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J Bacteriol. Nov 2006; 188(21): 7645–7651.
Published online Aug 25, 2006. doi:  10.1128/JB.00801-06
PMCID: PMC1636252

Neisseria gonorrhoeae DNA Recombination and Repair Enzymes Protect against Oxidative Damage Caused by Hydrogen Peroxide[down-pointing small open triangle]


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 (H2O2) 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 H2O2 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 H2O2. Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H2O2 resistance as well. Finally, we show that RecA protein levels did not increase as a result of H2O2 treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H2O2 resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H2O2.

Neisseria gonorrhoeae is the sole causative agent of the sexually transmitted disease gonorrhea. The hallmark of symptomatic gonococcal infection is a massive influx of activated neutrophils into the urethra, resulting in a purulent discharge consisting of neutrophils and N. gonorrhoeae (42, 31). Neutrophils kill bacteria through the combined action of antimicrobial proteins and reactive oxygen species (ROS) (37), perpetrating a potent bactericidal oxidative burst that generates substantial amounts of superoxide anion, hydroxyl radical, and hydrogen peroxide (H2O2). These ROS can damage proteins, lipids, carbohydrates, and DNA (53). The ability of N. gonorrhoeae to survive oxidative damage is illustrated by its ability to survive among neutrophils (31, 42, 45). During infection, N. gonorrhoeae is also likely to encounter H2O2 produced by commensal lactobacilli, which inhibit the growth of N. gonorrhoeae in vitro (61, 49). Since N. gonorrhoeae is an obligate human pathogen, it is not exposed to typical environmental stresses such as UV light, ionizing radiation, or chemical mutagens. Therefore, the preponderant type of DNA damage N. gonorrhoeae is likely to encounter is oxidative damage from neutrophils and commensal lactobacilli, as well as oxidative damage caused by free radicals evolved during the normal process of oxidative phorphorylation (10).

The gonococcal genome contains many genes predicted to be involved in a number of DNA repair pathways, including base excision repair, nucleotide excision repair, mismatch repair, and recombinational repair (15). Recombinational DNA repair has been studied extensively in N. gonorrhoeae and requires the recA (19) and recX (52) genes, which act in concert with either the RecBCD pathway (recB, recC, and recD genes) (25) or the RecF-like pathway (recO, recQ, recR, and recJ genes) (25, 46, 36). The Holliday junction processing enzymes encoded by recG, ruvA, ruvB, and ruvC also contribute to recombinational DNA repair in N. gonorrhoeae (36, 35). N. gonorrhoeae appears to use both DNA recombinational repair pathways simultaneously (25). This is in contrast to Escherichia coli, where mutants in the RecF pathway generally show phenotypes only in the context of recBC sbcBC mutations (21), leading to the conclusion that recombinational DNA repair is especially important for the repair of damaged DNA in N. gonorrhoeae (25).

In E. coli, recA (1) and many other DNA recombinational repair genes (12), have been shown to be important for the repair of oxidatively damaged DNA. E. coli RecA is important both directly for its functions in DNA repair and indirectly for its role in the induction of the SOS response of DNA repair (12, 18). However, since N. gonorrhoeae lacks a classical SOS response (2, 32), this indirect contribution of RecA to the repair of oxidatively damaged DNA is irrelevant in N. gonorrhoeae.

Microarray analysis has shown that only a single known DNA repair and recombination gene, recN, is upregulated after hydrogen peroxide treatment (51). The exact role of recN in DNA repair and recombination is unclear in any organism, but it appears to function in the repair of DNA double-strand breaks (29, 14, 33). Moreover, an N. gonorrhoeae recN mutant exhibits decreased survival to nalidixic acid (46) and hydrogen peroxide (51), both of which can result in DNA double-strand breaks.

Although several gonococcal genes have been identified that protect against oxidative damage, few of them are predicted to function in the repair of DNA. The katA gene product protects N. gonorrhoeae from H2O2 (48) by the reduction of H2O2 to H2O and O2. The ccp gene product (56) is also likely to act through the reduction of H2O2 (39), and the sco gene product may act as a thiol:disulfide oxidoreductase (38). Bacterioferritin (4) and azurin (60) appear to protect against oxidative stress by sequestering ions that exacerbate oxidative damage; a manganese uptake system (55) provides Mn ions that quench ROS; and the msrAB gene product repairs oxidatively damaged proteins (47). To date, only two genes that are involved in DNA repair and recombination have been found to protect against oxidative damage in N. gonorrhoeae. The previously described N. gonorrhoeae recN mutant (51) and a mutant inactivated in priA, which is involved in replication restart, both show decreased resistance to oxidative damaging agents (16). In contrast to E. coli recA (1), N. gonorrhoeae recA was reported to not protect against oxidative damage caused by H2O2 (9), suggesting that DNA repair and recombination enzymes may differ between N. gonorrhoeae and E. coli in their importance to the repair of oxidatively damaged DNA.

The majority of antioxidants identified thus far in N. gonorrhoeae do not function in DNA repair and N. gonorrhoeae recA has been demonstrated to not protect from oxidative damage (9). Therefore, one hypothesis is that DNA repair is not important for protection from oxidative damage. Alternatively, since recN is the only DNA recombination and repair gene upregulated in response to H2O2 and both recN (51) and priA (16) protect against oxidative damage, it is possible that DNA repair is important for the repair of oxidative damage, but constitutive levels of DNA recombination and repair enzymes provide sufficient damage protection. To test the importance of gonococcal DNA recombination and repair genes in conferring resistance to oxidative damage, we measured the resistance of a recA mutant (40) and of several mutants with defects in recombinational DNA repair enzymes (25, 52, 46, 36, 35). We show that RecA, in addition to the RecBCD and RecF-like recombinational repair pathways and Holliday junction processing enzymes, contribute to the survival of N. gonorrhoeae to oxidative damage.


Bacterial strains and growth conditions.

The gonococcal strains used in the present study were either strain FA1090 containing the recA gene under the control of its endogenous promoter or derivatives of this strain. FA1090recA4 is a recA loss-of-function mutation that contains a tetracycline resistance [Tetr] cassette inserted in the recA gene. FA1090recA6 contains an IPTG (isopropyl-β-d-thiogalactopyranoside)-regulatable gonococcal recA allele under the dual control of taclac and UV5 promoters and allows for the conditional control of recA expression and RecA-dependent processes (40). IPTG was used at a final concentration of 1 mM to provide induction of recA transcript (40). All strains contained the 1-81-S2 variant pilE sequence (41). Mutations in additional recombination genes were in the recA6 background. All mutant strains were previously constructed, and the mutations were validated phenotypically by complementation for their effect on DNA repair of UV damage and on nalidixic acid- or gamma-ray-induced double-strand breaks (25, 52, 46, 36, 35). N. gonorrhoeae strains were grown at 37°C, unless otherwise indicated. Strains were propagated on N. gonorrhoeae (GC) medium base (GCB; Difco) plus Kellogg supplements I (22.2 mM glucose, 0.68 mM glutamine, 0.45 mM cocarboxylase) and II [1.23 mM Fe(NO3)3] (13) at 37°C in 5% CO2 or in GC liquid (GCBL) medium (1.5% proteose peptone no. 3 [Difco], 0.4% K2HPO4, 0.1% KH2PO4, 0.1% NaCl) with Kellogg supplements I and II and 0.042% sodium bicarbonate. Unless explicitly stated, GCB and GCBL always contained Kellogg supplements I and II. All chemicals were obtained from Sigma unless otherwise indicated.

Hydrogen peroxide resistance assay.

N. gonorrhoeae strains were grown from freezer stocks for approximately 20 h, and ~10 colonies were passaged onto GCB. After 12 h, colonies were collected with a Dacron swab (Puritan), resuspended in GCBL at an optical density at 550 nm (OD550) of [congruent with]0.1, grown 16 h at 30°C in 15-ml conical tubes (Sarstedt) in a drum rotator, diluted to an OD550 of [congruent with]0.3, grown 2.5 to 3 h at 37°C, and diluted to OD550 of [congruent with]0.06 in GCBL containing 1 mM IPTG to induce recA expression in the recA6 background when appropriate. This culture was grown to mid-log phase (OD600 of [congruent with]0.5) and diluted 1:10 in GCBL containing IPTG where appropriate, and 1-ml aliquots were placed into 15-ml conical tubes. H2O2 (Sigma-Aldrich) was added to the tubes at a final concentration of 0, 10, 20, or 50 mM, and the tubes were placed in a drum rotator at 37°C for 15 min. Cultures were immediately serially diluted into plain GCBL (no Kellogg supplements) containing 10 μg of catalase/ml and plated onto GCB agar containing IPTG where appropriate. Piliated colonies were counted after ~20× growth, and the survival of each strain at the 10, 20, and 50 mM doses of H2O2 was calculated relative to survival at the 0 mM dose.

Isolation of protein from N. gonorrhoeae and Western blot analysis.

N. gonorrhoeae FA1090 and FA1090recA6 were grown in liquid culture as described above or on plates as previously described (52). To monitor the effect of H2O2 on N. gonorrhoeae RecA protein expression, cultures were diluted 1:10 into GCBL containing 1 mM IPTG, where appropriate, split in half, and half received 5 mM hydrogen peroxide and half was mock treated with water. Samples (10 ml) were taken after 15 and 30 min, and catalase (10 μg/ml [final concentration]; Sigma) was added to the samples to degrade any remaining H2O2, followed by a 1-min incubation at room temperature. Cultures were quickly cooled on ice and pelleted by centrifugation (4,000 × g for 10 min), and the supernatant was removed. Pellets were resuspended in 500 μl of phosphate-buffered saline (PBS), and a 20-μl sample of the supernatant was used for determining the protein concentration using the BCA protein assay (Pierce Chemicals). A 5× sodium dodecyl sulfate protein sample buffer was added to the remainder of the sample, the genomic DNA was sheared by repeated passage through a small-bore needle, and the sample was stored at −20°C. Equal amounts of total protein were loaded onto each lane of a 12% protein gel, which was prepared and run by standard techniques (23). Prestained markers (Bio-Rad) were included on all gels. Gels were blotted using CAPS buffer [10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (pH 11.0); 10% methanol] onto 0.45-μm-pore-size polyvinylidene difluoride membrane (Millipore) using a Bio-Rad transfer cell at 75 V for 1.5 h at 4°C. Western blots were developed according to the chemiluminescent (ECL) Western blotting protocol (Amersham Life Sciences) with the following modifications. Blots were blocked overnight at 4°C in a 5% solution of Carnation powdered milk (Nestle Foods) dissolved in washing buffer (Tris-buffered saline [pH 7.6], 0.1% Tween 20). Primary polyclonal anti-RecA antibodies (a gift from Mike Cox, University of Wisconsin-Madison) and secondary goat anti-rabbit immunoglobulin G antibodies conjugated to horseradish peroxidase (Chemicon) were diluted 1:5,000 and 1:25,000, respectively, in blocking solution and then incubated with shaking for 1 h at room temperature.


recA, but not recX, mutants show decreased resistance to hydrogen peroxide.

recA has been demonstrated to confer protection against oxidative damage in E. coli (1), Salmonella enterica serovar Typhimurium (3), and Lactococcus lactis (7). Since recA is essential for recombinational DNA repair in N. gonorrhoeae (20, 40), it was puzzling that an N. gonorrhoeae recA mutant was previously reported to be more resistant to H2O2 than the parent strain FA1090 (9). The H2O2 sensitivity of two independent recA mutant strains was measured to determine whether recombinational DNA repair is important for the repair of damage caused by H2O2. The H2O2 sensitivity of the parental strain FA1090 and FA1090recA4, which is a recA loss-of-function mutant containing a Tetr resistance cassette inserted in the recA gene (40), was assessed. FA1090recA4 was up to fourfold less resistant to H2O2 than the parent strain FA1090 (Fig. (Fig.1A),1A), as was an independent recA knockout construct containing an erythromycin resistance cassette, recA9 (40; data not shown).

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) ...

Since our current observation that a recA mutant showed decreased resistance to H2O2 was inconsistent with what has previously been reported (9), we wanted to verify our results by using a conditional recA mutant allele, FA1090recA6, where recA expression is controlled by IPTG and strains are phenotypically RecA in the absence of IPTG (40). FA1090recA6 expressing recA (+IPTG) exhibited the same resistance to H2O2 as strain FA1090, which has recA under the control of its endogenous promoter. In contrast, FA1090recA6 not expressing recA (−IPTG) showed the same resistance as the recA4 loss-of-function mutant (Fig. (Fig.1A).1A). Together, these data conclusively demonstrate that recA contributes to hydrogen peroxide resistance in N. gonorrhoeae.

The RecX protein inhibits RecA activity in vitro (57, 50, 6) and recX modulates a variety of RecA-dependent phenotypes in vivo (50, 44). An E. coli recX mutant shows decreased resistance to UV damage (50) and a Herbaspirillum seropedicae recX mutant shows decreased resistance to UV light and the mutagen methyl methanesulfonate (8). A Deinococcus radiodurans recX mutant shows an elevated frequency of recombination and decreased genetic stability (44) and appears to negatively regulate genes encoding the antioxidants catalase and superoxide dismutase through an unknown mechanism (43). Importantly, a N. gonorrhoeae recX mutant shows a small decrease in the ability to survive DNA damage caused by double-stand breaks (52), which is the major type of DNA damage resulting from treatment with hydrogen peroxide. However, the contribution of recX to oxidative damage resistance has not been measured in any organism. In the present study, an N. gonorrhoeae recX loss-of-function mutant showed H2O2 resistance nearly identical to that of the recA6 parent strain (Fig. (Fig.1A),1A), suggesting that recX does not contribute to the repair of oxidative damage by H2O2 in N. gonorrhoeae.

RecA protein levels are not upregulated in response to H2O2, and RecA expression is equivalent from its endogenous promoter and an IPTG-inducible promoter.

In many bacteria, recA and other genes of the SOS regulon are upregulated after H2O2 exposure (62, 30, 28, 27), likely as part of the SOS response. N. gonorrhoeae lacks a classical SOS response, evidenced by its lack of LexA and UmuD homologues, LexA boxes, and the lack of induction uvrA, uvrB, and recA transcripts in response to the DNA-damaging agents methyl methanesulfonate and UV light (2). To determine whether RecA protein levels were affected by H2O2 treatment and to determine the relative levels of RecA protein expression when recA was expressed from its endogenous promoter (strain FA1090) or from an IPTG-inducible promoter (strain FA1090recA6), Western blot analyses were performed. Strains FA1090 and FA1090recA6 (+IPTG) were grown in liquid culture, the culture was split, and half the culture was treated with 5 mM hydrogen peroxide, and the other half was left untreated. Aliquots were removed after 15 and 30 min, and the level of RecA protein expression was measured. Western blots containing equal amounts of total protein were probed with anti-RecA antiserum, and images were captured and analyzed by using a Gel-Imager (Alpha Innotech). RecA protein levels did not increase as a result of hydrogen peroxide treatment in either FA1090 (Fig. (Fig.1B)1B) or FA1090recA6 (data not shown). Comparison of the relative RecA protein levels in FA1090 and FA1090recA6 (induced with 1 mM IPTG) showed the RecA levels to be equivalent in these two strains when cultures were grown either in liquid or on solid media (Fig. (Fig.1C).1C). Together, these data show that RecA levels are comparable when recA is expressed from its endogenous promoter and from an IPTG-inducible lac promoter construct. They also demonstrate that RecA protein is not upregulated after hydrogen peroxide treatment. Since constitutive levels of RecA contributed to H2O2 resistance, we decided to expand our hypothesis by investigating the contribution of recombinational repair enzymes to H2O2 resistance.

The RecBCD pathway of DNA repair contributes to hydrogen peroxide resistance.

In E. coli, recombinational DNA repair is mediated by RecA in conjunction with either the RecBCD or the RecF pathway (21, 24). Although both pathways are important for H2O2 resistance in E. coli, the RecBCD pathway appears to be the predominant oxidative damage repair pathway (11, 12), probably due to the fact that the RecBCD enzyme processes DNA double-strand breaks (22). In N. gonorrhoeae, the two pathways have been shown to both be important for the repair of UV-induced lesions and DNA double-strand breaks caused by gamma irradiation or nalidixic acid, with most mutants showing a level of resistance intermediate to the parental strain and the recA mutant (25, 46, 36), but the roles of the RecBCD and RecF-like pathways in the repair of oxidative damage have not been previously tested.

To determine the roles of members of the RecBCD pathway in H2O2 resistance, the H2O2 resistance of strains with individual mutations in recB, recC, and recD was measured. The individual recBCD mutants showed decreased resistance to H2O2 relative to the parent strain at all levels of H2O2 tested, particularly at the 10 mM dose. These results demonstrate that the N. gonorrhoeae RecBCD pathway is important for the repair of DNA damage caused by hydrogen peroxide (Fig. (Fig.22).

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 ...

The RecF-like pathway of DNA repair contributes to hydrogen peroxide resistance.

To determine the roles of members of the RecF-like pathway (designated “RecF-like” since N. gonorrhoeae lacks a recF homolog) in H2O2 resistance, strains with individual mutations in recJ, recO, and recQ (25, 46) (Fig. (Fig.3)3) were exposed to H2O2, and the effect on survival was measured. All of the mutants showed the same pattern of sensitivity to H2O2, exhibiting only small decreases in resistance (twofold) relative to the parent strain FA1090recA6 at low levels of H2O2 (5 mM) but showing greater decreases in resistance (~10-fold) relative to the parent strain FA1090recA6 at the 50 mM dose of H2O2. These results mirror what is seen in E. coli, with mutants in the RecF pathway being more sensitive to H2O2 damage but not as sensitive as mutants in the RecBCD pathway (12).

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 ...

Branch migration machinery contributes to hydrogen peroxide resistance.

The resolution of DNA intermediates formed during recombinational DNA repair by both the RecBCD and the RecF-like pathways requires the action of proteins that catalyze both the translocation (RuvAB and RecG) and the cleavage (RuvC) of Holliday junctions (59). Mutation of the ruvA gene in E. coli renders strains more sensitive to killing by H2O2 (18). Therefore, the H2O2 resistance of strains containing mutations in genes that are responsible for branch migration (ruvA and recG) (36) and cleavage of Holliday junctions (ruvC) (35) was measured. All three mutants showed decreased resistance to all tested doses of H2O2, demonstrating that gene products involved in branch migration and Holliday junction resolution are also important for the repair of H2O2-damaged DNA in N. gonorrhoeae (Fig. (Fig.44).

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 ...


The data presented here represent the first comprehensive examination of the importance of DNA recombinational repair enzymes in resistance to oxidative damage by N. gonorrhoeae. We showed that recA, genes of the RecBCD and RecF-like recombination pathways, and genes whose products are involved in Holliday junction processing are all important for mediating repair of oxidative damage. Moreover, the data suggest that these genes are expressed at basal levels sufficient to mediate repair and do not need to be upregulated upon encountering DNA damage in order to function efficiently in N. gonorrhoeae.

In E. coli, RecA activity is central to the ability to repair damaged DNA. RecA participates directly in recombinational DNA repair (24), and RecA regulates the induction of the SOS response of DNA repair and mutagenesis (58). It has been demonstrated that increased expression of both recA (12) and other SOS genes such as ruvA (18) contribute to H2O2 survival. Our data indicate that there is no increase in N. gonorrhoeae RecA protein levels after the exposure of cells to H2O2. The lack of RecA protein induction and the overall lack of induction of DNA recombination and repair genes by hydrogen peroxide (51) are consistent with the well-documented lack of an SOS response in N. gonorrhoeae (2).

An N. gonorrhoeae recA mutant was previously shown to be 10-fold more resistant to H2O2 than the isogenic parent strain (9), yet we found a recA mutant to be ~4-fold less resistant to oxidative challenge by H2O2. One reason for this difference could be that the recA mutants in the previous study (9) were selected through subculture in liquid, perhaps allowing the accumulation of compensatory mutations that could yield the recA mutant cells more resistant to H2O2. We used a conditional recA mutant in the present study, allowing us to modulate recA expression in a single strain, to measure the effect of recA on hydrogen peroxide resistance, so the chance of compensatory mutations affecting the results was virtually nonexistent. These results were further corroborated by using the recA-null recA4 and recA9 mutants, thereby demonstrating conclusively that recA is important for survival to oxidative damage in N. gonorrhoeae. We also showed that the levels of RecA protein expressed from the endogenous recA promoter and from an IPTG-inducible promoter (40) are equivalent.

recX mutation had been previously shown to influence all RecA-mediated processes in N. gonorrhoeae, but its effect on DNA repair was the most subtle phenotype (52). In D. radiodurans, a recX loss-of-function mutant showed both higher activities of the antioxidant enzymes catalase and superoxide dismutase and higher transcript levels of the genes encoding these activities (43). However, the effect of recX mutation on H2O2 resistance was not measured in D. radiodurans. In the present study, the effect of recX mutation on survival of N. gonorrhoeae to H2O2 damage was negligible, and an effect on antioxidant enzymes was not measured. We therefore conclude either that RecX has no role in H2O2 resistance or that the role of N. gonorrhoeae RecX in H2O2 resistance is small enough to not be manifest in our assays, which is consistent with the previously observed subtle phenotype of a recX mutant in DNA repair.

Both the N. gonorrhoeae RecBCD and the RecF-like pathways are important for survival of oxidative damage, although the increased sensitivity of the RecBCD pathway mutants relative to the RecF-like pathway mutants suggests that RecBCD is the predominant repair pathway for oxidative damage in N. gonorrhoeae. This is similar to what was previously observed in E. coli (12) and is likely attributable to the fact that the RecBCD pathway repairs DNA double-strand breaks (22), which are the lesions most often resulting from exposure to oxidative damage (5). In E. coli, a recA single mutant is more sensitive to oxidative damage than a RecBCD or RecF pathway single mutant, and a recC recF double mutant shows the same sensitivity as a recA mutant (12). In contrast, the RecBCD pathway mutants of N. gonorrhoeae were as sensitive as a recA mutant, and a recB recO double mutant was no more sensitive than the recB single mutant (data not shown). These data suggest that the two recombinational repair pathways of N. gonorrhoeae may be intertwined during the repair of oxidative damage. The E. coli paradigm of recombinational repair is that the RecBCD pathway acts on double-strand breaks and the RecF pathway acts on single-strand DNA (ssDNA) gaps. Both double-strand breaks and ssDNA gaps result in stalled replication forks, which, if not repaired, will result in cell death. Our data suggest a model where the double-strand breaks caused by hydrogen peroxide are repaired by the RecBCD pathway and the ssDNA gaps which are repaired by the RecF-like pathway also feed into the RecBCD pathway through an unknown mechanism. It has been previously suggested that N. gonorrhoeae utilizes both DNA repair pathways simultaneously and that N. gonorrhoeae behaves like an E. coli recBC sbcBC mutant in its de facto use of the RecF-like pathway in wild-type cells (25). Interestingly, E. coli RecFOR proteins are needed for the repair of double-strand breaks in a recBC sbcBC mutant background (26), suggesting that there is cross talk between these pathways of DNA repair in E. coli as well.

Further steps common to both RecBCD- and RecF-catalyzed recombinational DNA repair are the migration and resolution of Holliday junctions, catalyzed by RuvABC and RecG, followed by replication restart. Replication restart in E. coli initiates through the action of DnaC, which reloads the DnaB helicase on stalled replication forks (34). Reloading of DnaB can occur either in a PriA-dependent manner, requiring PriB or PriC, as well as DnaT, or in a PriA-independent manner, requiring Rep, PriC, and possibly also DnaT. Studies on replication-restart in N. gonorrhoeae have demonstrated that N. gonorrhoeae lacks PriC and DnaT homologues (15), as well as a Rep-dependent replication restart pathway (17), suggesting that only the PriA-dependent pathway functions in replication restart in N. gonorrhoeae (16). The importance of replication restart in the repair of oxidative damage has not been investigated in E. coli. However, the sensitivity of an N. gonorrhoeae priA mutant to hydrogen peroxide (16) further demonstrates the importance of recombinational DNA repair in repairing oxidatively damaged DNA and predicts that additional genes involved in replication restart may be especially important for the protection of N. gonorrhoeae from oxidative damage.

Although we have demonstrated that RecA and DNA recombinational repair enzymes confer resistance to oxidative damage caused by H2O2, these mutants were not as sensitive as isogenic mutants inactivated in NGO1686 (51), which encodes an uncharacterized protein, or kat (catalase) (48) (E. Stohl, A. Criss, and H. Seifert, unpublished data), whose product directly degrades H2O2. Since oxidative damage is likely to be the main type of DNA damage N. gonorrhoeae is exposed to during infection of humans, possessing redundancy in genes conferring protection against H2O2 would be evolutionarily advantageous to this strict human pathogen. Moreover, the recent demonstration that N. gonorrhoeae is polyploid (54) suggests that, in the event of chromosomal damage, these additional copies of the chromosome could provide the genetic information present on the damaged copy, perhaps obviating the necessity of recombinational repair. Therefore, of the many mechanisms of resistance used by N. gonorrhoeae to combat oxidative insult, recombinational DNA repair appears to be one layer of resistance, and continued research will identify others.


We thank Michael Cox (University of Wisconsin-Madison) for providing anti-RecA antisera.

This study was supported by NIH grant RO1 AI044239.


[down-pointing small open triangle]Published ahead of print on 25 August 2006.


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