Associations between Bacillus subtilis sigmaB regulators in cell extracts.
Associations between Bacillus subtilis sigmaB regulators in cell extracts.
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DOI：
10.1099/mic.0.27421-0
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发表时间：
2004-12
期刊：
影响因子：
1.5
通讯作者：
Shrin Kuo;Shuyu Zhang;Robyn L Woodbury;W. Haldenwang
中科院分区：
文献类型：
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作者：
Shrin Kuo;Shuyu Zhang;Robyn L Woodbury;W. Haldenwang
枯草芽孢杆菌的一般应激调节是由 sigma(B) 转录因子的激活诱导的。 sigma(B) 的激活是其正调节因子 RsbV 被响应身体或营养应激的两种磷酸酶之一去磷酸化的结果。物理应激磷酸酶 (RsbU) 需要第二种蛋白质 (RsbT) 才能发挥作用。人们认为，压力会启动一个过程，触发由两种蛋白质 RsbR（和/或其旁系同源物）和 RsbS 的多个拷贝组成的大型抑制复合物释放 RsbT。驱动 RsbT 释放的应激信号尚不清楚，但在缺乏核糖体蛋白 L11 或核糖体相关蛋白 Obg 的枯草芽孢杆菌中，它无法发育。在枯草芽孢杆菌粗提物的凝胶过滤层析过程中，RsbR、RsbS、RsbT、Obg 和核糖体以常见的高分子质量级分洗脱。本文报告了通过检查野生型和突变型枯草芽孢杆菌以及工程化表达 RsbR、RsbS 和 RsbT 的大肠杆菌提取物中这些蛋白质之间的关联来研究这种共洗脱的基础。在枯草芽孢杆菌和大肠杆菌的提取物中均检测到与核糖体不同的大 RsbR/RsbS 复合物。在大肠杆菌中，当 RsbR 不存在时，高分子量形式的 RsbS 丰度较低，但在枯草芽孢杆菌中，只有当提取物中同时缺少 RsbR 及其主要旁系同源物时，这种形式的丰度才会较低。这一发现与以下观点一致：枯草芽孢杆菌中存在但大肠杆菌中不存在的 RsbR 旁系同源物可以替代此类复合物中的 RsbR。在所检查的任何提取物中，RsbT 均未与 RsbR/RsbS 结合，包括从具有 RsbS 变体 (RsbS59SA) 的枯草芽孢杆菌菌株制备的提取物，据信该变体与 RsbT 持续结合。人们发现 RsbT 的高分子质量形式对 Triton 敏感，并且其形成独立于任何其他枯草芽孢杆菌蛋白。这些可能代表 RsbT 聚集体。数据表明，核糖体/Obg 对 sigma(B) 激活的贡献并不涉及这些蛋白质和 Rsb 复合物之间稳定缔合的形成。此外，RsbT 与 RsbS/RsbR 的结合似乎比之前分析的形成抑制复合物的 Rsb 蛋白之间的结合更不稳定。这以及 RsbT 明显的聚集倾向表明，RsbT 具有内在的不稳定性，这可能在其调节中发挥作用。
The general stress regulon of Bacillus subtilis is induced by the activation of the sigma(B) transcription factor. Activation of sigma(B) occurs as a consequence of the dephosphorylation of its positive regulator RsbV by one of two phosphatases that respond to either physical or nutritional stress. The physical stress phosphatase (RsbU) requires a second protein (RsbT) for activity. Stress is thought to initiate a process that triggers the release of RsbT from a large inhibitory complex composed of multiple copies of two protein species, RsbR (and/or its paralogues) and RsbS. The stress-derived signal driving RsbT release is unknown, but it fails to develop in B. subtilis lacking either ribosome protein L11 or the ribosome-associated protein Obg. RsbR, RsbS, RsbT, Obg and ribosomes elute in common high-molecular-mass fractions during gel-filtration chromatography of crude B. subtilis extracts. This paper reports the investigation of the basis of this coelution by the examining of associations between these proteins in extracts prepared from wild-type and mutant B. subtilis, and Escherichia coli engineered to express RsbR, RsbS and RsbT. Large RsbR/RsbS complexes, distinct from ribosomes, were detected in extracts of both B. subtilis and E. coli. In E. coli, high-molecular-mass forms of RsbS were less abundant when RsbR was absent, but in B. subtilis, only when both RsbR and its principal paralogues were missing from the extract was this form less abundant. This finding is consistent with the notion that the RsbR paralogues, present in B. subtilis but not E. coli, can substitute for RsbR in such complexes. RsbT was not bound to RsbR/RsbS in any extract that was examined, including one prepared from a B. subtilis strain with an RsbS variant (RsbS59SA) that is believed to continuously associate with RsbT. The high-molecular-mass forms of RsbT were found to be Triton-sensitive and independent of any other B. subtilis protein for their formation. These probably represent RsbT aggregates. The data suggest that the contribution of ribosomes/Obg to sigma(B) activation does not involve formation of a stable association between these proteins and the Rsb complex. In addition, the binding of RsbT to RsbS/RsbR appears to be more labile than the binding between the previously analysed Rsb proteins which form inhibitory complexes. This, and the apparent proclivity of RsbT to aggregate, suggests an inherent instability in RsbT which may play a role in its regulation.