Award: OCE-1155290

Intellectual Merit. Fe(II) is widely available within ocean crust and marine sediments, and when Fe(II)-laden groundwater meets oxic ocean water, the Fe oxidizes, forming rust, i.e. Fe oxyhydroxides. This oxidation was largely assumed to be abiotic (purely chemical), but the discovery of the marine Fe(II)-oxidizing Zetaproteobacteria gave proof that the process can be driven by microbes. Furthermore, these marine Fe(II)-oxidizers are primary producers, supporting whole communities in dark systems like deep sea hydrothermal vents, as well as ecosystem engineers, using their rust waste products to form the mats in which they and others live. In this project, we sought to understand the diversity, ecology, function, and physical structure of Zetaproteobacterial communities. To do this, we colleced iron-rich microbial mat samples on three separate research expeditions to vent sites along the Mid-Atlantic Ridge; the active undersea volcano, Loihi Seamount, near Hawaii; and active seamounts and spreading centers along the Mariana forearc and backarc in the western Pacific. For comparison, we also sampled and isolated Zetaproteobacteria from near-shore environments in the Chesapeake Bay and Cape Henlopen on the Delaware Bay. Using a combination of field geochemistry, culturing, molecular biology, biochemistry and microscopy, we significantly expanded the known diversity, habitats, and functions of Zetaproteobacteria. Prior to this project, we knew that Zetaproteobacteria created beautiful Fe biominerals, in the form of twisted ribbon-like stalks and hollow sheaths, but we did not know how they came together to make a mat. Unlike typical biofilms, Fe mats are extremely delicate and tend to fall apart during sampling. Therefore, we developed new sampling and microscopy methods allowing us to visualize intact mat structures. Mat morphology correlated to niche: dendritic networks of stalks formed in steeper O2 gradients while woven fabrics of sheaths were associated with low to undetectable O2 gradients. Fe-biomineralized filaments, twisted stalks or hollow sheaths, formed the highly porous framework of each mat. The mat-formers are novel and uncommon Zetaproteobacteria. The distinctive architecture appears to help these Fe-oxidizers (1) remove Fe oxyhydroxide waste without entombing cells or clogging flow paths through the mat and (2) colonize niches where Fe(II) and O2 overlap. This work shows us and how mat morphology links to Zetaproteobacteria diversity and metabolism, and unique ways in which Zetaproteobacteria engineer their own habitats. In order to consistently track ecology, we needed to first create a taxonomic framework so that Zetaproteobacteria from different samples could be classified consistently. We wrote a program, Zetahunter, to classify based on a reference dataset of 16S rRNA gene sequences (a phylogenetic marker). We collected all available Zetaproteobacteria sequences from >150 studies to create this reference, and found that there are now 59 operational taxonomic units of Zetaproteobacteria (compared to 28 when last assessed in 2011). Some are cosmopolitan while others are specific to particular habitats. Furthermore, we found that groups (networks) of particular Zetaproteobacteria taxa tend to coexist within certain habitats, showing that Zetaproteobacteria have diversified to play complementary roles to one other. This led us to ask how diverse Zetaproteobacteria metabolisms are, and what defines and differentiates this taxonomic class? The roles and functions of microorganisms are rooted in their genomes, so we performed genome-resolved metagenomics and transcriptomics, which allowed us to assess the genetic potential of uncultured Zetaproteobacteria. From >170 genomes, including 74 of our own, remarkably, all Zetaproteobacteria taxonomic units had a putative Fe oxidation gene, cyc2, encoding for an outer membrane cytochrome. Cyc2 was also one of the most highly expressed genes, suggesting its importance, so we confirmed its function by inserting the gene in E. coli and showing that it conferred the Fe oxidation ability to a microbe normally unable to oxidize Fe. Because there were no other electron donors apparent within the Zetaproteobacteria genomes, this confirmed that the class is indeed defined by its ability to oxidize Fe. We observee variations in apparent oxygen tolerance and previously unrecognized anaerobic metabolisms, which could account for niche specialization in these gradient environments. We furthermore showed how Zetaproteobacteria interact with flanking community members to cycle C, N, and S, thus impacting the biogeochemistry of the deep sea. Broader Impacts. A better understanding of iron-oxidizing bacteria that include Zetaproteobacteria is of fundamental interest to scientists interested in areas of earth science and oceanography because they illustrate how microbes can influence geochemical cycling and mineral deposition. This work has contributed substantially to that understanding, and how these unique iron-oxidizing communities contribute to the production of biogenic iron oxides and how this may influence the iron budget of the global ocean. This work has provided research training to two postdoctoral research scientists, as well as either directly or indirectly, to half a dozen undergraduates. It has resulted in seven peer-reviewed scientific papers, and numerous presentations at national and international scientific meetings. Last Modified: 01/12/2018 Submitted by: Clara Chan