|FRAWLEY, ELAINE - WASH UNIV-ST. LOUIS
|LEE, RACHEL - WASH UNIV-ST. LOUIS
|ANGENENT, LARGUS - WASH UNIV-ST. LOUIS
|KRANZ, ROBERT - WASH UNIV-ST. LOUIS
Submitted to: American Society for Microbiology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/5/2008
Publication Date: 6/5/2008
Citation: Rosenbaum, M., Frawley, E.R., Lee, R.E., Angenent, L.T., Kranz, R.G. 2008. Recombinant engineering of Shewanella oneidensis MR-1 c-type cytochromes in Escherichia coli [abstract]. American Society for Microbiology. Paper No. 509.
Technical Abstract: Shewanella oneidensis is known to respire with extracellular solid metal oxides (i.e., iron, manganese, uranium) as a terminal electron acceptor. It has become the focus of intensive research not only due to its important bioremediation features, but also as a potential organism for biological electricity generation. For this, S. oneidensis respires with a carbon electrode as the terminal electron acceptor in microbial fuel cells (MFCs). While it is known that a number of c-type cytochromes conduct the electrons from the inner membrane through the periplasmic space to the cell surface, the individual role of these proteins and the minimum requirements for electron transfer to the outer membrane are not yet well understood. We developed a recombinant system in Escherichia coli for the expression of S. oneidensis proteins involved in this process: inner membrane cytochrome CymA, periplasmic cytochrome MtrA, outer membrane cytochrome MtrC, and MtrB, a non-cytochrome outer membrane protein. The S. oneidensis MR-1 genes cymA and mtrCAB were cloned into an arabinose inducible expression vector. The resulting clones were transformed into the E. coli delta ccm cell line RK103 in which the endogenous cytochrome c biosynthesis operon was knocked out. Cytochrome biogenesis is restored by expression of the eight E. coli systemI genes from the IPTG-inducible pRGK333 plasmid. With this inducible recombinant system in E. coli, the proteins involved in electron transport were expressed and characterized regarding localization and functionality. His6-tag modification of some of the proteins allowed their purification and individual characterization. This approach will enable us to determine the minimal set of proteins required for solid metal oxide reduction. An additional benefit is that E. coli is easy to manipulate and more metabolically diverse than S. oneidensis, which is limited to lactate as the carbon source for metal oxide reduction. Once the electron transport pathway has been engineered in E. coli, it should then be possible to optimize the electron flow through the pathway and develop more efficient MFCs using a wider variety of carbon sources.