Comparison of EHEC O157 PT 21/28 and PT32 strains.

<p>(A) Comparative genome hybridisation of six PT21/28 and six PT32 strains. From left to right the PT21/28 strains are: 09807, 13425, 09064, 11204, 16438 & 17482; the PT32 strains are: 11805, 16117, 177706, 17478, 17489 & 08997 (table S1). The heat map shown indicates relative hybridisation levels of the defined strains to the Stx2 phage genome and flanking gene sequences from the O157:H7 Sakai strain which does not contain a Stx2c prophage. Red through to orange indicates a positive hybridization signal, with yellow a weakening signal through to blue colouration indicative of poor hybridization. All the PT21/28 strains show good relative hybridization to the sequences especially the <i>gam</i>-<i>cII</i> immunity region that is indicated. This is not the case for the PT32 strains with only 1 showing positive hybridisation over this same region, indicating that the remaining 5 strains are unlikely to contain the Stx2 phage. This was confirmed for the strains using a PCR to detect Stx2 and Stx2c phage control regions (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#s4" target="_blank">Materials and Methods</a>), the results of which are shown under the heat map lanes. The Stx phage distribution was then determined by PCR for 60 strains (table S1), please see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#s2" target="_blank">Results</a> section. (B) Western blot analysis of a subset of PT21/28 and PT32 strains as defined (table S1). 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>. (C) Relative fluorescence levels from a LEE1-GFP reporter construct transformed into 14 PT21/28 and 16 PT32 strains with levels measured at OD₆₀₀ = 1. The strains with mean values are defined in table S1. The median level of fluorescence from the PT32 strains is significantly higher than for the PT21/28 (p<0.001), the variability in expression levels correlates with the Western blotting data in part B. Also included is the overall median level (horizontal dashed line) (D) LEE1-GFP expression levels compared between strains containing either one or both Stx2 or Stx2c prophages. Strains containing both Stx2 phages have a significantly reduced (P = 0.016) median level of LEE1 expression compared with strains containing only one type of Stx2 prophage as determined at OD₆₀₀ = 1 cultured in MEM-HEPES medium.