Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq analysis of Pseudomonas aeruginosa PAO1 cells infected with PhiKZ phage

ABSTRACT: 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.

ORGANISM(S): Pseudomonas aeruginosa

SUBMITTER: Bob Blasdel

PROVIDER: E-GEOD-58494 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Phages are viruses that infect prokaryotes and can shape microbial communitiesby lysis, thus offering applications in various fields. However, challengesexist in sampling, isolation and accurate prediction of the host specificity ofphages as well as in the identification of newly replicated virions in response toenvironmental challenges. A new workflow using biorthogonal non-canonicalamino acid tagging (BONCAT) and click chemistry (CC) allowed combinedanalysis of phages and their hosts, the identification of newly replicated virions,and the specific tagging of phages with biotin for affinity chromatography.Replication of phage λ in Escherichia coli was selected as a model for workflowdevelopment. Specific labeling of phage λ proteins with the non-canonicalamino acid 4-azido-L-homoalanine (AHA) during phage development in E. coliwas confirmed by LC–MS/MS. Subsequent tagging of AHA with fluorescentdyes via CC allowed the visualization of phages adsorbed to the cell surfaceby fluorescence microscopy. Flow cytometry enabled the automated detectionof these fluorescent phage-host complexes. Alternatively, AHA-labeled phageswere tagged with biotin for purification by affinity chromatography. Despitebiotinylation the tagged phages could be purified and were infectious afterpurification. Applying this approach to environmental samples would enablehost screening without cultivation. A flexible and powerful workflow for thedetection and enrichment of phages and their hosts in pure cultures has beenestablished. The developed method lays the groundwork for future workflowsthat could enable the isolation of phage-host complexes from diverse complexmicrobial communities using fluorescence-activated cell sorting or biotinpurification. The ability to expand and customize the workflow through thegrowing range of compounds for CC offers the potential to develop a versatiletoolbox in phage research. This work provides a starting point for these furtherstudies by providing a comprehensive standard operating procedure.

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RNA-Seq analysis of Pseudomonas aeruginosa PAO1 cells infected with PhiKZ phage

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