Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens.
Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens.
复制标题
根癌农杆菌感觉蛋白 VirA 的膜拓扑和功能分析。
作者：
L. Melchers;T. Regensburg;R. Bourret;N. Sedee;R. Schilperoort;P. Hooykaas
根癌农杆菌的virA基因编码一种内膜，该内膜介导毒力基因响应植物信号分子的转录激活。我们在这里报告跨膜信号传导中VirA的N-末端、C-末端和周质结构域的功能分析。首先，我们通过对碱性磷酸酶活性的分析表明VirA具有跨膜拓扑结构，这是由几个virA‐phoA基因融合决定的。其次，我们在这里报告了几个virA‐tar嵌合基因的构建，其中virA的3′编码区是保守的，以研究跨膜信号传导，以及一组virA缺失突变的构建。携带这些突变基因的农杆菌的VIR诱导行为和肿瘤诱导能力的分析结果不支持VirA周质域的化学感受器功能的现有模型。我们证明，VirA的周质结构域可以被大肠杆菌化学感受蛋白焦油的相应区域取代，甚至完全从VirA中删除而不丧失功能。在这里，我们提出了一个模型的病毒，其中涉及的第二跨膜结构域的受体功能和细胞内信号传导功能的细胞质结构域的病毒。此外，我们表明，VirA在乙酰丁香酮介导的vir诱导中决定pH和温度的敏感性方面发挥作用，我们提出了VirA周质结构域在检测外部pH条件中的作用。
The virA gene of Agrobacterium tumefaciens encodes an inner membrane that mediates the transcriptional activation of virulence genes in response to plant signal molecules. We report here a functional analysis of the N‐terminal, C‐terminal and periplasmic domains of VirA in transmembrane signalling. First, we show that VirA has a transmembrane topology by analysis of the alkaline phosphatase activities, determined by several virA‐phoA gene fusions. Second, we report here the construction of several virA‐tar chimeric genes, in which the 3′‐coding region of virA is conserved to study transmembrane signalling, as well as the construction of a set of virA deletion mutations. Results of analyses of vir induction behaviour and tumour inducing abilities of agrobacteria carrying these mutant genes do not support existing models for the chemoreceptor function of the VirA periplasmic domain. We demonstrate that the periplasmic domain of VirA can be either replaced by a corresponding region of the E.coli chemosensory protein Tar or even totally deleted from VirA without a loss of function. Here, we present a model of VirA which involves a receptor function for the second membrane‐spanning domain and an intracellular signalling function for the cytoplasmic domain of VirA. In addition, we show that VirA plays a role in determining the sensitivity for pH and temperature in acetosyringone‐mediated vir induction, and we propose a role for the VirA periplasmic domain in detection of the external pH conditions.