Graduate Thesis Or Dissertation
 

Phylogenetic characterization of the epibiotic bacteria associated with the hydrothermal vent polychaete Alvinella pompejana

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  • Symbiotic relationships of bacteria with higher organisms are commonly observed in nature; however, the functional role of these relationships is only rarely understood. This is particularly evident in epibiotic bacterial associations in the marine environment where the bacteria are often a diverse ensemble of microorganisms, thus complicating the identification of the functionally important members. Classical microbiological techniques, relying primarily on culturing these organisms, have provided an incomplete picture of these relationships. Molecular genetic techniques, focusing on the analyses of bacterial 16S rRNA sequences cloned directly from natural microbial populations, are now available which allow a more thorough examination of these associated bacterial populations. This study sought to characterize the epibiotic bacterial population associated with a very unique organism, Alvinella pompejana, using such a molecular approach. Alvinella pompejana is a polychaetous annelid that inhabits active deep-sea hydrothermal vent sites along the East Pacific Rise. This worm colonizes the walls of actively venting high temperature chimneys and is thought to be one of the most thermotolerant metazoans known. The chimney environment is characterized by high concentrations of sulfide and heavy metals in the vicinity of the worm colonies. A morphologically diverse epibiotic microflora is associated with the worm's dorsal integument, with a highly integrated filamentous morphotype clearly dominating the microbial biomass. It has been suggested that this bacterial population participates in either the nutrition of the worm or in detoxification of the worm's immediate environment; however, previous studies have been unable to confirm such a role. The primary goal of this study is to phylogenetically characterize the dominant epibionts through the analysis of 16S rRNA gene sequences. Nucleic acids were extracted from bacteria collected from the dorsal surface of Alvinella pompejana. 16S rRNA genes were amplified with universal bacterial primers by the polymerase chain reaction (PCR). These genes were subsequently cloned and the resulting clone library was screened by restriction fragment length polymorphism (RFLP) analysis to identify unique clone types. Thirty-two distinct clone families were found in the library. Four of these families were clearly dominant, representing over 65% of the library. The main assumption in this study is that the numerical dominance of the phylotypes in the starting population will be reflected in the clone library. Thus, representative clones from the four most abundant clone families were chosen for complete gene sequencing and phylogenetic analysis. These gene sequences were analyzed using a variety of phylogenetic inference methods and were found to be related to the newly established epsilon subdivision of the Proteobacteria. In future studies, these gene sequences will be used to construct specific oligodeoxynucleotide probes which can be used to confirm the morphology of the clone types in the epibiont population.
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