RUI: Molecular and AFM Analysis of a Novel Periplasmic Structure

这项研究的科学价值主要在于增进了对细菌结构的了解。现在很明显，细菌在细胞骨架元素方面比以前认为的要复杂得多，这一领域的原核研究在过去10年里取得了重要的成果。在初步的研究中，细胞质膜的质周表面有一个独特的曲线阵列。这个网络可能对细菌的代谢和生长具有重要的功能，对它的研究将扩大我们对原核过程的认识。利用原子力显微镜（AFM）成像，研究人员开发了一种渗透裂解方法，可以生成完整的亚细胞结构。虽然其中一些结构，如细胞幽灵，是已知的，但其他的是新的。特别是，一个球质体样结构已经被成像，在其表面上具有一个有组织的曲线阵列，表面上由蛋白质组成。曲线阵列由短的50-250纳米长，30-50纳米宽的片段组成，这些片段蜿蜒穿过细胞质膜的质周面。曲线阵列在本质上可能是动态的，因为当C. necator在最小的培养基中生长时，它是短弯曲段的形式，但当C. necator在丰富的培养基中生长时，初步实验表明它更像是一个网络。这个项目的重点是曲线阵列/网络的确认和进一步的结构表征。为了做到这一点，优化条件将用于获得SR球质体进行场发射扫描电镜和透射电镜分析，以确认阵列的存在并获得更准确的阵列成分的横向尺寸。由于该阵列最近也在大肠杆菌细胞幽灵的表面上进行了成像，因此提出了一种可能性，即该阵列是所有细菌细胞中都存在的结构。为此，将在不同的细菌种类中进行裂解实验，并利用原子力显微镜、扫描电镜和透射电镜寻找曲线表面网络存在的证据。更广泛的影响这项研究的更广泛的影响是在学生的科学教育领域，一般来说，来自不利的教育背景。虽然密歇根州立大学的学生非常有能力，但他们受到不合格的高中教育的阻碍，更重要的是，他们的职业抱负有限。对他们中的许多人来说，成为研究科学家的想法和成为宇航员的机会是一样的。参与这个研究项目不仅能教会他们研究的方法，还能向他们证明，从事科学研究并非遥不可及。

Intellectual merit The scientific merit of this research is primarily in an enhanced understanding of bacterial structure. It is now apparent that bacteria are much more complex with regards to cytoskeletal elements than previously thought and this area of prokaryotic research has yielded significant information in the last 10 years. A unique curvilinear array on the periplasmic surface of the cytoplasmic membrane has been, tentatively, identified in preliminary studies. It is possible that this network possesses significant functionalities for bacterial metabolism and growth and its study would expand our knowledge of prokaryotic processes.An osmotic lysis procedure has been developed for Cupriavidus necator that results in the generation of intact subcellular structures, as imaged using atomic force microscopy (AFM). While some of these structures, such as cell ghosts, are known, others are novel. In particular, a spheroplast-like structure has been imaged that possesses an organized curvilinear array on its surface, ostensibly comprised of protein. The curvilinear array is comprised of short 50-250 nm long, and 30-50 nm wide, segments snaking across the periplasmic face of the cytoplasmic membrane. The curvilinear array may be dynamic in nature because when C. necator is grown in minimal medium it is in the form of short curved segments, but when C. necator is grown in rich medium preliminary experiments suggest that it is more of a network. The focus of this project is the confirmation and further structural characterization of the curvilinear array/network. To do this optimized conditions will be used to obtain SR spheroplasts for field emission scanning electron microscopy and transmission electron microscopy analyses to confirm the existence of the array and obtain more accurate lateral dimensions for the array constituents. Because the array has recently also been imaged on the surface of Escherichia coli cell ghosts, it raises the possibility that the array is a structure found in all bacterial cells. To this end, lysis experiments will be conducted in different bacterial species and evidence for the existence of the curvilinear surface network will be sought employing AFM, SEM and TEM. Broader ImpactThe broader impact of this research is in the area of science education of students that are, generally, from disadvantaged educational backgrounds. While the students at MSU are extremely capable, they are hindered by substandard high school educations and, more importantly, limited career aspirations. For many of them the idea of becoming a research scientist is on a par with their chances of becoming an astronaut. Participation in this research project would not only educate them in the ways of research, it would prove to them that a career in scientific research is not unattainable.