GENE ANALYSIS OF SPIROCHETE PERIPLASMIC FLAGELLA

螺旋体周质鞭毛基因分析

• 批准号: 2391237
• 负责人: NYLES CHARON
• 金额: $ 10.86万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2391237
• 关键词: Treponema; bacterial genetics; cilium /flagellum motility; denaturing gradient gel electrophoresis; electroporation; flagellin; flagellum; gene targeting; mutant; protein structure function; transposon /insertion element; video microscopy; virulence; western blottings

DESCRIPTION (Adapted from applicant's abstract): Spirochetes are a medically significant but a remarkable poorly understood group of bacteria. These organisms cause a variety of important diseases including syphilis, Lyme disease, relapsing fever, and leptospirosis. They are also implicated in periodontal disease and human diarrheal disease. The present proposal aims to answer certain questions relating to the structure of spirochete periplasmic flagella (PFs) and motility. Spirochete PFs are the most complex of any bacterial flagellar filament. On the surface of the PFs is a protein sheath comprised of one to two FlaA protein species. The core is comprised of a polymer of three to four protein species referred to as FlaB proteins. The function of the individual PF proteins is not understood. To better understand the roles of these proteins in PF structure and motility, the applicant proposes to knock-out several genes involved in PF synthesis. The model system being used is Serpulina hyodysenteriae, formally known as Treponema hyodysenteriae. S. hyodysenteriae is the only spirochete species in which specific gene inactivation has been achieved. These spirochetes have PFs very similar to those found in Treponema pallidum, the causative agent for syphilis. T. pallidum still cannot be continuously cultured, let alone be manipulated for mutant analysis. Thus, because the PFs of both organisms are so similar, the results achieved with S. hyodysenteriae will be relevant to T. pallidum PF structure and motility. The applicant already has constructed three PF mutants, flaA1, flaB1, flaAlflaB1 by electroporation and allelic exchange. They hypothesize that these mutants have altered PF structure and that the altered PFs result in inefficient swimming. To test this, they will analyze PF structure and motility of the mutants, and make comparisons to the wild-type. To augment the above experiments, they will construct and analyze other motility mutants. These mutants include allelic exchange mutants in both the flaB2 gene, and the flgE gene. The latter gene encodes the hook gene, and mutations in this gene should result in cells completely deficient in PFs. The results obtained should lead to a clear understanding of the function of these proteins in PF structure and motility. The experiments proposed are the first to examine spirochete motility using a systematic molecular- genetic approach. They are especially relevant in that flagella and/or motility are implicated as a virulence factor for spirochetes and several other species of pathogenic bacteria.

描述（改编自申请人摘要）：螺旋体是一种 医学意义重大，但一个值得注意的知之甚少的群体， 细菌这些有机体引起多种重要疾病 包括梅毒、莱姆病、回归热和细螺旋体病。 它们还与牙周病和人类牙周炎有关 疾病本建议旨在回答有关下列方面的若干问题： 螺旋体周质鞭毛（PFs）的结构和运动性。 螺旋体的鞭毛是所有细菌鞭毛中最复杂的。 在PF的表面上是由一到两个 FlaA蛋白种类。该核由三至四元的聚合物组成， 这四种蛋白质被称为FlaB蛋白。的功能 单个PF蛋白还不清楚。为了更好地理解 这些蛋白质的PF结构和运动，申请人提出， 敲除几个参与PF合成的基因模型系统 被使用的是猪舌螺旋体，正式名称为密螺旋体 舌骨蕨亚科。S.猪链球菌是唯一一种 实现了特异性基因失活。这些螺旋体具有PF 与梅毒螺旋体中发现的非常相似， 梅毒T.苍白球仍不能继续培养， 单独操作用于突变体分析。因为两者的PF 微生物是如此相似，与S. hyodysenteriae 将与T相关。苍白球PF结构和运动。申请人 已经构建了三个PF突变体，flaA 1，flaB 1，flaAlflaB 1， 电穿孔和等位基因交换。他们假设这些突变体 已经改变了PF结构，并且改变的PF导致低效的 游泳.为了验证这一点，他们将分析PF的结构和运动性， 突变体，并与野生型进行比较。为了补充上述内容， 实验，他们将构建和分析其他运动突变体。 这些突变体包括flaB 2基因， 和flgE基因。后一种基因编码hook基因， 应该会导致细胞完全缺乏PFs。的 所取得的成果应有助于清楚地了解 这些蛋白质在PF结构和运动中的作用。提出的实验是 第一个用系统的分子生物学方法研究螺旋体运动的人， 遗传方法。它们尤其与鞭毛和/或 运动性被认为是螺旋体的毒力因子， 其他种类的致病菌。