Dufour A, Voelker U, Voelker A, Haldenwang W G
Department of Microbiology, University of Texas Health Science Center at San Antonio, Texas 78284-7758, USA.
J Bacteriol. 1996 Jul;178(13):3701-9 sigma. doi: 10.1128/jb.178.13.3701-9sigma.1996.
sigma B is a secondary sigma factor that controls the general stress response in Bacillus subtilis. sigma B-dependent genes are activated when sigma B is released from an inhibitory complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase. Two separate pathways, responding either to a drop in intracellular ATP levels or to environmental stress (e.g., heat, ethanol, or salt), cause the release of sigma B from RsbW. rsbR, rsbS, rsbT, and rsbU are four genes now recognized as the upstream half of an operon that includes sigB (sigma B) and its principal regulators. Using reporter gene assays, we find that none of these four genes are essential for stationary-phase (i.e., ATP-dependent) activation of sigma B, but rsbU and one or more of the genes contained within an rsbR,S,T deletion are needed for stress induction of sigma B. In other experiments, Western blot (immunoblot) analyses showed that the levels of RsbR, RsbS, Rsb, and RsbU, unlike those of the sigB operon's four downstream gene products (RsbV, RsbW, RsbX and sigma B), are not elevated during sigma B activation. Gel filtration and immunoprecipitation studies did not reveal the formation of complexes between any of the four upstream sigB operon products and the products of the downstream half of the operon. Much of the detectable RsbR, RsbS, RsbT, and RsbU did, however, fractionate as a large-molecular-mass (approximately 600-kDa) aggregate which was excluded from our gel filtration matrix. The downstream sigB operon products were not present in this excluded material. The unaggregated RsbR, RsbS, and RsbU, which were retarded by the gel matrix, elated from the column earlier than expected from their molecular weights. The RsbR and RsbS fractionation profile was consistent with homodimers (60 and 30 kDa, respectively), while the RsbU appeared larger, suggesting a protein complex of approximately 90 to 100 kDa.
σB是一种次要的σ因子，它控制枯草芽孢杆菌中的一般应激反应。当σB从与抗σB蛋白（RsbW）的抑制复合物中释放出来并自由与RNA聚合酶结合时，依赖σB的基因就会被激活。两条独立的途径，要么对细胞内ATP水平的下降做出反应，要么对环境应激（如热、乙醇或盐）做出反应，导致σB从RsbW中释放出来。rsbR、rsbS、rsbT和rsbU是四个基因，现在被认为是一个操纵子上游的一半，该操纵子包括sigB（σB）及其主要调节因子。使用报告基因分析，我们发现这四个基因中没有一个对于σB的稳定期（即依赖ATP）激活是必需的，但rsbU和rsbR、S、T缺失中包含的一个或多个基因是σB应激诱导所必需的。在其他实验中，蛋白质印迹（免疫印迹）分析表明，与sigB操纵子的四个下游基因产物（RsbV、RsbW、RsbX和σB）不同，RsbR、RsbS、Rsb和RsbU的水平在σB激活过程中没有升高。凝胶过滤和免疫沉淀研究没有揭示四个上游sigB操纵子产物中的任何一个与操纵子下游一半的产物之间形成复合物。然而，大部分可检测到的RsbR、RsbS、RsbT和RsbU确实以大分子质量（约600 kDa）聚集体的形式分级分离，该聚集体被排除在我们的凝胶过滤基质之外。下游sigB操纵子产物不存在于这种被排除的物质中。未聚集的RsbR、RsbS和RsbU被凝胶基质阻滞，比根据其分子量预期的时间更早地从柱中洗脱出来。RsbR和RsbS的分级分离图谱与同二聚体（分别为60和30 kDa）一致，而RsbU看起来更大，表明是一种约90至100 kDa的蛋白质复合物。
