Published: June 10, 2011 r2011 American Chemical Society 3386 dx.doi.org/10.1021/pr101294v | J. Proteome Res. 2011, 10, 3386–3398 ARTICLE pubs.acs.org/jpr Inverse Regulation in the Metabolic Genes pckA and metE Revealed by Proteomic Analysis of the Salmonella RcsCDB Regulon Alberto Paradela, †,‡ Javier F. Mariscotti, ‡,§ Rosana Navajas, † Antonio Ramos-Fern  andez, † Juan Pablo Albar, † and Francisco García-del Portillo* ,§ † Laboratorio de Prote omica, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain § Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain b S Supporting Information ABSTRACT: The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope stress. Signal transmission occurs from the sensor RcsC to the transcriptional regulator RcsB. Accessory proteins, such as IgaA, are known to adjust the response level. In a previous transcriptomic study, we uncovered 85 genes differentially expressed in Salmonella enterica serovar Typhimurium igaA mutants. Here, we extended these observations to proteomics by performing differential isotope-coded protein labeling (ICPL) followed by liquid chromatographyÀelectrospray ionization tandem mass spectrometry. Five-hundred five proteins were identified and quantified, with 75 of them displaying significant changes in response to alterations in the RcsCDB system. Divergent expression at the RNA and protein level was observed for the metabolic genes pckA and metE, involved in gluconeogenesis and methionine synthesis, respectively. When analyzed in diverse environmental conditions, including the intracellular niche of eukaryotic cells, inverse regulation was more evident for metE and in bacteria growing in defined minimal medium or to stationary phase. The RcsCDB system was also shown to repress the synthesis of the small RNA FnrS, previously reported to modulate metE expression. Collectively, these findings provide new insights into post-transcriptional regulatory mechanisms involving the RcsCDB system and its control over metabolic functions. KEYWORDS: Salmonella, RcsCDB, regulon, proteomics, transcriptomics, ICPL (isotope-coded protein labeling), MetE, FnrS ’ INTRODUCTION The proteome of any living organism is considered as a highly dynamic collection of proteins with alterations paralleled by those previously occurring at the transcriptome level. Recently, evidence has been found for multiple post-transcriptional regulatory me- chanisms occurring in both eukaryotic and prokaryotic systems. 1,2 In bacteria, protein translation is affected by varied mechanisms such as the RNase-mediated decay of the mRNA (mRNA) or the binding efficiency of ribosomes to a concrete Shine-Dalgarno site. 3 Other factors affecting translation rates include changes in the folding of the untranslated region (UTR) located at the 5 0 -end of some mRNAs. These changes occur upon binding of low-molec- ular weight effectors (amino acids, vitamins, coenzymes) in “riboswitches” structures or the intervention of small noncoding RNAs with capacity to perform base-pairing in these regions. Regulatory and degradative proteases with specificity for endogen- ous substrates also affect the amount of the ultimate protein product. 4À6 Large-scale proteomic and transcriptomic analyses allow to gain a global view of alterations resulting from a given stress or environ- mental change. When combined, these two experimental ap- proaches provide information not only about the dynamics of the transcriptional and translational machineries but also on post- transcriptional and post-translational regulatory mechanisms. De- spite being technically rather simple in microbial systems, combined Received: December 29, 2010