Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella : Implications for Virulence and Global Protein Translation Charles Ansong1., Hyunjin Yoon2., Steffen Porwollik4, Heather Mottaz-Brewer3, Brianne O. Petritis1, Navdeep Jaitly1, Joshua N. Adkins1, Michael McClelland4, Fred Heffron 2 , Richard D. Smith1* 1 Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, United States of America, 2 Department of Molecular Microbiology and Immunology, Oregon Health and Sciences University, Portland, Oregon, United States of America, 3 Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, United States of America, 4 The Sidney Kimmel Cancer Center, San Diego, California, United States of America Abstract Using sample-matched transcriptomics and proteomics measurements it is now possible to begin to understand the impact of post-transcriptional regulatory programs in Enterobacteria. In bacteria post-transcriptional regulation is mediated by relatively few identified RNA-binding protein factors including CsrA, Hfq and SmpB. A mutation in any one of these three genes, csrA, hfq, and smpB, in Salmonella is attenuated for mouse virulence and unable to survive in macrophages. CsrA has a clearly defined specificity based on binding to a specific mRNA sequence to inhibit translation. However, the proteins regulated by Hfq and SmpB are not as clearly defined. Previous work identified proteins regulated by hfq using purification of the RNA-protein complex with direct sequencing of the bound RNAs and found binding to a surprisingly large number of transcripts. In this report we have used global proteomics to directly identify proteins regulated by Hfq or SmpB by comparing protein abundance in the parent and isogenic hfq or smpB mutant. From these same samples we also prepared RNA for microarray analysis to determine if alteration of protein expression was mediated post-transcriptionally. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all possible Salmonella proteins, respectively, with limited correlation between transcription and protein expression. These proteins represent a broad spectrum of Salmonella proteins required for many biological processes including host cell invasion, motility, central metabolism, LPS biosynthesis, two-component regulatory systems, and fatty acid metabolism. Our results represent one of the first global analyses of post-transcriptional regulons in any organism and suggest that regulation at the translational level is widespread and plays an important role in virulence regulation and environmental adaptation for Salmonella. Citation: Ansong C, Yoon H, Porwollik S, Mottaz-Brewer H, Petritis BO, et al. (2009) Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation. PLoS ONE 4(3): e4809. doi:10.1371/journal.pone.0004809 Editor: Kirsten Nielsen, University of Minnesota, United States of America Received November 20, 2008; Accepted February 2, 2009; Published March 11, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: Portions of this research were supported by the National Institute of Allergy and Infectious Diseases NIH/DHHS through interagency agreement Y1-AI- 4894-01, and the National Center for Research Resources (RR18522). Portions of the work were performed in the Environmental Molecular Science Laboratory, a U.S. Department of Energy (DOE) national scientific user facility at Pacific Northwest National Laboratory (PNNL) in Richland, WA, using capabilities established by the DOE Office of Biological and Environmental Research. Battelle operates PNNL for the DOE under contract DE-AC05-76RLO01830. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rds@pnl.gov . These authors contributed equally to this work. Introduction Post-transcriptional regulation of gene expression by small noncoding RNAs (sRNAs) is widespread in both prokaryotes and eukaryotes and has only recently been recognized as playing a prominent role in the regulation of cellular processes. In bacteria, regulatory sRNAs act via base-pairing interactions with their target mRNAs to modulate their translation and/or decay [1,2], in a reaction typically dependent on the RNA-binding protein Hfq [3,4]. While the role of DNA-binding proteins, particularly transcription factors, in regulating gene expression at the level of transcription has received much attention, how RNA-binding proteins control gene expression at the post-transcriptional level remains unclear. The RNA-binding protein Hfq was initially shown to be the host factor required for replication of Qb RNA bacteriophage [5] and is a member of the Sm protein family of RNA binding proteins that also include eukaryotic proteins required for mRNA splicing [6,7]. Hfq is a highly conserved protein encoded within many bacterial genomes and at least one archaeon genome [7,8]. In bacteria, Hfq functions as a post-transcriptional regulator that modulates the stability and translation of mRNAs chiefly by facilitating sRNA-mRNA interactions [1,4,9]. Its broad role in regulation at the translational level has recently been hinted at by Sittka and co-workers [10]. The RNA-binding protein SmpB specifically interacts with the sRNA tmRNA (also known as ssrA and 10Sa) and is required for all known functions of tmRNA [11]. Indeed SmpB deletion mutants PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4809