Paraquat-mediated selection for mutations in the manganese-superoxide dismutase gene sodA

Abstract

We report the unexpected result that Escherichia coli isolates containing a multicopy plasmid (pDT1.5) carrying the manganese-superoxide dismutase gene sodA were more sensitive than the wild type to paraquatmediated growth inhibition. The pDTl.5 locus responsible for the paraquat-sensitive phenotype was delimited to a 0.6-kilobase segment by transposon Tn5 mutagenesis. Moreover, superoxide dismutase activity was the same as in the wild type in strains carrying pDT1.5::TnS insertions mapping to the 0.6-kilobase locus. These data identify the 0.6-kilobase segment as the locus of sod4 and establish an association between growth inhibition by paraquat and the function of the plasmid-borne sodA gene.Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) is thought to undergo oxidation-reduction cycling within cells, thereby generating intracellular superoxide radicals (5, 11) which may be toxic to cells at high concentrations. Manganese-superoxide dismutase (Mn-SOD) (14) activity is induced in the presence of paraquat and is most likely an important component of cellular defense against paraquat toxicity (10) as well as against superoxide radical-mediated toxicity in general (18).Because Escherichia coli strains carrying pDT1.5, a multicopy plasmid containing the Mn-SOD gene, sodA (21), display five times as much Mn-SOD activity as wild-type E. coli (21), we reasoned that pDT1.5 might confer a growth advantage to cells under conditions which produce high intracellular levels of superoxide radical. In contrast to our prediction, however, we report here that strains carrying pDT1.5 were more sensitive to paraquat than were wild-type E. coli strains. The paraquat-mediated growth inhibition phenotype conferred by pDT1.5 allowed us to screen for TnS insertion mutations in pDT1.5 which permitted growth on paraquat. These TnS insertions mapped to a 0.6-kilobase (kb) segment of pDT1.5 and reduced superoxide dismutase expression to wild-type levels. We were thus able to locate the sodA locus on pDT1.5 and to demonstrate an association between growth inhibition by paraquat and the function of the recombinant plasmid-borne sodA gene.( where appropriate, were diluted and allowed to simultaneously reenter the stationary phase. After about 3 h in the stationary phase they were again diluted and plated onto LB agar (1.8%) containing kanamycin or ampicillin or both where appropriate (at the same initial concentrations as above) and paraquat where indicated.For batch testing of transposon TnS insertion mutants, strains were grown overnight in 96-well microtiter dishes at 37°C for 24 h without shaking and reached densities of about 2 x 108 CFU/ml. With a multipronged replicating device to transfer the cultures, they were diluted and plated on LB at a density of about 105 CFU/cm2 (104 CFU per spot).For anaerobic experiments, plates were allowed to equilibrate for 24 h in an anaerobic chamber (Coy Laboratory Products, Inc., Ann Arbor, Mich.) containing an atmosphere of 85% N2, 10% C02, and 5% H2 (13) before inoculation with cells. The an...

Contribution of Oxidative Damage to Antimicrobial Lethality

“…1, 4, and 5) are consistent with several other reports. For example, detrimental rather than protective effects are observed with SOD overexpression in E. coli cells treated with a superoxide generator, paraquat (6,30). Since paraquat treatment increases superoxide levels (21), as does antimicrobial treatment (1,5), rapid conversion of excessive superoxide to H 2 O 2 by overexpressed SOD is likely to trigger lethal hydroxyl radical generation.…”

Mutagenic and lethal effects of near‐ultraviolet radiation (290–400 nm) on bacteria and phage

“…Interest-ingly, we have found that if cells are stressed by a number of agents (including heat), catalase in the medium will give higher colony counts (unpublished); H202 may be a common product of generalized cellular stress [Ames, 1983;Hartman, 1986;Richter and Loewen, 1981;Tyrrell, 1985;Vassilyadi and Archibald, 1985;Winguest et al, 19841. Superoxide dismutase (SOD) is a logical candidate as a NUV-recovery enzyme [Ahmad, 1981;Farr et al, 19861. We have found that the presence of a plasmid carrying an inducible Mn-superoxide dismutase (sodA) [Bloch and Ausubel, 1986;Carlioz and Touati, 19861 endows the cell with some resistance to NUV, but not FUV. This is of interest since it implicates 0, as a lethal radical.…”

“…Not only is there no correlation between NUV resistance and catalase activity, but in some cases the correlation is inverse. Mutants other than katF (ie, katE and katG') that are defective in catalase activity [Loewen, 1984;Loewen et al, 1983Loewen et al, , 1985a [Bloch and Ausubel, 1986; Carlioz and Touati, 19861 endows the cell with some resistance to NUV, but not FUV. This is of interest since it implicates 0, as a lethal radical.…”

The role of the cellular antioxidant defense in oxidant carcinogenesis.

“…It should be noted that evidence in the literature supports the notion that overexpression of SOD can sensitize rather than protect cells from oxidative stress. For example, a 5-fold increase in Mn-SOD or a 10-fold increase in Fe-SOD sensitized E. coli to paraquat toxicity (43,44). While transfection of Cu,Zn-SOD into HeLa cells resulted in overall resistance to paraquat, the degree of protection was not proportional to the increase in enzyme activity (45).…”