Validation of SPI-2 regulation 
SPI-2 encodes a type III secretion system and secreted effectors required for systemic mouse infection [8],[9]. To assess the effects of growth conditions on expression of the SPI-2 secretion apparatus we constructed a lacZ transcriptional fusion to ssaG, a component of the secretion apparatus, and tested expression in each mutant background under each of the four growth conditions. At the same time we determined transcript levels via quantitative real-time PCR (qRT-PCR), using transcripts from rpoD and gyrB as controls [51]. We observed that the results determined by these two methods matched closely (Figure S2) and that the level of transcription of ssaG was highest when Salmonella was grown in minimal acidic media. In agreement with the microarrays, the effect of these regulators on ssaG expression was minimal if the bacteria were grown in rich media (LB broth). Because the type III secretion system and associated virulence factors encoded within SPI-2 were most highly expressed in acidic minimal media we therefore focused on growth in this media and used qRT-PCR to measure transcript levels. To validate the transcriptional profiles we prepared RNA from mutants and parent bacteria grown in acidic minimal medium and used as template for qRT-PCR. Six promoters have been identified for the type III secretion system encoded within SPI-2 (see Figure 4; [52]). We monitored transcription of seven genes within SPI-2, covering each operon at least once, and used gyrB transcript as an internal control. We observed a decrease in transcription of all 7 genes in 11 of the 14 mutants. The three exceptions were spvR, fruR, and rpoS. Strains containing mutations in phoP/phoQ, ssrA/ssrB, slyA, and ompR/envZ showed at least 100-fold decreases in transcription of all SPI-2 genes (Figure 4). Mutations of ihf (himD) and csrA showed an intermediate level of 8-16-fold decreased transcription whereas hnr, rpoE, smpB, crp and hfq showed a modest decrease of 2-8-fold. The results of this analysis are generally concordant with the microarray results although the dynamic range was larger for qRT-PCR than for the microarrays as has been observed before [53]. Note that rpoE and hfq mutants showed a dramatic reduction in macrophage survival, but little difference in transcription of SPI-2 genes during growth in AMM. It has recently been reported that the translational regulator Hfq, regulates translation of RpoE explaining in part why the two mutations behave similarly [54]. Furthermore, Hfq regulates translation of more than 20% of all Salmonella proteins explaining why a mutation in this gene has such a dramatic phenotype ([54]; Charles Ansong, Hyunjin Yoon, Steffen Porwollik, Heather Mottaz-Brewer, Briana Ogata-Petritis, Navdeep Jaitly, Joshua N. Adkins, Michael McClelland, Fred Heffron, and Richard D. Smith; Global systems-level analysis of small RNA-mediated translational regulation: Implications for virulence and global protein translation; Submitted).
