The Mechanism of Chlorine Damage Using Enhanced Green Fluorescent Protein-Expressing Escherichia coli

Abstract

This study investigated how chlorine inactivates and damages Escherichia coli cells. E. coli that had transformed to express enhanced green fluorescent protein (EGFP) at the cytoplasm was treated with chlorine. Damage to the cell membrane and cell wall was analyzed by measuring the fluorescence intensity of the leaked EGFP, then accounting for the fluorescence deterioration. At pH 7, E. coli was lethally damaged after treatment with chlorine, but significant leakage of EGFP was not observed. In contrast, significant leakage of EGFP was observed at pH 9, even though E. coli was not as inactivated as it was at pH 7. Flow cytometry was used to confirm the fluorescence intensity of the remaining EGFP inside the cells. No significant fluorescence loss was observed in the cells at pH 7. However, at pH 9, the fluorescence intensity in the cells decreased, indicating leakage of EGFP. These results suggest that hypochlorous acid inactivates E. coli without damaging its cell membrane and cell wall, whereas the hypochlorite ion inactivates E. coli by damaging its cell membrane and cell wall. It was possible to confirm the chlorine damage mechanism on E. coli by measuring the fluorescence intensity of the leaked EGFP. Author Contributions: Conceptualization, M.M., M.O., and K.A.; methodology, M.M., M.O., and K.A.; validation, M.O. and K.A.; formal analysis, M.M.; investigation, M.M.; resources, M.O. and K.A.; data curation, M.M.; writing-original draft preparation, M.M.; writing-review and editing, M.O. and K.A.; visualization, M.M.; supervision, M.O. and K.A; project administration, M.O.; funding acquisition, M.O. and K.A.

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