have been observed to have the same phenotype as tmRNA deletion mutants [11]. tmRNA is found in a wide range of bacteria and in the mitochondria of some eukaryotes and uniquely exhibits properties of both tRNA and mRNA [12,13,14]. tmRNA functions as a part of the translational quality control process, recognizing and binding to ribosomes that become stalled. Once bound, tmRNA adds a peptide tag to the nascent partially synthesized protein chain which targets it for degradation by C-terminal- specific cellular proteases [15,16,17,18]. Usually classified as a housekeeping or repair system, a role for tmRNA as a regulatory sRNA has been recently suggested. Ranquet and Gottesman [19] report that the sRNA tmRNA plays a regulatory role in gene expression and is required for the correct high-level translation of RpoS. Recent reports suggest that both Hfq and SmpB play essential roles in bacterial pathogenesis. For example an Dhfq mutant derivative in the pathogen Salmonella enterica serovars Typhimurium (Salmonella Typhimurium) was found to attenuate virulence in mice and was impaired in the invasion of non-phagocytic cells [20]. Hfq was also reported to be essential for Vibrio cholerae virulence, with sRNAs in conjunction with Hfq suggested as likely critical regulators of Vibrio cholerae pathogenicity [21]. Robertson and Roop demonstrated that a Brucella abortus Dhfq mutant failed to replicate in cultured murine macrophages, and displayed attenu- ated virulence in a mouse model [22]. In another study, a smpB mutant of Salmonella Typhimurium exhibited defects in intra- macrophage survival, a model of Salmonella pathogenesis [23]. More recently mutation of tmRNA was shown to be responsible for a marked reduction in Salmonella Typhimurium virulence in BALB/c mice [24]. In Yersinia pseudotuberculosis mutations of smpB and tmRNA generated a bacterium that exhibited defects in survival and replication in macrophages and was avirulent in mice [25]. Collectively, these results demonstrate that Hfq and SmpB are essential for regulating virulence by controlling translation of key components. Identifying what proteins are actually regulated by Hfq and SmpB is an important step in understanding the impact of post- transcriptional gene regulation in Enterobacteria and more specifically how Hfq and SmpB control Salmonella gene expression at the post-transcriptional/ translational level and the role that this may play in Salmonella pathogenesis. Towards the global identification of Hfq targets, previous efforts have employed a combination of co-immunoprecipiatation (co-IP) and or micro- array analysis. Zhang et al. [26] used high-density microarrays to detect potential targets of Hfq co-immunoprecipitated with Hfq- specific antibodies. In a recent study Sittka et al. [10] used high- throughput pyrosequencing (HTPS) to detect potential targets of an epitope tagged-Hfq protein co-immunoprecipitated with commercially available antibodies, overcoming the limitations of the previous strategy; the need for custom microarrays and specialized antibodies. In another study [27] microarray analysis was used to identify changes in gene expression resulting from lack of Hfq, thus providing potential new target genes for sRNA regulation. While co- immunoprecipiatation has proven valuable in identifying targets of Hfq, and thus targets of translational regulation, one possible criticism of this approach 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. In addition, it is clear that a transcriptional analysis solely can not distinguish between transcriptional and translational/post-transcriptional regulatory effects. Using sample-matched transcriptomics and proteomics mea- surements it is possible to accurately characterize on a global scale Hfq, SmpB Targets in Salmonella control of gene expression at the post-transcriptional level. Here, we performed sample-matched global proteomics and transcrip- tional analyses together with cellular assays and animal studies to begin to understand how Hfq and SmpB control Salmonella gene expression at the post-transcriptional level and the role that this may play in Salmonella pathogenesis. Our global analyses revealed that a relatively high percentage of all the annotated Salmonella genes (.20%) are regulated post-transcriptionally either directly or indirectly. The extent of global regulation of translation is much greater than previously thought [10], with profound effects in all stages of Salmonella’s life cycle including a variety of house keeping pathways and known and novel virulence factors, in part explaining the requirement of both translational regulators for virulence in Salmonella. This study represents one of the first comparative global analyses of post-transcriptional regulation in any organism; the data suggest post-transcriptional regulation is widespread and plays an important role in virulence regulation and environmental adaptation for Salmonella. Results and Discussion Construction and growth characteristics of mutant and wildtype Salmonella Typhimurium strains Hfq and SmpB are essential for virulence in a variety of pathogenic bacteria including Salmonella Typhimurium [20,21,22,25]. To address the role of Hfq and SmpB in Salmonella Typhimurium pathogenesis and protein synthesis we first constructed non-polar deletion mutations of the hfq and smpB genes using a modified pKD13 derivative as the template. In this method [28], the coding region between the initiation methionine and the last seven amino acids of the gene was replaced with an inframe ‘‘scar’’ sequence that does not contain any stop codons. Functional disruption of the hfq and smpB mutant derivatives was first confirmed by PCR analysis after which the mutants were P22 transduced to a new genetic background to avoid the possibility of secondary mutations. Mutants that lacked either of these proteins showed impaired growth phenotype in acidic minimal media (AMM). The hfq-deficient strain also exhibited decreased growth rate in Luria-Bertani (LB) broth (Figure S1). These results suggest SmpB and Hfq are involved in broad cellular functions, including growth rate regulation. Dhfq and DsmpB Salmonella Typhimurium mutant strains are avirulent To directly assess the role of Hfq and SmpB in Salmonella Typhimurium pathogenesis, BALB/c mice were infected intra- peritoneally with 200 cfu of either Dhfq mutant strain, DsmpB mutant strain or wildtype strain and monitored for 21 days (Figure 1). The absence of smpB and hfq attenuated Salmonella virulence; mice infected with the DsmpB mutant strain died at 10 days post-infection while mice infected with the Dhfq mutant strain were fully resistant to the infection. Mice infected with wildtype strain exhibited typical symptoms of infection and died at around six days. These results are similar to those in previous reports [20,23]. Dhfq and DsmpB Salmonella Typhimurium mutant strains show defects in intra-macrophage proliferation As Salmonella’s ability to replicate within macrophages is essential for its virulence [29], we examined intracellular replication of Dhfq and DsmpB Salmonella Typhimurium mutant strains within the macrophage- like cell line RAW264.7. These cells were infected at a multiplicity of infection (m.o.i.) of about 1 PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4809