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

Abstract

Zetaproteobacteria 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 Acidithiobacillus ferrooxidans. However, Cyc2 function is not well verified in neutrophilic Fe oxidizers, particularly in Fe-oxidizing environments. Toward this, we analyzed genomes and metatranscriptomes of Zetaproteobacteria, 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 cyc2 and other model Fe oxidation pathway genes across all represented Zetaproteobacteria lineages. High expression of these genes was observed in diverse Zetaproteobacteria under multiple environmental conditions and in incubations. The putative Fe oxidase gene cyc2 was highly expressed in situ, often as the top expressed gene. The cyc2 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. IMPORTANCE 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, cyc2, in iron-rich hydrothermal vents, where iron-oxidizing microbes dominate. We pieced together diverse Zetaproteobacteria genomes, compared these genomes, and analyzed expression of cyc2 and other hypothetical iron oxidation genes. We show that cyc2 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.

Metagenome assembled genomes of novel taxa from an acid mine drainage environment

“…For sulfur cycling, we focus on dissimilatory sulfate reduction and sulfur oxidation by examining the presence or absence of dsr and sox genes. The metabolic potential for ferrous iron oxidation was based on the presence of Cyc2 like genes that may be involved in this process (56,57). None of the MAGs contain complete genomes and a gene that is absent in the MAG may be present in the taxon.…”

“…Cyc2 genes were identified using BLAST to identify genes homologous to the Cyc2 like cytochrome c involved in Fe(II) oxidation (56)(57)(58). A BLAST database was constructed using the Cyc2 retrieved previously (57) and the search was performed using an e-value of 1E-5.…”

“…Cyc2 genes were identified using BLAST to identify genes homologous to the Cyc2 like cytochrome c involved in Fe(II) oxidation (56)(57)(58). A BLAST database was constructed using the Cyc2 retrieved previously (57) and the search was performed using an e-value of 1E-5. To ensure that the sequences retrived with the BLAST search were homologous to the Cyc2-like protein involved in Fe(II) oxidation, retrieved sequences and those described previously (57) were aligned in MAFFT and a maximum likelihood tree was constructed as described above.…”

Microbial succession during the transition from active to inactive stages of deep-sea hydrothermal vent sulfide chimneys

“…Surprisingly, the overall microbial composition of the EPR-M chimney that only recently became inactive was highly similar to chimneys that had been extinct for much longer time periods [27,[29][30][31], suggesting that the microbial succession took place mainly during the early stages after venting ceases and that the microbial community stayed relatively stable afterwards over long temporal scales. This gene encodes an outer membrane cytochrome c as a candidate genetic marker for FeOB, which was demonstrated to mediate iron oxidation in the acidophilic FeOB Acidithiobacillus ferrooxidans and also found in all available genomes of neutrophilic FeOB [67,81,82]. Moreover, highly expressed cyc2 gene identified in the electroautotrophic Ca.…”

“…Here, the finding of prevalence of cyc2 gene in the "sulfide mineral" clade further suggests that iron oxidizing capability might play a critical role for these unique Nitrospirae to survive and success in the recently inactive chimneys. While cyc2 has been described to be involved in the oxidation of external electron donors, such as iron [67,81,82], which are very likely coming from the reduced metals making up the recently inactive sulfide chimneys, as well as other relatively reducing anoxic hydrothermal environments like buried subsurface massive sulfides (SMS). In supporting this hypothesis, a Nitrospirae MAG recovered from SMS in the Southern Mariana Trough was retrieved belonging to the "sulfide mineral" clade [65] (Additional file 1: Figure S6), suggesting that there are similar environmental features between the recently inactive chimneys and SMS, the conservation of metal sulfide minerals that are still not fully oxidatively weathered by permeating seawater probably is one of them.…”