Abstract
In recent studies performed by our group we identified and functionally characterized four ATP-binding cassette transporters involved in sulfur assimilation, three for alkane (SsuABC1, SsuABC2 and NrtTUWA), which is odd compared to other microorganisms, and one for sulfate, (SbpCysUWA). The sulfate uptake system has been the target of the doctoral project from Cristiane Pereira (FAPESP Proc.2013/16094-4) and it is directly related to this application. Functional results obtained so far revealed that sbpcysUWA operon is activated in vitro and in vivo, that the periplasmic binding protein Sbp binds sulfate but not molybdate, and that it showed increased thermal stability in the presence of the anion. A X. citri strain deleted for the cysU gene (encoding one of permeases) was constructed and showed deficiency in the production of xanthan gum and xanthomonadin, but not in bacterium growth. The three-dimensional structure of the Sbp was also solved in the presence of sulfate. All of this data combined with previous results obtained by our group supported the relevance of these transporters in X. citri, but still we do not know the range of susbtrates for each transporter and how is the mechanistic of the transport. Accordingly, this proposal aims to (i) the structural characterization of periplasmic-binding proteins NrtT and SsuA1; (ii) the spectroscopic studies to determine the range of substrates of the four proteins and comparison between them and, (iii) production of full carriers for interaction assays with the periplasmic proteins. This project will be conducted in collaboration with Dr. Dirk Jan Slotboom, leader of one of the most famous groups dedicated to the study of ABC transporters and characterization of membrane proteins, located at the University of Groningen, Netherlands. His group has experience in Sm-FRET, ITC, crystallization, expression and purification of membrane proteins, studies in proteoliposomes and others. The project will bring high-impact results in the area of interest as well as to the scientific community, as well as strengthening our group that will be benefit from learning new ways to deal with the structural and functional characterization of these proteins.
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