Presentation Information
[P01-129]Real-time monitoring of oxidative stress in Acinetobacter sp. Tol 5 using a genetically encoded fluorescent sensor
○Honoka Imai1, Shori Inoue1, Shogo Yoshimoto1, Katsutoshi Hori1 (1. Nagoya University (Japan))
Keywords:
Oxidative stress,Fluorescent sensor protein,Live-cell measurement,Bacteria
[Purpose]
Acinetobacter sp. Tol 5 is a highly adhesive gram-negative bacterium that rapidly autoaggregates and becomes immobilized on solid surfaces. In addition, it possesses a broad metabolic capacity toward volatile organic compounds. These traits make Tol 5 a promising platform for gas-phase bioprocesses and whole-cell applications [1]. However, its intracellular stress responses in these specialized states remain largely unexplored, and methods for real-time intracellular analysis have not been well established. Oxidative stress is particularly relevant because aromatic compound metabolism often involves oxygenase-mediated reactions that can lead to the unintended reactive oxygen species (ROS) generation [2], which damage essential cellular components such as proteins, lipids, and nucleic acids [3]. Although antioxidant systems have been described in some Acinetobacter strains [4], the redox dynamics in Tol 5 during substrate degradation remain unknown. This study aims to establish methods for analyzing intracellular oxidative stress in Tol 5 cells under various environmental and metabolic conditions.
[Method]
To establish an intracellular oxidative stress monitoring system in Tol 5, the genetically encoded fluorescent H2O2 sensor HyPer7 was expressed by plasmid transformation [5]. Sensor performance was validated by measuring the fluorescence excitation ratio (Ex490/Ex420) in response to exogenous H2O2 using a microplate reader. This system then tracked real-time redox dynamics during the metabolism of various substrates, including phenol, to assess its sensitivity and stability.
[Results]
HyPer7 showed a rapid response to exogenous H2O2 in Tol 5, demonstrating its functionality in this host. Phenol addition caused a concentration-dependent increase in the fluorescence ratio, whereas benzoic acid and ethanol had minimal effects within the tested concentration ranges. These findings show that HyPer7 enables sensitive detection of intracellular oxidative changes associated with phenol metabolism in Tol 5.
[Consideration]
The sustained fluorescence ratio elevation after phenol addition suggests that H2O2 generation during phenol metabolism exceeded the intracellular antioxidant capacity. The substrate-dependent response indicates that oxidative stress in Tol 5 is closely associated with metabolic activity. These results show that the HyPer7-based system can effectively track dynamic redox changes.
[Conclusion]
HyPer7 enables real-time monitoring of intracellular oxidative stress in Tol 5, revealing substrate-dependent redox changes and phenol-induced oxidative stress. This system provides a useful tool for studying intracellular stress responses in Tol 5.
[References]
[1] Usami et al., Green Chemistry., 2020. [2] Roma-Rodrigues et al., Journal of Proteomics., 2010. [3] Seixas et al., Frontiers in Genetics., 2022. [4] Yang et al., Nature Reviews., 2017. [5] Pak et al., Cell metabolism., 2020.
Acinetobacter sp. Tol 5 is a highly adhesive gram-negative bacterium that rapidly autoaggregates and becomes immobilized on solid surfaces. In addition, it possesses a broad metabolic capacity toward volatile organic compounds. These traits make Tol 5 a promising platform for gas-phase bioprocesses and whole-cell applications [1]. However, its intracellular stress responses in these specialized states remain largely unexplored, and methods for real-time intracellular analysis have not been well established. Oxidative stress is particularly relevant because aromatic compound metabolism often involves oxygenase-mediated reactions that can lead to the unintended reactive oxygen species (ROS) generation [2], which damage essential cellular components such as proteins, lipids, and nucleic acids [3]. Although antioxidant systems have been described in some Acinetobacter strains [4], the redox dynamics in Tol 5 during substrate degradation remain unknown. This study aims to establish methods for analyzing intracellular oxidative stress in Tol 5 cells under various environmental and metabolic conditions.
[Method]
To establish an intracellular oxidative stress monitoring system in Tol 5, the genetically encoded fluorescent H2O2 sensor HyPer7 was expressed by plasmid transformation [5]. Sensor performance was validated by measuring the fluorescence excitation ratio (Ex490/Ex420) in response to exogenous H2O2 using a microplate reader. This system then tracked real-time redox dynamics during the metabolism of various substrates, including phenol, to assess its sensitivity and stability.
[Results]
HyPer7 showed a rapid response to exogenous H2O2 in Tol 5, demonstrating its functionality in this host. Phenol addition caused a concentration-dependent increase in the fluorescence ratio, whereas benzoic acid and ethanol had minimal effects within the tested concentration ranges. These findings show that HyPer7 enables sensitive detection of intracellular oxidative changes associated with phenol metabolism in Tol 5.
[Consideration]
The sustained fluorescence ratio elevation after phenol addition suggests that H2O2 generation during phenol metabolism exceeded the intracellular antioxidant capacity. The substrate-dependent response indicates that oxidative stress in Tol 5 is closely associated with metabolic activity. These results show that the HyPer7-based system can effectively track dynamic redox changes.
[Conclusion]
HyPer7 enables real-time monitoring of intracellular oxidative stress in Tol 5, revealing substrate-dependent redox changes and phenol-induced oxidative stress. This system provides a useful tool for studying intracellular stress responses in Tol 5.
[References]
[1] Usami et al., Green Chemistry., 2020. [2] Roma-Rodrigues et al., Journal of Proteomics., 2010. [3] Seixas et al., Frontiers in Genetics., 2022. [4] Yang et al., Nature Reviews., 2017. [5] Pak et al., Cell metabolism., 2020.
Comment
To browse or post comments, you must log in.Log in