Nitrogen Regulation of Gene Expression in Bacillus subtilis

在革兰氏阳性产孢土壤细菌枯草芽孢杆菌中，在氮限制生长期间，一些渗透酶和氮降解酶的合成被去抑制。基因表达的这些变化促进了化合物的对数生长，这些化合物缓慢降解为含氮代谢中间体。在该项目中，正在研究双顺反子nrgAB操纵子对氮可用性的调节。在氮限制生长期间，nrgAB操纵子的表达被去阻遏超过4，000倍。nrgAB的氮调节（以及B中所有其他已知的氮调节基因）。枯草杆菌）的表达需要野生型谷氨酰胺合成酶蛋白，而不需要谷氨酰胺合成酶阻遏蛋白GlnR。因此，B.枯草芽孢杆菌中的Ntr由独特的全局调节系统介导，该系统不同于大肠杆菌中存在的Ntr调节系统。将通过缺失和突变分析鉴定nrgAB表达的氮调节所需的顺式作用位点nrgO。已知nrgAB表达的氮调节需要两种反式作用基因产物。谷氨酰胺合成酶（glnA）缺陷的突变体在过量氮的存在下生长期间表达去阻遏水平的nrgAB。将通过分离抑制glnA突变体中nrgAB去阻遏的突变来研究谷氨酰胺合成酶蛋白在氮调节中的作用。该实验应鉴定将谷氨酰胺合成酶依赖性氮信号传递给nrgAB调节蛋白的基因产物。其次，tnr突变体不能在任何氮源上生长期间去抑制nrgAB表达。因此，tnr基因产物可以介导nrgAB表达的氮调节。将对补充trn突变的DNA进行克隆和测序，以鉴定trn基因产物。将在凝胶迁移率变动试验中检查Tnr基因产物在nrgO位点结合的能力。了解谷氨酰胺合成酶蛋白在B的氮调节中的作用。枯草芽孢杆菌应该确定新的基因调控机制，并提供在革兰氏阳性菌的细胞代谢调节的见解。此外，这些研究应该为芽孢杆菌属中有用产物的生产提供遗传工具。在土壤中发酵或生长期间，其中含氮营养物相对缺乏。***

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. ***