Genomes to Life Contractor-Grantee Workshop I
Arlington, Virginia, February
9-12, 2003
A64
Progress in Development of Genetic Tools for Shewanella oneidensis MR-1
Margie Romine[1], Alex Beliaev[1,2], Greg Newton[1], Samantha Reed[1], Valerie Crusselle[1], Amber Alford[1], Peter Lu[1], Jim Fredrickson[1], Matthew Fields[2], Xiufeng Wan[2], Weimin Gao[2], Haichun Gao[2], Ting Li[2], Dawn Stanek[2], Dorothea K. Thompson[2], Jizhong Zhou[2], Achilles Kapanidis[3], Natalie Gassman[3], Ted Laurence[3], and Xiangxu Kong[3], and Shimon Weiss[3]
[1]Pacific Northwest National Lab (PNNL), [2]Oak Ridge National Lab (ORNL), and [3]University of California Los Angeles (UCLA)
The ability to Shewanella oneidensis MR-1 to reduce polyvalent metals and radionuclides has generated considerable interest in the potential role of this organism in biogeochemical cycling and in the bioremediation of contaminant metals and radionuclides. In spite of considerable effort, the details of MR-1’s electron transport system and the mechanisms by which it reduces metals and radionuclides remain unclear. The recent completion of the MR-1 genome sequence makes it possible to utilize targeted genetic approaches to identify the macromolecular components important to these processes and to interrogate the regulatory networks that lead to changes in the behavior of this organism in response changes in its environment. The Shewanella Federation, consisting of researchers from various academic institutions, national labs, and industry, was formed to jointly carry out these types of studies in MR-1.
Presented herein is a description of progress made in the development and use of selected tools for targeted mutagenesis of MR-1 and construction and expression of gene and promoter fusions in MR-1 by members of the Shewanella Federation. Two new vectors for targeted gene deletion have been constructed and used to generate 11 single and 4 double deletions of various MR-1 regulatory genes. A lower copy number vector encoding the GFP reporter has been identified and utilized to construct 63 promoter fusions. Methods to screen these reporters in a high through-put manner are being developed. A new Gateway vector, pBHG, was constructed for expression of his-tagged proteins in MR-1 and a new broad-host range vector was produced for expressing YFP gene fusions in MR-1. Ten YFP fusions to sensory proteins and respiratory genes have been constructed for using in live cell imaging. Twenty-nine his tag fusions have been constructed in pETDEST42 for production of proteins for subsequent purification and two have been transferred into pBGH for localization studies in MR-1. These genes fusions will be used for analyses, outlined here, of 1) protein-protein interactions using a newly developed confocal microscope and methodology, called ALEX (alternate-laser-excitation-based emission ratio) and 2) simultaneous topologic and spectroscopic features of living cells expressing fluorescently labeled proteins, at nanometer resolution, using AFM-enhanced two-photon confocal microscopy.
