Modulation of Bacterial Cell Division by (p)ppGpp

项目概要/摘要 在细菌中，细胞大小与营养物质的可用性呈正相关，与细菌的生长水平呈负相关。 关键的营养信号分子pppGpp和ppGpp（缩写为（p）ppGpp）。（p）ppGpp产生于 对环境营养限制的反应，主要功能是抑制生物合成和减缓生长。在 模式生物大肠杆菌，（p）ppGpp在转录和后 通过与RNA聚合酶（RNAP）和56个额外的细胞靶点的相互作用，在转录水平上进行了研究。 然而，（p）ppGpp有助于调节细胞大小的机制尚未完全了解。的 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 有证据表明，（p）ppGpp部分通过调节细胞大小之间的平衡， 这两个过程。（p）ppGpp水平的增加抑制了条件性细胞分裂的热敏感性 突变体并导致对美西林的抗性，美西林是一种靶向延伸机制（elongasome）的抗生素。 这些数据表明，（p）ppGpp积极影响细胞分裂机制（divisome）的活性。菌株 缺乏（p）ppGpp的细胞（（p）ppGpp 0）比野生型细胞长约30%，并且经常是丝状的。这些表型 在（p）ppGpp结合缺陷的RNAP突变体中没有重现，表明（p）ppGpp有助于 细胞大小通过转录后相互作用与它的其他结合伙伴之一。 我推测，（p）ppGpp通过与其受体的相互作用间接促进分裂体的组装和激活。 靶蛋白。阐明（p）ppGpp介导分裂体和延长酶体变化的分子基础 我提出了两个互补的目标。在目标1中，我将描述细胞内的改变的影响， （p）ppGpp浓度对细胞分裂机制的产生、组装和活化的影响。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 这些蛋白质的转录，翻译，装配，和活动的divisome组件（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 拟议工作的贡献是加深了对（p）ppGpp 调节细菌生理学。这一贡献是重要的，因为（p）ppGpp是 适应环境的细菌王国。这一建议也将增进我们的理解 （p）ppGpp在对临床上重要的β-内酰胺类抗生素的内在耐药性中的作用， 分裂体和延长体的关系。此外，F32奖学金将为我提供学习新知识的机会。 在显微镜和生物化学技术，探索新的概念途径，并获得额外的专业 培训，将准备我的职业生涯作为一个教授和独立调查员。

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.