Evidence that F-plasmid proteins TraV, TraK and TraB assemble. into an envelope-spanning structure in Escherichia coli
Evidence that F-plasmid proteins TraV, TraK and TraB assemble. into an envelope-spanning structure in Escherichia coli
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DOI：
10.1046/j.1365-2958.2001.02667.x
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发表时间：
2001-11-01
影响因子：
3.6
通讯作者：
Silverman, PM
中科院分区：
文献类型：
--
作者：
Harris, RL;Hombs, V;Silverman, PM
我们研究了F-质粒TraV外膜脂蛋白在F-菌毛组装中的作用。酵母双杂交分析与traV诱饵反复鉴定traK，这是预测编码周质蛋白，阳性猎物质粒。traK诱饵依次鉴定了traV和traB，后者被预测编码内膜蛋白。一种专门用来识别猎物的诱饵。几个额外的观察结果支持以下假设：TraV、TraK和TraB在大肠杆菌中形成复合物，该复合物跨越从外膜（TraV）通过周质（TraK）到内膜（TraB）的细胞包膜。首先，双杂交分析表明，TraV和TraB绑定到不同的TraK片段，如果需要TraK桥三元复合物。其次，用E. tra（+）细胞的外膜。与此相反，TraB与traV或traK突变体细胞的内膜分离，TraK出现在traV突变体的渗透压休克液中。这些结果与通过TraV脂蛋白锚定到外膜的TraV-TraK-TraB复合物一致。此外，在traK突变体细胞中，TraV未能积累到可检测的水平，TraB水平显著降低，这表明TraV和TraB必须与TraK相互作用，才能使任一蛋白质积累到其正常水平。TraK和TraV在traB 2 [Am]细胞中均有积累，但Western blot检测到TraB 2琥珀片段，序列分析表明该片段保留了酵母双杂交分析所提示的TraK结合结构域。我们建议，TraV是外膜的trans-envelope，Tra蛋白结构所需的F-菌毛的装配和可能的其他事件的共轭DNA转移的锚。
We have examined the role of the F-plasmid TraV outer membrane lipoprotein in the assembly of F-pili. Yeast two-hybrid analysis with a traV bait repeatedly identified traK, which is predicted to encode a periplasmic protein, among positive prey plasmids. A traK bait in turn identified traV and traB, which is predicted to encode an inner membrane protein. A traB bait exclusively identified traK preys. Several additional observations support the hypothesis that TraV, TraK and TraB form a complex in Escherichia coli that spans the cell envelope from the outer membrane (TraV) through the periplasm (TraK) to the inner membrane (TraB). First, two-hybrid analyses indicated that TraV and TraB bind to different TraK segments, as required if TraK bridges a ternary complex. Secondly, all three proteins fractionated with the E. coli outer membrane in tra(+) cells. In contrast, TraB fractionated with the inner membrane in traV or traK mutant cells, and TraK appeared in the osmotic shock fluid from the traV mutant. These results are consistent with a TraV-TraK-TraB complex anchored to the outer membrane via the TraV lipoprotein. Further, in traK mutant cells, TraV failed to accumulate to a detectable level, and the TraB level was significantly reduced, suggesting that TraV and TraB must interact with TraK for either protein to accumulate to its normal level. Both TraK and TraV accumulated in traB2[Am] cells; however, the TraB2 amber fragment could be detected by Western blot, and sequence analysis indicated that the fragment retained the TraK-binding domain suggested by yeast two-hybrid analysis. We propose that TraV is the outer membrane anchor for a trans-envelope, Tra protein structure required for the assembly of F-pili and possibly for other events of conjugal DNA transfer.