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

通过低温电子断层扫描技术研究微小细菌 SAR11 的 3D 结构

• 批准号: 7354995
• 负责人: DANIELA NICASTRO
• 金额: $ 0.94万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7354995
• 关键词: bacteria; ribosomes; tomography

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.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。sar11分支，其代表已被重新命名为泛层杆菌，是大约十年前通过克隆其16S核糖体RNA而被发现的。这些革兰氏阴性a-变形菌引起了科学家们的兴趣，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（如海洋碳循环）具有重要意义，但对这些海洋细菌的生理或生态学知之甚少。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来研究它们在冻水状态下的三维结构。将细胞培养到log-phase晚期，然后将其放入液态乙烷中快速冷冻。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软件包对单个倾斜系列图像进行对齐，重建，分析和可视化。层析重建清楚地揭示了Pelagibacter细胞的整体形态和独特的亚细胞组织。此外，在断层扫描的重要区域，分辨率足以区分大分子复合物，如肽聚糖层，核糖体样颗粒和毛状细丝。我们还分析了Pelagibacter中核糖体的细胞密度。结果表明，与其他细菌相比，这些小而生长缓慢的a-变形杆菌具有明显过量的核糖体。虽然我们仍在分析我们的数据，但这些发现表明，关于小细胞的组织和大分子结构的新见解将从这类研究中产生。