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

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。SAR11分支的代表被重新命名为Pelagibacter ubique，它是大约10年前通过克隆其16S核糖体RNA而被发现的。这些革兰氏阴性的a-蛋白细菌引起了科学家的兴趣，因为它们是迄今为止已知的最小的自主复制细胞之一，但它们是地球上最普遍、数量最多的微生物之一。尽管细菌组合对生物地球化学过程(例如海洋碳循环)很重要，但人们对这些海洋细菌的生理学或生态学知之甚少。为了更多地了解这些重要生物的生物学，我们正在使用冷冻电子断层扫描技术来研究它们在冷冻-水合状态下的三维结构。细胞生长到对数晚期，然后通过跳入液态乙烷进行快速冷冻。使用显微镜控制程序SerialEM，在Tecnai F-30显微镜上记录了几个单轴倾斜系列的玻璃化细胞。然后，使用IMOD、EM和AMIRA软件包对各个倾斜系列图像进行对准、重建、分析和可视化。断层重建清楚地显示了Pelagibacter细胞的整体形态和明显的亚细胞结构。此外，在断层图像的重要区域，分辨率足以区分大分子复合体，如肽聚糖层、核糖体样颗粒和菌毛样细丝。我们还在分析Pelagibacter中核糖体的细胞密度。结果表明，与其他细菌相比，这些生长缓慢的小型a-蛋白细菌具有明显的核糖体过剩。尽管我们仍在分析我们的数据，但这些发现表明，这类研究将对小细胞的组织和大分子结构产生新的见解。

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.