Project Summary: Bacteria that utilize iron-oxidation (Fe-oxidizing bacteria (FeOB)) as their primary energy source are abundant and diverse, despite being constrained to sub-oxic habitats on the fully oxygenated modern Earth. During much of Earth's history, probably starting around 3 billion years ago, when O2 levels were much lower or fluctuated between oxic and anoxic conditions, the oceans were replete with ferrous iron. It is highly likely that FeOB flourished. These bacteria could have played an important role in shaping conditions for life on the early Earth, furthermore, due to the overall abundance of iron on planets and planetary bodies, Fe redox chemistry could be a fundamental metabolism for extraterrestrial life. We also know that as a consequence of microbial growth on iron at circumneutral pH, FeOB produce unique organo-metallic nano-and micro-structures that are incorporated into the rock record, forming a biosignature for their existence and activity. Nonetheless, oxygen-dependent Fe-oxidation at circumneutral pH remains among the least well understood major lithotrophic metabolisms on Earth. The primary goal of this proposal is to develop a mechanistic understanding of microbial iron oxidation using comparative genomics and transcriptomics, as well as constrain the physiological solutions to this problem by assessing the diversity of mechanisms currently displayed by modern Fe-oxidizing bacteria. Within this context, a major technical challenge will be development of a robust pipeline for analyzing single cell transcriptomes from bacteria. Single cell transcriptomics is a novel approach that will provide information, not only about the genetic potential of uncultivated microbes, but also about what genes they are actually expressing, providing direct insight to important functions the bacteria are carrying out. In addition, in targeting a poorly understood metabolism like Fe-oxidation, transcriptomic analysis may provide important information as to the specific genes or gene families that are involved in energy conservation from Fe-oxidation. While the Single Cell Genomics Center at Bigelow has proven its success at acquiring DNA and genomic sequences from single cells, the acquisition of mRNA and determination of mRNA sequences has not been done before on environmental microorganisms. Thus, this approach will offer several technological challenges that, if successfully met, will greatly enhance our capacity to understand diverse microbial metabolisms, independent of our ability to cultivate the microbes responsible. In addition to this single cell transcriptomics approach, work will continue with the exploration of genomes from currently available single amplified genomes of both freshwater and marine FeOB, as well as analysis of in-situ habitats using more conventional metagenomic and metatranscriptome methods. Due to the unique evolutionary history of these two major groups of Fe-oxidizers, this kind of comparative analysis could well inform us about modes of microbial evolution related to iron metabolism. The FeOB are an excellent group of microbes to test these methods on due to their unique physiology, diverse, but conserved phylogeny and evolutionary history, as well there being a group of cultured representatives within a much larger group of uncultivated members. This project could contribute significantly to advancing our understanding of their biology, and, perhaps even more importantly provide new tools for astrobiologists, and other microbial ecologists and physiologists interested in learning about novel microbial processes.