THREE DIMENSIONAL RECONSTRUCTIONS OF ARCHAEA

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The phylogenetic tree of all known organisms comprises three distinct branches: Eucarya, Bacteria and Archaea. The Archaeal branch was only recently recognized as a coherent group that is phylogenetically distinct from Bacteria. Our knowledge of the fine structure of prokaryotic cells (Archaea and Bacteria) is significantly less than what we know about their biochemical and molecular organization. This is due primarily to limitations in preservation methods and, to some extent, in instrumentation. Advances in modern techniques of rapid freezing and freeze-substitution have overcome some of these problems, yielding elegant preservation of some prokaryotic organisms. Since molecular studies suggest that Archaea have more in common with Eukaryotes than with Bacteria, in terms of transcription, translation and other DNA related processing mechanisms, the morphology of these organisms may yield a wealth of biological information. Whole cell images of high-pressure frozen, freeze-substituted Sulfolobus solfataricus have been captured via serial section tomography. As a control, whole cell images of the bacterium Salmonella have been prepared in an identical manner. The whole cell tomograms have allowed the enumeration of several different structures in Sulfolobus, at least one of which varies according to the presence or absence of glucose in the medium in which the cells are grown. DNA preservation, previously a problem, is currently quite good with the addition of the sucrose polymer Ficoll-70 to the growth medium just prior to freezing. Lowicryl embedded Sulfolobus has been successfully probed for DNA and verified by tomographic ribosome exclusion models. Work will continue on the characterization of nuclear body structure of Sulfolobus and Salmonella during the next year. The principal rate determining step going forward is the labor intensive nature of segmentation.