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.
Highlights
Post-transcriptional regulation of gene expression by small noncoding RNAs is widespread in both prokaryotes and eukaryotes and has only recently been recognized as playing a prominent role in the regulation of cellular processes
Hfq and SmpB are essential for virulence in a variety of pathogenic bacteria including Salmonella Typhimurium [20,21,22,25]
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
Summary
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. 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]. 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 include eukaryotic proteins required for mRNA splicing [6,7]. Hfq functions as a post-transcriptional regulator that modulates the stability and translation of mRNAs 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]
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