Doty S L, Yu M C, Lundin J I, Heath J D, Nester E W
Department of Microbiology, University of Washington, Seattle 98195, USA.
J Bacteriol. 1996 Feb;178(4):961-70. doi: 10.1128/jb.178.4.961-970.1996.
The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.
跨膜传感器蛋白VirA在感受到高浓度乙酰丁香酮（AS）时会激活VirG。为了响应低水平的AS，VirA需要周质糖结合蛋白ChvE以及从植物伤口部位释放的单糖。为了更好地理解VirA如何感知这些诱导物，对C58 virA基因进行了随机诱变，并分离出14个在vir基因诱导方面存在缺陷且含有定位到VirA输入结构域突变的突变体。6个突变体在周质结构域三个广泛分离的区域存在单个错义突变。8个突变体在两亲性螺旋、TM1或TM2中或其附近存在突变。当将周质结构域中的4个突变引入相应的A6 virA序列时，会导致vir基因对葡萄糖的反应出现特定缺陷。这表明周质结构域的大部分对于与ChvE的相互作用或对其的反应是必需的。从周质结构域之外的区域选取3个突变，分别来自每个跨膜结构域和一个两亲性螺旋，将其构建到A6 virA中。这些突变体在vir基因对AS的反应方面存在缺陷。这些突变不影响VirA的稳定性或拓扑结构，也不阻止二聚化；因此，它们可能会干扰对AS的检测或将信号传递到激酶结构域。对C58 chvE突变体的表征表明，与A6 VirA不同，C58 VirA激活vir基因需要ChvE。
