Abstract
We measured the generation of hydroxyl radical (OH(.)) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron uptake (Fur) using electroparamagnetic resonance and gas chromatography-mass spectrometry with a selected-ion monitoring method. A specific signal corresponding to OH(.) generation and an increase in oxidative DNA lesions such as 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine were detected in the strain deficient in sodA sodB fur. We showed that iron metabolism deregulation in fur mutant produced a 2.5-fold iron overload. The sodA sodB fur strain was about 100-fold higher mutability than the wild-type strain. The mutation spectrum in the strain was found to induce GC --> TA and AT --> CG transversions predominantly. The hypermutability of the strain was suppressed by the tonB mutation which reduces iron transport. Thus, excess iron and excess superoxide were responsible for OH(.) generation, oxidative DNA lesion formation, and hypermutability in E. coli.
Highlights
We measured the generation of hydroxyl radical (OH1⁄7) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron uptake (Fur) using electroparamagnetic resonance and gas chromatography-mass spectrometry with a selected-ion monitoring method
A long-standing proposal for the mechanism of the first step of oxidative mutagenesis is the reaction of bases in DNA or nucleotides in the pool with OH1⁄7, a highly reactive reactive oxygen species, which can be generated during the HarberWeiss/Fenton reaction that consists of an iron reduction step by O2. and an OH1⁄7 generation step via the Fenton reaction, Fe(III) ϩ O2
Coli—Incubation of LB broth containing 4-POBN and ethanol without E. coli cells at 37 °C for 17 h produced the strong signal for the 4-POBN-C1⁄7H(CH3)OH spin adduct, which is in good agreement with the published values [19], indicating OH1⁄7 formation
Summary
Bacterial Strains, Plasmids, and Media—The bacterial strains used in this study were all derivatives of E. coli K-12. LB broth containing 70 units/ml catalase, 10 mM 4-POBN, and 170 mM ethanol was inoculated with 1 ϫ 107 cells/ml of culture of E. coli strains. Spontaneous Rifampicin-resistant Mutation—Overnight cultures of E. coli strains in LB broth were diluted 105-fold in phosphate buffer, 0.1-ml aliquots (103 cells) were inoculated into 5 ml of LB broth with appropriate antibiotics and incubated at 37 °C overnight. Measurement of Iron Contents—E. coli cells in overnight cultures prepared as described above were washed three times with phosphate buffer, and resuspended in the same volume of ultrapure H2O (Kanto Chemical Co. Inc., Tokyo). Analysis for Spontaneous E. coli supF Mutations—Individual cultures of GC4468, QC1726, and QC1736 cells containing pTN89 were grown in 5 ml of LB broth with appropriate antibiotics at 37 °C overnight. Selection of the mutant supF, calculation of mutation frequency, and DNA sequencing were carried out as described previously [29]
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