RecA mediates MgpB and MgpC phase and antigenic variation in Mycoplasma genitalium, but plays a minor role in DNA repair

Abstract

Mycoplasma genitalium, a sexually transmitted human pathogen, encodes MgpB and MgpC adhesins that undergo phase and antigenic variation through recombination with archived 'MgPar' donor sequences. The mechanism and molecular factors required for this genetic variation are poorly understood. In this study, we estimate that sequence variation at the mgpB/C locus occurs in vitro at a frequency of > 1.25 × 10(-4) events per genome per generation using a quantitative anchored PCR assay. This rate was dramatically reduced in a recA deletion mutant and increased in a complemented strain overexpressing RecA. Similarly, the frequency of haemadsorption-deficient phase variants was reduced in the recA mutant, but restored by complementation. Unlike Escherichia coli, inactivation of recA in M. genitalium had a minimal effect on survival after exposure to mitomycin C or UV irradiation. In contrast, a deletion mutant for the predicted nucleotide excision repair uvrC gene showed growth defects and was exquisitely sensitive to DNA damage. We conclude that M. genitalium RecA has a primary role in mgpB/C-MgPar recombination leading to antigenic and phase variation, yet plays a minor role in DNA repair. Our results also suggest that M. genitalium possesses an active nucleotide excision repair system, possibly representing the main DNA repair pathway in this minimal bacterium.

Fig. 1. Gene replacement at the MG_339 locus

A. Genome organization at the MG_339 (recA) locus and schematic representation of the construction of a recA deletion mutant by homologous recombination. Small arrows represent the primers used for the mutant screening and dotted lines show the predicted PCR products for the wild-type and ΔrecA mutant. B. PCR confirmation of the intended deletion at the recA locus. Genomic DNA from wild-type, ΔrecA and ΔrecA+recA strains were used as a template for PCR reactions using primers 5′mg339 and 3′mg340int (PCR 1) and 5′RTMG_339 and 3′mg339 (PCR 2). Primer sequences are shown in Table S1. PCR 1 detects replacement of the recA and MgPar 9 sequences by the chloramphenicol resistance gene (cat). PCR 2 demonstrates the absence of recA gene sequences. PCR products were electrophoresed on a 1 % agarose gel. Lane M: kb DNA ladder (Stratagene). C. Immunoblot analysis of wild-type, ΔrecA and ΔrecA+recA strains with polyclonal antibodies raised against M. genitalium RecA recombinant protein. Ten μg of whole cell extract was applied to each lane. Apparent molecular weights in kDa are indicated on the left side. The full-length blot is presented in Supplemental Fig. S2B.

Fig. 2. Gene variation at the mgpB/C locus

A. Schematic representation of the qPCR-based assay to monitor mgpB/C gene variation. As an example, the KLM-7 anchored PCR is illustrated. This PCR detects new KLM-7 variants using a primer (P1) targeting a conserved region in mgpC gene and a second primer (P2) matching unique sequences (represented by vertical lines) in MgPar 7. P1 and P2 correspond to 5′MgPar7(KLM) and 3′MgPar7(KLM) primers, listed in Table S1. Thus the KLM-7 anchored PCR detects specific variants generated by homologous recombination between MgPar 7 and the KLM variable region of the MgpC expression site. The different variable regions (B, EF, G and KLM) found in mgpB, mgpC and MgPar 7 are also indicated. B. Comparison of MG_339 gene expression profiles between G37C and ΔrecA+recA strains. Relative MG_339 transcripts levels were measured by quantitative RT-PCR normalized to 16S ribosomal RNA. The mean value ± standard deviation is shown from three independent experiments assayed in duplicate. Differences in MG_339 expression were found to be statistically significant (P=0.0032, two-tailed Student’s t-test). C to F. Frequency and accumulation of KLM-7 (C), B-24 (D), G-135 (E) and EF-2478 (F) variants after propagation of G37C, ΔrecA and ΔrecA+recA strains for 2, 4, 6, 8 and 10 passages. Note the different scale for each qPCR assay. The data are presented as the mean ± standard deviation from two independent experiments, with each sample assayed in duplicate.

Fig. 3

Growth curves of wild-type G37 and its derivatives strains, ΔrecA, ΔrecA+recA, ΔuvrC and ΔuvrC+uvrC. Growth was monitored over time by measuring the number of genomes by qPCR. Data is shown in a semi-logarithmic plot and represent the mean ± standard deviation of three separate experiments.

Fig. 4. Role of RecA in generating hemadsorption-deficient (HA⁻) phase variants

A. Frequency and accumulation of HA⁻ phase variants after propagation of G37C strain, ΔrecA and ΔrecA complemented mutant for 10 passages. The frequency of phase variants was calculated by dividing the number of colonies that failed to adsorb erythrocytes by the total number of colonies analyzed. The data are presented as the mean ± standard deviation from two independent experiments assayed in duplicate. The asterisk indicates a statistically significant difference when compared to G37C strain (P<0.001, two-tailed Student’s t-test). B. Representative image showing M. genitalium colonies after the HA assay. Arrows indicate phase variant colonies, which fail to adsorb erythrocytes.

Fig. 5

Survival of wild-type G37, ΔrecA, ΔuvrC and ΔuvrC complemented strains after (A) UV irradiation and (B) mitomycin C (MMC) treatment. Survivals are shown in semi-logarithmic plots and represent the mean ± standard deviation of three independent experiments. Survival of wild-type and ΔuvrC mutant strains differed significantly after UV and MMC treatment with P values of P<0.05 and P<0.1, respectively (two-tailed Student’s t-test). Differences between wild-type and ΔrecA mutant did not achieve statistical significance for any of the matched UV or MMC treatments.

Fig. 6. Gene replacement at the MG_206 locus

A. Genome organization at the MG_206 (uvrC) locus and schematic representation of the construction of a uvrC deletion mutant by homologous recombination. Small arrows represent the primers used for the mutant screening and dotted lines show the predicted PCR products for the wild-type and ΔuvrC mutant. B. PCR confirmation of the intended deletion at the uvrC locus. Genomic DNA from wild-type, ΔuvrC and ΔuvrC+uvrC strains were used as a template for PCR reactions using primers 5′mg206 and 3′mg206 (PCR 1) and 5′mg206int and 3′mg206int (PCR 2). Primer sequences are shown in Table S1. PCR 1 detects replacement of the uvrC gene by the tetM438 resistance gene. Note that the primers used in PCR 1 are the same as those employed for the complementation test, and thus two bands are detected in the ΔuvrC+uvrC strain. PCR 2 demonstrates the absence of uvrC gene sequences. PCR products were electrophoresed on a 1% agarose gel. Lane M: kb DNA ladder (Stratagene).