Introducing Differential RNAseq mapping to track the phage infection process for Pseudomonas virus ɸKZ
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ABSTRACT:
ORGANISM(S): Phikzvirus phiKZ Pseudomonas aeruginosa PAO1
PROVIDER: GSE153067 | GEO | 2020/11/13
REPOSITORIES: GEO
Similar Datasets
Project description:Pseudomonas aeruginosa bacteriophage PhiKZ is the type representative of the M-bM-^@M-^XgiantM-bM-^@M-^Y phage genus with unusually large virions and genomes. By unraveling the transcriptional map of the 280 kb genome to single-nucleotide resolution, we show that it encodes 369 genes organized in 134 operons, 20% more than originally annotated. Early transcription is initiated from 28 highly conserved AT-rich promoters distributed over the PhiKZ genome, all located on the same strand. Transcription of middle and late genes is dependent on protein synthesis and mediated by very poorly conserved middle (6) and late (16) promoters. As a result of massive PhiKZ transcription, halfway through infection only 1.5% of all mRNAs in the infected cell remain bacterial. Unique to PhiKZ is its ability to complete its infection in complete absence of bacterial RNA polymerase (RNAP) enzyme activity. Its transcription is performed by the consecutive action of two PhiKZ-encoded, non-canonical RNAPs, one of which is packed within the virion. This unique, rifampicin-resistant transcriptional machinery is conserved among giant viruses, seems to function without auxiliary factors and might have its origin preceding the split between Gram-positive and Gram-negative bacteria. Construction of transcription maps for the PhiKZ phage and analysis of differential expression of host and phage genes using RNA-Seq data from samples taken in duplicate at 0, 5, 10, 15, and 35 minutes into infection.
2014-06-14 | E-GEOD-58494 | biostudies-arrayexpress
Project description:Preliminary analyses of Pseudomonas aeruginosa cells infected with phage PhiKZ and a control at 20 minutes post infection.
2018-11-21 | PXD011520 | Pride
Project description:Pseudomonas aeruginosa bacteriophage PhiKZ is the type representative of the ‘giant’ phage genus with unusually large virions and genomes. By unraveling the transcriptional map of the 280 kb genome to single-nucleotide resolution, we show that it encodes 369 genes organized in 134 operons, 20% more than originally annotated. Early transcription is initiated from 28 highly conserved AT-rich promoters distributed over the PhiKZ genome, all located on the same strand. Transcription of middle and late genes is dependent on protein synthesis and mediated by very poorly conserved middle (6) and late (16) promoters. As a result of massive PhiKZ transcription, halfway through infection only 1.5% of all mRNAs in the infected cell remain bacterial. Unique to PhiKZ is its ability to complete its infection in complete absence of bacterial RNA polymerase (RNAP) enzyme activity. Its transcription is performed by the consecutive action of two PhiKZ-encoded, non-canonical RNAPs, one of which is packed within the virion. This unique, rifampicin-resistant transcriptional machinery is conserved among giant viruses, seems to function without auxiliary factors and might have its origin preceding the split between Gram-positive and Gram-negative bacteria.
2014-06-14 | GSE58494 | GEO
Project description:Introducing Differential RNAseq mapping to track the phage infection process for Pseudomonas virus ɸKZ
| PRJNA641380 | ENA
Project description:Large-genome bacteriophages (jumbo phages) of the Chimalliviriadae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA. Targeted knockdown of ChmC using mRNA-targeting Cas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.
2024-03-12 | GSE243675 | GEO
Project description:Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.
2024-11-26 | PXD049415 | Pride
Project description:Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.
2024-11-26 | PXD050275 | Pride
Project description:Assay of recombinant Pseudomonas phage PhiKZ gp54 with the prohead protease gp175. The gene for each protein was cloned into the expression plasmid DH10B. The assay was conducted in a lysate of E. coli DH10B cells, conditions in which PhiKZ gp175 had previously been shown to be active. Analyses of gel slices identified C-terminal processing of gp54 via the semi-tryptic peptide 558-FITAMDAAAAAAGVTVTME-576 in the gp54-plus-gp175 slices (predominantly slice 3) provided convincing evidence for proteolysis of gp54 in the presence of gp175.
2018-11-21 | PXD011519 | Pride
Project description:Large-genome bacteriophages (jumbo phages) of the Chimalliviriadae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.
2024-03-25 | PXD045260 | Pride
Project description: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 phage - alone 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.
| S-EPMC8244752 | biostudies-literature