Progress 06/01/22 to 05/31/23
Outputs
Target Audience:The target audience this period was the scientific community that wasreached via conference and seminar presentations. Changes/Problems:COVID-19 related issues delayed recruitment of students and progress on the project. We anticipate that we will request ano-cost extension to continue the project until 6/1/2025. What opportunities for training and professional development has the project provided?The project is the primary focus of two Ph.D. students, providing the reserach vichile for this eduation. Two NSF funded REUs, each one mentored by one of the Ph.D. student, worked on the project during 10 weeks over the summer. In addition, a visiting student PhD from Brazil,who is supported by her home state and is reseraching the role of siderophores in iron accumulation by esturine plants, has benefit from working in andcontriubted to the project. How have the results been disseminated to communities of interest?Presentations were given at the ASA, CSSA and SSSA Annual Meetings, the GSA annual meeting, and the University of Sao Paulo. What do you plan to do during the next reporting period to accomplish the goals?Following the enzymatic and fungal siderophore degradation experiments, we will characterize fungal enzymes involved in the siderophore degradation this summer together with an NSF REU student. The study will then be expanded to test siderophore degradation by root exudates and soil enzyme extracts. In final experiments, we will investigate siderophore degradation in plant-microbe cocultures in defined media and in soils. We plan to conduction adsorption isotherms for the siderophores DFOB, PDMA, and protochelin in both their unbound and iron chelated states to ferrihydrite, goethite, birnessite, kaolinite, montmorillonite, and organic matter, in order to understand how structure effects sorption. We also plan to expand our use of Ga-complexes in XAS and micro-XRF/XAS experiments to study the reactivity of PDMA and protochelin complexes in soil. Further plans include focusing on the reactions that occur with organic matter to determine whether soil organic matter acts as an iron source, or a siderophore sink. In addition, we currently have three papers in advanced preparation that we intend to submit in calendar year 2023.
Impacts
What was accomplished under these goals?
In the third year of the project we made substantial progress in our understanding of the interactions of siderophores with reactive soil components. Highlight of activities are as follow: We determined the stability constants of the model phytosiderophore proline 2′deoxymugineic acid (PDMA) with Mn, Co, Cu, Ni, and Zn to determine how these metals may impact iron acquisition. Furthermore, we determined that these stability constants correlate (log-log basis) with those of other phytosiderophores, the siderophore DFOB, and hydroxide. These relationships will allow for prediction of other constants and improve our understanding how these soil components may impact iron and other trace metal uptake. We have developed gallium as a spectroscopic tracer for siderophore complexes. Trivalent gallium is similar in size and makes complexes of comparable affinity to ferric iron with siderophores. We have thus utilized Ga-siderophore complexes in X-ray absorption spectroscopy (XAS) experiments so that we can follow the fate of the metal ion in siderophore complexes even in the presence of iron. We have built a spectral library of Ga-DFOB complexes interacting with mineral and DOM standards, as well as utilized spatially resolved micro X-ray absorption and micro-X-ray fluorescence spectroscopy to establish that we can track the fate of the complexes in soil. We have submitted a beamtime proposal to the Stanford Synchrotron Radiation Lightsource (SSRL) to expand this work. We have determined how a suite of extracellar enzymes break down siderophores and iron-siderophore metal complexes by UV-visible and mass spectrometry. To represent the wide variety of siderophore structures and chemistries, we tested three different structures, each characteristic for one of the three major chelating moieties found in siderophores (Fig. 1). Protochelin is a tris-catecholate siderophore, desferrioxamine B (DFOB) represents a tris-hydroxamate siderophore, and proline-2′- deoxymugineic acid (PDMA) is a carboxylate siderophore. Each of the siderophores was tested in its unbound form and as iron-siderophore complexes with each of the enzymes. We found that phenol oxidases are highly effective in the degradation of the catechol siderophore protochelin. Peroxidases were effective in both the degradation of protochelin and DFOB. Complexation of protochelin and DFOB with iron protected the siderophore and reduced the amount of degradation. PDMA was inert in all degradation reactions. Following these experiments we started testing degradation with cultures of fungi known to produce extracellular enzymes. We selected four fungi known to be prolific producers of extracellular enzymes for the acquisition of carbon, which can potentially degrade siderophores: (1, 2) Two strains of Phanerodontia chrysosporium, a white-rot fungus, (3) Linnemania elongata, a plant-growth-promoting fungus, and a new isolate from wheat and phenotypically in agreement with Drechslera biseptata. Of the four fungal cultures, only D. biseptata was able to efficiently break doewn protochelin and DFOB, while PDMA was only slightly degraded. Structures of breakdown products were determined in both the enzymatic and fungal degradation experiments using high-resolution LC-MS/MS. In summary, our work revealed siderophore breakdown by enzymes and selected fungi is possible, and particularly effective for protochelin, a representative of catechol siderophores. Hydroxamate siderophores, such as DFOB, were also degraded but their structure was protected when present as iron chelates. Siderophore breakdown products could be structurally characterized and were compromised or lost their ability to bind iron. The synthetic phytosiderophore analog PDMA remained relatively inert in all experiments.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Kohn, J., Evers, A., and Duckworth, O.W., Determining Stability Constants of Metal-Proline 2?Deoxymugineic Acid (PDMA) Chelates via Potentiometric and Spectrophotometric Titration, Geological Society of America Meeting, Denver, Colorado, October 9-12, 2022
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Evers, A. and Duckworth, O.W Understanding Critical Soil-Siderophore Antagonistic Interactions. ASA, CSSA, SSSA International Annual Meeting, Baltimore, Maryland. November 6-9 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
French, K., Chukwuma, E., and Baars, O. (2022) Enzymatic Degradation of Siderophores in Soil by Fungi. ASA, CSSA, SSSA International Annual Meeting, Baltimore, Maryland. November 6-9 2022.
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience:The target audience this period was the scientific community who was reached via a publication and a conference presentation. Changes/Problems:COVID-19 related issues delayed recruitment of students and progress on the project. We anticipate that the project would benefit from one or possibly two no-cost extensions. What opportunities for training and professional development has the project provided?Two PhD students (a female student in the Duckworth labanda minority student in the Baars lab) have recruited to the project and will use this project for thedissertationwork. Regular project meeting have occurred, and all three PI will serve on the mentoringcommittees for these students. How have the results been disseminated to communities of interest?A paper was published in Biometals and presenation given at theASA, CSSA and SSSA Annual Meetings. What do you plan to do during the next reporting period to accomplish the goals?We plan to accelerate the reach pace of the project as students advance in the project and COVID restriction loosen. We plan to systematically study the interactions of siderophore and siderophore-metal complexes with minerals and organic matter. We anticipate that this will also include submission of a beamtime proposal to a synchrotron facility lead by a student. Similarly, we plan to study the impact of extracellular enzymes on the degradation of siderophore, as well as the possible in activation of enzymes. These project will be complemented by the development of mass spectrometry pipelines to determine degradation products from these reactions. In addition, we have started two complementarycollaborations. The first involvesfaculty in material science interested in the reactivity of hydroxamate siderophores with organophosphates. We specifically will work with these faculty to better understand if siderophores can promote the degradation of xenobiotic organophosphates and phosphorus containing soil organic carbon. In the second collaboration, a visiting student from Brazil will seek to understand the impact of siderophores on the accumulation of iron by plants used for phytoremediation. Both of these projects leverage the knowledge associated with this project but are independently funded and will utilize resources from this project.
Impacts
What was accomplished under these goals?
Despite issue from COVID delays, we have made significant process toward the project. As noted, the there were delays in recruiting PhD students to the project. Additionally, both postdoctoral scholars working on the project moved on to other opportunities. Thus, a research scholar contributed to the project by studying the interactions of siderophores with other competing metals, resulting in a publication. Additionally, two PhD students have joined the project and started to conduct research on the interactions of siderophores and siderophore-metal complexes with mineral surfaces and extracellular enzymes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Doydora, S.A., Baars, O., Harrington, J.M., and Duckworth, O.W. (2022) Salicylate coordination in metal-protochelin complexes, Biometals, 35, 8798.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Doydora, S.A., Baars, O., Harrington, J.M., and Duckworth, O.W., Structure and Stability of Mo(VI)-Siderophore Complexes, Soil Science Society of America meeting, Salt Lake City, Utah, Nov 7-11 2021.
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:The target audience reached in this period was (1) the scietific community through a publication and (2) NC State University undergraduate students who received a "current topics" lecture based on this research project. Changes/Problems:The project started during a university shutdown due tocovid-19 policies. This delayed the start of our laboratory work and the recruitment of doctoral students to work on the project. What opportunities for training and professional development has the project provided?Recruiment of personel was delayed covid-19. Consequently, the intial phase of the project was carried out by existing postdoctoral scholars in the Duckworth and Baars laboratories, proving training for two female postdocs. For the fall, two PhD students have been recruited (1 female, 1 froman underrepresnted group). How have the results been disseminated to communities of interest?A paper was published inFrontiers in Microbiological Chemistry and Geomicrobiology. What do you plan to do during the next reporting period to accomplish the goals?Two PhD students supported by this projects will join us in the fall. A student in chemistry will continue to develop detection methods for siderophores, siderophore metal complexes, and breakdown products by liquid chromatography-mass spectrometry and liquid chromatography- inductively coupled plasma mass spectrometry. A student in soil science will continue experiments that probe the kinetics and breakdown products of siderophores with reactive soil components.
Impacts
What was accomplished under these goals?
NC State University experienced an extended shutdown due in 2020 to COVID-19. To continue progress on the project during a shutdown, we expanded analysis of a preliminary dataset used as the bast of the foundation of the proposal. Publication of a paper on methods of recovery and detection of siderophores in soils resulted from this effort. After the opening of facilities, we initiated initial experiments initiated to study the interactions of desferrioxamine B (DFOB) and iron-DFOB complexes with soil components
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Rai, V., Fisher, N., Duckworth, O.W., and Baars, O. (2020) Extraction and analysis of structurally diverse siderophores in soil, Frontiers in Microbiological Chemistry and Geomicrobiology, 11, 581508.
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