Bourg Gisèle, Sube Romain, O'Callaghan David, Patey Gilles
INSERM ESPRI 26, Université Montpellier 1 ERA 4204, Faculté de Médecine, Avenue Kennedy, CS 83021, 30908 Nîmes Cédex 2, France.
J Bacteriol. 2009 May;191(9):2985-92. doi: 10.1128/JB.01426-08. Epub 2009 Feb 27.
The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.
蛋白质VirB8在根癌土壤杆菌virB IV型分泌系统（T4SS）的组装和功能中起关键作用。根癌土壤杆菌和猪布鲁氏菌VirB8周质结构域的结构已被确定，定点诱变揭示了参与VirB8二聚化以及与VirB4和VirB10相互作用的氨基酸。我们之前已经表明，来自pSB102的VirB8同源物TraJ以及包含TraJ的细胞质和跨膜（TM）结构域与VirB8周质结构域的嵌合蛋白TraJB8，无法互补含有virB8基因框内缺失的猪布鲁氏菌突变体。这表明TraJ细胞质和TM结构域的存在可能会阻止VirB8在内膜中的二聚化或组装。通过细菌双杂交分析，我们发现VirB8、TraJ和嵌合体都能相互作用形成同二聚体和异二聚体。然而，TraJ的TM结构域的存在导致同二聚体和异二聚体中的相互作用更强。我们在野生型猪布鲁氏菌中表达了野生型和嵌合蛋白。携带TraJ TM结构域的蛋白质的存在具有显性负效应，导致毒力完全丧失。这表明T4SS是一种动态结构，强相互作用会阻碍正确组装和功能所需的空间灵活性。
