Introducing differential RNA-seq mapping to track the early infection phase forPseudomonasphage ɸKZ

Abstract

As part of the ongoing renaissance of phage biology, more phage genomes are becoming available through DNA sequencing. However, our understanding of the transcriptome architecture that allows these genomes to be expressed during host infection is generally poor. Transcription start sites (TSSs) and operons have been mapped for very few phages, and an annotated global RNA map of a phagealone or together with its infected host-is not available at all. Here, we applied differential RNA-seq (dRNA-seq) to study the early, host takeover phase of infection by assessing the transcriptome structure of Pseudomonas aeruginosa jumbo phage ɸKZ, a model phage for viral genetics and structural research. This map substantially expands the number of early expressed viral genes, defining TSSs that are active ten minutes after ɸKZ infection. Simultaneously, we record gene expression changes in the host transcriptome during this critical metabolism conversion. In addition to previously reported upregulation of genes associated with amino acid metabolism, we observe strong activation of genes with functions in biofilm formation (cdrAB) and iron storage (bfrB), as well as an activation of the antitoxin ParD. Conversely, ɸKZ infection rapidly down-regulates complexes IV and V of oxidative phosphorylation (atpCDGHF and cyoABCDE). Taken together, our data provide new insights into the transcriptional organization and infection process of the giant bacteriophage ɸKZ and adds a framework for the genome-wide transcriptomic analysis of phage-host interactions.

A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions

“…ncRNA OsiS may harbor a hidden small open reading frame that may drive sedimentation in 30S fractions. Terminator 5′phosphate-dependent exonuclease (TEX) treatment indicates a TEX-unaffected 5′ end downstream of the annotated TSS ( 51 ). Sequencing of RNA fragments recovered from monosomes (Ribo-seq [ 66 ] reanalyzed with HRIBO [ https://github.com/RickGelhausen/HRIBO ]) supports the possibility of a short translated fragment starting at a putative start codon that is out of frame with ptrB .…”

“…Stable noncoding RNA fragments overlapping with 59 or 39 untranslated regions (UTRs) constitute a potentially large class of bacterial riboregulators (47)(48)(49)(50). Recent experimental transcriptome annotation (51)(52)(53) suggested that also in the Pseudomonas strain used here, many such UTR-derived candidate ncRNAs exist and accumulate to considerable levels. Here, we observe that such UTR fragments often showed a different sedimentation profile compared to that of the respective coding sequence (CDS) of the same mRNA ( Fig.…”

“…Phage mRNAs are efficiently transferred from transcription to translation. At the early time point of UKZ infection used here, nearly all (366/376) annotated phage genes (NCBI nucleotide accession NC_004629.1) are expressed (Table S1) (30,51). For an initial glimpse at the sedimentation profiles of phage transcripts, we probed two well-expressed viral operons on Northern blots; specifically, operons 54 and 88 (30), which encode metabolic and structural proteins of the phage, respectively.…”

«PemIK (PemK/PemI) type II TA system from Klebsiella pneumoniae clinical strains inhibits lytic phage»

“…Thus, overexpression of the toxin, in the case of the phage vB_KpnP-VAC1, allowed the bacterium to protect themselves from phage infection by the action of the free toxin that inhibited phage infection. Furthermore, the recent study of Wicke et al (2020), demonstrated the reverse effect, i.e. enrichment of ParD antitoxin transcripts of the ParE/ParD type II TA system, leads to successful infection of the strain by the phage, as the viral DNA is protected from the deleterious action of the ParE toxin 32 .…”

“…Furthermore, the recent study of Wicke et al (2020), demonstrated the reverse effect, i.e. enrichment of ParD antitoxin transcripts of the ParE/ParD type II TA system, leads to successful infection of the strain by the phage, as the viral DNA is protected from the deleterious action of the ParE toxin 32 . Besides, it has been described that phage often harbor speci c protease inhibitors that can interfere with the degradation of antitoxins 33 .…”