A physiological and genomic investigation of dissimilatory phosphite oxidation in Desulfotignum phosphitoxidans strain FiPS-3 and in microbial enrichment cultures from wastewater treatment sludge

Phosphite (HPO32-) is a highly soluble, reduced phosphorus compound that is often overlooked in biogeochemical analyses. Although the oxidation of phosphite to phosphate is a highly exergonic process (Eo’ = -650 mV), phosphite is kinetically stable and can account for 10-30% of the total dissolved P in various environments. Its role as a phosphorus source for a variety of extant microorganisms has been known since the 1950s and the pathways involved in assimilatory phosphite oxidation (APO) have been well characterized. More recently it was demonstrated that phosphite could also act as an electron donor for energy metabolism in a process known as dissimilatory phosphite oxidation (DPO). The bacterium described in this study, Desulfotignum phosphitoxidans strain FiPS-3, was isolated from brackish sediments and is capable of growing by coupling phosphite oxidation to the reduction of either sulfate or carbon dioxide. FiPS-3 remains the only isolated organism capable of DPO and the prevalence of this metabolism in the environment is still unclear. This study, therefore, sought to investigate the genetic and physiological factors associated with DPO in FiPS-3 as well as to expand our understanding of this metabolism by enriching for novel environmental microorganisms capable of DPO./nThe first chapter of this dissertation is a published review paper (Figueroa & Coates 2016) that examines the current state of knowledge regarding the geochemistry of phosphite and the biology of phosphite oxidation. The chapter presents evidence suggesting that phosphite may have been involved in the development of early life and that it may be more prevalent on modern Earth than previously thought. Potential natural and anthropogenic sources of phosphite in the environment are discussed and the genetic and physiological properties thought to distinguish DPO from APO are explored./nThe second chapter of this dissertation (unpublished work) deals with my work using pure cultures of FiPS-3 to investigate DPO metabolism. Genomic analysis of FiPS-3 combined with physiological experiments led to improved growth of this strain and revealed its ability to grow aerobically. RNAseq analysis confirmed the importance of the ptx-ptd gene cluster under DPO conditions and suggested that the ptx and ptd portions of the cluster may constitute separate modules that are differentially regulated. Additionally, DPO-dependent biomineralization in FiPS-3 cultures was examined./nThe third chapter of this dissertation (unpublished work) documents my efforts to enrich for novel organisms capable of DPO from anaerobic wastewater treatment sludge. Enrichments with phosphite as the sole electron donor and carbon dioxide as the sole electron acceptor showed a decrease in phosphite with a concomitant increase in phosphate, which was not seen in killed controls. Phosphite oxidation was coupled to cellular growth and was enhanced by rumen fluid addition, while molybdate and sulfite were inhibitory. Community analysis revealed significant changes in the microbial population due to the presence of phosphite and identified a single bacterial operational taxonomic unit (OTU) whose abundance strongly correlated with phosphite oxidation. Phylogenetic analysis indicated that this OTU (designated Phox-21) belonged to a candidate order within the Deltaprotobacteria with no known cultured isolates./nThe fourth chapter of this dissertation (unpublished work) is a metagenomic analysis of the enrichment communities described in Chapter 3, focusing on the putative DPO-capable bacterium Phox-21. This analysis revealed the presence of a ptx-ptd cluster in Phox-21, similar to the one found in FiPS-3, which further supports the hypothesis that this organism is capable of DPO. The investigation also uncovered that Phox-21 has an incomplete Wood-Ljungdahl Pathway, which suggests it is capable of reducing carbon dioxide to formate as part of its energy metabolism but not of assimilating carbon dioxide into biomass. Furthermore, the metagenomic dataset provided insights on the metabolic capabilities of other community members, thus offering a wider ecological context for the role of DPO in this system./nThe fifth chapter of this dissertation summarizes the conclusions of this study and discusses the implications of this research for future investigations regarding the biology and geochemistry of DPO and other forms of reduced phosphorus metabolism.

Advisor: Coates, John D