Role for the RecBCD recombination pathway for pilE gene variation in repair-proficient Neisseria gonorrhoeae

Abstract

The role of the RecBCD recombination pathway in PilE antigenic variation in Neisseria gonorrhoeae is contentious and appears to be strain dependent. In this study, N. gonorrhoeae strain MS11 recB mutants were assessed for recombination/repair. MS11 recB mutants were found to be highly susceptible to DNA treatments that caused double-chain breaks and were severely impaired for growth; recB growth suppressor mutants arose at high frequencies. When the recombination/repair capacity of strain MS11 was compared to that of strains FA1090 and P9, innate differences were observed between the strains, with FA1090 and P9 rec(+) bacteria presenting pronounced recombination/repair defects. Consequently, MS11 recB mutants present a more robust phenotype than the other strains that were tested. In addition, MS11 recB mutants are also shown to be defective for pilE/pilS recombination. Moreover, pilE/pilS recombination is shown to proceed with gonococci that carry inverted pilE loci. Consequently, a novel RecBCD-mediated double-chain-break repair model for PilE antigenic variation is proposed.

FIG. 1.

CLUSTALW alignment of the amino acids that constitute the RecB nuclease active site. Regions of identity are indicated by stars and regions of similarity by dots. The sequences are as follows: N. gonorrhoeae strain MS11, GeneID no. 3282190 (NGON); Neisseria meningitidis strain FAM18, GeneID no. 4675668 (NMEN); Mycobacterium tuberculosis strain CDC151, GeneID no. 925060 (MTB); Escherichia coli strain K-12, GeneID no. 947286 (ECOLI); Haemophilus influenzae strain RdKW20, GeneID no. 950246 (HINF); Borrelia burgdorferi strain B31, GeneID no. 1195485 (BBURG); and Chlamydophila pneumoniae strain J138, GeneID no. 919511 (CPNEU). Also included are the homologous AddA proteins previously identified from Bacillus subtilis strain 168, GeneID no. 939793 (BSUB), and from Treponema pallidum strain Nichols, GeneID no. 2611603 (TPAL) (27).

FIG. 2.

Analysis of N. gonorrhoeae strain MS11 recB mutants. (A) Comparison of the growth characteristics of MS11 rec⁺, recB, recA, and recB suppressor mutants. (B) Exposure to nalidixic acid (0.5 μg/ml). Solid bar, rec⁺; hexed bar, proline minus mutant; clear bar, recB suppressor; stippled bar, recB; diagonally striped bar, recA; wavy bar, recB opaE::recB⁺. Error bars indicate standard deviations from the mean (n = 10). (C) Exposure to MMS. Solid bar, rec⁺; clear bar, recB suppressor; stippled bar, recB; diagonally striped bar, recA; shaded bar, recC; wavy bar, recB opaE::recB⁺. Data represent two experiments performed in triplicate. Error bars represent standard errors (n = 6).

FIG. 3.

Comparing the effects of DNA damage-causing reagents between N. gonorrhoeae strains MS11, FA1090, and P9. (A) Exposure to nalidixic acid (0.5 μg/ml). Solid bar, MS11 rec⁺; clear bar, MS11 recB suppressor; stippled bar, MS11 recB; diagonally striped bar, MS11 recA; squiggly bar, FA1090 rec⁺; dark diagonal bar, FA1090 recB; light diagonal bar, FA1090 recB_MS11. Strain P9 rec⁺ and recB mutants were unable to tolerate nalidixic acid exposure at this concentration. Error bars indicate standard deviations from the mean (n = 10). (B) Exposure to MMS. Dark stippled bars, strain MS11; hexed bars, strain FA1090; diagonal bars, strain P9. The + symbol reflects rec⁺ bacteria. Data represent two experiments performed in triplicate. Error bars represent standard errors (n = 6). (C) Exposure to UV irradiation. MS 11 rec⁺ (filled squares), FA 1090 rec⁺ (stars), P9 rec⁺ (crosses), MS11 recB (open circles), FA 1090 recB (closed circles), P9 recB (open squares), and MS 11 recA (open triangles). Data represent two experiments performed in triplicate. Standard error bars are omitted for clarity.

FIG. 4.

RT-PCR analysis assessing pilE/pilS recombination. (A) Schematic showing the relative locations of the oligonucleotide primers used in the assay. (B) Southern hybridizations of RT-PCR products using MS11 RNAs prepared from the wild type, recB mutants, recA mutants, and the recB intragenic growth suppressor. The blots were probed with primer 2 (top) or primer 3 (bottom). (C) Schematic showing the inverted pilE chromosomal context (not drawn to scale). S/C represents the Sma/Cla repeat located downstream of pilE. (D) Southern hybridizations of RT-PCR products performed on MS11 RNAs prepared from various MS11 strains. The inverted pilE locus is designated inv. The insertion (no. 1 to 4) mutants carry an erythromycin gene cassette at positions −192 (insertion 1), −221 (insertion 2), −336 (insertion 3), and −743 (insertion 4) relative to the ATG start codon. The blots were probed with primer 2 (top) or primer 3 (bottom).

FIG. 5.

Coconversion assay assessing pilE/pilS recombination. (A) XhoI linker DNA is located in the SmaI site in the Sma/Cla repeat downstream of the pilE locus. Following restriction of chromosomal DNA with SmaI, the XhoI linker can either be converted back to a wild-type configuration (1.4 kb; arrow) or remain in the mutated state (6 kb) if pilE/pilS recombination extends beyond the pilE locus. (A) Analysis of MS11 pilE variant 6 (9) and its recB derivatives. (B) Analysis of FA1090 and its recB derivatives. recB sup are intragenic growth suppressors derived from the cognate recB population. Each blot was probed with the pilE-specific probe 245. In each panel, lanes 1, 2, 3, and 4 represent independent mutants.

FIG. 6.

Double-chain-break repair model for pilE gene variation. The model is based on the yeast mating-type switching model in yeast (20). (A) The pilE locus is broken, and the ends are acted upon by the RecBCD nuclease yielding 3′ overhangs. (B) The single chain overhangs bind RecA protein, which then seeks homology and invades a homologous pilS gene copy. (C) Following invasion of the pilS gene copy, the 3′ end is extended by DNA polymerase, using the pilS gene copy as a template. (D) Following extension, the Holiday junctions are resolved (arrows). (E) Creation of a variant pilE gene consisting of novel pilS sequence that was obtained during the DNA polymerase extension.