H202 and Gamma Radiation

The tripeptide glutathione (L-y-glutamyl-L-cysteinylglycine) (GSH) is the predominant low-molecular-weight thiol in many organisms and has been proposed to protect cells from oxidative damage such as that formed by H202 and ionizing radiations (19). Escherichia coli deficient in GSH because of mutations in the gshA gene was found previously (3) to exhibit normal resistance to X irradiation in the presence of oxygen. Other workers reported (21) that E. coli gsh mutants lacked the protective effect of hypoxia during -y irradiation. At least one of the mutants employed previously contains a second mutation that affects its sensitivity to H202 (10), probably because it was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 3). To avoid this problem, we isolated transposon insertions in the gshA gene. Strains bearing a gshA::TnlO allele exhibit the expected hypersensitivities to thiol-specific reagents but are not at all defective in their resistance to oxidative damage, including y rays. The E. coli K-12 strains used were as follows: W3110 (gsh+) and NK5991 (W3110 srlD::TnlO) supplied by N. Kleckner; AB1157 (gsh') and BS53 (AB1157 gsh) from B. Sedgwick (24); TJ5 (AB1157 gshB) supplied by J. Fuchs; and KL164 (nalB14) from the Coli Genetic Stock Center (5). Strain W3110 was mutagenized by the lambda hop procedure (26) with a derivative of TnlO (element 9 of Way et al. [26], hereafter called TnlOkm) that confers kanamycin resistance (Kan9. Colonies were collected from kanamycin (50 ,ug/ml) selection plates and stored at -50°C in 25% glycerol. Thawed cells were centrifuged and suspended in M medium (M9 salts [20] with 0.1 mM CaCl2, 1.0 mM MgSO4, 1 llg of thiamine hydrochloride per ml, 0.025% Casamino Acids, and 0.1% glucose) and enriched for gshA mutants essentially by the procedure of Sedgwick and Robins (24) by using one 45-min and two 10-min treatments with 50 ,ug of MNNG per ml. Most (70%) of the isolates obtained in this way showed substantially increased resistance to MNNG when tested individually. Of the three randomly selected MNNGresistant isolates examined, all contained drastically diminished acid-soluble thiols (17). A putative gshA::TnJOkm mutation was transduced from one of these isolates (JTG1) into AB1157 by using bacteriophage P1 (20) and selection for Kanr. One Kanr transductant (JTG10) was selected at random for further study; the mutation it bears was designated gshA20: :TnlOkm. Cellular levels of reduced thiols are given in Table 1. These were quantitated by treating extracts (14) of thawed