Roles of the recJ and recN Genes in Homologous Recombination and ...

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and DNA Repair Pathways of Neisseria gonorrhoeae ... RecN protein is also required for RecBCD-dependent repair ..... Surprisingly, Hill has recently identified ...
JOURNAL OF BACTERIOLOGY, Feb. 2002, p. 919–927 0021-9193/02/$04.00⫹0 DOI: 10.1128/JB.184.4.919–927.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 184, No. 4

Roles of the recJ and recN Genes in Homologous Recombination and DNA Repair Pathways of Neisseria gonorrhoeae Eric P. Skaar, Matthew P. Lazio, and H. Steven Seifert* Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611 Received 4 October 2001/Accepted 14 November 2001

The paradigm of homologous recombination comes from Escherichia coli, where the genes involved have been segregated into pathways. In the human pathogen Neisseria gonorrhoeae (the gonococcus), the pathways of homologous recombination are being delineated. To investigate the roles of the gonococcal recN and recJ genes in the recombination-based processes of the gonococcus, these genes were inactivated in the N. gonorrhoeae strain FA1090. We report that both recN and recJ loss-of-function mutants show decreased DNA repair ability. In addition, the recJ mutant was decreased in pilus-dependent colony morphology variation frequency but not DNA transformation efficiency, while the recN mutant was decreased in DNA transformation efficiency but not pilus-dependent variation frequency. We were able to complement all of these deficiencies by supplying an ectopic functional copy of either recJ or recN at an irrelevant locus. These results describe the role of recJ and recN in the recombination-dependent processes of the gonococcus and further define the pathways of homologous recombination in this organism. The obligate human pathogen Neisseria gonorrhoeae uses homologous recombination to mediate a number of distinct cellular processes. In addition to a primary involvement in DNA replication and repair mechanisms, homologous recombination is required for natural DNA transformation and pilus antigenic variation (15, 16, 44, 54). As is the case in many bacterial cellular processes, much of what is understood about homologous recombination in bacteria comes from observations made in Escherichia coli. Investigations of the genes involved in the various recombination reactions of N. gonorrhoeae have revealed pathways that are similar to the homologous recombination pathways originally delineated in E. coli, with some differences. RecA-mediated homologous recombination in E. coli has traditionally been divided into two primary pathways, the RecBCD pathway and the RecF pathway (7, 13). Both pathways require RecA for homologous pairing and strand exchange (12, 19). In addition to RecA, a number of other proteins are involved in homologous recombination in E. coli. Subsequent division of these proteins into groups has been carried out based on the behavior of mutant strains in various genetic backgrounds. The major recombination pathway in E. coli is the RecBCD pathway, which is involved in conjugal and transductional recombination, DNA repair, and degradation of foreign DNA (14, 27, 57). The primary components of this pathway, in addition to RecA, are the products of the recB, recC, and recD genes. The alternative RecF pathway of recombination was identified by defining recombination events that occurred in the presence of the sbc suppressor mutations. These mutations suppress phenotypic effects caused by mutations in components of the RecBCD enzyme (21, 27). Subsequent research with E. coli has revealed that the RecF pathway

of homologous recombination confers UV resistance (21, 25, 33) and plasmid recombination (8, 25, 33) in a wild-type background and conjugal recombination and DNA repair in a recBC sbcBC background (27, 28). Proteins involved in the RecF pathway of E. coli include RecF, RecO, RecR, RecQ, and RecJ (31, 32, 58, 59). The RecN protein has been assigned to the RecF pathway of E. coli based on a 20- to 30-fold induction of recN in recBC sbcBC strains (38). However, the RecN protein is also required for RecBCD-dependent repair of double-strand breaks which arise as a result of DNA damage (37, 41, 43, 62). Additionally, in contrast to the other genes of the RecF pathway, mutations in recN do not restore resistance of E. coli to the absence of thymine known as “thymineless death” (36). Therefore, RecN does not fall cleanly into either pathway of recombination; instead, it may participate in both. Investigations into the recombination pathways in N. gonorrhoeae have revealed that the recombination pathways of E. coli are not completely generalizable to the gonococcus. This is not surprising based on the unique nature of gonococcal recombination-based processes such as the natural transformation of DNA and antigenic variation of the bacterial pilus. The gonococcal pilus is a hairlike appendage that extends from the cell surface. The major subunit of the pilus, pilin, is encoded by the pilE gene and is capable of undergoing high-frequency recombination-dependent antigenic variation (15, 16, 44, 56). There have been conflicting reports describing the involvement of the RecBCD enzyme in pilus antigenic variation (5, 34). Chaussee et al. (5) reported that a recD mutant showed increased pilus-dependent colony morphology changes, while Mehr and Seifert (34) reported that the gonococcal RecBCD enzyme is not involved in pilus-dependent colony morphology changes. Comparison of the RecF homologous recombination pathways of these bacteria is more complicated. Several potential homologues of E. coli proteins specific to the RecF pathway are encoded in the N. gonorrhoeae genome, including RecJ, RecN, RecO, RecQ, and RecR. However, the genome of N.

* Corresponding author. Mailing address: Department of Microbiology-Immunology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611. Phone: (312) 503-9788. Fax: (312) 503-1339. E-mail: [email protected]. 919

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gonorrhoeae strain FA1090 does not possess a potential RecF homologue, which typically defines this pathway. In addition, the gonococcal genes encoding the RecF pathway homologues studied to date, recO and recQ, are required for pilin antigenic variation, a process unique to the gonococcus. These distinctions between the RecF pathway processes and components of E. coli and N. gonorrhoeae have led to the designation of a RecF-like pathway in N. gonorrhoeae (34). In E. coli, many effects of mutations in the RecF pathway are observed only in a recBC mutant background containing sbcBC suppressor mutations (27, 28). Analysis of the partially complete Gonococcal Genome Sequencing Project (http://www.genome.ou.edu/gono .html) did not reveal the presence of a potential sbcB homologue, and a gene with only weak similarity to sbcC was identified. This led to the conclusion that the RecF-like pathway is involved in recombination-dependent processes of the gonococcus in a wild-type background (34). The phenotypes of these recO and recQ mutant strains suggest that other RecFlike pathway genes, such as recJ and recN, may play a role in pilin antigenic variation and DNA repair. In addition, a reported lack of involvement of recO and recQ in DNA transformation suggests a lack of involvement of the remaining RecF pathway homologues in this recombination-dependent process (34). Previously, a gonococcal recJ null mutant was reported to show sensitivity to both low-level UV irradiation and DNAalkylating reagents. In addition, it was reported that the recJ mutant strain did not show impairment in DNA transformation or pilin antigenic variation (20). Due to the reported lack of involvement of one member of the RecF-like pathway, recJ, in recombination efficiency at pilE, the author concluded that the RecF-like pathway was not likely to be involved in pilin variation in N. gonorrhoeae. This is in contrast to the previous observations that both RecF-like pathway proteins RecO and RecQ are necessary for wild-type levels of pilin antigenic variation in N. gonorrhoeae (34). The incongruence surrounding the role of RecJ in gonococcal pilin antigenic variation and an absence of information about the role of RecN in the biology of the gonococcus caused us to evaluate the role of these proteins in various recombination-dependent processes of the gonococcus. In this work, we report that in N. gonorrhoeae strain FA1090, both recJ and recN are involved in the repair of DNA damage. In contrast to previously reported findings, we demonstrate an involvement for recJ in pilin antigenic variation. In addition, we demonstrate that the recN gene product of the gonococcus is needed for efficient DNA transformation. These findings allow us to draw new conclusions about the role of RecJ and RecN in the biology of N. gonorrhoeae, as well as to further delineate the recombination pathways of this highly recombinogenic organism. MATERIALS AND METHODS 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 MS11⌬recJ::kan, which was

J. BACTERIOL. 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-␤-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⬘-CCGGA ACGGACGACCCCG-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⬘-CGA TTGGTCTGCTGTTGGGC-3⬘) and RecN2-Rev (5⬘-GCATATGCTCGGTCG TGTCG-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⬘-ATCCAAC TGA 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 MS11⌬recJ::kan into N. gonorrhoeae strain FA1090 or FA1090 recA6 created both strains FA1090⌬recJ::kan, and FA1090 recA6⌬recJ::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 chromo-

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some 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⫹, FA1090⌬recJrecJ⫹, and FA1090 recA6⌬recJrecJ⫹. 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-␮m-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 ␮l 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 ␮l of cells was added to 200 ␮l of GCBL containing 5 mM MgSO4, Kellogg supplements, 500 ng to 1 ␮g 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 Spcr transformants per CFU.

RESULTS Identification and cloning of the recN homologue. Several N. gonorrhoeae genes have been identified that are involved in

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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, FA1090⌬recJ:: kan, FA1090⌬recJ::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

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FIG. 1. The N. gonorrhoeae recN homologue. (A) Amino acid sequence alignment of RecN from E. coli and RecN from N. gonorrhoeae. Identical residues are boxed in black, and similar residues are boxed in gray. The N-terminal SMC domain encompassing the internal ATP/GTP binding site is shown as a dotted white bar. (B) Chromosomal map of the region containing the recN gene. Arrows represent the predicted direction of transcription. The gray open reading frame has homology to Chp from N. meningitidis (accession number NC_003112) the dotted gray open reading frames signify predicted tRNA genes. The recN gene is represented by the black arrow, and the N-terminal SMC domain encompassing the internal ATP/GTP binding site is shown as the dotted white bar.

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 FA1090⌬recJ::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 FA1090⌬recJ::kanrecJ⫹ showed levels of colony phase variation that were statistically increased over those in the recJ

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in the previous study and observed a similar decrease in pilusdependent 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 FA1090⌬recJ:: 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

FIG. 2. Mutations in recJ, but not recN, disrupt pilus antigenic variation. WT is wild-type strain FA1090 variant 1-81-S2 (46), and recA⫺ is FA1090 1-81-S2 recA9 with recA disrupted by an ermC knockout mutation (45). (A) Percent colony phase variation of N. gonorrhoeae. Error bars represent the standard error of the mean of at least three experiments. Statistically significant differences (P ⬍ 0.05) as determined by Student’s t test are indicated by asterisks for comparison to the wild type and by a pound sign for comparison to corresponding uncomplemented strains. (B) Kinetic variation assay of N. gonorrhoeae, measuring the number of sectors of a colony that undergo pilin-dependent colony variation over time. Error bars represent the standard error of the mean of at least four experiments analyzing 20 individual colonies per experiment.

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 pilusdependent 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 MS11⌬recJ::kan strain used

FIG. 3. Mutations in recJ and recN affect DNA repair capability. WT and recA are as described in the legend to fig. 2 (45, 46). (A) Relative survival after irradiation with UV light. Error bars represent the standard error of the mean of three experiments. (B) Relative survival on nalidixic acid. Error bars represent the standard error of the mean of three experiments done in duplicate. Statistically significant differences (P ⬍ 0.05) as determined by Student’s t test are indicated by asterisks for comparison to the wild type and by pound signs for comparison to strains carrying a mutation in either recJ or recN.

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(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 FA1090⌬recJ::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, FA1090⌬recJ::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). DISCUSSION The recN gene from N. gonorrhoeae strain FA1090 was identified, cloned, and insertionally inactivated. Phenotypic assays show that recN affects survival in response to DNA damage

FIG. 4. Mutations in recN decrease DNA transformation efficiency. WT and recA are as described in the legend to fig. 2 (45, 46). Both panels show transformation to spectinomycin resistance with genomic DNA containing a spectinomycin resistance marker. Error bars represent the standard error of the mean of five experiments done in triplicate for the wild-type strains, FA1090 recN::ermC, and FA1090 recN::ermCrecN and at least three experiments done in triplicate for the remaining strains. Statistically significant differences (P ⬍ 0.05) as determined by Student’s t test are indicated by asterisks for comparison to the wild type and by a pound sign for comparison to a strain carrying a mutation in recN. Formation of spontaneous Spc occurred at a frequency of ⬍10⫺8 as measured with a no-DNA control (data not shown).

and DNA transformation efficiency, but not pilin antigenic variation. This is in contrast to the recJ gene of N. gonorrhoeae strain FA1090, which we have shown to be involved in pilus antigenic variation and survival in response to DNA damage, but not in DNA transformation. This is the first report of a role for recN in any recombination processes of the gonococcus, and this work further delineates the recombination pathways of the gonococcus. Previous reports using the recJ mutation in strain MS11

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demonstrated an involvement in DNA repair but not in transformation or pilus-dependent colony variation (20). This conflicts with our observation that in strain FA1090, recJ is involved in pilus antigenic variation. This incongruence is not explained by strain differences, since we found that recJ mutations in strains MS11 (data not shown) and FA1090 were both decreased in pilus-based colony variation. The effect of a recJ mutation on pilus-dependent colony variation reported here may be the result of assay differences. In addition, the mutations in this work were tested in the recA6 genetic background, eliminating intrinsic differences in pilus-based colony morphology caused by the presence of different pilE sequences. Therefore, we are confident that the gonococcal RecF-like pathway is required for efficient pilin antigenic variation, as measured by pilus-dependent colony morphology changes. The recJ gene of E. coli encodes a single-stranded-DNA (ssDNA)-specific exonuclease that catalyzes 5⬘-to-3⬘ hydrolysis of ssDNA (4, 31). In E. coli, this protein plays a role in DNA repair, recombination, and prevention of mutation (9, 30, 31, 60). An involvement of the gonococcal recJ in DNA repair and pilin antigenic variation demonstrated a similar role for this protein in N. gonorrhoeae. This is not surprising based on the high degree of sequence identity between the gonococcal and E. coli RecJ proteins. In addition, all the motifs shown to be essential for RecJ exonuclease activity in E. coli are conserved in RecJ of N. gonorrhoeae (reference 50 and data not shown). Therefore, it is likely that this activity of RecJ is important for antigenic variation at pilE, probably by promoting the formation of ssDNA intermediates for recombination. The formation of ssDNA intermediates is postulated to play a role in current models describing antigenic variation at pilE (22). The biochemical function of the RecN protein is unknown. In E. coli, both conjugational and transductional recombination are severely reduced by a recN mutation in recBC sbcBC mutant backgrounds (29, 42), and recN is induced 20- to 30fold in this same mutant background (38). RecN plays a role in RecBCD-dependent repair of double-strand breaks caused by DNA damage (35, 38, 42, 63), as well as intramolecular recombination of DNA by the RecF pathway (23). The recN promoter in E. coli contains two SOS boxes (39), and defects in recN may result in an increase in the active form of RecA protein, RecAⴱ (6, 47). In addition, it has been postulated that recN strains may be constitutively active for SOS mutagenesis (11). It is difficult to determine the physiological significance in the gonococcus of recN involvement in the E. coli SOS response, due to an absence of a traditional SOS system in N. gonorrhoeae (3). We have found that the N-terminal region of most of the bacterial RecN proteins sequenced to date contains an ATP/ GTP binding domain within an SMC-like motif. SMC-like domains are involved in chromosomal scaffolding and segregation (49). It is possible that the function of RecN in homologous recombination is either structural or enzymatic or both. RecN may be involved in the proper positioning of the recombining segments of DNA, ensuring normal recombination. The observation that inactivation of this gene leads to a decreased transformation efficiency, as well as increased sensitivity to DNA-damaging agents, may be due to some defect in chromosomal partitioning or positioning during these recombination-dependent processes.

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TABLE 1. Involvement of gonococcal rec genes in recombination-dependent processes Involvement ina:

recA genes

Antigenic variation

DNA repair

DNA transformation

recA recX recBCD recN recO recQ recJ

⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺

a ⫹, gene shown to be involved in the recombination-dependent process; ⫺, gene shown not to be involved in the recombination-dependent process.

The data presented here enable us to compare homologous recombination in N. gonorrhoeae to what is known to occur in E. coli. Table 1 shows what is currently known about the involvement of the various rec genes in the recombination-based processes of the gonococcus. Similar to E. coli, recA is involved in all homologous recombination processes of the gonococcus. The gonococcal recX gene is also involved in all of these processes, possibly through some level of regulation of RecA activity (48). recO, recQ, and now recJ can be placed into the same pathway previously described in N. gonorrhoeae by Mehr and Seifert as the RecF-like pathway (34). This grouping is based on the involvement of these proteins in antigenic variation and DNA repair but not in DNA transformation. Finally, although RecBCD and RecN are both involved in DNA repair and the natural transformation of DNA, it is difficult to conclude if they are in the same recombination pathway due to difficulty in interpreting the results of the DNA transformation efficiency assay; this difficulty is caused by the growth defects of strains mutated in components of the RecBCD enzyme. Phenotypes of the recN mutant reveal that in the gonococcus, RecN cannot yet be segregated into either the RecBCD or the RecF-like pathways, underscoring the complex nature of the pathways of homologous recombination in this organism. With the Gonococcal Genome Sequencing Project 100% complete, we are able to confirm previous observations that N. gonorrhoeae does not contain sbcB or sbcC genes. This observation, in concert with the decreased DNA repair efficiency and pilin antigenic variation frequency of RecF-like pathway mutations, allows us to conclude that, in contrast to E. coli, mutations in sbcBC are not necessary to uncover the gonococcal processes dependent on the RecF-like pathway. The differential composition of the recombination-associated pathways, combined with the lack of a classical SOS response (3), shows that homologous recombination and DNA repair processes in the gonococcus and E. coli are not identical. ACKNOWLEDGMENTS We acknowledge the Gonococcal Genome Sequencing Project, B. A. Roe, L. Song, S. P. Lin, X. Yuan, S. Clifton, Tom Ducey, Lisa Lewis, and D. W. Dyer at the University of Oklahoma (supported by USPHS/NIH grant AI38399). We thank Kimberly Kline and Eric Sechman for critical reading of the manuscript. We also thank Stuart Hill for providing the MS11⌬recJ::kan strain. This work was supported by Public Health Service grant R01 AI33493. E.P.S. was partially supported by Public Health Service training grant T32 GM08061.

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