Formation of 8-oxoguanine in cellular DNA of Escherichia coli strains defective in different antioxidant defences

Abstract

This paper examines the relationship in Escherichia coli between the in vivo content of 8-oxoguanine (8-oxoG) in chromosomal DNA and deficiencies of various key antioxidant defences. The structural genes for catalases (katG and katE), cytosolic superoxide dismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase (fpg) were inactivated to obtain bacterial strains lacking the scavenger enzymes for H₂O₂ or O₂·⁻ or the DNA repair protein for 8-oxoG. Wild-type bacteria showed 5-fold increased sensitivity to both lethality and mutagenesis by H₂O₂ in K medium (1 % casamino acids and 1 % glucose), as compared with nutrient broth. This higher sensitivity was associated with increased chromosomal oxidative damage, estimated as the 8-oxodG content, and with a marked decrease in both catalase and SOD activities. Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayed increased 8-oxodG content in chromosomal DNA (2.8-fold that of the wild-type) when grown under standard aerated conditions. Comparatively, no significant difference in 8-oxodG content was observed in cells grown without aeration. Bacteria totally devoid of catalase activity (katG katE mutant) showed wild-type contents of 8-oxodG in chromosomal DNA when grown under aerated conditions. Nevertheless, the protective role of catalase in preventing formation of 8-oxodG in chromosomal DNA became evident under oxidative stress conditions: growth under hyperoxygenation and, particularly, following H₂O₂ exposure. Catalase deficiency resulted in a dramatic decrease in viability after H₂O₂ exposure. A deficiency of Fpg protein also sensitized E.coli to H₂O₂ lethality, though to lesser extent than a deficiency of catalase activity. However, the scavenger enzyme and the DNA repair protein protected equally against 8-oxoG formed in vivo upon H₂O₂ treatment.