REGULATION OF NITROGEN METABOLISM IN BACILLUS SUBTILIS

在革兰氏阳性产孢土壤细菌如枯草芽孢杆菌中，产孢的起始和抗生素的产生是对营养限制的响应。 在B中，许多碳和氮降解途径的表达也响应于养分的可利用性而受到调节。枯草杆菌。 我们的长期目标是了解该细菌中调节氮化合物利用的代谢信号和系统的网络。 B中与氮代谢相关的基因。枯草芽孢杆菌是由三个系统控制的，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，TnrA同系物，在过量氮生长的细胞中作为调节蛋白发挥作用。 CodY控制响应生长速率的基因表达。 在这个建议中，生物化学和遗传学的方法将被用来研究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TnrA位点的相互作用将在体外DNA结合实验中和使用体外转录系统进行检查。 野生型B.枯草杆菌谷氨酰胺合成酶（GS）蛋白是信号转导至TnrA蛋白所必需的。 野生型B.将在异源系统大肠杆菌细胞中检查枯草芽孢杆菌GS蛋白调节TnrA的调节活性。 将通过分离具有改变的nrgAB表达调节的突变体来寻找参与TnrA调节的反式作用因子。 将克隆和测序补充这些突变体的DNA。 通过TnrA和GlnR调节glnRA、tnrA和ureABC P3启动子转录的GlnR/TnrA位点将在遗传学上鉴定并在体外DNA结合实验中验证。通过确定TnrA是否是两个天冬酰胺酶基因ansB和ansZ的氮调节所需的，将寻找TnrA调节子中的其他基因。 这些研究将有助于确定新的范式调节革兰氏阳性土壤细菌的基因表达。 此外，它们将提供信息和遗传工具，用于优化芽孢杆菌属中医学和农业重要化合物的生产。通过商业发酵或在营养相对缺乏的土壤中生长。

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.