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Bacteria Synthesized Proteins for Mineral Affinity

This research explores the synthesis of specific proteins by bacteria that interact with minerals, and examines the forces and mechanisms controlling their interactions. The study aims to identify mineral-specific proteins and fabricate peptides with unique mineral-binding motifs. Living microbial cells are used as a lithographic tool in this investigation.

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Bacteria Synthesized Proteins for Mineral Affinity

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  1. MINERAL SPECIFIC PROTEINS SYNTHESIZED BY BACTERIA Steven Lower Ruchi Yongsunthon Brian Lower Michael Hochella, Jr. Ohio State University Virginia Tech ***This research has been published in Science, Geochimica et Cosmochimica Acta, Advances in Agronomy, Geomicrobiology Journal, and Eos.

  2. 400 nm bacterium-mineral interface interfacial forces and proteins *forces control, and are themselves modulated by, the expression of biopolymers on a bacterium’s surface G. Bowles

  3. Microorganisms Humans • # of cells • # of species • # years • 1030 109 • 106 100 • 109 106 Consider the following… A comparison of two organisms that work in the realm of the nanometer.

  4. Shewanella interactions with Fe oxyhydroxides metal reducing bacteria & oxides • primitive form of respiration using Fe(III) solids • remediation of organic and inorganic pollutants in surface and subsurface environments Shewanella Fe(III) mineral

  5. Fe(III) oxide H+ H+ e- O2 H2O NADH NAD+ ADP ATP outside outer membrane periplasm inner membrane inside

  6. pH Mr force microscopy 2D gel electrophoresis cell lithography phage display library Cell-material interface discover forces and proteins F~e -D theory mimic and utilize information

  7. living cell biological force microscopy laser lever mineral

  8. G x m1 x m2 (distance)2 Force-distance curves using different minerals, bacteria, and solutions approach retraction

  9. H R 6 d 2 electrostatic and van der Waals forces DLVO Theory 4 p s1 s2 R e - kd F(d) = e eo k s = surface charge R = radius e = dielectric constant eo= vacuum permittivity d = distance k = 1 / Debye length H= Hamaker constant R = radius d = distance ~(salt concentration) –1/2

  10. subsurface transport in the environment (approach measurements between G- bacterium and a silicate) green – low IS blue – high IS approach only

  11. Force-distance curves (retraction forces between Shewanella and AlOOH vs FeOOH) approach retraction Shew AlOOH FeOOH

  12. diaspore goethite (AlOOH) (FeOOH) + 39 7 - 26 6 aerobic + + - - anaerobic + 41 4 - 137 20 energy values between Shewanella and mineral (as function of oxygen concentration) attoJoules (10-18 J) Control experiment with nonviable cells ~6 aJ (did not change with mineral, oxygen concentration, or contact time)

  13. Fe(III) oxide outside outer membrane H+ periplasm e- H+ inner membrane O2 H2O NADH NAD+ inside ADP ATP

  14. d = distance or extension k = Boltzmann’s constant T = temperature b = persistence length (0.38nm Ca-Ca in protein) L = contour length (length of stretched protein chain) Protein folding/unfolding Worm-like Chain Model F(d) = (k T / b) [0.25 (1 – d / L)–2 – 0.25 + d / L]

  15. OM protein expression patternsShewanella andAlOOHorFeOOH protein signature observed in 80% of the data; only with goethite after some period of “recognition time”

  16. 2D Gel Electrophoresis of OM Proteins pH Fe+3 O2 Mr pH Mr excise proteins & fragment into peptides mass spec & database search extract membrane proteins compare with force signature culture cells separate proteins force microscopy

  17. terminal electron acceptor -O2vsFe(III) kDa 150 100 75 50 37 25 pH 4.0 5.0 6.0 7.0 8.0 9.0

  18. Cell-material interface pH Mr force microscopy 2D gel electrophoresis cell lithography phage display library discover forces and proteins F~e -D theory mimic and utilize information

  19. peptide phage library expose to target mineral wash away unbound phage elute bound phage “bio-panning” isolate clones, sequence DNA to find mineral-binding motif,

  20. protein cell substrate biological cell lithography biological cell lithography bacterium as living “pen” that produces and secretes genetically engineered proteins S-layer protein T. Beveridge

  21. bacterium-mineral interface nanoscale forces and proteins • quantify natural forces of affinity between inorganic crystalline phases and proteins synthesized by bacteria • use theoretical models and protein expression patterns to identify putative mineral specific proteins • mimic natural specificity by attempting to fabricate peptides with unique mineral-binding motifs • use living microbial cells as a lithographic tool

  22. Acknowledgements Acknowledgements Terry Beveridge, John Smit, Courtney Crummett, Graeme Bowles National Science Foundation (GEO & ENG) Department of Energy American Chemical Society ***This research has been published in Science, Geochimica et Cosmochimica Acta, Advances in Agronomy, Geomicrobiology Journal, and Eos. Steven Lower – Ohio State University – Lower.9@osu.edu

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