Glycan Expression Analysis using an Odyssey CLx Infrared Imaging System

项目总结 细菌细胞的形状具有基本的和医学上的重要性，通过以下方式有助于生存和毒力 影响营养物质的摄取、细胞与表面的附着、运动性、分化和对捕食或 宿主豁免权。此外，形态研究正在阐明以前未被认识到的相互作用 在推动细菌生长的各种生化事件中。简而言之，形态在生物进化中起着至关重要的作用 细菌生理学、生态学和发病机制，是了解细胞生物学的窗口。大多数细菌 通过制造肽聚糖细胞壁来确定它们的形状，干扰这种结构是 抗菌治疗最重要的靶点。尽管进行了几十年的工作，但在以下方面仍然存在巨大差距 了解肽聚糖的合成是如何与相关的代谢途径整合的。一次整合 重点是载体分子十一烯基磷酸(und-P)，它有助于合成肽多糖和其他 低聚糖。很明显，und-P及其衍生品形成了一个至关重要的联系，必须是 其特点是有助于对抗日益增长的抗生素耐药性。为此，我们提出如下建议 目标。目的1]确定Und-P管理如何改变细菌的生理和生化。Und-P和 它的同系物将许多化合物和聚合物运输到细胞的细胞膜上 生物界，包括古生物和真核生物。因为最终产物是由不同的 路线，载体必须在几个生化途径之间共享，但这是如何实现的 未知。然而，它是至关重要的--如果und-P被隔离在一条途径上，那么肽聚糖的可利用性就会减少 合成和细胞生长不良或死亡。我们将开发工具来监测活细胞中Und-P的数量 并将决定对und-P的竞争如何影响细菌的生长和每个途径的操作。 目的2]确定和表征新的和鲜为人知的Und-P利用途径。尽管有几个和-P- 依赖的路径是已知的，有许多几乎没有信息可用，毫无疑问， 还有一些我们一无所知的人。我们将对那些预测存在于大肠杆菌中的途径进行描述 并将启动基因筛查来寻找其他物种。目的3]鉴定和鉴定细胞形态突变体。 从历史上看，大多数形态突变是在研究其他东西时发现的。在这里，我们将 继续使用流式细胞术分离这种突变体，以确定影响细菌形态的新机制。 我们还将更全面地描述我们手中已有的突变体的特征。总而言之，这些工具和 这些方法将使我们能够更快、更深入地调查围绕着UD-P的奥秘 利用及其与基本代谢途径的相互作用。此外，这项工作还将创造一口井- 开发的细菌模型将在许多其他生物体中提供信息并加强类似的研究。

PROJECT SUMMARY Bacterial cell shape is of fundamental and medical importance, contributing to survival and virulence by influencing nutrient uptake, cell-to-surface attachment, motility, differentiation, and resistance to predation or host immunity. Furthermore, morphological studies are illuminating previously unrecognized interactions among diverse biochemical events that drive bacterial growth. In short, morphology plays crucial roles in bacterial physiology, ecology and pathogenesis, and serves as a window into cell biology. Most bacteria determine their shapes by making a peptidoglycan cell wall, and interfering with this structure is one of the most important targets for anti-bacterial therapy. Despite decades of work, there remain large gaps in understanding how peptidoglycan synthesis is integrated with related metabolic pathways. One integration point is the carrier molecule undecaprenyl-phosphate (Und-P), which helps synthesize peptidoglycan and other oligosaccharides. It has become clear that Und-P and its derivatives form a vital nexus that must be characterized in detail to help combat increasing antibiotic resistance. To this end we propose the following Aims. Aim 1] Determine how Und-P management alters bacterial physiology and biochemistry. Und-P and its homologues transport numerous compounds and polymers across the cytoplasmic membranes of cells in all biological kingdoms, including archaea and eukaryotes. Because the end-products are synthesized by diverse routes, the carrier must be shared among several biochemical pathways, but how this is accomplished is unknown. It is, however, vital – if Und-P is sequestered in one pathway, less is available for peptidoglycan synthesis and cells grow poorly or die. We will develop tools to monitor the amounts of Und-P in living cells and will determine how competition for Und-P affects bacterial growth and the operation of each pathway. Aim 2] Identify and characterize new and little-known Und-P-utilizing pathways. Although several Und-P- dependent pathways are known, there are many for which little information is available and, undoubtedly, others we know nothing about at all. We will characterize those pathways projected to exist in Escherichia coli and will initiate genetic screens to look for others. Aim 3] Identify and characterize cell shape mutants. Historically, most morphological mutants were discovered while studying something else. Here, we will continue to use flow cytometry to isolate such mutants, to identify new mechanisms that affect bacterial shape. We will also characterize more fully the mutants we already have in hand. In summary, these tools and approaches will enable us to investigate, faster and in greater depth, the mysteries surrounding Und-P utilization and its interactions with basic metabolic pathways. In addition, the work will create a well- developed bacterial model that will inform and enhance similar investigations in many other organisms.