Molecular Mechanisms of Phage Exclusion Systems

噬菌体排除系统的分子机制

• 批准号: 9506954
• 负责人: Ian Molineux
• 金额: $ 27万
• 依托单位: University of Texas at Austin
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-15 至 1999-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506954&HistoricalAwards=false
• 关键词: Molecular; Mechanisms; Phage; Exclusion; Systems

9506954 Molineux The specific goal of this research is to provide a complete understanding of the mechanism of F exclusion of T7 and related phages, an observation first made over 30 years ago. This exclusion system exhibits many physiological, and perhaps also mechanistic, similarities with other abortive infections, including rex and P22 sieB exclusion of other lambdoid phages, rex exclusion of T4 rII mutants, and Col Ib exclusion of T5 and related phages. These abortive infections are all characterized by a complex phenomenology with no underlying molecular model or rationale to describe their properties, and it has proven difficult to distinguish between the primary cause of the abortive infection and ancillary events that occur merely because the abortively infected cell is dying. At the physiological level, the dysfunctions that occur during an abortive phage infection also resemble those occurring during cell killing by some bacteriocins, during eukaryotic cell killing by some bacterial toxins, and during non-permissive infections of specific cell types by certain animal viruses. Many of these biological systems are only poorly understood at the molecular level, at least in part because the activities of the genes involved in causing cell death have not been defined. In order to fully understand the molecular basis of any exclusion system, it is necessary to know all of the genes involved. From previous work from this laboratory, the T7-coded targets of F exclusion are known to be genes 1.2 and 10; both genes interact with F pifA, the sole F determinant of exclusion, and in doing so kill the cell. Killing occurs in the absence of phage infection: cloning plasmids expressing either 1.2 or gene 10 as the only T7 gene is lethal to cells harboring pifA as their only F gene. A genetic selection for mutants of E. coli that tolerate co-expression of the T7 and F genes has been developed and it is expected that various different mutants will be isolated that do not support F exclusion of T7. An initial screen for such mutants resulted in the isolation of one strain that, when containing wild-type F or a multicopy plasmid expressing pifA, plates T7 at the same eop as an F- strain. Some other mutants isolated in this screen do not exclude (when containing F) T7 as efficiently as their isogenic parent strain and have acquired a phenotype of resistance to high levels of the antibiotic tetracycline, suggesting an involvement of cell membrane components in the abortive infection. A more detailed study of these mutants, and others to be obtained in a more extensive search, will provide the essential genetic framework necessary for a complete molecular understanding of this phage exclusion system. %%% The goal of this research is to provide a complete understanding of the mechanism where a bacterial conjugation factor (the F plasmid) prevents the successful infection of bacterial cells by the T7 phage, a bacterial virus. This study should impact the field of host-parasite interactions in general, since it is likely that the phage and plasmid genes involved in F exclusion of T7 interact with conserved cellular functions whose activities are compromised in many different biological settings. ***

9506954 Molineux这项研究的具体目标是完全理解F排除T7和相关噬菌体的机理，这是30年前首次进行的观察结果。 该排除系统表现出与其他流产感染的许多生理，也许还有机理的相似性，包括REX和P22 SIEB排除其他lambdoid Phages，REX排除T4 RII突变体以及Col IB排除T5和相关噬菌体。 这些堕胎感染的特征都是复杂的现象学，没有基本的分子模型或基本原理来描述其特性，事实证明，很难区分流产感染的主要原因和仅仅因为流产感染细胞而发生的辅助事件。 在生理水平上，流产噬菌体感染期间发生的功能障碍也类似于某些细菌毒素在细胞杀死过程中发生在某些细菌毒素的真核细胞杀死期间，以及某些动物病毒对特定细胞类型的非疗法感染期间。 这些生物系统中的许多仅在分子水平上才鲜为人知，至少部分是因为尚未定义引起细胞死亡的基因的活性。 为了充分了解任何排除系统的分子基础，有必要了解所有涉及的基因。 从该实验室的先前工作中，F排除的T7编码靶标已知基因1.2和10。这两个基因都与f pifa相互作用，f pifa是排除的唯一决定因素，并且这样做杀死了细胞。 在没有噬菌体感染的情况下，发生杀戮：克隆质粒表达1.2或基因10作为唯一的T7基因对携带PIFA作为其唯一F基因的细胞致死。 已经开发了耐受T7和F基因共表达的大肠杆菌突变体的遗传选择，并且预计将分离出各种不同的突变体，而不支持F排除T7。这种突变体的初始屏幕会导致一个菌株的分离，当包含野生型F或表达PIFA的多拷贝质粒时，与F菌株的EOP处T7平板T7。在此屏幕中分离的其他一些突变体（当包含F）T7不排除在同基因父母菌株的效率上，并且已经获得了对高水平的抗生素四环素的抗性表型，这表明细胞膜成分在多余的感染中受到参与。 对这些突变体的更详细的研究以及在更广泛的搜索中获得的其他研究将为对该噬菌体排除系统的完整分子理解提供必要的基本遗传框架。 %% %%这项研究的目的是完全了解细菌结合因子（F质粒）阻止细菌细胞（一种细菌病毒）成功感染细菌细胞的机制。 这项研究通常会影响宿主 - 寄生虫相互作用的领域，因为涉及F排除T7的噬菌体和质粒基因可能与在许多不同的生物环境中受到活性受到损害的保守细胞功能相互作用。 ***