CONTROL AND EXPRESSION OF TYPE 1 PILI IN E COLI

1型大肠杆菌和其他革兰氏阴性肠道细菌是 丝状的，由重复亚基组成的蛋白质附属物， pilin。 1型皮利介导细菌对甘露糖敏感的粘附， 多种真核细胞，并作为定植因子参与 肠外感染由E.大肠杆菌和肺炎克雷伯菌。 这个项目的长期目标是建立分子的性质， 遗传控制，装配1型皮利受体结合活性 在大肠大肠杆菌，并在这样做，有助于了解控制 超分子结构的组装和超分子的分子性质 受体与配体的相互作用 此外，希望这项工作将 从而更好地了解细菌粘附在 传染病的发病机理。 本文提出的具体研究 利用各种遗传和生物化学技术来辨别 分子的性质;（i）菌毛组装的控制。 菌毛组件 由与之相邻的基因（hyp）编码的反式作用多肽调节 pilA结构基因。 lacZ与pilA的融合表明， Hyp基因产物通过抑制 pilA。 拟议的研究利用条件致死的特点， hyp突变和pilA-lacZ融合以鉴定hyp基因中的位点 产物和pilA启动子中，影响pilA转录。 （二）菌毛 组装件. 至少有两个基因的产物，pilB和pilC参与 在菌毛组装中。 这项研究旨在确定这些 基因产物参与与菌毛蛋白的直接相互作用， 通过鉴定pilB和pilC中的突变，然后分离pilB和pilC中的突变， Pil+ pilA有病变的假回复突变体;（iii）受体结合。 菌毛蛋白与含有甘露糖的受体或真核细胞相互作用。 实验旨在检测菌毛蛋白参与的结构域， 利用pilA的核苷酸序列与受体结合， 采用定点诱变。 (iv)皮利的亚稳态性质 表情 在大肠杆菌中，皮利的表达以亚稳态方式调节。 大肠杆菌，导致有毛和无毛状态之间的变化。 这种变异将通过克隆1型毛化的基因来检验 来自大肠coli K12和E. coli Bam上的低拷贝数质粒， 确定哪些基因参与了纤毛的控制， 亚稳态方式。

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.