PAPM EAGER: High-throughput experimental methods to link mobile genetic elements with their bacterial hosts

基因在细菌之间的水平转移是抗生素耐药性传播的主要机制，但对自然细菌群落中的这一过程知之甚少。所谓的“灵活的”DNA，即基因组中水平获得的那部分，使细菌能够通过整合新的功能来迅速适应不断变化的环境条件。除了抗生素耐药性，这种柔性DNA上携带的功能还包括毒力因子、汞抗性、碳水化合物代谢或生物修复中有用的分解代谢基因。在某些情况下，生物体的基因组可以由50%以上的柔性DNA组成，例如在某些菌株的大肠杆菌和荧光假单胞菌中。最终，这可以扩大生物体的生态位，提供相对于其他生物体的竞争优势，或者改变它与宿主的关系。令人惊讶的是，比较微生物组研究总体上忽略了移动基因库作为变异的来源，主要调查了不同条件或队列中存在或不存在的物种。这主要是由于目前用于表征细菌群落的技术的局限性。为了满足这一需求，研究人员建议开发能够分析柔性DNA的工具。使用这些工具，他们可以检查这些基因水平转移的生态学和动力学。这项研究将催化在微生物学的所有领域发现细菌群落对扰动的弹性，特定生物体之间基因流动的障碍，以及水平基因转移在有机体进化中的作用。这对人类健康有重要影响，即抗生素耐药性的传播。国际生物研究所致力于将水平基因转移和来自该项目的数据纳入一门名为微生物组工程的课程的课程中。研究水平转移的DNA面临着几个挑战。这些基因可能存在于多个生物体中，基因组的灵活部分可能并不总是与生物体基因组的其余部分在物理上相连，就像噬菌体或质粒的情况一样。研究人员提议开发几种新的单细胞方法，可以检测可移动的遗传元素及其宿主细菌的身份。他们将开发这些方法，以便能够高通量和综合性地同时测试样本中的所有移动基因，而不是专注于单一基因或单一物种。使用新开发的技术，他们将进行几个概念验证实验，以检查移动基因携带的变异，并探索自然微生物群落中水平基因转移的动力学。

The horizontal transfer of genes between bacteria is the main mechanism by which antibiotic resistance spreads, yet little is known about this process in natural bacterial communities. The so-called "flexible" DNA, the portion of the genome that has been horizontally acquired, allows bacteria to rapidly adapt to changing environmental conditions by incorporating novel functions. In addition to antibiotic resistance, functions carried on this flexible DNA can include virulence factors, mercury resistance, carbohydrate metabolism or catabolic genes useful in bioremediation. In some cases, organisms' genomes can consist of upwards of 50% flexible DNA, such as in certain strains of Escherichia coli and Pseudomonas fluorescens. Ultimately, this can expand an organism's niche, provide a competitive edge against other organisms, or change its relationship with its host. Surprisingly, comparative microbiome studies have overall ignored the mobile gene pool as a source for variation, surveying mainly the species present or absent across different conditions or cohorts. This is mainly due to limitations of current technologies used to characterize bacterial communities. To address this need, the investigators propose to develop tools that enable analysis of the flexible DNA. Using these tools, they can then examine the ecology and dynamics of the horizontal transfer of these genes. This research will catalyze discovery across all fields of microbiology around the resiliency of bacterial communities in response to perturbation, barriers to gene flow between specific organisms and the role of horizontal gene transfer in organismal evolution. There are important implications for human health, namely the spread of antibiotic resistance. The PI is committed to incorporating horizontal gene transfer and data from this project into curriculum of a course entitled Engineering the Microbiome. Studying horizontally transferred DNA presents several challenges. These genes may be found in multiple organisms and the flexible portion of a genome may not always be physically linked to the rest of an organism's genome, as in the case of phage or plasmids. The investigators propose developing several new single-cell methods that can detect mobile genetic elements and the identities of their host bacteria. They will develop these methods so that they can be high-throughput and comprehensive, testing all of the mobile genes in a sample simultaneously, rather than focusing on a single gene or a single species. Using the newly developed technologies, they will perform several proof-of-concept experiments to examine variation in mobile gene carriage and probe the dynamics of horizontal gene transfer in natural microbial communities.