Isolation of Early Sporulation Genes

枯草芽孢杆菌是一种形成内孢子的细菌，是继大肠杆菌之后自然界研究得最好的生物体，并已被证明是其他革兰氏阳性细菌、病原体和非病原体的有用模型。芽孢杆菌属和梭菌属的内孢子形成。是对严重营养不足的反应因此，细胞感知的代谢信号是调节这种分化过程的关键组成部分。在最近的先前工作中，B.枯草杆菌CodY蛋白已被鉴定为代谢物感应阻遏物，其控制当细胞经历营养缺乏时打开的许多基因。其中一些基因对孢子形成至关重要，因为codY突变体在营养过剩的条件下形成孢子。长期以来人们已经认识到，当细胞从快速指数生长过渡到稳定期时，GTP的细胞内浓度下降。CodY现在已被证明对GTP的细胞内水平有反应，随着GTP浓度下降而失去作为阻遏物的活性。 CodY还对异亮氨酸和缬氨酸（两种支链氨基酸）有反应。这些新的发现表明CodY在细胞代谢和分化中起着令人惊讶的普遍作用。事实上，微阵列分析表明，数百个基因对CodY的缺失做出反应，因此是调节蛋白的直接或间接靶标。该提案旨在解决有关CodY的作用及其调节转录的机制的几个基本问题。该提案的具体目标是：（i）确定CodY在启动孢子形成中的作用;（ii）确定全球作用 CodY在代谢中的作用;（iii）确定CodY的分子结构及其与效应子的相互作用;（iv）表征CodY与其DNA靶标之间的相互作用;以及（v）鉴定介导从稳定期到孢子形成的转换的假设的第二信号传导途径。这项研究的结果有可能解决长期存在的谜团，例如为什么孢子形成的开始伴随着GTP池的短暂下降，并为稳定期和早期孢子形成事件的代谢调节提供新的见解。鉴于其他革兰氏阳性细菌中存在与CodY非常相似的蛋白质，该项目的发现可能会普遍应用于广泛的重要原核生物。

Bacillus subtilis, an endospore-forming bacterium, is, after Escherichia coli, nature's best studied organism and has proved to be a useful model for other gram-positive bacteria, pathogens and non-pathogens alike. Endosporulation in Bacillus and Clostridium spp. is a response to severe nutrient limitation. Thus, the metabolic signals that the cell perceives are a crucial component of the regulation of this differentiation process. In recent prior work, the B. subtilis CodY protein has been identified as a metabolite-sensing repressor that controls a number of genes that are turned on when cells experience nutrient deprivation. Some of these genes must be critical for sporulation, because a codY mutant sporulates under conditions of nutrient excess. It has long been recognized that the intracellular concentration of GTP drops when cells make the transition from rapid exponential growth to stationary phase. CodY has now been shown to respond to the intracellular level of GTP, losing activity as a repressor as the GTP concentration drops. CodY also responds to isoleucine and valine, two of the branched chain amino acids. These novel findings suggest that CodY plays a surprisingly general role in cellular metabolism and differentiation. In fact, microarray analysis has shown that hundreds of genes respond to the absence of CodY and are thus either direct or indirect targets of the regulatory protein. This proposal seeks to address several fundamental questions about the role of CodY and the mechanism by which it regulates transcription. The specific aims of the proposal are: (i) to determine the role of CodY in initiation of sporulation; (ii) to determine the global role of CodY in metabolism; (iii) to determine the molecular architecture of CodY and its interaction with effectors; (iv) to characterize the interaction between CodY and its DNA targets; and, (v) to identify a hypothetical second signaling pathway that mediates the switch from stationary phase to sporulation. The results of this research have the potential to resolve longstanding mysteries, such as why the onset of sporulation is accompanied by a transient drop in the GTP pool, and to provide new insight into the metabolic regulation of stationary phase and early sporulation events. Given the existence in other gram-positive bacteria of proteins that are remarkably similar to CodY, it is likely that the findings of this project will have general application to a broad group of important prokaryotes.