Isolation and characterization of a mutant strain of Chattonella marina with decreased production of superoxide anion

Chattonella mm ina is well known as the causative organism of red tide, and is highly toxic to fish. However, the toxic mechanism of C. marina has not been established. Recent studies demonstrated that C. marina generates reactive oxygen species (ROS) such as O-2(-) and H2O2. In this study, we attempted to establish mutant strains of C. marina using a chemical mutagen, ethyl methane sulfonate (EMS). After 48 h of treatment with 1 mg/ml of EMS at 26 degrees C, several viable cells were cloned, while more than 90% of C. marina cells died under these conditions. Among the strains isolated, one strain (mutant C) was significantly decreased in the production of O-2(-), but its growth rate was indistinguishable from the parental C. marina. Furthermore, superoxide dismutase (SOD) had almost no effect on the proliferation of mutant C, while SOD suppressed the growth of parental C. marina. In accordance with the lower level of O-2(-) generation, mutant C was less toxic to Vibrio alginolyticus in a plankton/bacteria co-culture system as compared do parental C. marina. In contrast, no significant difference in H2O2 production between mutant C and parental C. marina was observed. These results may provide experimental evidence for a direct connection between the toxic effect of C. marina against V. alginolyticus and the production of superoxide anion. Although the detailed mechanism of the production of superoxide anion by C. marina is still unclear, our mutant strain isolated in this study may be a good tool for the clarification of the mechanism of O-2(-) generation by C. marina.