Márquez-Magaña L M, Chamberlin M J
Division of Biochemsitry and Molecular Biology, University of California, Berkeley 94720.
J Bacteriol. 1994 Apr;176(8):2427-34. doi: 10.1128/jb.176.8.2427-2434.1994.
The sigma D factor of Bacillus subtilis is required for the transcription of the flagellin and motility genes as well as for wild-type chemotaxis. Southern blot and sequence analyses demonstrate that the structural gene for sigma D, sigD, is located immediately downstream of a region of DNA originally identified as the chemotaxis (che) locus and now renamed the fla/che region. In fact, sigD appears to be part of a very large operon (> 26 kb) containing genes which encode structural proteins that form the hook-basal body complex as well as regulatory proteins required for chemotaxis. Transposon insertions up to 24 kb upstream of sigD, within several of the genes for the hook-basal body components, give rise to only a moderate decrease in sigD expression. The transposon insertions, however, block sigma D activity as demonstrated by the lack of flagellin expression in strains bearing these insertions. These effects appear to arise from two types of regulation. In cis the transposon insertions appear to introduce a partial block to transcription of sigD from upstream promoter elements; in trans they disrupt genes whose gene products are required for sigma D activity. It appears that sigD transcription is initiated, at least in part, by a promoter many kilobases upstream of its translation start site and that transcription of the flagellin gene by sigma D is dependent on the formation of a functional hook-basal body complex. The possibility that sigD is part of the fla/che operon was further tested by the integration of an insertion plasmid, containing strong transcription terminators, 1.6 and 24 kb upstream of the sigD gene. In both cases, the introduction of the terminators resulted in a greater decrease of sigD expression than was caused by the plasmid sequences alone. These results indicate that wild-type transcription of sigD is dependent on promoter sequences > 24kb upstream of its structural gene and that the entire fla/che region forms a single operon.
枯草芽孢杆菌的σD因子是鞭毛蛋白和运动基因转录以及野生型趋化作用所必需的。Southern印迹和序列分析表明，σD的结构基因sigD位于最初被鉴定为趋化作用（che）位点、现重新命名为fla/che区域的DNA区域的紧邻下游。实际上，sigD似乎是一个非常大的操纵子（>26 kb）的一部分，该操纵子包含编码形成钩-基体复合体的结构蛋白以及趋化作用所需调节蛋白的基因。在sigD上游多达24 kb处、钩-基体组件的几个基因内的转座子插入，仅导致sigD表达适度下降。然而，如带有这些插入的菌株中鞭毛蛋白表达缺失所证明的，转座子插入会阻断σD活性。这些效应似乎源于两种类型的调控。顺式作用下，转座子插入似乎对来自上游启动子元件的sigD转录引入了部分阻断；反式作用下，它们破坏了其基因产物是σD活性所必需的基因。似乎sigD转录至少部分由其翻译起始位点上游许多千碱基处的一个启动子起始，并且σD对鞭毛蛋白基因的转录依赖于功能性钩-基体复合体的形成。通过在sigD基因上游1.6和24 kb处整合含有强转录终止子的插入质粒，进一步测试了sigD是fla/che操纵子一部分的可能性。在这两种情况下，终止子的引入导致sigD表达下降的幅度比单独的质粒序列所引起的更大。这些结果表明，sigD的野生型转录依赖于其结构基因上游>24 kb的启动子序列，并且整个fla/che区域形成一个单一操纵子。
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