Abstract
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.
Highlights
Homologous recombination (HR) is crucial for both maintenance of genome integrity and generation of diversity across all kingdoms of life
RecFOR and the maintenance of pneumococcal genome As expected from studies in other species, the RecFOR proteins appear important for pneumococcal genome maintenance, as deduced from the impact of inactivation of the corresponding genes on growth rate (S1 Fig.) and sensitivity to both methyl methanesulfonate and mitomycin C (S2 Fig.)
Quite unexpectedly, the RecFOR proteins appear involved in another facet of pneumococcal genome maintenance revealed by investigation of the impact of recO inactivation on the formation of merodiploids triggered by transformation
Summary
Homologous recombination (HR) is crucial for both maintenance of genome integrity and generation of diversity across all kingdoms of life. Transformation involves internalization of ssDNA fragments generated from exogenous double-stranded (ds) DNA substrate, which can be incorporated into the chromosome via HR This process generally occurs during a short time window called competence, during which all the proteins required for internalization and integration of ssDNA are produced (reviewed in [2,3]). Two main loaders are involved in genome maintenance, RecBCD targeting RecA to double strand DNA breaks (DSB) and RecFOR targeting it to ssDNA gaps. The latter was suggested as important for the restart of stalled replication forks, where such gaps are Author Summary
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