Department of Microbiology, University of Washington, Seattle, WA, USA.
mBio. 2013 May 7;4(3):e00242-13. doi: 10.1128/mBio.00242-13.
WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated.
Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.
WspR 是一种混合应答调节子 - 环二鸟苷酸二酯酶，它可被铜绿假单胞菌在表面生长时的 Wsp 信号转导复合物磷酸化。活性 WspR 产生环二鸟苷酸（c-di-GMP），继而刺激生物膜形成。在之前的工作中，我们发现当被磷酸化激活时，黄色荧光蛋白（YFP）标记的 WspR 形成簇，通过荧光显微镜可以在单个细胞中观察到。非磷酸化的 WspR 在细胞中弥散，不可见。因此，簇形成是 WspR 信号转导的一种测定方法。为了了解 WspR 如何以及为何形成亚细胞簇，我们分析了 WspR 的六个单一氨基酸变异体的簇形成和酶活性。一般来说，簇形成的增加与变异体的体内和体外双鸟苷酸环化酶活性的增加相关。此外，WspR 的比活性在体外强烈依赖于浓度，并且当用磷酸类似物铍氟化物处理 WspR 时，蛋白浓度对双鸟苷酸环化酶活性的影响被放大。簇形成似乎是磷酸化 WspR（WspR-P）的固有特性。这些结果支持了这样一种模型，即在表面刺激下，WspR-P 在体内形成亚细胞簇对于增强 WspR 的双鸟苷酸环化酶活性很重要。亚细胞簇的形成似乎是调节应答调节蛋白活性的另一种手段。
当被环境信号刺激时，细菌传感器蛋白通常会磷酸化同源应答调节蛋白。磷酸化的应答调节蛋白随后介导适当的适应性细胞反应。大约 6%的应答调节蛋白具有参与产生或降解环二鸟苷酸（c-di-GMP）的酶结构域，c-di-GMP 刺激细菌生物膜的形成。在这项工作中，我们检查了应答调节子 - 双鸟苷酸环化酶 WspR 的体内和体外行为。当响应与表面生长相关的信号而被磷酸化时，WspR 有形成寡聚体的趋势，在细胞中作为亚细胞簇可见。我们的结果表明，WspR-P 亚细胞簇的形成对于增强 WspR-P 的双鸟苷酸环化酶活性很重要，使其在 c-di-GMP 产生中更活跃。我们得出结论，寡聚体形成可视化作为亚细胞簇是调节应答调节子 - 双鸟苷酸环化酶活性的另一种机制。
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