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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. Work conducted in previous years has resulted in sample preparation protocols that routinely yield very high quality full-cell tomograms of these two organisms. Custom software created by Dr. Robertson automatically segments the tomographic image stacks for the major biomolecules which stain with heavy metals, (ribosomes, cell membrane, etc) in Salmonella and Sulfolobus. The automatic segmentation software provides the means to enumerate these biomolecules as the organisms respond to a variety of metabolic states such as heat shock, oxidative stress (exposure to hydrogen peroxide), or changes in growth medium (eg sugar/no sugar). The auto-segmentation software also generates structural bioinformatic models of the cells which may be used for in silico experimentation. The tomographic work has been complemented with immuno-EM of Lowicryl embedded Sulfolobus, has been successfully probed for DNA.