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Natural Transformation in Deinococcus Radiodurans: A Natural Transformation in Deinococcus radiodurans: A Genetic Analysis Reveals the Major Roles of DprA, DdrB, RecA, RecF, and RecO Proteins Solenne Ithurbide, Geneviève Coste, Johnny Lisboa, Nicolas Eugénie, Esma Bentchikou, Claire Bouthier de la Tour, Dominique Liger, Fabrice Confalonieri, Suzanne Sommer, Sophie Quevillon-Cheruel, et al. To cite this version: Solenne Ithurbide, Geneviève Coste, Johnny Lisboa, Nicolas Eugénie, Esma Bentchikou, et al.. Natural Transformation in Deinococcus radiodurans: A Genetic Analysis Reveals the Major Roles of DprA, DdrB, RecA, RecF, and RecO Proteins. Frontiers in Microbiology, Frontiers Media, 2020, 11, pp.1253. 10.3389/fmicb.2020.01253. hal-02898103 HAL Id: hal-02898103 https://hal.archives-ouvertes.fr/hal-02898103 Submitted on 3 Sep 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ORIGINAL RESEARCH published: 18 June 2020 doi: 10.3389/fmicb.2020.01253 Natural Transformation in Deinococcus radiodurans: A Genetic Edited by: Ludmila Chistoserdova, Analysis Reveals the Major Roles of University of Washington, United States DprA, DdrB, RecA, RecF, and RecO Reviewed by: Peter Graumann, Proteins University of Marburg, Germany John R. Battista, †‡ † ‡ Louisiana State University, Solenne Ithurbide , Geneviève Coste , Johnny Lisboa , Nicolas Eugénie, United States Esma Bentchikou, Claire Bouthier de la Tour, Dominique Liger, Fabrice Confalonieri, *Correspondence: Suzanne Sommer, Sophie Quevillon-Cheruel* and Pascale Servant* Sophie Quevillon-Cheruel Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France sophie.quevillon-cheruel@ i2bc.paris-saclay.fr Pascale Servant Horizontal gene transfer is a major driver of bacterial evolution and adaptation to [email protected] environmental stresses, occurring notably via transformation of naturally competent †These authors have contributed organisms. The Deinococcus radiodurans bacterium, characterized by its extreme equally to this work radioresistance, is also naturally competent. Here, we investigated the role of D. ‡Present address: Solenne Ithurbide, radiodurans players involved in different steps of natural transformation. First, we The Ithree Institute, University of identified the factors (PilQ, PilD, type IV pilins, PilB, PilT, ComEC-ComEA, and ComF) Technology Sydney, Ultimo, NSW, involved in DNA uptake and DNA translocation across the external and cytoplasmic Australia Johnny Lisboa, membranes and showed that the DNA-uptake machinery is similar to that described Group of Fish Immunology & in the Gram negative bacterium Vibrio cholerae. Then, we studied the involvement of Vaccinology, IBMC – Institute for Molecular and Cell Biology, i3S - recombination and DNA repair proteins, RecA, RecF,RecO, DprA, and DdrB into the DNA Institute for Research and Innovation processing steps of D. radiodurans transformation by plasmid and genomic DNA. The in Health, Porto, Portugal transformation frequency of the cells devoid of DprA, a highly conserved protein among Specialty section: competent species, strongly decreased but was not completely abolished whereas it This article was submitted to was completely abolished in dprA recF, dprA recO, and dprA ddrB double Evolutionary and Genomic mutants. We propose that RecF and RecO, belonging to the recombination mediator Microbiology, a section of the journal complex, and DdrB, a specific deinococcal DNA binding protein, can replace a function Frontiers in Microbiology played by DprA, or alternatively, act at a different step of recombination with DprA. We Received: 03 May 2019 also demonstrated that a dprA mutant is as resistant as wild type to various doses of Accepted: 18 May 2020 Published: 18 June 2020 γ-irradiation, suggesting that DprA, and potentially transformation, do not play a major Citation: role in D. radiodurans radioresistance. Ithurbide S, Coste G, Lisboa J, Keywords: Deinococcus radiodurans, natural transformation, DNA uptake, homologous recombination, DprA, Eugénie N, Bentchikou E, Bouthier de RecA, RecFOR, DdrB la Tour C, Liger D, Confalonieri F, Sommer S, Quevillon-Cheruel S and Servant P (2020) Natural INTRODUCTION Transformation in Deinococcus radiodurans: A Genetic Analysis Reveals the Major Roles of DprA, Natural transformation is a mode of horizontal gene transfer which contributes to the acquisition DdrB, RecA, RecF, and RecO of new properties and thus to genome evolution. Since the discovery of natural transformation Proteins. Front. Microbiol. 11:1253. of Streptococcus pneumoniae (Griffith, 1928), more than 85 species have now been shown to be doi: 10.3389/fmicb.2020.01253 naturally transformable (Johnston et al., 2014). This mechanism can be divided into three steps: Frontiers in Microbiology | www.frontiersin.org 1 June 2020 | Volume 11 | Article 1253 Ithurbide et al. Functions Involved in Deinococcus Transformation (1) External double-stranded DNA (dsDNA) capture followed 2010; Bouthier de la Tour et al., 2011; Sugiman-Marangos by the translocation of single-stranded DNA (ssDNA) into et al., 2016). Remarkably, it was shown that the cells devoid the cytosol. In most transformable bacteria (except for of DdrB were affected in the establishment of plasmid DNA Helicobacter pylori), dsDNA capture is dependent on type II during natural transformation, a process that requires pairing secretion or type IV pilus system proteins (Kruger and Stingl, of internalized plasmid single stranded DNA fragments, whereas 2011; Johnston et al., 2014; Matthey and Blokesch, 2016). The they were proficient in transformation by a chromosomal DNA translocation of ssDNA across the cytoplasmic membrane marker that integrates into the host chromosome through requires three components: the DNA receptor (ComEA) homologous recombination (Bouthier de la Tour et al., 2011). (Seitz et al., 2014), the permease/channel protein (ComEC) This study provided the first evidence linking Deinococcal (Pimentel and Zhang, 2018), and the ATPase protein (ComF) specific DNA repair proteins with natural transformation and (Diallo et al., 2017). raising numerous questions regarding the potential specifics of (2) Protection of the translocated ssDNA from degradation. transformation mechanisms in this species. Single-stranded DNA binding proteins (SSB) protect Here we identified, by genome analysis, candidate genes internalized ssDNA from degradation by nucleases and limit described in other bacterial species as being involved in different the loading of the RecA recombinase. In S. pneumoniae, SsbB transformation steps. We focused on genes that might be directly protects internalized ssDNA and maintains a DNA involved in the protection of the incoming ssDNA and its genome reservoir in the cell (Attaiech et al., 2011). DprA, a member integration or plasmid reconstitution. We showed that, as in of the recombination mediator proteins (RMP) family other bacteria, DprA plays a key role in DNA transformation and dedicated to natural bacterial transformation, interacts we highlighted the ability of the RecFOR complex and the DdrB with naked and SSB-coated ssDNA and also protects DNA protein to partially compensate the absence of DprA in the DNA (Mortier-Barriere et al., 2007; Quevillon-Cheruel et al., transformation process. 2012). (3) Integration into the chromosome of the incoming DNA RESULTS by homologous recombination or a reconstitution of an autonomous plasmid. The integration of the internalized Identification of D. radiodurans Genes ssDNA into the chromosome is catalyzed by the RecA Involved in DNA Uptake and DNA recombinase. Recombination mediators are required to load Translocation During Transformation RecA on SSB-coated ssDNA. DprA was reported in S. Although D. radiodurans is classified as a Gram positive pneumoniae as being a key partner of RecA, acting as a bacterium, it was shown that cells contain two membranes specific mediator for its loading on the incoming ssDNA (Thompson and Murray, 1981). We identified, by sequence (Mortier-Barriere et al., 2007; Quevillon-Cheruel et al., similarity, several D. radiodurans genes encoding type IV pilus 2012). The establishment of plasmid DNA requires a single biogenesis systems, type IV pili, and a fimbrial subunit (Table 1). strand annealing activity to pair internalized complementary The dr0774 gene encodes a protein belonging to the secretin plasmid DNA fragments in order to reconstitute a circular (PilQ) family, which forms, in V. cholerae, the entry pore through replicon in naturally transformable bacteria such as S. the outer membrane for the transforming DNA (Seitz and pneumoniae and Bacillus subtilis (Saunders and Guild, 1981; Blokesch, 2014). This protein was found to be one of the most Kidane et al., 2009). abundant D. radiodurans proteins of the inner/outer membrane The D. radiodurans bacterium, characterized by its extreme (Farci et al., 2014). In D. radiodurans, the dr0774 gene is the last resistance to the lethal effects of ionizing and ultraviolet gene of an operon encompassing pilM (dr0770), pilN (dr0771), radiations,
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