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, 188 (21), 7645-51

Neisseria Gonorrhoeae DNA Recombination and Repair Enzymes Protect Against Oxidative Damage Caused by Hydrogen Peroxide

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Neisseria Gonorrhoeae DNA Recombination and Repair Enzymes Protect Against Oxidative Damage Caused by Hydrogen Peroxide

Elizabeth A Stohl et al. J Bacteriol.

Abstract

The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H(2)O(2)) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H(2)O(2) damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2).

Figures

FIG. 1.
FIG. 1.
(A) Effect of recA and recX mutation on H2O2 resistance. Error bars represent the standard error of the mean of three to six experiments. FA1090, FA1090recA6 (+IPTG), and FA1090recX were statistically the same at all doses. FA1090recA6 (+IPTG) was statistically distinct from FA1090recA6 (−IPTG), at all doses (P < 0.05) as determined by the Student t test, and FA1090recA4 was statistically the same as FA1090recA6 (−IPTG), at all doses. (B and C) Western blot analyses of RecA levels. (B) RecA protein is not induced by H2O2. Strain FA1090 was grown as described and exposed to either no H2O2 (−) or 5 mM H2O2 (+) for 15 or 30 min. Then, 4 μg of total protein was run per lane and transferred to a polyvinylidene difluoride membrane with subsequent Western blot analysis with anti-RecA antisera using the ECL kit. (C) RecA protein is expressed at equivalent levels in strain FA1090 (FA) and FA1090recA6 (rA6) induced with IPTG when bacteria are grown in liquid or on solid media. Experiments for panels B and C were performed with three biological replicates on different days, and a representative blot is shown.
FIG. 2.
FIG. 2.
Effect of RecBCD pathway mutants on H2O2 resistance. Strains were grown in the presence of 1 mM IPTG to induce RecA expression. Error bars represent the standard error of the mean of two to six experiments. Error bars for the recC strain are dashed to aid in distinguishing strains. All strains were statistically different from the parent recA6 strain at the 50 mM dose (P < 0.02). FA1090recA4 is included for comparison.
FIG. 3.
FIG. 3.
Effect of RecF-like pathway mutants on H2O2 resistance. Strains were grown in the presence of 1 mM IPTG to induce RecA expression. Error bars represent the standard error of the mean of four to nine experiments. All strains were statistically different from the parent strain FA1090recA6 at the 20 mM dose (P < 0.04) and 50 mM dose (P ≤ 0.002). FA1090recA4 is included for comparison.
FIG. 4.
FIG. 4.
Effect of Holliday junction processing mutants on H2O2 resistance. Strains were grown in the presence of 1 mM IPTG to induce RecA expression. Error bars represent the standard error of the mean of four experiments. All strains were statistically different from the parent strain recA6 at the 50 mM dose (P < 0.05). FA1090recA4 is included for comparison.

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