Establishing a mechanistic basis for the plasmid acquisition cost

项目摘要/摘要 这个项目将调查以前未被研究的现象，称为质粒获取成本，在 水平基因转移(HGT)的背景。质粒转移是病原体适应的一种主要方式 环境应激源。这些质粒通常对细胞施加持续的负担，与持续的 表达的质粒性基因，称为质粒体适合度成本。它们还诱导了一种互补的， 在获得质粒后立即反映代谢适应的独立的、瞬时的负荷。 然而，尽管健身成本得到了很好的研究，但这些瞬变背后的机械性因素 目前尚不清楚这种影响，也就是指获得质粒的成本。这一信息对于 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，采购成本是新陈代谢的结果 在质粒转移后立即出现失调。我们将以两个具体目标来研究这一假设： (1)我们将通过进行详细的研究来阐明获得质粒成本的转录基础 Rp4结合质粒在标准实验室大肠杆菌中的鉴定 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 最初是使用这种组合完成的。具体地说，我们建立了一种新的实验方案，该方案可靠地 量化新质粒接受者的生长缺陷。在这里，我们将配对时间序列RNA-seq测量 用新陈代谢模型来阐明紧接在质粒之后发生的代谢失调 我们将使用一组合理选择的基因敲除菌株和 代表性的质粒组。总体而言，这将为观察到的采购成本建立一个机械性的解释。 (2)我们将使用自然的方法，在不同的质粒中确定获取成本的遗传决定因素 从环境和临床样本中分离出的相关质粒和菌株以及标准 实验室的质粒组。具体地说，我们将利用现有的全基因组序列 来自我们合作者的质粒，以及相应的样本，从两个物种中分离出质粒： 大肠埃希菌和肺炎克雷伯菌。然后我们将量化每个质粒的获取成本；多个变量 回归将被用来了解哪些质粒特征最能预测观察到的 收购成本。这将导致第一次严格量化在自然情况下获得质粒的成本 环境和临床中出现的质粒群。除了证明这一现象的普遍性外， 这些结果将为在更复杂的情况下预测和调节HGT动力学奠定基础 人口。此外，所有的质粒都将被提供，这将是一项宝贵的研究和 在更广泛的学术界使用的教育工具。

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.