Award Abstract # 1629439
Role of protein nanowires in metal cycling and mineralization

NSF Org: EAR
Division Of Earth Sciences
Recipient: MICHIGAN STATE UNIVERSITY
Initial Amendment Date: August 8, 2017
Latest Amendment Date: August 8, 2017
Award Number: 1629439
Award Instrument: Standard Grant
Program Manager: Enriqueta Barrera
EAR
 Division Of Earth Sciences
GEO
 Directorate for Geosciences
Start Date: September 1, 2017
End Date: August 31, 2021 (Estimated)
Total Intended Award Amount: $152,021.00
Total Awarded Amount to Date: $152,021.00
Funds Obligated to Date: FY 2017 = $152,021.00
History of Investigator:
  • Gemma Reguera (Principal Investigator)
    reguera@msu.edu
Recipient Sponsored Research Office: Michigan State University
426 AUDITORIUM RD RM 2
EAST LANSING
MI  US  48824-2600
(517)355-5040
Sponsor Congressional District: 07
Primary Place of Performance: Michigan State University
East Lansing
MI  US  48824-1000
Primary Place of Performance
Congressional District:
07
Unique Entity Identifier (UEI): R28EKN92ZTZ9
Parent UEI: VJKZC4D1JN36
NSF Program(s): Geobiology & Low-Temp Geochem
Primary Program Source: 01001718DB NSF RESEARCH & RELATED ACTIVIT
Program Reference Code(s):
Program Element Code(s): 729500
Award Agency Code: 4900
Fund Agency Code: 4900
Assistance Listing Number(s): 47.050

ABSTRACT

Geobacter bacteria are recognized as important agents in the cycling of iron and manganese. These bacteria "breath" iron and manganese oxides, a process that requires the cells to produce hair-like filaments (pili) to bind the minerals and discharge onto them electrons generated in their metabolism. The surface of these biological "nanowires" is decorated with pockets that could trap many other metals, particularly those that are positively charged (cationic metals). This suggests that the range of metals that Geobacter bacteria can cycle and mineralize is far greater than currently acknowledged. This project will investigate the metal spectrum that Geobacter cells can bind and mineralized with their conductive pili. This research will advance NSF's Mission "to promote the progress of science" by studying a novel form of microbial energy transduction that has global implications in nature. Not only are the metals targeted in these studies naturally abundant, they are also byproducts of many industrial activities and accumulate at toxic levels as highly mobile forms, which are rapidly introduced into the food chain and increase the risk of exposure. Investigating biological mechanisms for their immobilization will provide the fundamental knowledge needed to develop technologies for biomining and bioremediation, thus addressing a national need. The research is also intertwined with educational efforts directed at training young professionals at the interface of biology and geology, but with deep understanding of physics, chemistry, and engineering. Efforts are also aimed at training future educators and stirring their motivation to engage in outreach projects that promote science communication and inclusion.

This project will focus on Geobacter bacteria, the only microorganisms described to date that use protein nanowires as electronic conduits between the cell and extracellular metal electron acceptors. Each pilus fiber is an assembly of the same peptide subunit (the pilin) and exposes on its surface many carboxyl side chain ligands, which could bind cationic metals and position them optimally for their reduction. The investigator will test this in a series of experiments that evaluate the effect of piliation, pilus conductivity, and charge of the putative metal traps to bind and reductively precipitate trivalent cobalt (Co3+), divalent cadmium (Cd2+) and monovalent silver (Ag+) metal cations. These cationic metals are found in environments where iron reduction by Geobacter spp. is an active process, a process mediated by protein nanowires, and mineralized concomitantly to the reduction of iron oxides. This suggests that the Geobacter pili also mediate the reductive precipitation of the soluble metal cations, which one can assess in biological assays coupled to microscopic examination of the cells and bulk X-ray Absorption Near Edge Structure (XANES) spectroscopic analyses. The researcher will also collect the LIII-edge extended X-ray absorption fine structure (EXAFS) spectra from the pili-associated mineral to model the atomic coordination about the metal. This will allow to characterize the mineral phase and identify the nanowire ligands that are responsible for metal mineralization.

PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH

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Clark, M.M. and G. Reguera "Biology and biotechnology of microbial pilus nanowires." J Ind Microbiol Biotechnol , v.47 , 2020 10.1007/s10295-020-02312-5
Clark, M.M., Paxhia, M.D., Young, J.M., Manzella, M.P., and Reguera, G. "daptive synthesis of a rough lipopolysaccharide in Geobacter sulfurreducens for metal reduction and detoxification" Appl. Environ. Microbiol. , v.87 , 2021 , p.Aem009642 10.1128/aem.00964-21
Cosert, K. M., A. Castro-Forero, R.J. Steidl, R. J. Worden and G. Reguera "Bottom-up fabrication of protein nanowires via controlled self-assembly of recombinant Geobacter pilins." mBio , v.10 , 2019 10.1128/mBio.02721-19
Dulay, H, M. Tabares, K. Kashefi, and G. Reguera "Cobalt resistance via detoxification and mineralization in the iron-reducing bacterium Geobacter sulfurreducens" Front Microbiol , v.11 , 2020 10.3389/fmicb.2020.600463
K. M. Cosert and G. Reguera "Voltammetric study of conductive planar assemblies of Geobacter nanowire pilins unmasks their ability to bind and mineralize divalent cobalt" J Ind Microbiol Biotechnol , 2019 10.1007/s10295-019-02167-5
Tabares, M.D., R.H. Dulay and G. Reguera "Geobacter sulfurreducens. Trends Microbiol 28(4): 327-328, doi:10.1016/j.tim.2019.11.004." Trends Microbiol , v.28 , 2020 10.1016/j.tim.2019.11.004

PROJECT OUTCOMES REPORT

Disclaimer

This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.

This project investigated the spectrum of metals mineralized by bacteria in the genus Geobacter. These microbes produce hair-like conductive appendages known as pili to discharge respiratory electrons onto iron and manganese oxides. The pili-mediated reactions dissolve the oxides and release metal cations trapped in the minerals, including uranium, cobalt, cadmium, and other metals that can be potentially toxic to the cells. The identification on the pili surface of specialized motifs for binding divalent metal cations suggested that the nanowires could also be involved in the sequestration and reductive mineralization of a wide range of metals. Consistent with this, we demonstrated the ability of the pili to bind and reductively precipitate uranium to a mononuclear UIV mineral phase). This reaction harnessed energy for cell growth and prevented the infiltration of the toxic radionuclide. For more efficient protection, the cells coated themselves with a short (rough) lipopolysaccharide that sequestered the uranyl cation to prevent its permeation. The U-bound lipopolysaccharide was then released in outer membrane vesicles. This dual strategy mineralized 75% of the radionuclide at the pili and sequestered the rest in the LPS layer. To accelerate our research, we developed pilin-based conductive materials on electrodes and investigated interactions between the pili and other metals. The in vitro platforms demonstrated the reductive mineralization of cobalt at the pili’s metal traps. We used this information to guide in vivo studies that identified previously unknown pathways for the detoxification of CoII and its extracellular mineralization at the thermodynamic edge.

Intellectual merit: This research is innovative because Geobacter bacteria are the only microorganisms described to date that use protein nanowires as electronic conduits between the cell and extracellular metal electron acceptors. Despite their recognized role in metal cycling and mineralization, the true spectrum of metals cycled by Geobacter bacteria was not known at the beginning of the NSF-funded efforts. Capitalizing on the team’s expertise in microbial nanowires, biomineralization, and nanotechnology, the project led to the development of in vitro platforms that demonstrated the reductive precipitation of CoII as Co0 nanoparticles at the pilus metal traps. These studies guided in vivo experiments in the model representative Geobacter sulfurreducens that identified novel pathways for the detoxification and mineralization of metal cations such as uranium and cobalt. The detoxification of cobalt via mineralization, a previously unknown biological reaction, is particularly important in nature because it alleviates metal stress on Geobacter and preserves its ability to synthesize CoII cofactors (cobamides) for syntrophic partners. Some of the Geobacter partners are plant-associated and support essential plant functions. Thus, Geobacter control of cobalt availability indirectly impacts plant health and productivity.

Broader impacts: The research advanced NSF’s Mission “to promote the progress of science” by studying a novel form of microbial energy transduction of global relevance. Indeed, the cationic metals targeted in the studies are naturally abundant on Earth and their cycling contributes greatly to global planetary processes. The metals are also generated in many industrial activities, accumulating at toxic levels in highly mobile forms that risk human exposure. The identification of genetic markers for the biomineralization processes affords opportunities for in situ monitoring and predictive modeling of the fate of metal contaminants. This, in turn, is critical for effective bioremediation and long-term stewardship of contaminated sites. There is also economic interest in developing platforms for the reclamation of metals such as cobalt to reduce dependency on foreign and often vulnerable supply chains. Hence, findings from this work address the national need to advance technologies for the bioreclamation of critical metals. The ability of Geobacter to produce protein nanowires to respire and mineralize toxic metals is a novel concept in Geobiology that impacts several other fields such as Engineering, Nanotechnology, Public Health and Biomedicine. The research was therefore intertwined with educational efforts directed at training young professionals at the interface of biology and geology, but with deep understanding of physics, chemistry, and engineering. Efforts were also aimed at training future educators and stirring their motivation to engage in outreach projects that promoted science communication and inclusion, further broadening the impacts of the research.


Last Modified: 09/30/2021
Modified by: Gemma Reguera

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