<p>(A) Western blot analysis of paired <i>E. coli</i> O157 and O26 strains. EspD was detected from bacterial supernatants and EscJ and RecA from whole cell samples prepared as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#s4" target="_blank">Materials and Methods</a>. EDL is the sequenced EDL933 strain and this was originally compared with a published strain TUV93-0 (lane 4) that has lost both Stx1- and Stx2-encoding prophages. This strain demonstrates higher secretion levels and EscJ expression. The Stx2-encoding prophage and the Stx1-encoding prophage were deleted from EDL933 (lanes 2 and 3 respectively) leading to increased T3S. A marked Stx2 phage <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Dahan1" target="_blank">[67]</a> was conjugated into TUV93-0 expressing a cloned <i>cI</i> from Sakai Sp5 (lane 5) and this resulted in a reduction in T3S and EscJ expression. The final two lanes contain samples from a published isogenic pair of EHEC O26 strains <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Mellmann1" target="_blank">[47]</a> from which one has lost the Stx2 phage leading to a marked increase in T3S. (B–C) To quantify differences in T3S expression, the same strain sets as in part A, were transformed with a LEE1-GFP construct and fluorescence measured throughout the growth curve. This was repeated three times with one experiment shown in (B). A minimum of three values were determined from the expression curves for OD<sub>600</sub> = 0.9 and plotted in (C) as mean and 95% confidence intervals. *** : <i>p</i><0.001 for the TUV strains and <i>p</i> = 0.001 for the EDL strains compared. (D) The <i>E. coli</i> O26 pair of strains blotted in (A) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat-1002672-t001" target="_blank">table 1</a>) were transformed with the LEE1-GFP expression construct or a control plasmid and population fluorescence levels measured through the growth curve. Taken together the data demonstrates that lysogeny with an Stx2-encoding phage represses T3S expression.</p
