Cyclic Amp and Symbiosis in a Bioluminescent Bacterium

9408266费氏弧菌和海鞘乌贼(Euprymna scolope和E.morsei)之间的邓拉普互惠发光共生是确定定居所需微生物决定因素的模式系统。费氏弧菌的环核苷酸代谢可能在寄主-共生体相互作用中发挥重要作用，我们将从两个角度对其进行研究。周质3‘：5’-环核苷酸磷酸二酯酶(CpdPase)的作用将通过利用裂纹夜蛾CPDP缺失突变体对鱿鱼的定植试验来确定。将利用分子生物学方法制备V.fecheri CPDP缺失突变体。在共生的起始和维持阶段，将通过测量细菌中CPDP酶的活性、蛋白质水平和mRNA来确定CPDP基因的差异表达。微生物腺苷环化酶和cAMP受体蛋白在鱿鱼共生中的作用也将通过对鱿鱼共生弧菌菌株的cyA和CRP突变体的鱿鱼定植实验来观察。在这些研究中，将通过与大肠杆菌缺失突变体的互补，从费氏弧菌DNA文库中获得费氏弧菌的cyA和CRP基因。一旦获得，野生型费氏弧菌基因将被灭活，转座子插入将被用来产生CyA和CRP缺陷的鱿鱼-共生弧菌突变体。缺失CyA和CRP的突变体建立共生关系的能力的丧失将有助于我们了解乌贼定植的分子基础，并鉴定参与cAMP调控的基因。细菌和高等生物之间的共生关系支配着几乎所有的陆地和海洋生境。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式系统，可以为动物预防潜在致病细菌发展疾病的方式提供洞察。初步证据表明，环状AMP(CA MP)是细菌基因表达的调节因子，很可能在细菌与动物之间的相互作用中起着至关重要的作用。基因实验旨在确定这些相互作用，并了解cAMP是如何工作的。将构建缺乏周质cAMP磷酸二酯酶基因、在细菌细胞外分解cAMP的酶、腺苷环化酶形成的基因和/或cAMP受体蛋白基因的突变株，cAMP受体蛋白使细菌对cAMP做出反应。这些鱼类弧菌突变体将在鱿鱼身上进行测试，以衡量它们建立共生的能力。失去这种能力将有助于我们找到共生发展所必需的费氏弧菌基因。本项目由代谢生物化学(分子与细胞生物科学)和生态与进化生理学(综合生物学与神经科学)联合资助*

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) ***