Robust Stoichiometry of FliW-CsrA Governs Flagellin Homeostasis and Cytoplasmic Organization in Bacillus subtilis
Robust Stoichiometry of FliW-CsrA Governs Flagellin Homeostasis and Cytoplasmic Organization in Bacillus subtilis
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DOI：
10.1128/mbio.00533-19
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发表时间：
2019-05-01
期刊：
影响因子：
6.4
通讯作者：
Kearns, D. B.
中科院分区：
文献类型：
--
作者：
Oshiro, R. T.;Rajendren, S.;Kearns, D. B.
鞭毛蛋白(Hag)是枯草杆菌中含量最丰富的蛋白质之一。在这里，我们展示了每个鞭毛细丝由类似于12,000个HAG单体组装而成，并且有一个HAG细胞质池，限制在总数的5%。HAG被认为在翻译水平上受到FliW和同源二聚体RNA结合蛋白CSRA(CSRA(二聚体))伙伴转换机制的限制。我们进一步证明翻译抑制的机制是超敏的，因为HAG和FliW的比例为1：1，FliW和CSRA(二聚体)的抑制比例为1：1，CSRA(二聚体)和HAG的转录本的比例接近1：1。所有组分的等量将HAG输出的单分子检测耦合到补偿翻译，并导致细胞质HAG浓度在FliW水平附近振荡。我们发现，化学计量学是由遗传结构、翻译耦合和CSRA(二聚体)限制HAG转录积累的能力确保的。我们进一步表明，动态平衡可以防止HAG的过度聚集，否则可能会由于分子拥挤增加而导致细胞内结构的严重缺陷。我们注意到FliW-CSRA介导的结构动态平衡与一些毒素抗毒素系统的结构稳态有相似之处。在这里，我们证明了HAG-FliW-CSRA(二聚体)系统在近1：1：1的化学计量比下工作，并且该系统对扰动和对HAG细胞内浓度高度敏感。此外，限制细胞质HAG水平对于维持适当的细胞内结构是重要的，因为人工HAg的过度积累导致了普遍的空间缺陷和高频率的微细胞产生。HAG-FliW-CSRA系统在细菌系统发育的更深层次上是保守的，我们注意到HAG-FliW-CSRA“稳态模块”类似于毒素-抗毒素系统，以此类推，CSRA是“毒素”，FliW是“抗毒素”，而HAG是目标。
Flagellin (Hag) is one of the most abundant proteins in Bacillus subtilis. Here we show that each flagellar filament is assembled from similar to 12,000 Hag monomers and that there is a cytoplasmic pool of Hag that is restricted to 5% of the total. Hag is thought to be restricted at the level of translation by a partner-switching mechanism involving FliW and the homodimeric RNA-binding protein CsrA (CsrA(dimer)). We further show that the mechanism of translation inhibition is hypersensitive due to a 1:1 ratio of Hag to FliW, a 1:1 inhibitory ratio of FliW to CsrA(dimer), and a nearly 1:1 ratio of CsrA(dimer) to hag transcripts. Equimolarity of all components couples single-molecule detection of Hag export to compensatory translation and causes cytoplasmic Hag concentrations to oscillate around the level of FliW. We found that stoichiometry is ensured by genetic architecture, translational coupling, and the ability of CsrA(dimer) to restrict hag transcript accumulation. We further show that homeostasis prevents Hag hyperaccumulation that would otherwise cause severe defects in intracellular architecture, perhaps due to increased molecular crowding. We note that FliW-CsrA-mediated structural homeostasis has similarities to that seen with some toxinantitoxin systems.IMPORTANCE The intracellular concentration of flagellar filament protein Hag is restricted by the Hag-FliW-CsrA system in B. subtilis. Here we show that the Hag-FliW-CsrA(dimer) system functions at nearly 1:1:1 stoichiometry and that the system is both robust with respect to perturbation and hypersensitive to the Hag intracellular concentration. Moreover, restriction of cytoplasmic Hag levels is important for maintaining proper intracellular architecture, as artificial Hag hyperaccumulation led to generalized spatial defects and a high frequency of minicell production. The Hag-FliW-CsrA system is conserved in the deeper branches of bacterial phylogeny, and we note that the Hag-FliW-CsrA "homeostasis module" resembles a toxin-antitoxin system where, by analogy, CsrA is the "toxin," FliW is the "antitoxin," and Hag is the target.