RUI: Molecular and AFM Analysis of a Novel Periplasmic Structure

RUI：新型周质结构的分子和 AFM 分析

• 批准号: 1052127
• 负责人: Douglas Dennis
• 金额: $ 19.9万
• 依托单位: Morehead State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1052127&HistoricalAwards=false
• 关键词: RUI; Molecular; AFM; Analysis; Novel

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.

知识分子值得这项研究的科学优点主要是对细菌结构的增强理解。现在，显然，细菌在细胞骨架元素方面比以前认为的要复杂得多，而该领域的核研究领域在过去10年中产生了重要的信息。在初步研究中，暂时确定了细胞质膜周质表面上的独特曲线阵列。 该网络可能具有细菌代谢和生长的重要功能，其研究将扩大我们对原核生物过程的了解。为Cupriavidus Necator开发了一种渗透裂解程序，从而导致使用原子力显微镜（AFM）成像的完整亚细胞结构的产生。尽管其中一些结构，例如细胞鬼，但其他结构是新颖的。特别是，已经成像了带有类似的曲面的结构，该结构表面上有一个有组织的曲线阵列，表面上是由蛋白质组成的。曲线阵列由短的50-250 nm长，宽30-50 nm，缝在细胞质膜的周质脸上。曲线阵列本质上可能是动态的，因为当C. necator以最小的培养基生长时，它的形式是短弯曲段的形式，但是当C. necator在富含培养基的初步实验中生长时，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结构表征。为了完成这种优化的条件，将使用用于现场发射扫描电子显微镜和透射电子显微镜分析的SR球体，以确认阵列的存在并获得阵列成分的更准确的横向尺寸。由于最近在大肠杆菌细胞幽灵的表面成像了阵列，因此增加了阵列是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，并将寻求使用AFM，SEM和TEM的曲线表面网络存在的证据。这项研究的更广泛影响在于通常来自弱势教育背景的学生的科学教育领域。尽管MSU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，职业志向有限。对于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当。参与该研究项目不仅会以研究方式教育他们，还可以向他们证明，科学研究的职业并不是无法实现的。