Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly.
Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly.
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DOI：
10.1128/jvi.01492-16
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发表时间：
2016-11-15
影响因子：
5.4
通讯作者：
Black LW
中科院分区：
文献类型：
--
作者：
Thomas JA;Benítez Quintana AD;Bosch MA;Coll De Peña A;Aguilera E;Coulibaly A;Wu W;Osier MV;Hudson AO;Weintraub ST;Black LW
巨尾细菌病毒或噬菌体，例如铜绿假单胞菌噬菌体 phiKZ，具有包装成大型非典型病毒粒子的长基因组。人们对 phiKZ 和相关噬菌体生物学的许多方面知之甚少，主要是因为大多数蛋白质的功能尚不清楚。我们假设肠道沙门氏菌噬菌体 SPN3US 可能是解决这一知识空白的有用模型噬菌体。 240 kb SPN3US 基因组与 phiKZ 和相关噬菌体共享一组由 91 个基因组成的核心基因，其中~61 个是病毒体基因，这与病毒体复杂性是一种古老的保守特征的预期一致。对 18 个突变体进行核苷酸测序，可以将 13 个基因指定为必需基因，而这些信息无法通过基于序列的搜索来确定 11 个基因。对 64 和 241 两个 SPN3US 病毒体蛋白突变体进行蛋白质组分析，为巨噬菌体头的组成和组装提供了新的见解。 64 个突变体分析揭示了 SPN3US 头组装所需的所有遗传决定因素，以及 gp64 及其在相关噬菌体中的同源物可能的头尾连接作用，因为产生了无尾颗粒表型。对编码 RNA 聚合酶 β 亚基的 241 中的突变进行分析表明，如果没有该亚基，则不会有其他亚基组装到头部中，并且能够识别“缺失”的 β' 亚基结构域。这些发现支持 SPN3US 作为巨噬菌体研究的优秀模型，为未来分析其极不寻常的病毒体、宿主相互作用和进化奠定了基础。重要性 近年来，病毒学发生了范式转变，人们认识到在许多环境中都可以发现感染原核生物的极大病毒（巨型噬菌体）。一组与原型巨型噬菌体 phiKZ 相关的噬菌体引起了人们的极大兴趣，因为它们的病毒体是最复杂的原核病毒之一，并且具有生物防治和噬菌体治疗应用的潜力。我们对这些噬菌体生物学的理解是有限的，因为它们的大部分蛋白质尚未被表征和/或在没有任何实验验证的情况下被认为是推定的。在这项研究中，我们结合基因组学、遗传学和蛋白质组学分析了沙门氏菌噬菌体 SPN3US，并在此过程中揭示了有关巨噬菌体头部结构和组装以及病毒颗粒 RNA 聚合酶组成的新信息。我们的研究结果证明了 SPN3US 作为与 phiKZ 相关的不断增长的噬菌体群体的模型噬菌体的适用性。
Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage ϕKZ, have long genomes packaged into large, atypical virions. Many aspects of ϕKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with ϕKZ and related phages, ∼61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes an RNA polymerase β subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a “missing” β′ subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution. IMPORTANCE In recent years, there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage ϕKZ are of great interest due to their virions being among the most complex of prokaryotic viruses and their potential for biocontrol and phage therapy applications. Our understanding of the biology of these phages is limited, as a large proportion of their proteins have not been characterized and/or have been deemed putative without any experimental verification. In this study, we analyzed Salmonella phage SPN3US using a combination of genomics, genetics, and proteomics and in doing so revealed new information regarding giant phage head structure and assembly and virion RNA polymerase composition. Our findings demonstrate the suitability of SPN3US as a model phage for the growing group of phages related to ϕKZ.