Cellular Signals Mediating Symbiosis in Vibrio fischeri

9722972 DUNESTIC本项目将测试信号分子3'：5'-环腺苷酸（cAMP）和N-酰基高丝氨酸内酯（自诱导物）在费氏弧菌与乌贼Euprymna speopes的生物发光互惠共生中发挥关键作用的假设。两类突变体将被用于与离体共生的孵化鱿鱼的定殖试验中，以测试cAMP-CRP假设，腺苷酸环化酶和CRP缺陷的突变体，使用自杀载体pSMC 300构建，和cAMP-CRP调节蛋白缺陷的突变体，通过2-D凝胶电泳和通过转座子介导的lacZ插入诱变鉴定和分离。初步证据表明，费氏弧菌的周质3'：5'-CNP在其与大肠杆菌的共生中也起着关键作用。我是说...假设该酶介导细胞外的细菌代谢，宿主产生的cAMP在动物的光器官中，并且突变体的共生能力减弱将意味着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假设，将检查cpdP突变体的定殖起始能力和保留，并在动物发育期间的几个时间点定量3'：5 ' -CNP活性、CpdP蛋白和cpdP mRNA的水平。为了检验自诱导物假说，在鱿鱼定殖测定中使用两类突变体，即使用自杀载体pSMC 300和转座子诱变构建的两种费氏弧菌自诱导物酶LuxI和AinS中有缺陷的自诱导物缺陷突变体，以及自诱导物依赖性蛋白质产生中有缺陷的突变体，通过2-D凝胶电泳和通过转座子介导的lacZ插入突变鉴定和分离。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，细菌通过这些信号和反应感知和适应互惠宿主协会。 海洋B杆菌费氏弧菌和鱿鱼Euprymna spectopes之间的互利共生关系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现象在陆地和海洋生境中很常见，具有重大的环境意义，在某些情况下为生态系统提供了生物基础。因此，从对一个群丛的详细研究中获得的知识很可能提供广泛的重要生态学见解。 了解细菌信号和细菌-动物生物化学通讯对于理解互惠关系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具有根本的重要性。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中的核心重要性和它们对宿主相关性的可能重要性。

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.