Identification of a New Member of the Phage Shock Protein Response in Escherichia coli, the Phage Shock Protein G (PspG)статья из журнала
Аннотация: The phage shock protein operon (pspABCDE) of Escherichia coli is strongly up-regulated in response to overexpression of the filamentous phage secretin protein IV (pIV) and by many other stress conditions including defects in protein export. PspA has an established role in maintenance of the proton-motive force of the cell under stress conditions. Here we present evidence for a new member of the phage shock response in E. coli. Using transcriptional profiling, we show that the synthesis of pIV in E. coli leads to a highly restricted response limited to the up-regulation of the psp operon genes and yjbO. The psp operon and yjbO are also up-regulated in response to pIV in Salmonella enterica serovar Typhimurium. yjbO is a highly conserved gene found exclusively in bacteria that contain a psp operon but is physically unlinked to the psp operon. yjbO encodes a putative inner membrane protein that is co-controlled with the psp operon genes and is predicted to be an effector of the psp response in E. coli. We present evidence that yjbO expression is driven by σ54-RNA polymerase, activated by PspF and integration host factor, and negatively regulated by PspA. PspF specifically regulates only members of the PspF regulon: pspABCDE and yjbO. We found that increased expression of YjbO results in decreased motility of bacteria. Because yjbO is co-conserved and co-regulated with the psp operon and is a member of the phage shock protein F regulon, we propose that yjbO be renamed pspG. The phage shock protein operon (pspABCDE) of Escherichia coli is strongly up-regulated in response to overexpression of the filamentous phage secretin protein IV (pIV) and by many other stress conditions including defects in protein export. PspA has an established role in maintenance of the proton-motive force of the cell under stress conditions. Here we present evidence for a new member of the phage shock response in E. coli. Using transcriptional profiling, we show that the synthesis of pIV in E. coli leads to a highly restricted response limited to the up-regulation of the psp operon genes and yjbO. The psp operon and yjbO are also up-regulated in response to pIV in Salmonella enterica serovar Typhimurium. yjbO is a highly conserved gene found exclusively in bacteria that contain a psp operon but is physically unlinked to the psp operon. yjbO encodes a putative inner membrane protein that is co-controlled with the psp operon genes and is predicted to be an effector of the psp response in E. coli. We present evidence that yjbO expression is driven by σ54-RNA polymerase, activated by PspF and integration host factor, and negatively regulated by PspA. PspF specifically regulates only members of the PspF regulon: pspABCDE and yjbO. We found that increased expression of YjbO results in decreased motility of bacteria. Because yjbO is co-conserved and co-regulated with the psp operon and is a member of the phage shock protein F regulon, we propose that yjbO be renamed pspG. The phage shock protein operon (pspABCDE) was first characterized in Escherichia coli (1Brissette J.L. Russel M. Weiner L. Model P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 862-866Crossref PubMed Scopus (205) Google Scholar) and is highly conserved in many Gram-negative bacteria including several pathogens. There is good evidence that the psp genes are involved in protecting the bacterial cell during infectious processes. For example, pspC mutants of Yersinia enterocolitica are severely attenuated for virulence during infection (2Darwin A.J. Miller V.L. Mol. Microbiol. 1999; 32: 51-62Crossref PubMed Scopus (186) Google Scholar) and exhibit growth defects when the type III secretion system is expressed (3Darwin A.J. Miller V.L. Mol. Microbiol. 2001; 39: 429-444Crossref PubMed Scopus (80) Google Scholar). Significantly, the psp genes are among the most highly up-regulated genes in Salmonella typhimurium during macrophage infection (4Eriksson S. Lucchini S. Thompson A. Rhen M. Hinton J.C. Mol. Microbiol. 2003; 47: 103-118Crossref PubMed Scopus (714) Google Scholar). The psp operon is also up-regulated during swarming in S. typhimurium (5Wang Q. Frye J.G. McClelland M. Harshey R.M. Mol. Microbiol. 2004; 52: 169-187Crossref PubMed Scopus (178) Google Scholar) and during biofilm formation in E. coli (6Beloin C. Valle J. Latour-Lambert P. Faure P. Kzreminski M. Balestrino D. Haagensen J.A. Molin S. Prensier G. Arbeille B. Ghigo J.M. Mol. Microbiol. 2004; 51: 659-674Crossref PubMed Scopus (366) Google Scholar). Expression of the psp operon in E. coli is induced by protein IV (pIV), 1The abbreviations used are: pIV, protein IV; IHF, integration host factor; IPTG, isopropyl 1-thio-β-d-galactopyranoside; RT, reverse transcriptase. a secretin from filamentous phage f1 (1Brissette J.L. Russel M. Weiner L. Model P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 862-866Crossref PubMed Scopus (205) Google Scholar). pIV forms a pore in the bacterial outer membrane that is required for the assembly and export of filamentous phage (7Russel M. Kazmierczak B. J. Bacteriol. 1993; 175: 3998-4007Crossref PubMed Google Scholar, 8Opalka N. Beckmann R. Boisset N. Simon M.N. Russel M. Darst S.A. J. Mol. Biol. 2003; 325: 461-470Crossref PubMed Scopus (92) Google Scholar). The pIV protein is the founding member of a large family of bacterial secretins, all of which form large multimeric export channels in the outer membrane. Overexpression of several secretins, often components of the type II and type III bacterial secretion systems, has also been shown to induce expression of the psp operon (e.g. Refs. 7Russel M. Kazmierczak B. J. Bacteriol. 1993; 175: 3998-4007Crossref PubMed Google Scholar and 9Possot O. d'Enfert C. Reyss I. Pugsley A.P. Mol. Microbiol. 1992; 6: 95-105Crossref PubMed Scopus (89) Google Scholar) establishing that the response is not restricted to a phage protein. Expression of the psp operon can also be induced following overexpression of mutant forms of the outer membrane protein PhoE that are not efficiently secreted (10Kleerebezem M. Tommassen J. Mol. Microbiol. 1993; 7: 947-956Crossref PubMed Scopus (66) Google Scholar). PspA synthesis is switched on under conditions that block or reduce the efficiency of the export apparatus, for example, mutants in secA, secD, and secF (10Kleerebezem M. Tommassen J. Mol. Microbiol. 1993; 7: 947-956Crossref PubMed Scopus (66) Google Scholar) and depletion of YidC (11van der Laan M. Urbanus M.L. Ten Hagen-Jongman C.M. Nouwen N. Oudega B. Harms N. Driessen A.J. Luirink J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5801-5806Crossref PubMed Scopus (127) Google Scholar, 12Jones S.E. Lloyd L.J. Tan K.K. Buck M. J. Bacteriol. 2003; 185: 6707-6711Crossref PubMed Scopus (49) Google Scholar). Mutations in components of the twin-arginine translocation pathway also leads to PspA induction under anaerobic conditions (Ref. 13DeLisa M.P. Lee P. Palmer T. Georgiou G. J. Bacteriol. 2004; 186: 366-373Crossref PubMed Scopus (131) Google Scholar and see also Ref. 12Jones S.E. Lloyd L.J. Tan K.K. Buck M. J. Bacteriol. 2003; 185: 6707-6711Crossref PubMed Scopus (49) Google Scholar). Other more general stresses including extreme heat shock (50 °C), hyperosmotic shock, ethanol treatment (10%), and uncouplers of proton-motive force induce psp (reviewed in Ref. 14Model P. Jovanovic G. Dworkin J. Mol. Microbiol. 1997; 24: 255-261Crossref PubMed Scopus (175) Google Scholar). The common factor that may link psp-inducing stresses is their effect in dissipating proton-motive force. Indeed, it is significant that PspA, an effector protein of the phage shock response, is known to be involved in maintaining proton-motive force under stress conditions (15Kleerebezem M. Crielaard W. Tommassen J. EMBO J. 1996; 15: 162-171Crossref PubMed Scopus (159) Google Scholar). In addition to Psp protein homologues in other Gram-negative bacteria, a PspA homologue (VIPP1) has been found in Synechocystis, which is thought to be important in thylakoid formation, consistent with a role of PspA in sustaining membrane function (16Westphal S. Heins L. Soll J. Vothknecht U.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4243-4248Crossref PubMed Scopus (140) Google Scholar). Psp proteins mediate regulation of the psp operon (17Weiner L. Brissette J.L. Model P. Genes Dev. 1991; 5: 1912-1923Crossref PubMed Scopus (99) Google Scholar, 18Adams H. Teertstra W. Koster M. Tommassen J. FEBS Lett. 2002; 518: 173-176Crossref PubMed Scopus (46) Google Scholar). Transcription of the psp operon is driven by the σ54-RNA polymerase (σ54-RNAP) (17Weiner L. Brissette J.L. Model P. Genes Dev. 1991; 5: 1912-1923Crossref PubMed Scopus (99) Google Scholar), which is activated by the enhancer binding protein PspF (19Jovanovic G. Weiner L. Model P. J. Bacteriol. 1996; 178: 1936-1945Crossref PubMed Google Scholar) and facilitated by integration host factor (IHF) (20Jovanovic G. Model P. Mol. Microbiol. 1997; 25: 473-481Crossref PubMed Scopus (32) Google Scholar). The expression of PspF is negatively autogenously controlled (21Jovanovic G. Dworkin J. Model P. J. Bacteriol. 1997; 179: 5232-5237Crossref PubMed Google Scholar). PspA negatively regulates psp transcription by binding to the activator protein PspF (22Dworkin J. Jovanovic G. Model P. J. Bacteriol. 2000; 182: 311-319Crossref PubMed Scopus (79) Google Scholar, 23Elderkin S. Jones S. Schumacher J. Studholme D. Buck M. J. Mol. Biol. 2002; 320: 23-37Crossref PubMed Scopus (92) Google Scholar). Conversely, PspB and PspC act as positive regulators of psp operon transcription by overcoming the negative regulation imposed by PspA under specific inducing conditions (e.g. pIV) (17Weiner L. Brissette J.L. Model P. Genes Dev. 1991; 5: 1912-1923Crossref PubMed Scopus (99) Google Scholar, 24Brissette J.L. Weiner L. Ripmaster T.L. Model P. J. Mol. Biol. 1991; 220: 35-48Crossref PubMed Scopus (113) Google Scholar, 25Weiner L. Brissette J.L. Ramani N. Model P. Nucleic Acids Res. 1995; 23: 2030-2036Crossref PubMed Scopus (34) Google Scholar). Phenotypes of cells lacking the psp operon are very subtle and include reduced survival in stationary phase at alkaline pH and changed motility (14Model P. Jovanovic G. Dworkin J. Mol. Microbiol. 1997; 24: 255-261Crossref PubMed Scopus (175) Google Scholar). Here we have used whole genome transcriptional profiling to determine the global effect of pIV synthesis in E. coli. In the highly restricted response we have identified one new gene associated with the psp system, pspG (previously yjbO). pspG is physically unlinked with the psp operon but is co-conserved and co-regulated with the psp operon genes by σ54, PspF, IHF, and PspA. Several lines of evidence suggest that PspG is an effector of the phage shock system and not a regulator of psp expression. Bacterial Strains and Plasmids—The bacterial strains and plasmids used in this study are described in Table I. MG1655ΔpspA, MG1655ΔpspBC, and MG1655ΔpspF were constructed as described in Ref. 12Jones S.E. Lloyd L.J. Tan K.K. Buck M. J. Bacteriol. 2003; 185: 6707-6711Crossref PubMed Scopus (49) Google Scholar. MVA29 was constructed by transducing ΔpspABC::kn from J134 (17Weiner L. Brissette J.L. Model P. Genes Dev. 1991; 5: 1912-1923Crossref PubMed Scopus (99) Google Scholar) into MG1655. MVA19 was constructed by transducing ΔpspABC::kn from J134 and pspF::mTn10-tet from K1527 (19Jovanovic G. Weiner L. Model P. J. Bacteriol. 1996; 178: 1936-1945Crossref PubMed Google Scholar) into MG1655. MVA40 was constructed by transducing pspG::kn from JWK5716_1 (Km+) into MG1655. MVA42 was constructed by transducing pspG::kn from JWK5716_1 (Km+) into MG1655ΔpspA. Strains were grown aerobically with shaking at 37 °C. For microarray analyses, strains were grown to mid-exponential phase in N-C-minimal media (33Kustu S. Burton D. Garcia E. McCarter L. McFarland N. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4576-4580Crossref PubMed Scopus (53) Google Scholar) supplemented with 0.4% glucose as carbon source and 10 mm NH4Cl as nitrogen source. For all other experiments, strains were grown in Luria-Bertani (LB) media (34Sambrook J. Fritsch E. Maniatas T. Molecular Cloning, A Laboratory Manual. 2 Ed. Cold Spring Harbor Press Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Antibiotics were used at the following concentrations: ampicillin (ap), 100 μgml-1; chloramphenocol (cm), 25 μg ml-1; tetracycline (tet), 10 μg ml-1; and kanamycin (kn), 30 μgml-1. IPTG was added to a final concentration of 1 mm and arabinose was added to a final concentration of 0.4% when required unless otherwise stated. Transformations and P1vir transductions were performed as described in Ref. 35Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar.Table IBacterial strains and plasmidsStrain or plasmidGenotype or relevant characteristicsRef. or sourceBacterial strainsE. coliBW25113Wild-typeCGSC# 7739Gift from Hirotada Mori26Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google ScholarJWK3169_1 (Km+)BW25113 rpoN::knGift from Hirotada MoriJWK5716_1 (Km+)BW25113 pspG::knGift from Hirotada MoriMC1061lac −Gift from Majorie Russel (27Casadaban M.J. Cohen S.N. J. Mol. Biol. 1980; 138: 179-207Crossref PubMed Scopus (1753) Google Scholar)MC1068MC1061ΔhimA::Tn10 (tcr)A gift from Michael ChandlerMG1655Wild-typeCGSC No. 7740 (28Blattner F.R. Plunkett 3rd, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6056) Google Scholar)MG1655ΔpspAMG1655 ΔpspAThis workMG1655ΔpspBCMG1655 ΔpspBCThis workMVA29MG1655ΔpspABC::knThis workMVA19MG1655ΔpspABC::kn pspF::mTn10-tet (pspFΔHTH)This workMG1655ΔpspFMG1655 ΔpspFThis workMVA40MG1655 pspG::knThis workMVA42MG1655ΔpspA pspG::knThis workS. typhimuriumLT2Wild-type29McClelland M. Sanderson K.E. Spieth J. Clifton S.W. Latreille P. Courtney L. Porwollik S. Ali J. Dante M. Du F. Hou S. Layman D. Leonard S. Nguyen C. Scott K. Holmes A. Grewal N. Mulvaney E. Ryan E. Sun H. Florea L. Miller W. Stoneking T. Nhan M. Waterston R. Wilson R.K. Nature. 2001; 413: 852-856Crossref PubMed Scopus (1524) Google ScholarPlasmidspGZ119EHIPTH-inducible tac promoter expression vector. cmr.Gift from Marjorie Russel (30Lessl M. Balzer D. Lurz R. Waters V.L. Guiney D.G. Lanka E. J. Bacteriol. 1992; 174: 2493-2500Crossref PubMed Google Scholar)pPMR129pGZ119EH habouring pIV. cmrGift from Marjorie Russel (31Daefler S. Russel M. Model P. J. Mol. Biol. 1997; 266: 978-992Crossref PubMed Scopus (34) Google Scholar)pMR25lacZ transcriptional fusion vector. tetr12Jones S.E. Lloyd L.J. Tan K.K. Buck M. J. Bacteriol. 2003; 185: 6707-6711Crossref PubMed Scopus (49) Google ScholarpSJ1pMR25 with pspA promoter region plus the first 21 amino acids of PspA cloned into the mcs (EcoRI-EcoRI). tetr12Jones S.E. Lloyd L.J. Tan K.K. Buck M. J. Bacteriol. 2003; 185: 6707-6711Crossref PubMed Scopus (49) Google ScholarpMC1403lacZ translational fusion vector. apr32Casadaban M.J. Chou J. Cohen S.N. J. Bacteriol. 1980; 143: 971-980Crossref PubMed Google ScholarpLL1The pspG promoter region plus the first 6 amino acids of PspG cloned inframe into the mcs of pMC1403 (EcoRI-BamHI). aprThis workpLL2The pspG promoter region plus the first 6 amino acids of PspG cloned out of frame into the mcs of pMC1403 (EcoRI-BamHI). aprThis workpSLE1pspA promoter region (EcoRI-BamHI)cloned into the vector pTE103 (56Elliott T. Geiduschek E.P. Cell. 1984; 36: 211-219Abstract Full Text PDF PubMed Scopus (138) Google Scholar) apr23Elderkin S. Jones S. Schumacher J. Studholme D. Buck M. J. Mol. Biol. 2002; 320: 23-37Crossref PubMed Scopus (92) Google ScholarpJH2pspG promoter region (EcoRI-BamHI) subcloned from pLL1 into pTE103 aprThis workpBAD18-cmVector cmrA gift from Jonathan BeckwithpLL8pspG (XbaI-HindIII) cloned into the mcs of pBAD18-cm. cmrThis work Open table in a new tab Microarray Analysis—Growth of cultures was halted with 1/10 volume of 5% phenol in ethanol and RNA was extracted with hot phenol/SDS (36Lin-Chao S. Cohen S.N. Cell. 1991; 65: 1233-1242Abstract Full Text PDF PubMed Scopus (180) Google Scholar). RNA was treated with DNase I for 1 h at 37 °C. For the initial microarray experiments, RNA was fluorescently labeled during reverse transcription and cDNA was hybridized to E. coli PCR product microarrays according to S. Kustu and co-workers (37Zimmer D.P. Soupene E. Lee H.L. Wendisch V.F. Khodursky A.B. Peter B.J. Bender R.A. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14674-14679Crossref PubMed Scopus (291) Google Scholar). Hybridization, scanning, and normalization were carried out as described (38Khodursky A.B. Peter B.J. Cozzarelli N.R. Botstein D. Brown P.O. Yanofsky C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12170-12175Crossref PubMed Scopus (176) Google Scholar) and genome images were prepared (37Zimmer D.P. Soupene E. Lee H.L. Wendisch V.F. Khodursky A.B. Peter B.J. Bender R.A. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14674-14679Crossref PubMed Scopus (291) Google Scholar). Experiments were performed in duplicate with a dye swap. The microarray data for the pIV experiments were generated with fluorescently labeled genomic DNA as a reference channel in each experiment using E. coli and S. typhimurium PCR product microarrays printed at IFR (39Anjum M.F. Lucchini S. Thompson A. Hinton J.C. Woodward M.J. Infect. Immun. 2003; 71: 4674-4683Crossref PubMed Scopus (65) Google Scholar, 40Clements M.O. Eriksson S. Thompson A. Lucchini S. Hinton J.C. Normark S. Rhen M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8784-8789Crossref PubMed Scopus (140) Google Scholar, 41Thompson A. Lucchini S. Hinton J.C. Trends Microbiol. 2001; 9: 154-156Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Experiments were performed in quadruplicate, consisting of two biological replicates and two technical replicates. Microarray slides were scanned with a Genepix 4000B scanner (Axon Instruments). Fluorescent spot and local background intensities were quantified using Genepix Pro software. For labeling, hybridization, and data analysis protocols and details of statistical filtering procedures, see the online site (ifr.bbsrc.ac.uk/Safety/Microarrays/#Protocols). Further statistical analysis was carried out using Cyber-T (visitor.ics.uci.edu/genex/cybert/). RT-PCR—Qiagen® One-step RT-PCR kit was used according to the manufacturer' instructions to amplify pspA (20 cycles) and pspG (35 cycles) from RNA samples. For amplifying pspA the primers RT-PspA(a) (5′-CTCGCTTTGCCGACATCGTGAATG-3′) and RT-PspA(b) (5′-TGCCAGTTGTTCGCTGATTGCATC-3′) were used. For amplifying pspG the primers RT-PspG(a) (5′-GCTGGAACTACTTTTTGTGATTGG-3′) and RT-PspG(b) (5′-CGCCAGCGGTCATAACGCTGATAT-3′) were used. Western Blotting—Western blotting was carried out as described (23Elderkin S. Jones S. Schumacher J. Studholme D. Buck M. J. Mol. Biol. 2002; 320: 23-37Crossref PubMed Scopus (92) Google Scholar) using primary antibodies to PspA (12Jones S.E. Lloyd L.J. Tan K.K. Buck M. J. Bacteriol. 2003; 185: 6707-6711Crossref PubMed Scopus (49) Google Scholar) and pIV (a gift from Marjorie Russel). pIV antibodies were used at a 1:10,000 dilution with donkey anti-rabbit secondary antibodies (Amersham Biosciences). β-Galactosidase Assays—β-Galactosidase assays were carried out as described (35Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). Bioinformatics Methods—Fuzzpro (EMBOSS programs) was used to search for consensus sequences in regions of DNA by allowing small numbers of mismatches to be introduced to the search. DNase I Footprinting Assays—DNase I footprinting reactions (10 μl) were carried out at 37 °C in STA buffer (25 mm Tris acetate, pH 8.0, 8 mm magnesium acetate, 10 mm KCl, 1 mm dithiothreitol, 3.5% (w/v) polyethylene glycol 8000) essentially as described (42Burrows P.C. Severinov K. Ishihama A. Buck M. Wigneshweraraj S.R. J. Biol. Chem. 2003; 278: 29728-29743Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Briefly, 0-400 nm Eσ54 (reconstituted in situ with 1:2 molar ratio of E and σ54) or 0-1 μmE. coli PspF was incubated with 15 nm pLL1 for 10 min and treated with 1.75 × 10-3 units of DNase I (Amersham Biosciences) for 2 min. The DNase I reaction was quenched by the addition of DNase I stop buffer (400 mm NaCl, 30 mm EDTA, 1% SDS) and the DNA was purified using QIAquick spin columns (Qiagen) according to the manufacturer's instructions. DNase I protected regions were identified by primer extension PCR as described (42Burrows P.C. Severinov K. Ishihama A. Buck M. Wigneshweraraj S.R. J. Biol. Chem. 2003; 278: 29728-29743Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) using 0.5 μl of 1 μm γ-32P-end labeled primers pPspG1 (5′-GAACACGCGCTCAAACTGGTGGCGG-3′) (for σ54 binding) and pPspG2 (5′-CTGGCGCGCGGCAGTGGCGGC-3′) (for PspF binding). In Vitro Transcription Assay—In vitro transcription reactions (10 μl) were carried out as described (43Wigneshweraraj S.R. Nechaev S. Bordes P. Jones S. Cannon W. Severinov K. Buck M. Methods Enzymol. 2003; 370: 646-657Crossref PubMed Scopus (28) Google Scholar) with a 1:5 ratio RNAP to σ54 and with plasmids pSLE1 (pspA) or pJH2 (pspG). Motility Assay—Motility assays were carried out using motility agar (1% tryptone, 0.5% NaCl and 0.3% agar) plus the appropriate antibiotic. 2 μl of a fully grown LB overnight culture was pipetted into the motility agar, plates were incubated at 37 °C for 6 h, and zones of motility were measured in millimeters. pIV Secretin Stress Results in the Up-regulation of pspABCDE and pspG—To examine the transcriptional response to pIV-induced stress in E. coli wild type MG1655 cells containing the plasmids, pPMR129 (pIV) or pGZ119EH (vector control) were grown to mid-log phase, expression from the plasmids was induced with IPTG for 1 h, and cells were harvested for RNA extraction. The synthesis of pIV reached high levels after 1 h (see Supplementary Materials and Fig. 1) indicating that it should elicit a full cellular response but did not lead to reduced growth rates, or reduced yields of cells, indicating a lack of toxicity. Microarray analyses showed increased levels of psp operon transcripts in pIV-expressing cells compared with the vector control (Fig. 1A). We correlated activation of the pspA promoter with increased levels of the PspA protein using Western blotting (data not shown). Other than the psp operon genes, only a single gene, yjbO, showed a significant and sizeable up-regulation in response to pIV secretin stress (Fig. 1A; see Supplementary Materials Tables I and III). This data indicates that large transcriptional responses of E. coli to pIV are very rare and identify a new gene involved in the phage shock response, yjbO. We propose to rename this gene pspG. To confirm that transcript levels of pspG are increased in wild type MG1655 cells expressing pIV, RT-PCR was carried out on the RNA samples used for the microarray experiments. RT-PCR clearly demonstrates that pspG transcription is up-regulated, along with pspA transcription, in pIV-expressing MG1655 cells compared with the vector control (Fig. 2A). β-Galactosidase assays using a translational reporter for PspG (pLL1) confirm that PspG is produced in response to pIV in MC1061 cells (Fig. 2B). To determine whether the response to pIV detected in E. coli is conserved in other bacteria that contain the psp operon, a pIV expression experiment was carried out in Salmonella enterica serovar Typhimurium LT2. There is significant up-regulation of psp operon and pspG transcripts in the pIV-expressing S. typhimurium cells (Fig. 1B; see Supplementary Materials Tables I and III). The transcriptional response to pIV in S. typhimurium resembles that of E. coli in that the response to pIV secretin stress is highly restricted. Our comparative transcriptomic analysis of responses of E. coli and S. typhimurium to pIV shows that the common core of up-regulated genes are pspABCDE and pspG. Transcription of pspF does not show any change in response to pIV expression, consistent with control of PspF being exclusively at the level of activity (21Jovanovic G. Dworkin J. Model P. J. Bacteriol. 1997; 179: 5232-5237Crossref PubMed Google Scholar). Such a limited and specific response to pIV stress resembles the response of E. coli cells to IPTG, a gratuitous inducer of the E. coli lac operon. We performed a microarray experiment to show that IPTG only causes significant increased expression of lac operon genes in MG1655, no other transcriptome changes occur (see Supplementary Materials Table II). As with lac promoter activity induced by IPTG, the effect of pIV inducing stimulus under our growth conditions in E. coli appears to be close to gratuitous (44Monod J. Cohn M. Adv. Enzymol. Relat. Sub. Biochem. 1952; 13: 67-119PubMed Google Scholar). pspG Transcription Is Regulated by psp-encoded Proteins—To examine the effect of overexpression of the psp genes on the transcriptome, transcripts from cells lacking the negative regulator PspA (MG1655ΔpspA) were compared with transcripts from cells lacking the positive regulator PspF (MG1655ΔpspF). In MG1655ΔpspA, the psp operon is expressed at high levels because the negative regulator of its transcription has been removed. Conversely, in MG1655ΔpspF, expression of the psp operon is completely absent because the activator protein required for σ54-RNAP driven transcription has been removed. Levels of psp expression in the MG1655ΔpspA strain are therefore close to levels in wild type cells expressing pIV, but without the production of PspA. It is clear from Fig. 3 that pspBCDE is transcribed at high levels in MG1655ΔpspA compared with MG1655ΔpspF. As with the response of wild type MG1655 cells to pIV stress, there is very little transcriptional change across the whole genome in response to overexpression of psp genes in MG1655ΔpspA, with the clear exception of the gene pspG, which is strongly up-regulated. The level of expression from the pspA promoter and the pspG promoter was similar when we compared wild type cells expressing pIV to cells lacking PspA, the only known negative regulator of psp expression. This result establishes that pIV is a strong and effective inducing signal. We considered that the synthesis of pIV in mutants unable to mount a wild type Psp response might result in additional changes in the transcriptome to compensate for the inability of the cell to adapt to stress arising through the failure to express the psp genes. MG1655ΔpspA, MG1655ΔpspBC, and MG1655ΔpspF cells containing pPMR129 (pIV) and pGZ119-EH (vector control) were grown to mid-log phase and induced with IPTG. The synthesis of pIV reached high levels after 1 h (see Supplementary Materials Fig. 1), which is consistent with the observation that filamentous phage grow normally in psp mutants (1Brissette J.L. Russel M. Weiner L. Model P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 862-866Crossref PubMed Scopus (205) Google Scholar). The psp mutant strains did not show any growth defects on expression of pIV. Microarray analysis showed that synthesis of pIV in MG1655ΔpspA, MG1655ΔpspBC, and MG1655ΔpspF does not cause any pIV-dependent changes in the transcriptome attributable to the loss of PspA, PspBC, or PspF, respectively (see Supplementary Materials Table I). pspG was not further up-regulated in MG1655ΔpspA, MG1655ΔpspBC, or MG1655ΔpspF cells when expressing pIV, probably because the psp operon, and therefore pspG, were constitutively on in MG1655ΔpspA and always off in MG1655ΔpspBC and MG1655ΔpspF. This data shows that expression of pspG is negatively regulated by PspA and its transcription may be activated by PspF via a σ54 promoter and positively regulated by PspBC. The psp Operon and pspG Are Co-conserved and Co-regulated—pspG and pspFpspABCDE are not physically linked on the chromosome, but pspG is highly conserved among bacteria in which the psp operon is conserved. Furthermore, all bacteria containing a recognizable psp operon carry a pspG homologue, and pspG homologues are not present in bacteria lacking a psp operon. This shows that the pspG and psp loci are co-conserved. PspG is a small (∼9 kDa) highly hydrophobic protein that is predicted to be an inner membrane protein (enzim.hu/hmmtop/http://www.cbs.dtu.dk/services/TMHMM/). Because our experiments indicated that pspG transcription is regulated by the same elements that regulate psp operon transcription, we used a bioinformatic approach to search the pspG promoter region for the control elements that are present in the psp operon promoter, which are binding sites for σ54, PspF, and IHF. Using the program fuzzpro (EMBOSS programs) and the consensus sequence WWWTCAA[N4]TTR for IHF binding (45Hales L.M. Gumport R.I. Gardner J.F. J. Bacteriol. 1994; 176: 2999-3006Crossref PubMed Google Scholar) and sequences GGCACGCAAATTGT for σ54 binding and TAGTGTAATTCGCTAACT for PspF binding (based on the σ54 and PspF binding sites in the pspA promoter) (20Jovanovic G. Model P. Mol. Microbiol. 1997; 25: 473-481Crossref PubMed Scopus (32) Google Scholar, 46Dworkin J. Jovanovic G. Model P. J. Mol. Biol. 1997; 273: 377-388Crossref PubMed Scopus (35) Google Scholar) we found potential binding sites for σ54, IHF, and PspF (Fig. 4). Using the translational fusion for pspG (pLL1) we found that σ54-RNAP and activation by PspF are required in vivo for pIV-induced PspG expression. In the wild type strains the basal level of PspG expression is extremely low, but is up-regulated upon induction with pIV. In mutant strains for σ54 and PspF, pspG expression is abolished both before and after
Год издания: 2004
Авторы: Louise Lloyd, Susan Jones, Goran Jovanović, Prasad Gyaneshwar, Matthew D. Rolfe, Arthur R. Thompson, Jay C. D. Hinton, Martin Buck
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Bacteriophages and microbial interactions, Bacterial Genetics and Biotechnology, Escherichia coli research studies
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IMAGINE - Repository of the Institute of molecular genetics and genetic engineering (University of Belgrade) (PDF)
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IMAGINE - Repository of the Institute of molecular genetics and genetic engineering (University of Belgrade) (HTML)
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Том: 279
Выпуск: 53
Страницы: 55707–55714