Table 1. JCVI functional categories for the entire annotated genome and characterized proteome. Hfq, SmpB Targets in Salmonella Functional Categories Amino acid biosynthesis Biosynthesis of cofactors, prosthetic 163 groups, and carriers Cell envelope Cellular processes Central intermediary metabolism DNA metabolism Energy metabolism Fatty acid and phospholipid metabolism Hypothetical proteins Mobile and extrachromosomal element functions Protein fate Protein synthesis Purines, pyrimidines, nucleosides, 81 and nucleotides Regulatory functions Signal transduction Transcription Transport and binding proteins Unclassified Unknown function 130 469 286 168 155 546 73 78 221 184 375 304 26 52 517 332 670 Total from genome Total of Percent of Percent of observed observed genome proteome proteome Total of observed proteome Hfq SmpB % Hfq % SmpB regulated regulated regulated regulated 2.69 3.37 9.71 5.92 3.48 3.21 11.30 1.51 1.61 4.58 3.81 7.76 1.68 6.29 0.54 1.08 10.70 6.87 13.87 84 93 131 117 86 56 250 36 27 17 93 146 64 82 7 31 113 94 202 4.86 5.38 7.58 6.77 4.97 3.24 14.46 2.08 1.56 0.98 5.38 8.44 3.70 4.74 0.40 1.79 6.54 5.44 11.68 84 93 131 117 86 56 250 36 27 17 93 146 64 82 7 31 113 94 202 149 20 37 34 58 74 42 27 9 8 45 67 33 34 2 13 61 50 99 15 6 10 22 17 7 24 14 15 1 1 6 5 5 0 4 15 11 27 44.05 36.56 44.27 63.25 48.84 48.21 59.60 55.56 33.33 47.06 48.39 45.89 51.56 41.46 28.57 41.94 53.98 53.19 49.01 17.86 6.45 7.63 18.80 19.77 12.50 9.60 2.78 3.70 35.29 15.05 10.27 7.81 6.10 0.00 12.90 13.27 11.70 13.37 Genes and gene products are categorized by the JCVI (J. Craig Venter Institute), formerly TIGR (The Institute for Genomic Research), categorization system. Some proteins are annotated to multiple categories, accounting for the larger total protein count. doi:10.1371/journal.pone.0004809.t001 expected that a significant portion of the unobserved Salmonella proteome (2829 orfs) would also be regulated by Hfq, thus our assertion that the ,20% value may very well represent an underestimation. Similarly 189 proteins represents ,4% when compared to all annotated orfs but again this is likely an underestimate of proteins regulated by SmpB if accurate protein identifications could be made for the remaining 2829 orfs that are either expressed at levels too low to be detected or not expressed under the four growth conditions employed in this study. Classification of the Salmonella proteins deregulated by either Hfq or SmpB by the J. Craig Venter Institute (JCVI) functional categories [32] showed that Hfq modulated the expression of $40% of the observed proteins in 15 of the 19 functional categories annotated for Salmonella (Table 1). The ‘‘Cellular Processes’’ functional category represented the category with most genes impacted by Hfq (,63%). This category encompasses sub- categories including adaptations to atypical conditions, cell adhesion, pathogenesis, chemotaxis and motility, detoxification, toxin production and resistance, DNA transformation and cell division. Translational regulation of these genes permits more rapid adaptation than would be required if the genes had to be first transcribed. This observation may explain the acute attenuation in virulence observed for the Dhfq mutant as well as the previously observed invasion and motility phenotypes of an Dhfq mutant [10,20]. We validated a representative set of proteins by comparing our results with published results. Sittka et al. [20] investigated the proteome of an Dhfq mutant strain using 2D gel analysis, and found ,70 differentially expressed proteins in early stationary phase. Across all growth conditions we identified 67 of the ,70 differentially expressed proteins identified in that study. Further- more 51 of the ,70 Hfq-dependent proteins identified by Sittka et al. were also found to be differentially expressed in the present study. The direction of regulation of many of these proteins was in general agreement with the literature including OmpF, DppA, FliC, SurA, and LuxS as shown in Table S3. As best as we can approximate our LB Stat growth condition would most closely match the early stationary phase growth condition described by Sittka et al. [20] and hence one would expect minimal variability in a cross-comparison between both studies under these two growth conditions. Indeed the direction of regulation of 21 out of 22 differentially expressed proteins in LB Stat growth is in agreement with the literature providing validation. The exception was the SPI-1 protein SipA under LB Stat growth conditions. However under SPI-1 inducing conditions (LB Log) regulation of SPI-1 proteins by Hfq was generally consistent with the literature. To extend our validation of the proteomics results we also directly analyzed protein levels by Western blot analysis of selected proteins based on our proteomics results. Our proteomics results showed that HtrA protein levels were strongly up-regulated in the Dhfq mutant relative to wildtype in LB Log, while OsmY and STM1513 were strongly down-regulated in the Dhfq mutant relative to wildtype in AMM-1 and LB Stat respectively. Deletion of SmpB resulted in a significant decrease in protein expression levels of FliC under LB Log and a significant increase in the protein levels of YciF in AMM-2. Western blot analysis of the PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4809