v.2.5.0 as part of the NanoPack (113) and Porechop v.0.2.4 (https://github.com/rrwick/Porechop) (sequencing and filtering statistics in Tables S10 and S11 at https:// doi.org/10.5281/zenodo.8297777).

Illumina sequencing workflow
Whole-sample genomic DNA was extracted using Powersoil DNA Isolation Kit (QIAGEN) from frozen samples and sent to the Norwegian Sequencing Centre (University of Oslo, Norway) for shotgun metagenomic sequencing.A 150-bp paired-end sequencing was performed using an Illumina NovaSeq S4 flow cell.Raw reads were scanned for quality, duplication rate, and adapter contamination using FastQC v0.11.9 (https://github.com/s-andrews/FastQC), and concurrent visualization of the reports across samples was carried out in MultiQC (114).Strand-specific quality filtering methods recommended (115) were implemented through use of the "iu-filter-quality-minoche" script of the illumina-utils python package (116).Quality-filtered reads were subsequently cleaned of contaminating human DNA by mapping reads to the hg19 human genome with a mask applied to highly conserved genomic regions using the bbmap.shscript within the BBTools package (117) and human genome mask developed by Bushnell (available at https://drive.google.com/u/0/uc?id=0B3llHR93L14wd0pSSnFULUlhcUk).
Sequence reads were assembled by individual metagenomic sample with MEGAHIT v1.2.9 (118) using a minimum contig length of 1,000 bp.Reads from each sample were consecutively mapped to individual Illumina sample assemblies, effectively "co-map ping, " using Bowtie2 v.2.4.2 (119) then subsequent indexing with Samtools v. 1.11 (120).Binning and quality procedures were identical to those carried out as detailed for MinION sequencing with the exception of inclusion of MaxBin2 v 2.2.4 as an additional binning software used before implementation of DASTool.File manipulation, contig database creation, and profiles were accomplished with scripts from the Anvi'o v.7 platform (121).
Hybrid assembly of Nanopore and Illumina reads was also performed using meta SPAdes (128).This hybrid assembly had lower quality than wtdbg2 and MetaFlye only and Illumina-polished MetaFlye assemblies (see Table S12 at https://doi.org/10.5281/zenodo.8297777),with lower quality bins and fewer 16S sequences assigned to the genomes.Nanopore-only-based assembly generated only the most abundant Ghiorsea MAG, while the MetaFlye assembly polished with Illumina reads recovered two Ghiorsea MAGs, one of them the same species representative (>95% ANI) as the most abundant one in the Nanopore-only assembly and another one, less abundant.With this in mind, we decided to go forward with the MetaFlye long-read assembly polished with Illumina reads.Assembly read information and quality metrics are shown in Table S12 at https:// doi.org/10.5281/zenodo.8297777.Several other assembly and binning strategies were attempted and compared using QUAST v.5.0.2 with MetaQUAST output (129).
MAGs generated using MetaFlye, Pilon, and metaWRAP and the ones generated using only Illumina reads were dereplicated at 98% ANI using dRep v3.2.2 (138).These included all MAGs with at least 50% completeness and maximum 10% redundancy and all MAGs that had at least 0.5 coverage in the iron microbial mat metagenome (Fe Mat sample; 108 MEGAHIT Illumina MAGs and 19 MetaFlye, Pilon, and metaWRAP MAGs).The dereplica tion resulted in 111 MAGs.Relative abundances were calculated using the abundance output of relative coverage within one sample (Anvi'o v.7), and this was normalized to 1.

Taxonomic classification
The reconstructed MAGs were taxonomically classified using the genome taxonomy database tool kit gtdbtk v.2.3.2 (139) using the database GTDB 214 release.In addition, ZetaHunter v1.0.11 was used for assigning taxonomy to the 16S sequence of the Zetaproteobacteria MAGs (39), classifying sequences into Zetaproteobacteria opera tional taxonomic units at 97% similarity.Based on ZetaHunter cutoffs, we excluded all ZetaOTU classifications below 75% entropy.An overall taxonomic classification of Illumina metagenomic reads was performed with PhyloFLASH v.3.4 (140) based on 16S sequences using SILVA release 138 taxonomy as reference.

Genome database of Zetaproteobacteria
Zetaproteobacteria MAGs were reconstructed from samples of iron microbial mats, a chimney, and iron deposit at Fåvne (see Supplementary Material 1 Table S1 at https:// doi.org/10.5281/zenodo.8297777).The choice was made to concentrate efforts on the black smoker Fe Mat after identifying the presence of only the genus Ghiorsea and iron oxide sheaths since Fe Mat was the most precise sample of the iron microbial mat.All publicly available Zetaproteobacteria genomes (74) (taxid 580370) and corresponding metadata at NCBI GenBank were downloaded using ncbi-genome-download v.0.3.0 (https://github.com/kblin/ncbi-genome-download/)on the 19 October 2021.In addition, publicly available genomes of Zetaproteobacteria were downloaded from Genomes from Earth's Microbiome (141), Joint Genome Institute Integrated Microbial Genomes (JGI IMG), and from public repositories stated in selected studies (21,31,67,142).Additional genomes of Campylobacterota, Gammaproteobacteria, and Alphaproteobac teria closely related to the Fåvne MAGs were downloaded from NCBI as references.A threshold cutoff of high-and medium-quality genomes (min.50% completeness, max.10% redundancy) was used before further analysis.Phylogenomic analyses included 148 Zetaprotobacteria genomes in addition to the MAGs from this study.All selected genomes are presented in Table S4 at https://doi.org/10.5281/zenodo.8297777.
Optimal growth temperatures were predicted for Zetaproteobacteria MAGs (see Fig. S6 at https://doi.org/10.5281/zenodo.8297777)using genomic features and regression models (146).The models employed were Superkingdom Bacteria regression models that take into consideration the common absence of 16S sequence and genome incompleteness in MAGs.

Codon bias expression prediction
Codon bias gene expression levels were predicted using coRdon R package (v 1.8.0, https://github.com/BioinfoHR/coRdon),based on the measure independent of length and composition (MILC) and MILC-based expression level predictor values (164).

Phylogenetic tree of Cyc2
Cyc2 sequences were identified in MAGs present in the black smoker iron microbial mat and in all Zetaproteobacteria from all sampled Fåvne sites using FeGenie (162).Additional Cyc2 identifications were made from the top 10 hits using a blastp align ment based on the GenBank and NCBI_nr database.References were downloaded with BatchEntrez and reannotated as Cyc2 using FeGenie.Sequences shorter than 300 and longer than 600 amino acids were filtered out.Identical sequences were dereplicated using clustering with CD-HIT v4.8.1 (165).The resulting sequences of similar lengths were aligned using MAFFT L-INS-I v7.397 (148), manually checked in AliView v1.26 (166), and trimmed using trimAl v1.4.rev15 with -gt 0.7 (positions in the alignment with gaps in 30% or more of the sequences were removed) (149).A maximum likelihood phylogenetic tree was constructed with IQ-TREE v2.0.3 (150) using an alignment of 115 sequences with 388 positions and the best-fit model Q.pfam +F + I + I + R5, according to ModelFinder (151).Branch support values were calculated with standard bootstrapping with 1,000 iterations.The tree was rooted at midpoint.

Phylogenetic tree of Ni,Fe hydrogenase
The protein sequences of Ni,Fe large subunit hydrogenase 1d were identified from several sources: from the Fåvne iron microbial mat MAGs, the reference Zetaproteobacte ria, along with their closest relatives.Closest relatives were identified by BLAST Diamond annotations using HydDB (downloaded from HydDB webserver in February 2021) (70), the top 50 hits using an additional blastp alignment using GenBank and nr database, and blastp using a JGI IMG database gene search at 85% identity threshold (August 2022).Also, 213 Ni,Fe large subunit hydrogenase 1d reference sequences from HydDB were added (downloaded from HydDB webserver in March 2021).Sequences shorter than 460 amino acids were filtered out.The resulting sequences of similar lengths were aligned using MAFFT L-INS-i v7.397 (148), manually checked in AliView v1.26 (166), and trimmed using trimAl v1.4.rev15 with -gt 0.5 -cons 60.A phylogenetic tree was constructed with IQ-TREE v2.0.3 (150) using an alignment of 317 sequences with 595 positions, based on maximum likelihood and the best-fit model LG + I + I + R7, according to ModelFinder (151).Branch support values were calculated with standard bootstrapping with 1,000 iterations.Redundant sequences from several sources (NCBI, IMG JGI) were pruned, leaving only one sequence representative.The tree was rooted at midpoint.Environment data were pulled from available metadata and taxonomy from NCBI with corrections based on GTDB where genomes were present.

FIG 3
FIG3 Phylogeny of the large subunit of uptake Ni,Fe hydrogenase (hya; 1d).Phylogenetic tree of the large subunit of uptake Ni,Fe hydrogenase (hya; 1d) present in MAGs in the black smoker Fe mat and in all publicly available Zetaproteobacteria genomes, with closest relative reference using BLAST.Blue MAGs have been reconstructed from the Fåvne vent field.Black node circles mark branches with support values higher than 75% with standard bootstrapping and 1,000 iterations.Maximum likelihood tree with substitution model LG + I + I + R7.

FIG 4
FIG 4 Phylogeny of outer membrane cytochrome Cyc2.Phylogenetic tree of Fe oxidation cytochrome Cyc2 present in MAGs in the black smoker Fe mat including amino acid sequences from all publicly available Zetaproteobacteria genomes with closest relative references using BLAST.Blue labels are sequences from MAGs reconstructed from the Fåvne vent field in the current study.Support values for branches are calculated with standard bootstrapping and 1,000

FIG 6
FIG 6 Membrane complexes in Ghiorsea.Electrons coming from the oxidation of Fe 2+ are passed all the way to the high oxygen affinity terminal oxidase, leading to the generation of a proton motive force.Reverse electron transport is necessary to regenerate NADH needed for CO 2 fixation.NADH could also get replenished with the help of Ni,Fe uptake hydrogenase instead of the energy-intensive reverse electron transport.Hydrogenase can also donate electrons to the electron transport chain.ATP is generated by ATP synthase.A schematic representation of the metabolic potential of Ghiorsea, based on Ghiorsea MAGs from Fåvne and previous studies (40, 53).Created with BioRender.com.
, see Fig. S7 at https://doi.org/10.5281/zenodo.8297777)to assess the evolutionary relationships of encoded hydrogenases reveals the close relationship Based on Zetaproteobacteria distribution at Fåvne and encoded uptake hydrogenases, we demonstrate that H 2 availability indeed plays a role in the niche diversity of Zetaproteobacteria.Multiple species of Ghiorsea share the H 2 oxidation capacity in Fe mats at Fåvne, possibly sharing one niche.
mcoA mopA moxA FIG 5 Functional characterization of the top 25 abundant MAGs in the black smoker Fe mat.Distribution of genes involved in the utilization of a range of electron donors and electron acceptors.The number of genomes in each taxonomic class cluster is indicated in parenthesis, and the color gradient refers to the percentage of genomes per class that encode the genes.Average completeness and contamination values for each taxonomic class cluster are based on CheckM2 predictions.Top 25 most abundant MAGs account for 87% of MAG coverages.Research Article mSystems November/December 2023 Volume 8 Issue 6 10.1128/msystems.00543-238diversity.