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, septicemia, 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 transcriptome sequencing (RNAseq), confirms 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 that a currently unknown mechanism is responsible for the recombination of these phase-variable type I systems.