Bacterial cell wall synthesis, shape and septation

描述(由申请人提供)： 细菌表现出极端的形态多样性，这一事实自300多年前首次被人们看到以来就已知。然而，直到最近5-7年，人们才清楚地认识到，这些差异远远不只是表面上的。相反，细胞形状具有基础性和医学上的重要性，通过影响营养物质的摄取、细胞与表面的附着、运动性、分化以及对捕食和宿主免疫攻击的抵抗力来促进细菌的生存和毒力。形态也不是一成不变的：细菌投入大量资源来操纵它们的形状，以应对生长速度和营养状况的变化，并对抗生素和其他环境压力做出反应。总之，形态不是一种轻浮或中性的特征，而是在细菌细胞生物学、生态学和发病机制中发挥着至关重要的作用。尽管最近这类研究出现了复兴，但令人惊讶的是，我们对细胞如何创造和维持其形状知之甚少。我们所知道的是，大多数细菌都受到肽聚糖细胞壁的保护，而这一细胞壁的合成方式决定了细胞的形状。我们的长期目标是 了解肽聚糖的结构、合成、调节和功能含义，以及产生和修饰它的酶。为此，我们建议通过追求以下目标来扩大我们对细菌生理学和形态学的理解。目的1]应用新的方法，通过荧光激活细胞分选(FACS)丰富形态突变体，以确定新的形态机制和调控因子。人们对这些问题知之甚少的一个原因是，几乎所有的形态调节剂都是偶然发现的。在这里，我们将使用流式细胞仪作为一种遗传工具来丰富、分离和研究形态突变，以定向寻找调节细菌形状的新机制。[目的2]通过一种新设计的球体回收试验，鉴定和表征从头形成形状所需的机制。几乎所有关于细胞壁合成和形态的已知信息都涉及到保存或延长已存在的细胞壁的机制。当细胞的壁被严重破坏或完全移除时，细胞如何重新形成自己的形状，比如细菌遇到宿主免疫系统时，人们几乎不知道。我们发现，在后一种情况下的生存需要新的机制来支持或取代经典的维持途径。目的3]描述关键的FtsZ-肽聚糖反应，特别是那些将告诉我们：a)胞质周肽聚糖和青霉素结合蛋白如何在细胞分裂过程中控制细胞质Z环的几何形状；以及b)在细胞伸长到分裂的关键转变过程中胞质FtsZ如何触发肽聚糖的合成。这些鲜为人知的相互作用决定了细菌细胞的完整性、形状和繁殖。总之，这些新的工具和方法将使我们能够更快、更深入地调查仍然模糊我们对细菌生理学中一些最基本问题的理解的众多谜团。这项工作也将为其他生物的类似形态研究提供一个模型。

DESCRIPTION (provided by applicant): Bacteria exhibit extreme morphological diversity, a fact known since they were first visualized over 300 years ago. Yet only in the last 5-7 years has it become clear that these differences are much more than cosmetic. Instead, cell shape is of fundamental and medical importance, contributing to bacterial survival and virulence by influencing nutrient uptake, cell-to-surface attachment, motility, differentiation, and resistance to predation and host immune assaults. Nor is morphology static: bacteria invest significant resources to manipulate their shapes to cope with changes in growth rate and nutritional status and to respond to antibiotics and other environmental stresses. In short, morphology is not a frivolous or neutral characteristic, but plays crucial roles in bacterial cell biology, ecology and pathogenesis. Despite a recent renaissance in such studies, we understand surprisingly little about how cells create and maintain their shapes. What we do know is that most bacteria are protected by a peptidoglycan cell wall and that the way this wall is synthesized determines cell shape. Our long-term goal is to understand the structure, synthesis, regulation and functional implications of peptidoglycan and the enzymes that create and modify it. To that end, we propose to extend our understanding of bacterial physiology and morphology by pursuing the following Aims. Aim 1] Identify new morphological mechanisms and regulators by applying newly adapted methods for enriching shape mutants via fluorescence- activated cell sorting (FACS). One reason so little is known about these matters is because almost all morphological regulatory agents were discovered accidently. Here, we will use FACS as a genetic tool to enrich, isolate and study morphological mutants in a directed search for new mechanisms that regulate bacterial shape. Aim 2] Identify and characterize the mechanisms required for de novo shape generation by employing a newly devised Spheroplast Recovery assay. Virtually everything known about cell wall synthesis and morphology involves mechanisms that preserve or extend pre-existing walls. Little or nothing is known about how cells re-create their shapes when their walls are damaged severely or removed altogether, as occurs when bacteria encounter host immune systems. We find that survival in these latter circumstances demands new mechanisms to support or supplant the classic maintenance pathways. Aim 3] Characterize critical FtsZ- peptidoglycan reactions, particularly those that will tell us: a) how periplasmic peptidoglycan and penicillin binding proteins control the geometry of the cytoplasmic Z ring during cell division; and b) how cytoplasmic FtsZ triggers the synthesis of peptidoglycan during the critical transition from cell elongation to division. These poorly-understood interactions determine the integrity, shape and propagation of bacterial cells. In summary, these new tools and approaches will enable us to investigate, faster and in greater depth, the numerous mysteries that still obscure our understanding of some of the most basic issues in bacterial physiology. The work will also serve as a model for similar morphological investigations in other organisms.