Bacteria use different secretion systems for protein transportation into other bacteria, eukaryotic cells or for secretion to the extracellular medium. Among them, the type IV secretion system, T4SS, is perhaps the most versatile, being present in both Gram-positive and Gram-negative bacteria, being also capable of transporting protein-DNA complexes, in this way mediating horizontal gene transfer, an important mechanism for molecular evolution. T4SSs are also important virulence factors, used by some bacteria to release toxins into the host cell, such as Agrobacterium tumefaciens, Legionella pneumophila, Helicobacter pylori and Brucella suis. In recent years, our group has shown that Xanthomonas citri (and some other species of Xanthomonadaceae family, including the clinically relevant Stenotrophomonas spp) possess an atypical T4SS capable to kill other bacterial species through toxin secretion. The species Xanthomonas citri is of significant economic interest, since it is responsible for citrus canker, a disease affecting citrus characterized by lesions on the leaves and losses in productivity and fruit quality. The T4SS exhibits an outer membrane associated complex with 14-fold symmetry, composed by VirB7, VirB9 and VirB10, that connects by a flexible stalk to the inner membrane complex, composed by the N-terminal domain of VirB10, VirB3, VirB4, VirB6, VirB8, VirB11 and VirD4. In spite of our group success with the outer membrane complex, the inner membrane complex has so far proven to be a much more difficult target for structural studies, perhaps due to its expected dynamic nature and multiple modes of interaction of its components, all either integral or peripheral membrane proteins. The main goal of the present BEPE project is to express, purify and obtain structural insights regarding T4SS system inner membrane complex, and of its components individually. At the moment no high resolution data is available for inner membrane complexes of T4SS, although crystal structure have been determined for some individual components from T4SSs from other organisms. We expect that pursuing these lines of investigation will lead to a better understanding of general T4SS mechanisms as well as its alternative secretion functions. These studies also hold the potential to provide a basis for the development of new antibacterial drugs and bioremediation protocols.
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