Midcell Recruitment of the DNA Uptake and Virulence Nuclease, EndA, for Pneumococcal Transformation
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{"title"=>"Midcell Recruitment of the DNA Uptake and Virulence Nuclease, EndA, for Pneumococcal Transformation", "type"=>"journal", "authors"=>[{"first_name"=>"Matthieu J.", "last_name"=>"Bergé", "scopus_author_id"=>"7003575097"}, {"first_name"=>"Alain", "last_name"=>"Kamgoué", "scopus_author_id"=>"12800261800"}, {"first_name"=>"Bernard", "last_name"=>"Martin", "scopus_author_id"=>"7402931602"}, {"first_name"=>"Patrice", "last_name"=>"Polard", "scopus_author_id"=>"6603832546"}, {"first_name"=>"Nathalie", "last_name"=>"Campo", "scopus_author_id"=>"6604050878"}, {"first_name"=>"Jean Pierre", "last_name"=>"Claverys", "scopus_author_id"=>"7005212332"}], "year"=>2013, "source"=>"PLoS Pathogens", "identifiers"=>{"pui"=>"369904108", "issn"=>"15537366", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)", "doi"=>"10.1371/journal.ppat.1003596", "scopus"=>"2-s2.0-84884676627", "pmid"=>"24039578", "sgr"=>"84884676627"}, "id"=>"b121a574-2854-3974-bd24-b6e1e536d6e8", "abstract"=>"Genetic transformation, in which cells internalize exogenous DNA and integrate it into their chromosome, is widespread in the bacterial kingdom. It involves a specialized membrane-associated machinery for binding double-stranded (ds) DNA and uptake of single-stranded (ss) fragments. In the human pathogen Streptococcus pneumoniae, this machinery is specifically assembled at competence. The EndA nuclease, a constitutively expressed virulence factor, is recruited during competence to play the key role of converting dsDNA into ssDNA for uptake. Here we use fluorescence microscopy to show that EndA is uniformly distributed in the membrane of noncompetent cells and relocalizes at midcell during competence. This recruitment requires the dsDNA receptor ComEA. We also show that under 'static' binding conditions, i.e., in cells impaired for uptake, EndA and ComEA colocalize at midcell, together with fluorescent end-labelled dsDNA (Cy3-dsDNA). We conclude that midcell clustering of EndA reflects its recruitment to the DNA uptake machinery rather than its sequestration away from this machinery to protect transforming DNA from extensive degradation. In contrast, a fraction of ComEA molecules were located at cell poles post-competence, suggesting the pole as the site of degradation of the dsDNA receptor. In uptake-proficient cells, we used Cy3-dsDNA molecules enabling expression of a GFP fusion upon chromosomal integration to identify transformed cells as GFP producers 60-70 min after initial contact between DNA and competent cells. Recording of images since initial cell-DNA contact allowed us to look back to the uptake period for these transformed cells. Cy3-DNA foci were thus detected at the cell surface 10-11 min post-initial contact, all exclusively found at midcell, strongly suggesting that active uptake of transforming DNA takes place at this position in pneumococci. We discuss how midcell uptake could influence homology search, and the likelihood that midcell uptake is characteristic of cocci and/or the growth phase-dependency of competence.", "link"=>"http://www.mendeley.com/research/midcell-recruitment-dna-uptake-virulence-nuclease-enda-pneumococcal-transformation", "reader_count"=>48, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Researcher"=>12, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>21, "Student > Postgraduate"=>1, "Student > Master"=>6, "Student > Bachelor"=>2, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Researcher"=>12, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>21, "Student > Postgraduate"=>1, "Student > Master"=>6, "Student > Bachelor"=>2, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Engineering"=>1, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>31, "Medicine and Dentistry"=>1, "Physics and Astronomy"=>2, "Immunology and Microbiology"=>4}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>4}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>31}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>2}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"United States"=>2}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1191920"], "description"=>"<p>(<b>A</b>) YFP-EndA distribution in cells 10 min after addition (or not - noncompetent) of CSP with or without R304 DNA. Top: overlay of phase-contrast images and YFP-EndA. Bottom: deconvolved fluorescent images (<a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003596#s4\" target=\"_blank\">Materials and Methods</a>) of YFP-EndA. Scale bars: 1 µM. (<b>B</b>) EndA clusters in a subset of the population. Data are mean ± s.d. of 4 fields of hundreds cells from 3 independent experiments (2,563 competent and 2,823 noncompetent cells analyzed). (<b>C</b>) Distribution of foci along longitudinal axis in cells without constriction. Focus (n = 146) position is given in relative coordinates from 0 (pole) to 0.5 (midcell). P, pole; M, midcell. (<b>D</b>) Transformation proficiency (open diamonds), fraction of cells containing foci and focus position (histograms). Transformation proficiency was assayed by selecting for Sm<sup>R</sup> transformants as described in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003596#s4\" target=\"_blank\">Materials and Methods</a>. Color code (see inset cell diagram): red, midcell foci; white, intermediate position foci; orange, septal foci (S, septum); green, polar foci. (<b>E</b>) Fluorescence time-lapse microscopy of 6 competent YFP-EndA cells. Representative cells were extracted from a field containing 25 cells, all of which exhibited an EndA focus at some stage during the 25 minutes following CSP addition. Cells with foci are framed by blue squares.</p>", "links"=>[], "tags"=>["genetics", "Molecular genetics", "microbiology", "Bacterial pathogens", "Gram positive", "streptococci", "Microbial pathogens", "Molecular cell biology", "clusters", "competent"], "article_id"=>789509, "categories"=>["Biological Sciences"], "users"=>["Matthieu J. Bergé", "Alain Kamgoué", "Bernard Martin", "Patrice Polard", "Nathalie Campo", "Jean-Pierre Claverys"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003596.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_YFP_EndA_clusters_in_competent_pneumococci_/789509", "title"=>"YFP-EndA clusters in competent pneumococci.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-05 02:05:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/1191921"], "description"=>"<p>(<b>A</b>) Donor DNA binding at midcell in <i>endA</i><sup>−</sup> cells. Fluorescent images of cells incubated with 285-bp Cy3-DNA. Strains: R2811, <i>endA<sup>−</sup></i>; R3740, <i>endA<sup>−</sup> comEA<sup>−</sup></i>. (<b>B</b>) Effect of Cy3-DNA concentration on binding to <i>endA</i><sup>−</sup> cells. (<b>C</b>) Distribution of Cy3-DNA bound to <i>endA<sup>−</sup></i> cells. n, total number of DNA dots analyzed. See legend of <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003596#ppat-1003596-g001\" target=\"_blank\">Figure 1C</a> for details. (<b>D</b>) Co-localization of Cy3-DNA with EndA or ComEA. Strains: R3741, <i>gfp-endA</i><sup>0</sup>; R3606, <i>gfp-comEA endA</i><sup>−</sup> (GFP-ComEA fusion functionality assayed in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003596#ppat.1003596.s003\" target=\"_blank\">Figure S3A</a>). Arrows pinpoint the most prominent co-localization events. Scale bars: 1 µM.</p>", "links"=>[], "tags"=>["genetics", "Molecular genetics", "microbiology", "Bacterial pathogens", "Gram positive", "streptococci", "Microbial pathogens", "Molecular cell biology", "transforming", "comea", "enda", "subcellular"], "article_id"=>789510, "categories"=>["Biological Sciences"], "users"=>["Matthieu J. Bergé", "Alain Kamgoué", "Bernard Martin", "Patrice Polard", "Nathalie Campo", "Jean-Pierre Claverys"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003596.g002", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bound_transforming_DNA_ComEA_and_EndA_subcellular_localizations_/789510", "title"=>"Bound transforming DNA, ComEA and EndA subcellular localizations.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-05 02:05:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/1191934", "https://ndownloader.figshare.com/files/1191935", "https://ndownloader.figshare.com/files/1191936", "https://ndownloader.figshare.com/files/1191937", "https://ndownloader.figshare.com/files/1191938", "https://ndownloader.figshare.com/files/1191939", "https://ndownloader.figshare.com/files/1191940", "https://ndownloader.figshare.com/files/1191941", "https://ndownloader.figshare.com/files/1191942"], "description"=>"<div><p>Genetic transformation, in which cells internalize exogenous DNA and integrate it into their chromosome, is widespread in the bacterial kingdom. It involves a specialized membrane-associated machinery for binding double-stranded (ds) DNA and uptake of single-stranded (ss) fragments. In the human pathogen <i>Streptococcus pneumoniae</i>, this machinery is specifically assembled at competence. The EndA nuclease, a constitutively expressed virulence factor, is recruited during competence to play the key role of converting dsDNA into ssDNA for uptake. Here we use fluorescence microscopy to show that EndA is uniformly distributed in the membrane of noncompetent cells and relocalizes at midcell during competence. This recruitment requires the dsDNA receptor ComEA. We also show that under ‘static’ binding conditions, i.e., in cells impaired for uptake, EndA and ComEA colocalize at midcell, together with fluorescent end-labelled dsDNA (Cy3-dsDNA). We conclude that midcell clustering of EndA reflects its recruitment to the DNA uptake machinery rather than its sequestration away from this machinery to protect transforming DNA from extensive degradation. In contrast, a fraction of ComEA molecules were located at cell poles post-competence, suggesting the pole as the site of degradation of the dsDNA receptor. In uptake-proficient cells, we used Cy3-dsDNA molecules enabling expression of a GFP fusion upon chromosomal integration to identify transformed cells as GFP producers 60–70 min after initial contact between DNA and competent cells. Recording of images since initial cell-DNA contact allowed us to look back to the uptake period for these transformed cells. Cy3-DNA foci were thus detected at the cell surface 10–11 min post-initial contact, all exclusively found at midcell, strongly suggesting that active uptake of transforming DNA takes place at this position in pneumococci. We discuss how midcell uptake could influence homology search, and the likelihood that midcell uptake is characteristic of cocci and/or the growth phase-dependency of competence.</p></div>", "links"=>[], "tags"=>["genetics", "Molecular genetics", "microbiology", "Bacterial pathogens", "Gram positive", "streptococci", "Microbial pathogens", "Molecular cell biology", "recruitment", "dna", "uptake", "virulence", "pneumococcal"], "article_id"=>789523, "categories"=>["Biological Sciences"], "users"=>["Matthieu J. Bergé", "Alain Kamgoué", "Bernard Martin", "Patrice Polard", "Nathalie Campo", "Jean-Pierre Claverys"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003596.s001", "https://dx.doi.org/10.1371/journal.ppat.1003596.s002", "https://dx.doi.org/10.1371/journal.ppat.1003596.s003", "https://dx.doi.org/10.1371/journal.ppat.1003596.s004", "https://dx.doi.org/10.1371/journal.ppat.1003596.s005", "https://dx.doi.org/10.1371/journal.ppat.1003596.s006", "https://dx.doi.org/10.1371/journal.ppat.1003596.s007", "https://dx.doi.org/10.1371/journal.ppat.1003596.s008", "https://dx.doi.org/10.1371/journal.ppat.1003596.s009"], "stats"=>{"downloads"=>22, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Midcell_Recruitment_of_the_DNA_Uptake_and_Virulence_Nuclease_EndA_for_Pneumococcal_Transformation_/789523", "title"=>"Midcell Recruitment of the DNA Uptake and Virulence Nuclease, EndA, for Pneumococcal Transformation", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-09-05 02:05:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/1191924"], "description"=>"<p>(<b>A</b>) Transforming activity of Cy3-DNA. Wildtype cells were exposed to the indicated concentrations of a 4.3 kb fragment containing the Sm<sup>R</sup> mutation <i>rpsL41</i>, bearing or not a 5′ Cy3 label, and Sm<sup>R</sup> transformants were scored. Black, unlabelled donor DNA; red, Cy3-DNA. (<b>B</b>) Direct visualization of transformation. (<i>Left</i>) Schematic of transformation. Cy3-DNA, red dot; fluorescent FtsZ-GFP ring depicted in green. (<i>Right</i>) Fluorescence time-lapse microscopy. The Cy3 and GFP fluorescence images were respectively false colored red and green and overlaid on phase contrast images. Two representative cells are shown. Time after DNA addition indicated in min.</p>", "links"=>[], "tags"=>["genetics", "Molecular genetics", "microbiology", "Bacterial pathogens", "Gram positive", "streptococci", "Microbial pathogens", "Molecular cell biology", "visualization", "fluorescence"], "article_id"=>789513, "categories"=>["Biological Sciences"], "users"=>["Matthieu J. Bergé", "Alain Kamgoué", "Bernard Martin", "Patrice Polard", "Nathalie Campo", "Jean-Pierre Claverys"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003596.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Direct_visualization_of_transformation_by_fluorescence_microscopy_/789513", "title"=>"Direct visualization of transformation by fluorescence microscopy.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-05 02:05:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/1191922"], "description"=>"<p>(<b>A</b>) Transformation proficiency (open diamonds), fraction of cells containing GFP-ComEA foci and focus position (histograms). See <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003596#ppat-1003596-g001\" target=\"_blank\">Figure 1D</a> legend for details. (<b>B</b>) GFP-ComEA stability. Western-blot analysis of R2940 (<i>gfp-comEA</i>) extracts used anti-GFP antibodies. FL, full length fusion protein; asterisks, degradation products. (<b>C</b>) Persistence of Cy3-DNA fluorescence signal post-competence. Images of R3606 (<i>gfp-comEA endA</i><sup>−</sup>) cells 60 min after addition of CSP and 285 bp Cy3-DNA. Yellow arrows point to polar DNA molecules. (<b>D</b>) Stabilizing effect of persistent DNA on GFP-ComEA protein. Western-blot analysis of R3606 (<i>gfp-comEA</i>, <i>endA<sup>−</sup></i>) cells incubated with or without R304 chromosomal DNA. Samples were collected at the indicated time points (see legend of panel B for details).</p>", "links"=>[], "tags"=>["genetics", "Molecular genetics", "microbiology", "Bacterial pathogens", "Gram positive", "streptococci", "Microbial pathogens", "Molecular cell biology"], "article_id"=>789511, "categories"=>["Biological Sciences"], "users"=>["Matthieu J. Bergé", "Alain Kamgoué", "Bernard Martin", "Patrice Polard", "Nathalie Campo", "Jean-Pierre Claverys"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003596.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Localization_and_stability_of_GFP_ComEA_/789511", "title"=>"Localization and stability of GFP-ComEA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-05 02:05:15"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[269, 466, 588, 697, 800, 896, 988, 1076, 1165, 1254, 1340, 1417]}, {"subject_area"=>"/Biology and life sciences/Microbiology", "average_usage"=>[293, 503, 638, 755, 861, 960, 1056, 1146, 1239, 1323, 1403, 1491, 1568]}, {"subject_area"=>"/Biology and life sciences/Organisms", "average_usage"=>[281, 484, 611, 728, 835, 934, 1030, 1123, 1214, 1299, 1383, 1464]}, {"subject_area"=>"/Medicine and health sciences", "average_usage"=>[264, 460, 584, 692, 794, 887, 978, 1067, 1154, 1241, 1328, 1408, 1474]}, {"subject_area"=>"/Medicine and health sciences/Pathology and laboratory medicine", "average_usage"=>[267, 466, 592, 709, 806, 901, 989, 1075, 1162, 1254, 1342, 1424, 1486]}]}
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