Proteome remodelling by the stress sigma factor RpoS/ (cid:27) S in Salmonella: identification of small proteins and evidence for post-transcriptional regulation

The RpoS/ σ S sigma subunit of RNA polymerase is the master regulator of the general stress response in many Gram-negative bacteria. Extensive studies have been conducted on σ S -regulated gene expression at the transcriptional level. In contrast, very limited information regarding the impact of σ S on global protein production is available. In this study, we used a mass spectrometry-based proteomics approach to explore the wide σ S -dependent proteome of the human pathogen Salmonella enterica serovar Typhimurium. Our present goals were twofold: (1) to survey the protein changes associated with the Δ rpoS mutation and (2) to assess the coding capacity of σ S -dependent small RNAs. Our proteomics data, and complementary assays, unravelled the large impact of σ S on the Salmonella proteome, and validated expression and σ S regulation of twenty uncharacterized small proteins of 27 to 96 amino acids. Furthermore, a large number of genes regulated at the protein level only were identified, suggesting that post-transcriptional regulation is an important component of the σ S response. Novel aspects of σ S in the control of important catabolic pathways such as myo-inositol, L-fucose, propanediol, and ethanolamine were illuminated by this work, providing new insights into the physiological remodelling involved in bacterial adaptation to a non-actively growing state. Salmonella referred as or S . intracellular bacterial a wide including -transcriptome proteome-wide label-free quantification. lysed, with rLys-C and trypsin, the resulting peptides analysed by nLC-MS/MS. A statistical analysis of relative changes in protein abundance between the wild type strain and the Δ rpoS mutant was performed using Perseus, the companion software of MaxQuant (see Supplementary Methods for A protein was considered as being “present” in a strain, if observed in at least two of the three replicates. Proteins detected in less than two replicates of one strain and in at least two replicates of the other strain, were designated as “exclusive” to that latter strain. Two-sided T-tests of the base-2 logarithm (log 2 ) transformed intensity values were employed, using three False Discovery Rates (FDR of 5%, 1% and 0.1%) to identify differentially abundant proteins with different degrees of statistical significance. A complete list of all proteins, along with their relative abundance pattern and sequence coverage is provided in Supplementary Dataset S2. The (log 2 ) fold change of the level of each protein in the Δ rpoS mutant, with respect to that in wild type strain, is shown. Positive values indicate higher abundance in the mutant than the wild type. Negative values represent lower levels in the mutant than the wild type. centrifuged for 15 at and The in the lysates was determined using the DC Protein Assay kit (Bio-Rad). Integrity of the samples was checked by SDS-PAGE. This was done by resolving 10 μ g of all lysates on an SDS-polyacrylamide gel, followed by Coomassie staining. Absence of protein degradation and uniform intensity of major bands across all lysates were considered as indicators of sample integrity and accuracy of protein quantification.