铜绿假单胞菌PAO1中环鸟苷二磷酸代谢蛋白PA0575的生物学功能及作用研究

近年来，人们发现，新型第二信使环鸟苷二磷酸(c-di-GMP)在转录、转录后及翻译后水平上，对原核生物的生理过程有着重要的调控作用；其信号转导作用也拓展到了真核领域。细菌中环鸟苷二磷酸的代谢及胞内水平由两种酶共同决定：鸟苷酸环化酶（Diguanylate cyclase, DGCs）催化GTP生成c-di-GMP； 而磷酸二酯酶 （Phosphodiesterase，PDEs）则将c-di-GMP水解为GMP；它们统称为环鸟苷二磷酸代谢蛋白。我们的初步工作显示，模式菌株铜绿假单胞菌PAO1中，未经分析的环鸟苷二磷酸代谢蛋白PA0575作用复杂。在本申请中，我们将利用遗传学、分子生物学、结构生物学等手段，对PA0575进行深入研究，明确其催化功能，分析其生物学作用，探索其信号传递过程。我们还将尝试环鸟苷二磷酸的制备。我们的工作将有助于加深对环鸟苷二磷酸作用规律的认识，并拓展其未来的应用。

(3′,5′)-cyclic-di-guanosine monophosphate (c-di-GMP), a novel secondary messenger, regulates a plethora of physiological functions at the transcriptional, post-transcriptional and post-translational levels in bacteria. Recently, it has been demonstrated that c-di-GMP is also involved in signal transduction in eukaryotic organisms. In bacteria, there are two kinds of enzymes governing the c-di-GMP metabolism: diguanylate cyclases (DGCs) of GGDEF domains synthesize c-di-GMP from two GTP molecules; while phosphodiesterases (PDEs) of either the EAL or HD-GYP domain hydrolyze c-di-GMP. Both DGCs and PDEs are the c-di-GMP-metabolizing proteins. As a well-known model organism, P. aeruginosa PAO1 contains 38 c-di-GMP-metabolizing proteins. While a few of these proteins have been studied experimentally, the exact biological functions of the rest remain obscure. In current study, we will focus on PA0575, an uncharacterized protein from P. aeruginosa. Employing genetic, molecular biology and structural biology methods, we will characterize catalytic activity, and dissect biological functions of PA0575. We will also try to prepare c-di-GMP in tubes, to facilitate its applications in the future.