Genetic Control of Nitrate Respiration in E. coli

描述(申请人提供)：肠道细菌，如大肠杆菌和肠沙门氏菌，是兼性需氧菌，在无氧条件下培养时可以使用多种替代呼吸氧化剂。硝酸盐和亚硝酸盐是首选的替代品，厌氧呼吸酶基因的表达受到严格控制，以响应它们的可用性。这种控制是由两个相互作用的双成分调控系统来调节的。硝酸盐和亚硝酸盐控制着两个细胞质膜结合的感受器蛋白NarX和NarQ的自动磷酸化，而NarX和NarQ蛋白又控制着两个DNA结合反应调节蛋白NarL和NarP的磷酸化。因此，十几个操纵子上的转录启动根据电子受体的可用性而被激活或抑制。NAR调控网络是肠道细菌独有的；其他种类的变形杆菌(包括几种人类病原体)只包含NarQ-Narp系统(例如霍乱弧菌)或NarX-NarL系统(例如铜绿假单胞菌)。因此，拟议的研究广泛地为我们提供了对许多物种的厌氧生理学的理解，这些物种属于变形杆菌的伽马和贝塔亚类。这里提出的工作包括五个具体目标：(1)我们必须从生化细节上描述体外交叉调节的特征，以便主要基于体内观察来评估和扩展模型。(2)与E.P.Baldwin合作，研究反应调节因子NarL与DNA的协同结合。(3)与P.J.Kiley合作，我们将研究反应调节因子Narp的转录激活。(4)我们继续探索传感器NarX和NarQ对已定义的调控信号的反应。(5)与M.M.Igo合作，我们将确定其表达受NarX-NarL或NarQ-Narp系统控制的先前未知的靶基因。我们的总体目标是整合体内和体外的方法，以更好地了解无氧代谢的生理学。这项基础性研究与公众健康相关的领域是厌氧生理学和新陈代谢。蛋白质细菌中的大多数致病物种是兼性需氧菌或兼性厌氧菌，许多传染病涉及厌氧环境的定植，如哺乳动物的肠道。因此，来自模式物种大肠杆菌的结果加强了对广泛的病原体的了解，这些病原体对公共健康有重大影响。

DESCRIPTION (provided by applicant): Enterobacteria such as Escherichia coli and Salmonella enterica are facultative aerobes, and when cultured in the absence of oxygen can use a variety of alternative respiratory oxidants. Nitrate and nitrite are the preferred alternatives, and anaerobic respiratory enzyme gene expression is tightly controlled in response to their availability. This control is mediated by dual interacting two-component regulatory systems. Nitrate and nitrite control the autophosphorylation of two cytoplasmic membrane-bound sensor-kinases, the NarX and NarQ proteins, which in turn control the phosphorylation of two DNA-binding response regulators, the NarL and NarP proteins. Thus, transcription initiation at more than one dozen operons is activated or repressed according to electron acceptor availability. The Nar regulatory network is unique to enterobacteria; other species of proteobacteria (including several human pathogens) contain only the NarQ-NarP system (e.g., Vibrio cholerae) or the NarX-NarL system (e.g., Pseudomonas aeruginosa). The proposed studies therefore broadly inform our understanding of anaerobic physiology for many species within the gamma and beta subdivisions of the proteobacteria. Work proposed here comprises five specific aims: (1) We must characterize cross-regulation in vitro in biochemical detail in order to evaluate and extend models based primarily on in vivo observations. (2) In collaboration with E. P. Baldwin, we will study cooperative DNA binding by the response regulator NarL. (3) In collaboration with P. J. Kiley, we will study transcription activation by the response regulator NarP. (4) We continue to explore response to defined regulatory signals by the sensors NarX and NarQ. (5) In collaboration with M. M. Igo, we will identify previously-unknown target genes whose expression is controlled by the NarX-NarL or NarQ-NarP systems. Our overall goal is to integrate both in vivo and in vitro approaches to better understand the physiology of anaerobic metabolism. The relevance to public health of this fundamental research is in the realm of anaerobic physiology and metabolism. Most pathogenic species within the proteobacteria are facultative aerobes or facultative anaerobes, and many infectious diseases involve colonization of anaerobic environments such as the mammalian intestine. Thus, results from the model species E. coli enhance understanding for a broad range of pathogens that significantly impact public health.