Presentation Information
[P02-256]Analysis of cyanobacterial response and mutation induction mechanisms under grazing stress conditions
○Hana Nagashima1, Narumi Toda1, Akio Kuroda1, Ryuichi Hirota1 (1. Graduate School of Integrated Sciences for Life, Hiroshima University (Japan))
Keywords:
microalgae,cyanobacteria,predation,ROS,mutation frequency
Background and Objectives
Cyanobacteria are promising host for sustainable bioproduction because they can convert CO2 into biomass and valuable compounds through photosynthesis. However, large-scale outdoor cultivation is often threatened by contamination with predatory microorganisms, which can cause catastrophic damage and lead to significant losses of cyanobacterial biomass. We previously observed that co-cultivation of freshwater cyanobacterium Synechococcus elongatus PCC 7942 (PCC 7942) with the predatory protist Poterioochromonas malhamensis results in the emergence of cyanobacterial mutants exhibiting marked morphological changes. These morphological changes were shown to be caused by genetic mutations induced under prolonged grazing stress. Notably, the relatively rapid appearance of such mutants suggests that mutation frequency may increase under grazing stress. Reactive oxygen species (ROS) are known to cause DNA damage and oxidative impairment of photosynthetic systems and may therefore contribute to mutation promotion. In this study, we investigated intracellular ROS generation in cyanobacterial cells during co-culture with the predator. Furthermore, we quantified mutation rates under predation pressure using the acquisition of antibiotic resistance as an indicator and evaluated the physiological responses of cyanobacteria and the potential promotion of mutation under predation stress conditions.
Methods and Results
When PCC 7942 were co-cultured with P. malhamensis, pronounced aggregate formation was observed after approximately 2-3 days of cultivation, whereas no such aggregates were detected in axenic PCC 7942 cultures. Intracellular ROS were detected using 2',7'-dichlorofluorescein diacetate (DCFH-DA), and ROS accumulation was observed in PCC 7942 cells within the aggregates. To quantitatively evaluate the mutation rate under grazing stress, co-culture samples were plated on rifampicin-selective agar, and the frequency of rifampicin-resistant colonies was determined. Rifampicin targets the beta-subunit of RNA polymerase (rpoB), and spontaneous mutations in rpoB occur at a certain frequency, conferring rifampicin resistance. Therefore, measuring the frequency of rifampicin-resistant colonies enables estimation of the mutation rate (Sun H. et al., 2023, Nature Communications). Analysis of samples collected 24 h after the initiation of co-culture revealed that the mutation rate in the presence of the predator tended to be approximately 1.8-fold higher than that in the predator-free control. These results suggest that predator-induced oxidative stress may contribute to elevated mutation rates in cyanobacteria, potentially facilitating the emergence of elongated mutants that confer resistance to grazing pressure.
Cyanobacteria are promising host for sustainable bioproduction because they can convert CO2 into biomass and valuable compounds through photosynthesis. However, large-scale outdoor cultivation is often threatened by contamination with predatory microorganisms, which can cause catastrophic damage and lead to significant losses of cyanobacterial biomass. We previously observed that co-cultivation of freshwater cyanobacterium Synechococcus elongatus PCC 7942 (PCC 7942) with the predatory protist Poterioochromonas malhamensis results in the emergence of cyanobacterial mutants exhibiting marked morphological changes. These morphological changes were shown to be caused by genetic mutations induced under prolonged grazing stress. Notably, the relatively rapid appearance of such mutants suggests that mutation frequency may increase under grazing stress. Reactive oxygen species (ROS) are known to cause DNA damage and oxidative impairment of photosynthetic systems and may therefore contribute to mutation promotion. In this study, we investigated intracellular ROS generation in cyanobacterial cells during co-culture with the predator. Furthermore, we quantified mutation rates under predation pressure using the acquisition of antibiotic resistance as an indicator and evaluated the physiological responses of cyanobacteria and the potential promotion of mutation under predation stress conditions.
Methods and Results
When PCC 7942 were co-cultured with P. malhamensis, pronounced aggregate formation was observed after approximately 2-3 days of cultivation, whereas no such aggregates were detected in axenic PCC 7942 cultures. Intracellular ROS were detected using 2',7'-dichlorofluorescein diacetate (DCFH-DA), and ROS accumulation was observed in PCC 7942 cells within the aggregates. To quantitatively evaluate the mutation rate under grazing stress, co-culture samples were plated on rifampicin-selective agar, and the frequency of rifampicin-resistant colonies was determined. Rifampicin targets the beta-subunit of RNA polymerase (rpoB), and spontaneous mutations in rpoB occur at a certain frequency, conferring rifampicin resistance. Therefore, measuring the frequency of rifampicin-resistant colonies enables estimation of the mutation rate (Sun H. et al., 2023, Nature Communications). Analysis of samples collected 24 h after the initiation of co-culture revealed that the mutation rate in the presence of the predator tended to be approximately 1.8-fold higher than that in the predator-free control. These results suggest that predator-induced oxidative stress may contribute to elevated mutation rates in cyanobacteria, potentially facilitating the emergence of elongated mutants that confer resistance to grazing pressure.
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