Construction of transposon Tn3phoA:: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins
Construction of transposon Tn3phoA:: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins
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DOI：
10.1046/j.1365-2958.1998.00688.x
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发表时间：
1998-01-01
影响因子：
3.6
通讯作者：
Xie, YH
中科院分区：
文献类型：
--
作者：
Das, A;Xie, YH
蛋白质与大肠杆菌碱性磷酸酶的融合被广泛用于膜蛋白拓扑结构的分析。为了研究农杆菌T-DNA转移蛋白的拓扑结构，我们构建了转座子Tn 3 phoA。转座子以高频率移动到质粒中，转座后稳定，可产生phoA翻译融合物，并可直接用于分析目的细菌中的蛋白质拓扑结构。为了研究DNA转移蛋白，在我们的实验条件下，通过化学诱变构建phoA缺陷的农杆菌菌株。将含有virB和virD 4的质粒用作诱变的靶。28个独特的phoA阳性克隆，映射到8个virB基因分离。VirB 1、VirB 5、VirB 7、VirB 9和VirB 10中的多个插入表明这些蛋白主要面向周质。VirB 2、VirB 6和VirB 8中的插入允许鉴定它们的周质结构域。在VirB 3、VirB 4和VirB 11中未发现插入。这些蛋白质缺乏或具有短的周质结构域。也没有映射到VirD 4的插入。为了研究VirD 4拓扑结构，构建了靶向phoA融合体和随机lacZ融合体。融合蛋白的分析表明，VirD 4包含一个单一的周质结构域附近的N-末端，和大部分的蛋白位于细胞质中。提出了一个T-DNA转运孔的假设模型。
Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane proteins. To study the topology of the Agrobacterium T-DNA transfer proteins, we constructed a transposon, Tn3phoA. The transposon mobilizes into plasmids at a high frequency, is stable after transposition, can produce phoA translational fusions and can be used for the analysis of protein topology directly in the bacterium of interest. For studies on the DNA transfer proteins, an Agrobacterium strain deficient in phoA under our experimental conditions was constructed by chemical mutagenesis. A plasmid containing virB and virD4 was used as a target for mutagenesis. Twenty-eight unique phoA-positive clones that mapped to eight virB genes were isolated. Multiple insertions throughout VirB1, VirB5, VirB7, VirB9 and VirB10 indicated that these proteins primarily face the periplasm. Insertions in VirB2, VirB6 and VirB8 allowed the identification of their periplasmic domains. No insertions were found in VirB3, VirB4 and VirB11. These proteins either lack or have a short periplasmic domain. No insertions mapped to VirD4 either. To study VirD4 topology, targeted phoA fusions and random lacZ fusions were constructed. Analysis of the fusion proteins indicated that VirD4 contains a single periplasmic domain near the N-terminus, and most of the protein lies in the cytoplasm. A hypothetical model for the T-DNA transport pore is presented.