Presentation Information
[P04-506]Elucidation of Cell Death and RecA Activation Mechanisms in Prophage–Deficient Pseudomonas aeruginosa
○Miki Aramaki1, Ayaka Uehara2, Nobuhiko Nomura3,4,5,6, Masanori Toyofuku3,4,5 (1. Agro Biol. Res. Sci., Univ. Tsukuba (Japan), 2. Grad. Sch. Life Environ. Sci., Univ. Tsukuba (Japan), 3. Inst. Life Environ. Sci., Univ. Tsukuba (Japan), 4. MiCS, Univ. Tsukuba (Japan), 5. TIAR, Univ. Tsukuba (Japan), 6. TARA, Univ. Tsukuba (Japan))
Keywords:
prophage,RecA,cell death,phage-interaction,Pseudomonas aeruginosa
Purpose
In industrial applications, bacteriophages are generally eliminated due to their strong infectivity, which can cause horizontal gene transfer and host cell destruction. In contrast, prophages are widely conserved within host chromosomes and persist in a latent state. Although some prophages contribute to host defence mechanisms, most prophage functions remain unexplained, particularly in polylysogenic hosts. Interactions among coexisting prophages are poorly understood. Pseudomonas aeruginosa, an opportunistic pathogen widely distributed in natural environments, is a model for addressing this gap. Genome-wide analyses by our laboratory revealed that many publicly sequenced P. aeruginosa genomes harbor multiple prophages. In this study, we focused on the oceanic isolate Ocean-1187S, which is indicated to contain eight distinct prophage regions. In our previous work, at least two prophage regions actively produce infectious phage particles. To investigate the roles of these prophages, strains lacking individual prophage regions were constructed. Notably, deletion of one specific prophage, designated ΔOc87B, resulted in a marked increase in host cell death. Because prophage deletion-induced host cell death has not been reported previously, this study aimed to elucidate the underlying mechanism.
Method
To clarify the timing of cell death in Ocean-1187SΔOc87B, dead cells and extracellular DNA were stained with propidium iodide (PI). Bacterial growth (OD600) and PI fluorescence intensity were monitored over time for 24 hours using a plate reader. In addition, microscopic observation was performed after cultivation. Using the same experimental system, bacterial growth and PI fluorescence were compared among the wild-type, ΔOc87B, and ΔOc87BΔrecA strains. In addition, RNA sequencing was performed to comprehensively analyze transcriptional differences between the wild-type and ΔOc87B strains.
Results
Time-course analysis revealed that PI fluorescence in the ΔOc87B strain increased from the early stationary phase, indicating the onset of cell death, whereas fluorescence remained low in the wild-type strain. Microscopic observation revealed cell lysis in the ΔOc87B strain. Deletion of recA restored growth and suppressed PI fluorescence, demonstrating that cell death was RecA-dependent. RNA-seq analysis revealed increased transcription of two prophage regions distinct from ΔOc87B, namely Oc87C and Oc87D, in the ΔOc87B strain compared with the wild-type strain, while recA transcription was unchanged. In addition, whole-genome sequencing analysis showed a marked increase in DNA copy number within the Oc87C region, suggesting activation of additional prophages following Oc87B deletion.
Consideration
Together with the RecA-dependent cell death and transcriptional activation of other prophages, these findings suggest that deletion of a single prophage may induce the expression of coexisting prophages within the host genome.
Conclusion
We propose that deletion of Oc87B activates RecA, leading to induction of additional resident prophages and host cell lysis. Future studies using RecA reporter strains and systematic prophage deletion mutants will clarify this mechanism. Elucidating this regulatory cascade will advance our understanding of how multiple prophages are maintained within a single bacterial genome and reveal novel prophage-prophage and phage-host interaction mechanisms.
In industrial applications, bacteriophages are generally eliminated due to their strong infectivity, which can cause horizontal gene transfer and host cell destruction. In contrast, prophages are widely conserved within host chromosomes and persist in a latent state. Although some prophages contribute to host defence mechanisms, most prophage functions remain unexplained, particularly in polylysogenic hosts. Interactions among coexisting prophages are poorly understood. Pseudomonas aeruginosa, an opportunistic pathogen widely distributed in natural environments, is a model for addressing this gap. Genome-wide analyses by our laboratory revealed that many publicly sequenced P. aeruginosa genomes harbor multiple prophages. In this study, we focused on the oceanic isolate Ocean-1187S, which is indicated to contain eight distinct prophage regions. In our previous work, at least two prophage regions actively produce infectious phage particles. To investigate the roles of these prophages, strains lacking individual prophage regions were constructed. Notably, deletion of one specific prophage, designated ΔOc87B, resulted in a marked increase in host cell death. Because prophage deletion-induced host cell death has not been reported previously, this study aimed to elucidate the underlying mechanism.
Method
To clarify the timing of cell death in Ocean-1187SΔOc87B, dead cells and extracellular DNA were stained with propidium iodide (PI). Bacterial growth (OD600) and PI fluorescence intensity were monitored over time for 24 hours using a plate reader. In addition, microscopic observation was performed after cultivation. Using the same experimental system, bacterial growth and PI fluorescence were compared among the wild-type, ΔOc87B, and ΔOc87BΔrecA strains. In addition, RNA sequencing was performed to comprehensively analyze transcriptional differences between the wild-type and ΔOc87B strains.
Results
Time-course analysis revealed that PI fluorescence in the ΔOc87B strain increased from the early stationary phase, indicating the onset of cell death, whereas fluorescence remained low in the wild-type strain. Microscopic observation revealed cell lysis in the ΔOc87B strain. Deletion of recA restored growth and suppressed PI fluorescence, demonstrating that cell death was RecA-dependent. RNA-seq analysis revealed increased transcription of two prophage regions distinct from ΔOc87B, namely Oc87C and Oc87D, in the ΔOc87B strain compared with the wild-type strain, while recA transcription was unchanged. In addition, whole-genome sequencing analysis showed a marked increase in DNA copy number within the Oc87C region, suggesting activation of additional prophages following Oc87B deletion.
Consideration
Together with the RecA-dependent cell death and transcriptional activation of other prophages, these findings suggest that deletion of a single prophage may induce the expression of coexisting prophages within the host genome.
Conclusion
We propose that deletion of Oc87B activates RecA, leading to induction of additional resident prophages and host cell lysis. Future studies using RecA reporter strains and systematic prophage deletion mutants will clarify this mechanism. Elucidating this regulatory cascade will advance our understanding of how multiple prophages are maintained within a single bacterial genome and reveal novel prophage-prophage and phage-host interaction mechanisms.
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