Isolation and characterization of methyl viologen-sensitive mutants of Escherichia coli K-12

Abstract

Escherichia coli mutants sensitive to methyl viologen (MV), an active oxygen propagator, were isolated. Among them, the new genes mvrA and mvrB were mapped at 7 and 28 min on the E. coli linkage map, respectively. MV toxicity was exerted only in the presence of oxygen and was suppressed by the radical scavenger uric acid but not by the hydroxyl radical scavenger mannitol. The mvr mutants were sensitive only to MV and had a normal repair capacity for the MV-damaged DNA. From these results, these mutants were assumed to be related to the elimination of MV-specific toxic species. Gene mvrA was cloned into vector pBR322 and its sequence was determined. The mvrA gene, which was predicted to range in size from 600 to 900 base pairs (bp) by transposon TnlOOO insertion analysis, was identified to be 807 bp, with an approximately 60-bp promoter sequence carrying consensus sequences for the -35 region, the -10 region, and a ribosome-binding site. The MvrA protein deduced from the DNA sequence was 29.7 kilodaltons, which was in good agreement with the 29 kilodaltons of the MvrA protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after a maxicell labeling experiment.Active oxygens are produced in respiring cells and are assumed to be a major cause of oxygen toxicity (13,14). Since Escherichia coli cells are continually exposed to active oxygens during respiration as well as during exposure to ionizing radiation (35), they have defense mechanisms against them. These mechanisms include superoxide dismutase against the superoxide radical (29); peroxidase and catalase against hydrogen peroxide; and antioxidants and radical scavengers like glutathione, ascorbic acid, vitamin E, and uric acid. Nonetheless, a mutant strain of E. coli polA recB was found to be inviable due to the deficient repair of DNA damage produced by oxygen, an indication that DNA repair genes are required for the defense against oxygen toxicity (30). Thus, E. coli seems to have at least two kinds of defense mechanisms against oxygen toxicity: the elimination of active oxygens and the repair of damaged DNA. Since active oxygens are assumed to be a major cause of many important biological phenomena such as mutation, cancer (1, 37), and aging (8), it is of great importance to know the defense mechanisms against them. Methyl viologen (MV), or paraquat, is a powerful superoxide radical propagator (12,17,18). MV is readily reduced by a single electron transfer by NADPH diaphorase (18) or by NADPH cytochrome P-450 reductase (6) to a stable but oxygen-sensitive monocation radical. It rapidly reacts with oxygen, with the generation of truly toxic species, superoxide radical, and subsequently, hydrogen peroxide (12). The secondary reactions between these two species produces a hydroxyl radical (36,39,40). To elucidate the protection mechanisms against oxygen toxicity, mutants of E. coli sensitive to MV were isolated, their properties were examined, and the mutations were mapped. Its relevant gene mvrA was cloned and analyzed. MATERIALS AND M...

The Role of Ferredoxin‐NADP+ Reductase in the Concerted Cell Defense Against Oxidative Damage

“…Our results indicate that contrary to previous claims [17], the target of FNR detoxification is not the superoxide radical, but more likely a derivative originating from it. We also show that neither ferredoxin nor flavodoxin are involved in the FNR function as an antioxidant in E. coli cells.…”

“…The fpr mutants were also able to grow aerobically in M9 minimal medium at about half the rate of the corresponding wild-type, whereas the sodA sodB cells were not viable under these conditions (data not shown, but see [ 14,191). If the FNR and SOD systems were both involved in 0; detoxification, as claimed previously [17], their modus operundi appeared to be quite different.…”

“…This comparison provides a relative estimation of the protective potential of both antioxidant systems, since the relevant genes in the two strains had been knocked-out by insertional interruptions, without affecting the rest of the bacterial genome [14,191. The phenotypes of the mutants have been described previously [14,17,191, but the variety of conditions employed has precluded a quantitative evaluation of the three strains, as depicted in Fig. 2A.…”

“…In 1988, Morimyo used random mutagenesis to select a different group of E. coli mutants that were highly sensitive to methyl viologen toxicity. One of the mutant strains (mvrA) was used to clone an E. coli gene involved in the resistance against this radical propagator [17]. Several regions of the mvrA gene displayed significant similarity to plant ferredoxin-NADP' reductase (FNR) sequences [ 181.…”

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…DNA repair systems are essential for the aerobic lifestyle: mutants that lack both baseexcision repair and recombinational repair functions--recA xthA and polA recB strains, for example--are viable only when they are cultured in anaerobic media (84,85). Moreover, the mutation rates of wild-type E. coli are typically lower when cells are cultured anaerobically, and the spectrum of mutations shifts away from those that are associated with oxidative stress (86).…”