细菌外源基因抑制因子的结构与功能研究

In order to adapt new environments, bacteria frequently acquire new genes through horizontal transfer during evolution, such as the acquisition of virulence, antibiotic resistance or novel metabolic capabilities. However, foreign DNA sequences acquired are more likely to decrease rather than increase the fitness of the recipient bacteria. Therefore, many bacteria genera have evolved unique proteins to selectively bind to and silence expression from AT rich regions of the chromosome, which are highly likely acquired from a foreign source. As a result of their activity, xenogeneic silencers are the master regulators of horizontally acquired sequences, including many critical virulence and drug resistance genes in a large number of important bacterial pathogens, such as Mycobacteria tuberculosis, Vibrio cholera, Salmonella typhimurium, enteropathogeneic E. coli, and Pseudomonas aeruginosa. To date, four distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, MvaT of Pseudomonadaceae and Rok of Bacillaceae. We have elucidated that Lsr2 and H-NS recognize AT-rich DNA minor groove through a common AT-hook-like motif, although they are structurally different. Our work on the DNA-binding mechanism of Lsr2 and H-NS were published on PNAS in 2010 and 2011. It is interesting that no AT-hook-like motif is identified for the xenogeneic silencer MvaT and Rok, and their self-oligomerization mechanisms are poorly understood. The object of this study is to reveal the DNA-binding mechanism and oligomerzation mechanism of MvaT and Rok, which will help us to understand the structure-function relationship of these two xenogeneic silencers, as well as provide clues for the development of new antibiotic drugs.

基因水平转移对细菌的进化影响重大，很多毒力基因及抗生素抗性基因都是以这种方式被细菌获取。同时，这些基因的不恰当表达会降低细菌的生存能力。因此，许多细菌进化出外源基因抑制因子，选择性地结合并抑制外源基因的转录。外源基因抑制因子是许多毒力和耐药性基因的重要调控因子，在结核分枝杆菌、霍乱弧菌、致病性大肠杆菌和绿脓杆菌等病原菌中发挥重要作用。在细菌中已发现四类外源基因抑制因子：变形杆菌中的H-NS，分支杆菌中的Lsr2，假单胞菌中的MvaT，以及芽孢杆菌中的Rok。我们已阐明Lsr2和H-NS都运用了类似AT-hook的机理结合DNA，相关结果分别于2010和2011年发表在PNAS上。MvaT和Rok都不具有AT-hook类似结构，它们自聚合性质也缺少研究。本项研究旨在揭示MvaT和Rok的DNA结合机理以及自聚合机理，以期增进我们对细菌外源基因抑制因子结构和功能的认识，以辅助研发新的抗菌药物。

外源基因抑制因子在细菌进化过程中发挥着重要作用。它们可以识别并抑制细菌通过基因水平转移方式从环境中获取的外源基因，避免其不恰当的表达对细菌造成的不利影响。目前，已被人们发现的外源基因抑制因子从序列相似性上可以分为四类：广泛存在于α, β和γ-变形菌中的H-NS，放线菌中的Lsr2，假单胞菌和其他一些γ-变形菌中的MvaT，以及芽孢杆菌中的Rok。在之前的研究中，我们初步阐明了H-NS及Lsr2的DNA识别机制。而本项目则重点研究MvaT及Rok的DNA结合机理。我们利用蛋白质结合芯片深入研究了来自假单胞菌中的MvaT及来自枯草芽孢杆菌中的Rok的DNA结合特性，并利用核磁共振技术解析了二者C端结构域自由态及DNA结合态的溶液结构。.MvaT的C端结构域具有与H-NS类似的二级结构组成和折叠，但是不含有类似AT-hook的构象。MvaT C-端结构域N末端的Arg80和loop2上的Gly99和Asn100形成的“AT-pincer”结构插入到DNA的小沟中，并能够与碱基形成氢键。另外，由6个赖氨酸残基构成的“Lysine-network”与DNA的脱氧核糖磷酸骨架存在静电相互作用。体外和体内的突变研究表明，“AT-pincer”和“Lysine-network”对MvaT结合DNA的亲和力和转录抑制功能都是十分重要的。“AT-pincer”对DNA碱基的base readout，以及“Lysine-network”对DNA脱氧核糖磷酸骨架的shape readout，共同构成了MvaT识别高AT含量DNA的机理。.Rok的C端结构域为典型的winged helix结构域，但其DNA结合机制不同于其他已知winged helix蛋白，亦不同于其他外源基因抑制因子。位于α3螺旋N-末端的N154和T156残基以及Wing 1上的R174残基侧链插入DNA小沟，通过氢键相互作用识别碱基序列。四个赖氨酸残基侧链通过静电相互作用与DNA小沟两侧的磷酸基团结合，稳定蛋白与DNA复合体的结构。这些结构特征使得Rok倾向于结合含有T/AACTA或者连续多个柔性TpA序列的DNA分子，而对于A-tracts序列结合力较弱。相应的，枯草芽孢杆菌基因组富含A-tracts并且显著缺少T/AACTA序列，说明外源基因抑制因子与细菌基因组之间存在协同进化关系。.