The Shiga toxin (Stx)-Phage Encoded Ribosomal RNA Methyltransferase Regulates Stx-producing Escherichia coli (STEC) Virulence by Blocking Stx-Mediated Inactivation of Bacterial Ribosomes

Shiga toxin (Stx) produced and released after induction of Stx-encoding prophage resident within Shiga toxin producing E. coli (STEC) causes life-threatening illness. We previously identified that a two-subunit Stx prophage-encoded 16S rRNA methyltransferase, M.ECPA8\_3172P-PNB-2, which is both uniquely encoded by and commonly found in Stx2- encoding bacteriophage, regulates both prophage spontaneous induction and STEC virulence. We found here that sequential deletion of these two subunits leads to concomitant, progressive reduction in both prophage spontaneous induction and STEC virulence. This observation indicates that these outcomes are linked. The translation activity of extracts made from a Δ M.ECPA8\_3172P Δ PNB -2 Stx prophage-containing strain was lower that of extracts made from either the methyltransferase replete STEC strain or from a strain that did not contain a Stx-encoding prophage. We found that the Δ M.ECPA8\_3172P Δ PNB -2 STEC strain contained significantly fewer ribosomes that did the methyltransferase replete STEC strain. These observations suggested that the M.ECPA8\_3172P-PNB-2 methyltransferase may block Stx-mediated ribosome inactivation. Consistent with this idea, we found that translation extracts made from STEC expressing M.ECPA8\_3172P-PNB-2 are more resistant to Stx- mediated inactivation than are those made from Δ M.ECPA8\_3172P Δ PNB -2 STEC. These findings indicate the M.ECPA8\_3172P-PNB-2 methylation of 16S rRNA protects the ribosome from Stx-mediated inactivation, thereby allowing more phage and more Stx to be spontaneously produced. Direct 16S rRNA sequencing identified 4 putative M.ECPA8\_3172P-PNB-2 methylation sites, all of which map onto the RNA polymerase contacting surface of the 30S ribosome subunit in the expressome, suggesting the M.ECPA8_3172P-PNB-2 may protect the ribosome from inactivation by stabilizing this complex. ### Competing Interest Statement The authors have declared no competing interest.