希瓦氏菌硝酸还原多层次调控的分子机制

In Shewanella oneidensis, regulation of nitrate reduction is primarily under the control of a three-component system consisting of one sensing kinase and two DNA-binding responding regulators.A comparative analysis of genomes encoding nitrate reduction enzymes reveals that such a system is omnipresent, representing a novel mechanism for nitrate respirating bacteria to survive in the environments. Therefore, we propose to continue our investigation on this subject, in which we has accumulated an enormous amount of data and published the results on a high-impact Journal. Our focus will be posited on determining effects of the system on nitrate reduction, deciphering its regulatory mechanism at the molecular level, illustrate its status in evolution. In particular, we will first explore significance of each component of the systme in nitrate reduction by a mutational analysis. To this end, in-frame deletion mutants will be constructed and characterized. Second, we will access the essentility of phosphorylation of the DNA-binding regulators using purified proteins and point mutation strains. Third,with both in vivo and in vitro techniques, we will determine the phosphoryaltion pathway and identify the key genes under the control of this three-component system. Last, we will investigate how Crp as a higher level global regulator influences the expression of the systme and subquently nitrate reduction. We believe such a proposal, if granted, will definitely push forward our scientific development in the molecular microbiolgy field, and ensure our leading position in studies on this novel system in Shewanella.

S.oneidensis的硝酸还原途径由一个信号感应蛋白和两个DNA结合调控因子组成的三元系统调控。基因组比较分析发现这种三元系统在一些细菌中普遍存在，因而可能代表着一种适合这类细菌生存的新机制。这个申请中的课题旨在继续深入项目主持人在这方面的研究，进一步在生理生态上，分子生物学上，以及系统进化上对这种非经典的调控系统加深理解。具体地，我们要首先调查该系统各个组成蛋白对硝酸还原途径的影响；其次，弄清DNA结合调控因子磷酸化对其生物功能的必要性；再其次，全面地对这个系统进行体外和体内实验分析，以期了解这个系统的作用机理；最后通过研究此系统对无氧呼吸全局调控因子Crp的响应揭示Crp的作用机理。本课题的立项和实施，将有力推动我国以微生物为载体的分子生物学基础研究的发展，使我们在Shewanella的研究中继续保持国际领先。

Shewanella oneidensis 以其极端广谱的呼吸能力著称。该菌的硝酸还原途径具有很多新颖的特点，已被本课题负责人基本解析。在解析过程中，研究发现该菌硝酸还原途径也具有广泛的代表性：该菌硝酸还原途径由一个信号感应蛋白和两个DNA 结合调控因子组成的三元系统调控。基因组比较分析发现这种三元系统在一些细菌中普遍存在，因而可能代表着一种适合这类细菌生存的新机制。这个申请中的课题旨在继续深入项目主持人在这方面的研究，进一步在生理生态上，分子生物学上，以及系统进化上对这种非经典的调控系统加深理解。课题首先调查了该系统各个组成蛋白对硝酸还原途径的影响；其次弄清了DNA 结合调控因子磷酸化对其生物功能的必要性；接下来全面地对这个系统进行体外和体内实验分析；最后通过研究此系统对无氧呼吸全局调控因子Crp 的响应揭示Crp 的作用机理。在研究过程中，不但全面完成了计划任务，而且发现并在其它课题的交叉支持下弄清了几个长期困扰研究者的 问题，包括电子传递补救途径，亚硝酸的毒性及其机制，以及与之相关的细胞应答机制。这些成果极大地提升了对细菌的亚硝酸生物学的理解，为利用该菌进行含硝酸环境修复奠定基础。