The molecular mechanisms and organic effects on microaerophilic Fe(II) oxidation by Sideroxydans lithotrophicus ES-1
Date
2022
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Publisher
University of Delaware
Abstract
Iron (Fe) is the fourth most abundant element in the Earth’s crust and exists in both aqueous and solid phases in the environment. Microbes have been recognized to be the key drivers of Fe redox cycling, particularly in suboxic environments. Unlike the extensively studied microbial Fe(III) reduction, the microbial oxidation of different Fe(II) sources is less understood, which makes it difficult to evaluate the significance and geochemical influence of Fe(II)-oxidizing bacteria (FeOB) in the environment. In this dissertation, we chose a representative Fe(II)-oxidizing bacterium, Sideroxydans lithotrophicus ES-1, to study the physiology and mechanisms of microbial Fe(II) oxidation and quantify the microbial Fe(II) oxidation rate in the presence of different organics. ☐ Different types of organics are used in FeOB culturing and are present in the habitats of Fe(II)-oxidizers. Although the organics are known to affect iron oxidation kinetics differently, their effects on microbial iron oxidation have not been well quantified. In the first study presented in this dissertation, we added different representative organics into ES-1 culture and quantified their effects on biotic and abiotic Fe(II) oxidation in the low oxygen condition. We chose citrate and nitrilotriacetic acid (NTA) to represent the common chelators applied in FeOB culturing and Pahokee Peat Humic Acid (PPHA) and Suwannee River Fulvic Acid (SRFA) to represent the humic substances in the environment. We show that NTA accelerates abiotic oxidation and citrate has negligible effects, making citrate a better laboratory chelator in FeOB culturing. The humic substances only affect biotic Fe(II) oxidation, via a combination of chelation and electron transfer. PPHA accelerates biotic Fe(II) oxidation while SRFA decelerates or accelerates the rate depending on concentration. The specific nature of organic-Fe-microbe interactions may play key roles in environmental Fe(II) oxidation, which have cascading influences on the cycling of nutrients and contaminants that associate with Fe oxide minerals. ☐ S. lithotrophicus ES-1 is unique among the chemolithotrophic FeOB because of its metabolic versatility of growing on both aqueous Fe(II) and thiosulfate, which allows us to better constrain the genes specific to Fe(II) oxidation. In Chapter 3, we investigate the Fe(II) oxidation pathways using transcriptomics experiments validated with RT-qPCR. We explored the long-term gene expression response at different growth phases (over days-week) and expression changes during a short-term switch from thiosulfate to FeCl2 (90 min). To avoid the interference from a large amount of Fe(III) oxyhydroxides, citrate was added in the long-term experiment to chelate Fe(III), taking advantage of our Chapter 2 work. We showed that only one of the two Fe(II) oxidase candidate genes, cyc2 was Fe(II)-responsive, while the other one, mtoA was neither highly expressed nor differentially expressed, indicating the important role of Cyc2 in aqueous Fe(II) oxidation. We used gene expression profiles to further constrain the ES-1 Fe(II) oxidation pathway. Notably, alternative complex III, reverse electron transport and carbon fixation were all Fe(II)-responsive. This implies a direct connection between Fe(II) oxidation and carbon fixation, suggesting that CO2 is an important electron sink for Fe(II) oxidation. ☐ Previous research mostly focused on studying FeOB in aqueous Fe(II) culture despite the fact that most Fe(II) in the environment is associated with mineral structures. Therefore, in the third study, we grew ES-1 on the trioctahedral Fe(II)-bearing smectite, which is a common weathering product of basalts and forms a large Fe(II) pool that may support microbial growth. Our results showed that the cell growth of ES-1 relies on the oxidation of solid Fe(II) in smectite. X-ray absorption spectroscopy and Mössbauer spectroscopy showed biotic oxidation outcompete the abiotic oxidation and the resulting Fe(III) remains in the smectite structure. Differential gene and protein expression in smectite vs. Fe(II)-citrate culture showed the Mto pathway is directly involved in the solid Fe(II) oxidation. Cyc2 is indirectly involved in smectite oxidation by oxidizing the small amount of dissolved Fe(II), which is regenerable through the interfacial electron transfer with the solid Fe(II) in smectite. Our work shows that Fe(II)-oxidizers can grow on Fe(II) clays, which accommodate Fe(III) in the octahedral sheet. This makes smectite a renewable energy source for Fe-metabolizing bacteria, which are the key drivers of Fe cycling. ☐ Like smectite, magnetite is also widespread and contains both Fe(II) and Fe(III) to act as a potential electron donor for Fe(II)-oxidizers and electron acceptor for Fe(III)-reducers. Previous research has shown some FeOB can use Fe(II) in either biogenic or abiogenic magnetite as the electron donor, but the knowledge is still limited about the mechanisms of FeOB oxidizing the different types of magnetite. Quantitative proteomics were performed on ES-1 grown on biogenic magnetite, commercial magnetite, Fe(II)-citrate and thiosulfate. The results showed the proteins involved in the Mto pathway were only detected in the magnetite cultures while Cyc2 was expressed in all conditions but at higher abundance in the cultures with aqueous Fe(II). The proteomics results suggested Cyc2 is involved in aqueous Fe(II) oxidation and indirect solid Fe(II) oxidation and the Mto pathway is only responsive to solid Fe(II). These results suggest that FeOB use distinct pathways for oxidizing different Fe(II) forms, which benefits FeOB growth in their geochemical niches, where solid and aqueous Fe(II) coexist and interact. ☐ Overall, with the combination of chemical, physiological, mineralogical, and omics approaches, we clarified the mechanisms FeOB use to oxidize different substrates and how these microbes mediate mineral transformations. The findings in this dissertation study showed FeOB capabilities and adaptations to different Fe(II) sources, especially minerals, which expand their habitats and connect the Fe redox reactions with carbon cycling.
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Keywords
Sideroxydans lithotrophicus, Iron, Pahokee Peat Humic Acid, Metabolic versatility, Biotic oxidation