Jonas R M, Peters H K, Haldenwang W G
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.
J Bacteriol. 1990 Aug;172(8):4178-86. doi: 10.1128/jb.172.8.4178-4186.1990.
sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is synthesized from an inactive precursor protein (P31). The structural gene (sigE) for P31 was reengineered by oligonucleotide-directed mutagenesis to encode sigma E directly. The sequence specifying the first amino acid of sigma E (GGC) was placed immediately downstream of the initiating codon (ATG) of P31. The resulting sigE allele (sigE delta 84) encodes a sigma E-like protein which differs from the "processed product" by a single Met residue at its amino terminus. B. subtilis strains which carried this allele were Spo- and contained no detectable sigma E. The sigE delta 84 allele generated a product in Escherichia coli which, by quantitative Western immunoblot analysis, was present at 10 to 20% of the level of product (P31) obtained from a wild-type allele. A sigma E-like product was also not detected in two B. subtilis strains with missense mutations in the sequence encoding the processed region of P31. These results suggest that sigma E is a highly labile protein that is stabilized during its synthesis by an element of the precursor sequence. A mutant allele (sigE delta 48) which made an active sigma E-like protein in B. subtilis was isolated. This gene specified a product in which five amino acids, not derived from the P31 processed region, were joined to P31 at a position eight amino acids upstream of the processing site. The sigE delta 48 product was not processed, but it activated the sigma E -dependent spoIID promoter in vivo. The sigE delta 48 product therefore lost both an essential target for processing and a region which inhibited sigma sigma E activity. Cells which carried sig E delta 48 were Spo-. The basis of the sigE delta 48-dependent defect in sporulation is unknown, but the sigma E delta 48 activity appeared to persist beyond the time in development (4 h after onset sporulation) when wild-type sigma E activity declines. Thus, it may interfere with the proper regulation of late sporulation genes.
σE是枯草芽孢杆菌的一种芽孢形成特异性σ因子，由无活性的前体蛋白（P31）合成。通过寡核苷酸定向诱变对P31的结构基因（sigE）进行重新设计，以直接编码σE。指定σE第一个氨基酸（GGC）的序列紧接在P31起始密码子（ATG）的下游。所得的sigE等位基因（sigEΔ84）编码一种类σE蛋白，其在氨基末端与“加工产物”相差一个甲硫氨酸残基。携带该等位基因的枯草芽孢杆菌菌株为芽孢形成缺陷型（Spo-），且未检测到可检测的σE。sigEΔ84等位基因在大肠杆菌中产生一种产物，通过定量蛋白质免疫印迹分析，其含量为野生型等位基因获得的产物（P31）水平的10%至20%。在编码P31加工区域的序列中具有错义突变的两个枯草芽孢杆菌菌株中也未检测到类σE产物。这些结果表明，σE是一种高度不稳定的蛋白质，其在合成过程中通过前体序列的一个元件得以稳定。分离出一个在枯草芽孢杆菌中产生活性类σE蛋白的突变等位基因（sigEΔ48）。该基因指定一种产物，其中五个并非来自P31加工区域的氨基酸在加工位点上游八个氨基酸的位置与P31相连。sigEΔ48产物未被加工，但它在体内激活了依赖σE的spoIID启动子。因此，sigEΔ48产物既失去了加工的一个必需靶点，也失去了一个抑制σE活性的区域。携带sigEΔ48的细胞为芽孢形成缺陷型。sigEΔ48依赖性芽孢形成缺陷的基础尚不清楚，但sigEΔ48活性似乎在发育过程中（芽孢形成开始后4小时）野生型σE活性下降的时间之后仍然持续。因此，它可能会干扰后期芽孢形成基因的正确调控。
