Cellular Signals Mediating Symbiosis in Vibrio fischeri

本项目将验证信号分子3':5'-环AMP （cAMP）和n -酰基-同丝氨酸内酯（自诱导性因子）在费氏弧菌（Vibrio fischeri）与鱿鱼Euprymna scolopes的生物发光共生中起关键作用的假设。两类突变体将用于异共生孵化鱿鱼的定植试验，以验证cAMP-CRP假说，使用自杀载体pSMC300构建的腺苷酸环化酶和CRP缺陷突变体，以及cAMP-CRP调节蛋白缺陷突变体，通过2d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，fischeri的胞质外3':5'-CNP在其与E. scopes的共生中也起着关键作用。据推测，该酶可以介导动物光器官中宿主产生的细胞外cAMP的细菌代谢，而突变体的共生能力下降可能意味着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，将检测cpdP突变体的定植起始能力和保留能力，并在动物发育过程的几个点上量化3’：5’-CNP活性、cpdP蛋白和cpdP mRNA的水平。为了验证自诱导剂假说，两类突变体将用于鱿鱼定植试验，一种是利用自杀载体pSMC300和转座子诱变技术构建的fischeri自诱导剂合成酶LuxI和AinS存在缺陷的自诱导剂缺陷突变体，另一种是通过2-D凝胶电泳和转座子介导的lacZ插入诱变技术鉴定和分离的自诱导剂依赖蛋白产生缺陷的突变体。这项研究将为理解细菌感知和适应互惠宿主关联的细胞间和细胞内信号和反应的复杂网络奠定基础。海洋细菌费氏弧菌（Vibrio fischeri）与乌贼（Euprymna scolopes）之间的互利共生关系是确定细菌如何在动物宿主中定植和持续存在的重要模型系统。细菌和动物之间的共生关系在陆地和海洋生境中很常见，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，从一种关联的详细研究中获得的知识可能会提供广泛重要的生态学见解。了解细菌信号和细菌-动物的生化交流对于理解共生关系是如何成功建立和维持的，以及生物学上不同的生物体是如何合作生存和繁殖的，是至关重要的。因此，该项目侧重于关键的细菌细胞内和细胞间信号，因为它们在基因表达和宿主关联中可能具有重要意义。

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.