Cyclic Amp and Symbiosis in a Bioluminescent Bacterium

生物发光细菌中的循环放大和共生

• 批准号: 9408266
• 负责人: Paul Dunlap
• 金额: $ 27万
• 依托单位: Woods Hole Oceanographic Institution
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9408266&HistoricalAwards=false
• 关键词: Cyclic; Amp; Symbiosis; Bioluminescent; Bacterium

9408266 Dunlap Mutualistic luminescent symbiosis between the marine bacterium Vibrio fischeri and sepiolid squids (Euprymna scolopes and E. morsei) is a model system to identify microbial determinants needed for colonization. Cyclic nucleotide metabolism in V. fischeri, which may play a fundamental role in host-symbiont interactions, will be examined from two perpsectives. The role of periplasmic 3':5'-cyclic nucleotide phosphodiesterase (CpdPase) will be determined with colonization assay of the squid using V. fischeri cpdP deletion mutants. V. fischeri cpdP deletion mutants will be prepared using molecular biology methods. Differential expression of the cpdP gene will be determined measuring CpdP enzyme activity, protein levels, and mRNA in bacteria during the initiation and maintenance phases of the symbiosis. The role of microbial adenylate cyclase and cAMP receptor protein in squid symbiosis will be observed also using squid colonization experiments with cya and crp mutants from a squid-symbiotic V. fischeri strain. For these studies, V. fischeri cya and crp genes will be obtained from a V. fischeri DNA gene library by complementation with Escherichia coli deletion mutants. Once obtained, wild-type V. fischeri genes will be inactivated and transposon insertion will be employed to produce cya and crp defective squid-symbiotic V. fischeri mutants. Loss of ability of the cya and crp deficient mutants to establish symbiosis will help us understand the molecular basis of V. fischeri squid colonization and identify the cAMP regulated genes involved. %%% Symbiotic associations between bacteria and higher organisms dominate almost all terrestrial and marine habitats. Vibrio fischeri, a marine bacterium, forms a beneficial symbiosis with certain squids. The symbiosis is a useful model system that can provide insight into the ways which animals prevent potentially pathogenic bacteria from developing disease. Preliminary evidence indicates that cyclic AMP (cA MP), a regulator of bacterial gene expression, is likely to mediate bacteria-animal interactions critical to this symbiosis. Genetic experiments are designed to identify these interactions and learn how cAMP works. Mutants of V. fischeri will be constructed that lack the gene for periplasmic cAMP phosphodiesterase, the enzyme that breaks down cAMP outside the bacterial cell, the gene for adenylate cyclase formation, and/or the gene for cAMP receptor protein, which makes bacteria to respond to cAMP. These V. fischeri mutants will be tested in squid to measure their ability to establish symbiosis. Loss of this ability will help us find V. fischeri genes necessary for the symbiosis to develop. This project is supported jointly by programs of Metabolic Biochemistry (Molecular & Cellular Biosciences) and Ecological & Evolutionary Physiology (Integrative Biology and Neuroscience) ***

9408266 Dunlap海洋细菌Vibrio fischeri与乌贼类（Euprymna spanopes和E. Morsei）是鉴定定殖所需的微生物决定因子的模型系统。 本文从两个方面对费氏弧菌的环核苷酸代谢进行了研究，环核苷酸代谢可能在宿主-共生体相互作用中发挥重要作用。 将使用V. fischeri cpdP缺失突变体通过鱿鱼的定殖测定来确定周质3 '：5'-环核苷酸磷酸二酯酶（CpdP）的作用。 将使用分子生物学方法制备费氏弧菌cpdP缺失突变体。 将通过测量在共生的起始和维持阶段期间细菌中的CpdP酶活性、蛋白质水平和mRNA来确定cpdP基因的差异表达。 微生物腺苷酸环化酶和cAMP受体蛋白在鱿鱼共生中的作用也将使用鱿鱼定殖实验与cya和crp突变体从鱿鱼共生V. fischeri菌株观察。对于这些研究，将通过与大肠杆菌缺失突变体互补从费氏弧菌DNA基因文库获得费氏弧菌cya和crp基因。一旦获得，野生型费氏弧菌基因将被灭活，并且转座子插入将被用于产生cya和crp缺陷的鱿鱼共生费氏弧菌突变体。 cya和crp缺陷突变体建立共生关系能力的丧失将有助于我们理解费氏柔鱼定殖的分子基础，并确定相关的cAMP调控基因。 细菌和高等生物之间的共生关系几乎支配着所有的陆地和海洋栖息地。 费氏弧菌是一种海洋细菌，与某些鱿鱼形成有益的共生关系。 共生是一个有用的模型系统，可以深入了解动物预防潜在致病细菌发生疾病的方式。 初步证据表明，环腺苷酸（cAMP），细菌基因表达的调节，可能是介导的细菌-动物的相互作用，这种共生关系的关键。 遗传实验旨在确定这些相互作用并了解cAMP如何工作。 将构建缺乏周质cAMP磷酸二酯酶（在细菌细胞外分解cAMP的酶）基因、腺苷酸环化酶形成基因和/或cAMP受体蛋白基因的费氏弧菌突变体，所述cAMP受体蛋白使细菌对cAMP产生应答。 这些V. fischeri突变体将在鱿鱼中进行测试，以测量它们建立共生关系的能力。 这种能力的丧失将帮助我们找到共生关系发展所必需的费氏弧菌基因。 本项目由代谢生物化学（分子细胞生物科学）和生态进化生理学（综合生物学和神经科学）项目联合支持 *