Cyclic Amp and Symbiosis in a Bioluminescent Bacterium

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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) ***