Presentation Information
[P04-505]Physiological features of cell wall–deficient cells in Pseudomonas aeruginosa
○Natsuki Sugiyama1, Jun Harada1, Shusaku Kanematsu1, Nobuhiko Nomura2,3,4,5, Masanori Toyofuku2,3,4 (1. Grad. Sch. Life Environ. Sci., Univ. Tsukuba (Japan), 2. Fac. Life Environ. Sci., Univ. Tsukuba (Japan), 3. MiCS, Univ. Tsukuba (Japan), 4. TIAR, Univ. Tsukuba (Japan), 5. TARA, Univ. Tsukuba (Japan))
Keywords:
Cell Wall,Prophage,R-cell
[Purpose]
Most bacteria possess a cell wall mainly composed of peptidoglycan (PG), which maintains cell shape and protects cells from osmotic stress. The bacterial cell wall is also a primary target of β-lactam antibiotics and is therefore an important structure in antimicrobial therapy. We previously discovered that a subpopulation of Pseudomonas aeruginosa forms spherical cells lacking a cell wall, referred to as round cells (R-cells). R-cells are generated when the PG layer is degraded by endolysin under DNA damage–inducing stress conditions. Because R-cells occur only in a small fraction of the population, this morphology has largely been overlooked, and their physiological characteristics remain poorly understood. Therefore, this study aimed to elucidate the properties and potential functions of R-cells in P. aeruginosa.
[Method]
P. aeruginosa PAO1 was used in this study. R-cell formation was induced by expressing hol and lys genes under an arabinose inducible promoter. Intracellular reactive oxygen species (ROS) were measured using a fluorescent probe. Transcriptomic analysis was performed by RNA sequencing to compare gene expression between rod-shaped cells and R-cells.
[Results]
Live-cell imaging revealed that R-cells were capable of fusing with each other. In addition, some R-cells reverted to rod-shaped cells and resumed proliferation. However, the mechanism underlying this regrowth remains unclear. RNA-seq analysis comparing rod-shaped cells and R-cells showed that genes involved in glycolysis were upregulated in R-cells. Because enhanced glycolysis can promote the accumulation of reactive oxygen species (ROS), intracellular ROS levels were examined. Fluorescence imaging revealed that ROS accumulated in R-cells compared with rod-shaped cells. Since ROS can exert cytotoxic effects, ROS accumulation may inhibit cell division in R-cells. To further examine the role of ROS, we tested whether R-cells could form under anaerobic conditions where ROS generation is reduced. As a result, R-cells were also observed under anaerobic conditions.
[Consideration]
These results suggest that R-cells exhibit a physiological state distinct from that of rod-shaped cells. The upregulation of glycolysis-related genes and the accumulation of intracellular ROS indicate metabolic changes during R-cell formation. Because excessive ROS can interfere with cellular processes, ROS accumulation may influence the proliferative behavior of R-cells.
[Conclusion]
In this study, we characterized the physiological features of R-cells in P. aeruginosa. Our results indicate that R-cells show distinct metabolic states associated with glycolysis activation and intracellular ROS accumulation. As genes required for the transition to R-cells are conserved in many bacteria, R-cell formation may represent a widespread phenomenon among bacteria.
Most bacteria possess a cell wall mainly composed of peptidoglycan (PG), which maintains cell shape and protects cells from osmotic stress. The bacterial cell wall is also a primary target of β-lactam antibiotics and is therefore an important structure in antimicrobial therapy. We previously discovered that a subpopulation of Pseudomonas aeruginosa forms spherical cells lacking a cell wall, referred to as round cells (R-cells). R-cells are generated when the PG layer is degraded by endolysin under DNA damage–inducing stress conditions. Because R-cells occur only in a small fraction of the population, this morphology has largely been overlooked, and their physiological characteristics remain poorly understood. Therefore, this study aimed to elucidate the properties and potential functions of R-cells in P. aeruginosa.
[Method]
P. aeruginosa PAO1 was used in this study. R-cell formation was induced by expressing hol and lys genes under an arabinose inducible promoter. Intracellular reactive oxygen species (ROS) were measured using a fluorescent probe. Transcriptomic analysis was performed by RNA sequencing to compare gene expression between rod-shaped cells and R-cells.
[Results]
Live-cell imaging revealed that R-cells were capable of fusing with each other. In addition, some R-cells reverted to rod-shaped cells and resumed proliferation. However, the mechanism underlying this regrowth remains unclear. RNA-seq analysis comparing rod-shaped cells and R-cells showed that genes involved in glycolysis were upregulated in R-cells. Because enhanced glycolysis can promote the accumulation of reactive oxygen species (ROS), intracellular ROS levels were examined. Fluorescence imaging revealed that ROS accumulated in R-cells compared with rod-shaped cells. Since ROS can exert cytotoxic effects, ROS accumulation may inhibit cell division in R-cells. To further examine the role of ROS, we tested whether R-cells could form under anaerobic conditions where ROS generation is reduced. As a result, R-cells were also observed under anaerobic conditions.
[Consideration]
These results suggest that R-cells exhibit a physiological state distinct from that of rod-shaped cells. The upregulation of glycolysis-related genes and the accumulation of intracellular ROS indicate metabolic changes during R-cell formation. Because excessive ROS can interfere with cellular processes, ROS accumulation may influence the proliferative behavior of R-cells.
[Conclusion]
In this study, we characterized the physiological features of R-cells in P. aeruginosa. Our results indicate that R-cells show distinct metabolic states associated with glycolysis activation and intracellular ROS accumulation. As genes required for the transition to R-cells are conserved in many bacteria, R-cell formation may represent a widespread phenomenon among bacteria.
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