CAREER: Structural Basis of Bacterial Conjugation

职业：细菌接合的结构基础

• 批准号: 9733655
• 负责人: Joel Schildbach
• 金额: $ 50万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9733655&HistoricalAwards=false
• 关键词: CAREER; Structural; Basis; Bacterial; Conjugation

Schildbach 9733655 F factor, like all conjugative plasmids, directs its transfer from one bacterium to another, even between species. While the genetics of F factor have been studied extensively, little is known about the structural basis of conjugal transfer. The investigator is mounting a long-term effort to describe F factor conjugation in molecular detail. The initial research will concentrate on a complex of three proteins and plasmid DNA that facilitates DNA cleavage with strand and sequence specificity. Of these proteins, only plasmid-encoded TraI possesses nucleolytic activity. Integration host factor (IHF) and plasmid-encoded TraY are required for efficient TraI activity, although their roles are unknown. To better define TraY activity, the investigator will identify TraY amino acids involved in DNA recognition through site-directed mutagenesis, and will characterize the F plasmid DNA sequence bound by TraY. The binding of IHF and TraY to DNA will be assessed for cooperativity, and for its effect on DNA conformation. Crystallization of TraY will also be attempted, in preparation for eventual structure determination by X-ray crystallography. This research, coupled with teaching in lecture and seminar courses, is intended to demonstrate to graduate and undergraduate students the value of examining biological systems using interdisciplinary studies that include structural biology. Conjugation is an efficient mechanism for transferring DNA between even unrelated bacterial species. Consequently, acquisition of antibiotic resistance genes by pathogenic bacteria frequently occurs through conjugal transfer. Drugs that inhibit conjugation, given in concert with regular antibiotic treatment, may render antibiotics more effective and may reduce the incidence of antibiotic resistance. Design of such inhibitors requires an understanding of the structures and mechanisms of the proteins and DNA involved in conjugation. The proposed activities will provide understanding of one step i n bacterial conjugation, as well as instill in students an appreciation of the value and design of such studies.

Schildbach 9733655 F因子，像所有接合质粒一样，指导它从一种细菌转移到另一种细菌，甚至在物种之间。 虽然F因子的遗传学已被广泛研究，但对接合转移的结构基础知之甚少。 研究人员正在进行长期的努力，以描述F因子结合的分子细节。 最初的研究将集中在三种蛋白质和质粒DNA的复合物上，该复合物有助于DNA切割具有链和序列特异性。 在这些蛋白质中，只有质粒编码的TraI具有溶核活性。 整合宿主因子（IHF）和质粒编码的TraY是高效TraI活性所必需的，尽管它们的作用尚不清楚。 为了更好地定义TraY活性，研究者将通过定点诱变鉴定参与DNA识别的TraY氨基酸，并表征TraY结合的F质粒DNA序列。 将评估IHF和TraY与DNA的结合的协同性及其对DNA构象的影响。 还将尝试TraY的结晶，为通过X射线晶体学进行最终结构测定做准备。这项研究，再加上在讲座和研讨会课程教学，旨在证明研究生和本科生的价值，检查生物系统使用跨学科研究，包括结构生物学。 接合是在甚至不相关的细菌物种之间转移DNA的有效机制。 因此，病原菌获得抗生素抗性基因经常通过接合转移发生。 抑制结合的药物，与常规抗生素治疗相结合，可能使抗生素更有效，并可能减少抗生素耐药性的发生。 这种抑制剂的设计需要了解参与缀合的蛋白质和DNA的结构和机制。 建议的活动将使学生了解细菌接合的一个步骤，并向学生灌输对此类研究的价值和设计的理解。