Modulation of Bacterial Cell Division by (p)ppGpp

项目摘要/摘要 在细菌中，细胞大小与营养可利用性呈正相关，与 关键营养信号分子pppGpp和ppGpp(缩写为(P)ppGpp)。(P)ppGpp生产于 对环境营养限制的反应，其功能主要是抑制生物合成和缓慢生长。在……里面 模式生物大肠杆菌，(P)ppGpp在转录和转录后调节细胞生理。 通过与RNA聚合酶(RNAP)和另外56个细胞靶点的相互作用来调节转录水平。 然而，(P)ppGpp对调节细胞大小的作用机制尚不完全清楚。这个 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。几个 一些证据表明，(P)ppGpp对细胞大小的影响部分是通过调节 这两个过程。(P)ppGpp水平的增加抑制了条件细胞分裂的热敏感性 突变并导致对甲氧西林产生抗药性，甲氧西林是一种针对伸长机械(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)的活动有积极的影响。菌株 缺少(P)ppGpp((P)ppGpp0)的细胞比野生型细胞长约30%，且常呈丝状。这些表型 在(P)ppGpp结合缺陷的RNAP突变体中没有概括，这表明(P)ppGpp有助于 通过转录后与其其他结合伙伴之一相互作用的细胞大小。 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 靶蛋白。阐明(P)ppGpp介导的分裂体和拉长体变化的分子基础 关于活动，我提出了两个相辅相成的目标。在目标1中，我将描述细胞内变化的影响 (P)ppGpp浓度，用于细胞分裂机制的生产、组装和激活。在《目标2》中，我会 筛选增加细胞长度所需的(P)ppGpp结合蛋白。然后我会确定它的效果 这些蛋白质对分裂体组分的转录、翻译、组装和活性的影响(子目标2b) 并且，在子目标2c中，确定候选蛋白质调节细胞分裂的机制。预期中的 拟议工作的贡献是加深了对(P)ppGpp 调节细菌的生理。这一贡献意义重大，因为(P)ppGpp是 整个细菌界的环境适应。这项建议也将增进我们对 (P)ppGpp在临床上重要的β-内酰胺类抗生素的内在耐药性中的作用 指分裂体和拉长体。此外，F32奖学金将为我提供学习新知识的机会 显微镜和生物化学方面的技术，探索新的概念途径，并获得额外的专业知识 培训将为我作为教授和独立调查员的职业生涯做好准备。

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.