VIBRIO CHOLERAE 01 EPS--STRUCTURE, REGULATION & FUNCTION

描述（改编自申请人摘要）：流行病学 观察表明，引起亚洲霍乱的霍乱弧菌O 1存在， 作为水生植物群的土著成员， 疾病 最近，斯考尼克实验室的研究人员分离出了一种新的 胞外多糖（EPS）来自V. 霍乱O 1埃尔托尔。EPS的化学表征表明， 与O 1 LPS和O 139荚膜多糖不同， 形态学研究表明，它介导生物膜的形成， 作为细菌间基质，导致细菌聚集并附着在 无生命的表面 纯化的EPS还保护光滑的殖民地变体 氯的杀菌作用导致的同一种菌株。 插入 皱纹变体的诱变导致了迄今为止的鉴定， 未知的基因簇，命名为vps，具有共享同源性的序列 与其他物种的多糖生物合成基因。 另一类 突变体持续插入与双组分调控基因同源的基因中， 系统. 这些初步数据导致了这样的假设，即每股收益 表达赋予在环境水生栖息地中的生存优势。 为了验证这一假设，Schoolnik博士建议完成结构 表征EPS;表征vps基因簇;制备敲除 选择的VPS基因的突变体，并测试这些突变体的能力， 在模拟自然水生栖息地的实验性小宇宙中生存。 博士Schoolnik的研究小组还将确定生理因素， EPS的表达，并研究这些信号如何激活vps基因转录。 最后，在孟加拉国进行的合作研究中，他们将尝试 了解霍乱弧菌O 1是否在自然感染的水中表达EPS 源 综合起来，这些研究应该能解释为什么 该物种继续作为“重新出现”的传染媒介引起流行病。 拨款建议分为三个具体目标：（1）继续 检查EPS的结构和生物合成， 物理/化学结构表征，并通过识别基因 vps基因簇的突变分析和互补 克隆 (2)利用vps基因研究vps基因的表达调控 融合体为了确定VPS基因在不同生长条件下的表达， 包括生物膜形成、鼠肠道生长和藻类共培养。 鉴定调控基因介导的这种调控的分离， 突变体的调节改变。 (3)研究EPS的功能， 确定VPS突变体在各种条件下对存活的影响， 并确定存在于V上的EPS和O 1 LPS的相对丰度。 孟加拉国生物膜层中发现的霍乱。 确定是否存在 皱纹形态型和EPS产量之间的直接相关性。

DESCRIPTION (Adapted from the applicant's abstract): Epidemiological observations suggest that V. cholerae O1, that cause Asiatic cholera, exists as an indigenous member of the aquatic flora between outbreaks of human disease. Recently investigators in the Schoolnik lab have isolated a novel extracellular polysaccharide (EPS) from the rugose colonial variant of V. cholerae O1 El Tor. Chemical characterization of EPS shows that it is distinct from the O1 LPS and the O139 capsular polysaccharide and morphological studies suggest that it mediates biofilm formation by serving as an interbacterial matrix that causes bacteria to aggregate and attach to inanimate surfaces. Purified EPS also protects the smooth colonial variant of the same strain from the bactericidal effects of chlorine. Insertion mutagenesis of the rugose variant led to the identification of a heretofore unknown cluster of genes, designated vps, with sequences that share homology with polysaccharide biosynthetic genes from other species. Another class of mutants sustained insertions in genes homologous to two-component regulatory systems. These preliminary data have led to the hypothesis that EPS expression confers a survival advantage in environmental aquatic habitats. To test this hypothesis Dr. Schoolnik proposes to complete the structural characterization of EPS; characterize the vps gene cluster; prepare knockout mutants of selected vps genes and test these mutants for their capacity to survive in experimental microcosms that simulate natural aquatic habitats. Dr. Schoolnik's group will also identify physiologic factors that control EPS expression and study how these signals activate vps gene transcription. Finally, in collaborative studies conducted in Bangladesh, they will attempt to learn if V. cholerae O1 expresses EPS in naturally infected water sources. Taken together, these studies should shed some light on why this species continues to cause epidemics as a "re-emerging" infectious agent. The grant proposal is divided into three specific aims:(1) Continue to examine the structure and biosynthesis of EPS by completing the physical/chemical structural characterization, and by identifying the genes of the vps gene cluster by mutational analysis and complementation on clones. (2) Study the regulation of vps gene expression using vps gene fusions to ascertain vps gene expression under different growth conditions, including biofilm formation, murine intestinal growth, and algal co-culture. Identify regulatory genes that mediate this regulation by the isolation of mutants with altered regulation. (3) Study the function of EPS by determining the effects of vps mutants on survival under various conditions, and ascertaining the relative abundance of EPS and O1 LPS present on V. cholerae found in biofilm layers in Bangladesh. Determine if there is a direct correlation between the rugose morphotype and EPS production.