Understanding bacterial epitranscriptomics on the basis of Escherichia coli – T4 phage interactions

基于病毒杀死细菌的潜力，病毒劫持宿主遗传机制的机制引起了极大的兴趣。除了对噬菌体结构和基因组的了解外，对其转录和翻译机制的研究还很薄弱。T4噬菌体使用三种ADP-核糖基转移酶（ADPRT）（Alt、ModA和ModB）对蛋白质进行后修饰。ADPRT将ADP-核糖片段从普遍存在的辅酶烟酰胺腺嘌呤二核苷酸（NAD）转移到特定的受体蛋白，称为ADP-核糖基化。最近，NAD被鉴定为细菌和高等生物中RNA分子的5 '-修饰。NAD作为一种新的RNA修饰的鉴定可能会在所有生命王国中实现一种新的细胞调节的“表观转录组学”层。在这里，我们报告说，噬菌体T4 ADPRT不仅接受NAD，但也NAD-RNA作为底物，从而共价连接整个RNA链的受体蛋白质的“RNA化”反应。我们的工作首次证实了RNA修饰和翻译后蛋白质修饰之间的直接联系。考虑到ADPRTs在病毒感染过程中对核糖体和RNA聚合酶活性的调节作用，ADP-核糖基化和RNA修饰可能在细菌生物学中具有深远的意义。应用NAD或NAD-RNA等小分子调控细菌的转录和翻译，为细胞组织和病毒感染周期的调控提供了一个新的表观转录组学概念。coli相互作用。这个跨学科的基础研究项目将首次深入了解E.大肠杆菌T4噬菌体感染。通过结合不同的下一代测序（NGS）策略和蛋白质组学方法，我们将研究ADP-核糖基化和RNA基化对E. coli-T4噬菌体相互作用。对于这些研究，我们可以利用我们小组已经建立的一系列方法。在我们的项目中，我们使用仅表达一种功能性ADPRT的T4噬菌体突变体，为了解T4 ADPRT在病毒感染周期中的功能作用奠定了基础。此外，我们研究如何翻译后蛋白质修饰调节细菌和噬菌体转录组使用NGS。最后，我们描述了ADP-核糖基化和RNA化对细菌翻译器调节的影响。这项研究将拓宽我们对T4噬菌体（Alt，ModA和ModB）的ADPRT如何调节细菌关键机制并抑制细菌防御机制以引发有效感染的理解。

Based on the potential of phages to kill bacteria, the mechanisms by which viruses hijack their host’s genetic machinery are of enormous interest. Besides the knowledge about phage structure and genome, the dynamics of its transcription and translation apparatus remain poorly understood.For the specific temporal reprogramming of the E. coli transcriptional and translational apparatus, the T4 phage uses three ADP-ribosyltransferases (ADPRTs) (Alt, ModA and ModB) that post-translationally modify proteins. ADPRTs transfer an ADP-ribose fragment from the ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) to specific acceptor proteins, called ADP-ribosylation. Recently, NAD was identified as a 5’-modification of RNA molecules in bacteria and higher organisms. The identification of NAD as a new RNA-modification might implement a new ‘epitranscriptomic’ layer of cellular regulation in all kingdoms of life. Here, we report that bacteriophage T4 ADPRTs accept not only NAD, but also NAD-RNA as substrate, thereby covalently linking entire RNA chains to acceptor proteins in an “RNAylation” reaction. Our work exemplifies the first direct connection between RNA modification and posttranslational protein modificationConsidering the significant role of ADPRTs for the regulation of ribosome and RNA polymerase activities during viral infections, ADP-ribosylation and RNAylation may have far-reaching implications in bacterial biology. The application of small molecules like NAD or NAD-RNA to regulate transcription and translation in bacteria provides a new epitranscriptomic concept for the cellular organisation and regulation of the viral infection cycle.Here, we propose to characterise the bacterial epitranscriptomics on the basis of T4 phage – E. coli interaction. This inter-disciplinary, fundamental research project will provide first insights into the temporal modulation of the transcriptome and translatome of E. coli infected with T4 phage. By combining different next generation sequencing (NGS) strategies and proteomic approaches, we will study the molecular influence of ADP-ribosylation and RNAylation on the central cellular processes of transcription and translation during E. coli – T4 phage interactions. For these studies, we can make use of a range of methods already established in our group. In our project, we set the foundation to understand the functional role of T4 ADPRTs during the viral infection cycle using T4 phage mutants that express only one functional ADPRT. Moreover, we study how post-translational protein modifications regulate the bacterial and phage transcriptome using NGS. Finally, we characterize the influence of ADP-ribosylation and RNAylation on the regulation of the bacterial translation apparatus. This study will broaden our understanding of how the ADPRTs of the T4 phage (Alt, ModA, and ModB) modulate the bacterial key mechanisms and inhibit bacterial defence mechanisms in order to trigger an effective infection.