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

Direct Visualization of Horizontal Gene Transfer by Transformation in Live Pneumococcal Cells Using Microfluidics

Natural genetic transformation is a programmed mechanism of horizontal gene transfer in bacteria. It requires the development of competence, a specialized physiological state during which proteins involved in DNA uptake and chromosomal integration are produced. In Streptococcus pneumoniae, competence is transient. It is controlled by a secreted peptide pheromone, the competence-stimulating peptide (CSP) that triggers the sequential transcription of two sets of genes termed early and late competence genes, respectively. Here, we used a microfluidic system with fluorescence microscopy to monitor pneumococcal competence development and transformation, in live cells at the single cell level. We present the conditions to grow this microaerophilic bacterium under continuous flow, with a similar doubling time as in batch liquid culture. We show that perfusion of CSP in the microfluidic chamber results in the same reduction of the growth rate of individual cells as observed in competent pneumococcal cultures. We also describe newly designed fluorescent reporters to distinguish the expression of competence genes with temporally distinct expression profiles. Finally, we exploit the microfluidic technology to inject both CSP and transforming DNA in the microfluidic channels and perform near real time-tracking of transformation in live cells. We show that this approach is well suited to investigating the onset of pneumococcal competence together with the appearance and the fate of transformants in individual cells

Identification of the Major Protein Component of the Pneumococcal Eclipse Complex▿ †

During genetic transformation of Streptococcus pneumoniae, single strands from native donor DNA enter competent cells, where they associate with an unidentified protein with a molecular mass of 15 to 20 kDa to form the eclipse complex. Using Western blotting, we identify the principal protein cofractionating with donor DNA in this complex as SsbB

Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum

A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette. In discontinuous cultures, time course profiles of β-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C. acetobutylicum. Furthermore, a new inducible gene expression system was developed in C. acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system

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

A novel protein fold and extreme domain swapping in the dimeric TorD chaperone from Shewanella massilia

TorD is the cytoplasmic chaperone involved in the maturation of the molybdoenzyme TorA prior to the translocation of the folded protein into the periplasm. The X-ray structure at 2.4 A resolution of the TorD dimer reveals extreme domain swapping between the two subunits. The all-helical architecture of the globular domains within the intertwined molecular dimer shows no similarity with known protein structures. According to sequence similarities, this new fold probably represents the architecture of the chaperones associated with the bacterial DMSO/TMAO reductases and also that of proteins of yet unknown functions. The occurrence of multiple oligomeric forms and the chaperone activity of both monomeric and dimeric TorD raise questions about the possible biological role of domain swapping in this protein

Growth phase impacts the kinetics of deletion-junction loss in transformed cultures.

<p>(A) Experiment in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004934#pgen-1004934-g003" target="_blank">Fig. 3D</a> repeated with different time-points after DNA addition. (B) Growth curves representing growth of wildtype and <i>recO^- xerS^-</i> cells during experiment in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004934#pgen-1004934-g003" target="_blank">Fig. 3D</a> and panel A. (C) Repeat of experiment in panel A but with cells diluted 10-fold 20 min after DNA addition, and later time points taken. <i>xerS</i> and <i>recO</i> mutants were included to determine the kinetics of deletion junction disappearance in these two backgrounds. (D) Growth curves representing growth of wildtype and <i>recO^- xerS^-</i> cells during experiment in panel C. Strains used in all four panels: wildtype; R246, <i>recO^- xerS^-</i>; R3873.</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