Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus.
Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus.
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DOI：
10.1371/journal.pone.0195449
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Zylicz-Stachula A
中科院分区：
文献类型：
--
作者：
Skowron PM;Kropinski AM;Zebrowska J;Janus L;Szemiako K;Czajkowska E;Maciejewska N;Skowron M;Łoś J;Łoś M;Zylicz-Stachula A
噬菌体 TP-84 是一种具有历史意义的特征明确的噬菌体。它是管状病毒科的成员，感染许多嗜热地芽孢杆菌（芽孢杆菌）嗜热脂肪芽孢杆菌菌株。其 47.7 kbp 双链 DNA 基因组显示存在 81 个编码 4 kDa 或更大多肽的编码序列 (CDS)。有趣的是，所有 CDS 的方向都相同，指向一条 DNA 链的主导转录方向。基于同源搜索，可以将假设的功能分配给 31 个 CDS。在噬菌体基因组上没有发现 RNA 或 DNA 聚合酶编码基因，表明 TP-84 依赖于宿主的转录和复制酶。 TP84 基因组与 CDS 紧密堆积在一起，通常间隔几个到几十个 bp，或者经常重叠。该基因组包含五个推定的启动子样序列，与宿主启动子共有序列相似，并允许存在 2 bp 错配。此外，还检测到十个假定的与 rho 无关的终止子。由于基因组序列与任何先前表征的噬菌体基本上没有相似性，因此 TP-84 应被视为 Siphoviridae 家族中未定义属的新物种。因此，新的Tp84病毒属的分类学建议已被国际病毒分类委员会接受。通过确认 33 种 TP-84 蛋白来验证生物信息学基因组分析，其中包括：a) 在大肠杆菌中克隆选定的 CDS，编码 DNA 单链结合蛋白（SSB；基因 TP84_63），b) 重组 TP-84 SSB 的纯化和功能测定，该重组 TP-84 SSB 已被证明可以改善 PCR 反应，c) TP-84 噬菌体的质谱 (MS) 分析衣壳蛋白，d) TP-84 内溶素活性的纯化，e) 对感染时程的宿主细胞进行 MS 分析。
Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the Siphoviridae, and infects a number of thermophilic Geobacillus (Bacillus) stearothermophilus strains. Its’ 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs. No RNA or DNA polymerase-coding genes were found on the bacteriophage genome indicating that TP-84 relies on the host’s transcriptional and replication enzymes. The TP84 genome is tightly packed with CDSs, typically spaced by several-to-tens of bp or often overlapping. The genome contains five putative promoter-like sequences showing similarity to the host promoter consensus sequence and allowing for a 2-bp mismatch. In addition, ten putative rho-independent terminators were detected. Because the genome sequence shows essentially no similarity to any previously characterised bacteriophage, TP-84 should be considered a new species in an undefined genus within the Siphoviridae family. Thus a taxonomic proposal of a new Tp84virus genus has been accepted by the International Committee on Taxonomy of Viruses. The bioinformatics genome analysis was verified by confirmation of 33 TP-84 proteins, which included: a) cloning of a selected CDS in Escherichia coli, coding for a DNA single-stranded binding protein (SSB; gene TP84_63), b) purification and functional assays of the recombinant TP-84 SSB, which has been shown to improve PCR reactions, c) mass spectrometric (MS) analysis of TP-84 bacteriophage capsid proteins, d) purification of TP-84 endolysin activity, e) MS analysis of the host cells from infection time course.
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