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.