Virus-Host interaction of E. coli DksA-deficient cells infected by phage P1vir

Bacteriophage P1 along with ? and T4 phages are among the best described bacterial viruses in molecular biology. For years, P1 features as well as its life cycle have been studied and its complete genome was published. Undeciphered phenomenon of improved P1vir lytic development in the absence of DksA protein in cell engaged us to more holistic experimental approach. Bacterial wild type and dksA strains were cultured to OD600 = 0.2. Next, P1vir was added, samples were withdrawn at 0, 10 and 30 minutes after P1vir infection. Total RNA was isolated and checked for quality using the Bioanalyzer 2100. The sequencing run was conducted on the Illumina NovaSeq6000 platform. 30 million pair-end reads per samples were assessed with 101 pb read length. Reference P1 phage genome sequence and annotations were downloaded from GenBank. We have discovered many changes in virus transcriptome. For instance: downregulation of phage genes encoding the main repressor of lysogeny C1 or proteins triggering cell lysis (e.g., lysozyme, holin) and upregulation of genes encoding antiholins in dksA mutant. This results support our gentle lysis hypothesis – less efficient lysis, combined with minor improvements of phage development which may lead to higher phage yield in DksA-devoid cells. We have observed upregulated expression of phage genes responsible for virion-parts production in the dksA mutant. Interestingly, expression of lysogeny-related c8 gene is upregulated in the dksA mutant. We speculate that P1vir developing in the dksA host is at the brink of lysogeny but is unable to established it and eventually enters the lytic pathway. We also found some interesting events in host cells upon infection. P1vir is taking control of the cellular protein, sugar and lipid metabolism in both, the wild type and dksA mutant hosts. However, in dksA mutant several genes involved in sulfur metabolism were uniquely upregulated. It remains unclear if this associates with obtaining new energy sources or with global reprograming via H2S signaling functions. Generally, the hosts are reacting by activating SOS response or upregulating the heat shock proteins. But we also found downregulation of proteolysis which was unique for the dksA strain. We believe that this extensive and comprehensive study not only finds reasonable explanations for the improved P1vir development in dksA strain, but also makes a great contribution to the field of P1 phage biology. Funding: This research was funded by the National Science Center, Poland (grant PRELUDIUM 2013/09/N/NZ2/01899 to G.M.C.) Overall design: To obtain a widespread and holistic overview of the virus–host interactions, we analyzed the transcriptome of both, the P1vir phage and the dksA and wild type hosts during the infection. Total RNA sequencing was performed to assess the changes in gene expression pattern at 0, 10 and 30 min of P1vir infection

噬菌体P1（Bacteriophage P1）与λ噬菌体和T4噬菌体一同，是分子生物学（molecular biology）领域中研究最为透彻的细菌病毒之一。多年来，学界对P1的特性及其生命周期开展了大量研究，其完整基因组序列也已发表。我们在研究中发现，宿主细胞缺失DksA蛋白（DksA protein）时，P1vir的裂解发育过程会得到增强，这一尚未被阐明的现象促使我们采用更为全面的实验策略展开探究。
将细菌野生型菌株与dksA突变株培养至光密度600nm（OD600）=0.2。随后接种P1vir，分别在感染后0、10和30分钟采集样本。提取总RNA（total RNA），并使用生物分析仪2100（Bioanalyzer 2100）对RNA质量进行检测。测序工作在Illumina NovaSeq6000测序平台上完成，每个样本获得3000万条双端读段（pair-end reads），读长为101 bp。从GenBank数据库（GenBank）下载P1噬菌体的参考基因组序列及注释信息。
我们在病毒转录组（transcriptome）中发现了诸多表达变化。例如，在dksA突变株中，编码溶源性（lysogeny）主要阻遏蛋白C1的噬菌体基因、触发细胞裂解的蛋白（如溶菌酶、holin（holin））的表达均出现下调，而编码antiholins（antiholins）的基因则呈现上调。上述研究结果支持我们提出的缓裂解假说：在缺失DksA蛋白的细胞中，噬菌体裂解效率降低，同时其发育过程得到小幅优化，这或许可使噬菌体的最终产量得到提升。我们还观察到，在dksA突变株中，负责噬菌体颗粒组分（virion-parts）合成的噬菌体基因表达出现上调。值得注意的是，与溶源性相关的c8基因在dksA突变株中的表达量同样出现上调。我们推测，在dksA宿主细胞中增殖的P1vir处于溶源性的临界状态，但无法完成溶源化建立，最终仍进入裂解途径。
我们还在感染后的宿主细胞中发现了一些有趣的现象。无论是野生型宿主还是dksA突变株宿主，P1vir均会调控宿主细胞的蛋白质、糖类及脂质代谢过程。但在dksA突变株中，若干参与硫代谢（sulfur metabolism）的基因出现了特异性上调。目前尚不清楚这一现象是否与获取新的能量来源有关，或是通过硫化氢（H2S）信号通路实现的全局重编程相关。总体而言，宿主细胞会通过激活SOS应答（SOS response）或上调热休克蛋白（heat shock proteins）的表达来应对感染。但我们同时发现，蛋白水解（proteolysis）过程出现下调，这一现象仅在dksA菌株中特异性存在。
我们认为，这项全面且深入的研究不仅为dksA菌株中P1vir发育增强的现象提供了合理的解释，同时也为P1噬菌体生物学领域的研究作出了重要贡献。
资助说明：本研究由波兰国家科学中心（National Science Center, Poland）资助（项目编号：PRELUDIUM 2013/09/N/NZ2/01899，获得者：G.M.C.）
实验整体设计：为全面解析病毒与宿主的相互作用，我们分析了P1vir噬菌体、dksA突变株及野生型宿主在感染过程中的转录组表达情况。通过总RNA测序，我们评估了P1vir感染后0、10和30分钟时的基因表达模式变化。