Mutation of the priA gene of Neisseria gonorrhoeae affects DNA transformation and DNA repair

Abstract

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.

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 motif, and thick black underlines designate highly conserved residues. Conserved helicase motifs are underlined and labeled. A, B, and VI indicate the Walker-A, -B, and motif VI domains. The dashed line denotes the cysteine-rich, zinc finger domain. (B) Chromosomal map of the region containing the priA gene and the complement strain. Arrows indicate the predicted direction of transcription. An open reading frame with homology to DsbC of E. coli is represented by the gray arrow. The open reading frame depicted by the white arrow does not share homology to any known protein.

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 stereomicroscope observations after 24 h of growth on solid media. (B) Growth measured as CFU per colony over time. Error bars represent the standard error of the mean of two experiments, one done in duplicate and the other triplicate (n = 5). (C) Viability of cells in mid-log phase. The percentage of cells staining positive with propidium iodide compared to the total number were counted. Error bars represent the standard error of the mean of three experiments.

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 grown without IPTG and therefore lacks RecA expression. (A) A mutation in priA does not disrupt pilin antigenic variation. The percentage of pilE sequences that differed from the starting pilE sequence is graphed. Error bars represent the standard error of the mean of the percent variant pilE from five separate starter colonies (a total of 181 to 196 colonies per strain were sequenced for each time point shown). (B) A mutation in priA decreases DNA transformation efficiency. Error bars represent the standard error of the mean of eight experiments done in duplicate or triplicate. Statistical differences (P = 0.066) as determined by the Student's t test are indicated by asterisks for comparison to parental. A pound sign indicates a value statistically indistinguishable (P = 0.25) from the parental strain.

FIG. 4.

The N. gonorrhoeae priA mutant is deficient in DNA repair. Strains are as described in the legend for Fig. 3. (A) Relative survival after irradiation with UV light. Error bars represent the standard error of the mean of three experiments performed in duplicate or triplicate (n = 8). Statistically significant differences (P < 0.05) as determined by Student's t test are indicated by asterisks for comparison to the parental strain. (B) Relative survival to double strand breaks induced by nalidixic acid. Error bars represent the standard error of the mean of three experiments performed in duplicate or triplicate (n = 8). Statistically significant differences (P = 0.005) as determined by Student's t test are indicated by the pound sign for comparison to the complement strain. DS, double strand.

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 determined by Student's t test are indicated by asterisks for comparison to the parental strain. (B) Relative survival after exposure to cumene hydroperoxide. Error bars represent the standard error of the mean of four experiments performed in duplicate (n = 8). Statistically significant differences (P < 0.05) as determined by Student's t test are indicated by asterisks for comparison to the parental strain.

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 Streptococcus pneumoniae (6), or processed into a partially single-stranded molecule once there (4). The thick lines indicate linear donor transforming DNA, while thin lines indicate recipient chromosomal DNA. I. The 3′ end of donor transforming DNA (large arrowhead) invades homologous recipient DNA in a RecA-dependent manner forming a D-loop. (A) PriA binds to the D-loop and directly acts to process this structure into a mature recombinant chromosome. (B) The D-loop is processed by one or more unknown endonucleases (open triangles) to create nicked or gapped DNA that would disrupt an incoming replication fork (left). Replication restart would require homologous recombination factors and subsequent PriA-mediated replication restart (31, 34).