Nitrogen Regulation of Gene Expression in Bacillus subtilis

革兰氏阳性产孢土壤细菌枯草芽孢杆菌在限氮生长过程中，一些透气孔和氮降解酶的合成受到抑制。这些基因表达的变化促进了化合物的对数生长，这些化合物缓慢地降解为含氮代谢中间体。在本项目中，我们正在研究双双电子nrgAB操纵子对氮有效性的响应。在氮限制生长过程中，nrgAB操纵子的表达降低了4000倍以上。nrgAB（以及枯草芽孢杆菌中所有其他已知的氮调节基因）表达的氮调节需要野生型谷氨酰胺合成酶蛋白，而不是谷氨酰胺合成酶抑制蛋白GlnR。因此，枯草芽孢杆菌中的氮调控是由一个独特的全球调控系统介导的，该系统与大肠杆菌中的Ntr调控系统不同。nrgAB表达氮调控所需的顺式作用位点nrgO将通过缺失和突变分析确定。已知氮调控nrgAB表达需要两个反式基因产物。缺乏谷氨酰胺合成酶（glnA）的突变体在过量氮存在下的生长过程中表达低水平的nrgAB。谷氨酰胺合成酶蛋白在氮调控中的作用将通过分离glnA突变体中抑制nrgAB抑制的突变来研究。本实验应确定将谷氨酰胺合成酶依赖的氮信号转导到nrgAB调节蛋白的基因产物。其次，在任何氮源的生长过程中，tnr突变体都不能抑制nrgAB的表达。因此，tnr基因产物可能介导氮对nrgAB表达的调控。将克隆与trn突变互补的DNA并对其进行测序，以鉴定trn基因产物。Tnr基因产物结合nrgO位点的能力将在凝胶迁移转移试验中进行检验。了解谷氨酰胺合成酶蛋白在枯草芽孢杆菌氮调控中的作用，有助于发现基因调控的新机制，并为革兰氏阳性菌的细胞代谢调控提供新的见解。此外，这些研究应该为芽孢杆菌在含氮养分相对匮乏的土壤中发酵或生长过程中生产有用产品提供遗传工具。＊＊＊

Fisher 9408094 In the gram-positive sporulating soil bacterium Bacillus subtilis, the synthesis of some permeases and nitrogen degradative enzymes is derepressed during nitrogen-limited growth. These changes in gene expression promote logarithmic growth on compounds which are slowly degraded to nitrogen-containing metabolic intermediates. In this project, the regulation of the dicistronic nrgAB operon in response to nitrogen availability is being studied. Expression of the nrgAB operon is derepressed over 4,000-fold during nitrogen-limited growth. Nitrogen regulation of nrgAB ( and of all other known nitrogen-regulated genes in B. subtilis) expression requires the wild-type glutamine synthetase protein, butnot the glutamine synthetase repressor protein, GlnR. Thus, nitrogen regulation in B. subtilis is mediated by a unique global regulatory system which is distinct from the Ntr regulatory system present in Escherichia coli. The cis-acting site(s), nrgO, required for nitrogen-regulation of nrgAB expression will be identified by deletion and mutational analysis. Two trans-acting gene products are known to be required for nitrogen regualation of nrgAB expression. Mutants deficient in glutamine synthetase (glnA) express derepressed levels of nrgAB during growth in the presence of excess nitrogen. The role of the glutamine synthetase protein in nitrogen regulation will be studied by isolating mutations which suppress the nrgAB derepression in glnA mutants. This experiment should identify gene products which transduce the glutamine synthetase-dependent nitrogen signal to the nrgAB regulatory proteins. Secondly, tnr mutants are unable to derepress nrgAB expression during growth on any nitrogen source. Thus, the tnr gene product(s) may mediate nitrogen regulation of nrgAB expression. DNA complementing the trn mutations will be cloned and sequenced to identify the trn gene products. The ability of the Tnr gene products to bind at the nrgO site will be examined in gel mobility shift assays . %%% An understanding of the role of the glutamine synthetase protein in nitrogen regulation in B. subtilis should identify novel mechanisms of gene regulation and provide insight into the regulation of cellular metabolism in gram-positive bacteria. In addition, these studies should provide genetic tools for the production of useful products in Bacillus spp. during fermentation or growth in the soil, where nitrogen-containing nutrients are relatively scarce. ***