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J Bacteriol. 2005 Aug; 187(15): 5347–5355.
PMCID: PMC1196015

Mutation of the priA Gene of Neisseria gonorrhoeae Affects DNA Transformation and DNA Repair


In Escherichia coli, PriA is central to the restart of chromosomal replication when replication fork progression is disrupted and is also involved in homologous recombination and DNA repair. To investigate the role of PriA in recombination and repair in Neisseria gonorrhoeae, we identified, cloned, and insertionally inactivated the gonococcal priA homologue. The priA mutant showed a growth deficiency and decreased DNA repair capability and was completely for deficient in DNA transformation compared to the isogenic parental strain. The priA mutant was also more sensitive to the oxidative damaging agents H2O2 and cumene hydroperoxide compared to the parental strain. These phenotypes were complemented by supplying a functional copy of priA elsewhere in the chromosome. The N. gonorrhoeae priA mutant showed no alteration in the frequency of pilin antigenic variation. We conclude that PriA participates in DNA repair and DNA transformation processes but not in pilin antigenic variation.

Neisseria gonorrhoeae (gonococcus) is the causative agent of the sexually transmitted disease gonorrhea. The hallmark of acute gonococcal infection is a purulent discharge consisting of neutrophils, which are associated with the inflammatory response mounted against the gonococcal infection (55). Neutrophils perpetrate an oxygen-dependent bactericidal attack with the release of numerous highly reactive compounds, including superoxide and H2O2, resulting in DNA and protein damage in the microbe. N. gonorrhoeae may also encounter H2O2 produced by commensal organisms often associated with gonococci in vivo (72). N. gonorrhoeae is highly adapted to survive oxidative damage, as evidenced by the ability of gonococci to survive within and among neutrophils (40, 55). In addition, since N. gonorrhoeae is an obligate human pathogen and is unlikely to be subject to damage by chemical and physical agents in the environment (e.g., UV and ionizing radiation, chemical mutagens, desiccation), it is probable that a major source of DNA damage stems from free radicals endogenously generated during aerobic respiration (63). Consistent with this is the fact that gonococci possess a number of defenses to counteract oxidative damage, including catalase (59), cytochrome c oxidase (51), cytochrome c peroxidase (52, 67), peptide methionine sulfoxide reductase (58), and a Mn-dependent superoxide quenching mechanism (66).

To maintain genome integrity in the face of DNA damage, N. gonorrhoeae has developed various strategies to either reverse, excise, or tolerate DNA damage products via a network of DNA repair mechanisms including base excision repair, nucleotide excision repair, mismatch repair, and recombinational repair (18). Recombinational repair is well studied in N. gonorrhoeae and requires the recA (21) and recX (62) genes, along with either the RecBCD pathway components recB, recC, and recD (33) or the RecF pathway components recO, recR, recQ, and recJ (33, 50, 57). The branch migration genes recG and ruvA also contribute to recombinational DNA repair in N. gonorrhoeae (50). The use of multiple genetic pathways for recombinational DNA repair in N. gonorrhoeae distinguishes it from Escherichia coli, which utilizes the RecF DNA repair pathway only in a mutant recBC sbcBC background (22, 25). This difference may reflect an increased importance of recombinational repair pathways in N. gonorrhoeae since this organism lacks an SOS response (2).

In addition to DNA repair functions, homologous recombination is also required for pilin antigenic variation and DNA transformation in N. gonorrhoeae. Gonococci evade not only the host's innate immune response by resisting polymorphonuclear leukocyte killing but also the host's adaptive immune response by employing a large number of highly variable surface components; both of these immune evasion strategies are believed to aid in the successful transmission of the organism. The gonococcal pilus is one such variable structure, which undergoes antigenic variation at frequencies greater than 4 × 10−3 per CFU per generation (A. K. Criss, K. A. Kline, and H. S. Seifert, unpublished data). Antigenic variation occurs via a unidirectional homologous recombination event between the pilin coding gene (pilE) and one of multiple unexpressed pilin copies (pilS) found at various sites throughout the chromosome. During pilin antigenic variation, a variable portion of a pilS copy replaces the corresponding portion of pilE, recombining at short regions of identity shared between the silent and expressed genes (16). Pilin antigenic variation is dependent on recA (21), the recF-like pathway (33, 57), rdgC (32), and rep (19) as well as the ruvABC and recG branch migration pathways (50).

DNA transformation is responsible for horizontal transfer of chromosomal alleles among the members of the Neisseria genus and within each species (37, 60). N. gonorrhoeae is naturally competent for transformation at all stages of growth (1). Extracellular DNA is taken up into the bacterium in a DNA uptake sequence-dependent manner (11, 12). Numerous gene products are involved in DNA uptake and competence, including pilE, pilQ, and pilT, involved in elaboration of the type IV pilus, and the competence-associated factors comE, comA, comP, comL, tpc, and dca (5). Once transforming homologous DNA is taken up into the cell, it is recombined into the chromosome, a process which requires recA (21) and relies partially on the recBCD-encoded enzyme (33), recN (57), and rep (19) but is independent of the recF-like pathway of recombination (33, 57).

Bacteria possess many systems which accurately repair damaged DNA prior to chromosomal replication. However, if DNA damage is not dealt with prior to the arrival of a replisome, the DNA lesion can disrupt the replication fork. Left unrepaired, blocks of replication fork progression are lethal to the cell. Replication fork collapse is estimated to occur in between 15 to 50% of E. coli cells in the absence of exogenous DNA damage (9) and is thought to be due in part to intracellular oxidative damage arising during normal aerobic growth (8, 63). One mechanism to restart replication in an origin-independent manner is initiated by the helicase PriA. PriA was originally discovered for its role in phage ΦX174 DNA replication (49, 69) and replication of a subset of plasmids in E. coli (23, 27). priA mutants were subsequently shown to exhibit reduced viability, increased sensitivity to UV irradiation and agents of double-strand breaks, increased cell filamentation, and reduced abilities to undergo homologous recombination (20, 23, 39). Surprisingly, the role of priA in survival of E. coli to oxidative stress has never been reported. Based on these collective observations, Kogoma and colleagues proposed that PriA was involved in replisome reloading on D-loops created at recombination intermediates (20). This hypothesis was supported by observations that PriA-mediated replication fork assembly could rescue arrested replication forks (26), data showing that PriA binds D-loops with high specificity (30, 38), and studies characterizing the genetic pathways of replication restart (45). Therefore, it is well-accepted that replication restart allows for chromosome replication after DNA damage and promotes cell survival.

In order to understand the role of PriA in N. gonorrhoeae, the gonococcal priA homologue was identified, disrupted, and assessed for its role in the homologous recombination-mediated processes of pilin antigenic variation, DNA transformation, and DNA repair. N. gonorrhoeae priA is not involved in pilin antigenic variation but plays a critical role in DNA transformation and DNA repair. In particular, priA is important for resisting killing by oxidative damaging agents.


Bacterial strains and growth conditions.

E. coli One Shot TOP10 competent cells (Invitrogen) were grown on Luria-Bertani (LB) broth or agar at 37°C and used to propagate plasmids. Plate media contained 15 g of agar per liter. Gonococcal strains were grown on Gc Medium Base (Difco) plus Kellogg supplements (GCB) [22.2 mM glucose, 0.68 mM glutamine, 0.45 mM cocarboxylase, 1.23 mM Fe(NO3)3; all from Sigma] (17) at 37°C in 5% CO2 or in N. gonorrhoeae liquid (GCBL) medium (1.5% protease peptone no. 3 [Difco], 0.4% K2HPO4, 0.1% KH2PO4, 0.1% NaCl) with Kellogg supplements and 0.042% sodium bicarbonate. For growth of N. gonorrhoeae strains in liquid medium, gonococci were grown from freezer stocks on GCB solid medium for approximately 20 h, and ~10 colonies were passaged onto GCB plates. After 12 h, colonies were collected with a Dacron swab (Puritan), resuspended in GCBL at an optical density of 550 nm (OD550) of ~0.1, grown 12 h, diluted 1:5 to an OD550 of ~0.3, grown 2.5 to 3 h, and diluted 1:10 to an OD550 of ~0.07. This culture was grown to mid-log phase (OD600 [congruent with] 0.5) for use in assays. Liquid growth of N. gonorrhoeae was carried out in 15-ml conical tubes (Sarstedt) rotating in a drum rotor.

Gonococcal strains in this study were derivatives of strain FA1090 variant 1-81-S2 (54) with matched pilE sequences and matched opacity colony types. Mutations were created in a recA6 background which contains an isopropyl β-d-thiogalactopyranoside (IPTG)-regulatable gonococcal recA allele and allows for control of recA expression and recA-dependent processes (53). IPTG (Diagnostic Chemicals Limited) was used at 1 mM to provide maximal induction of recA transcription (53), which yields RecA expression levels and RecA-dependent phenotypes (H2O2 and UV sensitivities and DNA transformation efficiency) similar to that of strains with a wild-type recA gene (E. A. Stohl and H. S. Seifert, unpublished data).

Antibiotics were added at the following concentration for E. coli: chloramphenicol, 15 mg/liter; kanamycin, 50 mg/liter; erythromycin, 250 mg/liter. For N. gonorrhoeae strain FA1090, the antibiotics were added as follows: chloramphenicol, 2 mg/liter; erythromycin, 1 mg/liter; kanamycin, 40 mg/liter; nalidixic acid, 1.5 mg/liter.

DNA manipulations and analysis.

Standard procedures were performed as described previously (44). Plasmid DNA was isolated from strains of E. coli using plasmid isolation kits (QIAGEN Inc.). Enzymes were used as specified by the manufacturer (New England Biolabs Inc.). Southern blot analysis was performed by transferring DNA to a Magnagraph nylon membrane (Micro Separations Inc.) and hybridization with digoxigenin-labeled dUTP probes and chemiluminescent substrate from the DIG System (Boehringer Mannheim) according to the manufacturer's instructions. pilE sequences were determined by amplifying pilE from the chromosome of lysed cells with primers PILRBS and SP3A (54) using Taq polymerase (Promega). The resulting PCR product was sequenced with PILRBS and/or SP3A.

Sequencing reactions were performed using CEQ Dye Terminator Cycle Sequencing Quick Start kit and a CEQ 2000XL automated sequencer (Beckman Coulter) according to the manufacturer's instructions. DNA analysis was performed using Lasergene software (DNASTAR, Inc.) and VectorNTI software (Informax, Inc.). BLAST searches were performed, and the N. gonorrhoeae strain FA1090 Gonococcal Genome Sequencing Project (43) was accessed to identify the PriA homologue.

Plasmid and mutation construction.

Plasmid pGcPriA was created by cloning a DNA fragment containing the predicted priA gene that was PCR amplified from N. gonorrhoeae strain FA1090 genomic DNA with primers PriAF4 (5′-TTCTTCGGTCAGGTTTTTGC-3′) and PriAR4 (5′-CCAAACTCGACTACCCGAAA-3′) into the pCR2.1-TOPO plasmid (Invitrogen). This plasmid was cut within priA using SacII to create staggered ends compatible with BsiEI-cut ends. The ermC gene was cut out of pErmUP using BsiEI and inserted into the priA gene of pGcPriA to create pGcPriA::ErmUP. Plasmid pErmUP was created by cloning the ermC gene amplified from pHSS24 (68) using ErmAInUP (5′-GCCGTCTGAATCTTTTATTCAATAATCGCATCAG-3′) (the gonococcal uptake sequence required for efficient uptake of DNA is underlined) and ErmBIn (5′-ACAAAAAATAGGTACACGAAAAAC-3′).

To create a strain for complementation analysis, pGCC6::GcPriA was constructed. The priA gene, including its own promoter, was PCR amplified from N. gonorrhoeae strain FA1090 genomic DNA using PriA5′comp (5′-AGCTTTGTTTAAACTTGGAAAAGTGGCATTGTATC-3′) with the PmeI site underlined and PriA3′comp (5′-CCTTAATTAATCACCGTTCCCGAGATTCT-3′) with the PacI site underlined. The resulting ~2.2-kb PCR product was inserted into pCRBlunt (Invitrogen) to create pGcPriAPmePac. The entire gene was sequenced to ensure accuracy. Plasmid pGcPriAPmePac was digested with PmeI and PacI to release priA. The priA gene was subcloned into plasmid pGCC6, digested with PmeI and PacI, at an intergenic chromosomal site located between the lctP and aspC genes (32, 33) and linked to a chloramphenicol resistance cassette to create pGCC6::GcPriA.

The gonococcal priA mutant strain was created by spot transformation of N. gonorrhoeae strain FA1090 variant 1-81-S2 recA6 with pGcPriA::ErmUP to create N. gonorrhoeae strain FA1090priA::erm. Briefly, N. gonorrhoeae organisms were streaked for confluence on GCB plates containing IPTG for RecA induction. Liquid spots containing an excess of donor DNA, 5 mM MgSO4, and Kellogg's supplements were spotted onto the plates and allowed to air dry. After 20 h at 37°C in 5% CO2, gonococci grown on the spots were collected using a Dacron swab (Puritan) and plated on erythromycin-containing GCB. N. gonorrhoeae strain FA1090priA::erm was spot transformed with pGCC6::GcPriA to create the FA1090priA::erm/priA+ complement strain. The genotypes of strainsFA1090priA::erm and FA1090priA::erm/priA+ were confirmed by Southern blot of ClaI-digested DNA using probes against priA, erm, and cat (for the priA complement strain). The pilE genes were sequenced to ensure all strains contained the same pilE gene sequence as the parental strain.

Growth assay.

Visualization of colony morphology was performed using a Nikon SMZ-10A stereomicroscope and recorded using a Polaroid camera model DMC le. Strains imaged were grown on the same plate to eliminate an effect of agar depth on colony size measurement.

The gonococcal growth curve was assessed on solid medium because reproducible growth in liquid medium is difficult to achieve. The inability to dilute N. gonorrhoeae below 105 CFU/ml and restore growth contributes to this difficulty. Growth on solid medium was assessed by picking and suspending 10 N. gonorrhoeae colonies in GCBL medium, plating serial dilutions, and determining mean CFU per colony.

Cell viability assay.

The viability of N. gonorrhoeae was measured using the Live/Dead BacLight bacterial viability kit (Molecular Probes). Briefly, N. gonorrhoeae was grown to mid-log phase, and 1 ml was pelleted at 14,000 rpm for 5 min. Gonococci were washed and resuspended in 1 ml buffer (0.1 M morpholinepropanesulfonic acid, 1 mM MgCl2, pH 7.2) and stained with 3 μl of a 1:1 mixture of SYTO9 and propidium iodide at room temperature for 15 min. Red and green fluorescence was visualized by fluorescence microscopy.

Antigenic variation assay.

N. gonorrhoeae strains were streaked for isolated colonies on plates containing IPTG and incubated at 37°C in 5% CO2 for 20 h. Five isolated piliated “starter” colonies per strain were selected and passaged onto separate plates without IPTG to turn off antigenic variation and “lock” in place any sequence changes that occurred at pilE. Approximately 40 colonies that arose from each starter colony were passaged two more times to ensure clonal populations, and the pilE gene of each colony was sequenced as described above (A. K. Criss, K. A. Kline, and H. S. Seifert, unpublished).

DNA transformation assay.

DNA transformation efficiency was determined in liquid medium by standard techniques (33). A total of 50 ng of plasmid pSY6 DNA carrying a gyrB mutation resulting in nalidixic acid resistance (Nalr) (61) was incubated with ~107 CFU/ml of N. gonorrhoeae in GCBL plus Kellogg's supplements and IPTG where appropriate for 15 min at 37°C, followed by the addition of 1 U of RQ1 DNase (Promega) and an additional 5 min of incubation at 37° to degrade extracellular DNA. The mix was then diluted 1:10 into GCBL plus Kellogg's supplements and IPTG where appropriate and incubated for 4 h. All incubations were carried out at 37°C in 5% CO2. Efficiency was expressed as the mean number of Nalr transformants per CFU.

UV sensitivity assay.

Serial dilutions of N. gonorrhoeae were plated on solid media and exposed to 0, 20, 40, 60, or 80 J/m2, and the surviving CFU at each dose were compared to the unexposed CFU.

Nalidixic acid survival assay.

Nalidixic acid (Nal) causes DNA double-strand breaks by partially inhibiting DNA gyrase (10, 29). Sensitivity to the toxic effects of nalidixic acid-induced double-strand breaks was measured by growing gonococcal strains for 24 h on GCB with IPTG, collecting organisms with a Dacron swab, and resuspending them in 1 ml of GCBL. Cultures were serially diluted, and 0.1 ml of the undiluted culture and of 10−1 and 10−2 dilutions were plated onto GCB containing 1 mM IPTG and 0.75 mg/liter nalidixic acid. Total CFU were calculated by plating spot dilutions of the cultures onto GCB agar. Colonies were counted after ~40 h of growth. Sensitivity to nalidixic acid is expressed as the ratio of survivors to the total CFU for each strain.

H202 sensitivity assay.

N. gonorrhoeae was grown to mid-log phase (OD600 [congruent with] 0.5) in liquid medium as described above and diluted 1:10 into GCBL, and 5-ml aliquots were placed into 15-ml conical tubes (Sarstedt). H2O2 (Sigma Aldrich) was added to the tubes at final concentrations of 0, 5, 10, 20, or 50 mM, and tubes were placed in a drum rotator for 15 min. Cultures were immediately serially diluted into GCBL containing 10 μg/ml catalase and spot-plated onto GCB agar. Colonies were counted after ~24 h of growth, except for FA1090 priA::erm, which was counted at 40 h of growth. Survival of each strain at the 5, 10, 20, and 50 mM dose of H2O2 was calculated relative to survival at the 0 mM dose.

Cumene hydroperoxide sensitivity assay.

N. gonorrhoeae was grown to mid-log phase in liquid medium diluted 1:10 in GCBL, and 1-ml aliquots were placed in Eppendorf tubes. Cumene hydroperoxide (Aldrich) was dissolved in ethanol and added to the tubes at final concentrations of 0.005% and 0.01%. The tubes were incubated for 15 min without rotation. Cultures were diluted, plated, and counted as described above. Relative survival was calculated compared to survival of untreated cells. Ethanol treatment alone had no effect on gonococcal growth (data not shown).


Identification and mutagenesis of the priA gene.

In order to begin investigating the role of priA in N. gonorrhoeae, the nucleotide sequence of the approximately 2,000-bp E. coli K12 priA gene (3) was used in a BLASTN search of the N. gonorrhoeae strain FA1090 genome (43). BLAST analysis using default values yielded several gene products that share sequence homology with E. coli PriA, including N. gonorrhoeae PriA, Mfd, and RecG with e-values of e-123, 2e-11, and 5e-7, respectively. The PriA homologue with the highest e-value and greatest sequence homology is encoded by a 2,187-bp gene predicted to encode a polypeptide of 54% similarity and 38% identity to E. coli PriA (Fig. (Fig.1A).1A). Several conserved motifs have been described for E. coli PriA, all of which are also present in N. gonorrhoeae PriA, including an N-terminal D-loop recognition domain (64), Walker-A, Walker-B, and QxxGRxGR motifs (24, 65, 70), which respectively correspond to motifs I, II, and VI described for the SFII family of DEAD/DEXH RNA helicases (13), as well as a zinc finger domain (71) (Fig. (Fig.1A).1A). Based on the high degree of sequence similarity to E. coli PriA and the presence of conserved motifs, this predicted protein was determined to be the N. gonorrhoeae PriA homologue. The deduced protein is 729 amino acids in length, with a predicted molecular mass of 81,118 Da.

FIG. 1.
The N. gonorrhoeae priA homologue. (A) Amino acid sequence alignment of PriA from E. coli (Ec) and PriA from N. gonorrhoeae (Gc). Identical residues are boxed in black, and similar residues are boxed in gray. Thick gray underlines denote the D-loop recognition ...

Immediately upstream and divergently transcribed from priA is a gene predicted to encode DsbC (35, 56), a thiol:disulfide interchange protein required for disulfide bond formation in some periplasmic proteins (Fig. (Fig.1B).1B). A gene encoding an uncharacterized, Neisseria-specific protein lies downstream, and in the opposite orientation, of priA, and its stop codon is 51 bp from the priA stop codon. N. gonorrhoeae priA was inactivated by inserting a gene encoding erythromycin resistance at codon 187. The gene encoding erythromycin resistance reads in the same direction of priA. The priA mutation is causal to all of the phenotypes observed in this study, since each could be complemented with a functional copy of priA expressed elsewhere on the gonococcal chromosome.

The N. gonorrhoeae priA mutant exhibits a growth defect.

It has been reported that E. coli priA mutants form smaller colonies and exhibit a 10- to 100-fold reduction in viability as measured by colony formation relative to OD (23, 39). Similarly, disruption of the gonococcal priA gene yielded visibly smaller colonies (Fig. (Fig.2A).2A). In contrast, N. gonorrhoeae priA and parental strains grown in liquid to matched ODs gave rise to similar numbers of CFUs (data not shown). Since it is difficult to achieve reproducible or long-term growth curves in liquid medium, whereas gonococci grow consistently and reliably on solid medium (our unpublished observations), the growth defect of the N. gonorrhoeae priA mutant was quantified by collecting colonies over time and measuring CFU arising per colony. Comparing CFU per colony at numerous time points during growth, it was observed that the priA mutant strain yielded approximately 50- to 10,000-fold fewer CFU/colony than the parental strain, depending on the time point selected, and that the growth defect could be fully restored by supplying a functional copy of priA elsewhere on the chromosome (priA/priA+) (Fig. (Fig.2B2B).

FIG. 2.
N. gonorrhoeae priA mutants exhibit a growth defect. The parental strain is strain FA1090 variant 1-81-S2 recA6. All strains were grown in 1 mM IPTG for maximal RecA expression. (A) Colony morphology of parental and priA mutant strains. Representative ...

To test whether the extreme growth defect of the gonococcal priA mutant was due to decreased viability of the cells, the Live/Dead staining assay (Molecular Probes) was performed. Propidium iodide is only taken up by cells with compromised membrane integrity, therefore staining with this dye allows for the measurement of nonviable cells. In cultures grown to mid-log phase, the percentage of nonviable cells was approximately 17% for the parental strain. The number of nonviable cells observed in the priA mutant strain and the priA complement strain were equivalent to the parental strain (Fig. (Fig.2C).2C). Thus, the growth defect of the priA mutant is not caused by increased cell death but results from a reduced growth rate. In addition, no gross differences in cellular morphology between the priA mutant and parental strain were observed by fluorescence microscopy for either the viable or nonviable cells (data not shown).

Gonococcal priA is involved in DNA transformation but not pilin antigenic variation.

Since E. coli priA mutants show reduced frequencies of P1 transduction, the role of N. gonorrhoeae priA in the homologous recombination-mediated processes of DNA transformation and pilin antigenic variation was tested. Pilin antigenic variation is commonly assessed using phenotypic assays. However, it was possible that the growth defect of the priA mutant might bias phenotypic measurements of colony variation. Therefore, a molecular assay was developed to measure pilin variation in which the pilE gene was sequenced to detect changes occurring during antigenic variation. Decreases in antigenic variation can be detected using this assay, since no pilE sequence variants are detected in an antigenic variation-deficient mutant (data not shown). Recombination at pilE was measured after identical times of growth for each strain. In addition, to account for the growth difference between the priA mutant and the parental strain, recombination was also compared at times matched for CFU/colony. Regardless of the time point examined, the priA mutant gave rise to similar frequencies of recombined variant pilE genes as the parental strain (Fig. (Fig.3A).3A). Therefore, loss of priA does not in any way alter the process of pilin antigenic variation.

FIG. 3.
Homologous recombination is differentially affected in a priA mutant. The parental strain is strain FA1090 variant 1-81-S2 recA6. Parental, priA, and priA/priA+ strains were grown in 1 mM IPTG for maximal RecA expression. recA is a parental gene ...

A role for gonococcal priA in the homologous recombination-mediated process of DNA transformation was also tested. Transformation assays were performed by incubating N. gonorrhoeae with transforming DNA containing a gyrB allele which confers nalidixic acid resistance. The transformation efficiency was measured as the frequency with which the gyrB allele recombined into the chromosome and subsequent Nalr colonies arose relative to overall CFU. A 10,000-fold reduction in DNA transformation efficiency of the priA mutant compared to the parental strain was observed. The transformation efficiency was restored to parental levels with the priA complement strain, indicating that the observed transformation defect of the priA mutant was not due to a secondary mutation or to polar effects on other genes. The decreased transformation frequency of the priA mutant was similar to that of a recA mutant (Fig. (Fig.4B)4B) and was near the level at which spontaneous mutations arose (data not shown). From these data we conclude that priA is essential for DNA transformation.

FIG. 4.
The N. gonorrhoeae priA mutant is deficient in DNA repair. Strains are as described in the legend for Fig. Fig.3.3. (A) Relative survival after irradiation with UV light. Error bars represent the standard error of the mean of three experiments ...

priA is involved in DNA repair in N. gonorrhoeae.

In E. coli, priA mutants display increased sensitivities to UV light and double-strand breaks induced by gamma irradiation (23). To test whether N. gonorrhoeae priA plays a role in DNA repair, gonococci were exposed to increasing doses of UV light. The priA mutant was approximately 10-fold more sensitive to UV light than the parental strain at all levels of UV exposure (Fig. (Fig.4A).4A). This repair defect was fully restored in the priA complement strain. We also tested whether priA is involved in the repair of double-strand DNA breaks caused by exposure to nalidixic acid. Nalidixic acid has been shown in E. coli to produce double-strand breaks in DNA by inhibiting the ligation function of gyrase (10, 29). We have found a direct correlation between DNA repair capabilities and survival to nalidixic acid exposure in N. gonorrhoeae and have shown that survival is not due to a change in spontaneous mutation rate giving rise to Nalr (57, 62). Similar to the recA mutant, the gonococcal priA mutant displayed a greater than 10,000-fold reduction in ability to repair double-strand breaks (Fig. (Fig.4B).4B). Survival after nalidixic acid exposure was partially restored in the priA/priA+ complement strain to a level statistically different from the priA mutant. The reason for the partial complementation phenotype observed only in this assay is unclear but suggests that PriA-mediated double-strand break repair is sensitive to slight differences in priA expression levels that are likely to exist between the parent and complement strains. From these studies, we conclude that priA is involved in most DNA repair processes in N. gonorrhoeae.

It is unlikely that N. gonorrhoeae encounters UV light during its life cycle within the human body. In addition, it has been suggested that the low viability of E. coli priA mutants in the absence of exogenous DNA damaging agents is due in part to intracellular oxidative respiration (8). Therefore, the ability of the gonococcal priA mutant to repair oxidative induced DNA damage was analyzed. The priA mutant was nearly 50-fold more sensitive to H2O2 exposure and approximately 10-fold more sensitive to the organic peroxide cumene hydroperoxide (Fig. (Fig.5).5). However, priA expression is not altered more than twofold after exposure to H2O2, showing that its expression is not significantly altered by oxidation (E. A. Stohl and H. S. Seifert, personal communication). Therefore, we conclude that oxidative damage of DNA results in stalled replication forks and that priA and replication restart are important to reinitiate replication in N. gonorrhoeae.

FIG. 5.
The N. gonorrhoeae priA mutant is more sensitive to oxidative damage. (A) Relative survival after exposure to H2O2. Error bars represent the standard error of the mean of three experiments. Statistically significant differences (P < 0.05) as ...


While PriA was originally characterized for its role in ΦX174 phage replication, its role in chromosomal replication restart has been the focus of most recent studies (9, 31, 34). The E. coli replication restart mechanisms require reloading of the DnaB helicase by DnaC, which is targeted to branched structures at the stalled replication fork either by a PriA-dependent pathway that utilizes PriB or PriC and DnaT or by a PriA-independent pathway that uses Rep, PriC, and possibly DnaT (45). N. gonorrhoeae differs from E. coli in that N. gonorrhoeae does not possess homologues of PriC or DnaT (18) and does not have a Rep-dependent replication restart pathway (19), indicating that the overall genetic and biochemical pathway mediating replication restart must be different in the gonococcus than in other bacteria. Furthermore, although PriB and PriC are thought to be redundant in E. coli, the cell requires one of these proteins to be present since an E. coli double mutant is not viable (45, 47). Since gonococci lack priC, one would predict that priB may be essential in N. gonorrhoeae. Indeed, it has been impossible to insertionally inactivate the gonococcal priB gene using the same methods used here to successfully mutate priA (data not shown).

Gonococcal priA mutants produce colonies that are significantly smaller than colonies of the parental strain. The reduced colony size was not surprising, as it has been reported that E. coli and B. subtilis priA mutant strains also form small colonies (23, 39, 41). The N. gonorrhoeae priA mutant strain contains 50- to 10,000-fold fewer CFU/colony compared to the parental strain after identical times of growth. Two hypotheses can explain the priA mutant growth defect: (i) the priA mutant cells are growing at a normal rate but are less viable, or (ii) the priA mutant grows more slowly. The growth defect cannot be explained by increased cell death, since both the mutant and parental strains have a similar percentage of nonviable cells and gave rise to similar CFU when matched for OD. In contrast, E. coli priA mutants were reported to exhibit reduced cell viability. In E. coli, a portion of priA mutant cells are filamentous, likely due to the increase in basal levels of SOS induction (23, 27, 28, 39, 48). It is possible that nonviable filamentous cells contribute to the observed growth defect in E. coli. In contrast, the gonococcal priA mutant does not exhibit a filamentous cellular morphology, consistent with the observation that N. gonorrhoeae does not have an SOS response (2). Therefore, we conclude that the growth defect of the gonococcal priA mutant is due solely to a decrease in growth rate.

The homologous recombination-mediated process of pilin antigenic variation in N. gonorrhoeae requires numerous factors, including the RecFOR-mediated recombination pathway (33, 57), RdgC (32) and branch migration machinery (50), which are also linked to replication restart in E. coli (7, 14, 15, 28, 36, 42, 46). In addition, a link between replication and antigenic variation has been postulated (16). Therefore, we tested whether priA is also associated with these recombination processes in N. gonorrhoeae and whether it plays a role in pilin antigenic variation. Sequence analysis of variant pilin genes revealed no effect of a priA mutation on pilE recombination, and thus we conclude that pilin antigenic variation is a PriA-independent process. These data show replication and antigenic variation are not linked or at least are not linked to replication restart.

An E. coli priA mutant displays a 50-fold decrease in P1 transduction frequencies (27, 48). Similarly, the gonococcal priA mutant demonstrated a complete loss in transformability, showing that priA is critical for DNA transformation. Two hypotheses can explain the role of priA in gonococcal DNA transformation. It is possible that PriA recognizes a branched structure formed between the transforming and resident DNA molecules and provides an activity that contributes directly to the incorporation of the transforming DNA into the chromosome (Fig. (Fig.6A).6A). This action could be mediated directly by the PriA helicase activity or a localized replication program. Alternatively, recombinant structures formed during transformation could cause replication fork collapse (Fig. (Fig.6B).6B). In the absence of priA, chromosomal replication blocked by recombination intermediates would not be restarted, removing potentially transformed progeny from the population. Regardless of how PriA functions in promoting transformation, the priA mutant is one of the few that have been isolated which reduces transformation levels to that of a recA mutant and shows a pivotal role for this gene product in genetic exchange.

FIG. 6.
Models for PriA involvement in DNA transformation in N. gonorrhoeae. In both models, it is likely that single-stranded donor DNA is either imported into the cytoplasm directly, as is the case in the naturally competent bacteria Bacillus subtilis and ...

As observed in E. coli priA mutants, the N. gonorrhoeae priA mutant displayed increased sensitivity to UV irradiation. The sensitivity of the N. gonorrhoeae priA mutant is not as severe as that of a recA mutant strain; however, the approximately 10-fold decrease observed is similar to that seen in E. coli priA mutants (23, 48). It is likely that efficient UV repair systems can usually repair UV-mediated DNA lesions, and it is only a subset of lesions that interferes with replication fork progression requiring PriA activity after fork collapse.

Because N. gonorrhoeae lives exclusively in the human body and a hallmark of infection is neutrophil influx, this facultative aerobic organism is likely to frequently encounter oxidative stress from both internal and external sources. Therefore, a role for N. gonorrhoeae priA in survival to oxidative damage was tested. The gonococcal priA mutant strain is much more sensitive to H2O2 and somewhat more sensitive to cumene hydroperoxide than the parental strain. Although gonococci possess numerous mechanisms to respond to oxidative stress, the decreased survival of a priA mutant to oxidative damaging agents indicates that replication restart serves as an additional defense for countering the effects of toxic oxygen species. These findings provide the first direct evidence for a role of priA and replication restart in resistance to oxidative damage. In addition, of the known oxidative defense mechanisms in N. gonorrhoeae, this is the only one predicted to deal directly with damaged DNA. These data are consistent with the notion that oxygen radicals arising during normal aerobic respiration may interfere with chromosomal replication and provide an explanation for the importance of priA in cells in the absence of exogenous DNA-damaging agents. Additionally, this work has uncovered a new way by which N. gonorrhoeae might survive in the presence of the oxidative burst of phagocytic cells.

It is interesting that the strongest effect of the N. gonorrhoeae priA mutant was seen in DNA transformation, the only gonococcal homologous recombination-mediated process that relies solely on the RecBCD pathway, whereas priA had no effect on antigenic variation which only utilizes the RecF-like pathway. DNA repair in N. gonorrhoeae relies partially on the RecBCD pathway, and priA has a partial effect on this process. Since it is thought that the RecBCD pathway acts at double-strand DNA breaks and the RecF pathway acts at single-strand DNA gaps, it is possible that replication restart functions only at double-strand breaks in N. gonorrhoeae. It is interesting to note that N. gonorrhoeae recB, recC, and recD mutants exhibit similar phenotypes as the gonococcal priA mutant, namely a growth defect and intermediate levels of UV sensitivity and DNA transformation when transforming DNA is not limiting. Overall, the phenotypes associated with a gonococcal priA mutant are consistent with the biological functions of a PriA-dependent replication restart pathway demonstrated in E. coli. In contrast, we have shown that the alternative replication restart pathway that is dependent on Rep and PriC does not function in N. gonorrhoeae (18, 19). The differences in the viability phenotype and in the repertoire of factors that mediate replication restart in N. gonorrhoeae indicate that evolutionary pressures on the human-specific pathogen N. gonorrhoeae differ significantly from other microbes which occupy multiple environmental niches, and this pressure modifies the basic process of replication in this organism.


We are grateful to Alison Criss and Elizabeth Stohl for critical reading of the manuscript.

This work was supported by NIH grants R37 AI44239 and R01 AI33493. K.A.K. was partially supported by Public Health Service training grant T32 GM08061.


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