A novel Methylomirabilota methanotroph potentially couples methane oxidation to iodate reduction

Impact statement Methane oxidizing microbes play a key role in reducing the emission of this potent greenhouse gas to the atmosphere. The known versatility of the recently discovered anaerobic Methylomirabilota methanotrophs is limited. Here, we report a novel uncultured Methylomirabilis species, Candidatus Methylomirabilis iodofontis, with the genetic potential of iodate respiration from biofilm in iodine‐rich cavern spring water. Star‐like cells resembling Methylomirabilis oxyfera were directly observed from the biofilm and a high‐quality metagenome‐assembled genome (MAG) of Ca. M. iodofontis was assembled. In addition to oxygenic denitrification and aerobic methane oxidation pathways, the M. iodofontis MAG also indicated its iodate‐reducing potential, a capability that would enable the bacterium to use iodate other than nitrite as an electron acceptor, a hitherto unrecognized metabolic potential of Methylomirabilota methanotrophs. The results advance the current understanding of the ecophysiology of anaerobic Methylomirabilota methanotrophs and may suggest an additional methane sink, especially in iodate‐rich ecosystems.

Methane oxidizing microbes are essential in controlling methane emissions from various environments.In addition to aerobic methanotrophs within the Proteobacteria and Verrucomicrobiota, anaerobic methantrophic archaea (the ANMEs) and bacteria within the Methylomirabilota (previously NC10 phylum), capable of anaerobic oxidation of methane (AOM), have been discovered during the last two decades.ANME archaea are suggested to oxidize methane via reverse methanogenesis 1 , using different electron acceptors, such as sulfate 2 , iron oxides 3 , nitrate and nitrite 4,5 , with or without a syntrophic partner.In contrast, bacteria within the methanotrophic Methylomirabilota oxidize methane via a canonical methane monooxygenase-dependent aerobic pathway, exclusively using nitrite as electron acceptor 6,7 .Methylomirabilota methanotrophs are proposed to generate their own intracellular oxygen supply via nitric oxide (NO) dismutation into O 2 and N 2 , catalyzed by a putative NO dismutase 8 .NO dismutase (nod) genes are widely distributed among diverse microbial lineages 9 .In addition to this peculiar metabolism, Methylomirabilis oxyfera was reported to display a characteristic polygonal cell shape in electron micrographs 10 .However, it remains to be shown whether other Methylomirabilota methanotrophs also show similar morphologies.
The diversity of Methylomirabilota methanotrophs as inferred from functional marker genes, such as particulate methane monooxygenase (pmoA) 11 or nod genes 12 seems limited, especially in comparison to the diversity of Methylomirabilota derived from 16S rRNA sequences 12,13 .Hitherto, the dominant bacteria in denitrifying AOM cultures, for example [14][15][16] , as well as environmental microbes with supposed denitrifying methane-oxidizing capability 17,18 , were all closely related to M. oxyfera.Other environmental metagenome-assembled genomes (MAGs) affiliated with the Methylomirabilota phylum did not indicate a denitrifying potential linked to methanotrophy 19,20 .Recently, denitrifying AOM enrichment cultures containing Methylomirabilota bacteria were reported to reduce selenate 21 or chlorate 22 under methane oxidation.However, there was no direct evidence for the involvement of Methylomirabilota in these processes.Hence, our current understanding of the diversity and metabolic versatility of Methylomirabilota methanotrophs remains very limited.
Here, we report the MAG of a novel Methylomirabilota bacterium, Candidatus Methylomirabilis iodofontis, from methane-oxidizing biofilms sampled under iodine-rich mineral water in a subsurface spring cavern in Sulzbrunn, Germany.Iodine-rich (>20 mg l −1 ) formation water from the subalpine Lower Marine Molasse enters the spring together with thermogenic methane, which accumulated up to 3000 ppm in the undisturbed microoxic cavern atmosphere 23 .Within the submersed biofilm at the cavern wall, transmission electron microscopy revealed peculiar star-shaped microbial morphologies, resembling that of M. oxyfera (Figure 1A).In addition, 16S rRNA gene sequences related to that of Methylomirabilis spp.were retrieved via targeted PCR and cloning (Figure S1), consistent with previous results of 16S rRNA gene amplicon sequencing of the respective submersed biofilms, where reads of the Methylomirabilota (NC10) accounted for up to 10% 23 .These lines of evidence all indicate the presence of Methylomirabilis methanotrophs in the cave biofilm.
Therefore, we sequenced the metagenome of the submersed biofilm and assembled a putative Methylomirabilota genomic bin (bin48), which was over 70% complete and with very low contamination (1.52%) (Table S1).In total, 4780 Methylomirabilota 16S rRNA gene reads were retrieved, accounting for 14.3% of all 16S rRNA reads detected in the metagenomic library, representing one of the most abundant (sub)phylum-level populations (Table S2).All Methylomirabilota 16S rRNA reads were assembled into one consensus fulllength 16S rRNA gene, which showed >99% similarity to that of Methylomirabilis limnetica (Figure S1).Yet, the pairwise average amino acid identity (AAI) and the average nucleotide identity (ANI) between M. limnetica genome and bin48 were only 85.8% and 91.3%, respectively, suggesting the newly binned MAG to represent a novel Methylomirabilis species, which was tentatively named Ca.M. iodofontis.Phylogenomic analysis based on 121 concatenated protein markers further supported that M. iodofontis was closely related to other Methylomirabilis species, forming a monophyletic clade within the order Methylomirabilales of the Methylomirabilota phylum (Figure 1B).
In the MAG of M. iodofontis, a pyrroloquinoline quinone (PQQ)-dependent methanol-dehydrogenase and a formatedehydrogenase highly similar to those in M. oxyfera and M. limnetica were also present.However, a particulate methane monooxygenase (pMMO) operon was missing (Table 1), possibly due to the incompleteness of the MAG.The presence of a complete methane-oxidizing pathway in the MAG was statistically assessed using MetaPOAP 24 , and the falsepositive and false-negative probabilities were 7.524e−10 and 0.069, respectively, suggesting that the pMMO genes are likely present in the source genome.Moreover, M. iodofontis harbored a complete Calvin−Benson−Bassham (CBB) cycle, except for the Rubisco small unit gene (Table 1), indicating an autotrophic lifestyle like M. oxyfera 25 .The Rubisco large subunit of M. iodofontis clustered closely to that of other Methylomirabilis spp., all falling in the type IC/D group (Figure S2).The high similarity between M. iodofontis and other Methylomirabilis methanotrophs on the whole-genome level as well as for key methane-oxidizing enzyme genes (Table 1) also strongly argues for a methane-oxidizing capability in M. iodofontis.Like other Methylomirabilis species, a complete oxygenic denitrification pathway was present, although a second nod (DAMO2434-like) gene 12 was not identified in the MAG (Table 1).Yet, nod-targeted PCR and cloning recovered two Nod clusters as known for other Methylomirabilis spp., and a distantly related Nod (Figure 1C), indicating that the M. iodofontis genome likely also harbors two distinct nod gene homologs.The M. iodofontis Nod possessed all characteristic substitutions known for other Nod sequences (Figure S3).In comparison, reconstructed genomes of other members of the Methylomirabilales 19,20 , distantly related to Methylomirabilis spp., neither indicated methane oxidation nor oxygenic denitrification capacities (Figure 1B).Likely, the denitrifying methanotrophic lifestyle is restricted to the genus Methylomirabilis within the Methylomirabilota.
Interestingly, the cave spring water only contained low nitrate concentrations (<0.2 mg l −1 ) and nitrite was undetectable 23 .Thus, a potential for respiring other electron acceptors by M. iodofontis was assessed within the MAG.Remarkably, the corresponding MAG also harbored a gene cluster encoding cytochrome c peroxidases (IdrP1 and IdrP2) and an iodate reductase (IdrBA), the activity of which was recently demonstrated for Pseudomonas sp.SCT 26 and Denitromonas sp.IR-12 27 .The GC content and sequencing depth of the contig (bin48_25), where the iodate reductase gene cluster was located, was comparable to that of other contigs in the MAG, supporting its origin from M. iodofontis (Figure S4).Phylogenetic analysis demonstrated that the catalytic subunit of the iodate reductase (IdrA) of M. iodofontis was clearly placed within a cluster of iodate reductases (Figure 1D).The organization of this iodate reductase gene cluster (idrP2,P1,B,A) in Ca.M. iodofontis was also the same as in Pseudomonas sp.SCT and Denitromons sp.IR-12 (Figure 1D).This organization seems characteristic among iodate reductases, distinct from more distantly related arsenite oxidases and periplasmic nitrate reductase encoding gene clusters 27 .These results strongly suggest that M. iodofontis carries a functional iodate reductase.Notably, M. iodofontis  iodate reductase genes had no significant hits in genomes of other Methylomirabilis species (Table 1).An incomplete operon (idrP1,B,A) was detected on a contig of another subsurface Methylomirabilota MAG (GCA_001771285.1)(Figure 1B), which also belonged to the order Methylomirabilales but was not placed within the Methylomirabilis clade and lacked oxygenic denitrification and methane oxidation pathways (Figure 1B).This may indicate that M. iodofontis could have acquired iodate reductase genes via lateral gene transfer, as also proposed for other iodate-reducing bacteria 27 .SignalP analysis 28 revealed that both IdrP1 and IdrP2 possess the Sec and IdrB possesses a twin-arginine translocation (TAT) signal peptide, suggesting a periplasmic location of the M. iodofontis iodate reductase.This was also shown for Pseudomonas sp.SCT and Denitromonas sp.IR-12 26,27 .It has been proposed that in Denitromonas sp.IR-12, IdrAB first reduces iodate to hypoiodous acid (HIO), which is chemically unstable and undergoes abiotic disproportionation to I − and IO 3

−
. The latter is subsequently cycled back to the enzymatic reduction 27 .In Pseudomonas sp.SCT, iodate reduction by IdrAB to hydrogen peroxide (H 2 O 2 ) and HIO was proposed.The resulting H 2 O 2 is detoxified by cytochrome c peroxidase (IdrP1 and IdrP2) to water and HIO is presumably disproportionated into O 2 and iodide by a chlorite dismutase like (Cld-like) enzyme 26 .Both Denitromonas sp.IR-12 and Pseudomonas sp.SCT oxidize acetate to fuel iodate reduction; however, the potential electron donor for this reaction in M. iodofontis is still unclear.Notably, iodate reduction via the second proposed pathway would also allow for an oxygen-dependent methane oxidation in M. iodofontis (Figure 1E), via the following redox reaction: In summary, we report the MAG of a novel, yet uncultured Methylomirabilota methanotroph, Ca.M. iodofontis.Consistent with the specific biogeochemical setting of the iodineand methane-rich mineral spring cave, genetic and phylogenomic analyses suggest a capacity for methane oxidation, oxygenic denitrification, as well as iodate reduction in M. iodofontis (Figure 1E).This expands our perspective of the metabolic versatility of Methylomirabilota methanotrophs.Due to the ubiquity of iodate in ocean waters 29 , such ecophysiologies might be widely distributed and represent an overlooked methane sink in marine ecosystems.

Figure 1 .
Figure 1.Cell morphology, phylogenetic analysis, gene cluster organization, and key respiratory pathways.(A) TEM image of Methylomirabilis oxyfera-shaped cell from the submersed biofilm.(B) Phylogenomic analysis of Methylomirabilota phylum bacteria and MAGs, including Methylomirabilis iodofontis and other Methylomirabilis species and Rocubacteriales.(C) Nod phylogenetic tree including cloned Nod sequences from submersed biofilm and assembled Nod in Candidatus Methylomirabilis iodofontis genome.(D) Phylogenetic tree of the catalytic subunit of iodate reductase (IdrA), arsenite oxidase (AioA), and periplasmic nitrate reductase (NapA).IdrA encoded in the M. iodofontis is in bold, and the gene cluster organization of iodate reductase in Pseudomonas sp.SCT, Denitromonas sp.IR-12 and Ca.M. iodofontis, and arsenite oxidase, nitrate reductase in other microbes are shown.(E) Key respiratory pathways in M. iodofontis according to genetic analysis.Both proposed iodate reduction routes taking place in periplasmic space are illustrated.
metagenome-derived physiology of M. iodofontis clearly awaits validation via labeling experiments in biofilm samples and enrichment cultures under laboratory conditions.

Table 1 .
Key metabolic pathways and CBB cycle-associated genes in Ca.Methylomirabilis iodofontis.The two Nod sequences have 26 residual overlap and can be assembled, resulting in one complete M. iodofontis Nod.ND, no significant similarity found.
a Based on amino acid sequence.b