Meta-omics Reveal Gallionellaceae and Rhodanobacter Species as Interdependent Key Players for Fe(II) Oxidation and Nitrate Reduction in the Autotrophic Enrichment Culture KS

Nitrate reduction coupled to Fe(II) oxidation (NRFO) has been recognized as an environmentally important microbial process in many freshwater ecosystems. However, well-characterized examples of autotrophic nitrate-reducing Fe(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 Fe(II)-oxidizing enrichment culture KS growing under autotrophic or heterotrophic conditions and originating from 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 Gallionellaceae sp. and Rhodanobacter sp. transcripts and proteins likely involved in Fe(II) oxidation (e.g., mtoAB, cyc2, and mofA), denitrification (e.g., napGHI), and oxidative phosphorylation (e.g., respiratory chain complexes I to 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 performed 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 Fe(II)-oxidizing bacteria, which might resemble microbial interactions in freshwater environments. IMPORTANCE Nitrate-reducing Fe(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 Fe(II)-oxidizing bacteria is rarely investigated and not fully understood. The most prominent model system for this type of study is the enrichment culture KS. To gain insights into the metabolism of nitrate reduction coupled to Fe(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 Fe(II) oxidizers in culture KS. Our work demonstrates that autotrophic nitrate reduction coupled to Fe(II) oxidation is not performed by an individual strain but is a cooperation of at least two members of the bacterial community in culture KS. These findings serve as a foundation for our understanding of nitrate-reducing Fe(II)-oxidizing bacteria in the environment.