Microbes are numerous, widespread, and metabolically-versatile organisms, impacting virtually every environment on Earth?s surface. Iron-oxidizing microorganisms have likely been oxidizing, or ?rusting,? the Earth for billions of years. In near-neutral pH environments, they form iron oxyhydroxide minerals, which adsorb metals, organics, and other nutrients, affecting element bioavailability, carbon cycling, and soil and water quality. Iron-oxidizing microbes are predicted to be active in low oxygen, ferrous iron-rich environments found in near-neutral pH aquifers and soils, where abiotic oxidation rates are slow. However, few microorganisms have been unambiguously shown to oxidize iron at neutral pH (which is most common in the environment). Therefore we know little about the diversity, genetics, and biochemistry of iron-oxidizers that function at neutral pH. 

In this project, we showed that neutrophilic (neutral pH-loving) iron-oxidizers are widespread, occurring in novel environments such as a stratified estuary and coastal aquifer, in addition to the well-known environments of streams and iron-rich hydrothermal vents. We isolated new neutrophilic iron-oxidizers, and demonstrated Fe oxidation ability for existing isolates, which expanded the diversity of known FeOB. Freshwater FeOB are famous for making distinctive mineralized filaments like twisted stalks, similar to the first known FeOB Gallionella ferruginea. Our two new stalk-forming isolates are related to G. ferruginea, but form a new genus Ferriphaselus (?iron bean?) within the family Gallionellaceae. We also isolated two new coastal marine FeOB, Mariprofundus aestuarium and M. ferrinatatus, the first pelagic marine FeOB, from the Cheseapeake Bay. All of these new isolates gave us the opportunity to investigate the genomes for genes involved in Fe oxidation. When comparing all available marine and terrestrial neutrophilic iron-oxidizer genomes, we found one specific gene in common ? cyc2. These sequences are distantly related to one from an acidophilic iron-oxidizer, Acidithiobacillus ferrooxidans, in which Cyc2 has been shown to oxidize iron, suggesting that the neutrophiles? Cyc2 may also oxidize Fe. Our protein structure modelling predicts that this is an unusual structure consisting of a fused cytochrome-porin, which would make it an outer membrane cytochrome. This overall structure is similar to the known iron-reducing porin-cytochrome complex MtrCAB, which also has an iron-oxidizing homolog, MtoAB; however, Cyc2 is distinct in its simpler structure, with one heme-binding motif fused to a smaller porin, suggesting a distinct role in iron transformations.

To investigate the function of the neutrophilic iron-oxidizer cyc2, we first studied its expression in different iron-oxidizing environments. If Cyc2 is an Fe oxidase, we would expect high expression of cyc2 in such environments. We analyzed metatranscriptomes from two ecosystems dominated by Fe-oxidizing bacteria: the Loihi Seamount (Hawaii) hydrothermal vent Fe microbial mat and an Fe-rich alluvial aquifer in Rifle, Colorado (reanalysis of Jewell et al., 2016 dataset). In both environments, the putative Fe oxidase gene cyc2 was highly expressed in situ, often as the highest expressed gene. Furthermore, the cyc2 gene increased in expression in Loihi iron microbial mat incubations after Fe(II) amendment, with corresponding increases in expression of carbon fixation and central metabolism genes. Our re-analysis of the Rifle aquifer dataset shows that the cyc2 gene was expressed at much higher levels than mtoA, by two orders of magnitude. Thus, while both putative Fe oxidases were expressed when Fe oxidation was stimulated, cyc2 was clearly preferentially expressed. Our results support a Cyc2-based Fe oxidation pathway and demonstrate its significance in both marine and terrestrial Fe-mineralizing environments. Initial studies on heterologously-expressed Cyc2 provide additional evidence of function. In all, the work to date suggests that cyc2 could be a broadly-distributed genetic marker for Fe oxidation.

This project provided training for four undergraduates, four graduate students, and four postdocs, including seven female trainees, in geomicrobiological techniques, including culturing, biochemistry, and bioinformatics. Four theses were produced, and the PI earned tenure. We used research project sites and data in a problem-based learning Geomicrobiology class for undergraduates and early graduate students. We also developed a Microbes in the Wild outreach curriculum at the Serviam Academy in Wilmington, DE. This workshop introduce 5^th to 8th grade girls to geo/environmental microbiology using a combination of culturing, microscopy, DNA extractions, and other hands on activities. Other outreach included science communication education at Havre de Grace high school, public outreach at our annual marine science festival Coast Day and a 4H camp, public community-oriented presentations, and school tours of our Biotechnology institute.

Last Modified: 07/10/2019
Modified by: Clara Chan