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Molecular characterization of the type II secretion system of the phytopathogenic bacterium Dickeya dadantii : structural and functional studies of the interaction of OutC and OutD

Abstract : The type II secretion system (T2SS) is widely exploited by Gram-negative bacteria to secrete diverse virulence factors from the periplasm into the extra-cellular milieu. The phytopathogenic bacterium Dickeya dadanti (ex. Erwinia chrysanthemi) uses this system, named Out, to secrete several cell-wall degrading enzymes that cause soft-rot disease of many plants. The two core components of the Out system, the inner membrane protein OutC and the secretin OutD, which forms a secretion pore in the outer membrane, are involved in secretion specificity. The interaction between OutC and OutD could assure the structural and functional integrity of the secretion system by connecting the two membranes. To understand structure-function relationships between these two components and characterize their interaction sites, we applied an integrative approach involving in vivo cysteine scanning and disulfide cross-linking analysis, truncation analysis of OutC and OutD combined with in vitro GST pull-down, and structural analysis of these proteins and of their interactions by NMR. Our results indicate the presence of at least three interacting sites between the periplasmic regions of OutC and OutD and suggest a β-strand addition mechanism for these interactions. We demonstrated that one site of the HR domain of OutC can interact with two distinct sites of OutD suggesting an alternative mode of their interactions. The presence of exoproteins or/and the inner membrane components of the system OutE-L-M differently alters the affinity of the three OutC-OutD interacting sites. We suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machinery between different functional states. To study the mechanism of the targeting and assembly of the secretin OutD into the outer membrane, we exploited the interactions between OutD and two auxiliary proteins, i.e., the inner membrane protein OutB and the outer membrane lipoprotein OutS. We showed a direct interaction between the periplasmic domain of OutB and the N0 domain of OutD. Structure-function analysis of OutS-OutD complex shows that the pilotin OutS binds tightly to 18 residues close to the C-terminus of the secretin subunit causing this unstructured region to become helical on forming the complex. This work allows us to better understand the assembly and function mechanism of the type II secretion system.
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https://tel.archives-ouvertes.fr/tel-00790733
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  • HAL Id : tel-00790733, version 1

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Xiaohui Wang. Molecular characterization of the type II secretion system of the phytopathogenic bacterium Dickeya dadantii : structural and functional studies of the interaction of OutC and OutD. Agricultural sciences. INSA de Lyon, 2012. English. ⟨NNT : 2012ISAL0010⟩. ⟨tel-00790733⟩

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