Gaidenko Tatiana A, Bie Xiaomei, Baldwin Enoch P, Price Chester W
Departments of Microbiology, University of California, Davis, California, USA.
J Bacteriol. 2012 Aug;194(15):3913-21. doi: 10.1128/JB.00583-12. Epub 2012 May 18.
The stressosome is a 1.8-MDa cytoplasmic complex that conveys environmental signals to the σ(B) stress factor of Bacillus subtilis. A functionally irreducible complex contains multiple copies of three proteins: the RsbRA coantagonist, RsbS antagonist, and RsbT serine-threonine kinase. Homologues of these proteins are coencoded in different genome contexts in diverse bacteria, forming a versatile sensing and transmission module called RST after its common constituents. However, the signaling pathway within the stressosome itself is not well defined. The N-terminal, nonheme globin domains of RsbRA project from the stressosome and are presumed to channel sensory input to the C-terminal STAS domains that form the complex core. A conserved, 13-residue α-helical linker connects these domains. We probed the in vivo role of the linker using alanine scanning mutagenesis, assaying stressosome output in B. subtilis via a σ(B)-dependent reporter fusion. Substitutions at four conserved residues increased output 4- to 30-fold in unstressed cells, whereas substitutions at four nonconserved residues significantly decreased output. The periodicity of these effects supports a model in which RsbRA functions as a dimer in vivo, with the linkers forming parallel paired helices via a conserved interface. The periodicity further suggests that the opposite, nonconserved faces make additional contacts important for efficient stressosome operation. These results establish that the linker influences stressosome output under steady-state conditions. However, the stress response phenotypes of representative linker substitutions provide less support for the notion that the N-terminal globin domain senses acute environmental challenge and transmits this information via the linker helix.
应激小体是一种1.8兆道尔顿的细胞质复合物，可将环境信号传递给枯草芽孢杆菌的σ(B)应激因子。一个功能上不可简化的复合物包含三种蛋白质的多个拷贝：RsbRA拮抗剂、RsbS拮抗剂和RsbT丝氨酸-苏氨酸激酶。这些蛋白质的同源物在不同细菌的不同基因组环境中共同编码，根据其共同成分形成一个通用的传感和传递模块，称为RST。然而，应激小体本身的信号通路尚不清楚。RsbRA的N端非血红素球蛋白结构域从应激小体中伸出，推测可将感觉输入传递到形成复合物核心的C端STAS结构域。一个保守的13个残基的α-螺旋连接子连接这些结构域。我们使用丙氨酸扫描诱变探究了连接子在体内的作用，通过一个依赖σ(B)的报告基因融合来检测枯草芽孢杆菌中的应激小体输出。在四个保守残基处的替换在未受应激的细胞中使输出增加了4至30倍，而在四个非保守残基处的替换显著降低了输出。这些效应的周期性支持了一个模型，即RsbRA在体内作为二聚体发挥作用，连接子通过一个保守界面形成平行配对的螺旋。这种周期性进一步表明，相对的非保守面进行的额外接触对于应激小体的有效运作很重要。这些结果表明，连接子在稳态条件下影响应激小体的输出。然而，代表性连接子替换的应激反应表型对N端球蛋白结构域感知急性环境挑战并通过连接子螺旋传递该信息这一观点的支持较少。
