Roles of the recJ and recN Genes in Homologous Recombination and DNA Repair Pathways of Neisseria gonorrhoeae

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 co-carboxylase, 1.23 mM Fe(NO₃)₃; all from Sigma] (24) at 37°C in 5% CO₂. Gonococcal strains used in this study were derivatives of strain FA1090 variant 1-81-S2 with the 1-81-S2 pilE sequences (1-81-S2) (46), with the exception of strain MS11ΔrecJ::kan, which was used to obtain donor DNA for creation of the recJ mutant strains. All mutations were placed in both a wild-type recA background, and a recA6 background. 1-81-S2 recA6 strains were created by transformation of 1-81-S2 with plasmid DNA carrying an isopropyl-β-d-thiogalactopyranoside (IPTG)-regulatable gonococcal recA allele, recA6, which allows control of recA and subsequent control of recA-dependent recombination and repair processes including pilin antigenic variation (45). An IPTG concentration of 1 mM in the media allows for maximal induction of recA transcription and restoration of transformation competence to near-wild-type levels.

Antibiotics were added at the following concentration for E. coli: chloramphenicol, 30 mg/liter; erythromycin, 250 mg/liter. For N. gonorrhoeae the antibiotic concentrations were as follows: erythromycin, 1 mg/liter, kanamycin, 30 mg/liter; spectinomycin, 50 mg/liter; chloramphenicol, 2 mg/liter. IPTG (Diagnostic Chemicals Ltd.) was supplied at 1 mM (for gonococci) in solid agar or liquid medium.

DNA manipulations and analysis.

Standard procedures were performed as described by Sambrook et al. (40). Plasmid DNA was isolated from strains of E. coli by using plasmid kits (Qiagen Inc.). Enzymes were used as specified by the manufacturers (Promega Corp. and New England Biolabs). E. coli strains were transformed by electroporation using the Gene Pulser II electroporation system (Bio-Rad Laboratories) as specified by the manufacturer. For Southern blot analysis, DNA was transferred to Magnagraph nylon membrane as specified by the manufacturer (Micro Separations Inc.). Sequencing reactions were performed using the Big-Dye Terminator cycle-sequencing kit (Perkin-Elmer Corp.), and sequencing products were separated on an ABI model 377 automated DNA sequencer. DNA sequence analysis was performed using Lasergene software (DNASTAR, Inc.) and VectorNTI software (Informax, Inc.). PCR fragments were purified using the Qiaquick PCR purification kit (Qiagen). The BLAST program (1) was used to search the National Center for Biotechnology Information nonredundant database. The N. gonorrhoeae strain FA1090 Gonococcal Genome Sequencing Project (accession no. AE004969 http://www.genome.ou.edu/gono.html) was accessed to identify the RecN homologue. pilE sequences were determined by amplifying pilE from the chromosome with primers PILRBS (5"-GGCTTTCCCCTTTCAATTAGGAG-3") and SP3A (5"-CCGGAACGGACGACCCCG-3") (46) using Taq polymerase and sequencing the resulting PCR product with CONST-F2 (5"-TACCAAGACTACACCGCCCG-3") (46).

Plasmid and mutation construction.

Plasmid pBluntRecN was constructed by cloning a DNA fragment containing the predicted recN gene PCR amplified from N. gonorrhoeae strain FA1090 genomic DNA with primers RecN2-IN (5"-CGATTGGTCTGCTGTTGGGC-3") and RecN2-Rev (5"-GCATATGCTCGGTCGTGTCG-3") into the pBlunt plasmid (Invitrogen). This plasmid was cut with BsgI, the staggered ends were filled in with T4 polymerase, and the ermC gene was inserted internal to the recN gene to create pBluntRecNErmC. In addition, pBluntRecNCat2-9 was created by insertion of the cat2-9 gene at the same site internal to recN. Plasmids pGCC6RecN and pGCC6RecJ were constructed by PCR amplification using Pfu polymerase (Stragagene) of recJ with primers GCRecJForward (5"-TCCTCAGACAAATGAACGG-3") and GCRecJReverse (5"-TATTGCGTCCCTAAGAAGG-3") or recN with primers GCRecNForward (5"-GTTGATATTGCTTTTGTCGG-3") and GCRecNReverse (5"-ATCCAACTGA GCTACGG-3"), with subsequent cloning into the PmeI site of plasmid pGCC6 (I. J. Mehr, J. Edwards, M. Apicella, and H. S. Seifert, unpublished data).

Gonococcal mutant strains were created by spot transformation of N. gonorrhoeae strain FA1090 variant 1-81-S2 with donor DNA. Briefly, gonococci were streaked for confluency on GCB plates. Liquid spots containing excess donor DNA, 5 mM MgSO₄, and Kellogg's supplements were spotted onto the plates and allowed to dry. After 18 h at 37°C under 5% CO₂, the spots were collected with Dacron swabs (Puritan) and plated on the appropriate antibiotic. Successful transformation of the mutation from N. gonorrhoeae strain MS11ΔrecJ::kan into N. gonorrhoeae strain FA1090 or FA1090 recA6 created both strains FA1090ΔrecJ::kan, and FA1090 recA6ΔrecJ::kan. Plasmid pBluntRecNErmC was transformed into N. gonorrhoeae strain FA1090 and FA1090 recA6, and strains FA1090 recN::ermC and FA1090 recA6recN::ermC were created through allelic replacement of the recN gene. All mutant strains were confirmed by PCR and Southern blotting.

To create strains to complement the recJ and recN mutations, we took advantage of the Neisserial Insertional Complementation System (NICS) (Mehr et al., unpublished). This system allows for second-locus complementation of the interrupted genes and allows us to circumvent the fact that the gonococcus does not readily support plasmids. Briefly, both recJ and recN were PCR amplified from the gonococcal chromosome, cloned into the vector pGCC6, and sequenced for accuracy. This vector contains a region of the gonococcal chromosome that is not involved in antigenic variation at pilE, flanking a multicloning site. The recJ or recN genes are cloned into the vector under the control of lac operator sequences, allowing for IPTG-regulatable control of the recJ or recN genes. Successful cloning of recJ and recN into pGCC6 and subsequent transformation into the N. gonorrhoeae mutant strains with consequent allelic replacement created strains FA1090 recN::ermCrecN⁺, FA1090 recA6recN::ermCrecN⁺, FA1090ΔrecJrecJ⁺, and FA1090 recA6ΔrecJrecJ⁺. Construction of these strains was confirmed by both PCR and Southern blotting.

Antigenic variation assays. (i) Pilus-dependent colony variation frequency assay.

The variation assay was performed as described previously (61). N. gonorrhoeae was passaged twice from frozen stocks onto GCB medium to obtain isolated piliated gonococci. After 24 h, gonococci were collected with a Dacron swab and suspended in 1 ml of GCBL (1.5% Proteose Peptone 3, 0.4% K₂HPO₄, 0.1% KH₂PO₄, 0.1% NaCl with Kellogg’s supplements, 0.042% NaHCO₃). The liquid suspension was passed through a 1.2-μm-pore-size filter to remove clumps and isolate individual monococci and diplococci. This ensured that each colony that was scored for phenotype arose from an individual unit. Dilutions were plated onto GCB medium in triplicate, and incubated at 37°C at 5% CO₂. The colony morphology was scored 19 hrs after plating. The frequency of phase variation from the pilus-plus to pilus-minus colony phenotype was determined by dividing the number of colonies with the nonpiliated phenotype (as measured in a stereomicroscope) by the total number of colonies. These colony morphology changes are pilus dependent and reflect a recombination event at the pilin expression locus, pilE (51-53, 55). Therefore, the pilus-dependent colony variation frequency assay is a surrogate measure of pilin antigenic variation. The frequency per cell per generation was determined by dividing the above-determined frequency of phase variation by the calculated number of generations for which the colonies had grown.

(ii) Kinetic pilus-dependent colony variation assay.

The kinetic colony phase variation assay was performed as follows. N. gonorrhoeae strains were struck for isolated colonies on GCB plates containing IPTG and incubated at 37°C under 5% CO₂. Isolated colonies were screened at 18, 20, 22, 24, and 26 h for the presence of sectors of variation indicative of pilus variation. Scores ranging from 0 to 4 were assigned to isolated colonies based on the number of sectors of variation present per colony (E. V. Sechman and H. S. Seifert, unpublished data). Colonies that contained more than four sectors of variation were given a score of 4. Analysis was performed on at least 20 colonies per screen per experiment, and the assay was performed at least three times per strain.

DNA repair assays. (i) UV sensitivity assay.

The UV sensitivity of gonococcal strains was assayed as previously described (26). Gonococci were incubated for 24 h on GCB plates (in the presence of IPTG for recA6 and NICS derivatives), collected with a Dacron swab, and suspended in GCBL. Serial dilutions were made and exposed to UV radiation at 0, 2, 4, 6, or 8 J/m² in a Stratagene UV Stratalinker 1800. Relative resistance was expressed as percent survival at 2, 4, 6, and 8 J/m² compared to the number of CFU at 0 J/m².

(ii) Nalidixic acid survival assay.

Nalidixic acid causes DNA double-strand breaks by partially inhibiting DNA gyrase (10). Sensitivity to the toxic effects of nalidixic acid 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 100 μl of the undiluted culture and of 10⁻¹ and 10⁻² dilutions were plated onto GCB-IPTG agar containing nalidixic acid. Total CFU per milliliter were calculated by plating spot dilutions of the cultures onto GCB agar. Colonies were counted after 36 h of growth. Sensitivity to nalidixic acid was expressed as Nal CFU divided by total CFU for each strain.

DNA transformation assays.

DNA transformation efficiency was determined in liquid media in 12-well tissue culture plates. Gonococcal strains were grown on GCB for 18 h, collected with a Dacron swab, and suspended to a density of approximately 10⁸ CFU/ml in 1 ml of GCBL with 1 mM IPTG to induce the expression of genes under the control of lac operator sequences. Then 20 μl of cells was added to 200 μl of GCBL containing 5 mM MgSO₄, Kellogg supplements, 500 ng to 1 μg of chromosomal DNA encoding Spc, and 1 mM IPTG. After 10 min at 37°C, the transformation mix was diluted into 2 ml of the amended GCBL described above and incubated at 37°C in 5% CO₂ for 5 h without agitation. The transformation mix was serially diluted from 10⁻¹ to 10⁻⁶ and spot plated on GCB and GCB plus spectinomycin to determine total CFU and to detect transformants, respectively. Transformation efficiency was expressed as the mean number of Spc^r transformants per CFU.

Identification and cloning of the recN homologue.

Several N. gonorrhoeae genes have been identified that are involved in homologous recombination. The components of the gonococcal homologous recombination pathways are critical to DNA repair, the natural transformation of DNA, and pilin antigenic variation. To augment our investigations into the various recombination pathways of the gonococcus, we searched for a homologue of an additional RecF-like pathway gene, recN, in the gonococcus. By using the nucleotide sequence of the E. coli recN gene (39) in a BLASTN search of the N. gonorrhoeae genome sequence (http://www.genome.ou.edu/gono.html), a similar sequence of 1,674 nucleotides was identified. This fragment encoded a putative peptide that was 37% identical and 54% similar to E. coli RecN (Fig. 1). The majority of the identity and similarity between the two proteins resides in the N- and C-terminal portions of the protein, and there is more divergence throughout the middle of the open reading frame. The PROSITE (2) and BLOCKS (17, 18) programs were used to identify potential functional motifs in the amino acid sequence. This analysis revealed the presence of an N-terminal SMC domain, typically involved in chromosome scaffolding and partitioning within the conserved N-terminal portion of the RecN sequences. In addition, internal to the SMC domain, an ATP/GTP-binding-site motif that is conserved in many repair proteins (49) was identified in the N-terminal region of the amino acid sequence (Fig. 1). Based on the high degree of similarity to E. coli RecN, we determined this putative protein to be the N. gonorrhoeae RecN. The deduced protein is 557 amino acids in length, with a predicted molecular mass of 61,131 Das, and a pI of 5.185.

A conserved hypothetical protein of unknown function is predicted to be encoded 187 bp upstream of the recN ATG. Two putative tRNA genes arranged in tandem are predicted 114 bp downstream of the potential stop codon of recN (Fig. 1B). Alignment of the N. gonorrhoeae RecN with 27 homologues from different gram-negative and gram-positive bacterial species revealed conservation over the entire reading frame, with some notable exceptions being a larger RecN protein in Deinococcus radiodurans and a smaller RecN protein in Bacillus stearothermophilus (data not shown).

Mutations in recJ, but not recN, affect pilus antigenic variation.

The RecO and RecQ proteins of N. gonorrhoeae are required for antigenic variation and DNA repair in the gonococcal strain FA1090 (34). Based on these observations, we hypothesized that other homologues of E. coli RecF pathway proteins such as RecJ and RecN might also be involved in these processes. We tested whether the RecF-like pathway genes recJ and recN are involved in pilin antigenic variation in N. gonorrhoeae by using assays that measure pilus-dependent colony morphology changes that are dependent on the recombination reactions that are the basis of antigenic variation (see Materials and Methods). Comparisons of pilin antigenic variation were performed using strains FA1090, FA1090ΔrecJ::kan, FA1090ΔrecJ::kanrecJ⁺, FA1090 recN::ermC, FA1090 recN::ermCrecN⁺, and FA1090 recA9, which contains a recA gene disrupted by ermC to create a knockout mutation (45). In addition, all the above strains except FA1090 recA9 were analyzed in a recA6 background, in which the expression of recA is under the control of lac operator sequences, allowing for the regulatable expression of recA with IPTG. In contrast to previous findings (20), comparisons of pilin antigenic variation using a colony morphology-based variation assay revealed that inactivation of recJ leads to an approximately fivefold reduction in pilus-dependent colony variation. This level of variation was greater than that of the recA mutant strain FA1090 recA9 (Fig. 2A). This result was in contrast to that for the gonococcal strains containing the recN mutation, which showed wild-type levels of pilus-dependent colony variation (Fig. 2A). To confirm this observation, a kinetic variation assay was used that quantifies the number of phase variation events within a colony over time (Sechman and Seifert unpublished). The kinetic assay also revealed that the mutation of recJ but not that of recN resulted in a decrease in the number of sectors that underwent pilus-dependent variation over time. Again, the amount of observed variation in the recJ mutant strain remained above that of the recA mutant strain FA1090 recA9. This result was observed in both a wild-type recA background (Fig. 2B) and a recA6 background (data not shown). Inactivation of either recJ or recN did not impact the growth rate of N. gonorrhoeae as measured by the ability of FA1090ΔrecJ::kan, and FA1090 recN::ermC to form colonies on semisolid media at a size and rate consistent with those of FA1090 (data not shown), eliminating the possibility that the differences in colony variation frequency were due to differences in the growth rates of the mutant strains.

To ensure that the observed differences in the frequency and rate of colony variation were not due to polar effects on downstream genes, we inserted functional copies of either recJ or recN under the control of lac operator sequences at an irrelevant intergenic locus in the N. gonorrhoeae chromosome. Strain FA1090ΔrecJ::kanrecJ⁺ showed levels of colony phase variation that were statistically increased over those in the recJ mutant in both wild-type recA and recA6 backgrounds (Fig. 2A). In addition, providing a functional copy of recJ in this alternate locus resulted in complementation of the kinetic variation score back to wild-type levels (Fig. 2B). The levels of variation between FA1090 recN::ermC and FA1090 recN::ermCrecN⁺ were statistically indistinguishable from one another. Thus, recJ but not recN of N. gonorrhoeae is involved in pilus-dependent colony variation. Surprisingly, Hill has recently identified the recJ gene of N. gonorrhoeae and reported that a knockout mutation inhibits DNA repair capabilities but not antigenic variation at pilE in the gonococcal strain MS11 (20). To eliminate the possibility that this reported lack of effect is due to strain differences, we performed the above kinetic colony variation assay on the original MS11ΔrecJ::kan strain used in the previous study and observed a similar decrease in pilus-dependent colony variation (data not shown). Therefore, we conclude that recJ is involved in pilin antigenic variation of the gonococcus.

Mutation of either the recJ or recN gene disrupts DNA repair.

To assess the involvement of both RecJ and RecN in the repair of DNA damage, we first measured the sensitivity of the mutant strains to DNA damage caused by UV irradiation. In wild-type recA and recA6 backgrounds, both FA1090ΔrecJ::kan and FA1090 recN::ermC demonstrated a substantially decreased survival in response to to UV irradiation compared with that of FA1090. This decreased survival after UV irradiation approached that of the recA mutant strain FA1090 recA9 (Fig. 3A). Next, we sought to determine the level of involvement of RecJ and RecN in the repair of double-strand DNA breaks caused by exposure to nalidixic acid. Nalidixic acid introduces double-strand breaks into DNA by inhibiting gyrase (10). We have shown that cells able to survive in the presence of nalidixic acid are not simply revertants (data not shown). Therefore, this is a measure of DNA repair and not of mutation rate. As in the UV resistance assay, inactivation of either recJ or recN resulted in a statistically significant decrease in survival after exposure to nalidixic acid. This decreased survival is approximately 1 and 3 log units lower than the wild-type survival for FA1090ΔrecJ::kan and FA1090 recN::ermC, respectively (Fig. 3B). Unlike the UV resistance assay, the nalidixic acid survival rate of all the recJ and recN mutant strains was well above the nalidixic acid survival rate of FA1090 recA9. The strains containing intact recJ or recN genes provided in trans, FA1090ΔrecJ::kanrecJ⁺ and FA1090 recN::ermCrecN⁺, displayed increases in resistance to UV irradiation and nalidixic acid survival that were statistically increased over those of the corresponding strains containing the inactivated alleles (Fig. 3B). Similar results were obtained when these mutants were compared in a recA6 background (data not shown).

Mutations in recN, but not recJ, disrupt natural DNA transformation.

The RecBCD enzyme complex, but not RecO and RecQ, is involved in the transformation of DNA in N. gonorrhoeae. This observation implies that the RecF-like pathway does not participate in DNA transformation. To test this hypothesis, we evaluated the roles of recJ and recN in the DNA transformation efficiency of the gonococcus. While strains lacking a functional recJ gene maintained near-wild-type levels of DNA transformation efficiency, strains mutated in recN showed an approximately 1-log-unit decrease in DNA transformation efficiency. The recN mutant strains possessed transformation efficiencies considerably higher than those of the recA mutant strain recA9. Strain FA1090 recN::ermCrecN⁺ showed wild-type levels of DNA transformation efficiency, indicating that the effect observed in the recN mutant strain is not due to polar effects on downstream genes (Fig. 4A). The partial recN transformation phenotype was also observed in a recA6 background (data not shown).

The observation that recN and recBCD are involved in DNA transformation in the gonococcus led us to investigate whether RecN and RecBCD act in concert in DNA transformation. To determine if recN and the recBCD genes act in the same recombination pathway, double-mutant strains FA1090 recN::cat2-9recB::ermC and FA1090 recN::cat2-9recD::ermC were created. Strains mutated in both recN and either the recB or recD component of the RecBCD enzyme consistently showed a more pronounced decrease in DNA transformation efficiency than did strains mutated in recN or the corresponding member of the RecBCD enzyme complex alone. This decrease did not reach statistical significance, and therefore it is impossible to draw definite conclusions. One reason for this situation is the severe growth defects observed in strains inactivated for either recB or recD (data not shown). However, the reduction in frequencies in both double mutants indicates that RecN and RecBCD could act in separate pathways during DNA transformation (Fig. 4B).