# Discovery of Eremiobacterota with nifH homologues in tundra soil

Igor S Pessi, Tom O Delmont, Jonathan P Zehr, Jenni Hultman

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Igor S Pessi, Tom O Delmont, Jonathan P Zehr, Jenni Hultman. Discovery of Eremiobacterota with nifH homologues in tundra soil. Environmental Microbiology Reports, 2024, 16 (3), pp.e13277. 10.1111/1758-2229.13277. hal-04776164

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# Discovery of Eremiobacterota with *nifH* homologues in tundra soil

Igor S. Pessi<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> ID

Tom O. Delmont<fnref n="4" /> ID

Jonathan P. Zehr<fnref n="5" /> ID

Jenni Hultman<fnref n="1" />,<fnref n="2" />,<fnref n="6" /> ID

**Correspondence**

Jenni Hultman, Department of Microbiology, University of Helsinki, Helsinki, Finland.
Email: jenni.hultman@helsinki.fi

**Funding information**

Research Council of Finland, Grant/Award Number: 335354

**Abstract**

We describe the genome of an Eremiobacterota population from tundra soil that contains the minimal set of *nif* genes needed to fix atmospheric N₂. This putative diazotroph population, which we name *Candidatus* Lamibacter sapmiensis, links for the first time Eremiobacterota and N₂ fixation. The integrity of the genome and its *nif* genes are well supported by both environmental and taxonomic signals. Ca. Lamibacter sapmiensis contains three *nifH* homologues and the complementary set of *nifDKENB* genes that are needed to assemble a functional nitrogenase. The putative diazotrophic role of Ca. Lamibacter sapmiensis is supported by the presence of genes that regulate N₂ fixation and other genes involved in downstream processes such as ammonia assimilation. Similar to other Eremiobacterota, Ca. Lamibacter sapmiensis encodes the potential for atmospheric chemo-synthesis via CO₂ fixation coupled with H₂ and CO oxidation. Interestingly, the presence of a N₂O reductase indicates that this population could play a role as a N₂O sink in tundra soils. Due to the lack of activity data, it remains uncertain if Ca. Lamibacter sapmiensis is able to assemble a functional nitrogenase and participate in N₂ fixation. Confirmation of this ability would be a testament to the great metabolic versatility of Eremiobacterota, which appears to underlie their ecological success in cold and oligotrophic environments.

The conversion of atmospheric N₂ to ammonia by microbial diazotrophs represents the largest external source of nitrogen in tundra soils (Larmola et al., 2014; Stewart et al., 2014; Yin et al., 2022). Tundra N₂ fixation rates are generally low but are predicted to increase with the rise in atmospheric temperatures and precipitation levels associated with anthropogenic climate change (Stewart et al., 2013; Stewart et al., 2014). Greater inputs from N₂ fixation coupled with faster rates of organic matter mineralization will increase the pool of bioavailable nitrogen in tundra soils and potentially lead to higher greenhouse gas emissions, particularly N₂O (Hugelius et al., 2020; Stewart et al., 2013; Virkkala et al., 2024). More knowledge on the microbial drivers of N₂ fixation is thus essential for a better

understanding of the dynamics of greenhouse gas production in tundra soils.

Here, we investigated the diversity of microbial diazotrophs in the tundra by leveraging a catalogue of 796 manually curated metagenome-assembled genomes (MAGs) from soils in Kilpisjärvi, northern Finland (Pessi, Viitamäki, et al., 2022) (see Methods in Supplementary information). The Kilpisjärvi MAGs represent populations from different soil ecosystems (from bare soil to water-logged fens) and encompass a high diversity of Archaea and Bacteria (Pessi, Rutanen, & Hultman, 2022; Pessi, Viitamäki, et al., 2022). We searched the 796 Kilpisjärvi MAGs using a hidden Markov model of the *nifH* gene, which encodes the dinitrogenase reductase subunit of the nitrogenase enzyme

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2024 The Author(s). *Environmental Microbiology Reports* published by John Wiley & Sons Ltd.

<footnote n="1">¹Department of Microbiology, University of Helsinki, Helsinki, Finland</footnote>

<footnote n="2">²Helsinki Institute of Sustainability Science (HELSUS), University of Helsinki, Helsinki, Finland</footnote>

<footnote n="3">³Marine and Freshwater Solutions, Finnish Environment Institute (Syke), Helsinki, Finland</footnote>

<footnote n="4">⁴Génomique Métabolique, Genoscope, Evry, France</footnote>

<footnote n="5">⁵Ocean Sciences Department, University of California Santa Cruz, Santa Cruz, California, USA</footnote>

<footnote n="6">⁶Natural Resources Unit, Natural Resources Institute Finland (Luke), Helsinki, Finland</footnote>
and is the most widely used marker for N₂ fixation (Dos Santos et al., 2012; Koirala & Brözel, 2021; Zehr et al., 2003). We found *nifH* homologues in 26 MAGs, most of which are affiliated with groups that include known diazotrophs such as Alpha- and Gammaproteobacteria and methanogenic Archaea (see 'Kilpisjärvi MAGs with nifH homologs' in Supplementary information).

Surprisingly, we identified *nifH* homologues in the MAG KWL-0264 assigned to Eremiobacterota, a phylum for which no diazotrophic activity and/or nitrogenase sequences have been previously reported. The validity of the MAG as a whole and the inclusion of *nifH* homologues are supported by several features including homogeneous GC content, uniform coverage in multiple metagenomes, and clear taxonomical signal for Eremiobacterota across contigs, including those containing the *nifH* homologues (Figure 1A; see also 'The integrity of the Eremiobacterota MAG KWL-0264' in Supplementary information). Phylogenomic analysis with other publicly available genomes (n = 302) confirmed the affiliation of KWL-0264 with Eremiobacterota, placing it alongside other MAGs from polar environments in the family Baltobacteraceae (Figure 1B). The phylogenetic placement and low average nucleotide identity (ANI) with other Eremiobacterota genomes indicate that KWL-0264 represents a distinct lineage in this phylum. Given its higher environmental signal in minerotrophic fens compared to upland soils (see 'Distribution of nifH MAGs' in Supplementary information), we

propose the name *Candidatus* Lamibacter sapmiensis (N.L. fem. n. *lama*, a bog or fen; N.L. masc. n. *bacter*, a rod; N.L. masc. n. *Lamibacter*, a rod that grows on bogs or fens; N.L. masc. adj. *sapmiensis*, pertaining to Sápmi, the cultural region inhabited by the Sámi people in Fennoscandia).

The presence of *nifH* homologues and other related genes suggests that Ca. Lamibacter sapmiensis plays a role in N₂ fixation in tundra soils (Figure 2; see also 'The *nifH* homologs of KWL-0264' in Supplementary information). The MAG KWL-0264 contains three *nifH* homologues, which is not uncommon among known diazotrophs (Zehr et al., 2003), and also includes the additional *nifDKENB* genes that are deemed necessary for the assembly of a functional nitrogenase (Dos Santos et al., 2012; Koirala & Brözel, 2021). Importantly, one of the *nifH* homologues (*nifH*₁) encodes a nitrogenase affiliated with Cluster III, which is a diverse group of canonical nitrogenases mostly from anaerobic Bacteria and Archaea with demonstrated diazotrophic activities (Dos Santos et al., 2012; Koirala & Brözel, 2021; Zehr et al., 2003). Further hints towards a role in N₂ fixation is given by the occurrence of *nifl*₁ and *nifl*₂ downstream of *nifH*₁. These genes are usually found in diazotrophs containing Cluster III nitrogenases and encode a type of GlnB/PII protein that inactivates N₂ fixation when ammonia is available (Kessler et al., 2001; Zehr et al., 2003). It is important to note that the *nifH*₁ homologue is not co-located with *nifDK*, but this is likely due to low genome contiguity rather

<figure><img src="image_3.png" /><figcaption>**FIGURE 1** (A) Representation of the metagenome-assembled genome (MAG) KWL-0264 (*Candidatus* Lamibacter sapmiensis). The four external rings show the mean coverage of each contig across four fen metagenomes from Kilpisjärvi, northern Finland. The innermost blue ring (taxonomic signal) represents the proportion of genes in each contig that had the best match with another Eremiobacterota sequence in the GenBank nr database. MAG completion and redundancy was estimated based on the presence of 71 single-copy genes with *anvi'o* v7.1. (B) Phylogenomic analysis of Ca. Lamibacter sapmiensis alongside other publicly available Eremiobacterota genomes (n = 302). Maximum-likelihood tree (LG + I + G model) based on a concatenated alignment of 71 single-copy genes. Bootstrap support is ≥95% unless shown. The tree is rooted at midpoint. The average nucleotide identity (ANI) between Ca. Lamibacter sapmiensis and close neighbours is indicated.</figcaption></figure>
than gene loss. The other two *nifH* homologues of KWL-0264 (*nifH*₂ and *nifH*₃) encode Cluster IV nitrogenases, which have been largely thought as paralogs that do not participate in N₂ fixation (Dos Santos et al., 2012; Koirala & Brözel, 2021; Zehr et al., 2003). However, this view has been challenged by the reported diazotrophic growth of *Endomicrobium proavi-tum* with a Cluster IV nitrogenase (Zheng et al., 2016). Importantly, the contig with the *nifH*₂ homologue con-tains the complete set of *nifDKENB* genes, suggesting the capacity to assemble a functional nitrogenase. Fur-ther support for a role of Ca. Lamibacter sapmiensis in N₂ fixation is given by the occurrence, near all three *nifH* homologues, of several genes that are involved in nitrogen and amino acid transport and metabolism. These include genes for the GS-GOGAT pathway of ammonia assimilation (*glnA* and *gltD*), ABC-type branched-chain amino acid (*livFGHKM*) and nitrate (*tauABC*) transporters, and amino acid biosynthesis (*dapD*, *aspB*, *cysE*, *argA*, and *metC*).

Further gene annotation and metabolic reconstruc-
tion revealed many potential traits of Ca. Lamibacter
sapmiensis that might be beneficial in mineratrophic
fens and other oligotrophic environments (Figure 3; see
also 'Reconstruction of the metabolic potential of KWL-
0264' in Supplementary information). First, the potential
to fix N₂ represents an ecological advantage in northern
fens and peatlands in general, where most of the nitro-
gen is bound to the organic matter and thus not readily
available for growth (Hugelius et al., 2020; Yin
et al., 2022). Second, KWL-0264 encodes the potential
to fix CO₂ using energy obtained from the oxidation of
H₂ and CO. This metabolic strategy, known as atmo-
spheric chemosynthesis, has been reported for other
Eremiobacterota, and appears to underlie their ecologi-
cal success in cold and oligotrophic ecosystems such
as tundra soils, polar deserts, and peatlands (Ji
et al., 2017; Ji et al., 2021; Woodcroft et al., 2018; Yabe
et al., 2022). Third, the potential to oxidize H₂ can help
alleviate the high energetic cost of N₂ fixation, as it

<figure><img src="image_4.png" /><figcaption>FIG URE 2 Representation of the three contigs harbouring *nifH* homologues in *Candidatus* Lamibacter sapmiensis KWL-0264. Genes highlighted with a blue rectangle had the best match with another Eremiobacterota sequence in the GenBank *nr* database.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**FIG URE 3** Simplified representation of the metabolic potential of Ca. Lamibacter sapmiensis KWL-0264. Only selected pathways discussed in the text are shown. Figure created with **BioRender**.</figcaption></figure>
allows the recovery of part of the spent energy by recy-
cling the H₂ that is produced during the process
(Greening et al., 2016). Finally, KWL-0264 encodes a
clade II N₂O reductase and thus the potential for anaer-
obic respiration via the reduction of N₂O to N₂, which is
favoured in the typically anoxic environment of water-
logged fens (Pessi, Viitamäki, et al., 2022). Moreover, it
can be speculated that the reduction of N₂O to N₂ might
also be used as a strategy to fuel N₂ fixation. The ability
to reduce N₂O indicates a potential role of this Eremio-
bacterota population in mitigating the emission of this
potent greenhouse gas, as has been shown for other
non-denitrifying N₂O reducers (Jones et al., 2013).

After more than 20 years since the first description
of Eremiobacterota as 'candidate phylum WPS-2' from
contaminated soil (Nogales et al., 2001), the recovery
of a MAG harbouring *nifH* homologues suggests that
N₂ fixation might also be a part of the metabolic reper-
toire of this phylum. At present, it remains unclear if the
genomic constitution of Ca. Lamibacter sapmiensis can
lead to a nitrogenase with function in N₂ fixation. Fur-
ther studies should focus on isolating this population
in vitro, recovering other closely related genomes (pref-
erentially using long-read sequencing technologies),
and providing evidence of N₂ fixation activity using, for
example, rate measurements coupled with (meta)tran-
scriptomics and/or stable isotope probing.

AUTHOR CONTRIBUTIONS

**Igor S. Pessi:** Conceptualization (equal); data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); visualization (lead); writing – original draft (lead). **Tom O. Delmont:** Conceptualization (equal); visualization (supporting); writing – review and editing (equal). **Jonathan P. Zehr:** Methodology (supporting); writing – review and editing (equal). **Jenni Hultman:** Conceptualization (equal); funding acquisition (lead); investigation (supporting); project administration (lead); resources (equal); writing – review and editing (equal).

ACKNOWLEDGEMENTS

We would like to acknowledge the CSC—IT Centre for
Science, Finland for providing the computing
resources, and Antti Karkman, Eeva Eronen-Rasimus,
and Hermanni Kaartokallio for comments. We also
thank the two anonymous reviewers for the critical eval-
uation of our manuscript. This study was funded by the
Academy of Finland (project 335354) and the Univer-
sity of Helsinki.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

DATA AVAILABILITY STATEMENT

The raw metagenomic data and the 796 Kilpisjärvi
MAGs including Ca. Lamibacter sapmiensis KWL-0264

can be found in the European Nucleotide Archive
(ENA) under the project PRJEB41762. The MAGs are
also available from FigShare (doi.org/10.6084/m9.
figshare.19722505). The detailed bioinformatics work-
flow used in this study can be found in github.com/
ArcticMicrobialEcology/Candidatus-Lamibacter-
sapmiensis.

ORCID

Igor S. Pessi https://orcid.org/0000-0001-5926-7496

Tom O. Delmont https://orcid.org/0000-0001-7053-7848

Jonathan P. Zehr https://orcid.org/0000-0002-5691-5408

Jenni Hultman https://orcid.org/0000-0002-3431-1785

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Pessi, I.S., Delmont, T.O., Zehr, J.P. & Hultman, J. (2024) Discovery of Eremiobacterota with *nifH* homologues in tundra soil. *Environmental Microbiology Reports*, 16(3), e13277. Available from: https://doi.org/10.1111/1758-2229.13277