CONTROL AND EXPRESSION OF TYPE 1 PILI IN E COLI

1型大肠埃希氏菌和其他革兰氏阴性肠杆菌 由重复的亚单位组成的丝状蛋白质附属物， 皮林。1型菌毛介导细菌对甘露糖敏感的黏附 多种真核细胞，作为一种定植因子，参与了 由大肠杆菌和肺炎克雷伯氏菌引起的肠外感染。 这个项目的长期目标是确定 I型菌毛的遗传控制、组装和受体结合活性 在大肠杆菌中，并通过这样做，有助于理解控制 以及超分子结构的组装和分子性质 受体与配体之间的相互作用。此外，希望这项工作将 使我们更好地理解细菌黏附在细菌感染中的作用 传染病的发病机制。本文提出了具体的研究建议 利用各种遗传和生化技术来识别 分子性质；(I)菌毛组装的控制。Pilus组件是 受邻近基因(Hyp)编码的反式作用多肽调节 结构基因(Pila)。Lacz和Pila的融合表明 Hyp基因产物通过抑制转录调节梅毒 皮拉。建议的研究利用了条件性致命性 利用Hyp突变和Pila-LacZ融合技术确定Hyp基因的位点 产物和Pila启动子中影响Pila转录的基因。(Ii)Pilus 集合。至少有两个基因的产物--PilB和PILC参与其中 在Pilus组装中。这项拟议的研究旨在确定这些 基因产物参与了与菌毛蛋白的直接相互作用 鉴定PilB和PILC突变并分离的聚合反应 PIL+假逆变体在PILA中有病变；(Iii)受体结合。 菌毛素与含有甘露糖的受体或真核细胞相互作用。 实验旨在检测参与Pilin结构域的 利用Pila和Pila的核苷酸序列与受体结合 采用定点突变。(Iv)菌毛的亚稳性 表情。Pili在E. Coli，导致纤毛状态和非纤毛状态之间的差异。 这种变异将通过克隆1型花粉症的基因进行检测。 从E.coliK12和E.coliBam转移到低拷贝数的质粒上 确定哪些基因参与皮下感染是受 亚稳的方式。

Type 1 of Escherichia coli and other gram negative enteric bacteria are filamentous, proteinaceous appendages composed of a repeating subunit, pilin. Type 1 pili mediate a mannose sensitive adhension of bacteria to a variety of eucaryotic cells and are involved, as a colonization factor, in extraintestinal infections caused by E. coli and Klebsiella pneumoniae. The long-term goal of this project is to establish the molecular nature of the genetic control, assembly an receptor-binding activity of Type 1 pili in E. coli and in doing so, contribute to the understanding of the control and assembly of supramolecular structures and the molecular nature of receptor-ligand interactions in general. Also, it is hoped this work will lead to a better understanding of the role of bacterial adhesion in the pathogenesis of infectious disease. The specific research herein proposed utilizes a variety of genetic and biochemical techniques to discern the molecular nature of; (i) The control of pilus assembly. Pilus assembly is regulated by a trans-acting polypeptide encoded by a gene (hyp) adjacent to the structural gene (pilA). Fusions of lacZ with pilA have suggested that the hyp gene product regulates piliation by repressing transcription of pilA. The proposed research utilizes the conditional lethal character of hyp mutations and pilA-lacZ fusions to identify sites in the hyp gene product and in the pilA promotor that effect pilA transcription. (ii)Pilus assembly. The products of at least two genes, pilB and pilC are involved in pilus assembly. The proposed research is designed to determine if these gene products are involved in direct interaction with pilin during polymerization by identifying mutations in pilB and pilC and then isolating Pil+ pseudorevertants having lesions in pilA; (iii) Receptor binding. Pilin interacts with a mannose containing receptor or eucaryotic cells. Experiments are designed to detect the domains of pilin involved in receptor binding by taking advantage of the nucleotide sequence of pilA and employing site-directed mutagenesis. (iv) The metastable nature of pili expression. Pili expression is regulated in a metastable manner in E. coli, resulting in variation between piliated and non-piliated states. This variation will be examined by cloning the genes for type 1 piliation from E. coli K12 and E. coli Bam onto a low copy number plasmid and determining which of the genes involved in piliation is controlled in a metastable manner.