Figure 1. Comparison of survival in mice. Five BALB/c mice were i.p. infected with 200 cfu of Salmonella for each strain; the wild type strain, the Dhfq mutant strain and the DsmpB mutant strain, and monitored for 21 days. Alive mice were counted and shown in % survival. doi:10.1371/journal.pone.0004809.g001 bacteria per cell with stationary phase bacteria grown overnight in LB broth as described in Materials and Methods. Extracellular bacteria were removed by washing and treatment with gentamicin. After infection the cells were lysed at various times by rapid resuspension in PBS containing 1% Triton X-100. Intracellular survival/replication results (Figure 2) are consistent with earlier reports that suggest Dhfq and DsmpB Salmonella Typhimurium mutant strains are essential for intra-macrophage proliferation [22,23,25]. Global effects of Hfq and SmpB mutation on Salmonella Typhimurium chromosome LC-MS-based proteomics was used to obtain a general overview of the effects of Hfq and SmpB on global protein translation in Salmonella. The differences in protein expression between parent and isogenic mutant was assessed via hypothesis testing, specifically a comprehensive ANOVA scheme included in the computer program DAnTE [30]. Relative changes in protein abundance between DsmpB or Dhfq mutant strains and a wild-type strain were evaluated across four differing growth conditions. These included logarithmic (log) and stationary (stat) phase cultures and growth in two variations of an acidic, low magnesium [Mg2+] minimal media (AMM), to approximate conditions encountered in the hosts’s phagocytes (Materials and Methods). Across all experimental datasets we observed a total of 1621 Salmonella Typhimurium proteins (Table S1), corresponding to ,36% coverage of predicted protein coding regions. These proteins represent a broad spectrum of gene products from the genetic translation (Table 1), with proteins of unknown or unclassified function representing the largest functional category as might be expected. Across each of the four growth conditions examined our analysis revealed that Hfq had a far more significant effect on the Salmonella Typhimurium proteome than SmpB. Overall, in comparison to expression in the parent strain, Hfq and SmpB altered, ,50% (781proteins) and ,12% (189 proteins) of the observed proteome (1621 proteins) respectively, as determined by an ANOVA analysis (p,0.05) using the computer program DAnTE [30] (Table S1). When compared to the entire Salmonella proteome of 4450 annotated open reading frames (orf; [31]), 781 proteins represents ,20% of the entire Salmonella genome. This is in agreement with a recent study that found that Hfq affects 18% of the Salmonella genome [10]. Considering the reported pleitropic nature of deletion of hfq on the biology of Salmonella Typhimurium it is Hfq, SmpB Targets in Salmonella Figure 2. Comparison of survival within macrophage cells. RAW264.7 cells were infected with Salmonella cells grown overnight in LB via MOI of 50. After infection initiation by centrifuging the bacteria onto the cell monolayers at 1,0006g for 5 min, cells were incubated at 37uC with 5% CO2 for 30 min, treated with gentamicin (100 mg/ml) for 1 h, and incubated in DMEM with gentamicin (20 mg/ml) for the remainder of the test. Macrophage cells were lysed using 1% Triton-X at 1.5 h (A) and 18 h (B) post-infection and the intracellular bacteria were enumerated by serial dilution of cell lysates. The data shown represents the mean and standard deviation of three individual experiments. doi:10.1371/journal.pone.0004809.g002 likely that a proportion of the changes in protein abundances maybe a non-specific and/or secondary consequence of hfq deletion. That is, not every deregulated protein in the hfq mutant strain is necessarily directly regulated by Hfq. Indeed analysis of the correlation between Hfq-dependent proteins identified in this study and Hfq-associated mRNAs identified by Sittka et al. [10] showed an approximately 25% overlap (Table S2), similar to the ,30% overlap observed between data from a transcriptional analysis of an Dhfq mutant strain and data from a co-IP analysis of Hfq-associated mRNAs [10]. We report that Hfq directly or indirectly modulates ,20% of the Salmonella genome. However this most likely is an underesti- mate of the true number of proteins that are regulated by Hfq, if accurate protein identifications could be made for the remaining 2829 orfs of the entire Salmonella proteome (4450 orfs) that are either expressed at levels too low to be detected or not expressed under the four growth conditions employed in this study. Considering that we observe 36% (1621 orfs) of the entire Salmonella proteome (4450 orfs) and that 50% (781 orfs) of this observed proteome (1621 orfs) is modulated by Hfq, it would be PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4809