Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Mar;185(6):1942-50.
doi: 10.1128/JB.185.6.1942-1950.2003.

High levels of intracellular cysteine promote oxidative DNA damage by driving the fenton reaction

Sunny Park  1 , James A Imlay
Affiliations

High levels of intracellular cysteine promote oxidative DNA damage by driving the fenton reaction

Sunny Park et al. J Bacteriol. 2003 Mar.

Abstract

Escherichia coli is generally resistant to H(2)O(2), with >75% of cells surviving a 3-min challenge with 2.5 mM H(2)O(2). However, when cells were cultured with poor sulfur sources and then exposed to cystine, they transiently exhibited a greatly increased susceptibility to H(2)O(2), with <1% surviving the challenge. Cell death was due to an unusually rapid rate of DNA damage, as indicated by their filamentation, a high rate of mutation among the survivors, and DNA lesions by a direct assay. Cell-permeable iron chelators eliminated sensitivity, indicating that intracellular free iron mediated the conversion of H(2)O(2) into a hydroxyl radical, the direct effector of DNA damage. The cystine treatment caused a temporary loss of cysteine homeostasis, with intracellular pools increasing about eightfold. In vitro analysis demonstrated that cysteine reduces ferric iron with exceptional speed. This action permits free iron to redox cycle rapidly in the presence of H(2)O(2), thereby augmenting the rate at which hydroxyl radicals are formed. During routine growth, cells maintain small cysteine pools, and cysteine is not a major contributor to DNA damage. Thus, the homeostatic control of cysteine levels is important in conferring resistance to oxidants. More generally, this study provides a new example of a situation in which the vulnerability of cells to oxidative DNA damage is strongly affected by their physiological state.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
A cystine pulse transiently sensitizes cells to H2O2. (A) E. coli AB1157 (wild-type) cells were grown to log phase in minimal medium containing sulfate (circles) or cystine (squares). Cystine (0.5 mM) was added (solid symbols) or not added (open symbols) to the cells, and 3 min later, 2.5 mM H2O2 was added. At intervals, cells were diluted, and viability was determined by colony formation. (B) AB1157 cells were grown to log phase in medium containing sulfate. At time zero, cystine (0.5 mM) was added. At time points, aliquots were removed and challenged for 3 min with 2.5 mM H2O2.
FIG. 2.
FIG. 2.
Cystine-H2O2 treatment generates abundant DNA damage. Log-phase E. coli AB1157 cells were treated with either 0.5 mM cystine, 2.5 mM H2O2, or cystine and H2O2, or left untreated as a control (con). Total genomic DNA was isolated, and qPCR was performed, using equivalent amounts of template DNA for each reaction mixture. PCR products were scanned, and the relative fluorescence was normalized to the untreated control. Values are the means and standard deviations (error bars) from three experiments.
FIG. 3.
FIG. 3.
Cystine-H2O2 treatment accelerates mutagenesis. E. coli AB1157 cells were grown to log phase and exposed for 3 min to 0.5 mM cystine, 0.1 mM H2O2, or both cystine and H2O2 or left untreated as a control (con). After 3 min, catalase was added to scavenge H2O2, and cells were spread on LB plates containing thymine or thymine and TMP. Values are the means and standard deviations (error bars) from three experiments. Note that the scale is exponential.
FIG. 4.
FIG. 4.
Conditions that confer H2O2 sensitivity. Different E. coli strains and treatments were studied and are grouped in sets of bars. For bars 1 to 3, AB1157 cells were grown to the log phase and treated with iron chelators for 5 min (none [bar 1], 1 mM dipyridyl [bar 2], and 20 mM desferrioxamine [bar 3]) before the addition of 0.5 mM cystine and 2.5 mM H2O2. For bars 4 to 11, AB1157 cells were treated with a 0.5 mM concentration of an alternative sulfur species (cysteine [bar 4], homocystine [bar 5], sulfide [bar 6], thiosulfate [bar 7], sulfite [bar 8], GSH [bar 9], GSSG [bar 10], and DTT [bar 11],) instead of cystine. For bars 12 and 13, AB1157 cells were grown in minimal medium containing cystine until the cells reached early log phase, and then the cells were washed twice and suspended in minimal medium containing sulfate for 1 h. For bar 13, 20 μg of chloramphenicol per μl was present during the period of growth on sulfate. Cystine was then added, and the cells were challenged with H2O2 for 3 min. For bars 14 and 15, BW25113 and SP53 (cysB) cells were grown in minimal medium containing djenkolic acid and tested for cystine-mediated H2O2 sensitivity. JTG10 (gshA) cells (bar 16) and WP840 (gor) cells (bar 17) were grown in minimal medium containing sulfate and tested for cystine-mediated H2O2 sensitivity. Values are the means and standard deviations (error bars) from five samples.
FIG. 5.
FIG. 5.
Evidence that H2O2 sensitivity requires cystine uptake. E. coli AB1157 cells were grown in minimal medium containing sulfate until they reached log phase, and then they were exposed to different concentrations of d- or l-cystine for 3 min before 2.5 mM H2O2 was added. Viability was determined after 3 min of H2O2 exposure.
FIG. 6.
FIG. 6.
GSH-deficient cells have less DNA damage than wild-type cells. E. coli JTG10 cells (gshA) were treated with both cystine and H2O2. Total genomic DNA was isolated, and qPCR was performed using four different amounts of template DNA. Data shown are for cells before (stippled bars) and after (gray bars) cystine-H2O2 treatment. Compare with the GSH+ parent (Fig. 2, cystine+H2O2 bar).
FIG. 7.
FIG. 7.
Cystine treatment increases cysteine content. E. coli AB1157 (wild-type) (A) and JTG10 (gshA) (B) cells were grown in minimal medium containing sulfate until they reached log phase, and the cells were treated with 0.5 mM cystine for 3 min (thick line) or not treated (thin line). Acid-soluble thiols were isolated, labeled with mBBr fluorescent dye, and separated by HPLC. Cysteine and GSH peaks are indicated.
FIG. 8.
FIG. 8.
Cysteine reduces ferric iron better than GSH does. Ferric chloride (10 μM) was mixed in anaerobic 20 mM Tris-HCl (pH 7.4) with 3 mM cysteine or GSH. At each time point, aliquots were removed and ferrous iron was assayed.
FIG. 9.
FIG. 9.
Cysteine efficiently drives Fenton-mediated DNA damage in vitro. In an anaerobic Coy chamber, 33 ng of pACYC184 plasmid was mixed in 3.5 mM NaHCO3 buffer (pH 7.2) with 10 μM FeCl3, 20 μM GSH or cysteine, and 50 μM H2O2. At each time point, the reaction was stopped by adding catalase. The reaction mixture was run in a 1% agarose gel. RF, relaxed form; SF, supercoiled form.
FIG. 10.
FIG. 10.
Mechanism of cystine-mediated H2O2 sensitivity. The CysB protein, which is activated during growth on relatively poor sulfur sources, stimulates the concerted import and reduction of cystine. Reduced GSH is necessary for this process. When saturating cystine is provided to erstwhile sulfur-poor cells, intracellular cysteine pools transiently rise to supranormal levels. Free iron catalyzes electron transfer from excess cysteine to H2O2, generating hydroxyl radicals.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alikhanian, S. I., T. S. Iljina, E. S. Kaliaeva, S. V. Kameneva, and V. V. Sukodolec. 1965. Mutants of Escherichia coli K12 lacking thymine. Nature 206:848-849. - PubMed
    1. Anderson, M. E. 1985. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113:548-555. - PubMed
    1. Au, D. C.-T., G. N. Green, and R. B. Gennis. 1984. Role of quinones in the branch of the Escherichia coli respiratory chain that terminates in cytochrome o. J. Bacteriol. 157:122-125. - PMC - PubMed
    1. Bachmann, B. J. 1987. Derivations and genotypes of some mutant derivatives of Escherichia coli K-12, p. 1190-1219. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C.
    1. Baptist, E. W., and N. M. Kredich. 1977. Regulation of l-cystine transport in Salmonella typhimurium. J. Bacteriol. 131:111-118. - PMC - PubMed

Publication types

MeSH terms