REGULATION OF NITROGEN METABOLISM IN BACILLUS SUBTILIS

革兰氏阳性产孢菌如枯草芽孢杆菌的产孢量和抗生素的产生是对营养限制的反应。在枯草杆菌中，许多碳和氮降解途径的表达也受到营养可利用性的调节。我们的长期目标是了解调节这种细菌中氮化合物利用的代谢信号和系统的网络。枯草杆菌中参与氮代谢的基因由三个系统控制，每个系统调节一个独特的基因子集，以响应不同的营养条件。TnrA调节发生在氮限制生长期间。GlnR是TnrA的同系物，在生长在过量氮的细胞中起到调节蛋白的作用。Cody控制基因表达以响应生长速度。在这项建议中，将使用生化和遗传学方法来研究TnrA调节基因表达的分子机制。纯化的TnrA蛋白与野生型和突变型TnrA位点的相互作用将通过体外DNA结合实验和体外转录系统进行检测。野生型枯草杆菌谷氨酰胺合成酶(GS)蛋白是TnrA蛋白信号转导所必需的。野生型枯草杆菌GS蛋白调节TnrA调节活性的能力将在一个异源系统--大肠杆菌细胞中进行检测。参与TnrA调控的反式作用因子将通过分离nrgAB表达调节发生变化的突变株来寻找。补充这些突变体的DNA将被克隆和测序。调节TnrA和GlnR转录glnRA、tnrA和ureABC P3启动子的GlnR/TnrA位点将从基因上得到鉴定，并在体外DNA结合实验中得到验证。将通过确定TnrA是否需要TnrA来调节两个天冬酰胺酶基因ansB和ansZ的氮来寻找TnrA调节子中的其他基因。这些研究应该有助于确定控制革兰氏阳性土壤细菌基因表达的新范例。此外，它们还将为优化芽孢杆菌属中医学和农业上重要化合物的生产提供信息和遗传工具。通过商业发酵或在养分相对稀缺的土壤中生长期间。

The initiation of sporulation and the production of antibiotics in Gram-positive sporulating soil bacteria such as Bacillus subtilis occurs in response to nutrient limitation. The expression of many carbon and nitrogen degradative pathways is also regulated in response to nutrient availability in B. subtilis. Our long term objective is to understand the network of metabolic signals and systems regulating the utilization of nitrogen compounds in this bacterium. The genes involved in nitrogen metabolism in B. subtilis are controlled by three systems, each of which regulates an unique subset of genes in response to different nutritional conditions. TnrA regulation occurs during nitrogen-limited growth. GlnR, the TnrA homolog, functions as a regulatory protein in cells grown with excess nitrogen. CodY controls gene expression in response to growth rate. In this proposal, biochemical and genetic approaches will be used to investigate the molecular mechanism by which TnrA regulates gene expression. The interaction of the purified TnrA protein with wild-type and mutant TnrA sites will be examined in in vitro DNA binding experiments and using an in vitro transcription system. The wild-type B. subtilis glutamine synthetase (GS) protein is required for signal transduction to the TnrA protein. The ability of the wild-type B. subtilis GS protein to regulate the TnrA regulatory activity will be examined in a heterologous system, Escherichia coli cells. trans-acting factors involved in TnrA regulation will be sought by isolating mutants with altered regulation of nrgAB expression. DNA complementing these mutants will be cloned and sequenced. The GlnR/TnrA sites regulating the transcription of the glnRA, tnrA and ureABC P3 promoters by TnrA and GlnR will be identified genetically and verified in in vitro DNA binding experiments. Additional genes in the TnrA regulon will be sought by determining whether TnrA is required for the nitrogen regulation of two asparaginase genes, ansB and ansZ. These studies should help define the novel paradigms regulating gene expression in Gram-positive soil bacteria. In addition, they will provide both information and genetic tools for optimizing the production of medically and agriculturally important compounds in Bacilli spp. by commercial fermentation or during growth in the soil, where nutrients are relatively scarce.