Role of the Single-Stranded DNA–Binding Protein SsbB in Pneumococcal Transformation: Maintenance of a Reservoir for Genetic Plasticity

Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB−) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs' acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ∼1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison d'être of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell

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

RecFOR is not required for pneumococcal transformation but together with XerS for resolution of chromosome dimers frequently formed in the process.

Homologous recombination (HR) is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes. This process initiates from single-stranded (ss) DNA and is driven by a universal recombinase, which promotes strand exchange between homologous sequences. The bacterial recombinase, RecA, is loaded onto ssDNA by recombinase loaders, RecBCD and RecFOR for genome maintenance. DprA was recently proposed as a third loader dedicated to genetic transformation. Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae. We firstly established that RecFOR proteins are not required for plasmid transformation, strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process. We then observed no reduction in chromosomal transformation using a PCR fragment as donor, contrasting with the 10,000-fold drop in dprA- cells and demonstrating that RecFOR play no role in transformation. However, a ∼1.45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants. To account for this limited deficit, we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency (>30% of cells) formation of chromosome dimers as an intermediate in the generation of tandem duplications, and that RecFOR were crucial for dimer resolution. We validated this hypothesis, showing that the site-specific recombinase XerS was also crucial for dimer resolution. An even higher frequency of dimer formation (>80% of cells) was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA, which contains numerous inversions compared to pneumococcal chromosome, each potentially promoting dimerization. In the absence of RecFOR and XerS, dimers persist, as confirmed by DAPI staining, and can limit the efficiency of transformation, since resulting in loss of transformant chromosome. These findings strengthen the view that different HR machineries exist for genome maintenance and transformation in pneumococci. These observations presumably apply to most naturally transformable species

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

Organization of the <i>dpnII</i> locus.

<p>Schematic representation of the locus in <i>S. pneumoniae</i>, with the identity of the competence-induced promoter and DpnA start codons indicated. Open circle, terminator. P<i>_dpn</i> and P_X, σ⁷⁰ and σ^X dependent promoters, respectively; L and S, start codon of <i>dpnA_L</i> and <i>dpnA_S</i>, respectively; <i>cin</i> box, σ^X binding site; rbs, ribosome-binding site. The limits of the deletion internal to <i>dpnA</i> (Δ) previously constructed <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003178#ppat.1003178-Cerritelli1" target="_blank">[13]</a> and used in this study are indicated.</p

Presence of 19A and 19F serotypes in both DpnI and DpnII populations.

*<p>pneumococcal isolates represent assembled pneumococcal genomes. Dpn identity of other available sequenced pneumococcal genomes with known serotypes can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003178#ppat.1003178.s001" target="_blank">Table S1</a>.</p>†<p>capsule regulatory module identity determined by BLAST against <i>wze</i> sequence from blue and red references defined by Varvio and colleagues <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003178#ppat.1003178-Varvio1" target="_blank">[21]</a>. Values in brackets refer to % nucleotide sequence identity with D39 <i>wze</i> (blue module); followed by % identity with TIGR4 <i>wze</i> (red module).</p>‡<p>Two <i>S. mitis</i> sequenced isolates arbitrarily chosen to demonstrate presence of both <i>Dpn</i>I and <i>Dpn</i>II systems in <i>S. mitis</i> population.</p>§<p>87% identity to CSP2.</p

Information on heterology cassettes used in this study.

*<p>^C and ^A indicate, respectively, the co-transcribed and reverse orientation of an inserted mini-transposon antibiotic resistance cassette with respect to the target gene.</p>†<p>See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003178#ppat.1003178.s002" target="_blank">Table S2</a>.</p>‡<p><i>cps2E</i>::<i>spc7</i>^C cassette transformed into R6-derived strains R3087 and R3088 (with 8 kb deletion in capsule locus).</p>§<p><i>cps2E</i>::<i>spc7</i>^C cassette transformed into <i>cps</i>::<i>kan</i> mutant strains R3148 and R3149 (with 21 kb capsule locus deleted).</p