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首页> 外文期刊>Journal of bacteriology >Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii
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Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii

机译：探索配体结合对潘氏泛酸亚种的酰基-高丝氨酸内酯阻碍的转录因子EsaR的影响。斯图瓦蒂

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摘要

The quorum-sensing regulator EsaR from Pantoea stewartii subsp. stewartii is a LuxR homologue that is inactivated by acyl-homoserine lactone (AHL). In the corn pathogen P. stewartii, production of exopolysaccharide (EPS) is repressed by EsaR at low cell densities. However, at high cell densities when high concentrations of its cognate AHL signal are present, EsaR is inactivated and derepression of EPS production occurs. Thus, EsaR responds to AHL in a manner opposite to that of most LuxR family members. Depending on the position of its binding site within target promoters, EsaR serves as either a repressor or activator in the absence rather than in the presence of its AHL ligand. The effect of AHL on LuxR homologues has been difficult to study in vitro because AHL is required for purification and stability. EsaR, however, can be purified without AHL enabling an in vitro analysis of the response of the protein to ligand. Western immunoblots and pulse-chase experiments demonstrated that EsaR is stable in vivo in the absence or presence of AHL. Limited in vitro proteolytic digestions of a biologically active His-MBP tagged version of EsaR highlighted intradomain and interdomain conformational changes that occur in the protein in response to AHL. Gel filtration chromatography of the full-length fusion protein and cross-linking of the N-terminal domain both suggest that this conformational change does not impact the multimeric state of the protein. These findings provide greater insight into the diverse mechanisms for AHL responsiveness found within the LuxR family.
机译：Pantoea stewartii亚种的群体感应调节剂EsaR。 stewartii是由酰基高丝氨酸内酯（AHL）灭活的LuxR同源物。在玉米病原体P. stewartii中，EsaR在低细胞密度下抑制胞外多糖（EPS）的产生。但是，在高细胞密度下，当存在高浓度的其同源AHL信号时，EsaR会失活，并且会降低EPS的产生。因此，EsaR以与大多数LuxR家族成员相反的方式响应AHL。根据其结合位点在靶启动子中的位置，EsaR在不存在而不是在其AHL配体存在的情况下充当阻遏物或激活物。 AHL对LuxR同源物的影响很难在体外进行研究，因为AHL是纯化和稳定性所必需的。然而，可以在没有AHL的情况下纯化EsaR，从而能够对蛋白质对配体的反应进行体外分析。西方的免疫印迹和脉冲追踪实验表明，在不存在或存在AHL的情况下，EsaR在体内是稳定的。 EsaR的具有生物活性的His-MBP标签版本的有限的体外蛋白水解消化突出了响应AHL的蛋白质中发生的域内和域间构象变化。全长融合蛋白的凝胶过滤色谱法和N末端结构域的交联都表明这种构象变化不会影响该蛋白的多聚体状态。这些发现为LuxR家族中发现的AHL应答的多种机制提供了更深入的了解。

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