Collaborative Research: Molecular Dissection of an Epibiotic Symbiosis in a Highly Thermotolerant Metazoan

细菌与真核宿主之间紧密结合的共生关系在自然界中比比皆是。这在海洋系统中尤其如此，因为新的关联经常被发现。无论细菌是寄生在宿主外部还是内共生，这种关联的功能作用仍然知之甚少。该研究项目旨在了解与深海热液喷口周围高温环境中的Alvinella pompejana （Alvinella polychaete）相关的多种表观细菌种群的结构和功能。多毛体被认为是已知的最耐热的真核生物。该项目的主要论点认为，在这种共生关系的进化范围内，共生体和宿主已经进化出独特的适应能力，以在这种恶劣的环境中生存。本研究项目的主要目的是对混合种群中优势共生体的代谢特征进行表征，以确定它们在共生中的功能作用。具体而言，该项目将：1)种群中优势共生体的系统发育特征；2)确定这些共生体在原位的空间取向和代谢状态；3)确定共生体种群结构内的代谢能力和活动分离。为了实现这些目标，两种强大的分子技术将被整合起来，这两种技术的发展理念是，来自自然生态系统的细菌，如细胞器，可以在没有培养的情况下进行研究。过去的研究已经证明了核酸探测技术在原位识别和定位目标序列方面具有很高的特异性。细菌人工染色体（BAC）载体通过以“环境文库”的形式保存其基因组内容来表征混合海洋微生物群落的效用最近得到了证实。本项目建议将这些技术结合起来，从混合种群中对优势共生体进行遗传解剖，并原位鉴定和量化共生体中代谢重要基因的表达。这些技术的描述性质不仅将为这种共生的生态提供有价值的见解，而且将提供赋予适应极端热液喷口环境的新基因的特征。＊＊＊

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. ***