This month's Genome Watch explores how genetic variation in restriction–modification systems from Streptococcus pneumoniae contributes to the defence against phages and to bacterial virulence.
Restriction–modification (R–M) systems protect bacteria against the integration of foreign DNA from, for example, phages. These systems rely on restriction enzymes that cleave foreign unmethylated DNA at specific short sequence motifs; methylation of 'self' DNA protects the bacterial genome from its own restriction enzymes. Importantly, high frequency inversions in genes coding for R–M systems in Bacteroides fragilis have been shown to affect the cleavage specificity of these systems1. Now, Croucher et al.2 and Manso et al.3 describe a similar strategy in Streptococcus pneumoniae, and show that these R–M systems are not limited to the defence against phages but can also regulate the expression of genes involved in bacterial virulence.
Croucher et al. used a collection of 616 S. pneumoniae draft genomes to study the variation of mobile genetic elements across different lineages. They found that although integrative and conjugative elements (ICEs) were stably associated with specific S. pneumoniae lineages, prophage elements were highly variable across the collection, even within a single lineage. These data indicate a high rate of phage transmission among the strains and led the authors to hypothesize that S. pneumoniae has evolved a mechanism that prevents the spread of prophage elements through the population, which could otherwise result in overwhelming disruptive integration into the genome. In agreement with this hypothesis, Croucher et al. detected inversions in the hsdS locus — which encodes the specificity domain of a type I R–M system — that created six different alleles (named alleles A–F), each with a different target specificity. This locus rearranged rapidly enough that even a single colony contained a mixture of possible sequence arrangements, suggesting that this R–M system is effective at limiting the integration of diverse phages.
In addition to its role in preventing DNA integration, it has been proposed that the changes in methylation that result from the variation in R–M systems can also alter bacterial gene expression and influence virulence4. To investigate this possibility, Manso et al. constructed S. pneumoniae mutants that exclusively express each of the six possible hsdS alleles (A–F). By using single-molecule real-time (SMRT) sequencing to determine the methylation patterns across the genome of the different mutants, they identified unique motifs that are recognized by each of the specificity domains. To determine whether the different methylation patterns of the mutants carrying different hsdS alleles affected gene expression, Manso et al. then carried out RNA sequencing and observed a substantial downregulation of the capsule operon in mutants expressing the B allele of hsdS.
Increased capsule production by S. pneumoniae leads to the formation of opaque colonies, which are associated with invasive disease, as opposed to transparent colonies, which are associated with asymptomatic carriage. Indeed, bacterial mutants expressing the B allele of hsdS yielded only 7% of opaque colonies, whereas all of the colonies of bacterial mutants expressing the A allele of hsdS were opaque. The functional relevance of the variation in the hsdS locus was further confirmed in vivo, following the infection of mice with different S. pneumoniae mutants; capsule-producing mutants expressing the A allele caused invasive infection, but were unable to stably colonize the nasopharynx, whereas the opposite was true for the mutants expressing the B allele. These observations suggest that this R–M system could be the first genetic factor to be associated with virulence of S. pneumoniae.
Taken together, these two studies highlight the importance of R–M systems both in the defence against phages and in regulating the expression of virulence genes, and suggest that this method of gene regulation could also have similarly important roles in additional bacterial species — a key focus for future studies.
Cerdeno-Tarraga, A. M. et al. Extensive DNA inversions in the B. fragilis genome control variable gene expression. Science 307, 1463–1465 (2005).
Croucher, N. J. et al. Diversification of bacterial genome content through distinct mechanisms over different timescales. Nature Commun. 5, 5471 (2014).
Manso, A. S. et al. A random six-phase switch regulates pneumococcal virulence via global epigenetic changes. Nature Commun. 5, 5055 (2014).
Dybvig, K., Sitaraman, R. & French, C. T. A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements. Proc. Natl Acad. Sci. USA 95, 13923–13928 (1998).
R.A.G. received her Ph.D. stipend from Pfizer, received funding to attend conferences from Pfizer and co-supervises a Ph.D. student funded by Pfizer. J.L. declares no competing interests.
About this article
Cite this article
Lees, J., Gladstone, R. R–M systems go on the offensive. Nat Rev Microbiol 13, 131 (2015). https://doi.org/10.1038/nrmicro3435