Wang Lijuan, Perpich John, Driks Adam, Kroos Lee
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
J Bacteriol. 2007 Dec;189(23):8467-73. doi: 10.1128/JB.01285-07. Epub 2007 Sep 21.
In the mother cell of sporulating Bacillus subtilis, a regulatory network functions to control gene expression. Four transcription factors act sequentially in the order sigma(E), SpoIIID, sigma(K), GerE. sigma(E) and sigma(K) direct RNA polymerase to transcribe different regulons. SpoIIID and GerE are DNA-binding proteins that activate or repress transcription of many genes. Several negative regulatory loops add complexity to the network. First, transcriptionally active sigma(K) RNA polymerase inhibits early sporulation gene expression, resulting in reduced accumulation of sigma(E) and SpoIIID late during sporulation. Second, GerE represses sigK transcription, reducing sigma(K) accumulation about twofold. Third, SpoIIID represses cotC, which encodes a spore coat protein, delaying its transcription by sigma(K) RNA polymerase. Partially circumventing the first feedback loop, by engineering cells to maintain the SpoIIID level late during sporulation, causes spore defects. Here, the effects of circumventing the second feedback loop, by mutating the GerE binding sites in the sigK promoter region, are reported. Accumulation of pro-sigma(K) and sigma(K) was increased, but no spore defects were detected. Expression of sigma(K)-dependent reporter fusions was altered, increasing the expression of gerE-lacZ and cotC-lacZ and decreasing the expression of cotD-lacZ. Because these effects on gene expression were opposite those observed when the SpoIIID level was maintained late during sporulation, cells were engineered to both maintain the SpoIIID level and have elevated sigK expression late during sporulation. This restored the expression of sigma(K)-dependent reporters to wild-type levels, and no spore defects were observed. Hence, circumventing the second feedback loop suppressed the effects of perturbing the first feedback loop. By feeding information back into the network, these two loops appear to optimize target gene expression and increase network robustness. Circumventing the third regulatory loop, by engineering cells to express cotC about 2 h earlier than normal, did not cause a detectable spore defect.
在枯草芽孢杆菌的芽孢形成母细胞中，一个调控网络发挥作用来控制基因表达。四个转录因子按σ（E）、SpoIIID、σ（K）、GerE的顺序依次起作用。σ（E）和σ（K）引导RNA聚合酶转录不同的调控子。SpoIIID和GerE是DNA结合蛋白，可激活或抑制许多基因的转录。几个负调控环增加了网络的复杂性。首先，转录活性的σ（K）RNA聚合酶抑制早期芽孢形成基因的表达，导致芽孢形成后期σ（E）和SpoIIID的积累减少。其次，GerE抑制sigK转录，使σ（K）的积累减少约两倍。第三，SpoIIID抑制cotC（其编码一种芽孢衣蛋白），延迟其由σ（K）RNA聚合酶进行的转录。通过改造细胞使其在芽孢形成后期维持SpoIIID水平来部分规避第一个反馈环，会导致芽孢缺陷。在此，报告了通过突变sigK启动子区域中的GerE结合位点来规避第二个反馈环的效果。前体σ（K）和σ（K）的积累增加，但未检测到芽孢缺陷。σ（K）依赖性报告融合体的表达发生了改变，gerE - lacZ和cotC - lacZ的表达增加，而cotD - lacZ的表达减少。因为这些对基因表达的影响与在芽孢形成后期维持SpoIIID水平时观察到的相反，所以对细胞进行改造，使其在芽孢形成后期既维持SpoIIID水平又提高sigK表达。这将σ（K）依赖性报告基因的表达恢复到野生型水平，并且未观察到芽孢缺陷。因此，规避第二个反馈环抑制了干扰第一个反馈环的影响。通过将信息反馈到网络中，这两个环似乎优化了靶基因的表达并提高了网络的稳健性。通过改造细胞使其比正常情况提前约2小时表达cotC来规避第三个调控环，未导致可检测到的芽孢缺陷。
