Mobilome Genomics: Large Plasmids of Diverse Prokaryotic Groups

智力优势：所有的原核生物（最简单的单细胞生物，包括细菌和古细菌）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给其他简单的微生物。在过去十年中完成的所有原核生物基因组测序中都可以看到HGT发生的证据。在不同的原核生物进化过程中，这些HGT事件的可能起源和时间，以及这些转移基因现在在它们的过继宿主中所起的作用，现在受到了很多关注。转移的基因通常聚集在主要的细胞染色体上，作为斑块或“岛”，其中还包含众所周知的移动遗传元件（MGEs）的痕迹，包括质粒（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），它们是基因转移和聚集的媒介。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/或改变它们所处的环境。这种特殊的移动元素遗传特征包括光合作用基因、农药降解酶基因和对人类、动物或植物有害的毒素基因。尽管MGE在原核生物适应和进化中具有重要意义，但我们对MGE本身的DNA序列数据还不到对整个细菌染色体的DNA序列数据的4%，而且只有20%的MGE序列信息来自在基因转移中起主要作用的大型移动质粒。Summers的研究小组长期以来一直在研究质粒基因，最近他们发明了一种快速、廉价、简单的方法，从单个大质粒中纯化高质量的DNA，从而克服了质粒测序的一个主要瓶颈。萨默斯将与不同原核生物群体的生物学专家合作，使用这种方法和其他新技术，对来自不同原核生物的多达4个大质粒（每个50,000碱基对）进行快速DNA测序，这些质粒在温室气体产生、海洋硫循环、热液喷口群落、鱿鱼发光、生物燃料产生和农业动植物疾病中发挥着不同的重要作用。这些质粒编码的基因将通过现有的计算机方法以及为本项目开发的新的质粒特异性方法进行鉴定。原始和注释的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：本项目在两个方面具有更大的影响。首先，原核生物的染色体DNA序列被认为是了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要的圆形染色体，很少包括通常从实验室培养的原核生物中丢失的原始移动质粒。因此，许多研究人员假设他们的生物体从来没有质粒。但直接从自然界分离出来的原核生物通常含有几个质粒，大小从几到几百个碱基不等。该项目将是世界上第一个在广泛研究的原核生物的新鲜天然分离物中故意检查大质粒基因组的项目。这些结果至少可以改变研究这些细菌的研究人员的工作模型，提醒他们注意与他们研究的特定功能有关的潜在的可移动遗传元素，最好的情况是，它将吸引其他研究人员在他们自己的生物体中扩大质粒的研究。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实验和计算机分析的实践经验，培养有抱负的科学家对原核生物进化中水平基因转移的重要性的认识。

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.