Thrust 4: Whole Cell and Cytochrome Biological Force Microscopy (LDRD: Cytochrome and Whole Cell Interactions wtih Iron Oxides)
EMSL Project ID
13494
Abstract
This LDRD project is the fourth one associated with the Biogeochemistry Grand Challenge (BGC). Its overall goal is to develop new capabilities and materials as needed to understand the molecular scale biogeochemistry associated with electron transfer at the organism-mineral interface. Two primary activities will be pursued to accomplish this goal. First, new applications of scanning probe microscopy will be developed to characterize the nanometer-scale spatial location of cytochromes on bacterial surfaces, to quantify the interaction forces between purified recombinant cytochromes and mineral surfaces and between individual cytochromes and other proteins in complexes, and to measure integrated adhesion forces between whole organisms and mineral surfaces of known structure. Second, new model oxide experimental materials will be developed (both synthetic and natural) that can be used to study the molecular biogeochemistry of bacterial iron reduction, and new surface science preparation methods will be developed to allow the imaging of surface structure and Fe valence beneath single microorganisms, bacterial colonies, and/or biofilms with state-of-the-art instrumental techniques. These capabilities are essential to the successful completion of the BGC. Biological force microscopy will be used to quantitatively measure forces at the interface between S. oneidensis and iron oxide surfaces being investigated by the BGC team (including those in activity 2 below). Previous applications of this technique (e.g., Lower et al., 2001) will be extended to include single cell microscopy and the mounting of small, oriented Fe(III) oxide coupons on the AFM tip to probe microbe-mineral attraction and adhesion forces. BFM will be performed on strains of S. oneidensis that have been engineered to over express a particular protein (e.g., MtrB or MtrC) or strains in which the gene encoding the protein has been deleted. These force data will be examined for evidence of unique force features indicative of the interaction between a specific outer membrane protein (e.g., MtrB and MtrC) and mineral surfaces. Subsequent force experiments will be conducted on knockout strains of S. oneidensis deficient in protein trafficking and secretion proteins (e.g., GspD and GspE) to determine the roles these proteins play in mediating contact with a mineral surface.
In addition to whole cell-mineral BFM experiments, protein-AFM will be preformed between purified recombinant proteins mtrA, mtrB, or mtrC and an iron oxide surface or polyclonal antibody in-vitro. The resulting force data will be compared to the whole cell BFM data to search for distinctive force signatures of mtrB or mtrC, if they indeed exist. Furthermore, since it is believed that mtrA, mtrB, mtrC, and possibly omcA form protein complexes in-vivo, we will attempt to probe their protein-protein interactions in-vitro by performing AFM between selected purified proteins themselves (e.g., AFM preformed between mtrB of omcA linked to a substrate and mtrC linked to an AFM tip). Sophisticated modeling approaches (beyond such models as the worm-like chain model) will likely have to be developed in collaboration with Kevin Rosso (BGC LDRD Project #3) to interpret the complex intermolecular force data that will result.
Control BFM experiments will be preformed between S. oneidensis and aluminum oxide surfaces, between S. oneidensis and a non-functionalized AFM tip (a bare silicon nitride tip or a tip coated with a non-reactive polyclonal antibody), or between S. oneidensis and a functionalized tip (e.g., a tip coated with polyclonal Anti-mtrB or Anti-mtrC) in which free mtrB, mtrC, or protease has also been introduced into the AFM fluid cell. Similar control experiments will be conducted for protein-AFM experiments, as well as experiments with proteins having a similar molecular mass (e.g., bovine serum albumin) to mtrB and mtrC.
1d.) Artificial Membranes for Cytochrome Function Studies
A model system will be developed that simulates key physicochemical attributes of the outer membrane of gram negative bacteria (such as S. oneidensis) to enable mechanistic studies of the redox reactivity of hydrophobic outer membrane proteins. To this end, we propose to develop a new hybrid membrane system based on polymeric lipobeads (Jin et. al., 1996) in which lipopolysaccharide (LPS) is conjugated to an acetylated polymeric bead to produce a mechanically stable system that has structural similarity to the outer membrane of S. oneidensis. Acylated hydrogel beads will be formed in collaboration with Dr. Ping Lee (University of Toronto) and will be treated with purified LPS (obtained from commercial sources) to produce a LPS-lipid shell around the hydrogel core. Conditions will be optimized (e.g., using different size fatty acid chains for surface acylation) to produce lipobeads having a uniform LPS-lipid anchored shell, size, and morphology. Characterization of the LPS-lipobeads and incorporation of outer membrane proteins (e.g., MtrB and MtrC) will be monitored using surface plasmon resonance spectroscopy and laser scanning confocal fluorescence microscopy. Other techniques including membrane fragments and artificial planar bilayer membranes (e.g., Gutsman et al., 1999) will also be considered.
Project Details
Project type
Grand Challenge
Start Date
2005-02-08
End Date
2008-02-12
Status
Closed
Released Data Link
Team
Principal Investigator
Related Publications
Bose, S, M. F. Hochella Jr., Y. A. Gorby, D. W. Kennedy, D. E. McCready, A. S. Madden, and B. H. Lower. 2009. "Bioreduction of hematite nanoparticles by the dissimilatory iron reducing bacterium Shewanella oneidensis MR-1." Geochimica et Cosmochimica Acta 73: 962-976.
Brian H. Lower, Liang Shi, Ruchi Yongsunthon, Timothy C. Droubay, David E. McCready, and Steven K. Lower (2007). “Specific bonds between an iron oxide surface and outer membrane cytochromes MtrC and OmcA from Shewanella oneidensis MR-1,” J. Bacteriol. 189:4944-4952.
Eggleston CM, J Voros, L Shi, BH Lower, TC Droubay, and PJ Colberg. 2008. "Binding and Direct Electrochemistry of OmcA, an Outer-Membrane Cytochrome from an Iron Reducing Bacterium, with Oxide Electrodes: A candidate Biofuel Cell System." Inorganica Chimica Acta 361(3):769-777. doi:10.1016/j.ica.2007.07.015
High-affinity binding and direct electron transfer to solid metals by the Shewanella oneidensis MR-1 outer membrane c-type cytochrome OmcA, 2006. Xiong YJ, Shi L, Chen BW, Mayer MU, Lower BH, Bose S, Hochella MF, Fredrickson JK, Squier TC. JACS 128 (43): 13978-13979.
Lower B. H., R. Yongsunthon, L. Shi, L. Wildling, H. J. Gruber, N. S. Wigginton, C. L. Reardon, G. E. Pinchuk, T. C. Droubay, J-F. Boily, and S. K. Lower. 2009. "Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of Shewanella oneidensis MR-1." Applied and Environmental Microbiology 75:2931-2935.
Nicholas S. Wigginton, Kevin M. Rosso, Brian H. Lower, Liang Shi, and Michael F. Hochella, Jr. (2007). “Scanning Tunneling Microscopy and Spectroscopy of Bacterial Outer Membrane Cytochromes,” Geochim. et Cosmochim. Acta 71:543-555.
Saumyaditya Bose, Michael F. Hochella Jr., Yuri A. Gorby, David W. Kennedy, David E. McCready, Andrew S. Madden, and Brian H. Lower (2009). Bioreduction of Hematite Nanoparticles by the Dissimilatory Iron Reducing Bacterium Shewanella oneidensis MR-1. Geochimica et Cosmochimica Acta (in press).
Shi L, Chen BW, Wang ZM, Elias DA, Mayer MU, Gorby YA, Ni S, Lower BH, Kennedy DW, Wunschel DS, Mottaz HM, Marshall MJ, Hill EA, Beliaev AS, Zachara JM, Fredrickson JK, Squier TC. 2006. "Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1." J. Bacteriol.188 (13): 4705-4714.