The opacity ratio of ST556 derivatives.

<div><p>Human pathogen <i>Streptococcus pneumoniae</i> forms multiple epigenetically and phenotypically distinct intra-populations by invertase PsrA-driven inversions of DNA methyltransferase <i>hsdS</i> genes in the colony opacity-determinant (<i>cod</i>) locus. As manifested by phase switch between opaque and transparent colonies, different genome methylation patterns or epigenomes confer pathogenesis-associated traits, but it is unknown how the pathogen controls the <i>hsdS</i> inversion orientations. Here, we report our finding of the SpxA1-TenA toxin-antitoxin (TA) system that regulates the orientations of <i>hsdS</i> inversions, and thereby bacterial epigenome and associated traits (e.g., colony opacity) by targeting pneumococcal protein synthesis. SpxA1 and TenA were found to constitute a highly conserved type II TA system in <i>S</i>. <i>pneumoniae</i>, primarily based on the observation that overexpressing toxin TenA led to growth arrest in <i>E</i>. <i>coli</i> and enhanced autolysis in <i>S</i>. <i>pneumoniae</i>, and the antitoxin SpxA1 repressed the transcription of the <i>spxA1-tenA</i> operon. When the transcription of <i>tenA</i> was de-repressed by a spontaneous AT di-nucleotide insertion/deletion in the promoter region of the <i>spxA1-tenA</i> operon, TenA bound to the ribosome maturation factor RimM, and thereby reduced the cellular level of alternative sigma factor ComX (known for the activation of natural transformation-associated genes). Attenuation of ComX expression in turn enhanced the transcription of the invertase gene <i>psrA</i>, which favored the formation of the transparent colony phase-associated <i>hsdS</i> allelic configurations in the <i>cod</i> locus. Phenotypically, moderate expression of TenA dramatically reshaped pneumococcal epigenome and colony opacity. Because spontaneous variations frequently occur during bacterial growth in the number of the AT di-nucleotides in the promoter region of the <i>spxA1-tenA</i> operon, this locus acts as a programmed genetic switch that generates pneumococcal subpopulations with epigenetic and phenotypic diversity.</p></div>