Establishing a mechanistic basis for the plasmid acquisition cost

项目总结/摘要 这个项目将调查以前研究不足的现象称为质粒收购成本，在 水平基因转移（horizontal gene transfer，HGT）质粒转移是病原体适应的主要方式 环境压力源这些质粒通常对细胞施加持续的负荷，与持续的细胞毒性相关。 质粒基因的表达，称为质粒适应度成本。它们还诱导一种互补的， 独立的、瞬时的负荷，反映了质粒获得后立即发生的代谢适应。 然而，尽管健身成本得到了很好的研究，这些短暂的机械因素 影响，称为质粒获取成本，目前尚不清楚。这些信息对于 发展对病原体动态和潜在下游干预策略的基本见解。 根据我们的初步数据，我们的中心假设是，收购成本的产生是由于新陈代谢 质粒转移后立即发生失调。我们将通过两个具体目标来研究这一假设： (1)我们将通过进行详细的分析，阐明质粒获取成本的转录基础。 在标准实验室大肠杆菌中充分表征的RP 4接合质粒的表征 我们注意到，我们的初步工作说明了购置成本的存在和程度， 最初使用这种组合。具体来说，我们建立了一个新的实验方案， 量化新质粒受体中的生长缺陷。在这里，我们将配对时间序列RNA-seq测量 用代谢建模来阐明质粒转染后立即发生的代谢失调， 我们将使用一组合理选择的基因敲除菌株来验证这些结果， 代表性质粒。总的来说，这将为观察到的购置成本建立一个机械解释。 (2)我们将确定不同质粒中获得成本的遗传决定因素， 从环境和临床样品中分离的相关质粒和菌株，以及标准品 实验室质粒具体来说，我们将利用可用的全基因组序列， 我们的合作者的质粒，沿着相应的样品，从两个物种中分离质粒： E.大肠杆菌和肺炎克雷伯菌。然后，我们将量化每个质粒的获取成本;多个变量 回归将用于了解哪些质粒特征最能预测观察到的结果 购置成本。这将导致自然界中质粒获取成本的第一次严格量化。 存在环境和临床质粒。除了证明这种现象的普遍性之外， 这些结果将作为预测和调节更复杂的HGT动力学的基础 人口。此外，所有质粒都将提供，这将成为一项宝贵的研究， 在整个学术界的教育工具。

Project Summary/Abstract This project will investigate the previously understudied phenomenon termed plasmid acquisition cost, in the context of horizontal gene transfer (HGT). Plasmid transfer is a dominant way that pathogens adapt to environmental stressors. These plasmids often exert a sustained burden on cells, associated with continued expression of plasmid genes, known as the plasmid fitness cost. They also induce a complementary, independent, transient burden reflective of the metabolic adaptation immediately following plasmid acquisition. However, whereas fitness costs are well-studied, the mechanistic factors underlying these transient effects, referred to as the plasmid acquisition costs, are currently unknown. This information is critical to developing fundamental insights into pathogen dynamics and potential downstream interventional strategies. Based on our preliminary data, our central hypothesis is that acquisition costs arise as a result of metabolic dysregulation immediately following plasmid transfer. We will investigate this hypothesis with two Specific Aims: (1) We will elucidate the transcriptional basis of the plasmid acquisition cost, by undertaking a detailed characterization of the well-characterized RP4 conjugative plasmid in a standard laboratory Escherichia coli strain; we note that our preliminary work illustrating the existence and extent of acquisition costs was originally done using this combination. Specifically, we established a novel experimental protocol that reliably quantifies the growth defect in new plasmid recipients. Here, we will pair time series RNA-seq measurements with metabolic modeling to elucidate the metabolic dysregulation that occurs immediately following plasmid acquisition; we will validate these results using a rationally selected set of gene knockout strains and representative plasmids. Overall, this will establish a mechanistic explanation for observed acquisition costs. (2) We will determine the genetic determinants of the acquisition cost in diverse plasmids, using naturally relevant plasmids and strains isolated from both environmental and clinical samples, as well as standard laboratory plasmids. Specifically, we will leverage available whole genome sequences with assembled plasmids from our collaborators, along with the corresponding samples, to isolate plasmids from two species: E. coli and Klebsiella pneumoniae. We will then quantify acquisition costs for each plasmid; multiple variable regression will be used to understand which plasmid characteristics are most predictive of observed acquisition costs. This will result in the first rigorous quantification of plasmid acquisition costs in naturally occurring environmental and clinical plasmids. Beyond demonstrating the generality of this phenomenon, these results will serve as the foundation for predicting and modulating HGT dynamics in more complex populations. Moreover, all plasmids will be made available, which will serve as an invaluable research and educational tool throughout the wider academic community.