Fernandez D, Spudich G M, Zhou X R, Christie P J
Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, 77030, USA.
J Bacteriol. 1996 Jun;178(11):3168-76. doi: 10.1128/jb.178.11.3168-3176.1996.
The Agrobacterium tumefaciens virB7 gene product is a lipoprotein whose function is required for the transmission of oncogenic T-DNA to susceptible plant cells. Three lines of study provided evidence that VirB7 interacts with and stabilizes other VirB proteins during the assembly of the putative T-complex transport apparatus. First, a precise deletion of virB7 from the pTiA6NC plasmid of wild-type strain A348 was correlated with significant reductions in the steady-state levels of several VirB proteins, including VirB4, VirB9, VirB10, and VirB11; trans expression of virB7 in the delta virB7 mutant partially restored the levels of these proteins, and trans coexpression of virB7 and virB8 fully restored the levels of these proteins to wild-type levels. Second, modulation of VirB7 levels resulted in corresponding changes in the levels of other VirB proteins in the following cell types: (i) a delta virB7 mutant expressing virB7 and virB8 from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Plac and other virB genes from acetosyringone (AS)-inducible PvirB; (ii) a delta virB operon mutant expressing virB7 and virB8 from Plac and virB9, virB10, and virB11 from PvirB; and (iii) a delta virB operon mutant expressing virB7 from IPTG-inducible Pklac and virB9 from an AS-inducible PvirB. Third, the synthesis of a VirB7::PhoA fusion protein in strain A348 was correlated with a significant reduction in the steady-state levels of VirB4, VirB5, and VirB7 through VirB11; these cells also exhibited a severely attenuated virulence phenotype, indicating that synthesis of the fusion protein perturbs the assembly of VirB proteins into a stabilized protein complex required for T-complex transport. Extracts of AS-induced cells electrophoresed under nonreducing conditions possessed undetectable levels of the 32-kDa VirB9 and 4.5-kDa VirB7 monomers and instead possessed a 36-kDa complex that cross-reacted with both VirB7 and VirB9 antisera and accumulated as a function of virB7 expression. Our results are consistent with a model in which VirB7 stabilizes VirB9 by formation of a covalent intermolecular cross-link; in turn, the VirB7-VirB9 heterodimer promotes the assembly of a functional T-complex transport machinery.
根癌土壤杆菌的virB7基因产物是一种脂蛋白，其功能是致癌性T-DNA传递至易感植物细胞所必需的。三项研究表明，在假定的T复合物转运装置组装过程中，VirB7与其他VirB蛋白相互作用并使其稳定。首先，从野生型菌株A348的pTiA6NC质粒中精确缺失virB7，与几种VirB蛋白（包括VirB4、VirB9、VirB10和VirB11）的稳态水平显著降低相关；在delta virB7突变体中反式表达virB7可部分恢复这些蛋白的水平，而virB7和virB8的反式共表达可将这些蛋白的水平完全恢复至野生型水平。其次，在以下细胞类型中，VirB7水平的调节导致其他VirB蛋白水平发生相应变化：(i) 一个delta virB7突变体，其从异丙基-β-D-硫代半乳糖苷（IPTG）诱导的Plac表达virB7和virB8，从乙酰丁香酮（AS）诱导的PvirB表达其他virB基因；(ii) 一个delta virB操纵子突变体，其从Plac表达virB7和virB8，从PvirB表达virB9、virB10和virB11；(iii) 一个delta virB操纵子突变体，其从IPTG诱导的Pklac表达virB7，从AS诱导的PvirB表达virB9。第三，在菌株A348中合成VirB7::PhoA融合蛋白，与VirB4、VirB5以及VirB7至VirB11的稳态水平显著降低相关；这些细胞还表现出严重减弱的毒力表型，表明融合蛋白的合成扰乱了VirB蛋白组装成T复合物转运所需的稳定蛋白复合物。在非还原条件下电泳分析AS诱导细胞的提取物，未检测到32 kDa的VirB9和4.5 kDa的VirB7单体，而是检测到一种36 kDa的复合物，该复合物与VirB7和VirB9抗血清均发生交叉反应，并随virB7表达而积累。我们的结果与一个模型一致，即VirB7通过形成共价分子间交联来稳定VirB9；反过来，VirB7-VirB9异二聚体促进功能性T复合物转运机制的组装。
