Recombination of the phase variable spnIII locus is independent of all known pneumococcal site-specific recombinases.

Streptococcus pneumoniae is one of the world's leading bacterial pathogens, causing pneumonia, septicaemia and meningitis. In recent years it has been shown that genetic rearrangements in a type I restriction-modification system (SpnIII) can impact colony morphology and gene expression. By generating a large panel of mutant strains, we have confirmed a previously reported result that the CreX (also known as IvrR and PsrA) recombinase found within the locus is not essential for hsdS inversions. In addition, mutants of homologous recombination pathways also undergo hsdS inversions. In this work we have shown that these genetic rearrangements, which result in different patterns of genome methylation, occur across a wide variety of serotypes and sequence types including two strains (a 19F and a 6B strain) naturally lacking CreX. Our gene expression analysis, by RNAseq, confirm that the level of creX expression is impacted by these genomic rearrangements. In addition, we have shown that the frequency of hsdS recombination is temperature dependent. Most importantly we have demonstrated that the other known pneumococcal site-specific recombinases XerD, XerS and SPD_0921 are not involved in spnIII recombination, suggesting a currently unknown mechanism is responsible for the recombination of these phase variable type I systems.ImportanceStreptococcus pneumoniae is a leading cause of pneumonia, septicaemia and meningitis. The discovery that genetic rearrangements in a type I restriction modification locus can impact gene regulation and colony morphology have led to a new understanding of how this pathogen switches from harmless coloniser to invasive pathogen. These rearrangements, which alter the DNA specificity of the type I restriction modification enzyme, occur across many different pneumococcal serotypes and sequence types, and in the absence of all known pneumococcal site-specific recombinases. This finding suggests that this is a truly global mechanism of pneumococcal gene regulation and the need for further investigation of mechanisms of site specific recombination.