[11,16]. However, results from our global proteomics analysis indicate that SmpB directly or indirectly modulates the expression of at least 4% of the entire Salmonella Typhimurium proteome determined from ANOVA analysis (p,0.05) (Table S1). The majority of genes encoding these proteins showed no changes in mRNA level thus it is interesting to speculate an additional role for SmpB as a general post-transcriptional/translational regulator in Salmonella Typhimurium. Few of the genes identified are regulated by RpoS again suggesting that this is a direct translational effect and not indirect transcriptional. The RpoS regulon in Salmonella Typhimurium has been initially characterized, identifying 13 conserved RpoS-regulated genes [48]. From our global proteomics analysis we were able to observe gene products for 8 of the 13 conserved RpoS-regulated genes, of which only a single protein, YciF, was regulated by SmpB. We note however that a more detailed and rigorous analysis is required to confirm the speculated global regulatory role of SmpB. Examination of proteins modulated by SmpB revealed a number of SmpB regulated cellular processes. SmpB regulates histidine biosynthesis in AMM-1, affecting HisDGCBHAF. Proteins involved in motility in Salmonella Typhimurium also showed down-regulation during growth in LB media. This set of proteins included the chemotaxis proteins CheZ, CheY, CheW, CheA and Tsr as well as the flagellar proteins FliC, FliD, FliG and FljB. This suggests that a more thorough examination of the role of SmpB in the regulation of cell motility and histidine biosynthesis is warranted. Western blot analysis of FliC relative protein levels validated results from the proteomics analysis (Figure S2B). Proteins co-regulated by Hfq and SmpB We were interested in determining whether there was overlap in translational regulation between Hfq and SmpB i.e., the extent of cross-talk or level of co-ordination if any between these two translational regulators (Table S1). For example it was possible that Hfq directly modulates translation of smpB and therefore all of the effects observed for smpB could be related to Hfq. For logarithmic phase growth conditions, the expression pattern of 242 proteins appeared to exhibit a dependence on Hfq, while 37 proteins exhibited a dependency on SmpB. Twenty proteins overlapped both sets, including the motility proteins FliC, CheA, CheZ and Tsr. Under stationary phase growth conditions 270 proteins appeared to be under control of Hfq and 27 proteins under control of SmpB. Nine proteins, including the NADPH- dependent quinine oxidoreductase Qor, TolC involved in LPS biosynthesis, and a putative hydrolase YbeM, overlapped. Of the 390 proteins regulated by Hfq and 50 proteins regulated by SmpB in growth in AMM-2, 15 proteins were co-regulated by both Hfq and SmpB, including the invasion protein PrgI, MdoH involved in the synthesis of membrane-derived oligosaccharide, and a putative periplasmic protein CreA. Under growth in AMM-1 267 proteins appeared regulated by Hfq and the expression pattern of 86 proteins exhibited a dependence on SmpB. Twenty four of these proteins were co-regulated by both Hfq and SmpB. This subset included a putative serine/threonine protein kinase, a putative intracellular proteinase YhbO, SrfJ, a known Salmonella Typhi- murium virulence protein, and the stress protein HtpG. These results also confirm that there is no direct translational control of smpB by Hfq. Summary Post-transcriptional regulation has not been directly examined on a global scale by proteomic methods. This work has focused on two translational regulators in Salmonella (Hfq and SmpB) that are Hfq, SmpB Targets in Salmonella required for virulence. In order to accurately characterize global control of gene expression at the post-transcriptional level it is essential to measure global changes in both transcript and protein levels. We performed a sample-matched global proteomics and transcriptional analysis to examine the broad impacts of deletion mutants of hfq and smpB on global protein and transcript abundance levels to begin to understand how Hfq and SmpB mediate the control of global gene expression at the post-transcriptional level. Using a sample-matched procedure our strategy simultaneously measures changes in global transcript and protein abundance levels allowing for a clear distinction between the transcriptional and post- transcriptional effects of Hfq or SmpB. Furthermore, our strategy requires no tagging and or isolation of Hfq or SmpB precluding the possibility of spurious results introduced by tagging and or co- immunoprecipitation processes. Previous attempts to identify processes/targets regulated by Hfq, and thus targets of sRNA regulation, have relied on transcript-centric approaches. In one study microarray analysis was used to identify changes in gene expression resulting from lack of Hfq, thus providing potential new target genes for sRNA regulation [27]. High-density oligonucleotide microarrays were used to detect sRNA and other targets of Hfq co-immunoprecip- itated with Hfq-specific antibodies [26]. Most recently high- throughput pyrosequencing (HTPS) was used to detect message and sRNA bound to Hfq following immunoprecipitation [10]. The result of this work was very surprising namely that Hfq bound to 20% of all Salmonella mRNA transcripts and at least 64 small RNA molecules. One possible criticism of this work is that the RNA co- immunoprecipitated with Hfq may be brought down simply because Hfq strongly binds to RNA molecules and that the binding is somewhat non-specific. Our efforts presented here support the conclusions of that study and suggest that an even larger number of Salmonella proteins are translationally regulated than the 20% suggested by these authors, when accessed across a larger number of growth conditions. We have compared wildtype and isogenic hfq and smpB derivatives with matched samples to examine both transcription and translation by global proteomics. There were some cases in which a change in protein level could be related to a change in transcription but this was the exception. These results suggest that while feedback mechanisms between protein abundance and transcript levels exist, the majority of the effects observed here for both hfq and smpB are a result of direct modulation of post-transcriptional events. These results have been validated by Western blotting to a number of proteins. The amount of translational regulation varied with growth conditions. During growth in rich media approximately 14% of all proteins observed showed differences in abundance where as in acidic minimal media the result was ,21%. Taking into account all growth conditions about ,50% of all observed proteins are regulated by Hfq an astonishingly high number. These results may explain the very high LD50 of the hfq mutant strain (.109 i.p.) and its growth defect even in rich media. It has recently been shown that half of all the known sRNAs in Salmonella associate with Hfq [10]. It is interesting to note that in adaptive evolution experiments using a chemostat to continuously alter grow conditions often results in changes in Hfq (Bernard Palsson, UCSD, personal communication). It has long been known that many proteins are transcriptionally regulated. These results and the results recently described by Sittka et al. [10] demonstrate that post-transcrip- tional/translational regulation plays as large a role in controlling the myriad of bacterial processes as does transcriptional regulation. It is the precisely this ability to rapidly adjust to changing conditions in less time than it takes to make an RNA molecule that makes this a pivotal regulatory process. PLoS ONE | www.plosone.org 8 March 2009 | Volume 4 | Issue 3 | e4809