Department of Microbiology, One Shields Avenue, University of California, Davis, California 95616, USA.
J Bacteriol. 2011 Jul;193(14):3588-97. doi: 10.1128/JB.00060-11. Epub 2011 May 20.
The stressosome is a multiprotein, 1.8-MDa icosahedral complex that transmits diverse environmental signals to activate the general stress response of Bacillus subtilis. The way in which it senses these cues and the pathway of signal propagation within the stressosome itself are poorly understood. The stressosome core consists of four members of the RsbR coantagonist family together with the RsbS antagonist; its cryo-electron microscopy (cryoEM) image suggests that the N-terminal domains of the RsbR proteins form homodimers positioned to act as sensors on the stressosome surface. Here we probe the role of the N-terminal domain of the prototype coantagonist RsbRA by making structure-based amino acid substitutions in potential interaction surfaces. To unmask the phenotypes caused by single-copy rsbRA mutations, we constructed strains lacking the other three members of the RsbR coantagonist family and assayed system output using a reporter fusion. Effects of five individual alanine substitutions in the prominent dimer groove did not match predictions from an earlier in vitro assay, indicating that the in vivo assay was necessary to assess their influence on signaling. Additional substitutions expected to negatively affect domain dimerization had substantial impact, whereas those that sampled other prominent surface features had no consequence. Notably, even mutations resulting in significantly altered phenotypes raised the basal level of system output only in unstressed cells and had little effect on the magnitude of subsequent stress signaling. Our results provide evidence that the N-terminal domain of the RsbRA coantagonist affects stressosome function but offer no direct support for the hypothesis that it is a signal sensor.
应激体是一种多蛋白、1.8MDa 的二十面体复合物，可将多种环境信号传递到枯草芽孢杆菌中，激活一般应激反应。目前，人们对其感知这些信号的方式以及应激体内部信号传递途径知之甚少。应激体核心由 RsbR 共拮抗剂家族的四个成员和 RsbS 拮抗剂组成；其低温电子显微镜（cryoEM）图像表明，RsbR 蛋白的 N 端结构域形成同源二聚体，位于应激体表面充当传感器。在这里，我们通过在潜在相互作用表面上进行基于结构的氨基酸取代来探究原型共拮抗剂 RsbRA 的 N 端结构域的作用。为了揭示单拷贝 rsbRA 突变引起的表型，我们构建了缺乏 RsbR 共拮抗剂家族其他三个成员的菌株，并使用报告融合物检测系统输出。在突出的二聚体凹槽中进行的五个单独丙氨酸取代的效果与早期的体外测定结果不符，表明体内测定对于评估它们对信号传递的影响是必要的。预计会对结构域二聚化产生负面影响的其他取代具有重大影响，而那些采样其他突出表面特征的取代则没有影响。值得注意的是，即使是导致表型明显改变的突变，也只能在未受应激的细胞中提高系统输出的基础水平，而对随后的应激信号强度几乎没有影响。我们的研究结果提供了证据，表明 RsbRA 共拮抗剂的 N 端结构域影响应激体功能，但没有直接支持它是信号传感器的假设。
