The nitric oxide response of Escherichia coli

一氧化氮(NO)是高等真核生物中重要的信号分子，是对抗入侵病原体的有毒分子中的一员。NO也是硝酸盐和亚硝酸盐呼吸的副产物，因此，像大肠杆菌这样的硝酸盐呼吸细菌会暴露在内源性NO中。因此，对NO的生物学反应在真核生物和原核生物中都广泛存在，并引起了人们的极大兴趣。在大肠杆菌和相关的肠道细菌中，对NO的反应的一个主要调节因子是最近被描述的转录激活因子NorR。NorR通过与Norv启动子区域的位点结合并激活由含有替代的sigma 54因子的RNA聚合酶催化的转录，激活由Norv基因编码的Favorubredoxin(将NO还原为一氧化二氮)的表达。在反硝化细菌Ralstonia eutroa中，NorR调控编码NO还原酶的NorB基因的表达，以及命名为NorA的基因的表达，该基因功能未知，但在硝酸盐呼吸生物的基因组中广泛存在。本研究的总体目标是进一步了解NorR反应NO和激活Norv表达的机制，鉴定NorR调节子的新成员，并探索NorA(YtfE)基因在大肠杆菌中的作用。具体目标是：(1)确定NorR中与NO感受有关的残基；(2)利用无偏见的全基因组搜索，在大肠杆菌基因组中寻找更多受NorR调控的基因；(3)评估NorR基因启动子区域的三个NorR结合位点和两个IHF结合位点的单独作用；以及(4)检验NO诱导的ytfE基因产物在硝酸盐和亚硝酸盐呼吸过程中具有生理作用的假设，并确定ytfE表达的调节因子。这项研究的更广泛影响将涉及博士后、研究生和本科生研究人员的培训和教学，以及促进更广泛社区的科学教育的外联活动。

Nitric oxide (NO) is an important signaling molecule in higher eukaryotes, and is a member of the arsenal of toxic molecules deployed against invading pathogens. NO is also generated as a byproduct of nitrate and nitrite respiration, so nitrate-respiring bacteria such as Escherichia coli are exposed to endogenously-generated NO. Thus, biological responses to NO are widespread in both eukaryotes and prokaryotes, and attract considerable interest. In E. coli and related enteric bacteria, a major regulator of the response to NO is the recently described transcriptional activator designated NorR. NorR activates the expression of a flavorubredoxin (that reduces NO to nitrous oxide) encoded by the norV gene, by binding to sites in the promoter region of norV and activating transcription catalyzed by RNA polymerase containing the alternative sigma 54 factor. In the denitrifying bacterium Ralstonia eutropha, NorR regulates expression of the norB gene encoding NO reductase, as well as a gene designated norA that is of unknown function but is widespread in the genomes of nitrate respiring organisms. The overall goals of the research are to further understanding of the mechanism by which NorR responds to NO and activates norV expression, to identify new members of the NorR regulon, and to explore the role of the norA (ytfE) gene in E. coli. Specific objectives are: (1) to identify residues in NorR that are involved in sensing NO; (2) to identify additional NorR-regulated genes in the E. coli genome, making use of an unbiased genome-wide search; (3) to evaluate the individual roles of the three NorR binding sites and the two IHF binding sites in the norV promoter region, and (4) to test the hypothesis that the product of the NO-inducible ytfE gene has a physiological role during nitrate and nitrite respiration, and to identify the regulator of ytfE expression. The broader impact of this research will involve the training and teaching of post-doctoral, graduate and undergraduate researchers, and outreach to promote the science eduction of the broader community.