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Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics.

Krishnamurthi, Revathy; González-Tortuero, E; Plahe, Grace; Goodhead, Ian B; Fothergill, Joanne L; James, Chloë E; Allison, Heather E

Authors

Revathy Krishnamurthi

E González-Tortuero

Grace Plahe

Joanne L Fothergill

Heather E Allison



Abstract

Temperate phages are found integrated as prophages in the majority of bacterial genomes. Some prophages are cryptic and fixed in the bacterial chromosome, but others are active and can be triggered into a replicative form either spontaneously or by exposure to inducing factors. Prophages are commonly associated with the ability to confer toxin production or other virulence-associated traits on their host cell. More recent studies have shown they can play a much bigger role in altering the physiology of their hosts. The technique described here has enabled us to investigate how prophages affect gene expression in the opportunistic bacterium Pseudomonas aeruginosa. In this work, the growth of the wild-type P. aeruginosa strain PAO1 was compared with that of isogenic lysogens carrying different combinations of prophages from the Liverpool Epidemic Strain (LES) LESB58. In a lysogen culture, a proportion of bacterial cells will be supporting lytic bacteriophage replication (spontaneous induction) with a high level of expression per cell oflate phage genes, such as those associated with the assembly of phage particles,thus masking the low-level gene expression associated with lysogen-restricted geneexpression. The impact of spontaneous induction can thus obscure prophage gene expression across a lysogen population. Growth profiling experiments were used to identify spontaneous induction, which was minimal during the early exponential growth phase. This study reports how to prepare sample cultures during the early exponential growth phase and how to set up adequate controls despite low cell numbers. These protocols ensure the reliable and reproducible comparison of wild-type and lysogenic bacteria under various conditions, thus improving the transcriptomic profiling of prophage genomes and aiding in the identification of previously unrecognized prophage functions.

Citation

Krishnamurthi, R., González-Tortuero, E., Plahe, G., Goodhead, I. B., Fothergill, J. L., James, C. E., & Allison, H. E. (in press). Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics. Journal of Visualized Experiments, 10.3791/64945. https://doi.org/10.3791/64945

Journal Article Type Article
Acceptance Date May 11, 2023
Online Publication Date Jan 5, 2024
Deposit Date May 12, 2023
Publicly Available Date Jan 6, 2026
Journal Journal of visualized experiments : JoVE
Electronic ISSN 1940-087X
Publisher Journal of Visualized Experiments
Peer Reviewed Peer Reviewed
Issue 203
Pages 10.3791/64945
DOI https://doi.org/10.3791/64945
Keywords Bacteriophages - genetics, Gene Expression Profiling, Chromosomes, Bacterial, Bacterial Typing Techniques, Cell Count
Publisher URL https://www.jove.com/journal