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.

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence

“…To identify the genetic basis for the lack of transformability in the 11 nontransformable isolates, we performed DNA sequencing analysis of 24 pneumococcal genes that are required for natural transformation, including endA and the 23 essential com genes (24,25). This analysis revealed that five of the nontransformable isolates (TH880, TH2813, TH2861, TH2863, and TH2893) harbored potential loss-of-function mutations and unique nucleotide polymorphisms in the coding sequences of one or more essential com genes ( Table 2).…”

“…Many of the late com genes are involved in binding, processing, uptake, and recombination of foreign DNA (19,20). Although a previous study identified 124 genes in strain TIGR4 that are induced by CSP treatment (23), only 23 of the CSP-inducible genes are essential for natural transformation (24,25). Finally, the constitutively expressed gene endA, coding for a nuclease, is also required for DNA uptake (25).…”

“…Although a previous study identified 124 genes in strain TIGR4 that are induced by CSP treatment (23), only 23 of the CSP-inducible genes are essential for natural transformation (24,25). Finally, the constitutively expressed gene endA, coding for a nuclease, is also required for DNA uptake (25). Together, these genes are usually referred to as essential com genes (23).…”

Enterococcus faecalisreadily colonizes the entire gastrointestinal tract and forms biofilms in a germ-free mouse model

“…Cyan fluorescent protein ( CFP) construct Using a previously described genetic markerless insertion system, 45 a codon-optimized CFP sequence recently reported by Berg e et al 46 to be stable in Gram positive cocci was inserted into the chromosome of E. faecalis OG1RF under constitutive expression using the lactococcal P23 promoter system 47 creating OG1RF::p23 cfp (OG1RF cfp C ). From a series of CFP-expressing transconjugants, mutants were empirically screened by IFM to select those with the best combination of fluorescent intensity, stability, and resistance to photobleaching; candidates were checked for basic metabolic and phenotypic growth to ensure no significant differences from parental strain prior to general experimental use.…”

The Influence of Biofilms in the Biology of Plasmids

“…Recently, improved fluorescent reporter proteins, and highly sensitive and specific reagents for fluorescent staining were used to demonstrate localization of the competence machinery and visualize individual genetic transformation events in Streptococcus pneumoniae . (46). Similar approaches hold great promise for analysis of plasmid copy number and conjugation in enterococci and staphylococci growing in biofilms.…”