FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2006182849> ?p ?o ?g. }

• W2006182849 endingPage "11948" @default.
• W2006182849 startingPage "11940" @default.
• W2006182849 abstract "Although the m-xylene-responsive σ54 promoter Pu of Pseudomonas putida mt-2, borne by the TOL plasmid pWWO, is one of the strongest known promoters in vivo, its base-line level in the absence of its aromatic inducer is below the limit of any detection procedure. This is unusual because regulatory networks (such as the one to which Pu belongs) can hardly escape the noise caused by intrinsic fluctuations in background transcription, including that transmitted from upstream promoters. This study provides genetic evidence that the upstream-activating sequences (UAS), which serve as the binding sites for the pWW0-encoded XylR protein (the m-xylene-responsive σ54-dependent activator of Pu), isolate expression of the upper TOL genes from any adventitious transcriptional flow originating further upstream. An in vivo test system was developed in which different segments of the Pu promoter were examined for the inhibition of incoming transcription products from an upstream promoter in P. putida and Escherichia coli. Minimal transcription filter ability was located within a 105-bp fragment encompassing the UAS of Pu. Although S1 nuclease assays showed that the UAS prevented the buildup of downstream transcripts, the mechanism seems to diverge from a typical termination system. This was shown by the fact that the UAS did not halt transcription in vitro and that the filter effect could not be relieved by the anti-termination system of λ phage. Because the Pu promoter lies adjacent to the edge of a transposon in pWW0, the preset transcriptional filter in the UAS may isolate the upper TOL operon from undue expression after random insertion of the mobile genetic element in a new replicon. Although the m-xylene-responsive σ54 promoter Pu of Pseudomonas putida mt-2, borne by the TOL plasmid pWWO, is one of the strongest known promoters in vivo, its base-line level in the absence of its aromatic inducer is below the limit of any detection procedure. This is unusual because regulatory networks (such as the one to which Pu belongs) can hardly escape the noise caused by intrinsic fluctuations in background transcription, including that transmitted from upstream promoters. This study provides genetic evidence that the upstream-activating sequences (UAS), which serve as the binding sites for the pWW0-encoded XylR protein (the m-xylene-responsive σ54-dependent activator of Pu), isolate expression of the upper TOL genes from any adventitious transcriptional flow originating further upstream. An in vivo test system was developed in which different segments of the Pu promoter were examined for the inhibition of incoming transcription products from an upstream promoter in P. putida and Escherichia coli. Minimal transcription filter ability was located within a 105-bp fragment encompassing the UAS of Pu. Although S1 nuclease assays showed that the UAS prevented the buildup of downstream transcripts, the mechanism seems to diverge from a typical termination system. This was shown by the fact that the UAS did not halt transcription in vitro and that the filter effect could not be relieved by the anti-termination system of λ phage. Because the Pu promoter lies adjacent to the edge of a transposon in pWW0, the preset transcriptional filter in the UAS may isolate the upper TOL operon from undue expression after random insertion of the mobile genetic element in a new replicon. Bacterial promoters form part of regulatory networks through which signals are propagated faithfully from one member to the next (1Hooshangi S. Thiberge S. Weiss R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3581-3586Crossref PubMed Scopus (383) Google Scholar, 2Pedraza J.M. van Oudenaarden A. Science. 2005; 307: 1965-1969Crossref PubMed Scopus (626) Google Scholar). This course of events is frequently affected by fluctuations brought about by variations in the pool of housekeeping regulatory proteins (the most common of which is RNAP 2The abbreviations used are: RNAP, RNA polymerase; UAS, upstream-activating sequences; IPTG, isopropyl 1-thio-β-d-galactopyranoside; ssDNA, single-stranded DNA; IHF, integration host factor; RT, reverse transcriptase; 3MBA, 3-methylbenzyl alcohol; nt, nucleotide. 2The abbreviations used are: RNAP, RNA polymerase; UAS, upstream-activating sequences; IPTG, isopropyl 1-thio-β-d-galactopyranoside; ssDNA, single-stranded DNA; IHF, integration host factor; RT, reverse transcriptase; 3MBA, 3-methylbenzyl alcohol; nt, nucleotide. and its σ factors) as well as by changes in environmental conditions (3Xia Y. Yu H. Jansen R. Seringhaus M. Baxter S. Greenbaum D. Zhao H. Gerstein M. Annu. Rev. Biochem. 2004; 73: 1051-1087Crossref PubMed Scopus (118) Google Scholar, 4Rao C.V. Wolf D.M. Arkin A.P. Nature. 2002; 420: 231-237Crossref PubMed Scopus (795) Google Scholar, 5Elowitz M.B. Levine A.J. Siggia E.D. Swain P.S. Science. 2002; 297: 1183-1186Crossref PubMed Scopus (3797) Google Scholar). Although noise is intrinsically associated with molecular events involving few components, how cells keep regulatory noise within limits is still unknown (4Rao C.V. Wolf D.M. Arkin A.P. Nature. 2002; 420: 231-237Crossref PubMed Scopus (795) Google Scholar). Although cells may occasionally gain from the biological consequences of random fluctuations in gene expression, noise may end up destroying biological circuits. However, bacteria appear to control noise in natural gene networks and thus avoid regulatory and metabolic chaos (1Hooshangi S. Thiberge S. Weiss R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3581-3586Crossref PubMed Scopus (383) Google Scholar, 2Pedraza J.M. van Oudenaarden A. Science. 2005; 307: 1965-1969Crossref PubMed Scopus (626) Google Scholar). Although some cellular mechanisms can tolerate transcriptional noise, the same may be detrimental in scenarios in which carefully coordinated gene community behavior is necessary. For example, regulatory circuits that control the expression of metabolic programs for the biodegradation of pollutants in soil bacteria require the suppression of transcriptional noise if these organisms are to survive initial exposure (6Cases I. de Lorenzo V. Nat. Rev. Microbiol. 2005; 3: 105-118Crossref PubMed Scopus (145) Google Scholar). How cells organize their transcriptional response can be examined by analyzing the biodegradation of m-xylene by Pseudomonas putida mt-2, a function encoded by the catabolic TOL plasmid pWW0 which it carries (7Ramos J.L. Marques S. Timmis K.N. Annu. Rev. Microbiol. 1997; 51: 341-373Crossref PubMed Scopus (269) Google Scholar). Individual cells failing to demonstrate the required catabolic ability at any given time or location are surely displaced by fitter members of a community (8Delgado A. Duque E. Ramos J.L. Microb. Releases. 1992; 1: 23-28PubMed Google Scholar, 9Ramos J.L. Duque E. Ramos-Gonzalez M.I. Appl. Environ. Microbiol. 1991; 57: 260-266Crossref PubMed Google Scholar). When P. putida mt-2 is challenged with m-xylene in the medium, much of the available transcriptional machinery is reassigned to allow the bacterium to endure this general stress (10Velazquez F. Parro V. de Lorenzo V. Mol. Microbiol. 2006; 57: 1557-1559Crossref Scopus (22) Google Scholar, 79Dominguez-Cuevas P. Gonzalez-Pastor J.E. Marques S. Ramos J.L. de Lorenzo V. J. Biol. Chem. 2006; 10.1074/jbc.m509848200PubMed Google Scholar). In theory this could reduce the availability of the RNAP and other transcription factors necessary for expression of the xyl genes borne by plasmid pWW0, thereby making the corresponding catabolic promoters more sensitive to cell-to-cell variations. However, this is not the case because TOL genes seem to be equally expressed in all cells under these conditions (11Moller S. Sternberg C. Andersen J.B. Christensen B.B. Ramos J.L. Givskov M. Molin S. Appl. Environ. Microbiol. 1998; 64: 721-732Crossref PubMed Google Scholar). These features of the TOL plasmid prompted us to examine in more detail the aspects of the catabolic promoters that might be related to noise suppression. A remarkable feature of this system is the blend of extraordinary transcriptional capacity with an extremely low basal expression of Pu, the main m-xylene-responsive promoter of pWW0 (Fig. 1). Although the output of β-galactosidase from a chromosomal Pu-lacZ fusion reaches 10,000-15,000 Miller units in P. putida cells exposed for a short time to m-xylene, promoter activity in noninduced cells is below the detection limits of this reporter (12Cases I. de Lorenzo V. Perez-Martin J. Mol. Microbiol. 1996; 19: 7-17Crossref PubMed Scopus (80) Google Scholar). Pu belongs to the class of promoters that depends on the alternative σ factor, σ54, and is activated at a distance by the toluene-responsive activator XylR (13Perez-Martin J. de Lorenzo V. J. Mol. Biol. 1996; 258: 562-574Crossref PubMed Scopus (42) Google Scholar, 14Devos D. Garmendia J. de Lorenzo V. Valencia A. Environ. Microbiol. 2002; 4: 29-41Crossref PubMed Scopus (28) Google Scholar). This involves the binding of the regulator to upstream-activating sequences (UAS) and the looping out of the complex into close proximity to the σ54-containing form of RNA polymerase bound to the -12/-24 region of the promoter. This event is assisted by the binding of the integration host factor (IHF) to the region between the UAS and the σ54 RNAP attachment site (Fig. 1). This facilitates contact between distant proteins and aids in the recruitment of σ54-RNAP to -12/-24 (15Bertoni G. Fujita N. Ishihama A. de Lorenzo V. EMBO J. 1998; 17: 5120-5128Crossref PubMed Scopus (64) Google Scholar, 16Macchi R. Montesissa L. Murakami K. Ishihama A. De Lorenzo V. Bertoni G. J. Biol. Chem. 2003; 278: 27695-27702Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Moreover, IHF enhances the specificity of Pu for its legitimate activator XylR (17Perez-Martin J. de Lorenzo V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7277-7281Crossref PubMed Scopus (43) Google Scholar). During the course of experiments on the expression of plasmid-encoded proteins in maxicells, it was noticed that the Pu promoter is endowed with the capacity to stop transcription that originated upstream (18Harayama S. Rekik M. Wubbolts M. Rose K. Leppik R.A. Timmis K.N. J. Bacteriol. 1989; 171: 5048-5055Crossref PubMed Google Scholar). At that time, however, the phenomenon was not understood, other than being a nuisance when trying to determine the gene content of different fragments of pWW0, and it was given no biological significance. In this work, we address in detail the ability of the Pu promoter to inhibit readthrough transcription from upstream promoters. The results show that this activity can be traced to an ∼100-bp DNA sequence that spans the binding site for the activator protein XylR. Moreover, the mechanism of inhibition is unlike a typical transcription termination event. It is also argued that such an effect is instrumental in protecting the regulatory sub-network of the TOL system from the transcriptional noise of the host. Strains, Plasmids, and General Methods—P. putida KT2442, a rifampicin-resistant derivative of the reference P. putida strain KT2440 (19Regenhardt D. Heuer H. Heim S. Fernandez D.U. Strompl C. Moore E.R. Timmis K.N. Environ. Microbiol. 2002; 4: 912-915Crossref PubMed Scopus (140) Google Scholar), was used as a host for the reporter DNA segments indicated in each case. Escherichia coli CC118 (Δara-leu araD lacX74 galE galK phoA thi1 rpsE rpoB argE-Am recA1; see Ref. 20de Lorenzo V. Herrero M. Jakubzik U. Timmis K.N. J. Bacteriol. 1990; 172: 6568-6572Crossref PubMed Scopus (1209) Google Scholar) was employed as a genetically reliable host for in vivo transcription assays. Two derivatives of this strain were used for different purposes: E. coli CC118 Pu-lacZ, with its chromosomal insertion of mini-Tn5 Sm Pu-lacZ, was used to monitor the activity of the Pu promoter in single copy gene dosage by measuring the output of β-galactosidase (21de Lorenzo V. Herrero M. Metzke M. Timmis K.N. EMBO J. 1991; 10: 1159-1167Crossref PubMed Scopus (114) Google Scholar), and E. coli CC118 λpir (22de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (742) Google Scholar) was used as a recipient for all mini-transposon delivery vectors with a π protein-dependent R6K origin of replication. The mobilizing strain E. coli S17-1 λpir (22de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (742) Google Scholar) was used for conjugal transfer. This strain expresses the replication π protein as well as the tra genes of the broad host range plasmid RP4 (which encodes functions for conjugal transfer of plasmids endowed with an oriT sequence; see Ref. 22de Lorenzo V. Timmis K.N. Methods Enzymol. 1994; 235: 386-405Crossref PubMed Scopus (742) Google Scholar). The plasmids used in this work were as follows: pUG11 (a kind gift of C. Kane), which consists of the N gene of the λ phage under the control of the left promoter (PLλ), cloned in vector pUC18; pCI857, a Kmr plasmid derived from pMC931 (i.e. it has a p15A origin of replication) in which the thermosensitive variant of the λ repressor is expressed through its own native promoter (23Remaut E. Tsao H. Fiers W. Gene (Amst.). 1983; 22: 103-113Crossref PubMed Scopus (240) Google Scholar); and pFHR, a derivative of the Cmr monocopy vector pVDL8 (24Cebolla A. Guzman C. de Lorenzo V. Appl. Environ. Microbiol. 1996; 62: 214-220Crossref PubMed Google Scholar), which has a 1.9-kb segment of the TOL plasmid spanning the xylR gene downstream of its native promoter Pr. Predictions of secondary mRNA structures were generated with the mfold program (25Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10151) Google Scholar). Assembly of Hybrid Mini-transposons—Recombinant DNA techniques were performed as described previously (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Fig. 1 shows the DNA segments from Pu employed for the various constructs described below, all derived from pCG2Pu (27Carmona M. Fernandez S. Rodriguez M.J. de Lorenzo V. J. Bacteriol. 2004; 187: 125-134Crossref Scopus (13) Google Scholar), an Apr ori ColE1 ori M13 phagemid derived from vector pCG2 containing an EcoRI-BamHI fragment of pEZ9 (21de Lorenzo V. Herrero M. Metzke M. Timmis K.N. EMBO J. 1991; 10: 1159-1167Crossref PubMed Scopus (114) Google Scholar) spanning positions -211 to +107 of the promoter. The U-containing, single-stranded pCG2Pu DNA was employed as the substrate for the introduction of new EcoRI sites at locations -44 (pFH14), -106 (pFH15), and both -158 and -106 (pFH23) by using the method of Kunkel et al. (28Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4557) Google Scholar). Digestion of these plasmids with EcoRI gave rise to the various Pu fragments shown in Fig. 1: UAS/IHF, UAS-DP, UAS-P, and UAS-D. Each of these was separately cloned at the single EcoRI site in the lacZ vector pUJ8 (20de Lorenzo V. Herrero M. Jakubzik U. Timmis K.N. J. Bacteriol. 1990; 172: 6568-6572Crossref PubMed Scopus (1209) Google Scholar). The resulting plasmids were then digested with NotI, which released fragments encompassing each of the Pu inserts followed by an intact trp::lacZ reporter and a T7 terminator. A hybrid Kmr mini-transposon delivery plasmid was then constructed based in vector pCNB5 (29de Lorenzo V. Eltis L. Kessler B. Timmis K.N. Gene (Amst.). 1993; 123: 17-24Crossref PubMed Scopus (372) Google Scholar). In this plasmid the single NotI site is downstream of the strong hybrid trp/lac promoter, Ptrc, accompanied in cis by lacIq (thereby allowing the expression unit to be induced by IPTG), all assembled within the boundaries of a mini-Tn5. The NotI fragments from the pUJ8 derivatives described above were then inserted at the single NotI site in pCNB5, thereby generating a succession of functional elements as follows: lacIq/Ptrc → UAS → trp::lacZ → tT7. The designations of the resulting transposon delivery plasmids were pFH19C (control, pCNB5::Ptrc → lacZ), pFH19 (pCNB5::Ptrc → UAS/DP → lacZ), pFH36 (pCNB5::Ptrc → UAS-D → lacZ), and pFH37 (pCNB5::Ptrc → UAS-P → lacZ). At various stages of the process, automated DNA sequencing in an Applied Biosystems device verified the inclusion of the cloned inserts and DNA fragments. Mobilization and Transposition—To generate P. putida and E. coli strains carrying the reporter DNA segments specified in each case, each of the pCNB5 derivatives mentioned above was transformed into the mobilizing bacterial strain E. coli S17-1 λpir and then passed by conjugation into the target cells using a filter-mating technique (21de Lorenzo V. Herrero M. Metzke M. Timmis K.N. EMBO J. 1991; 10: 1159-1167Crossref PubMed Scopus (114) Google Scholar). After 8 h of incubation at 30 °C on LB plates, the cells were washed with 10 mm MgSO4 and plated on either M9 citrate medium with 50 μg/ml kanamycin for Pseudomonas or LB with 50 μg/ml rifampicin and kanamycin for E. coli. Exconjugants were then screened for the lacZ+phenotype accompanied by the loss of the piperacillin/ampicillin marker to confirm the correct insertion of the reporter construct in the mini-transposon vector (21de Lorenzo V. Herrero M. Metzke M. Timmis K.N. EMBO J. 1991; 10: 1159-1167Crossref PubMed Scopus (114) Google Scholar). The result was the insertion of the mini-Tn5 vectors with the built in functional segments indicated in Fig. 2 (top) into the chromosome of either P. putida KT2442 or E. coli CC118. The designations of the resulting strains were as follows: P. putida 19C (control, KT2442::mini-Tn5 Km [lacIq Ptrc → lacZ]), P. putida 19 (KT2442::mini-Tn5 Km [lacIq Ptrc → UAS/DP → lacZ]), P. putida 36 (KT2442::mini-Tn5 Km [lacIq Ptrc → UAS-D → lacZ]), P. putida 37 (KT2442::mini-Tn5 Km [lacIq Ptrc → UAS-P → lacZ]). The corresponding insertions in E. coli CC118 originated strains CC118-19C, CC118-19, CC118-36, and CC118-37. The organization of such insertions (Figs. 2 and 3) shields the reporter cassettes from readthrough transcription from upstream and downstream host promoters, thus minimizing positional effects on the mobile element.FIGURE 3Inhibition of readthrough transcription by the UAS of Pu in the presence or absence of the cognate binding factor XylR. The E. coli CC118 derivatives employed in the experiments of (panels a-d) have chromosomal insertions of the hybrid mini-Tn5 Km transposon (top), which bear the UAS/DP segment (Fig. 1) containing the entire UAS between Ptrc and trp::lacZ (see “Materials and Methods”). In addition, the strains used in c and d were transformed with the single-copy xylR+ plasmid pFHR. Bacteria were grown at 30 °C in LB medium to an A600 of ∼0.5, at which point 1 mm IPTG was added. In panel d, 1 mm of 3MBA, an inducer of XylR, was also added. Accumulation of β-galactosidase was measured 4 h later. Panel e shows a control experiment in which E. coli CC118, Pu-lacZ transformed or not with pFHR, was grown and induced under the same conditions as represented in panels a-d. Note that in panels b-d, the reporter strain is insensitive to the presence of XylR and 3MBA, whereas Pu becomes strongly induced under the same conditions in E. coli CC118 Pu-lacZ (pFHR) in panel e. XylR is represented in the top diagram as a hexamer binding the UAS in the chromosome of the different strains.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Construction of Plasmids for Anti-termination Assays—The test plasmids for examining N-mediated anti-termination were assembled in vector pVTR-A (30Perez-Martin J. de Lorenzo V. Gene (Amst.). 1996; 172: 81-86Crossref PubMed Scopus (31) Google Scholar). This is a single-copy Cmr plasmid in which a lacIq plus the hybrid trp/lac promoter, Ptrc, is followed by a multiple cloning site. pVTR-A was digested with BamHI and SalI and a BglII-SalI DNA fragment from pIZ820 (31Macian F. Perez-Roger I. Armengod M.E. Gene (Amst.). 1994; 145: 17-24Crossref PubMed Scopus (40) Google Scholar) containing the fusion galK::lacZ inserted as a reporter. This gave rise to the reference plasmid pK1 (lacIq Ptrc → galK::lacZ). A 180-bp segment of Pu spanning the UAS and the IHF site (Fig. 1) was then amplified by PCR using primers AUAS-L (5′-GCCCGGGTACCCGCGATGAACCTT-3′) and AUAS-R (5′-GCTTATACCGATC CCGGGTTTCA-3′) (restriction sites in bold). This added flanking KpnI and XmaI ends to the PCR fragment, which was then inserted at the corresponding sites of pK1, generating pL1. Alternatively, a synthetic TR2 λ phage terminator (32Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 33Wilson K.S. von Hippel P.H. J. Mol. Biol. 1994; 244: 36-51Crossref PubMed Scopus (51) Google Scholar, 34Yarnell W.S. Roberts J.W. Science. 1999; 284: 611-615Crossref PubMed Scopus (263) Google Scholar) was produced by hybridizing oligonucleotides TER-A (5′-AATTCAATAACAGGCCTGCTGGTAATCGCAGGCCTTTTTATTTGGT-3′) and TER-B (5′-AATTACCAAATAAAAAGGCCTGCGATTACCAGCAGGCCTGTTATTG-3′). The resulting 46-bp linker contained the TR2 terminator sequence flanked by one downstream AATT overhang (compatible with EcoRI but unable to regenerate this restriction site upon ligation) and a genuine upstream EcoRI site. Subsequent cloning of this linker at the single EcoRI site of pL1 gave rise to pK2, which keeps a free EcoRI site between Ptrc and the TR2 terminator. The λ phage nut sequence (35Das A. Wolska K. Cell. 1984; 38: 165-173Abstract Full Text PDF PubMed Scopus (56) Google Scholar, 36DeVito J. Das A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8660-8664Crossref PubMed Scopus (72) Google Scholar) was amplified with primers NutL (5′-GGAATTCCTAATAACCCCGCTCTTACA-3′) and NutR (5′-GGAATTCCTGGTTTAATTTGATGCCCT-3′) as a 69-bp EcoRI fragment (55 bp corresponding to nut). This fragment was subsequently cloned into pK2 and pL1, originating test plasmids pK3 (lacIq Ptrc → nut → λ-TR2 → galK::lacZ) and pL1N (lacIq Ptrc → nut → UAS/DP → galK::lacZ), respectively. Growth and Induction Conditions—Unless indicated, bacteria were cultured at 30 °C in rich LB medium supplemented, when required, with ampicillin (150 μg/ml), chloramphenicol (30 μg/ml), or kanamycin (50 μg/ml). Typical induction experiments consisted of growing the cells under study to an absorbance of 0.05-0.5 at 600 nm (A600). At this point, 0.1-1.0 mm IPTG was added, and when specified, the growth temperature was raised to 42 °C. Where indicated, 1 mm of the upper TOL pathway inducer 3-methylbenzyl alcohol (3MBA) was added to the cultures. Four hours after induction, P. putida and E. coli cells were collected, permeabilized with chloroform and SDS, and subjected to β-galactosidase assays (79Dominguez-Cuevas P. Gonzalez-Pastor J.E. Marques S. Ramos J.L. de Lorenzo V. J. Biol. Chem. 2006; 10.1074/jbc.m509848200PubMed Google Scholar) to determine the output of the lacZ fusions under the conditions mentioned in each case. The linearity of the assay within the range of cell densities and the development of the reaction with o-nitrophenyl β-d-galactopyranoside were verified in all cases. The β-galactosidase activity values given throughout this paper are the mean of at least three independent experiments conducted in duplicate (deviations ≤15%). In Vitro Transcription Assays—The DNA fragments for transcription in vitro were generated by amplification of relevant portions of pFH19C (no UAS), pFH19 (UAS/DP), and pFH36 (UAS-D) with oligonucleotides TER2 (5′-CACTCCCGTTCTGGATAATG-3′) and TER3 (5′-CACGATGCGTCCGGCGTAGA-3′). These primers hybridized 51 bp upstream of the transcription initiation site of the Ptrc promoter and immediately downstream of the BamHI, respectively, which precedes the reporter trp::lacZ gene (see above). The resulting fragments were purified and used as linear DNA templates at a concentration of 20 ng/ml in transcription reactions performed in 25 mm Tris-HCl, pH 7.5, 50 mm KCl, 10 mm MgCl2 following a standard technique (37Meiklejohn A.L. Gralla J.D. Cell. 1985; 43: 769-776Abstract Full Text PDF PubMed Scopus (20) Google Scholar). Briefly, cold ATP, CTP, and GTP were added to the reaction mixture at a concentration of 10 mm; UTP was used at only 1 mm. The cold NTPs and the DNA were mixed in a volume of 25 μl, and 1 μl of RNasin (10 units/μl) and 1 μCi of [α-32P]UTP (3000 Ci/mmol) were added. Four units of E. coli RNA polymerase (a kind gift from F. Rojo) were then added, and the reactions were incubated for 15 min at 37 °C. Heparin (10 μg/ml) was added and the incubation continued for 2 min to complete ongoing transcription rounds. Finally, the reactions were stopped with 25 μl of TE buffer, 1 μl of 0.5 m EDTA and 1 μl of 10 μg/μl carrier tRNA. Samples were filtered through 1 ml of Sephadex G-50 equilibrated with TE buffer and precipitated with ethanol. The sediment was resuspended in loading buffer with 90% formamide and electrophoresed in a denaturing 8 m urea, 6% polyacrylamide gel, and the transcripts were visualized by autoradiography. S1 Nuclease Assays—The method described by Sambrook et al. (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) was used for extracting total RNA from E. coli cells. The RNA was treated with DNase to eliminate any DNA contamination and further treated with phenol/chloroform treatment to eliminate residues of the enzyme. To produce the labeled single-stranded DNA (ssDNA) probe used in the experiments, plasmid pK1 (see above) was cleaved with XmaI, hybridized with the oligonucleotide LacS1 (5′-GGTGTGAGTGGCAGGGTAGCC-3′) labeled at the 5′ end with 32P, and subjected to 30 cycles of linear amplification with Taq polymerase, similar to the procedure described by Ding et al. (38Ding Q. Kusano S. Villarejo M. Ishihama A. Mol. Microbiol. 1995; 16: 649-656Crossref PubMed Scopus (68) Google Scholar). Because the 5′ end of the labeled primer binds 63 nucleotides downstream of the galK::lacZ fusion, the ssDNA produced by the amplification spanned positions -245 (5′ end) to +63 (3′ end) of the galK::lacZ coding sequence. For the S1 nuclease protection assays, 50 μg of RNA from each sample were hybridized with an excess of the labeled ssDNA probe, digested with S1, and processed as described by Ausubel et al. (39Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates/Wiley-Interscience, New York1989Google Scholar). Samples were loaded onto a DNA sequencing gel with 7 m urea, run at high voltage, and dried. Autoradiographic images were acquired on x-ray film. Semi-quantitative RT-PCR—Two micrograms of total RNA extracted from the E. coli cells under analysis were retrotranscribed using the first-strand cDNA synthesis kit (Amersham Biosciences). 10-Fold serial dilutions of the resulting product were used as templates for a standard PCR with primers PSREV (5′-ATGAGCTGTTGACAATTAATCATC-3′) and GALK (5′-CGGTGGCGGAGCGCAGCAGAGG-3′). These hybridize just downstream of the Ptrc promoter and the leading sequence of the reporter gene, respectively, thereby amplifying transcript segments from pK1 (147 bp), pK3 (262 bp), and pL1N (373 bp). The PCR was set at 30 cycles of 30 s at 90 °C, 30 s at 60 °C, and 30 s at 72 °C followed by a final 10-min extension at 72 °C. The UAS of the Pu Promoter Inhibited Readthrough Transcription— Initial observations on the ability of the whole σ54 Pu promoter of the TOL plasmid, or parts of it, to impede the progress of upstream transcripts were made in maxicells (18Harayama S. Rekik M. Wubbolts M. Rose K. Leppik R.A. Timmis K.N. J. Bacteriol. 1989; 171: 5048-5055Crossref PubMed Google Scholar). In these assays, DNA sequences downstream of the native SmaI site at -205 bp from the transcription initiation site of Pu (Fig. 1) altogether prevented the expression of genes from a PL promoter of λ phage artificially placed upstream. Although little importance was given at that time, this prompted us to re-examine the phenomenon from a fresh perspective. Experiments were therefore performed to confirm this fact and to trace its origin to a minimum of elements. For this, we created new EcoRI sites at several places in the DNA sequence encompassing the Pu promoter, from its SmaI site (-205) all the way down to the HaeIII site at + 93. This generated four main EcoRI segments designated UAS/IHF (167 bp, including the UAS for the cognate activator of Pu, the XylR protein, and the IHF site), UAS-DP (105 bp, containing both the distal UAS-D and the proximal UAS-P), UAS-P (52 bp, the proximal UAS only), and UAS-D (53 bp, the distal UAS-only). Each of these fragments was cloned in front of a promoterless lacZ gene and placed downstream of an IPTG/lacIq-controlled Ptrc promoter within a mini-Tn5 transposon vector. The resulting DNA segments gave rise to a reporter system (Fig. 2, top) consisting of the DNA under examination placed between a strong IPTG-inducible promoter and lacZ. As explained below, this provided a dependable genetic test for the inhibition of incoming transcription from Ptrc by DNA sequences located between the promoter and the β-galactosidase gene. By using these constructs, attempts were made to certify the expression inhibition phenomenon observed in P. putida and to verify its schoichiometry (single-copy gene dosage). Each of the hybrid mini-transposons bearing the Pu portions mentioned above were separately inserted into the chromosome of P. putida KT2442, along with a control devoid of Pu. The resulting strains were then subjected to β-" @default.
• W2006182849 created "2016-06-24" @default.
• W2006182849 creator A5040213406 @default.
• W2006182849 creator A5067795768 @default.
• W2006182849 creator A5088742946 @default.
• W2006182849 date "2006-04-01" @default.
• W2006182849 modified "2023-09-30" @default.
• W2006182849 title "The Upstream-activating Sequences of the σ54 Promoter Pu of Pseudomonas putida Filter Transcription Readthrough from Upstream Genes" @default.
• W2006182849 cites W1500916922 @default.
• W2006182849 cites W1517645475 @default.
• W2006182849 cites W1577856817 @default.
• W2006182849 cites W1602283030 @default.
• W2006182849 cites W1636002209 @default.
• W2006182849 cites W1749353139 @default.
• W2006182849 cites W1749993378 @default.
• W2006182849 cites W1918959617 @default.
• W2006182849 cites W1927923848 @default.
• W2006182849 cites W193267154 @default.
• W2006182849 cites W1970686860 @default.
• W2006182849 cites W1971073048 @default.
• W2006182849 cites W1974021446 @default.
• W2006182849 cites W1974488432 @default.
• W2006182849 cites W1977452155 @default.
• W2006182849 cites W1982805829 @default.
• W2006182849 cites W1987568156 @default.
• W2006182849 cites W1988791304 @default.
• W2006182849 cites W1989639494 @default.
• W2006182849 cites W1991713013 @default.
• W2006182849 cites W1994596486 @default.
• W2006182849 cites W1998379753 @default.
• W2006182849 cites W2007858891 @default.
• W2006182849 cites W2008497514 @default.
• W2006182849 cites W2011487388 @default.
• W2006182849 cites W2013544986 @default.
• W2006182849 cites W2014839221 @default.
• W2006182849 cites W2019432777 @default.
• W2006182849 cites W2019812876 @default.
• W2006182849 cites W2021263163 @default.
• W2006182849 cites W2021871572 @default.
• W2006182849 cites W2023135381 @default.
• W2006182849 cites W2023457952 @default.
• W2006182849 cites W2024583069 @default.
• W2006182849 cites W2026095016 @default.
• W2006182849 cites W2030002597 @default.
• W2006182849 cites W2035336351 @default.
• W2006182849 cites W2035578856 @default.
• W2006182849 cites W2038749101 @default.
• W2006182849 cites W2040662033 @default.
• W2006182849 cites W2040736005 @default.
• W2006182849 cites W2042245212 @default.
• W2006182849 cites W2059927405 @default.
• W2006182849 cites W2064537018 @default.
• W2006182849 cites W2065663947 @default.
• W2006182849 cites W2082971321 @default.
• W2006182849 cites W2094362432 @default.
• W2006182849 cites W2095293755 @default.
• W2006182849 cites W2098720265 @default.
• W2006182849 cites W2107342968 @default.
• W2006182849 cites W2110071847 @default.
• W2006182849 cites W2118781875 @default.
• W2006182849 cites W2122282339 @default.
• W2006182849 cites W2124001616 @default.
• W2006182849 cites W2128181506 @default.
• W2006182849 cites W2128458628 @default.
• W2006182849 cites W2131113503 @default.
• W2006182849 cites W2131285745 @default.
• W2006182849 cites W2131706188 @default.
• W2006182849 cites W2134613767 @default.
• W2006182849 cites W2139924741 @default.
• W2006182849 cites W2141157874 @default.
• W2006182849 cites W2150247421 @default.
• W2006182849 cites W2156050394 @default.
• W2006182849 cites W2159850373 @default.
• W2006182849 cites W2160183974 @default.
• W2006182849 cites W2162667777 @default.
• W2006182849 cites W2164826029 @default.
• W2006182849 cites W2165289894 @default.
• W2006182849 cites W2166756426 @default.
• W2006182849 cites W2170323088 @default.
• W2006182849 cites W2171280873 @default.
• W2006182849 cites W2888511473 @default.
• W2006182849 cites W31306344 @default.
• W2006182849 doi "https://doi.org/10.1074/jbc.m511782200" @default.
• W2006182849 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16510445" @default.
• W2006182849 hasPublicationYear "2006" @default.
• W2006182849 type Work @default.
• W2006182849 sameAs 2006182849 @default.
• W2006182849 citedByCount "9" @default.
• W2006182849 countsByYear W20061828492012 @default.
• W2006182849 countsByYear W20061828492017 @default.
• W2006182849 countsByYear W20061828492019 @default.
• W2006182849 countsByYear W20061828492020 @default.
• W2006182849 crossrefType "journal-article" @default.
• W2006182849 hasAuthorship W2006182849A5040213406 @default.
• W2006182849 hasAuthorship W2006182849A5067795768 @default.
• W2006182849 hasAuthorship W2006182849A5088742946 @default.
• W2006182849 hasBestOaLocation W20061828491 @default.
• W2006182849 hasConcept C101762097 @default.

• next