title: RecFOR is not required for pneumococcal transformation but together with XerS for resolution of chromosome dimers frequently formed in the process.
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.
authors.affiliation: Centre National de la Recherche Scientifique, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France.
authors.forename: Calum
authors.initials: C
authors.lastname: Johnston
authors.affiliation: Centre National de la Recherche Scientifique, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Mortier-Barrière
authors.affiliation: Centre National de la Recherche Scientifique, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France.
authors.forename: Chantal
authors.initials: C
authors.lastname: Granadel
authors.affiliation: Centre National de la Recherche Scientifique, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France.
authors.forename: Patrice
authors.initials: P
authors.lastname: Polard
authors.affiliation: Centre National de la Recherche Scientifique, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Martin
authors.affiliation: Centre National de la Recherche Scientifique, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Claverys
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D002875:Chromosomes / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D043211:Exodeoxyribonuclease V / Q000235:genetics*; D059765:Homologous Recombination / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D017354:Point Mutation; D011938:Rec A Recombinases / Q000235:genetics; D045522:Recombinases / Q000235:genetics*; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000472:pathogenicity; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; C093815:DprA protein, bacteria; D008565:Membrane Proteins; D045522:Recombinases; C000599016:XerS tyrosine recombinase, Streptococcus pneumoniae R6; D004254:DNA Nucleotidyltransferases; D011938:Rec A Recombinases; C077570:Site-specific recombinase; D043211:Exodeoxyribonuclease V