An Investigation of the Prevalence, Diversity, Evolution, Physiology, and Community Dynamics of Dissimilatory Phosphite Oxidizing Microorganisms

Phosphite is the most energetically favorable chemotrophic electron donor known, with a half-cell redox potential (Eo′) of −650 mV for the HPO42−/HPO32− couple. Dissimilatory phosphite oxidizing microorganisms (DPOM) harness the free energy from phosphite oxidation to support cellular growth and are universally capable of CO2 fixation. The prevalence of this unique metabolism remained largely uncharacterized since its discovery in 2000, as only two species had been identified in three discrete locations prior to the writing of this dissertation. A false notion of rarity has consequently limited our understanding of the diversity and environmental distribution of DPOM. However, phosphite has been detected in several environments at concentrations that suggest a contemporary P redox cycle that might sustain a greater diversity of DPOM than is currently recognized. This dissertation significantly expands the known diversity of DPOM and uses enrichments and genome-resolved metagenomics to describe their prevalence, diversity, evolutionary history, and metabolic potential. Physiological studies further describe the biochemistry and community dynamics of cultures that perform dissimilatory phosphite oxidation (DPO). Chapter 1 of this dissertation reviews our current understanding of phosphite biogeochemistry and DPOM. I begin with a historical review of phosphorus research to demonstrate the significant role phosphorus plays in both geochemical and anthropogenic contexts. Phosphorus redox chemistry is then reviewed to support a subsequent discussion on the geochemistry and distribution of reduced phosphorus species, with particular emphasis on the DPO substrate of phosphite. Establishing our understanding of phosphite distribution aids later discussions on the potential impact of DPOM on the global phosphorus cycle, for which I also review our current understanding of biological phosphite utilization, including assimilatory phosphite oxidation (APO) and DPO. This chapter ends with a review of DPOM diversity and biochemistry, which briefly outlines the status of DPOM research that had been published prior to the work presented in this dissertation. Chapter 1 is intended to prime the reader for subsequent chapters, whose separate introductions explore the pertinent aspects of DPOM in more detail. Upon initiating this thesis work, the only DPOM known were Desulfotignum phosphitoxidans FiPS-3 and Phosphitivorax anaerolimi Phox-21, of which only FiPS-3 had been isolated. Chapter 2 describes the establishment and cultivation of phosphite-enriched microbial communities. I focus on the methodology that successfully identified 21 additional DPOMs, whose metagenomic data supports later chapters’ analyses of DPOM prevalence, diversity, evolution, and metabolic potential. I also describe the cultivation and characterization of a highly enriched phosphite oxidizing culture (HEPO), which is used for the physiological studies of subsequent chapters. In Chapter 3, the genomes of those DPOM that were enriched in Chapter 2 are used to identify DPO marker genes, whose sequences are used to build profile hidden Markov models (pHMMs). Select marker genes are compared with over 17,000 publicly available metagenomes, and it is found that DPO metabolism exists globally in diverse anoxic environments, including wastewaters, sediments, and subsurface aquifers. By assigning taxonomy to metagenome-assembled genomes of DPOM, we find that DPOMs are also phylogenetically diverse, spanning six classes of bacteria, including the Negativicutes, Desulfotomaculia, Synergistia, Syntrophia, Desulfobacteria, and Desulfomonilia_A. By comparing DPOM taxonomy to the phylogeny of DPO marker genes, we conclude that DPO metabolism likely has ancient evolutionary origins predating the split of monoderm and diderm bacteria. Chapter 4 discusses the physiology and metabolism of DPOM. The metagenomes of the DPOM enriched in Chapter 2 are analyzed using DRAM, a novel software that profiles the metabolic potential of microbial genomes. In this chapter, these DRAM predictions are used to refine and expand upon current models for DPOM energy conservation. I pay particular attention to the way in which DPOM appear to utilize CO2 as both an electron acceptor for energy conservation and as a carbon source for biomass generation. Physiological studies using the HEPO culture established in Chapter 2 are leveraged to test hypotheses generated from our DRAM analyses, with primary emphasis on the identification of the CO2-reduction end-product. Throughout this dissertation, physiological experiments are designed to understand the activity of the DPOM that exist within the HEPO culture. When interpreting results, it is therefore important to remember that the HEPO culture is a mixed microbial community. Chapter 5 aims to characterize and understand the potential interactions that may be occurring between DPOM and the non-DPOM community members. I consider the viability of alternative symbiotic relationships and pursue the hypothesis that DPO activity is dependent on nutrient-exchange with the non-DPOM community. I explore the potential role of alternative community members, with particular attention given to methanogens and those community members with high relative abundance. I then discuss the potential role of certain nutrients that may be exchanged, including corrinoids, coenzyme M (CoM), and S-adenosyl L-methionine (SAM). Finally, Chapter 6 summarizes the main conclusions of this dissertation and highlights the future research that will be needed to resolve the many questions about DPOM that remain unanswered.

Advisor: Coates, John D