Nitrate reduction coupled to iron(II) oxidation (NRFO) has been recognized as an environmentally important microbial process in many freshwater ecosystems. However, well-characterized examples of autotrophic nitrate-reducing iron(II)-oxidizing bacteria are rare and their pathway of electron transfer as well as their interaction with flanking community members remain largely unknown. Here, we applied meta-omics (i.e., metagenomics, metatranscriptomics and metaproteomics) to the nitrate-reducing iron(II)-oxidizing enrichment culture KS, growing under autotrophic compared to heterotrophic conditions, and originating from a freshwater sediment. We constructed four metagenome-assembled genomes with an estimated completeness of ≥95%, including the key players of NRFO in culture KS, identified as Gallionellaceae sp. and Rhodanobacter sp. The presence of Gallionellaceae sp. and Rhodanobacter sp. transcripts and proteins likely involved in iron(II) oxidation (e.g., mtoAB, cyc2, mofA), denitrification (e.g., napGHI) and oxidative phosphorylation (e.g., respiratory chain complexes I-V), along with Gallionellaceae sp. transcripts and proteins for carbon fixation (e.g., rbcL) were detected. Overall, our results indicate that in culture KS, the Gallionellaceae sp. and Rhodanobacter sp. are interdependent: while Gallionellaceae sp. fixes CO2 and provides organic compounds for Rhodanobacter sp., Rhodanobacter sp. likely detoxifies NO through NO reduction and completes denitrification, which cannot be done by Gallionellaceae sp. alone. Additionally, the transcripts and partial proteins of cbb3- and aa3- type cytochrome c suggest the possibility for a microaerophilic lifestyle of the Gallionellaceae sp., yet culture KS grows under anoxic conditions. Our findings demonstrate that autotrophic NRFO is performed through cooperation among denitrifying and iron(II)-oxidizing bacteria, which might resemble microbial interactions in freshwater environments.
Importance
Nitrate-reducing iron(II)-oxidizing bacteria are widespread in the environment, contribute to nitrate removal, and influence the fate of the greenhouse gases nitrous oxide and carbon dioxide. The autotrophic growth of nitrate-reducing iron(II)-oxidizing bacteria is rarely investigated and not fully understood. The most prominent model system for this type of studies is the enrichment culture KS. To gain insights into the metabolism of nitrate reduction coupled to iron(II) oxidation in the absence of organic carbon and oxygen, we performed metagenomic, metatranscriptomic and metaproteomic analyses of culture KS, and identified Gallionellaceae sp. and Rhodanobacter sp. as interdependent key iron(II)-oxidizers in culture KS. Our work demonstrates that autotrophic nitrate reduction coupled to iron(II) oxidation is not performed by an individual strain but a cooperation of at least two members of the bacterial community in culture KS. These findings serve as foundation for our understanding of nitrate-reducing iron(II)-oxidizing bacteria in the environment.