Programmed Protection of Foreign DNA from Restriction Allows Pathogenicity Island Exchange during Pneumococcal Transformation

Calum Johnston,Bernard Martin,Jean-Pierre Claverys,Chantal Granadel,Patrice Polard,Michael S. Gilmore

doi:10.1371/journal.ppat.1003178

Abstract

In bacteria, transformation and restriction-modification (R-M) systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen Streptococcus pneumoniae is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds) DNA. S. pneumoniae DpnII strains possess DpnM, which methylates dsDNA to protect it from DpnII restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss) DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from DpnII restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in dpnA mutant cells and provide evidence that the specific induction of dpnA during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the first evidence for a mechanism that actively promotes genetic diversity of S. pneumoniae through programmed protection and incorporation of foreign DNA.

Highlights

While sexual reproduction, which is crucial for genetic diversity in eukaryotes, is lacking in bacteria, genetic transformation is regarded as a substitute [1]
Error bars are calculated from triplicate repeats of experiments (N = 3)
Ss-methylases in other species methylases specific for ssDNA such as DpnA appear to be rare, a few others exist. Both DpnI and DpnII are present in Streptococcus mitis, a species closely-related to S. pneumoniae, and we propose that DpnB/DpnA play a similar dual role of protection from bacteriophage attack and maintenance of genetic exchange

Summary

Introduction

While sexual reproduction, which is crucial for genetic diversity in eukaryotes, is lacking in bacteria, genetic transformation is regarded as a substitute [1]. Genetic transformation proceeds through the internalization of single stranded (ss) DNA fragments created from an exogenous double stranded (ds) DNA substrate, which are incorporated into the genome by homology This forms a heteroduplex of one strand of host DNA associated with complementary exogenous ssDNA, which is resolved by replication, producing one wild-type daughter chromosome, and one possessing the mutation originally present on the exogenous DNA. Current pneumococcal vaccines target the polysaccharide capsule, which is considered the main pneumococcal virulence factor, and of which over 90 different serotypes exist [5] These serotype loci are almost equivalent in size to a single pneumococcal genome, demonstrating the high levels of genetic diversity present in the pneumococcal population. These loci occupy the same position in the genome, between the dexB and aliA genes which provide flanking homology for chromosomal integration of heterologous capsule sequences

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