Bacteriophages are highly abundant viruses of bacteria. The major role of phages in shaping bacterial communities and their emerging medical potential as antibacterial agents has triggered a rebirth of phage research. To understand the molecular mechanisms by which phages hijack their host, omics technologies can provide novel insights into the organization of transcriptional and translational events occurring during the infection process. In this study, we apply transcriptomics and proteomics to characterize the temporal patterns of transcription and protein synthesis during the T4 phage infection of E. coli. We investigated the stability of E. coli-originated transcripts and proteins in the course of infection, identifying the degradation of E. coli transcripts and the preservation of the host proteome. Moreover, the correlation between the phage transcriptome and proteome reveals specific T4 phage mRNAs and proteins that are temporally decoupled, suggesting post-transcriptional and translational regulation mechanisms. This study provides the first comprehensive insights into the molecular takeover of E. coli by bacteriophage T4. This data set represents a valuable resource for future studies seeking to study molecular and regulatory events during infection. We created a user-friendly online tool, POTATO4, which is available to the scientific community and allows access to gene expression patterns for E. coli and T4 genes.

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The mechanisms by which viruses hijack their host's genetic machinery are of enormous current interest. One mechanism is adenosine diphosphate (ADP) ribosylation, where ADP-ribosyltransferases (ARTs) transfer an ADP-ribose fragment from the ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) to acceptor proteins. When bacteriophage T4 infects Escherichia coli, three different ARTs reprogram the host's transcriptional and translational apparatus. Recently, NAD was identified as a 5'-modification of cellular RNA molecules in bacteria and higher organisms. Here, we report that bacteriophage T4 ARTs accept not only NAD, but also NAD-RNA as substrate, thereby covalently linking entire RNA chains to acceptor proteins in an "RNAylation" reaction. One of these ARTs, ModB, efficiently RNAylates its host protein target, ribosomal protein S1, at arginine residues and strongly prefers NAD-RNA over NAD. Mutation of a single arginine at position 139 abolishes ADP-ribosylation and RNAylation. Overexpression of mammalian ADP-ribosylarginine hydrolase 1 (ARH1), which cleaves arginine-phosphoribose bonds, shows a decelerated lysis of E. coli when infected with T4. Our findings not only challenge the established views of the phage replication cycle, but also reveal a distinct biological role of NAD-RNA, namely activation of the RNA for enzymatic transfer. Our work exemplifies the first direct connection between RNA modification and post-translational protein modification. As ARTs play important roles in different viral infections, as well as in antiviral defence by the host, RNAylation may have far-reaching implications.

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