Molecular Evolution and Life Cycle of Wolbachia Bacteriohage

项目总结： 摘要细管沃尔巴克氏菌是多细胞动物中最常见的专性胞内细菌之一。通过 这会导致节肢动物发生一系列不同的生殖变化，从而有利于受感染的雌性动物的健康 细菌已经通过数十万种物种在世界范围内传播。一种独特的基因组 沃尔巴克氏菌的特征是过多的可移动DNA，包括温和的双链DNA 噬菌体，称为WO-B，没有已知的功能。我们的长期目标是阐明这些机制， 该噬菌体与沃尔巴克氏菌生物学的相关性及其应用。具体的假设是 噬菌体是沃尔巴克氏菌的一种可移动的遗传寄生虫，能够整合并杀死沃尔巴克氏菌。 以确保其自身复制为自私的元素。我们的假设基于四个观察：第一，WO-B 广泛存在，在感染节肢动物的两个主要沃尔巴克氏菌谱系中，感染的菌株占所有菌株的%。 其次，序列分析表明WO-B重组和沃尔巴克氏菌之间的水平传播 共感染同一宿主。第三，WO-B的存在与诱导能力无关 生殖改变。第四，从纯化的昆虫匀浆中观察到WO-B颗粒 在裂解的沃尔巴克氏菌细胞内，证明WO-B是活性的，并可能杀死沃尔巴克氏菌。基于这些 观察到，这一建议的实验重点是沃尔巴克氏菌的分子进化和捕食 噬菌体WO-B。具体目标是： 1.确定形成沃尔巴克氏噬菌体的分子进化力。通过完成这一目标，我们 我将回答三个相关问题：(目标1A)专有细胞内生态位如何影响噬菌体基因组 进化论？我们将确定专性胞内细菌的独特生态条件是如何 影响WO-B的遗传多样性，我们将把它与自由生活中的噬菌体基因组进化规律进行比较 细菌。(目标1B)哪些类别的噬菌体基因正在被选择，它们与基因功能是否相关 如整合、裂解、裂解、毒力或结构？(目标1C)数量、多样性和 噬菌体基因在沃尔巴克氏菌联合感染中横向转移的功能？我们的初步结果 建议猖獗的水平噬菌体转移与强大的净化选择相结合。 2.确定噬菌体是否杀死沃尔巴克氏菌，从而调节宿主内的密度和沃尔巴克氏菌的影响 在主机上。在这里，我们将回答两个与噬菌体功能有关的问题：(目标2A)在受控实验室 环境，裂解噬菌体捕食沃尔巴克氏菌内共生体吗？(目标2B)环境压力是否会引发 噬菌体裂解以及这种裂解的变化是否对应于沃尔巴克氏菌密度的变化以及与 主持人？我们提出这些子目标，以替代流行的观点，即噬菌体可能适应 沃尔巴克氏杆菌改变节肢动物繁殖的能力。我们的初步研究表明，它们的主要作用是 捕食沃尔巴克氏菌。项目说明： 沃尔巴克氏噬菌体的研究可能会通过增加对如何使用 噬菌体作为(I)将转基因特征传播到节肢动物传播载体的管道，以及(Ii)作为一种治疗 用于清除丝虫病沃尔巴克氏菌和相关人类病理的工具

Project Summary: Wolbachia pipientis is one of the most prevalent obligate, intracellular bacteria in multicellular animals. By causing a diverse array of reproductive alterations in arthropods that favor the fitness of infected females, this bacterium has spread worldwide through hundreds of thousands of species. One of the unique genomic features to Wolbachia is an overabundance of mobile DNA including a temperate double-stranded DNA bacteriophage, called WO-B, that has no known function. Our long term goal is to elucidate the mechanisms, relevance, and applications of this bacteriophage to the biology of Wolbachia. The specific hypothesis is that the bacteriophage is a mobile genetic parasite of Wolbachia, capable of integrating into and killing Wolbachia to ensure its own replication as a selfish element. We base that hypothesis on four observations: First, WO-B is widespread and infects 89% of all strains in the two major Wolbachia lineages that infect arthropods. Second, sequence analyses specify WO-B recombination and horizontal transmission between Wolbachia that coinfect the same host. Third, the presence of WO-B does not correlate with the capacity to induce reproductive alterations. Fourth, observations of WO-B particles from purified insect homogenates and from within lysed Wolbachia cells demonstrate that WO-B is active and may kill Wolbachia. Based on these observations, the experimental focus of this proposal is on the molecular evolution and predation of Wolbachia phage WO-B. The specific aims are to: 1. Determine the molecular evolutionary forces shaping Wolbachia bacteriophage. By completing this aim, we will answer three related questions: (Aim 1A) How does an obligate intracellular niche affect phage genome evolution? We will determine how the distinctive ecological conditions of an obligate intracellular bacterium affect genetic diversity in WO-B and we will compare this to the rules of phage genome evolution in free-living bacteria. (Aim 1B) What classes of phage genes are under selection and do they correlate with gene functions such as integration, lysis, lysogeny, virulence, or structure? (Aim 1C) What are the number, diversity, and functions of phage genes that laterally transfer to and from Wolbachia coinfections? Our preliminary results suggest rampant horizontal phage transfer coupled with strong purifying selection. 2. Determine if bacteriophage kill Wolbachia and thereby regulate intrahost densities and Wolbachia's effects on hosts. Here, we will answer two questions related to phage function: (Aim 2A) In a controlled laboratory environment, do lytic phages prey on Wolbachia endosymbionts? (Aim 2B) Does environmental stress trigger phage lysis and does this variation in lysis correspond to variation in Wolbachia densities and interactions with the host? We propose these subaims as an alternative to the prevailing view that phage may be adaptive to Wolbachia's ability to modify arthropod reproduction. Our preliminary studies suggest their principal role is predation of Wolbachia. Project Narrative: The study of Wolbachia pipientis phage may impact public health by increasing knowledge on how to employ the phage as (i) a conduit for spreading transgenic traits into arthropod-borne vectors and (ii) as a therapeutic tool for removing filarial Wolbachia and associated human pathologies