relative protein levels of each of HtrA, OsmY, STM1513, FliC, and YciF (Figure S2) served to further verify our global proteomics measurements. As transcript levels are often equated to protein expression levels, it was of particular interest to determine whether genes that showed significant changes in protein expression also exhibited significant changes in transcript levels. Examination of transcript levels using microarray analysis ($2-fold change) revealed a more modest effect on Salmonella transcription, with ,11% (492 genes) and ,8% (370 genes) of the Salmonella Typhimurium genome (4550 genes; [31]) under control of Hfq and SmpB, respectively (Table S4). The result for hfq transcriptional analysis is in general agreement with previous studies for Salmonella [10] and Pseudomonas aeruginosa [33] where ,20% and ,15% of all genes, respectively, were regulated by Hfq, but slightly higher than the number of Hfq-dependent genes reported for E. coli [27] and Vibrio cholerae [21], ,6% each. In light of the fact that these studies employed varying growth conditions it is not possible to perform a rigorous direct comparison across these diverse datasets. However it is worth noting that our observation that Hfq strongly regulates flagellar genes and SPI-1 TTSS genes, discussed below, is consistent with previous results in Salmonella [10,20]. Overall, the transcription data showed fewer discernable patterns of regulation by Hfq or SmpB relative to the proteome analysis, and comparison across all growth conditions revealed little overlap in proteins and transcripts regulated by either Hfq or SmpB (Figure 3). Of the 781 proteins and 492 transcripts regulated by Hfq, 113 genes overlapped both sets. For SmpB-regulated proteins (189) and transcripts (370) there was an overlap of 20 genes. Given the fact that the present study specifically examines the effects of two Hfq, SmpB Targets in Salmonella translational regulators, which decouple transcription and trans- lation, a lack of correlation between transcriptomics and proteomics might be expected. Overall Hfq has a much stronger global effect on Salmonella protein translation than SmpB which is in agreement with the large number of regulatory sRNAs that serve as guides for Hfq translational modulation as opposed to the single regulatory sRNA (i.e. tmRNA) that requires SmpB for activity. Hfq control of biological processes While the role of Hfq as a global regulator has become increasingly appreciated over the past few years, the actual extent of Hfq regulation of translation remains unknown. We observed changes in expression for ,50% of the observed Salmonella Typhimurium proteome based on an ANOVA analysis (p,0.05) (Table S1). The proteins affected represent a broad spectrum of Salmonella proteins required for many biological processes including general metabolism, the translational machinery, stress response, and virulence-assocaited processes as described next. General metabolism. Proteome profiling data suggested Hfq regulates nucleoside (DeoABC) and nucleotide metabolism (CarAB, PurUBCMHAD, Ndk) under AMM conditions, as well as regulates ribose metabolism and transport (RbsABK) under AMM-2. Hfq also appears to play a significant role in central metabolism based on observed differential regulation of numerous proteins involved in glycolysis/gluconeogenesis, the TCA pathway, pyruvate decarboxylation and the pentose phosphate shunt (see Table S1). Other metabolic processes regulated by Hfq include Fatty acid metabolism (FadA, FadB, FabA, FabZ, fbab, AccB, Acs, FabG, FabF, FabI, FabB); Arginine catabolism Figure 3. Summary of transcriptional and translational control functions of Hfq and SmpB. Venn diagram showing overlap between transcripts and proteins regulated by Hfq (Panel A), between transcripts and proteins regulated by SmpB (Panel B) and between proteins co- regulated by Hfq and SmpB (Panel C) across all growth conditions. doi:10.1371/journal.pone.0004809.g003 PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4809