Aerobic and anaerobic iron oxidizers together drive denitrification and carbon cycling at marine iron-rich hydrothermal vents

Abstract

In principle, iron oxidation can fuel significant primary productivity and nutrient cycling in dark environments such as the deep sea. However, we have an extremely limited understanding of the ecology of iron-based ecosystems, and thus the linkages between iron oxidation, carbon cycling, and nitrate reduction. Here we investigate iron microbial mats from hydrothermal vents at Lō'ihi Seamount, Hawai'i, using genome-resolved metagenomics and metatranscriptomics to reconstruct potential microbial roles and interactions. Our results show that the aerobic iron-oxidizing Zetaproteobacteria are the primary producers, concentrated at the oxic mat surface. Their fixed carbon supports heterotrophs deeper in the mat, notably the second most abundant organism, Candidatus Ferristratum sp. (uncultivated gen. nov.) from the uncharacterized DTB120 phylum. Candidatus Ferristratum sp., described using nine high-quality metagenome-assembled genomes with similar distributions of genes, expressed nitrate reduction genes narGH and the iron oxidation gene cyc2 in situ and in response to Fe(II) in a shipboard incubation, suggesting it is an anaerobic nitrate-reducing iron oxidizer. Candidatus Ferristratum sp. lacks a full denitrification pathway, relying on Zetaproteobacteria to remove intermediates like nitrite. Thus, at Lō'ihi, anaerobic iron oxidizers coexist with and are dependent on aerobic iron oxidizers. In total, our work shows how key community members work together to connect iron oxidation with carbon and nitrogen cycling, thus driving the biogeochemistry of exported fluids.

Fig. 1. Lōʻihi Seamount iron mat microbial community composition based on 16S rRNA gene, metagenome (MG), and metatranscriptome (MT) relative abundance.

Zetaproteobacteria and Candidatus Ferristratum sp. were the most abundant taxa. The 16S rRNA gene surveyed the bacterial population only. Viral expression based on viral contigs identified from VirSorter. S6 expression shown for pre-Fe(II) addition sample only.

Fig. 2. Gene expression for carbon fixation, electron donors and electron acceptors within the iron mat.

Heatmaps show the total normalized gene expression (TPM) for a carbon fixation, b electron donors and c electron acceptors, with d pie charts showing the relative taxonomic contribution to expression. Genes that lacked expression that were still found within MAGs are indicated with gray boxes. Pathways that were not represented in a given sample remain white. Carbon fixation genes were expressed primarily by the Zetaproteobacteria (CBB), though other organisms were also involved: unbinned Methanoperedens sp. (WL) in S1, unbinned Nitrospirae (rTCA and WL) in S6, and Gammaproteobacteria Thiotrichales (CBB) and Actinobacteria (WL) in S19. In addition to iron oxidation, methane oxidation was another significant source of energy in S1, carried out by the unbinned Archaea Methanoperedens (mcrABG) and by the Gammaproteobacteria (pmoABC).

Fig. 3. Cyc2 maximum likelihood phylogenetic tree (300 bootstraps) showing the relative placement of Cyc2 belonging to Candidatus Ferristratum sp. MAGs identified in this study.

The cyc2 genes found within the Candidatus Ferristratum sp. MAGs form a monophyletic clade (100% bootstrap support) distinct from the Zetaproteobacteria.

Fig. 4. Nitrogen cycling gene expression within the iron mat.

Heatmap shows the total normalized gene expression (TPM) for different metabolic pathways related to nitrogen cycling (a), with pie charts of relative taxonomic contribution to expression also shown (b). Genes that lacked expression that were still found within MAGs are indicated with gray boxes. Pathways that were not represented in a given sample remain white.

Fig. 5. Gene expression patterns for major taxa during the S6 Fe(II) addition experiment.

Maximum normalized gene expression (Max Norm. TPM) totals shown for organisms (a) and genes of interest (b, c) for the Zetaproteobacteria (left) and Candidatus Ferristratum sp. (right). Genes related to Fe(II) oxidation (cyc2, ccoNO, narG) (b) are separated from genes involved in denitrification (narG, nirK, eNOR, cNOR, nosZ) (c). Data are shown for the sample pre-Fe(II) addition, as well as at 10 min intervals starting 2 min after Fe(II) addition. Organism maximum TPM values: All Zetaproteobacteria (621,257), all Candidatus Ferristratum sp. (106,842), all viruses (37,024). Zetaproteobacteria gene maximum TPM values: cyc2 (3,683), ccoNO (826), nirK (787), eNOR (31), nosZ (218). Candidatus Ferristratum sp. gene maximum TPM values: cyc2 (110), narG (1,330), cNOR (106).

Fig. 6. Heatmap showing the log₂ total normalized gene expression (TPM) for different metabolic pathways related to iron, nitrogen, and oxygen cycling.

Expression is shown for representative Zetaproteobacteria MAGs (left) and near-complete Candidatus Ferristratum sp. MAGs from S6/S19 and the only Candidatus Ferristratum sp. MAG from S1 (right). MAG percent completeness indicated in parentheses. Genes that lacked expression that were still found within MAGs are indicated with gray boxes. Pathways that were not represented in a given MAG remain white.

Fig. 7. Cartoon showing the contribution of different members of the microbial community to iron, carbon, and nitrogen cycling within the aerobic/anaerobic gradient of an iron mat.

The Zetaproteobacteria influence nearly every metabolic process in the mat, and support/are supported by a diverse flanking community, including the anaerobic Candidatus Ferristratum sp. The mat community is also supported by other metabolisms, such as oxidation of methane and H₂.

Fig. 8. Cartoon model showing potential C and N cycling between the aerobic Zetaproteobacteria and anaerobic Candidatus Ferristratum sp. within the gradient of the iron mat.

Letters denote either different Zetaproteobacteria taxa or different potential metabolic strategies in a single Candidatus Ferristratum sp. cell. Zetaproteobacteria A (ZA): Capable of Fe(II) oxidation using oxygen, assimilating nitrate, reducing nitrite, and fixing carbon. ZB: Capable of reducing nitrogen intermediates. All have NOR, though ones with NIR don’t have NOS. (see Fig. 6). ZC: A few Zetaproteobacteria with NapA may be able to couple nitrate reduction with iron oxidation (these Zetaproteobacteria also capable of the metabolism in ZA). Candidatus Ferristratum A: Capable of nitrate reduction coupled to iron oxidation. Candidatus Ferristratum B: Capable of nitrate reduction coupled to organic C oxidation.