(AstABCD); and Propanoite catabolism (PrpBCDE). Examination of the transcript levels of the corresponding genes showed no significant change in mRNA expression levels relative to wildtype, suggesting that Hfq provides most of the control of these pathways for the growth conditions monitored. Translation. Hfq also appears to play a role in the regulation of the translational machinery itself presumably as a feedback mechanism to maintain tight control over the amount of ribosomal synthesis. Several ribosomal proteins including RpsM, RpsG, RpmD, and RplN exhibited a marked reduction in both protein and mRNA levels in the Dhfq mutant strain compared to the wildtype under LB Stat growth condition. One hallmark of growth under nutrient poor conditions is lower expression of the machinery necessary to synthesize proteins, including tRNA synthetases and other factors. In fact all of the aminoacyl-tRNA synthetases (IleS, ProS, CysS, GlnS, AspS, ArgS, GltX, AlaS, TrpS, GlyS, GlyQ) observed were up-regulated in the Dhfq mutant relative to the wild type without a concurrent change in transcription. This suggests that Hfq normally represses translation of these genes under conditions that mimics the intracellular environment (AMM). Stress response. Hfq was observed to modulate numerous proteins involved in envelope stress under AMM-2 conditions, including FkpA, SurA, HtrA, NlpB, NmpC, ClpA, SlyD, RseA, RseB, RpoE, OmpF, and HtpG, which were all up-regulated in the Dhfq mutant strain. These observations are in agreement with the recently suggested role of Hfq in envelope stress based on lacZ translational fusions [34] as well as the observation that rpoE is translationally regulated by Hfq [10]. Proteins involved in oxidative stress (NarY, Qor, Dps, WraB, OxyR, GrxA, Gor, GrxB, KatG, KatE, SodC, SodB, AhpF); acid stress (CadA); and starvation (SspA, SspB) were all also differentially regulated by Hfq (Table S1). Collectively, these results point to a central role for Hfq in regulating stress response in Salmonella Typhimurium. Many of these genes are required for virulence as discussed in more detail below. For example rpoE has been shown to be required for the virulence of Salmonella Typhimurium [35]. Two RpoE-regulated genes, htrA and surA, have also been shown to be involved in Salmonella virulence [35,36,37]. The OxyR-regulated gene dps was demonstrated to contribute to Salmonella oxidative stress resistance, survival in macrophages, and virulence in mice [38]. Recent studies have shown the periplasmic Cu, Zn-superoxide dismutase sodC to contribute to Salmonella virulence in mice [39,40]. Virulence-associated. LPS (lipopolysaccharide) has long been known as an essential component of virulence of Gram negative pathogens and, more recently, the agonist for toll like receptor 4. Hfq differentially regulated the expression of several proteins involved in lipopolysaccharides (LPS) biosynthesis in acidic minimal media. LpxA and LpxD involved in Lipid A synthesis, TolC involved in LPS Core completion, as well as proteins involved in O-antigen synthesis (RfbACKMGJU), appeared as up-regulated in the Dhfq mutant strain suggesting that Hfq may normally down regulate expression within the host. Complementary transcriptional analysis revealed no change in the corresponding mRNA levels relative to the wildtype. Reduced expression of LPS and alterations in LPS modification has been reported for intracellular Salmonella [41,42,43]. Our results suggest Hfq plays a role in the post-transcriptional regulation of several two component systems including three that have been shown to be essential for virulence in a variety of animal models; SsrA/B, PhoP/Q, and OmpR/EnvZ. Two component systems are widespread methods of sensing external conditions and responding by altering gene expression in prokaryotes, eukaryotes and archaea. Well known two component regulators respond to Hfq, SmpB Targets in Salmonella osmolarity, nitrogen and oxygen limitation [44,45]. As such, they are critical to the ability of pathogenic bacteria to adapt and survive following environmental changes. In addition to the three mentioned above Hfq negatively modulates PhoB involved in the phosphate starvation response, RstA, NarP, RcsB, BasR, and CpxR. Microarray analysis showed no effect on transcription of these genes. Salmonella motility enhances cell contact thereby increasing host cell invasion. A recent study showed that an Dhfq mutant that was non-motile exhibited an invasion defect [20]. Analysis of protein expression in the Dhfq strain showed reduced expression of SPI-1 TTSS components and the flagellar subunit protein FliC compared to the wildtype Salmonella strain. Consistent with this previous study, we observed reduced expression of SPI-1 invasion proteins and numerous motility proteins in the Dhfq mutant strain primarily in the log phase growth condition compared to the wildtype strain. Among the SPI-1 invasion proteins were PrgI, SptP, SipD, SicA, InvC and InvG, and the motility proteins FliC, FliT, FliB, CheZ, CheY, and CheW. Microarray results revealed a reduction in mRNA expression of the SPI-1 TTSS and several flagellar proteins, including FliC, FliO, and FliN grown under LB Log growth conditions. This reduction in mRNA expression suggests that Hfq regulates motility and invasion at the transcription level, presumably by modulating one of the upstream regulators. Regulatory elements are generally not made at high levels and thus difficult to identify and quantify. Analysis of our data however demonstrated the upstream regulator for motility flgM [46] was poorly expressed in an Dhfq mutant. Propanediol utilization operon. The cob operon is required for the de novo biosynthesis of vitamin B12 in Salmonella Typhimurium. Propanediol, derived from rhamnose and fucose catabolism, is degraded by products of the propanediol utilization (pdu) operon (immediately adjacent to the cob operon) in a vitamin B12 dependent manner. This allows Salmonella Typhimurium to grow using propanediol as a sole carbon source and because it is required for growth in macrophages this may be the preferred intracellular metabolite [47]. Results from our proteomics analysis revealed that Hfq regulates the cob-pdu operon in AMM-2 (Figure 4A) and stationary phase (Figure 4D) growth conditions, with several products of the cob-pdu operon exhibiting increased expression compared to wild type. Among these were PduA, PduB, PduG, PduL, PduM and PduT as well as CobT, CbiH, CbiF, CbiC, and CbiL. This was not apparent in the DsmpB mutant strain in which cob-pdu protein levels are the same as the parent (Figure 4A and 4D). Western blot analysis of PduA and PduE relative protein levels validated results from the proteomics analysis (Figures 4B, 4C and 4E, 4F). The strong regulation of Pdu protein expression by Hfq suggests the pdu operon to be a potential target of sRNA regulation and that correct coordinate expression may be essential for activity. Both microarray analysis and confirmative qRT-PCR assays showed that RNA levels were unchanged for pduA and pduE genes in the Dhfq mutant strain compared to the wildtype strain (Figure 5), thus showing that Hfq represses translation in these two environmental conditions. The data presented here suggests Hfq likely plays a substantial role in the regulation of the protein expression of the cob-pdu operon, required for intra-macrophage replication [47], via post-transcriptional regulatory mechanisms. SmpB control of biological processes SmpB is thought to be limited to trans-translation but has been shown to increase the translation of a single protein - the stationary phase sigma factor RpoS [19]. The normal function of SmpB is in quality control to free ribosomes stalled on defective mRNAs PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4809