Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk, Poland.
BioVentures Institute Ltd., Poznan, Poland.
PLoS One. 2018 Apr 6;13(4):e0195449. doi: 10.1371/journal.pone.0195449. eCollection 2018.
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.
TP-84 噬菌体是一种具有历史意义的特征明确的噬菌体。它是 Siphoviridae 科的成员，感染许多嗜热的 Geobacillus（芽孢杆菌）stearothermophilus 菌株。其 47.7kbp 的双链 DNA 基因组显示存在 81 个编码序列（CDS），编码 4kDa 或更大的多肽。有趣的是，所有 CDS 都指向同一个方向，指向一条 DNA 链的主要转录方向。基于同源性搜索，可以为 31 个 CDSs 分配一个假设的功能。噬菌体基因组上未发现 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 基因组紧密包装有 CDSs，通常间隔几个到几十个碱基对，或者经常重叠。基因组包含五个假定的启动子样序列，与宿主启动子的保守序列相似，允许有 2 个碱基对的错配。此外，还检测到十个假定的 rho 非依赖性终止子。由于基因组序列与任何以前描述的噬菌体基本上没有相似性，TP-84 应被视为 Siphoviridae 家族中未定义属的新物种。因此，国际病毒分类学委员会已接受了一种新的 Tp84virus 属的分类建议。通过确认 33 种 TP-84 蛋白，对生物信息学基因组分析进行了验证，这些蛋白包括：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）纯化和功能测定重组 TP-84 SSB，已证明其可改善 PCR 反应，c）TP-84 噬菌体衣壳蛋白的质谱（MS）分析，d）TP-84 内切酶活性的纯化，e）感染时间过程中宿主细胞的 MS 分析。
