Detection and Metabolism of Phenolic Compounds by Agrobacterium tumefaciens

与真核宿主相关的抽象细菌通常具有复杂的机制来检测宿主释放的化学信号。这一主题在植物病原体根癌农杆菌中得到了很好的研究，它检测到几类植物释放的信号。这个项目的重点是对一类从植物伤口处释放的酚类化合物的感知。这些化合物是由一个由跨膜组氨酸激酶VirA和细胞质反应调节因子Virg组成的双组分系统检测到的，该系统直接诱导VIR调节子。VIR调节子的产物介导致癌DNA片段从细菌转移到宿主植物细胞的细胞核，导致冠胆肿瘤。VirA是一种二聚体蛋白，有四个结构域：激酶本身和三个调节激酶活性的结构域。在过去，通过系统地在基因水平上消除每个区域的功能，将功能分配给每个区域。在这个项目中，基因技术将被用来创造只包含一个磷酸盐和一个磷酸盐的VirA异二聚体，每个结构域的作用将在体内确定。可溶的病毒片段将用于酚类诱导剂的结合以及酚类反应的自磷酸化和磷酸转移反应。Winans的实验室最近确定了VIR调节子(VirH)中的一个操纵子指导了酚类VIR诱导剂的破坏。例如，野生型菌株可以使阿魏酸(一种强烈的vir基因诱导剂)去甲基化，产生咖啡酸(一种非诱导剂)，而virH2突变株则无法进行这种转化。无论相关的酚类化合物是VirH2的底物还是密切相关的蛋白质VirH1的底物，都将被解决。他们还将测试这些反应的产物是否会被其他根癌农杆菌蛋白进一步分解。最后，他们将学习这些酶在植物定植中扮演的生物学角色。所有病原体都必须检测到它们的宿主并做出适当的反应。检测通常是使用嗅觉机制(基本上是通过嗅觉)进行的。这个项目将提供使用遗传和生化技术来了解特定的植物病原体(根癌农杆菌)如何检测寄主植物。最近发现农杆菌可以破坏这些信号分子，这个项目将研究是所有类型的这些分子都被破坏，还是只有某些类型的分子被破坏。它还将研究这些反应对这种细菌定植宿主能力的总体影响。这些研究将提供有关宿主-病原体相互作用的基本信息，这将有助于对抗这种病原体和其他病原体。

Winans, Stephen C.MCB-9904917Abstract Bacteria that live in association with eukaryotic hosts often have intricate mechanisms to detect host-released chemical signals. This theme is well-studied in the plant pathogen Agrobacterium tumefaciens, which detects several classes of plant-released signals. This project focuses on the perception of a class of phenolic compounds that are released from plant wound sites. These compounds are detected by a two-component system consisting of the transmembrane histidine kinase VirA and the cytoplasmic response regulator VirG, which direct the induction of the vir regulon. Products of the vir regulon mediate the transfer of oncogenic DNA fragments from the bacterium to the nuclei of host plant cells, resulting in crown gall tumors. VirA is a dimeric protein that has four domains: the kinase itself and three domains that modulate kinase activity. In the past, functions have been assigned to each domain by systematically ablating each one at the genetic level. In this project, genetic techniques will be used to create VirA heterodimers containing just one phosphodonor andone phosphorecipient and the role of each domain will be determined in vivo. Soluble VirA fragments will be tested for binding of phenolic inducers and phenolic-responsive autophosphorylation and phosphotransfer reactions. The Winans' laboratory recently determined that one of the operons in the vir regulon (virH) directs the destruction of phenolic vir-inducers. For example, wild type strains can demethylate ferulic acid (a strong vir gene inducer), creating caffeic acid (a noninducer), while virH2 mutants fail to carry out this conversion. Whether related phenolic compounds are substrates for VirH2 or for the closely related protein VirH1 will be addressed. They will also test whether the products of these reactions are further catabolized by other A. tumefaciens proteins. Finally, they will learn what biological role these enzymes play in plant colonization. All pathogens must detect their hosts and make appropriate responses. Detection is generally carried out using olfactory mechanisms (essentially, by sense of smell). This project will provide use genetic and biochemical techniques to learn how a particular plant pathogen (Agrobacterium tumefaciens) detects host plants. It was recently discovered that Agrobacterium can destroy these signal molecules, and this project willstudy whether all types of these molecules are destroyed, or only certain ones. It will also study the overall effects of these reactions on the ability of this bacterium to colonize its host. These studies will provide fundamental information about host-pathogen interactions that will be useful in combating this and other pathogens.