Cellular Signals Mediating Symbiosis in Vibrio fischeri

介导费氏弧菌共生的细胞信号

• 批准号: 9722972
• 负责人: Paul Dunlap
• 金额: --
• 依托单位: University of Maryland Biotechnology Institute
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-15 至 2001-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9722972&HistoricalAwards=false
• 关键词: Cellular; Signals; Mediating; Symbiosis; Vibrio

9722972 DUNLAP This project will test the hypotheses that the signal molecules 3' :5'-cyclic AMP (cAMP) and N-acyl-homoserine lactones (autoinducers) play key roles in the bioluminescent mutualism of Vibrio fischeri with the sepiolid squid Euprymna scolopes. Two classes of mutants will be used in colonization assays with aposymbiotic hatchling squid to test the cAMP-CRP hypothesis, mutants defective in adenylate cyclase and CRP, constructed using the suicide vector pSMC300, and mutants defective in cAMP-CRP-regulated proteins, identified and isolated by 2-D gel electrophoresis and through transposon-mediated lacZ insertional mutagenesis. Preliminary evidence suggests that the periplasmic 3' :5'-CNP of V.fischeri also plays a key role in its symbiosis with E. scolopes. The enzyme is hypothesized to mediate bacterial metabolism of extracellular, host-produced cAMP in the animal's light organ, and diminished symbiosis competency of the mutant would imply that cAMP is a growth substrate or key signal for the bacteria in the light organ. To address 3' :5 ' -CNP hypothesis, colonization initiation capability and retention of the cpdP mutant will be examined and levels of 3' :5 ' -CNP activity, CpdP protein, and cpdP mRNA will be quantified at several points during development of the animal. To test the autoinducer hypothesis, two classes of mutants will be used in squid colonization assays, an autoinducer-deficient mutant defective in the two V. fischeri autoinducer synthases, LuxI and AinS, constructed using the suicide vector pSMC300 and transposon mutagenesis, and mutants defective in production of autoinducer-dependent proteins, identified and isolated by 2-D gel electrophoresis and through transposon-mediated lacZ insertional mutagenesis. This research will form a foundation for understanding the complex network of inter- and intra-cellular signals and responses through which a bacterium senses and adapts to mutualistic host association. The mutually beneficial symbiosis between the marine b acterium Vibrio fischeri and the squid Euprymna scolopes serves as an important model system for determining how a bacterium colonizes and persists in an animal host. Symbiosis between bacteria and animals is common in terrestrial and marine habitats and has a substantial environmental importance, in some cases providing the biological foundation for an ecosystem. Consequently, knowledge gained from detailed study of one assocation is likely to provide widely significant ecological insight. Knowledge of the bacterial signals and the bacterium-animal biochemical communication is of fundamental importance for understanding how mutualisms are successfully established and maintained and how biologically different organisms cooperate to survive and reproduce. This project consequently focuses on key bacterial intracellular and intercellular signals, because of their central importance in gene expression and their likely importance for host-association.

9722972 DUNLAP 该项目将测试以下假设：信号分子 3':5'-环 AMP (cAMP) 和 N-酰基高丝氨酸内酯（自诱导剂）在费氏弧菌与海泡乌贼 Euprymna scolopes 的生物发光互利作用中发挥关键作用。两类突变体将用于非共生幼体鱿鱼的定植测定中，以测试 cAMP-CRP 假说，腺苷酸环化酶和 CRP 缺陷突变体，使用自杀载体 pSMC300 构建，以及 cAMP-CRP 调节蛋白缺陷突变体，通过 2-D 凝胶电泳和转座子介导的 lacZ 插入诱变进行鉴定和分离。初步证据表明，V.fischeri 的周质 3':5'-CNP 在其与 E. scolopes 的共生中也发挥着关键作用。假设该酶介导动物光器官中细胞外宿主产生的 cAMP 的细菌代谢，突变体共生能力的减弱意味着 cAMP 是光器官中细菌的生长底物或关键信号。为了解决 3':5'-CNP 假说，将检查 cpdP 突变体的定植起始能力和保留，并在动物发育过程中的几个点对 3':5'-CNP 活性、CpdP 蛋白和 cpdP mRNA 的水平进行定量。为了检验自诱导剂假说，将在鱿鱼定植测定中使用两类突变体，一种是在两种费氏弧菌自诱导剂合酶 LuxI 和 AinS 中存在缺陷的自诱导剂缺陷型突变体，使用自杀载体 pSMC300 和转座子诱变构建，另一种是在产生自诱导剂依赖性蛋白方面存在缺陷的突变体，由以下方法鉴定和分离： 二维凝胶电泳和转座子介导的 lacZ 插入突变。这项研究将为理解细胞间和细胞内信号和反应的复杂网络奠定基础，细菌通过该网络感知和适应互利的宿主关联。 海洋细菌费氏弧菌和乌贼 Euprymna scolopes 之间的互利共生是确定细菌如何在动物宿主中定殖和持续存在的重要模型系统。 细菌和动物之间的共生在陆地和海洋栖息地中很常见，并且具有重大的环境重要性，在某些情况下为生态系统提供了生物基础。因此，从对某一协会的详细研究中获得的知识可能会提供具有广泛意义的生态见解。 了解细菌信号和细菌-动物生化通讯对于理解互利共生如何成功建立和维持以及生物学上不同的生物体如何合作生存和繁殖至关重要。因此，该项目重点关注关键的细菌细胞内和细胞间信号，因为它们在基因表达中至关重要，并且可能对宿主关联也很重要。