Streptococcus pneumoniae (the pneumococcus) is the world’s foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or ‘phases’) that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.

Epigenetic modifications in bacteria, such as DNA methylation, have been shown to affect gene regulation, thereby generating cells that are isogenic but with distinctly different phenotypes. Restriction-modification (RM) systems contain prototypic methylases that are responsible for much of bacterial DNA methylation. This review focuses on a distinctive group of type I RM loci that , through phase variation, can modify their methylation target specificity and can thereby switch bacteria between alternative patterns of DNA methylation. Phase variation occurs at the level of the target recognition domains of the hsdS (specificity) gene via reversible recombination processes acting upon multiple hsdS alleles. We describe the global distribution of such loci throughout the prokaryotic kingdom and highlight the differences in loci structure across the various bacterial species. Although RM systems are often considered simply as an evolutionary response to bacteriophages, these multi-hsdS type I systems have also shown the capacity to change bacterial phenotypes. The ability of these RM systems to allow bacteria to reversibly switch between different physiological states, combined with the existence of such loci across many species of medical and industrial importance, highlights the potential of phase-variable DNA methylation to act as a global regulatory mechanism in bacteria.

Phase-variation of Type I restriction-modification systems can rapidly alter the sequence motifs they target, diversifying both the epigenetic patterns and endonuclease activity within clonally descended populations. Here, we characterize the Streptococcus pneumoniae SpnIV phase-variable Type I RMS, encoded by the translocating variable restriction (tvr) locus, to identify its target motifs, mechanism and regulation of phase variation, and effects on exchange of sequence through transformation. The specificity-determining hsdS genes were shuffled through a recombinase-mediated excision-reintegration mechanism involving circular intermediate molecules, guided by two types of direct repeat. The rate of rearrangements was limited by an attenuator and toxin-antitoxin system homologs that inhibited recombinase gene transcription. Target motifs for both the SpnIV, and multiple Type II, MTases were identified through methylation-sensitive sequencing of a panel of recombinase-null mutants. This demonstrated the species-wide diversity observed at the tvr locus can likely specify nine different methylation patterns. This will reduce sequence exchange in this diverse species, as the native form of the SpnIV RMS was demonstrated to inhibit the acquisition of genomic islands by transformation. Hence the tvr locus can drive variation in genome methylation both within and between strains, and limits the genomic plasticity of S. pneumoniae.

Virus-host interactions are regulated by complex coevolutionary dynamics. In Streptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phage-host interaction through the strong upregulation of the nrdR nucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogen S. pneumoniae. IMPORTANCE With antimicrobial drug resistance becoming an increasing burden on human health, much attention has been focused on the potential use of bacteriophages and their enzymes as therapeutics. However, the investigations into the physiology of the complex interactions of bacteriophages with their hosts have attracted far less attention, in comparison. This work describes the molecular characterization of the infectious cycle of a bacteriophage in the important human pathogen Streptococcus pneumoniae and explores the intricate relationship between phase-variable host defense mechanisms and the virus. This is the first report showing how a phase-variable type I restriction-modification system is involved in bacteriophage restriction while it also provides an additional level of infection control through abortive infection.

Type I restriction-modification (R-M) systems consist of a DNA endonuclease (HsdR, HsdM and HsdS subunits) and methyltransferase (HsdM and HsdS subunits). The hsdS sequences flanked by inverted repeats (referred to as epigenetic invertons) in certain Type I R-M systems undergo invertase-catalyzed inversions. Previous studies in Streptococcus pneumoniae have shown that hsdS inversions within clonal populations produce subpopulations with profound differences in the methylome, cellular physiology and virulence. In this study, we bioinformatically identified six major clades of the tyrosine and serine family invertases homologs from 16 bacterial phyla, which potentially catalyze hsdS inversions in the epigenetic invertons. In particular, the epigenetic invertons are highly enriched in host-associated bacteria. We further verified hsdS inversions in the Type I R-M systems of four representative host-associated bacteria and found that each of the resultant hsdS allelic variants specifies methylation of a unique DNA sequence. In addition, transcriptome analysis revealed that hsdS allelic variations in Enterococcus faecalis exert significant impact on gene expression. These findings indicate that epigenetic switches driven by invertases in the epigenetic invertons broadly operate in the host-associated bacteria, which may broadly contribute to bacterial host adaptation and virulence beyond the role of the Type I R-M systems against phage infection.