FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2091296993> ?p ?o ?g. }

• W2091296993 endingPage "5747" @default.
• W2091296993 startingPage "5738" @default.
• W2091296993 abstract "The temperature-dependent regulation of Shigella virulence genes is believed to be accomplished at the transcriptional stage by the regulators VirF and InvE. Several lines of evidence herein described indicate that post-transcriptional regulation of InvE expression plays a key role in the temperature-dependent regulation of virulence gene expression: (i) a considerable amount of invE mRNA continues to be transcribed under low temperature conditions, where the production of InvE protein is tightly repressed; (ii) the stability of invE mRNA markedly decreases, because its decay rate is significantly increased under the repressing conditions. Strikingly, in the hfq mutant of Shigella sonnei, a considerable amount of InvE protein was produced even at low temperature. This increase in the InvE level was found to be associated with the improved stability of invE mRNA, in agreement with the finding that the RNA chaperon Hfq influences post-transcriptional regulations of various genes. Consistently, overexpression of the Hfq protein decreased the production of InvE protein even under the expressing condition at 37 °C. The binding in vitro of purified Hfq protein to invE RNA was shown to be stronger at 30 °C than at 37 °C in two experiments, gel shift analysis and surface plasmon resonance (Biacore) analysis. These results altogether suggest that Hfq plays an important role in the temperature-dependent regulation of invE expression at the post-transcriptional step. The temperature-dependent regulation of Shigella virulence genes is believed to be accomplished at the transcriptional stage by the regulators VirF and InvE. Several lines of evidence herein described indicate that post-transcriptional regulation of InvE expression plays a key role in the temperature-dependent regulation of virulence gene expression: (i) a considerable amount of invE mRNA continues to be transcribed under low temperature conditions, where the production of InvE protein is tightly repressed; (ii) the stability of invE mRNA markedly decreases, because its decay rate is significantly increased under the repressing conditions. Strikingly, in the hfq mutant of Shigella sonnei, a considerable amount of InvE protein was produced even at low temperature. This increase in the InvE level was found to be associated with the improved stability of invE mRNA, in agreement with the finding that the RNA chaperon Hfq influences post-transcriptional regulations of various genes. Consistently, overexpression of the Hfq protein decreased the production of InvE protein even under the expressing condition at 37 °C. The binding in vitro of purified Hfq protein to invE RNA was shown to be stronger at 30 °C than at 37 °C in two experiments, gel shift analysis and surface plasmon resonance (Biacore) analysis. These results altogether suggest that Hfq plays an important role in the temperature-dependent regulation of invE expression at the post-transcriptional step. Type III secretion system (TTSS) 2The abbreviations used are:TTSSType III secretion systemIPTGisopropyl-1-thio-β-d-galactosideRTreverse transcription.2The abbreviations used are:TTSSType III secretion systemIPTGisopropyl-1-thio-β-d-galactosideRTreverse transcription. plays a key role in the expression of virulence by pathogenic Shigella. The expression of TTSS is tightly regulated by temperature in such a way that it is high at 37 °C but not at 30 °C (1Maurelli A.T. Sansonetti P.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2820-2824Crossref PubMed Scopus (148) Google Scholar, 2Maurelli A.T. Blackmon B. Curtiss R. II I Infect. Immun. 1984; 43: 195-201Crossref PubMed Google Scholar). This regulation apparently fits the life cycle of Shigella, because the expression of virulence genes is needed for its invasion and propagation in host animals, but the expression of virulence genes might be a potential burden for its survival in natural environment. Type III secretion system isopropyl-1-thio-β-d-galactoside reverse transcription. Type III secretion system isopropyl-1-thio-β-d-galactoside reverse transcription. The TTSS-associated genes of Shigella are encoded on the virulence plasmid and controlled by two regulator proteins VirF and InvE (VirB) (3Kato J. Ito K. Nakamura A. Watanabe H. Infect. Immun. 1989; 57: 1391-1398Crossref PubMed Google Scholar, 4Tobe T. Yoshikawa M. Mizuno T. Sasakawa C. J. Bacteriol. 1993; 175: 6142-6149Crossref PubMed Google Scholar). VirF, an AraC type transcriptional regulator, activates transcription of the invE (virB) gene (3Kato J. Ito K. Nakamura A. Watanabe H. Infect. Immun. 1989; 57: 1391-1398Crossref PubMed Google Scholar, 5Adler B. Sasakawa C. Tobe T. Makino S. Komatsu K. Yoshikawa M. Mol. Microbiol. 1989; 3: 627-635Crossref PubMed Scopus (135) Google Scholar, 6Watanabe H. Arakawa E. Ito K. Kato J. Nakamura A. J. Bacteriol. 1990; 172: 619-629Crossref PubMed Google Scholar, 7Nakayama S. Watanabe H. J. Bacteriol. 1995; 177: 5062-5069Crossref PubMed Google Scholar). The second regulatory protein, InvE, a homolog of a plasmid-partitioning factor, ParB (6Watanabe H. Arakawa E. Ito K. Kato J. Nakamura A. J. Bacteriol. 1990; 172: 619-629Crossref PubMed Google Scholar), with DNA binding activity (8Taniya T. Mitobe J. Nakayama S. Mingshan Q. Okuda K. Watanabe H. J. Bacteriol. 2003; 185: 5158-5165Crossref PubMed Scopus (26) Google Scholar), activates transcription of the mxi-spa and ipa genes encoding TTSS components by replacement of a global repressor H-NS, a histone-like DNA-binding protein (9Beloin C. Dorman C.J. Mol. Microbiol. 2003; 47: 825-838Crossref PubMed Scopus (74) Google Scholar). Various mechanisms have been proposed to explain the temperature-dependent regulation of TTSS (10Dorman C.J. Porter M.E. Mol. Microbiol. 1998; 29: 677-684Crossref PubMed Scopus (119) Google Scholar). For instance, a mutation of the hns gene in Shigella flexneri was reported to result in increased TTSS expression at the repressing temperature 30 °C (11Dorman C.J. Bhriain N.N. Higgins C.F. Nature. 1990; 344: 789-792Crossref PubMed Scopus (167) Google Scholar). The H-NS protein is now accepted as one of the key factors for temperature-dependent gene expression in various Gram-negative bacterias through the structural change of its oligomer formation at high temperature (12Ono S. Goldberg M.D. Olsson T. Esposito D. Hinton J.C. Ladbury J.E. Biochem. J. 2005; 391: 203-213Crossref PubMed Scopus (115) Google Scholar). In fact, the hns mutation increases the expression of virF promoter-lacZ fusion, and the addition of H-NS protein into the in vitro transcription system results in decreased transcription from the virF promoter in a temperature-dependent manner (13Falconi M. Colonna B. Prosseda G. Micheli G. Gualerzi C.O. EMBO J. 1998; 17: 7033-7043Crossref PubMed Scopus (223) Google Scholar). On the contrary, detailed analysis for transcription in vivo of virF and invE genes indicated that significant amounts of virF and invE mRNA are transcribed, albeit at a reduced level, at 30 °C in both wild-type S. flexneri and hns mutant at various growth phases (14Porter M.E. Dorman C.J. J. Bacteriol. 1997; 179: 6537-6550Crossref PubMed Google Scholar). Previously, we isolated a cpxA mutant, in which the expression of TTSS genes was significantly decreased. Characterization of the cpxA mutant revealed that the expression of InvE protein is controlled at the post-transcriptional level. Analysis with both transcriptional and translational fusion of invE-lacZ reporter plasmids showed that a sufficient amount of invE mRNA is transcribed, but the protein expression is repressed in the cpxA mutant (15Mitobe J. Arakawa E. Watanabe H. J. Bacteriol. 2005; 187: 107-113Crossref PubMed Scopus (37) Google Scholar). Finding of the post-transcriptional regulation in InvE expression allowed us to reexamine the transcription of the invE gene under the repressing condition at 30 °C. Using the invE-lacZ transcriptional fusion plasmid, a considerable level of β-galactosidase activity was detected at 30 °C, but with use of the invE translational fusion plasmid, a similar level of the β-galactosidase activity was not detected at 30 °C. Taking the results together, we propose the concept of post-transcriptional regulation for InvE expression. Supporting this model, an RNA chaperone, Hfq, was found to exert marked influence on the expression of the invE gene. Sequencing—The hfq gene of Shigella sonnei HW383 was sequenced by ABI-PRISM™ 310 genetic analyzer (PerkinElmer Life Sciences), using oligonucleotide primers (see supplemental Table S1) hfq1 and hfq2. The sequenced hfq gene in S. sonnei HW383 was identical to that of Escherichia coli K-12 strain, submitted to the DNA sequence data base DDBJ™ with the accession number AB293541. The upstream region of virF gene in S. sonnei HW383 was sequenced using oligonucleotide primers virF1 and virF2 (GenBank™/EBI Data Bank accession number AB300612). The sequence containing a putative transposase was fully identical to that of S. sonnei strain Ss046 (nucleotides 38934-37955 of GenBank™/EBI Data Bank accession number CP000039) and Shigella boydii strain Sb227 (nucleotides 37617-38596 of GenBank™/EBI Data Bank accession number CP000037). Expression Plasmids—The hfq gene (nucleotides 8002-8368 of Genbank™/EBI Data Bank Accession number AE000489) was amplified by PCR from genomic DNA of E. coli K-12 strain BW25113 (16Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11136) Google Scholar) using the primers hfq3 and hfq4. The amplified DNA product was digested with the restriction enzymes NcoI and XbaI, ligated into the plasmid pTrc99A (17Amann E. Ochs B. Abel K.J. Gene (Amst.). 1988; 69: 301-315Crossref PubMed Scopus (877) Google Scholar) (Genbank™/EBI Data Bank accession number U13872) to produce pTrc-hfq. The same DNA sequence was amplified with primers hfq5 and hfq6. The amplified DNA product was digested with the restriction enzymes NdeI and XhoI, ligated into the plasmid pET22b (Novagen, Madison, WI) without the addition of the histidine-tag sequence to produce pET-hfq. For construction of pBAD-invE, the invE sequence (nucleotides 209-1190 of Genbank™/EBI Data Bank accession number M33790) was amplified using oligonucleotide primers invE1 and invE2. The amplified DNA product was digested with the restriction enzymes NheI and HindIII, ligated into the plasmid pBAD24 (18Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3941) Google Scholar) (Genbank™/EBI Data Bank Accession number X81837) to produce pBAD-invE. The integrity of the sequences in the above plasmids were checked by a PerkinElmer Life Sciences 310 DNA sequencer. All primers were synthesized by Grainer Co. (Tokyo, Japan). Construction of Mutant Strains—For the construction of deletion mutants for hfq, hns, and invE genes, a PCR-based gene disruption technique was applied for wild-type S. sonnei strain MS390 as described previously (15Mitobe J. Arakawa E. Watanabe H. J. Bacteriol. 2005; 187: 107-113Crossref PubMed Scopus (37) Google Scholar, 16Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11136) Google Scholar). A kanamycin-resistant gene cassette in pKD13 (16Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11136) Google Scholar) was amplified with the following primers: for MS4831, hfq7 and hfq8; for MS4841, hns1 and hns2; and for MS1632, invE3 and invE4. The kanamycin-resistant gene cassette was removed as described (16Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11136) Google Scholar). Integrity of the nonpolar deletion sequences was confirmed by DNA sequencing. For construction of the hfq::aphA deletion mutants of S. sonnei MS4835 and S. flexneri MF4835, a three-step PCR-based gene disruption technique (19Derbise A. Lesic B. Dacheux D. Ghigo J.M. Carniel E. FEMS Immunol. Med. Microbiol. 2003; 38: 113-116Crossref PubMed Scopus (199) Google Scholar) was applied for both wild-type strains of S. sonnei MS390 and S. flexneri 2457T (20Wei J. Goldberg M.B. Burland V. Venkatesan M.M. Deng W. Fournier G. Mayhew G.F. Plunkett G. II I Rose D.J. Darling A. Mau B. Perna N.T. Payne S.M. Runyen-Janecky L.J. Zhou S. Schwartz D.C. Blattner F.R. Infect. Immun. 2003; 71: 2775-2786Crossref PubMed Scopus (331) Google Scholar), respectively, using primers hfqU1, hfqU2, hfqD1, and hfqD2. A kanamycin-resistant gene cassette aphA was amplified from pTH18ks5 (21Hashimoto-Gotoh T. Yamaguchi M. Yasojima K. Tsujimura A. Wakabayashi Y. Watanabe Y. Gene (Amst.). 2000; 241: 185-191Crossref PubMed Scopus (61) Google Scholar) using Km1 and Km2 primers. For construction of the S. sonnei pinvE::paraBAD mutant MS5512, a modification of the three-step PCR-based gene replacement technique (22Roux A. Beloin C. Ghigo J.M. J. Bacteriol. 2005; 187: 1001-1013Crossref PubMed Scopus (89) Google Scholar) was applied for MS390 using invEU1, invEU2, invED1, and invED2 primers. The gene cassette, composed of the chloramphenicol acetyltransferase gene, the araC repressor, and the promoter region for the araBAD operon (nucleotides 70188-71339 of GenBank™/EBI Data Bank accession number AP009048.1), was amplified from an E. coli strain BW25113 chloramphenicol acetyltransferase-araC (22Roux A. Beloin C. Ghigo J.M. J. Bacteriol. 2005; 187: 1001-1013Crossref PubMed Scopus (89) Google Scholar), using the araC1 and araC2 primers (see Fig. 2A). After construction of all mutants, the presence of the virulence plasmid that encodes the form I antigen was confirmed by agglutination with diagnostic antiserum for S. sonnei (Denka Seiken, Tokyo, Japan) or PCR of the invE gene for S. flexneri 2457T using primers invE1 and invE2. At least two independent deletion mutants were constructed for each deletion, which showed the same results for all experiments. Bacterial strains and plasmids used in this study are listed in Table 1.TABLE 1Bacterial strains and plasmids used in this studyBacterial strain or plasmidGenotypeSource/ReferenceE. coli K-12BW25113Ref. 16S. sonneiHW383S. sonnei wild-type strain (Tcr)Ref. 6HW506S. sonnei HW383 without pSS120 plasmid (noninvasive)Ref. 6MS390HW383 (Tcs)Ref. 15MS1632MS390 ΔinvEThis studyMS2830MS390 ΔcpxR (cpxR: chromosomal activator of virF gene)Ref. 15MS4831MS390 ΔhfqThis studyMS4835MS390 Δhfq::aphAThis studyMS4841MS390 ΔhnsThis studyMS5512MS390 ΔpinvE::paraBADThis studyS. flexneri2457TS. flexneri 2a wild-type strainRef. 20MF48352457T Δhfq::aphAThis studyPlasmidspBAD24Ref. 18pBAD-invEPCR-amplified invE gene was cloned into pBAD24 (Apr)This studypET22bNovagenpET-hfqPCR-amplified hfq gene was cloned into pET22b (Apr)This studypHW848virF-lacZ translational fusion plasmid (Cmr)Ref. 7pJM4320invE-lacZYA transcriptional fusion in pTH18cs5 (Cmr)Ref. 15pJM4321invE-lacZYA translational fusion in pTH18cs5 (Cmr)Ref. 15pTH18cs5pSC101 ori, low copy plasmid (Cmr)Ref. 21pTH18ks5pSC101 ori, low copy plasmid (Kmr)Ref. 21pTrc99ARef. 17pTrc-hfqPCR-amplified hfq gene was cloned into pTrc99A (Apr)This study Open table in a new tab Bacterial Culture Condition—Luria-Bertani (LB) medium (LB Lenox; Difco) was used for bacterial growth. Concentrations of antibiotics were as follows: ampicillin, 50 μg/ml; chloramphenicol, 12.5 μg/ml; rifampicin, 200 μg/ml (Sigma). Usage of antibiotics is specified by the figure legends for each experiment. For all experiments, each strain was inoculated into 2 ml of LB medium and grown overnight at 30 °C with shaking (150 rpm) in a water bath. The precultures were diluted 50-fold for the 30 °C culture and 100-fold for the 37 °C culture to give similar incubation periods (2.5-3 h) in a pair of 5-ml fresh LB medium samples. The samples were incubated at both 30 and 37 °C with shaking at 150 rpm, monitored for turbidity at 600 nm by a spectrophotometer (Spectronic™ 20+, Shimadzu, Kyoto, Japan), and harvested at A600 = 0.8. Each 50 μl of the whole culture was used for measurement of β-galactosidase activities as previously described (23Miller J.H. 3rd Ed. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 72-74Google Scholar), or 10 μl of the whole culture was subjected to Western blotting with 10% SDS-PAGE as previously described (24Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2002Google Scholar). IpaB and InvE proteins were detected by anti-IpaB monoclonal antibody (25Ito K. Nakajima T. Sasaki T. Watanabe H. Microbiol. Immunol. 1991; 35: 335-341Crossref PubMed Scopus (5) Google Scholar) or anti-InvE polyclonal antibody (15Mitobe J. Arakawa E. Watanabe H. J. Bacteriol. 2005; 187: 107-113Crossref PubMed Scopus (37) Google Scholar). Hfq and H-NS proteins were detected as previously described (26Azam T.A. Ishihama A. J. Biol. Chem. 1999; 274: 33105-33113Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 27Jishage M. Ishihama A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4953-4958Crossref PubMed Scopus (152) Google Scholar). RNA Preparation and Detection—Each 2 ml of the whole culture was quickly mixed with 150 μl of 5% (v/v) water-saturated phenol in ethanol (28Bhagwat A.A. Phadke R.P. Wheeler D. Kalantre S. Gudipati M. Bhagwat M. J. Microbiol. Methods. 2003; 55: 399-409Crossref PubMed Scopus (33) Google Scholar). The bacterial cells were immediately collected by centrifugation at 12,000 × g for 1 min, and total RNA was purified by 1 ml of a phenol containing the RNA extraction reagent ISOGEN™ (Nippon Gene, Tokyo, Japan). For measurement of invE mRNA stability, S. sonnei strains were inoculated into 35 ml of LB medium without antibiotics and allowed to grow until A600 reached 0.8. After rifampicin (Sigma) was added to give a final concentration of 200 μg/ml, the culture continued to incubate at the initial temperature. An aliquot of the cultures (2 ml) was harvested every 2 min for preparation of RNA samples. The total RNA pellets were dissolved in 40 μl of nuclease-free water (Invitrogen) and measured for the concentration by spectrophotometer ND-1000™ (Nano-Drop Technologies, Wilmington, DE). The concentration of the RNA samples was adjusted to 50 ng/μl, and then the residual DNA was digested by TURBO DNA-free™ kit (Ambion, Austin, TX) for 30 min at 37 °C. For reverse transcription (RT)-PCR, each 100 ng of total RNA was amplified by Titan™ one-tube RT-PCR kit (Roche Applied Science). Conditions for RT-PCR were as follows: 60 °C for 30 min; 94 °C for 2 min; and 26 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 90 s. For detection of virF mRNA and 6 S RNA, two additional cycles (for a total of 28 cycles) of PCR were employed. Primers used for RT-PCR for virF mRNA were virF-RT1 and virF-RT2. Primers used for invE transcript were invE-RT1 and invE-RT2. Primers used for RT-PCR for 6 S RNA transcript were ssrS-RT1 and ssrS-RT2. Each 10 μl of the PCR product was subjected to electrophoresis in a 1% agarose gel containing 50 μg/ml ethidium bromide. For real time PCR analysis, cDNA was synthesized from 100 ng of total RNA in 10 μl of Transcriptor™ first strand cDNA synthesis kit (Roche Applied Science) for 30 min at 60 °C. Each cDNA was analyzed in a triplicated manner by ABI Prism 2000 thirmal cycler with a Perfect real time™ PCR kit (Takara Japan) in a 25-μl reaction containing 2 μl of cDNA, 120 nm concentration each of invE84F, invE156R, ssrS36F, and ssrS72R primers, and a 32 nm concentration each of invE100T and ssrS65T Taqman™ probes. Samples were amplified by 40 cycles of 95 °C for 5 s and 60 °C for 31 s. Primers and Taqman™ probes were designed by ABI PRISM primer design software and synthesized by ABI Japan. The amounts of invE mRNA and 6 S RNA were determined by ABI prism evaluation software using a standard curve provided by five serial dilutions of cDNA from the sample of time 0 at 37 °C. The amount of invE-mRNA was normalized by the amount of 6 S RNA transcript as an internal control. Relative values against the sample of time 0 at 37 °C were blotted on the semilog plot. The RNA preparation and real time PCR analysis were repeated three times, and similar trends of results were obtained. Invasion Assay—Bacterial invasion into HeLa cells was tested using the gentamicin protection assay. Overnight precultures (2 ml of LB-ampicillin) were inoculated in 5 ml of brain-heart infusion broth (Difco). After incubation at 37 °C for 40 min, IPTG was added to a final concentration of 0.1 or 1 mm, and the incubation was continued for an additional 80 min at 37 °C. HeLa cells were grown on glass coverslips to 60% confluence in antibiotic-free Dulbecco's modified eagle medium (Invitrogen) containing 10% fetal calf serum (Invitrogen). Cells were then infected with bacteria grown in BHI at 37 °C at a multiplicity of infection of 100 per cell and centrifuged at 700 × g for 10 min. After cells were incubated at 37 °C in a CO2 incubator for 20 min, gentamicin (Wako) was added to the culture medium at a final concentration of 200 μg/ml, and cells were incubated further at 37 °C in a CO2 incubator for 15 min. After incubation, cells were washed twice with phosphate-buffered saline and lysed in phosphate-buffered saline containing 0.5 ml of 0.5% Triton X-100. A 100-μl aliquot of the lysates was plated onto LB plates and incubated at 37 °C overnight. Colonies grown on LB plates were counted. For the reliability of results, each sample determination was performed in triplicate. The result of a representative assay is shown in Table 2.TABLE 2Efficiency of invasionBacterial strainIPTGNumber of invasionsmmHW50600MS3900114 ± 16MS4831 (pTrc99A)0.1538 ± 118MS4831 (pTrc99A)1518 ± 165MS4831 (pTrc-hfq)0.1228 ± 22MS4831 (pTrc-hfq)1141 ± 54 Open table in a new tab Purification of Hfq Protein—An E. coli strain, BL21(DE3), carrying pET-hfq was grown in 1 liter of LB medium 37 °C for 4 h as described (26Azam T.A. Ishihama A. J. Biol. Chem. 1999; 274: 33105-33113Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Hfq protein was purified with a 10-ml bed volume of Buthyl-Toyopeal™-650 hydrophobic interaction medium (Tosoh, Tokyo, Japan) in a 35-ml Econo Column™ (Bio-Rad) using an Econo System™ gradient maker (Bio-Rad) as described (29Vassilieva I.M. Rouzanov M.V. Zelinskaya N.V. Moll I. Blasi U. Garber M.B. Biochemistry (Mosc.). 2002; 67: 1293-1297Crossref PubMed Scopus (15) Google Scholar). Gel Shift Analysis—Two RNA probes encoding 75- and 140-nucleotide invE sequences from the transcription start site were prepared by in vitro transcription of T7 RNA polymerase (Roche Applied Science). The DNA templates for the T7 transcription were prepared by PCR amplification of the invE sequence in MS390 with primer pairs of invE-T7F and invE75R (for the 75-nucleotide RNA) or invE-T7F and invE140R (for the 140-nucleotide RNA). The T7 transcription mixtures were treated by RNase-free DNase I (Roche Applied Science) for digestion of the DNA template at 37 °C 15 min and purified by phenol/chloroform and a Nuc-away™ spin column (Ambion). The 5′-end of purified RNA was dephosphorylated by alkaline phosphatase in the Kinase Max™ labeling kit (Ambion), and 2 pmol of the RNA was labeled by [γ-32P]ATP (catalog number AA0018; GE Healthcare) with T4 polynucleotide kinase in the kit. The labeled RNA was purified by phenol/chloroform and a Nuc-away™ spin column (Ambion) equilibrated with an RNA binding buffer: 10 mm Tris-HCl, pH 7.5, 100 mm NH4Cl, 5 mm magnesium acetate, 0.1 mm dithiothreitol (30Afonyushkin T. Vecerek B. Moll I. Blasi U. Kaberdin V.R. Nucleic Acids Res. 2005; 33: 1678-1689Crossref PubMed Scopus (139) Google Scholar). Each 20 fmol of the labeled RNA was used for probes of gel shift analysis. Hfq protein (0, 1, 2, 4, 8, and 16 nm calculated for the Hfq hexamer) was mixed with the probe in 10 μl of the RNA binding buffer at 30 or 37 °C for 10 min. Then the samples were mixed with 2 μl of 5× hi-Density TBE Sample Buffer™ (catalog number LC6678; Invitrogen), subjected to electrophoresis with 6% Novex™ DNA retardation gel (catalog number EC63652; Invitrogen) in 0.5× TBE at 30 °C for 80 min or 37 °C for 70 min. Electrophoresis was performed in an XCell SureLock™ mini-cell electrophoresis tank (catalog number EI0001) with a glass circulation pipe from Nihon Eido Co. (Tokyo, Japan), which was placed in a water bath with a circulation pump (Haake, Germany), heated at the indicated temperature. The gel was fixed by 10% methanol, 10% acetate and dried on filter paper. The signals on the gel were visualized on x-ray film RX-U (Fuji Film, Tokyo, Japan) with an intensifying screen at -80 °C for 4-16 h. Surface Plasmon Resonance (Biacore) Analysis—The Biacore 2000 system and sensor chip SA (research grade; Biacore International AB) were used for the binding assay. For preparation of the RNA-immobilized sensor tip, 2 pmol of the in vitro transcribed invE RNA used for gel shift analysis (140 nucleotides) were labeled with 0.2 mm biotin-11-ATP (catalog number NEL544; PerkinElmer Life Sciences) by 1 unit of E. coli poly(A) polymerase (Ambion) for 10 min at 37 °C as described (31Akabayov B. Henn A. Elbaum M. Sagi I. IEEE Trans. Nanobiosci. 2003; 2: 70-74Crossref PubMed Scopus (1) Google Scholar). The labeled invE RNA was extracted by phenol/chloroform, chloroform and purified by the Nuc-away™ spin column (Ambion) two times. Before binding of the RNA, the sensor tip was treated with 50 mm NaOH, 1 m NaCl at a flow rate of 20 μl/min for 1 min three times. The invE RNA was diluted in a high salt buffer (10 mm Tris-HCl, pH 7.4, 1 m NaCl) and captured on flow cell 2 of the streptavidin-coated sensor chip at a flow rate of 10 μl/min until a change of at least 100-150 resonance units was detectable. The base line of resonance intensity was then allowed to stabilize for at least 15 min. A binding assay was performed at 30 and 37 °C. Purified Hfq protein was diluted in the binding buffer (see above) to give a final concentration of 0, 1, 2, 4, or 8 nm, calculated for the Hfq hexamer, and subsequently injected for 3 min onto two flow cells (flow cell 1, blank as a control; flow cell 2, invE RNA) at a flow rate of 20 μl/min. The nonbinding protein was washed out from the specific RNA-protein by the flow of binding buffer for an additional 700 s. Bound Hfq protein was efficiently removed from the complex by a solution of 2 m NaCl for 2 min at a flow rate of 20 μl/min. The response from the reference cell (flow cell 1, blank) was subtracted from the response from flow cell 2 (biotin-invE RNA) to correct a nonspecific binding. We attempted to determine the Kd value using BIAevaluation™ version 3.1 software (Biacore International AB), but the interaction did not fit the 1:1 Langmuir fitting model because of a multimeric binding between the protein and RNA as observed in the gel shift analysis. The measurement of CD spectroscopy (32Chowdhury S. Ragaz C. Kreuger E. Narberhaus F. J. Biol. Chem. 2003; 278: 47915-47921Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) was performed by Toray Research Center (Kamakura, Japan). The 140-nucleotide invE RNA was diluted to 4 μg/ml in the same buffer used for the gel shift analysis, and 80 μl of the sample was subjected to the CD analyzer, J-820 (Nippon Bunko Inc.), in a 10-mm microcell at 30 and 37 °C; values were integrated from 16 measurements. Transcription of the virF gene encoding the upstream regulator of the invE gene was first examined in the wild-type strain of S. sonnei MS390. For the accurate measurement of intact mRNA, the whole culture was directly mixed with an acidic phenol/ethanol solution for quantitative isolation of mRNA (28Bhagwat A.A. Phadke R.P. Wheeler D. Kalantre S. Gudipati M. Bhagwat M. J. Microbiol. Methods. 2003; 55: 399-409Crossref PubMed Scopus (33) Google Scholar), and RT-PCR rather than Northern analysis was employed for the detection of full-length intact mRNA. The level of virF mRNA in the wild-type S. sonnei strain grown in LB medium was almost the same between 30 and 37 °C (Fig. 1A, virF-mRNA), and the level of β-galactosidase activity in wild-type S. sonnei strain MS390 encoded by a virFTL-lacZ translational fusion plasmid (7Nakayama S. Watanabe H. J. Bacteriol. 1995; 177: 5062-5069Crossref PubMed Google Scholar) was also similar between 30 and 37 °C (Fig. 1B, graph 1). Previously, however, Porter and Dorman (14Porter M.E. Dorman C.J. J. Bacteriol. 1997; 179: 6537-6550Crossref PubMed Google Scholar) reported that virF transcription in S. flexneri strain 2457T at 30 °C was 15-25% of the level at 37 °C (14Porter M.E. Dorman C.J. J. Bacteriol. 1997; 179: 6537-6550Crossref PubMed Google Scholar). In order to identify the molecular basis of this difference, we sequenced the upstream region of the virF promoter of S. sonnei MS390 and found an insertion of a transposon sequence (GenBank™ accession number AB300612). The difference in temperature-dependent transcription of the virF gene between the two wild-type strains might be related to the presence or absence of this insertion sequence. In addition to the intact virF mRNA, we detected an additional band of shorter length by Northern analysis (see “Discussion”) (15Mitobe J. Arakawa E. Watanabe H. J. Bacteriol. 2005; 187: 107-113Crossref PubMed Scopus (37) Google Scholar). Temperature-dependent regulation of the invE gene, which is under the control of VirF, was next examined by Western blotting and RT-PCR. The production of InvE protein was almost completely repressed at 30 °C (Fig. 1A, lane 1), but under the same culture conditions, a significant amount of invE mRNA was detected by RT-PCR (Fig. 1A, invE-mRNA). The amount of invE transcript at 30 °C was then measured" @default.
• W2091296993 created "2016-06-24" @default.
• W2091296993 creator A5010867165 @default.
• W2091296993 creator A5061595177 @default.
• W2091296993 creator A5063001437 @default.
• W2091296993 creator A5078541763 @default.
• W2091296993 date "2008-02-01" @default.
• W2091296993 modified "2023-09-30" @default.
• W2091296993 title "Involvement of RNA-binding Protein Hfq in the Post-transcriptional Regulation of invE Gene Expression in Shigella sonnei" @default.
• W2091296993 cites W1517198674 @default.
• W2091296993 cites W1551176833 @default.
• W2091296993 cites W1571932130 @default.
• W2091296993 cites W1586391720 @default.
• W2091296993 cites W1667785400 @default.
• W2091296993 cites W1955204755 @default.
• W2091296993 cites W1960007027 @default.
• W2091296993 cites W1967924074 @default.
• W2091296993 cites W1971359499 @default.
• W2091296993 cites W1977406857 @default.
• W2091296993 cites W1984463832 @default.
• W2091296993 cites W1988037107 @default.
• W2091296993 cites W1989781175 @default.
• W2091296993 cites W1999794292 @default.
• W2091296993 cites W2005318857 @default.
• W2091296993 cites W2026856977 @default.
• W2091296993 cites W2033194967 @default.
• W2091296993 cites W2037295768 @default.
• W2091296993 cites W2039270960 @default.
• W2091296993 cites W2044913844 @default.
• W2091296993 cites W2049737239 @default.
• W2091296993 cites W2053925448 @default.
• W2091296993 cites W2065335949 @default.
• W2091296993 cites W2077441699 @default.
• W2091296993 cites W2080098971 @default.
• W2091296993 cites W2087821919 @default.
• W2091296993 cites W2096974471 @default.
• W2091296993 cites W2099940763 @default.
• W2091296993 cites W2100231871 @default.
• W2091296993 cites W2111578570 @default.
• W2091296993 cites W2115033904 @default.
• W2091296993 cites W2116137883 @default.
• W2091296993 cites W2123038177 @default.
• W2091296993 cites W2134696956 @default.
• W2091296993 cites W2135453467 @default.
• W2091296993 cites W2136659582 @default.
• W2091296993 cites W2137141622 @default.
• W2091296993 cites W2142136966 @default.
• W2091296993 cites W2142863488 @default.
• W2091296993 cites W2144713625 @default.
• W2091296993 cites W2145757131 @default.
• W2091296993 cites W2150990196 @default.
• W2091296993 cites W2154425268 @default.
• W2091296993 cites W2155432444 @default.
• W2091296993 cites W2159850373 @default.
• W2091296993 cites W2160084442 @default.
• W2091296993 cites W2164663386 @default.
• W2091296993 cites W2165698410 @default.
• W2091296993 cites W236057538 @default.
• W2091296993 cites W2766341694 @default.
• W2091296993 cites W4238678686 @default.
• W2091296993 doi "https://doi.org/10.1074/jbc.m710108200" @default.
• W2091296993 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18156173" @default.
• W2091296993 hasPublicationYear "2008" @default.
• W2091296993 type Work @default.
• W2091296993 sameAs 2091296993 @default.
• W2091296993 citedByCount "31" @default.
• W2091296993 countsByYear W20912969932012 @default.
• W2091296993 countsByYear W20912969932013 @default.
• W2091296993 countsByYear W20912969932014 @default.
• W2091296993 countsByYear W20912969932015 @default.
• W2091296993 countsByYear W20912969932016 @default.
• W2091296993 countsByYear W20912969932017 @default.
• W2091296993 countsByYear W20912969932018 @default.
• W2091296993 countsByYear W20912969932020 @default.
• W2091296993 countsByYear W20912969932023 @default.
• W2091296993 crossrefType "journal-article" @default.
• W2091296993 hasAuthorship W2091296993A5010867165 @default.
• W2091296993 hasAuthorship W2091296993A5061595177 @default.
• W2091296993 hasAuthorship W2091296993A5063001437 @default.
• W2091296993 hasAuthorship W2091296993A5078541763 @default.
• W2091296993 hasBestOaLocation W20912969931 @default.
• W2091296993 hasConcept C104317684 @default.
• W2091296993 hasConcept C150194340 @default.
• W2091296993 hasConcept C153911025 @default.
• W2091296993 hasConcept C165864922 @default.
• W2091296993 hasConcept C170344550 @default.
• W2091296993 hasConcept C178809742 @default.
• W2091296993 hasConcept C27153228 @default.
• W2091296993 hasConcept C2776986154 @default.
• W2091296993 hasConcept C2911111840 @default.
• W2091296993 hasConcept C41282012 @default.
• W2091296993 hasConcept C54355233 @default.
• W2091296993 hasConcept C547475151 @default.
• W2091296993 hasConcept C67705224 @default.
• W2091296993 hasConcept C86803240 @default.
• W2091296993 hasConcept C95444343 @default.
• W2091296993 hasConceptScore W2091296993C104317684 @default.
• W2091296993 hasConceptScore W2091296993C150194340 @default.

• next