Enhancing the E. coli Plasmid Genome Database and the Tools for its Effective Use

禽类致病性大肠杆菌(APEC)引起大肠杆菌病，这是一种经济破坏性疾病，对该国的家禽养殖业有重大影响。此外，有证据表明，污染零售家禽的亚太经合组织可能是引起人类尿路感染和其他肠道外疾病的食源性大肠杆菌。尽管APEC很重要，但人们对这种有机体致病、抵抗治疗和在环境中持续存在的能力背后的机制知之甚少。然而，科学家最近报告说，质粒在APEC中广泛存在，可能是APEC病毒型的定义特征。质粒是染色体外的DNA分子，在“细菌性交”期间经常在细菌之间转移。APEC质粒已被证明拥有许多使大肠杆菌能够致病和抵抗抗生素和重金属的基因，例如用作消毒剂的那些。这些质粒的可传播性以及它们在APEC禽类生产环境和零售禽类中的普遍存在表明，这些质粒可以作为其他细菌病原体的“致病”基因的储存库，包括那些引起人类疾病的病原体。不幸的是，由于缺乏完整的质粒基因组序列，以及研究质粒的工具的不足，科学家们对质粒对疾病的贡献的理解受到了阻碍。本研究试图通过提供已知的最常见的APEC质粒的高质量序列，培训新一代科学家进行质粒序列分析，并开发可广泛用于大肠杆菌质粒分析的新的大肠杆菌质粒基因组数据库工具来解决这些问题。该项目的直接目标是获得7个最常见的APEC质粒的完全注释的基因组序列。这些序列将使高通量功能基因组研究成为可能，加快以假设为导向的大肠杆菌病发病机制研究，并提供对这些质粒和质粒转移在塑造APEC和其他代价高昂且致命的病原体基因组中的作用的洞察。人们认为，这项拟议的研究将使人们更好地了解未来如何控制质粒转移，以改善动物、人类和环境的健康。具体地说，研究人员将：1)构建7个APEC质粒的基因组文库，并对每个质粒进行随机散弹枪测序，以获得8倍的覆盖率；2)组装草稿序列，填补序列空白并解决歧义；3)注释所有质粒基因组并将其与已知序列进行比较；4)对密西西比州立大学的学生进行生物信息学和基因组学方面的培训；以及5)向生产者、家禽兽医、家禽保健研究人员和其他对大肠杆菌病预防和治疗感兴趣的科学家传播该项目的信息和材料，包括研究APEC和其他细菌病原体之间的致病能力和抗药性发展中的质粒转移对致病能力和耐药性的影响。

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, an economically devastating disease, which has major impact on this country's poultry industry. Also, there is evidence that APEC, contaminating retail poultry, may be a food-borne source of E. coli causing urinary tract infections and other extraintestinal diseases in human beings. Despite the importance of APEC, little has been known about the mechanisms underlying this organism's abilities to cause disease, resist therapy, and persist in the environment. However, scientists have recently reported that plasmids are widespread among APEC and may be the defining trait of the APEC pathotype. Plasmids are extrachromosomal DNA molecules that are frequently transferred among bacteria during "bacterial sex". APEC plasmids have been shown to possess many of the genes which enable E. coli to cause disease and resist antibiotics and heavy metals, such as those used as disinfectants. The transmissibility of these plasmids and their ubiquity among APEC in the poultry production environment and on retail poultry suggest that these plasmids could serve as a reservoir of "disease-enhancing" genes for other bacterial pathogens, including those causing disease in humans. Unfortunately, scientists' understanding of plasmids' contributions to disease have been hindered by the lack of complete plasmid genomic sequences available and the inadequacies of the tools with which plasmids can be studied. The present study seeks to remedy these problems by providing high quality sequences of the most common APEC plasmids known, training a new generation of scientists in plasmid sequence analysis, and development of a new E. coli Plasmid Genome Database tool that is available for widespread use for analysis of E. coli plasmids. The immediate objective of this project is to obtain fully annotated genomic sequences of 7 of the most commonly occurring APEC plasmids. These sequences will enable high-throughput functional genomic investigations, accelerate hypothesis-driven research on the pathogenesis of colibacillosis, and provide insight into the role of these plasmids and plasmid transfer in sculpting the genome of APEC and other costly and deadly pathogens. It is thought that the proposed study will enable better understanding of how plasmid transfer can be controlled in the future to the betterment of animal, human, and environmental health. Specifically, the investigators will: 1) construct genomic libraries of 7 APEC plasmids and conduct random shotgun sequencing of each to 8-fold coverage; 2) assemble the draft sequences, close sequence gaps, and resolve ambiguities; 3) annotate all plasmid genomes and compare them to known sequences; 4) train students at Mississippi State University in bioinformatics and genomics; and 5) disseminate information and materials from this project to producers, poultry veterinarians, poultry health researchers, and other scientists with interests in colibacillosis prevention and treatment, plasmids, and the effects of plasmid transfer on development of disease-causing ability and antimicrobial resistance among APEC and other bacterial pathogens.