RpoE is involved in transcriptional activity of a subset of virulence genes 
In order to confirm the effect of sigmaE on the expression of a broad range of SsrB-regulated virulence genes, we used wild type and DeltarpoE cells and integrated into the chromosome individually six single-copy transcriptional fusions representing promoters for four classes of SsrB-dependent genes or operons
((i) type III secretion effector operon (sseA); (ii) structural operon I (ssaB); (iii) structural operon II (ssaG); (iv and v) effectors encoded outside of SPI-2 (sseL and sifA); and (vi) integrated virulence genes unlinked to SPI-2 (srfN) [9].
Transcriptional fusions to lacZ of the sseA, ssaB, ssaG, sseL, sifA and srfN promoters (mapped previously; [9,24] were integrated into the chromosome of wild type and DeltarpoE cells and then grown in the SsrB-activating medium LPM.
The activity of each promoter was measured using a beta-galactosidase assay during exponential growth.
Although DeltarpoE was observed to have a slightly prolonged lag phase relative to wild type cells under these experimental conditions, at later time points the mutant grew similarly to wild type.
To account for any differences in growth kinetics of the cultures, all data was normalized to the optical density at 600 nm of the culture, which permitted direct comparisons.
In wild type cells, promoter activity from all the transcriptional fusions was high, as expected, because LPM medium is highly inducing for SsrB activity [21].
In contrast, promoter activity for sseA, ssaB, and sifA decreased in the rpoE mutant compared to wild type cells (Figure 2A, B and 2D), whereas promoter activity from the ssaG and srfN reporters was upregulated in the rpoE mutant (Figure 2C and 2F).
beta-galactosidase activity observed from the sseL reporter was unaltered in the rpoE deletion compared to that in wild type cells (Figure 2E).
These data are consistent with the protein levels detected for these gene products.
Together, these data indicate that sigmaE can have a variable and bidirectional effect on SsrB-regulated virulence genes.
