RadC, a Misleading Name?▿ †

The pfam04002 annotation describes RadC as a bacterial DNA repair protein. Although the radC gene is expressed specifically during competence for genetic transformation in Streptococcus pneumoniae, we report that radC mutants exhibit normal uptake and processing of transforming DNA. They also display normal sensitivity to DNA-damaging agents, providing no support for the rad epithet

Programmed Protection of Foreign DNA from Restriction Allows Pathogenicity Island Exchange during Pneumococcal Transformation

<div><p>In bacteria, transformation and restriction-modification (R-M) systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen <em>Streptococcus pneumoniae</em> is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds) DNA. <em>S. pneumoniae</em> DpnII strains possess DpnM, which methylates dsDNA to protect it from <em>Dpn</em>II restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss) DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from <em>Dpn</em>II restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in <em>dpnA</em> mutant cells and provide evidence that the specific induction of <em>dpnA</em> during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the first evidence for a mechanism that actively promotes genetic diversity of <em>S. pneumoniae</em> through programmed protection and incorporation of foreign DNA.</p> </div

Importance of DpnA-mediated protection from restriction is dependent on the number of GATC sites present in the heterologous cassette.

<p>(A) Relationship between heterology length and <i>dpnA⁻</i> to <i>dpnA</i>⁺ ratio. Ratios represent transformation efficiency in <i>dpnA⁻</i> compared to <i>dpnA⁺</i>. me⁺ donor DNA, closed diamonds. me⁰ DNA, open squares. Error bars are calculated from triplicate repeats of experiments (N = 3). Cassettes transferred and donor strains can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003178#ppat-1003178-t001" target="_blank">Table 1</a>. Recipient strains: R3087 (DpnII); R3088 (DpnII, <i>dpnA⁻</i>); R3163 (DpnII) and R3164 (DpnII, <i>dpnA⁻</i>) were used for transfer of <i>fcsR</i>::<i>ermAM1</i>^C to overcome antibiotic incompatibilities. To create the 21.7 kb heterology, the following recipient strains were used, which lacked the fully capsule locus: R3148 (DpnII, <i>cps</i>::<i>kan</i>) and R3149 (DpnII, <i>dpnA⁻</i>, <i>cps</i>::<i>kan</i>). (B) Relationship between number of GATC sites in donor heterologous cassette and <i>dpnA⁻</i> to <i>dpnA⁺</i> ratio. Same experiment as in panel A, but with ratios plotted against GATC sites in the heterology. (C) Comparison of transformation efficiency of <i>glnR</i>::<i>kan22</i>^C cassettes with varying number of GATC sites. me⁺ donor DNA was not tested. Error bars as in panel A legend. Strains used: donor, R3154 (DpnI, <i>glnR</i>::<i>kan22</i>^{C (8)}, <i>rpsL41</i>), R3238 (DpnI, <i>glnR</i>::<i>kan22</i>^{C (3)}, <i>rpsL41</i>) and R3239 (DpnI, <i>glnR</i>::<i>kan22</i>^{C (6)}, <i>rpsL41</i>); recipient, R3087 (DpnII) and R3088 (DpnII, <i>dpnA⁻</i>).</p

Diagrammatic representation of the impact of R-M systems on transformation.

<p>(A) Integration of a point mutation carried by me⁰ donor DNA is not affected by restriction. Color code: green, transforming (donor) DNA; black, host DNA; grey, displaced recipient strand; red, newly replicated DNA. me⁺ DNA shown as closed circles and me⁰ DNA as open circles; point mutation as R in donor and s for recipient. (B) Integrated me⁺ heterologous donor DNA is resistant to <i>Dpn</i>II, which is unable to cleave me^+/0 dsDNA produced by replication. Color code as above; thick red, neosynthesized complement to heterology. (C) Integrated me⁰ heterologous donor DNA is sensitive to restrictase (e.g., <i>Dpn</i>II) because me⁰ dsDNA is produced by replication. We hypothesize that DpnA, which, unlike DpnM, acts on ssDNA, protects the transformation intermediate by ensuring production of me^+/0 dsDNA after replication (as in panel B).</p

High frequency formation of chromosome dimers upon transformation with <i>S. mitis</i> chromosomal DNA.

<p>(A) <i>S. mitis</i> DNA impacts transformation frequency of <i>recO</i>, <i>xerS</i> and <i>recO xerS</i> mutant cells. Comparison of transformation efficiency using as donor a mixture of PCR fragment carrying the <i>rpsL41</i> mutation (Sm^R) and <i>S. mitis</i> chromosomal DNA. As a slight competition for uptake is observed between the PCR fragment and <i>S. mitis</i> chromosomal DNA in wildtype cells (reducing efficiency compared to PCR fragment alone by 1.6-fold), we adjusted fold reduction in wildtype to 1 and normalized to this same factor in all mutant cells. PCR concentration: 1.5 ng mL⁻¹; chromosomal DNA concentration: 1 µg mL⁻¹. Strains used: wildtype; R246, <i>recO^-</i>; R3170, <i>xerS^-</i>; R3214, <i>recO^- xerS^-</i>; R3873. (B) Monitoring ploidy through DAPI staining in cells transformed with <i>S. mitis</i> chromosomal DNA and grown to stationary phase (blue bars), compared to non-transformed competent cells (grey bars). Samples were taken at 160 min (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004934#pgen-1004934-g004" target="_blank">Fig. 4B</a>). No difference in DAPI fluorescence intensity profile was observed in wildtype cells. Relative fluorescence intensity calculated by dividing total cell intensity by cell area. Results shown are representative of three individual experiments. Strain used: wildtype, R246. (C) Monitoring ploidy through DAPI staining in <i>recO^- xerS^-</i> cells transformed with <i>S. mitis</i> chromosomal DNA and grown to stationary phase. Two individual sets of data showing with non-transformed cells (light and dark grey) and transformed cells (two shades of blue). A shift in intensity profile towards greater fluorescence was observed with cells transformed with <i>S. mitis</i> DNA (compare blue and grey bars). Sampling and analysis as in panel B. Strain used: <i>recO^- xerS^-</i>, R3873. (D) Monitoring ploidy through DAPI staining in <i>recO^- xerS^-</i> cells transformed with <i>S. mitis</i> chromosomal DNA and maintained in exponential growth (blue bars), compared to non-transformed competent cells (grey bars). Samples were taken at 80 min (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004934#pgen-1004934-g004" target="_blank">Fig. 4D</a>). A shift in intensity profile towards greater fluorescence was observed with cells transformed with <i>S. mitis</i> DNA (compare blue and grey bars). Same calculations and strain as in panel C.</p

RecO and the generation of merodiploids by transformation.

<p>(A) Diagrammatic representation of the formation of merodiploids by transformation. This model involves ‘alternative pairing’ of a repeat sequence (R₁) within the transforming ssDNA, i.e. pairing not with its chromosomal counterpart but with a similar repeat (R₂) on one arm of a partially replicated recipient chromosome, coupled with ‘normal pairing’ of the non-repeat flanking ssDNA (A) on the other chromosome arm (next to the true chromosomal counterpart of R₁). This bridges the two chromosome arms, creating a chromosome dimer. It is of note that this dimer differs from 'simple' chromosome dimers made of two directly repeated monomers. Resolution of this 'rearranged' chromosome dimer generates one merodiploid chromosome with the region between repeats duplicated and another chromosome lacking this region <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004934#pgen.1004934-Johnston2" target="_blank">[27]</a> (panel B). (B) Chromosome dimer resolution can be mediated by XerS or by homologous recombination, where RecA could be loaded by RecO. The duplicated region is shown in green. Δ, deletion; †, abortive chromosome. (C) Stimulation of merodiploid formation by transformation in wildtype cells (R246). (D) Stimulation of merodiploid formation by transformation in <i>recO</i> mutant cells (R3170).</p