Midcell recruitment of the DNA uptake and virulence nuclease, EndA, for pneumococcal transformation

PLoS Pathog. 2013;9(9):e1003596. doi: 10.1371/journal.ppat.1003596. Epub 2013 Sep 5.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Chromosomes, Bacterial / genetics
  • Chromosomes, Bacterial / metabolism*
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism*
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Humans
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Streptococcus pneumoniae / genetics
  • Streptococcus pneumoniae / metabolism*
  • Transformation, Bacterial / physiology*
  • Virulence Factors / genetics
  • Virulence Factors / metabolism*

Substances

  • Bacterial Proteins
  • ComE protein, Streptococcus pneumoniae
  • DNA, Single-Stranded
  • Membrane Proteins
  • Virulence Factors
  • Endodeoxyribonucleases
  • endA protein, bacteria

Grants and funding

This work was supported in part by grants from the Association pour la Recherche sur le Cancer (ARC) and the Fonds Européen de Développement Régional (FEDER). MJB was the recipient of a doctoral fellowship from the French Ministère de la Recherche (2010–2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.