Role of the Rep helicase gene in homologous recombination in Neisseria gonorrhoeae

Abstract

In Escherichia coli, the Rep helicase has been implicated in replication fork progression, replication restart, homologous recombination, and DNA repair. We show that a Neisseria gonorrhoeae rep mutant is deficient in the homologous-recombination-mediated processes of DNA transformation and pilus-based colony variation but not in DNA repair.

FIG. 1.

N. gonorrhoeae rep homologue. (A) Amino acid sequence alignment of Rep from E. coli (Ec) and Rep from N. gonorrhoeae (Gc). Identical residues are boxed in black, and similar residues are boxed in gray. Conserved helicase motifs are underlined and labeled. A conserved DEX(D/H) helicase motif is indicated TXGX, and black circles are over residues of E. coli Rep involved in single-stranded-DNA contact (23). (B) Chromosomal map of the region containing the rep gene and the complement strain. The rep gene (black arrow) was PCR amplified from N. gonorrhoeae genomic DNA with primers RepF2 (5′-CGTGGCAAATGCTCAAAAA-3′) and RepR1 (5′-GCCTCCAAATTTCCACAGAA-3′), cloned into plasmid pCR2.1-TOPO (Invitrogen), and cut within the predicted Rep open reading frame with MfeI, and the tetM gene tagged with the gonococcal uptake sequence required for efficient uptake of DNA (11) was inserted. This plasmid was used to transform N. gonorrhoeae (transformants were selected on Gc medium base (GCB) containing 1 mg of tetracycline per liter), creating the gonococcal rep mutant strain. The triangle indicates the site and orientation of the inserted tetM gene. The rep gene was cloned downstream of a dual taclac promoter (hatched box) and inserted into the neisserial intergenic complementation site (NCIS), a chromosomal site located between the lctP and aspC genes (27, 28) (linked to a chloramphenicol resistance cassette allowing for selection on GCB containing 2 mg of chloramphenicol per liter) to create the rep/rep⁺ complement strain. Gonococcal strains in this study were derivatives of strain FA1090 variant 1-81-S2 (39) containing an IPTG (isopropyl-β-d-thiogalactopyranoside)-regulatable recA allele (38) and have matched pilE sequences as described previously (41). Large arrows indicate the predicted direction of transcription. The white open reading frame 74 bp downstream of and transcribed in the orientation opposite to that of the rep stop codon has homology to DinP (31), which is involved in SOS-induced mutagenesis in E. coli (19, 45). The gray open reading frame, whose stop codon is 23 bp upstream of the rep ATG, has homology to AroD from E. coli. AroD is believed to be involved in the biosynthesis of aromatic amino acids (10). Small arrows (numbered 1 to 5) in aroD and rep were used for RT-PCR operon analysis.

FIG. 2.

A mutation in rep decreases DNA transformation efficiency. Parental strain 1-81-S2 recA6 containing the IPTG-regulatable recA allele (38) is a variant of parental strain FA1090. All strains were grown in 1 mM IPTG for maximal recA induction, which yields RecA expression levels similar to those of strains with a wild-type recA allele (E. A. Stohl and H. S. Seifert, unpublished data). DNA transformation efficiency was determined as previously described (5). Transformation to nalidixic acid resistance occurred when plasmid DNA containing gyrB with a mutation which yields Nal^r was recombined into the gonococcal chromosome. Transformants were selected on GCB plates containing 1.5 mg of nalidixic acid per liter. Error bars represent the standard errors of the means of results of three experiments done in duplicate or triplicate (n = 6 to 9). Statistically significant differences (P < 0.05) as determined by Student's t test are indicated by asterisks for comparison to results with the parental strain and by a pound sign for comparison to the strain carrying the rep mutation.

FIG. 3.

A mutation in rep disrupts pilin antigenic variation. Strains are as described in the legend for Fig. 2, and assays were performed as previously described (40). (A) Percent colony phase variation of N. gonorrhoeae. Error bars represent the standard errors of the means of results of four experiments performed at least in duplicate (n = 10 to 11). (B) Kinetic variation assay, measuring the average number of pilus-dependent colony morphology changes that occur over time. Error bars represent the standard errors of the means of results of three experiments done at least in triplicate (n = 10 to 12). Statistically significant differences (P < 0.05) as determined by Student's t test are indicated by asterisks for comparison to the parental strain and by a pound sign for comparison to the strain carrying the rep mutation.