PilM/N/O/P Proteins Form an Inner Membrane Complex That Affects the Stability of the Pseudomonas aeruginosa Type IV Pilus Secretin
PilM/N/O/P Proteins Form an Inner Membrane Complex That Affects the Stability of the Pseudomonas aeruginosa Type IV Pilus Secretin
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DOI：
10.1016/j.jmb.2009.09.034
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发表时间：
2009-11-20
影响因子：
5.6
通讯作者：
Burrows, L. L.
中科院分区：
文献类型：
--
作者：
Ayers, M.;Sampaleanu, L. M.;Burrows, L. L.
高度保守的PIL/M/N/O/P/Q基因簇是IV型PILI型细胞表面表达和相关抽搐运动所需的核心基因组之一。除了PILQ亚基的多聚体外膜秘书素外，该基因簇编码的产品的特定功能的表征很差。相关细菌型H分泌系统中的直系同源蛋白已被证明可以相互作用以形成蛋白质分泌所需的内膜复合物。在这项研究中，我们提供的证据表明PILM/N/O/P蛋白会形成功能等效的IVA PILUS复合物。我们使用铜绿假单胞菌作为模型生物，我们发现所有四种蛋白质（包括名义上的细胞质PILM）共定位于内膜。通过Western印迹分析进行的稳定性研究表明，一个成分的损失对推定复合物的其他成员的水平产生负面影响。此外，互补研究表明，组件的化学计量对于正确形成体内稳定复合物很重要。我们提供的证据表明，由于PILQ稳定性在不存在的情况下对PILQ稳定性产生了负面影响，因此需要完整的内膜复合物才能最佳地形成IVA Pilus型系统的外膜复合物。最后，我们表明，在没有PILIN亚基的情况下，内膜复合物的膜结合成分的水平受到PILR/S两个组分系统的负调节，这表明PILR/S在感知Pilriation中的作用单元的状态。 （c）2009 Elsevier Ltd.保留所有权利。
The highly conserved pil/M/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. With the exception of the outer membrane secretin, a multimer of PilQ subunits, the specific functions of the products encoded by this gene cluster are poorly characterized. Orthologous proteins in the related bacterial type H secretion system have been shown to interact to form an inner membrane complex required for protein secretion. In this study, we provide evidence that the PilM/N/O/P proteins form a functionally equivalent type IVa pilus complex. Using Pseudomonas aeruginosa as model organism, we found that all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane. Stability studies via Western blot analyses revealed that loss of one component has a negative impact on the levels of other members of the putative complex. Furthermore, complementation studies revealed that the stoichiometry of the components is important for the correct formation of a stable complex in vivo. We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa, as PilQ stability is negatively affected in its absence. Finally, we show that, in the absence of the pilin subunit, the levels of membrane-bound components of the inner membrane complex are negatively regulated by the PilR/S two-component system, suggesting a role for PilR/S in sensing the piliation status of the cell. (C) 2009 Elsevier Ltd. All rights reserved.