Activity of the Escherichia coli mutT mutator allele in an anaerobic environment

Abstract

Mutation frequencies for an Escherichia coli mutT strain were measured in both aerobic and anaerobic environments. When cells were grown in a rich medium (L broth), mutation frequencies were similar in both aerobic and anaerobic conditions. In contrast, when grown in a minimal medium, mutT anaerobic mutation frequencies were reduced dramatically compared with aerobic values, which were similar to L broth frequencies. L broth mutT cultures treated with a commercial enzyme complex that reduces free oxygen in the medium also showed strongly reduced anaerobic mutation frequencies. These results indicate that the biological role of the MutT protein is to prevent oxidative damage from becoming mutagenic.Although the mutT mutator allele was the first to be described in Escherichia coli (25), its mechanism of activity has remained unclear until recently. An early key to the nature of mutT activity was the observation that this mutator causes exclusively A* T--C G transversions (27). These transversions arise through A* G mispairings rather than T-C intermediates (2, 23). The MutT protein catalyzes the hydrolysis of nucleoside triphosphates with a preference for dGTP (5, 6), and it was suggested that the biological role of MutT might be to hydrolyze dGTP before it could mispair with a template adenine during DNA replication (5). Recently, Maki and Sekiguchi (17) showed that 8-oxo-7,8-dihydro-2'-dGTP (8-oxodGTP) is more readily hydrolyzed to the monophosphate than dGTP and hypothesized that the MutT protein might act to remove 8-oxodGTP from the nucleotide pool during DNA replication because of its increased tendency to mispair with template adenine compared with that of dGTP (8,16,17,22).8-OxodGTP may be produced in the cell from dGTP by ionizing radiation and oxygen radical-producing chemicals (13-15) as well as from physiological and biochemical processes occurring during normal cellular metabolism (4). It is widely accepted that active partial reduction products of oxygen, i.e., superoxide, hydrogen peroxide, and hydroxyl radical, produced during aerobic respiration, are capable of directly (hydroxyl radical) or indirectly (superoxide radical and hydrogen peroxide) forming oxidative products with DNA and precursor molecules (3, 10, 11). 8-oxodGTP may be among the most abundant of these oxidative products in both bacteria and animals (21, 24). mutT homologs have been found in other bacteria (7,12,18), and a similar nucleoside triphosphatase activity has been found in human cells (20), suggesting that misincorporation of 8-oxodGTP may be a common problem.If MutT does function to remove 8-oxodGTP from nucleotide pools, it might be expected that mutT strains would show lower mutation frequencies in an anaerobic environment than in an aerobic environment. We tested this prediction by measuring mutT mutation frequencies inside an anaerobic * Corresponding author. Phone: (408) Cells were grown in L broth supplemented with glucose (1% tryptone, 0.5% yeast extract, 0.1% glucose, and 0.5% NaCl) or in minimal medium (0.2% glu...

Elevated mutation rate in mutT bacteria during starvation: evidence for DNA turnover?

“…However, although the bacteria in this study were starved of a required amino acid, they were not starved of glucose, and it is possible that the metabolism of glucose yields more oxidative species when protein synthesis is inhibited. Fowler et al (11) have reported that under anaerobic conditions the level of oxidative species, as reflected in the mutation rate of mutT bacteria, is influenced by the richness of the growth medium, illustrating that differences may occur under different nutrient conditions; however, it is not possible to extrapolate from these results to those reported here. While a difference in the relative level of 8-oxo-dGTP is possible (a subject worthy of study), it is unlikely that this would entirely explain the difference in the mutation rate.…”

DNA precursor metabolism and genomic stability

“…Second, Fowler et al (104) reported that under certain anaerobic growth conditions, where nt oxidation should not be occurring, mutT mutant bacteria retained the mutator phenotype. Third, Einolf and Guengerich (105,106) reported that 8-oxo-dGTP is a poorer substrate than dGTP, by several orders of magnitude, for several different DNA polymerases tested.…”

Antibiotics induce redox-related physiological alterations as part of their lethality

“…To ensure the most stringent anaerobic conditions achievable, we did not remove the culture vessels from the chamber during anaerobic drug incubations (50) or plate the bacteria aerobically before counting colonies (49). Previous observations using mutT mutants have noted equivalent mutation frequencies when grown either aerobically or anaerobically in undefined rich (but not in minimal) medium (94), indicating that undefined rich medium may promote confounding redox reactions under anaerobic conditions. Consequently, we chose Neidhardt fully defined complete medium, which contains only sulfate as a potential terminal electron acceptor (95), for anaerobic culturing.…”