Validating the Cyc2 Neutrophilic Iron Oxidation Pathway Using Meta-omics of Zetaproteobacteria Iron Mats at Marine Hydrothermal Vents

create extensive iron (Fe) oxide mats at marine hydrothermal vents, making them an ideal model for microbial Fe oxidation at circumneutral pH. Comparison of neutrophilic Fe oxidizer isolate genomes has revealed a hypothetical Fe oxidation pathway, featuring a homolog of the Fe oxidase Cyc2 from However, Cyc2 function is not well verified in neutrophilic Fe oxidizers, particularly in Fe-oxidizing environments. Toward this, we analyzed genomes and metatranscriptomes of , using 53 new high-quality metagenome-assembled genomes reconstructed from Fe mats at Mid-Atlantic Ridge, Mariana Backarc, and Loihi Seamount (Hawaii) hydrothermal vents. Phylogenetic analysis demonstrated conservation of Cyc2 sequences among most neutrophilic Fe oxidizers, suggesting a common function. We confirmed the widespread distribution of and other model Fe oxidation pathway genes across all represented lineages. High expression of these genes was observed in diverse under multiple environmental conditions and in incubations. The putative Fe oxidase gene was highly expressed , often as the top expressed gene. The gene showed increased expression in Fe(II)-amended incubations, with corresponding increases in carbon fixation and central metabolism gene expression. These results substantiate the Cyc2-based Fe oxidation pathway in neutrophiles and demonstrate its significance in marine Fe-mineralizing environments. Iron oxides are important components of our soil, water supplies, and ecosystems, as they sequester nutrients, carbon, and metals. Microorganisms can form iron oxides, but it is unclear whether this is a significant mechanism in the environment. Unlike other major microbial energy metabolisms, there is no marker gene for iron oxidation, hindering our ability to track these microbes. Here, we investigate a promising possible iron oxidation gene, , in iron-rich hydrothermal vents, where iron-oxidizing microbes dominate. We pieced together diverse genomes, compared these genomes, and analyzed expression of and other hypothetical iron oxidation genes. We show that is widespread among iron oxidizers and is highly expressed and potentially regulated, making it a good marker for the capacity for iron oxidation and potentially a marker for activity. These findings will help us understand and potentially quantify the impacts of neutrophilic iron oxidizers in a wide variety of marine and terrestrial environments.