Characterization of the structure, dynamics, function and interactions of components from the Type IV secretion system of Xanthomonas citri by solution nuclear magnetic resonance

Abstract

Bacteria use specialized secretion systems to translocate substrates to the environment or to other cells. Secretion systems are supramolecular complexes located at the bacterial cell wall. The Type IV Secretion System (T4SS) is involved in translocation of virulence factors, bacterial conjugation and uptake or release of DNA. Xanthomonas citri pv. citri (Xac) is a gram-negative bacterium that infects citrus plants causing a disease called "citrus canker". Xac has two T4SSs, one encoded by the chromosomal DNA and another encoded by the mega plasmid pXAC64. Xac's chromosomal T4SS is involved in killing gram-negative bacteria through the translocation of toxins, creating in this way an adaptive advantage with respect to other bacteria that populate the same environment. While a large amount of structural information about the conjugative T4SSs was obtained in the recent years, little is known about T4SSs that perform other functions. The T4SS is generally composed by 12 proteins, VirB1 to VirB11 and VirD4. Single particle cryo electron microscopy and X-ray crystallography data showed that the T4SS channel corresponds to a ring-shaped supramolecular complex, formed by 14 repetitions of the VirB7-VirB9-VirB10 heterotrimer. Our group has been studying the structure of different components of Xac's chromosomally encoded T4SS. We solved the three-dimensional structure of Xac-VirB7 and showed that, in contrast to the canonical VirB7, it contains a C-terminal globular domain (Souza et al., 2011). The main goal of the present proposal is to describe the solution NMR structure of a complex formed by the C-terminal domain of Xac-VirB9 (Xac-VirB9CT) and a peptide derived from the N-terminal portion of Xac-VirB7 (Xac-VirB7NT). In vivo bacterial competition assays will be performed in order to assess the importance of the interaction between VirB7 and VirB9 for the functioning of the T4SS. Additionally, we will characterize Xac-VirB10 by solution NMR. The study of the N-terminal domain of VirB10 is of particular interest because it makes part of the inner layer of the T4SS's channel, about which there is not high-resolution structural information to date. We expect that the successful completion of this project will allow for a better understanding of Xac's T4SS architecture and functioning. (AU)