Insights into the Organization of an ECF-Type Transporter by Fluorescence Lifetime and Anisotropy Microscopy on Living E. Coli

BioMNY (Rhodobacter capsulatus) is an ATP dependent, ECF-type biotin importer, consisting of two different transmembrane domains BioY, BioN and ATPase cassettes BioM. The organization and stoichiometry of the transporter subunits is still a matter of debate (Erkens, 2011; Berntsson, 2012; Erkens, 2012).To obtain insights into the stoichiometry of the transporter components we used spectrometric, lifetime-based HeteroFRET (Finkenwirth, 2010) and static anisotropy-based HomoFRET (Kirsch, 2012) approaches. To this aim, certain transporter components, tagged with a fluorescence donor or acceptor, were co-expressed in recombinant E. coli. The measurements led to the postulation of a BioM2N?Y2 stoichiometry in living bacteria (in accordance with Neubauer, 2009, 2011).Recently, we expanded the Hetero and HomoFRET approach form the spectroscopic to the microscopic level (Ziomkowska, 2012). The use of imaging techniques for microbial cells has the same advantages as for mammalian cells: expression level and localization of the protein can be validated in a single cell during the measurement. Our experiments show that the spectroscopic data can be reproduced in imaging experiments and will be used for the ongoing research. Nevertheless, the orientation of the probe has to be taken into account for anisotropy microscopy. To the best of our knowledge, we present the first FRET-Imaging approach on living bacteria, a tool likely to be used for interaction studies in any E. coli expression system.