An Agrobacterium VirB10 Mutation Conferring a Type IV Secretion System Gating Defect
An Agrobacterium VirB10 Mutation Conferring a Type IV Secretion System Gating Defect
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DOI：
10.1128/jb.00038-11
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发表时间：
2011-05-01
影响因子：
3.2
通讯作者：
Christie, Peter J.
中科院分区：
文献类型：
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作者：
Banta, Lois M.;Kerr, Jennifer E.;Christie, Peter J.
在VIRB/VirD4 IV型分泌系统(T4SS)的生物发生过程中，农杆菌VirB7、VirB9和VirB10形成了一个“核心复合体”。VirB10跨越细胞膜，响应VIRB/D4 ATPase对ATP能量消耗的感应，经历DNA跨外膜(OM)转移所需的构象变化。在这里，我们测试了一个模型，在该模型中，VirB10通过筛选突变来调节底物通道，这些突变允许独立于靶细胞接触而将VirE2分泌底物不受调节地释放到细胞表面。一个突变，G272R，使VirE2释放，也使VirB10构象对细胞ATP耗尽不敏感。值得注意的是，G272R不影响底物向靶细胞的转移(Tra(+))，但确实阻断了菌毛的产生(Pil(+))。G272R突变株对万古霉素和十二烷基硫酸钠的敏感性增强，但不能非特异性地释放周质蛋白或截短其分泌信号的病毒E2。G272在VirB10同源物中高度保守，包括pKM101 Traf，在Traf的X射线结构中，对应的Gly残基位于一个称为天线投影(AP)的α螺旋结构域附近，这与OM孔的形成有关。AP部分缺失突变(Delta AP)也赋予Tra(+)Pil(-)表型；然而，该突变不允许VirE2表面暴露，而是允许毛细蛋白单体或短低聚物释放到环境中。我们认为(I)G272R扰乱了核心室中调节底物通过OM的门控机制，(Ii)G272R和Delta AP突变在菌毛生物发生途径的不同步骤阻止菌毛的产生。
Agrobacterium VirB7, VirB9, and VirB10 form a "core complex" during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra(+)) but did block pilus production (Pil(+)). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an alpha-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (Delta AP) also confers a Tra(+) Pil(-) phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and Delta AP mutations block pilus production at distinct steps of the pilus biogenesis pathway.