The Escherichia coli S2P intramembrane protease RseP regulates ferric citrate uptake by cleaving the sigma factor regulator FecR

Escherichia coli RseP, a member of the site-2 protease family intramembrane proteases, is involved in activation σE extracytoplasmic stress response and elimination signal peptides from cytoplasmic membrane. However, whether RseP has additional cellular functions unclear. In this study, we used mass spectrometry–based quantitative proteomic analysis to search for new substrates that might reveal unknown physiological roles RseP. Our data showed levels several Fec system proteins encoded by fecABCDE operon (fec operon) were significantly decreased an RseP-deficient strain. The responsible uptake ferric citrate, transcription fec controlled FecI, alternative sigma factor, its regulator FecR, single-pass transmembrane protein. Assays with expression reporter demonstrated proteolytic activity essential citrate–dependent upregulation operon. Analysis using FecR protein FecR-derived model undergoes sequential processing at membrane participates last step generate N-terminal fragment FecI. A shortened construct was not dependent on activation, confirming cleavage sufficient role study unveiled E. performs proteolysis novel regulating iron citrate transport system. While bacterial membranes act as barrier protect cell extrinsic damages caused various xenobiotics hazardous changes environmental conditions, they must mediate only selective import nutrients other small molecules but also transduction signals external milieu adapt changes. variety mechanisms exist transmit information across Among them, regulated (RIP) crucial mechanism conserved among all kingdoms (1Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Regulated proteolysis: control bacteria humans.Cell. 2000; 100: 391-398Abstract Full Text PDF PubMed Google Scholar, 2Wolfe Intramembrane proteolysis.Chem. Rev. 2009; 109: 1599-1612Crossref Scopus (90) Scholar). RIP, class proteases called (IMPs) (TM) signaling through target proteins. 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Although rseP growth, deleted absence functional rseA strains, constitutively activated thus, exclude possible resulting RseP-dependent response. strains grown mid-log phase L broth containing 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), inducer plasmid-encoded derivatives, subject nanoLC/MS/MS analysis. As result, 13,815 derived 1419 (E. K-12). proteins, 17 exhibited significant (p < 0.05) increase, 41 decrease samples RseP(E23Q)-expressing RseP(WT)-expressing (Table Table S1). Gene ontology enrichment DAVID (44Huang D.W. Sherman B.T. Lempicki R.A. Bioinformatics tools: Paths toward lists.Nucleic Acids Res. 1-13Crossref (8810) 45Huang Systematic integrative lists bioinformatics resources.Nat. Protoc. 4: 44-57Crossref (22148) former included groups related TonB box receptor beta strand, (Fold Enrichment > 10, S2). [CyoE (heme O synthase (46Saiki Mogi Anraku Heme cyoE cytochrome BO encodes protoheme IX farnesyltransferase.Biochem. Commun. 1992; 1491-1497Crossref (75) 47Saiki vitro heme synthesis 1993; 268: 26041-26044Abstract Scholar)), AmtB (ammonium transporter (48Soupene He Kustu Ammonia enteric bacteria: Physiological ammonium/methylammonium B (AmtB) protein.Proc. 95: 7030-7034Crossref (142) ZupT (heavy metal divalent cation (49Grass Wong M.D. Rosen B.P. R.L. Rensing Zn(II) 184: 864-866Crossref (123) 50Grass Franke Taudte Nies D.H. Kucharski L.M. Maguire M.E. permease broad spectrum.J. 187: 1604-1611Crossref (147) Scholar))], fold change greater than (Fig. 1). accumulated under condition, unlikely top group FecA, FecD, FecE, 2. system, systems induced availability iron. indicates should responses (e.g., HurR (21King-Lyons FpvR FoxR (51Bastiaansen K.C. Otero-Asman J.R. Luirink Bitter W. Llamas M.A. Processing anti-sigma prior recognition autoproteolytic domains.Environ. 17: 3263-3277Crossref (16) HxuR, HasS (52Otero-Asman García-García A.I. Civantos Quesada J.M. possesses distinct sensing host molecule haem.Environ. 21: 4629-4647Crossref (10) putida IutY (53Bastiaansen Ibañez Ramos Prc activity.Environ. 2433-2443Crossref (21) further examined involvement expression.Table 1Significantly changed (p-value depending RsePLog2 (E23Q/WT)-Log10 p-valueGene namesProtein namesLocationUpregulated 2.203.80cyoEProtoheme farnesyltransferaseIM 1.542.94amtBAmmonia channelIM 1.141.55zupTZinc ZupTM 0.851.73ycfJUncharacterized YcfJM 0.631.67fiuCatecholate siderophore FiuOM 0.621.65hdeBAcid chaperone HdeBPeri 0.611.76osmBOsmotically inducible lipoprotein BM 0.582.01mdtAMultidrug resistance MdtAIM 0.571.85cirAColicin I receptorOM 0.551.41wzcTyrosine-protein kinase wzcIM 0.521.38glnBNitrogen P-II 1- 0.481.56yjiYInner YjiYIM 0.461.46rsePRegulator sigma-E RsePIM 0.461.49fepAFerrienterobactin 0.441.72yqiCUncharacterized YqiCCyto 0.441.77zntALead, cadmium, mercury-transporting ATPaseIM 0.392.18mdtBMultidrug MdtBIMDownregulated −3.304.15fecDFe(3+) FecDIM −3.274.42fecAFe(3+) FecAOM −1.803.90fecEFe(3+) ATP-binding FecEIM −0.762.06yeeRInner YeeRIM −0.721.76znuAHigh-affinity ZnuAPeri −0.711.39fluAntigen 43; Antigen 43 alpha chain; chainPeri −0.691.47sucDSuccinyl-CoA ligase [ADP-forming] subunit alpha- −0.641.39yaaAUPF0246 YaaA- −0.581.34putABifunctional PutA; Proline dehydrogenase; Delta-1-pyrroline-5-carboxylate dehydrogenase- −0.561.51cstACarbon starvation AIM −0.541.48truBtRNA pseudouridine B- −0.501.48purNPhosphoribosylglycinamide formyltransferase- −0.481.32rluBRibosomal −0.441.50accAAcetyl-coenzyme carboxylase carboxyl transferase alphaCyto −0.441.62ycbXUncharacterized YcbX- −0.431.39thrSThreonine--tRNA ligaseCyto −0.431.45thiItRNA sulfurtransferaseCyto −0.411.48gltACitrate synthase- −0.411.45sodBSuperoxide dismutase [Fe]- −0.401.78hrpAATP-dependent RNA helicase HrpA- −0.401.48rneRibonuclease ECyto −0.392.19pepPXaa-Pro aminopeptidaseCyto −0.391.51serCPhosphoserine aminotransferaseCyto −0.391.71ybeDUPF0250 YbeD- −0.391.64selBSelenocysteine-specific elongation factorCyto −0.381.44degPPeriplasmic endoprotease DegPIM −0.351.41tehBTellurite methyltransferaseCyto −0.351.38gmhAPhosphoheptose isomeraseCyto −0.341.70oppFOligopeptide OppFIM −0.331.42uvrAUvrABC ACyto −0.311.34rlmJRibosomal l