9317334 STEIN Closely integrated symbiotic associations between bacteria and eukaryotic hosts abound in nature. This is particularly the case in marine systems where novel associations are being routinely discovered. Whether the bacteria reside externally to the host or endosymbiotically the functional role of the association have remained poorly understood. This research project seeks to understand the structure and function of the diverse epibiotic bacterial populations found associated with the Alvinellid polychaete, Alvinella pompejana, which inhabits the high temperature environment surrounding deep-sea hydrothermal vents. The polychaetes are considered the most thermotolerant eukaryotes known. The primary thesis of the project maintains that within the evolutionary confines of this symbiosis the symbiont and host have evolved unique adaptations to survive this harsh environment. The primary objective of this research project is to metabolically characterize the predominant symbionts within the mixed population in order to determine their functional role in the symbiosis. Specifically the project will: 1) phylogenetically characterize the dominant symbionts in the population, 2) determine the spatial orientation and metabolic state of these symbiont in situ, 3) determine the metabolic capabilities and segregation of activity within the structure of the symbiont population. In order to achieve these goals two powerful molecular techniques which were developed with the philosophy that bacteria from natural ecosystems, like organelles, can be studied without cultivation will be integrated. Past studies have demonstrated the utility of nucleic acid probing technology to identify and localize target sequences in situ with a high degree of specificity. The utility of Bacterial Artificial Chromosome (BAC) vectors to characterize mixed marine microbial communities by preserving their genomic content in the form of "environmental libraries" has recently bee n demonstrated. This project proposes to combine these technologies to genetically dissect the predominant symbionts from the mixed population and to identify and quantify in situ the expression of metabolically important genes implicated in the symbiosis. The descriptive nature of these techniques will provide not only valuable insights into the ecology of this symbiosis but will provide characterization of novel genes that confer adaptation to the extremes of the hydrothermal vent environment. ***
