Project description:The 280-kB giant bacteriophage PHIKZ expresses hundreds of transcripts upon infection of Pseudomonas aeruginosa. To illuminate which transcripts are the very first and abundant ones, we extracted RNA after infection and sequenced the transcripts. The early expressed genes promise to be important for surpassing the host defence systems and to reprogram the host machinery towards phage replication. We discovered a number of transcripts that encode for proteins which we found in an independent study to be associated with large complexes. In essence, early genes represent exciting opportunities to filter for important factors that could also engage in large complexes.
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.
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.
Project description:ChmA forms the phage nucleus in PhiKZ-infected PAO1 cells, we aimed to illuminate the effects on the transcriptome upon knockdown (KD) of ChmA translation through anti-sense-based technology. We identified phage transcripts that are affected in level upon KD of ChmA and clustering allowed to group these together that likely are expressed at a similar stage and dependency in the phage replication cycle.
Project description:We aimed to illuminate the effects on the transcriptome upon translation knockdown (KD) of core genes and other characterised genes or early expressed genes in PHIKZ through anti-sense-based technology. We identified phage transcripts that are affected in level upon KD and clustering allowed to group these together that likely are expressed at a similar stage and dependency in the phage replication cycle. The read coverage can point to alterations in transcripts and mode of action for several candidates that were knocked down.