title: Midcell recruitment of the DNA uptake and virulence nuclease, EndA, for pneumococcal transformation.
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.
authors.affiliation: Centre National de la Recherche Scientifique, LMGM-UMR5100, Toulouse, France ; Université de Toulouse, Université Paul Sabatier, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France.
authors.forename: Matthieu J
authors.initials: MJ
authors.lastname: Bergé
authors.forename: Alain
authors.initials: A
authors.lastname: Kamgoué
authors.forename: Bernard
authors.initials: B
authors.lastname: Martin
authors.forename: Patrice
authors.initials: P
authors.lastname: Polard
authors.forename: Nathalie
authors.initials: N
authors.lastname: Campo
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Claverys
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial / Q000502:physiology*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C103179:ComE protein, Streptococcus pneumoniae; D004277:DNA, Single-Stranded; D008565:Membrane Proteins; D037521:Virulence Factors; D004706:Endodeoxyribonucleases; C064668:endA protein, bacteria
references.citation: Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11140-4
references.pmid: 7479953
references.citation: Annu Rev Microbiol. 1999;53:217-44
references.pmid: 10547691
references.citation: Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1184-9
references.pmid: 20080542
references.citation: Res Microbiol. 2000 Jul-Aug;151(6):487-91
references.pmid: 10961464
references.citation: Mol Microbiol. 1998 Jul;29(1):75-83
references.pmid: 9701804
references.citation: Nucleic Acids Res. 2011 Jan;39(2):623-34
references.pmid: 20846957
references.citation: Mol Microbiol. 2002 Jul;45(2):411-21
references.pmid: 12123453
references.citation: J Biol Chem. 2006 Aug 4;281(31):21720-21727
references.pmid: 16751195
references.citation: Mol Gen Genet. 1988 Aug;213(2-3):444-8
references.pmid: 3185510
references.citation: J Mol Biol. 1990 Jun 20;213(4):727-38
references.pmid: 2359120
references.citation: J Biol Chem. 1993 Mar 15;268(8):5594-9
references.pmid: 8449922
references.citation: Science. 2005 Dec 2;310(5753):1456-60
references.pmid: 16322448
references.citation: J Bacteriol. 1999 Mar;181(5):1555-61
references.pmid: 10049388
references.citation: Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):E1035-44
references.pmid: 23440217
references.citation: J Biol Chem. 1986 Oct 5;261(28):13087-96
references.pmid: 3020024
references.citation: Mol Microbiol. 1994 Jul;13(2):197-205
references.pmid: 7984101
references.citation: Mol Microbiol. 2011 May;80(4):860-7
references.pmid: 21435041
references.citation: Nat Rev Microbiol. 2007 Mar;5(3):219-29
references.pmid: 17277796
references.citation: Mol Microbiol. 2004 Aug;53(4):1161-75
references.pmid: 15306019
references.citation: Cell. 2005 Jul 15;122(1):73-84
references.pmid: 16009134
references.citation: J Bacteriol. 2009 Feb;191(3):949-58
references.pmid: 19028902
references.citation: Mol Microbiol. 2004 Nov;54(3):783-94
references.pmid: 15491367
references.citation: PLoS Pathog. 2013;9(6):e1003473
references.pmid: 23825953
references.citation: BMC Microbiol. 2007 Nov 28;7:105
references.pmid: 18045469
references.citation: J Vis Exp. 2011 Jul 28;(53):
references.pmid: 21841760
references.citation: Mol Microbiol. 2004 Feb;51(4):1051-70
references.pmid: 14763980
references.citation: Mol Microbiol. 2011 May;80(3):612-27
references.pmid: 21414037
references.citation: J Bacteriol. 2012 Jul;194(14):3661-9
references.pmid: 22582279
references.citation: Microbiology (Reading). 2006 Feb;152(Pt 2):343-349
references.pmid: 16436422
references.citation: Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8710-5
references.pmid: 15928084
references.citation: FEMS Microbiol Rev. 2009 May;33(3):643-56
references.pmid: 19228200
references.citation: J Bacteriol. 2011 Apr;193(7):1633-42
references.pmid: 21278288
references.citation: Mol Microbiol. 2007 Jul;65(2):454-64
references.pmid: 17630974
references.citation: J Bacteriol. 1999 Aug;181(16):5004-16
references.pmid: 10438773
references.citation: Nucleic Acids Res. 2011 Apr;39(7):2943-53
references.pmid: 21113026
references.citation: Curr Biol. 2006 Feb 21;16(4):401-7
references.pmid: 16488875
references.citation: Cell Mol Life Sci. 2003 Nov;60(11):2371-88
references.pmid: 14625683
references.citation: Mol Microbiol. 2004 Feb;51(4):1071-86
references.pmid: 14763981
references.citation: Nat Rev Microbiol. 2011 Dec 28;10(2):123-36
references.pmid: 22203377
references.citation: PLoS Genet. 2011 Jun;7(6):e1002156
references.pmid: 21738490
references.citation: J Bacteriol. 1975 Jul;123(1):222-32
references.pmid: 237879
references.citation: Nature. 2012 Feb 08;482(7385):423-7
references.pmid: 22318518
references.citation: Annu Rev Microbiol. 2006;60:451-75
references.pmid: 16771651
references.citation: Mol Microbiol. 2005 Feb;55(3):881-96
references.pmid: 15661011
references.citation: J Bacteriol. 1975 Dec;124(3):1321-9
references.pmid: 366
references.citation: Cell. 2005 Jul 15;122(1):59-71
references.pmid: 16009133
references.citation: Mol Microbiol. 2013 Jan;87(2):394-411
references.pmid: 23216914
references.citation: J Mol Biol. 1980 Aug 5;141(2):133-46
references.pmid: 6255165
references.citation: Mol Microbiol. 2010 Mar;75(6):1513-28
references.pmid: 20180906