3D STRUCTURE OF TINY BACTERIUM SAR11 STUDIED BY CRYO-ELECTRON TOMOGRAPHY

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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 SAR11-clade, whose representatives have been renamed Pelagibacter ubique, was discovered about a decade ago by the cloning of its 16S ribosomal RNA. These gram-negative a-proteobacteria have since intrigued scientists, because they are among the smallest autonomously replicating cells so far known, yet they are one of the most ubiquitous and numerically abundant microorganisms on the planet. Despite the importance of bacterial assemblages to biogeochemical processes (e.g. the oceanic carbon cycle), little is known about the physiology or ecology of these marine bacteria. To learn more about the biology of these important organisms we are using cryo-electron tomography to study their 3-D structure in the frozen-hydrated state. The cells were grown to late log-phase and then rapidly frozen by plunging into liquid ethane. Several single-axis tilt series of vitrified cells have been recorded on a Tecnai F-30 microscope, using the microscope control program, SerialEM. Individual tilt series images have then been aligned, reconstructed, analyzed and visualized using the IMOD, EM and AMIRA software packages. Tomographic reconstructions clearly reveal the overall morphology and distinct subcellular organization of the Pelagibacter cells. Furthermore in significant areas of the tomograms the resolution is sufficient to distinguish macromolecular complexes, such as peptidoglycan-layers, ribosome-like particles and pilus-like filaments. We are also analyzing the cellular density of ribosomes in Pelagibacter. The results indicate that compared to other bacteria, these small and slow-growing a-proteobacteria have an apparent excess of ribosomes. Although we are still analyzing our data, these findings indicate that novel insights about the organization and macromolecular structure of small cells will arise from studies of this kind.