Cellular Signals Mediating Symbiosis in Vibrio fischeri

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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.