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School of Biological Sciences

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College of Sciences

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Center for the Study of Systems Biology

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Molecular mechanisms of microbial pathways for environmental contaminant remediation

(Georgia Institute of Technology, 2023-01-13) Toporek, Yael Jordan

This thesis examines the molecular mechanism of alternate strategies for remediation of contaminated environments. Radioiodine, perfluoroalkyl substances (PFAS), and 1,4-dioxane represent emerging contaminants of national concern. For example, microbially catalyzed reductive methylation of 129IO3- has received recent attention as an alternate strategy for remediation of radioiodine-contaminated environments. This thesis identified enzymes required for IO3- reduction coupled to organic acid oxidation in the facultative anaerobe Shewanella oneidensis: cytoplasmic electron donors are oxidized, and the electrons are transferred through the periplasm via cytochromes of the metal-reducing pathway to extracellular dimethylsulfoxide (DMSO) reductase, which directly reduces IO3- to iodide (I-) as an alternate substrate. Future work aims to investigate the apparent import of I- back to the cytoplasm, where it is putatively methylated and volatilized by a promiscuous thiopurine methyltransferase, presenting a potential strategy for bioremediation of radioiodine. In the case of PFAS, the industrial surfactant and flame retardant perfluorooctanoic acid (PFOA) has been designated as an emerging contaminant. In the present study, the microbially driven Fenton reaction (MFR) was employed to attempt degradation of PFOA by cycling between aerobic and anaerobic ferric iron (Fe(III))-reducing conditions. Under aerobic conditions, S. oneidensis reduced molecular oxygen (O2) to hydrogen peroxide (H2O2), while under anaerobic conditions, S. oneidensis reduced Fe(III) to Fe(II). During aerobic-to-anaerobic transition periods, Fe(II) and H2O2 interacted chemically via the Fenton reaction to produce contaminant-degrading hydroxyl (HO•) radicals, which in turn interacted with PFOA. PFOA concentrations, however, remained unchanged, which most likely reflects the stability of carbon-fluorine bonds and consequent inability of HO• radicals to oxidatively degrade PFOA. Finally, the present study aimed to determine the redox conditions of the intracellular environment during oxidative stress in S. oneidensis from aerobic respiration and H2O2 stress. In contrast to S. oneidensis anaerobic respiration, aerobic respiration is understudied, but is a key contributor to MFR in degrading organic and chlorinated environmental contaminants like 1,4-dioxane. This work describes the native and perturbed redox environment of the S. oneidensis cytoplasm, as well as the contribution of individual genes, particularly catalases and peroxidases, to intracellular H2O2 scavenging rates using the genetically-encoded ratiometric fluorescent sensor HyPer-3 as a reporter. As measured by HyPer-3, deletion of one or more catalases and peroxidases resulted in dramatic changes in the redox condition of the cytoplasm, while other H2O2-scavenging enzymes provided overlapping H2O2 scavenging activity to combat H2O2 challenges. Based on cytoplasmic HyPer-3 redox signals, results from the present study indicated that periplasmic PgpD, cytoplasmic KatB, and previously overlooked cytoplasmic KatG1 and KatG2 provide first- and second-line defenses to protect against exogenous H2O2 challenges in minimal growth medium.
Novel microbial platform for degradation of hazardous organic contaminants and production of sustainable bioplastics

(Georgia Institute of Technology, 2016-04-15) Sekar, Ramanan

Improper disposal of 1,4-dioxane and the chlorinated organic solvents trichloroethylene (TCE) and tetrachloroethylene (PCE) has resulted in widespread contamination of soil and groundwater. In the present study, a novel microbially-driven Fenton reaction system was designed to generate hydroxyl (HO) radicals for simultaneous degradation of source zone levels of single, binary, and ternary mixtures of TCE, PCE, and 1,4-dioxane. The new Fenton reaction system was driven by the Fe(III)-reducing facultative anaerobe Shewanella oneidensis amended with lactate, Fe(III), and contaminant mixtures and exposed to alternating anaerobic and aerobic conditions. The novel microbially-driven Fenton reaction system successfully degraded TCE, PCE, and 1,4-dioxane either as single contaminants or as binary and ternary mixtures. Degradation of lignocellulosic biomass was also demonstrated through the novel microbially driven fenton reaction by S. oneidensis. In this study, we have developed a new method that combines both pretreatment and saccharification of cellulose and xylan in a microbially driven fenton reaction. The combined pretreatment and saccharification method for cellulose and xylan developed did not involve the addition of acid, alkali compounds or the use of hydrolyzing enzymes thus being an economically feasible process to directly produce simple fermentable sugars from cellulose and xylan. Microbial Fe(III) reduction is a dominant anaerobic respiratory process in soil and sediments, which suggests that the microbially driven fenton reaction may play an important role in the degradation of decaying plant and woody materials in the natural environment. The expansion of metabolic capability to convert D-xylose to a useful product such as PHB can be beneficial in biotechnological applications to couple multiple carbon sources such as glucose, glycerol and D-xylose by S.oneidensis to improve efficiency of electricity generation, biofuel production and bioremediation of toxic contaminants.
Bacterial iron and manganese reduction driven by organic sulfur electron shuttles

(Georgia Institute of Technology, 2015-04-08) Cooper, Rebecca Elizabeth

Dissimilatory metal-reducing bacteria (DMRB) play an important role in the biogeochemical cycling of metals. DMRB are unique in that they possess the ability to couple metal reduction with their metabolism. Microbial Fe(III) respiration is a central component of a variety of environmentally important processes, including the biogeochemical cycling of iron and carbon in redox stratified water and sediments, the bioremediation of radionuclide-contaminated water, the degradation of toxic hazardous pollutants, and the generation of electricity in microbial fuel cells. Despite this environmental and evolutionary importance, the molecular mechanism of microbial Fe(III) respiration is poorly understood. Current models of the molecular mechanism of microbial metal respiration are based on direct enzymatic, Fe(III) solubilization, and electron shuttling pathways. Fe(III) oxides are solid at circumneutral pH and therefore unable to come into direct contact with the microbial inner membrane, these bacteria must utilize an alternative strategy for iron reduction. Reduced organic compounds such as thiols are prominent in natural environments where DMRB are found. These thiol compounds are redox reactive and are capable of abiotically reducing Fe(III) oxides at high rates S. oneidensis wild-type and ΔluxS anaerobic biofilm formation phenotypes were examined under a variety of electron donor-electron acceptor pairs, including lactate or formate as the electron donor and fumarate, thiosulfate, or Fe(III) oxide-coated silica surfaces as the terminal electron acceptor. The rates of biofilm formation under the aforementioned growth conditions as well as in the presence of exogenous thiol compounds indicate that ∆luxS formed biofilms at rates only 5-10% of the wild-type strain and ∆luxS biofilm formation rates were restored to wild-type levels by addition of a variety of exogenous compounds including cysteine, glutathione, homocysteine, methionine, serine, and homoserine. Cell adsorption isotherm analyses results indicate that wild-type is can attach to the surface of hematite particles attachment , but ΔluxS is unable to attach the hematite surfaces. These results indicate that biofilm formation is not required for Fe(III) oxide reduction by S. oneidensis ∆luxS anaerobic biofilm formation rates were restored to wild-type levels by addition of exogenous auntoinducer-2 (AI-2), a by-product of homocysteine production in the Activated Methyl Cycle. This discovery led to subsequent experiments performed to detect the production and utilization of AI-2 by wild-type and ∆luxS strains under aerobic and anaerobic conditions. AI-2 production experiments showed wild-type, but not ΔluxS, was capable of producing AI-2. The addition of exogenous S. oneidensis and Vibrio harveyi-produced AI-2 to wild-type and ∆luxS resulted in the swift depletion of AI-2 from the media. These results provide evidence that S. oneidensis can produce AI-2 and subsequently utilize its’ own AI-2 as well as AI-2 produced by other bacteria as a carbon and electron source in the absence of preferred carbon sources. S. oneidensis produces and secretes a suite of extracellular thiols under anaerobic Fe(III)-reducing and Mn(III) and Mn(IV)-reducing conditions including cysteine, homocysteine, glutathione, and cyteamine. Exogenous thiols produced by S. oneidensis are intermediates of the Activated Methyl Cycle (AMC) and Transulfurylation Pathway (TSP). Reduced and oxidized thiols were detected, indicating that the thiols are in a constant state of flux between the reduced and oxidized forms and that the concentration of reduced thiols to its’ oxidized counterpart is indicative of the state of metal reduction by the microorganisms. Respiratory phenotypes Based on Fe(III) and Mn(IV) respiratory phenotypes observed in the AMC and TSP pathway mutants (∆luxS, ∆metB, ∆metC and ∆metY) we can infer that cysteine, glutathione, and cysteamine contribute to metal reduction by serving as efficient electron shuttling molecules, while homocysteine is critical for maintenance of the AMC, propagation of thiol biosynthesis, and maintenance of cellular metabolism via the AMC intermediate SAM. Furthermore, these findings suggest that all metal-reducing bacteria require thiol formation to reduce solid metal oxides. Direct contact mechanism is not the dominant means through electrons are transferred and metals are reduced, instead electron shuttles are the maid reduction mechanism.
Novel pathway for microbial FE(III) reduction: electron shuttling through naturally occurring thiols

(Georgia Institute of Technology, 2014-02-17) Wee, Seng Kew

The g-proteobacterium Shewanella oneidensis MR-1 reduces a wide range of terminal electron acceptors, including solid Fe(III) oxides. Pathways for Fe(III) oxide reduction by S. oneidensis include non-reductive (organic ligand-promoted) solubilization reactions, and either direct enzymatic, or indirect electron shuttling pathways. Results of the present study expand the spectrum of electron acceptors reduced by S. oneidensis to include the naturally occurring disulfide compounds cystine, oxidized glutathione, dithiodiglycolate, dithoidiproponiate and cystamine. Subsequent electron shuttling experiments demonstrated that S. oneidensis employs the reduced (thiol) form of the disulfide compounds (cysteine, reduced glutathione, mercaptoacetate, mercaptopropionate, and 2-nitro-5-thiobenzoate, cystamine) as electron shuttles to transfer electrons to extracellular Fe(III) oxides. The results of the present study indicate that microbial disulfide reduction may represent an important electron-shuttling pathway for electron transfer to Fe(III) oxides in anaerobic marine and freshwater environments.
A novel mode of bacterial respiration: iron solubilization prior to electron transfer

(Georgia Institute of Technology, 2010-11-11) Fennessey, Christine Michelle

Microbial iron respiration contributes significantly to the biogeochemical cycling of metals and may be one of the earliest respiratory processes to have evolved on early earth. Metal-respiring microbes also hold great potential for use in microbial fuel cells for the generation of "green" energy and for remediation of radionuclides in contaminated environments. Despite its significance in global metal cycling processes, the molecular mechanism of Fe(III) respiration has yet to be determined. Unlike many other terminal electron acceptors, Fe(III) is a solid at circumneutral pH and, therefore, cannot come into direct contact with the microbial inner membrane: the site of terminal electron transfer in gram-negative bacteria. It is postulated that metal-respiring organisms have developed alternate strategies for the reduction of solid iron. One such strategy involves the production of an Fe(III)-solublizing ligand by the metal-respiring bacteria which solubilizes the Fe(III) prior to respiration, rendering the metal more easily accessible to the Fe(III) reductase complex. In this study, the genes involved in the solubilization of Fe(III) by the gram-negative dissimilatory metal reducing bacteria Shewanella oneidensis MR-1 were determined using random mutagenesis to generate mutations in the wild-type genome and high-throughput square-wave voltammetry to screen for the attenuation of Fe(III) production in the mutants. Two mutants unable to solubilize Fe(III) were identified and designated d29 and d64. After mutation complementation analysis, it was determined that the point mutations were both located in type II secretion genes: gspG and gspE respectively, indicating that the type II secretion system is required for Fe(III) solubilization prior to respiration. It was also hypothesized that the ligand produced for Fe(III) solubilization during dissimilatory Fe(III) respiration was a siderophore: a small Fe(III)-chelating molecule produced by the cells for the assimilation of Fe(III) for growth. A siderophore biosynthesis gene (SO3031) and a siderophore ferric reductase gene (SO3034) were deleted in frame and the resultant mutants screened to determine whether they were capable of Fe(III) solubilization and reduction during anaerobic Fe(III) respiration. Both mutants retained Fe(III) solubilization and reduction activity, indicating that the siderophore Fe(III) assimilatory system is distinct from the Fe(III) solubilization system utilized during Fe(III) respiration. The work presented here is significant in that it describes a rapid screening method for identifying Fe(III) solubilization mutants, reports on the involvement of the type II secretion system in Fe(III) solubilization during iron respiration, and finally demonstrates that a dissimilatory metal reducing bacteria synthesizes and secretes Fe(III)-chelating molecules which are distinct from Fe(III)-siderophores.
Molecular mechanisms of microbial iron respiration by Shewanella oneidensis MR-1

(Georgia Institute of Technology, 2010-04-05) Burns, Justin Lee

Metal-respiring bacteria occupy a central position in a variety of environmentally important processes including the biogeochemical cycling of metals and carbon, biocorrosion of steel surfaces, bioremediation of radionuclide-contaminated aquifers, and electricity generation in microbial fuel cells. Metal-respiring bacteria are presented, however, with a unique physiological challenge: they are required to respire anaerobically on electron acceptors (e.g., Fe(III) oxides, elemental sulfur) that are highly insoluble at circumneutral pH and unable to enter the cell and contact inner membrane-localized respiratory systems. To overcome these physiological problems, metal-respiring bacteria are postulated to employ a variety of novel respiratory strategies not found in other bacteria, including 1) direct enzymatic reduction at the cell surface, 2) electron shuttling between the cell and metal surfaces, and 3) metal solubilization by bacterially-produced organic ligands followed by respiration of the soluble organic-metal complexes. This work highlights my latest findings on the genetic and enzymatic mechanism of metal respiration by Shewanella oneidensis, a facultative anaerobe ubiquitous to redox-stratified natural waters and sediments.
Cytochrome c maturation and redox homeostasis in uranium-reducing bacterium Shewanella putrefaciens

(Georgia Institute of Technology, 2007-10-11) Dale, Jason Robert

Microbial metal reduction contributes to biogeochemical cycling, and reductive precipitation provides the basis for bioremediation strategies designed to immobilize radionuclide contaminants present in the subsurface. Facultatively anaerobic ×-proteobacteria of the genus Shewanella are present in many aquatic and terrestrial environments and are capable of respiration on a wide range of compounds as terminal electron acceptor including transition metals, uranium and transuranics. S. putrefaciens is readily cultivated in the laboratory and a genetic system was recently developed to study U(VI) reduction in this organism. U(VI) reduction-deficient S. putrefaciens point mutant Urr14 (hereafter referred to as CCMB1) was found to retain the ability to respire several alternate electron acceptors. In the present study, CCMB1 was tested on a suite of electron acceptors and found to retain growth on electron acceptors with high reduction potential (E¡¬0) [O2, Fe(III)-citrate, Mn(IV), Mn(III)-pyrophosphate, NO3-] but was impaired for anaerobic growth on electron acceptors with low E¡¬0 [NO2-, U(VI), dimethyl sulfoxide, trimethylamine N-oxide, fumarate, ×-FeOOH, SO32-, S2O32-]. Genetic complementation and sequencing analysis revealed that CCMB1 contained a point mutation (H108Y) in a CcmB homolog, an ABC transporter permease subunit required for c-type cytochrome maturation in E. coli. The periplasmic space of CCMB1 contained low levels of cytochrome c and elevated levels of free thiol equivalents (-SH), an indication that redox homeostasis was disrupted. Anaerobic growth ability, but not cytochrome c maturation activity, was restored to CCMB1 by adding exogenous disulfide bond-containing compounds (e.g., cystine) to the growth medium. To test the possibility that CcmB transports heme from the cytoplasm to the periplasm in S. putrefaciens, H108 was replaced with alanine, leucine, methionine and lysine residues via site-directed mutagenesis. Anaerobic growth, cytochrome c biosynthesis or redox homeostasis was disrupted in each of the site-directed mutants except H108M. The results of this study demonstrate, for the first time, that S. putrefaciens requires CcmB to produce c-type cytochromes under U(VI)-reducing conditions and maintain redox homeostasis during growth on electron acceptors with low E¡¬0. The present study is the first to examine CcmB activity during growth on electron acceptors with widely-ranging E¡¬0, and the results suggest that cytochrome c or free heme maintains periplasmic redox poise during growth on electron acceptors with E¡¬0 < 0.36V such as in the subsurface engineered for rapid U(VI) reduction or anoxic environments dominated by sulfate-reducing bacteria. A mechanism for CcmB heme translocation across the S. putrefaciens cytoplasmic membrane via heme coordination by H108 is proposed.
Differentiating between living and non-living prokaryotic cells : development, evaluation, and application of a modified vital stain and probe method (mVSP)

(Georgia Institute of Technology, 2002-08) Howard-Jones, Michelle Hope
Molecular mechanism and biogeochemical controls of Fe(III) reduction

(Georgia Institute of Technology, 2002-05) Moore, Charles Michael
Dissimilatory FE(III) reduction by Shewanella putrefaciens : biochemical and genetic analysis

(Georgia Institute of Technology, 2002-05) Haller, Carolyn A.