GLU-255 OUTSIDE THE PREDICTED CHVE BINDING-SITE IN VIRA IS CRUCIAL FOR SUGAR ENHANCEMENT OF ACETOSYRINGONE PERCEPTION BY AGROBACTERIUM-TUMEFACIENS
GLU-255 OUTSIDE THE PREDICTED CHVE BINDING-SITE IN VIRA IS CRUCIAL FOR SUGAR ENHANCEMENT OF ACETOSYRINGONE PERCEPTION BY AGROBACTERIUM-TUMEFACIENS
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DOI：
10.1128/jb.176.11.3242-3249.1994
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发表时间：
1994-06-01
影响因子：
3.2
通讯作者：
BINNS, AN
中科院分区：
文献类型：
--
作者：
BANTA, LM;JOERGER, RD;BINNS, AN
根癌农杆菌病毒调节剂的转录激活受到酚类物质（例如乙酰丁香酮（AS））、某些单糖和受伤植物细胞产生的酸性条件的调节。跨膜蛋白 VirA 充当环境传感器，在感知这些刺激时介导信号转导。尽管 VirA 的周质结构域并不是 AS 依赖性 vir 基因诱导所必需的，但它是与周质糖结合蛋白 ChvE 相互作用所必需的，从而导致糖诱导的酚类敏感性增强。在本报告中，我们证明周质结构域内但预测的 ChvE 结合区域外的突变可以极大地改变 VirA 对 AS 的敏感性。使用定点诱变，我们描述了三个单独的氨基酸在糖依赖性 AS 敏感性中的作用，并将诱导表型与表达 VirA 突变版本的菌株的致瘤能力相关联。用亮氨酸替代 Glu-255 会消除糖的增强，而用天冬氨酸替代则会产生野生型表型。该残基位于预测的 ChvE 结合位点之外，因此识别出 VirA 周质结构域的一个新区域，对于增强碳水化合物对 vir 基因的诱导至关重要。在没有诱导糖的情况下，野生型 VirA 蛋白似乎会受到某种形式的抑制，从而抑制转录激活的最大水平；周质区域内的缺失缓解了这种抑制。
Transcriptional activation of the Agrobacterium tumefaciens vir regulon is regulated by phenolics such as acetosyringone (AS), certain monosaccharides, and acidic conditions produced by wounded plant cells. The transmembrane protein VirA acts as an environmental sensor, mediating signal transduction upon perception of these stimuli. Although the periplasmic domain of VirA is not absolutely required for AS-dependent vir gene induction, it is needed for interactions with the periplasmic sugar-binding protein ChvE that result in sugar-induced enhancement of phenolic sensitivity. In this report, we demonstrate that mutations within the periplasmic domain but outside the predicted ChvE binding region can drastically alter the sensitivity of VirA to AS. Using site-directed mutagenesis, we have characterized the roles of three individual amino acids in sugar-dependent AS sensitivity and have correlated the induction phenotype with the tumorigenic capacity of strains expressing mutant versions of VirA. Substitution of leucine for Glu-255 abolishes sugar enhancement while replacement with aspartic acid results in a wild-type phenotype. This residue lies outside the predicted ChvE binding site and thus identifies a new region of the VirA periplasmic domain crucial for the enhancement of vir gene induction by carbohydrates. In the absence of inducing sugar, wild-type VirA protein appears to be subject to some form of inhibition that suppresses the maximal level of transcriptional activation; deletions within the periplasmic region relieve this suppression.