Mobilome Genomics: Large Plasmids of Diverse Prokaryotic Groups

智力优势：所有原核生物(最简单的单细胞生物体，包括细菌和古生菌)都可以通过一种称为水平基因转移(HGT)的过程将它们的许多基因直接转移到其他简单的微生物中。在过去十年测序的所有已完成的原核生物基因组中，都可以看到发生了HGT的证据。现在人们致力于了解这些HGT事件在不同原核生物进化过程中的可能来源和时间，以及这些转移的基因现在在它们的收养宿主中所起的作用。转移的基因通常以补丁或“岛”的形式聚集在主要的细胞染色体上，其中还含有众所周知的可移动遗传元件(MGES)的痕迹，其中包括作为基因转移和聚集的媒介的质粒(独立复制的小DNA片段)、噬菌体(细菌病毒)和转座子(能够随机重组或“跳跃基因”的遗传片段)。除了它们在细胞染色体之间移动基因的作用外，这些MGES本身通常携带特殊的基因，使它们的细胞宿主能够适应和/或改变它们生活的环境。这种特殊的可移动元素携带的特征包括光合作用、农药降解酶和危害人类、动物或植物的毒素的基因。尽管MGES在原核适应和进化中具有重要意义，但我们拥有的MGES本身的DNA序列数据不到整个细菌染色体的4%，并且只有20%的MGE序列信息来自在基因转移中起主要作用的大的可移动质粒。萨默斯的团队长期以来一直在研究质粒基因，最近通过设计一种快速、廉价和容易的方法从单个大的质粒中提纯高质量的DNA，克服了质粒测序中的一个主要瓶颈。萨默斯与一组不同原核生物的生物学专家合作，将使用这种方法和其他新技术对来自一组不同原核生物的多达4个大质粒(每个50,000个碱基对)进行快速DNA测序，这些原核生物在温室气体产生、海洋硫磺循环、热液喷口群落、鱿鱼发光、生物燃料生成以及农业动植物疾病中都具有不同的重要性。这些质粒编码的基因将通过现有的计算机方法以及为该项目开发的新的特定于质粒的方法来识别。原始数据和注释数据将存储在公共数据库中，供MGE研究人员和其他对进化感兴趣的人分析。更广泛的影响：该项目在两个领域有更大的影响。首先，原核生物的染色体DNA序列被认为是理解这些生物在自然界中所有功能的决定性信息。然而，由于这些序列来自实验室长期携带的菌株，大多数只覆盖单个主循环染色体，很少包括实验室培养的原核生物中通常丢失的原始可移动质粒。因此，许多研究人员认为他们的生物体永远不会有质粒。但在直接从自然界分离出来的情况下，原核生物通常含有几个大小从几个千碱基到数百个千碱基的质粒。该项目将是世界上第一个刻意研究大量原核生物新鲜天然分离物中大质粒基因组的项目。这一结果至少可以改变研究这些细菌的研究人员的工作模式，让他们注意到他们研究的特定功能涉及的潜在移动遗传元素，充其量它将吸引其他研究人员扩大对他们自己生物体中的质粒的研究。其次，该项目包括研究生和本科生培训部分，旨在通过对这些现象的板凳和计算机分析的实践经验，让有抱负的科学家认识到水平基因转移在原核生物进化中的重要性。

Intellectual Merit: All prokaryotes (the simplest single-celled organisms, including both bacteria and archaea) can transfer many of their genes directly to other simple microbes in a process called horizontal gene transfer (HGT). Evidence that HGT has occurred is visible in all of the completed procaryotic genomes sequenced in the last decade. Much attention is now devoted to understanding the possible origins and timing of these HGT events during the evolution of different prokaryotes as well as the roles that such transferred genes now play in their adoptive hosts. Transferred genes are often clustered on the major cellular chromosome as patches or "islands" that also contain traces of well-known mobile genetic elements (MGEs) including plasmids (small independently replicating DNA segments), bacteriophages (bacterial viruses) and transposons (genetic segments capable of random recombination or "jumping genes") that were the agents of the gene transfer and clustering. In addition to their roles in moving genes between cellular chromosomes, these MGEs themselves often carry specialized genes that allow their cellular hosts to adapt to and/or alter the environment in which they live. Such specialized mobile-element-borne traits include genes for photosynthesis, for pesticide degrading enzymes, and for toxins that harm humans, animals, or plants. Despite the significance of MGEs in prokaryotic adaptation and evolution, we have less than 4% as much DNA sequence data for the MGEs themselves as we do for whole bacterial chromosomes and only 20% of MGE sequence information comes from the large, mobile plasmids that play a major role in gene transfer. The Summers' group has long studied plasmid genes and recently overcame a major bottle-neck in plasmid sequencing by devising a fast, cheap, and easy method to purify high-quality DNA from individual large plasmids. With co-investigators expert in the biology of a diverse group of prokaryotes, Summers will use this method and other novel techniques for rapid DNA sequencing of up to 4 large plasmids ( 50,000 basepairs each) each from a diverse set of prokaryotes that are variously important in greenhouse gas production, oceanic sulfur cycling, hydrothermal vent communities, luminescence in squid, biofuel generation, and diseases of agricultural animals and plants. The genes encoded by these plasmids will be identified by existing computer methods as well as new plasmid-specific methods to be developed for this project. The raw and annotated data will be deposited in public databases for analysis by MGE researchers and others interested in evolution. Broader Impacts: This project has larger implications in two areas. Firstly, the chromosomal DNA sequences of prokaryotes are considered the definitive information for understanding all the functions of these organisms in nature. However, since these sequences were derived from strains long-carried in the laboratory, most only cover the single major circular chromosome and few include the original mobile plasmids which are generally lost from lab-cultivated prokaryotes. Thus, many investigators assume their organism never has plasmids. But on direct isolation from nature prokaryotes typically contain several plasmids ranging from a few to hundreds of kilobases in size. This project will be the first worldwide to deliberately examine the genomes of large plasmids in fresh natural isolates of a wide range of otherwise well-studied prokaryotes. The results could at minimum alter the working models of investigators studying those bacteria by alerting them to potential mobile genetic elements involved in the specific function they study and at best it will attract other investigators to expand the study of plasmids in their own organisms. Secondly, the project includes graduate and undergraduate training components designed to inoculate aspiring scientists with an appreciation for the significance of horizontal gene transfer in procaryotic evolution via hands-on experience with both bench and computer analyses of these phenomena.