Shiga toxin (Stx) released by Stx-producing E. coli (STEC), can cause life-threatening illness. The synthesis and release of Stx requires induction of Stx-encoding prophage present in all STEC genomes. The STEC strains PA2 and PA8 harbor nearly identical Stx-encoding prophages, yet exhibit differing spontaneous induction frequencies and virulence.
ϕPA2 and ϕPA8 each bear an IS629 insertion element but in different locations. This element lies in an intergenic region in ϕPA8. In ϕPA2, it disrupts a gene predicted to encode a DNA adenine methyltransferase (M.EcoPA8). Expressing wild-type M.EcoPA8 in STEC::ϕPA2 restores both prophage induction frequency and virulence to STEC::ϕPA8 levels, but an active site mutant does not. Thus M.EcoPA8 methyltransferase activity governs prophage spontaneous induction and STEC virulence.
Full, specific in vitro methyltransferase activity of M.EcoPA8 requires complexation with a second protein, PNB-2. Sequential deletion of the genes encoding these proteins leads to concomitant, progressive reductions in spontaneous induction and STEC virulence, showing that these phenotypes are interconnected.
We found that despite its annotation as a DNA adenine Mtase, the M.EcoPA8-PNB-2 holoenzyme specifically methylates 16S rRNA, not DNA. A ΔM.ecoPA8 Δpnb-2 STEC strain has fewer ribosomes than the M.EcoPA8-PNB-2 replete STEC strain. The translation activity of extracts made from a ΔM.EcoPA8 ΔPNB-2 STEC strain is lower than that of extracts from STEC::ϕPA8 or from a strain without any Stx-encoding prophage. Significantly, translation extracts from M.EcoPA8 replete STEC exhibit greater resistance to Stx-mediated inactivation compared to STEC strains lacking M.EcoPA8. Collectively, these findings suggest that M.EcoPA8-mediated 16S rRNA methylation safeguards ribosomes from Stx inactivation, enhancing prophage and Stx production, resulting in PA8's enhanced virulence.
16S rRNA sequencing identified 4 putative methylation sites all mapping onto the RNA polymerase-contacting face of the 30S ribosome subunit in the expressome. We suggest M.EcoPA8 -PNB-2 16S rRNA methylation protects the ribosome from Stx inactivation by stabilizing this complex.