Project description:The global transcriptional profiles of Pseudomonas aeruginosa phages LUZ19, LUZ24, YuA, PAK_P3, 14-1 and phiKZ was obtained using the long read RNA sequencing technique ONT-cappable-seq. Using this approach we obtained a comprehensive genome-wide map of viral transcription start sites, terminators and transcription units.
Project description:The global transcriptional profile of novel T7-like Pseudomonas aeruginosa phage LUZ100 was obtained using the long read RNA sequencing technique ONT-cappable-seq. Using this approach we obtained a comprehensive genome-wide map of viral transcription start sites, terminators and transcription units and gained new insights in the molecular mechanisms of transcriptional regulation of T7-like temperate phages.
Project description:We developed ONT-cappable-seq, a specialized long-read RNA sequencing technique that allows end-to-end sequencing of primary prokaryotic transcripts using the Nanopore sequencing platform. We applied ONT-cappable-seq to study the transcriptional landscape of Pseudomonas aeruginosa phage LUZ7, leading to a comprehensive genome-wide map of viral transcription start sites, terminators and complex operon structures that fine-regulate gene expression. At the same time, it provides new insights in the RNA biology of LUZ7 and paves the way for more in depth transcription studies that can help unveil the complex layers of phage-host interactions.
Project description:The global transcriptional profile of Pseudomonas chlororaphis infecting phage 201f2-1 was obtained using the long-read RNA sequencing technique ONT-cappable-seq. this resulted in a comprehensive genome-wide map of viral transcription start and termination sites. In addition, we were able to identify different transcription units and gained new insights in the molecular mechanisms of of transcriptional regulation of members of the Phikzvirus.
Project description:Virulent bacteriophages (or phages) are viruses that specifically infect and lyse a bacterial host. When multiple phages co-infect a bacterial host, the extent of lysis, dynamics of bacteria-phage and phage-phage interactions are expected to vary. The objective of this study is to identify the factors influencing the interaction of two virulent phages with different Pseudomonas aeruginosa growth states (planktonic, an infected epithelial cell line, and biofilm) by measuring the bacterial time-kill and individual phage replication kinetics. A single administration of phages effectively reduced P. aeruginosa viability in planktonic conditions and infected human lung cell cultures, but phage-resistant variants subsequently emerged. In static biofilms, the phage combination displayed initial inhibition of biofilm dispersal, but sustained control was achieved only by combining phages and meropenem antibiotic. In contrast, adherent biofilms showed tolerance to phage and/or meropenem, suggesting a spatiotemporal variation in the phage-bacterial interaction. The kinetics of adsorption of each phage to P. aeruginosa during single- or co-administration were comparable. However, the phage with the shorter lysis time depleted bacterial resources early and selected a specific nucleotide polymorphism that conferred a competitive disadvantage and cross-resistance to the second phage. The extent and strength of this phage-phage competition and genetic loci conferring phage resistance, are, however, P. aeruginosa genotype dependent. Nevertheless, adding phages sequentially resulted in their unimpeded replication with no significant increase in bacterial host lysis. These results highlight the interrelatedness of phage-phage competition, phage resistance and specific bacterial growth state (planktonic/biofilm) in shaping the interplay among P. aeruginosa and virulent phages.
Project description:Bacteriophages (hereafter “phages”) are ubiquitous predators of bacteria in the natural world, but interest is growing in their development into antibacterial therapy as complement or replacement for antibiotics. However, bacteria have evolved a huge variety of anti-phage defense systems allowing them to resist phage lysis to a greater or lesser extent, and in pathogenic bacteria these inevitably impact phage therapy outcomes. In addition to dedicated phage defense systems, some aspects of the general stress response also impact phage susceptibility, but the details of this are not well known. In order to elucidate these factors in the opportunistic pathogen Pseudomonas aeruginosa, we used the laboratory-conditioned strain PAO1 as host for phage infection experiments as it is naturally poor in dedicated phage defense systems. Screening by transposon insertion sequencing indicated that the uncharacterized operon PA3040-PA3042 was potentially associated with resistance to lytic phages. However, we found that its primary role appeared to be in regulating biofilm formation. Its expression was highly growth-phase dependent and responsive to phage infection and cell envelope stress.
Project description:Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial infections. Its ability to survive under different conditions relies on a complex regulatory network engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use the available resources. P. aeruginosa PA3973 encodes a putative TetR family transcriptional regulator, with a helix-turn-helix motif involved in DNA binding. We applied transcriptome profiling (RNA-seq), and genome-wide identification of binding sites using ChIP-seq to unravel the biological role of PA3973.