Beijersbergen A, Smith S J, Hooykaas P J
Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, The Netherlands.
Plasmid. 1994 Sep;32(2):212-8. doi: 10.1006/plas.1994.1057.
Agrobacterium tumefaciens causes tumors on plants by transferring part of its Ti (tumor inducing) plasmid, the T-DNA or transferred DNA, to plant cells. The virB operon, the largest operon of the virulence (Vir) region located on the Ti plasmid, is necessary for tumor induction on all plant species. Previously, the complete nucleotide sequences of the virB operons of several Ti plasmids of A. tumefaciens were determined. The 11 predicted proteins mostly have signal sequences and/or hydrophobic domains. Hence, these proteins are thought to be located in or transported over the agrobacterial inner membrane. The VirB proteins are suggested to form a pore structure in the membrane through which the T-DNA-protein complex is transported. To obtain direct evidence for transport of these proteins over the inner membrane, we made fusions between genes of the virB operon and the gene for the enzyme alkaline phosphatase. Here we show the localization of several VirB proteins in the bacterial membrane and predict the topology of the membrane-localized VirB proteins. The finding of a fusion between the VirB7 protein and the enzyme alkaline phosphatase provides the first evidence for the expression of the small virB7 gene. The VirB2 protein shows similarity with the TraA propilin of the Escherichia coli F plasmid. Here we show that the predicted topology of the VirB2 protein in the inner membrane is identical to that of the TraA protein. Therefore, we hypothesize that the VirB2 protein is part of an agrobacterial pilus-like structure.
根癌土壤杆菌通过将其Ti（致瘤）质粒的一部分，即T-DNA或转移DNA，转移到植物细胞中而导致植物产生肿瘤。virB操纵子是位于Ti质粒上的毒性（Vir）区域中最大的操纵子，对于所有植物物种的肿瘤诱导都是必需的。此前，已确定了根癌土壤杆菌几种Ti质粒的virB操纵子的完整核苷酸序列。预测的11种蛋白质大多具有信号序列和/或疏水区。因此，这些蛋白质被认为位于农杆菌内膜中或在内膜上运输。有人提出VirB蛋白在膜中形成孔结构，T-DNA-蛋白质复合物通过该孔结构运输。为了获得这些蛋白质在内膜上运输的直接证据，我们构建了virB操纵子基因与碱性磷酸酶基因之间的融合体。在这里，我们展示了几种VirB蛋白在细菌膜中的定位，并预测了膜定位的VirB蛋白的拓扑结构。VirB7蛋白与碱性磷酸酶的融合体的发现为小virB7基因的表达提供了首个证据。VirB2蛋白与大肠杆菌F质粒的TraA前菌素相似。在这里，我们表明VirB2蛋白在内膜中的预测拓扑结构与TraA蛋白相同。因此，我们假设VirB2蛋白是农杆菌菌毛样结构的一部分。
