RUI: Molecular and AFM Analysis of a Novel Periplasmic Structure

智力价值这项研究的科学价值主要在于加强对细菌结构的理解。现在很明显，细菌在细胞骨架元素方面比之前认为的要复杂得多，这一原核生物研究领域在过去10年里产生了重要的信息。在初步研究中，在细胞质膜的周质表面上初步鉴定出一个独特的曲线排列。这个网络可能对细菌的新陈代谢和生长具有重要的功能，它的研究将扩大我们对原核过程的了解。一种针对坏死型铜绿假单胞菌的渗透裂解程序已经开发出来，导致产生完整的亚细胞结构，如原子力显微镜(AFM)所示。虽然这些结构中的一些是已知的，如细胞幽灵，但其他结构是新的。特别是，成像的球体样结构在其表面拥有一个有组织的曲线阵列，表面上由蛋白质组成。该曲线阵列由50-250 nm长和30-50 nm宽的短片段组成，这些片段蜿蜒穿过细胞膜的周质面。曲线阵列在本质上可能是动态的，因为当在最小的介质中生长时，它是短曲线段的形式，但当在丰富的介质中生长时，初步实验表明它更像是一个网络。该项目的重点是曲线阵列/网络的确认和进一步的结构表征。为此，优化的条件将被用来获得SR球体，用于场发射、扫描电子显微镜和透射电子显微镜分析，以证实阵列的存在，并获得更准确的阵列成分的横向尺寸。由于该阵列最近也被成像在大肠杆菌细胞幽灵的表面，这增加了该阵列是在所有细菌细胞中发现的结构的可能性。为此，将在不同的细菌物种中进行裂解实验，并将利用AFM、扫描电子显微镜和电子显微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，这些学生通常来自弱势教育背景。虽然密歇根州立大学的学生非常有能力，但他们受到了不符合标准的高中教育的阻碍，更重要的是，他们的职业抱负有限。对他们中的许多人来说，成为一名研究科学家的想法与他们成为宇航员的机会相同。参与这项研究项目不仅会对他们进行研究方面的教育，还会向他们证明，从事科学研究并不是遥不可及的。

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.