Modulation of Bacterial Cell Division by (p)ppGpp

(p)ppGpp 对细菌细胞分裂的调节

• 批准号: 10458524
• 负责人: Sarah Emily Anderson
• 金额: $ 6.76万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10458524
• 关键词: Actins; Affect; Alleles; Anabolism; Animal Model; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Physiology; Binding; Binding Proteins; Biochemistry; Cell Size; Cell Wall; Cell division; Cell physiology; Cells; Clinical; DNA-Directed RNA Polymerase; Data; Defect; Down-Regulation; Enzymes; Equilibrium; Escherichia coli; Fellowship; Filament; Genetic Transcription; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Lead; Learning; Length; Life; Maintenance; Mediating; Microscopy; Modeling; Molecular; Molecular Machines; Monobactams; Mutation; Nucleotides; Nutrient; Nutritional; Phenotype; Process; Production; Proteins; Regulation of Cell Size; Research; Research Personnel; Resistance; Rod; Role; Signaling Molecule; Stress; Techniques; Testing; Training; Translations; Tubulin; Work; antimicrobial; beta-Lactam Resistance; beta-Lactams; career; cell growth; cell type; cellular targeting; environmental adaptation; knock-down; mutant; professor; response; scaffold

PROJECT SUMMARY/ABSTRACT In bacteria, cell size positively correlates with nutrient availability and negatively correlates with levels of the key nutritional signaling molecules pppGpp and ppGpp (abbreviated (p)ppGpp). (p)ppGpp is produced in response to environmental nutrient limitation and functions primarily to inhibit biosynthesis and slow growth. In the model organism Escherichia coli, (p)ppGpp modulates cell physiology at both the transcriptional and post- transcriptional levels through interactions with RNA polymerase (RNAP) and 56 additional cellular targets. However, the mechanism by which (p)ppGpp contributes to regulation of cell size is not fully understood. The balance between cell division and elongation is a major determinant of size in rod-shaped bacteria. Several pieces of evidence suggest that (p)ppGpp contributes to cell size in part by modulating the balance between these two processes. Increases in (p)ppGpp levels suppress the heat sensitivity of conditional cell division mutants and leads to resistance to mecillinam, an antibiotic targeting the elongation machinery (elongasome). These data suggest that (p)ppGpp positively affects activity of the cell division machinery (divisome). Strains lacking (p)ppGpp ((p)ppGpp0) are ~30% longer than wild-type cells and frequently filament. These phenotypes are not recapitulated in RNAP mutants defective for (p)ppGpp binding, suggesting that (p)ppGpp contributes to cell size through a post-transcriptional interaction with one of its other binding partners. I hypothesize that (p)ppGpp indirectly promotes divisome assembly and activation via interaction with its target proteins. To illuminate the molecular basis of (p)ppGpp mediated changes in divisome and elongasome activity, I propose two complementary aims. In Aim 1, I will characterize the effects of alterations in intracellular (p)ppGpp concentration on production, assembly, and activation of the cell division machinery. In Aim 2, I will screen for (p)ppGpp binding proteins that are required to increase cell length. I will then determine the effect of these proteins on the transcription, translation, assembly, and activity of divisome components (Sub-aim 2b) and, in Sub-aim 2c, determine the mechanism by which candidate proteins modulate cell division. The expected contribution of the proposed work is an enhanced understanding of the mechanisms by which (p)ppGpp modulates bacterial physiology. This contribution is significant because (p)ppGpp is a key component of environmental adaptation throughout the bacterial kingdom. This proposal will also enhance our understanding of (p)ppGpp’s role in intrinsic resistance to the clinically important β-lactam antibiotics, which target components of the divisome and elongasome. In addition, this F32 fellowship will provide me with opportunities to learn new techniques in microscopy and biochemistry, explore new conceptual avenues, and obtain additional professional training that will prepare me for a career as a professor and independent investigator.

项目概要/摘要 在细菌中，细胞大小与营养可用性呈正相关，与营养水平呈负相关。 关键营养信号分子 pppGpp 和 ppGpp（缩写为 (p)ppGpp）。 (p)ppGpp 产生于 对环境养分限制的反应，主要作用是抑制生物合成和减缓生长。在 模式生物大肠杆菌 (p)ppGpp 在转录和转录后调节细胞生理学 通过与 RNA 聚合酶 (RNAP) 和 56 个其他细胞靶标的相互作用来调节转录水平。 然而，(p)ppGpp 调节细胞大小的机制尚不完全清楚。这 细胞分裂和伸长之间的平衡是杆状细菌大小的主要决定因素。一些 多项证据表明，(p)ppGpp 部分通过调节细胞大小之间的平衡来影响细胞大小。 这两个过程。 (p)ppGpp 水平的增加抑制条件细胞分裂的热敏感性 突变体并导致对美西林（mecillinam）的耐药性，美西林是一种针对伸长机制（伸长体）的抗生素。 这些数据表明 (p)ppGpp 对细胞分裂机制（分裂体）的活性产生积极影响。菌株 缺乏 (p)ppGpp ((p)ppGpp0) 的细胞比野生型细胞长约 30%，并且通常呈丝状。这些表型 在 (p)ppGpp 结合缺陷的 RNAP 突变体中没有重演，表明 (p)ppGpp 有助于 通过与其他结合伙伴之一的转录后相互作用来改变细胞大小。 我假设 (p)ppGpp 通过与其相互作用间接促进分裂体的组装和激活 目标蛋白质。阐明 (p)ppGpp 介导的分裂体和延长体变化的分子基础 活动中，我提出了两个互补的目标。在目标 1 中，我将描述细胞内变化的影响 (p)ppGpp 专注于细胞分裂机制的产生、组装和激活。在目标 2 中，我将 筛选增加细胞长度所需的 (p)ppGpp 结合蛋白。然后我将确定效果 这些蛋白质对分裂体成分的转录、翻译、组装和活性的影响（子目标 2b） 在子目标 2c 中，确定候选蛋白调节细胞分裂的机制。预期的 拟议工作的贡献是加深了对 (p)ppGpp 机制的理解 调节细菌生理学。这一贡献意义重大，因为 (p)ppGpp 是 整个细菌王国的环境适应。这个建议也将加深我们的理解 (p)ppGpp 在临床上重要的 β-内酰胺抗生素的内在耐药性中的作用，其目标成分 分裂和细长的。此外，这个F32奖学金将为我提供学习新知识的机会 显微镜和生物化学技术，探索新的概念途径，并获得更多专业知识 培训将使我为教授和独立调查员的职业生涯做好准备。