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Dive into the research topics where Patrick N. Reardon is active.

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Featured researches published by Patrick N. Reardon.


Journal of Biological Chemistry | 2013

Structure of the Type IVa Major Pilin from the Electrically Conductive Bacterial Nanowires of Geobacter sulfurreducens

Patrick N. Reardon; Karl T. Mueller

Background: PilA is the major type IVa pilin that forms the conductive nanowires of Geobacter sulfurreducens. Results: We report the atomic resolution structure of PilA determined with solution state NMR spectroscopy. Conclusion: The Geobacter sulfurreducens PilA adopts a long, kinked α-helix with a dynamic C-terminal region. Significance: The structure provides a foundation to build a model of the bacterial nanowire. Several species of δ proteobacteria are capable of reducing insoluble metal oxides as well as other extracellular electron acceptors. These bacteria play a critical role in the cycling of minerals in subsurface environments, sediments, and groundwater. In some species of bacteria such as Geobacter sulfurreducens, the transport of electrons is proposed to be facilitated by filamentous fibers that are referred to as bacterial nanowires. These nanowires are polymeric assemblies of proteins belonging to the type IVa family of pilin proteins and are mainly comprised of one subunit protein, PilA. Here, we report the high resolution solution NMR structure of the PilA protein from G. sulfurreducens determined in detergent micelles. The protein is >85% α-helical and exhibits similar architecture to the N-terminal regions of other non-conductive type IVa pilins. The detergent micelle interacts with the first 21 amino acids of the protein, indicating that this region likely associates with the bacterial inner membrane prior to fiber formation. A model of the G. sulfurreducens pilus fiber is proposed based on docking of this structure into the fiber model of the type IVa pilin from Neisseria gonorrhoeae. This model provides insight into the organization of aromatic amino acids that are important for electrical conduction.


Langmuir | 2016

Protein–Mineral Interactions: Molecular Dynamics Simulations Capture Importance of Variations in Mineral Surface Composition and Structure

Amity Andersen; Patrick N. Reardon; Stephany S. Chacon; Nikolla Qafoku; Nancy M. Washton; Markus Kleber

Molecular dynamics simulations, conventional and metadynamics, were performed to determine the interaction of model protein Gb1 over kaolinite (001), Na(+)-montmorillonite (001), Ca(2+)-montmorillonite (001), goethite (100), and Na(+)-birnessite (001) mineral surfaces. Gb1, a small (56 residue) protein with a well-characterized solution-state nuclear magnetic resonance (NMR) structure and having α-helix, 4-fold β-sheet, and hydrophobic core features, is used as a model protein to study protein soil mineral interactions and gain insights on structural changes and potential degradation of protein. From our simulations, we observe little change to the hydrated Gb1 structure over the kaolinite, montmorillonite, and goethite surfaces relative to its solvated structure without these mineral surfaces present. Over the Na(+)-birnessite basal surface, however, the Gb1 structure is highly disturbed as a result of interaction with this birnessite surface. Unraveling of the Gb1 β-sheet at specific turns and a partial unraveling of the α-helix is observed over birnessite, which suggests specific vulnerable residue sites for oxidation or hydrolysis possibly leading to fragmentation.


Environmental Science & Technology | 2016

Abiotic protein fragmentation by manganese oxide: Implications for a mechanism to supply soil biota with oligopeptides

Patrick N. Reardon; Stephany S. Chacon; Eric D. Walter; Mark E. Bowden; Nancy M. Washton; Markus Kleber

The ability of plants and microorganisms to take up organic nitrogen in the form of free amino acids and oligopeptides has received increasing attention over the last two decades, yet the mechanisms for the formation of such compounds in soil environments remain poorly understood. We used Nuclear Magnetic Resonance (NMR) and Electron Paramagnetic Resonance (EPR) spectroscopies to distinguish the reaction of a model protein with a pedogenic oxide (Birnessite, MnO2) from its response to a phyllosilicate (Kaolinite). Our data demonstrate that birnessite fragments the model protein while kaolinite does not, resulting in soluble peptides that would be available to soil biota and confirming the existence of an abiotic pathway for the formation of organic nitrogen compounds for direct uptake by plants and microorganisms. The absence of reduced Mn(II) in the solution suggests that birnessite acts as a catalyst rather than an oxidant in this reaction. NMR and EPR spectroscopies are shown to be valuable tools to observe these reactions and capture the extent of protein transformation together with the extent of mineral response.


Analytical Chemistry | 2016

3D TOCSY-HSQC NMR for Metabolic Flux Analysis Using Non-Uniform Sampling

Patrick N. Reardon; Carrie L. Marean-Reardon; Melanie A. Bukovec; Brian E. Coggins; Nancy G. Isern

(13)C-Metabolic Flux Analysis ((13)C-MFA) is rapidly being recognized as the authoritative method for determining fluxes through metabolic networks. Site-specific (13)C enrichment information obtained using NMR spectroscopy is a valuable input for (13)C-MFA experiments. Chemical shift overlaps in the 1D or 2D NMR experiments typically used for (13)C-MFA frequently hinder assignment and quantitation of site-specific (13)C enrichment. Here we propose the use of a 3D TOCSY-HSQC experiment for (13)C-MFA. We employ Non-Uniform Sampling (NUS) to reduce the acquisition time of the experiment to a few hours, making it practical for use in (13)C-MFA experiments. Our data show that the NUS experiment is linear and quantitative. Identification of metabolites in complex mixtures, such as a biomass hydrolysate, is simplified by virtue of the (13)C chemical shift obtained in the experiment. In addition, the experiment reports (13)C-labeling information that reveals the position specific labeling of subsets of isotopomers. The information provided by this technique will enable more accurate estimation of metabolic fluxes in large metabolic networks.


Frontiers in Microbiology | 2015

Regulation of electron transfer processes affects phototrophic mat structure and activity.

Phuc Thi Ha; Ryan S. Renslow; Erhan Atci; Patrick N. Reardon; Stephen R. Lindemann; James K. Fredrickson; Douglas R. Call; Haluk Beyenal

Phototrophic microbial mats are among the most diverse ecosystems in nature. These systems undergo daily cycles in redox potential caused by variations in light energy input and metabolic interactions among the microbial species. In this work, solid electrodes with controlled potentials were placed under mats to study the electron transfer processes between the electrode and the microbial mat. The phototrophic microbial mat was harvested from Hot Lake, a hypersaline, epsomitic lake located near Oroville (Washington, USA). We operated two reactors: graphite electrodes were polarized at potentials of -700 mVAg/AgCl [cathodic (CAT) mat system] and +300 mVAg/AgCl [anodic (AN) mat system] and the electron transfer rates between the electrode and mat were monitored. We observed a diel cycle of electron transfer rates for both AN and CAT mat systems. Interestingly, the CAT mats generated the highest reducing current at the same time points that the AN mats showed the highest oxidizing current. To characterize the physicochemical factors influencing electron transfer processes, we measured depth profiles of dissolved oxygen (DO) and sulfide in the mats using microelectrodes. We further demonstrated that the mat-to-electrode and electrode-to-mat electron transfer rates were light- and temperature-dependent. Using nuclear magnetic resonance (NMR) imaging, we determined that the electrode potential regulated the diffusivity and porosity of the microbial mats. Both porosity and diffusivity were higher in the CAT mats than in the AN mats. We also used NMR spectroscopy for high-resolution quantitative metabolite analysis and found that the CAT mats had significantly higher concentrations of osmoprotectants such as betaine and trehalose. Subsequently, we performed amplicon sequencing across the V4 region of the 16S rRNA gene of incubated mats to understand the impact of electrode potential on microbial community structure. These data suggested that variation in the electrochemical conditions under which mats were generated significantly impacted the relative abundances of mat members and mat metabolism.


Environmental Science & Technology | 2018

Degradation of Glyphosate by Mn-Oxide May Bypass Sarcosine and Form Glycine Directly after C–N Bond Cleavage

Hui Li; Adam F. Wallace; Mingjing Sun; Patrick N. Reardon; Deb P. Jaisi

Glyphosate is the active ingredient of the common herbicide Roundup. The increasing presence of glyphosate and its byproducts has raised concerns about its potential impact on the environment and human health. In this research, we investigated abiotic pathways of glyphosate degradation as catalyzed by birnessite under aerobic and neutral pH conditions to determine whether certain pathways have the potential to generate less harmful intermediate products. Nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography (HPLC) were utilized to identify and quantify reaction products, and density functional theory (DFT) calculations were used to investigate the bond critical point (BCP) properties of the C-N bond in glyphosate and Mn(IV)-complexed glyphosate. We found that sarcosine, the commonly recognized precursor to glycine, was not present at detectable levels in any of our experiments despite the fact that its half-life (∼13.6 h) was greater than our sampling intervals. Abiotic degradation of glyphosate largely followed the glycine pathway rather than the AMPA (aminomethylphosphonic acid) pathway. Preferential cleavage of the phosphonate adjacent C-N bond to form glycine directly was also supported by our BCP analysis, which revealed that this C-N bond was disproportionately affected by the interaction of glyphosate with Mn(IV). Overall, these results provide useful insights into the potential pathways through which glyphosate may degrade via relatively benign intermediates.


Journal of Geophysical Research | 2017

Water column particulate matter: A key contributor to phosphorus regeneration in a coastal eutrophic environment, the Chesapeake Bay

Jiying Li; Patrick N. Reardon; James P. McKinley; Sunendra R. Joshi; Yuge Bai; Kristi Bear; Deb P. Jaisi

Particulate phosphorus (PP) in the water column is an essential component of phosphorus (P) cycling in the Chesapeake Bay because P often limits primary productivity, yet its composition and transformation remain undercharacterized. To understand the mobilization of PP and P sequestration in the water column, we studied seasonal variations in particulate organic and inorganic P species at three sites in the Chesapeake Bay, using chemical extractions, 1-D (31P) and 2-D (1H-31P) NMR spectroscopies, and electron microprobe analyses. Our results suggest that an average of 9% and 50% of water column PP was recycled in shallow and deep sites, respectively, primarily through remineralization of organic P, which was 3 times higher than Fe-bound P remobilization. P recycling efficiency was highest in the warm and anoxic seasons. Organic P compositions and concentrations responded strongly to seasonal and redox variations: orthophosphate monoesters and diesters, and diester-to-monoester ratios (D/M) decreased with depth; both esters and D/M ratios were lower in the anoxic waters in July and September. In contrast, pyrophosphate concentration increased with depth and polyphosphate concentration was high in anoxic seasons. Our analyses suggest the presence of Ca-phosphate minerals (Ca-P) in the water column but with concentrations comparable to sediment Ca-P. It is unclear, however, whether authigenic precipitation occurred in the water column or resuspended from sediments. Overall, these results reveal the dominance of internal P cycling particularly via organic P remineralization and controlling P availability in the water column of the Chesapeake Bay.


Biotechnology and Bioengineering | 2018

Structural and metabolic responses of Staphylococcus aureus biofilms to hyperosmotic and antibiotic stress

Mia Mae Kiamco; Abdelrhman Mohamed; Patrick N. Reardon; Carrie L. Marean-Reardon; Wrya Moh Aframehr; Douglas R. Call; Haluk Beyenal; Ryan S. Renslow

Biofilms alter their metabolism in response to environmental stress. This study explores the effect of a hyperosmotic agent–antibiotic treatment on the metabolism of Staphylococcus aureus biofilms through the use of nuclear magnetic resonance (NMR) techniques. To determine the metabolic activity of S. aureus, we quantified the concentrations of metabolites in spent medium using high‐resolution NMR spectroscopy. Biofilm porosity, thickness, biovolume, and relative diffusion coefficient depth profiles were obtained using NMR microimaging. Dissolved oxygen concentration was measured to determine the availability of oxygen within the biofilm. Under vancomycin‐only treatment, the biofilm communities switched to fermentation under anaerobic condition, as evidenced by high concentrations of formate (7.4 ± 2.7 mM), acetate (13.1 ± 0.9 mM), and lactate (3.0 ± 0.8 mM), and there was no detectable dissolved oxygen in the biofilm. In addition, we observed the highest consumption of pyruvate (0.19 mM remaining from an initial 40 mM concentration), the sole carbon source, under the vancomycin‐only treatment. On the other hand, relative effective diffusion coefficients increased from 0.73 ± 0.08 to 0.88 ± 0.08 under vancomycin‐only treatment but decreased from 0.71 ± 0.04 to 0.60 ± 0.07 under maltodextrin‐only and from 0.73 ± 0.06 to 0.56 ± 0.08 under combined treatments. There was an increase in biovolume, from 2.5 ± 1 mm3 to 7 ± 1 mm3, under the vancomycin‐only treatment, while the maltodextrin‐only and combined treatments showed no significant change in biovolume over time. This indicated that physical biofilm growth was halted during maltodextrin‐only and combined treatments.


Scientific Reports | 2018

Carbohydrates protect protein against abiotic fragmentation by soil minerals

Patrick N. Reardon; Eric D. Walter; Carrie L. Marean-Reardon; Chad W. Lawrence; Markus Kleber; Nancy M. Washton

The degradation and turnover of soil organic matter is an important part of global carbon cycling and of particular importance with respect to attempts to predict the response of ecosystems to global climate change. Thus, it is important to mechanistically understand the processes by which organic matter can be degraded in the soil environment, including contact with reactive or catalytic mineral surfaces. We have characterized the outcome of the interaction of two minerals, birnessite and kaolinite, with two disaccharides, cellobiose and trehalose. These results show that birnessite reacts with and degrades the carbohydrates, while kaolinite does not. The reaction of disaccharides with birnessite produces Mn(II), indicating that degradation of the disaccharides is the result of their oxidation by birnessite. Furthermore, we find that both sugars can inhibit the degradation of a model protein by birnessite, demonstrating that the presence of one organic constituent can impact abiotic degradation of another. Therefore, both the reactivity of the mineral matrix and the presence of certain organic constituents influence the outcomes of abiotic degradation. These results suggest the possibility that microorganisms may be able to control the functionality of exoenzymes through the concomitant excretion of protective organic substances, such as those found in biofilms.


Journal of Cheminformatics | 2018

An automated framework for NMR chemical shift calculations of small organic molecules

Yasemin Yesiltepe; Jamie R. Nuñez; Sean M. Colby; Dennis G. Thomas; Mark I. Borkum; Patrick N. Reardon; Nancy M. Washton; Thomas O. Metz; Justin G. Teeguarden; Niranjan Govind; Ryan S. Renslow

AbstractWhen using nuclear magnetic resonance (NMR) to assist in chemical identification in complex samples, researchers commonly rely on databases for chemical shift spectra. However, authentic standards are typically depended upon to build libraries experimentally. Considering complex biological samples, such as blood and soil, the entirety of NMR spectra required for all possible compounds would be infeasible to ascertain due to limitations of available standards and experimental processing time. As an alternative, we introduce the in silico Chemical Library Engine (ISiCLE) NMR chemical shift module to accurately and automatically calculate NMR chemical shifts of small organic molecules through use of quantum chemical calculations. ISiCLE performs density functional theory (DFT)-based calculations for predicting chemical properties—specifically NMR chemical shifts in this manuscript—via the open source, high-performance computational chemistry software, NWChem. ISiCLE calculates the NMR chemical shifts of sets of molecules using any available combination of DFT method, solvent, and NMR-active nuclei, using both user-selected reference compounds and/or linear regression methods. Calculated NMR chemical shifts are provided to the user for each molecule, along with comparisons with respect to a number of metrics commonly used in the literature. Here, we demonstrate ISiCLE using a set of 312 molecules, ranging in size up to 90 carbon atoms. For each, calculation of NMR chemical shifts have been performed with 8 different levels of DFT theory, and with solvation effects using the implicit solvent Conductor-like Screening Model. The DFT method dependence of the calculated chemical shifts have been systematically investigated through benchmarking and subsequently compared to experimental data available in the literature. Furthermore, ISiCLE has been applied to a set of 80 methylcyclohexane conformers, combined via Boltzmann weighting and compared to experimental values. We demonstrate that our protocol shows promise in the automation of chemical shift calculations and, ultimately, the expansion of chemical shift libraries.

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Nancy M. Washton

Environmental Molecular Sciences Laboratory

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Carrie L. Marean-Reardon

Environmental Molecular Sciences Laboratory

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Douglas R. Call

Washington State University

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Eric D. Walter

Pacific Northwest National Laboratory

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Haluk Beyenal

Washington State University Spokane

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Karl T. Mueller

Environmental Molecular Sciences Laboratory

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Erhan Atci

Washington State University

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