Isolation and characterization of methyl viologen-resistant synechocystis sp. PCC 6803 mutants

Abstract

Methyl viologen (MV) is a widely used herbicide that competes electrons from photosystem I and induces the formation of active radical species causing cellular oxidative stress in plants and photosynthetic cyanobacteria. However, the exact target molecule of methyl viologen in the cells has yet to be identified. This study aimed to identify the mutation(s) that lead to MV resistance in cyanobacterium Synechocystis sp. PCC 6803. Synechocystis wild type was cultured for 30 days to reach the stationary-growth phase to allow the occurrence of spontaneous mutations in aged cells. Approximately 109 cells were spread on an agar medium containing high MV concentrations (10-40 µM), which could completely inhibit cell growth. After 30-40 days, spontaneous MV-resistant mutants up to 23, 176 and 2,936 colonies were obtained from agar plates containing 40, 20 and 10 µM of MV, respectively. MV-resistant mutations were subsequently identified in seven selected MV-resistant mutants using genome sequencing. Mutant A9 contained the stop codon gained mutation that disrupted the function of the cntO_1 gene encoding the TonB-dependent receptor involved in the transport of positively charged ions across the cell membrane. Thus, this TonB-dependent receptor might transport MV to the cell, and the disrupted function leads to MV resistance. Mutant C14 and C21 contained the frameshift mutation that abolished the function of the menH_2 gene encoding triacylglycerol lipase (enzyme generating diacylglycerol, the main component of cell membrane), suggesting that this gene disruption may affect MV import to the cells. Moreover, mutant A9, A11, B1, B14, C10, C14 and C21 contained one amino acid alteration (53Met-->Lys) in a small hypothetical protein (a 92-amino-acid protein product of the gene FMAMFGPO_01147) with an unknown function. Protein structure prediction showed that the structure of this hypothetical protein was significantly changed upon 53Met-->Lys substitution. Additionally, the mutations in genes associated with the biosynthesis of cell-membrane component, the biosynthesis of cell-wall peptidoglycan, the biofilm formation, and the antioxidant defense were found in the MV-resistant mutants A9, C14 and C21. In conclusion, the mechanisms of MV resistance found in this study is related to the alteration of MV transport and the modified composition of the cell membrane. This data provides basic information on spontaneous MV resistance in an aquatic photosynthetic microbe.