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• W2000873407 abstract "The four RpoN factors of Rhodobacter sphaeroides are functionally specialized. In this bacterium, RpoN1 and RpoN2 are specifically required for the transcription of the nitrogen fixation and flagellar genes, respectively. Analysis of the promoter sequences recognized by each of these RpoN proteins revealed some significant differences. To investigate the functional relevance of these differences, the flagellar promoter fliOp was sequentially mutagenized to resemble the nitrogen fixation promoter nifUp. Our results indicate that the promoter sequences recognized by these sigma factors have diverged enough so that particular positions of the promoter sequence are differentially recognized. In this regard, we demonstrate that the identity of the -11-position is critical for promoter discrimination by RpoN1 and RpoN2. Accordingly, purified RpoN proteins with a deletion of Region I, which has been involved in the recognition of the -11-position, did not show differential binding of fliOp and nifUp promoters. Substitution of the flagellar enhancer region located upstream fliOp by the enhancer region of nifUp allowed us to demonstrate that RpoN1 and RpoN2 interact specifically with their respective activator protein. In conclusion, two different molecular mechanisms underlie the transcriptional specialization of these sigma factors. The four RpoN factors of Rhodobacter sphaeroides are functionally specialized. In this bacterium, RpoN1 and RpoN2 are specifically required for the transcription of the nitrogen fixation and flagellar genes, respectively. Analysis of the promoter sequences recognized by each of these RpoN proteins revealed some significant differences. To investigate the functional relevance of these differences, the flagellar promoter fliOp was sequentially mutagenized to resemble the nitrogen fixation promoter nifUp. Our results indicate that the promoter sequences recognized by these sigma factors have diverged enough so that particular positions of the promoter sequence are differentially recognized. In this regard, we demonstrate that the identity of the -11-position is critical for promoter discrimination by RpoN1 and RpoN2. Accordingly, purified RpoN proteins with a deletion of Region I, which has been involved in the recognition of the -11-position, did not show differential binding of fliOp and nifUp promoters. Substitution of the flagellar enhancer region located upstream fliOp by the enhancer region of nifUp allowed us to demonstrate that RpoN1 and RpoN2 interact specifically with their respective activator protein. In conclusion, two different molecular mechanisms underlie the transcriptional specialization of these sigma factors. In eubacteria, the sigma subunit of the RNA polymerase is responsible for recognizing the promoter sequence to initiate transcription (1Burgess R.R. Travers A.A. FASEB J. 1970; 29: 1164-1169Google Scholar, 2Burgess R.R. Travers A.A. Dunn J.J. Bautz E.K.F. Nature. 1969; 221: 43-46Crossref PubMed Scopus (637) Google Scholar). A large number of sigma factors have been described in eubacteria (1Burgess R.R. Travers A.A. FASEB J. 1970; 29: 1164-1169Google Scholar, 3Mittenhuber G. J. Mol. Microbiol. Biotechnol. 2002; 4: 77-91PubMed Google Scholar, 4Wosten M.M.S.M. FEMS Microbiol. Rev. 1998; 22: 127-150Crossref PubMed Scopus (306) Google Scholar). In general, all of them can be grouped in two different families: the family of sigma-70 that in Escherichia coli includes σ70, σ32, σ24, σS, and σ 28 and the family of sigma-54, of which σ54 is the only member (5Helmann J.D. Chamberlin M.J. Annu. Rev. Biochem. 1988; 57: 839-872Crossref PubMed Scopus (715) Google Scholar, 6Ishihama A. Annu. Rev. Microbiol. 2000; 54: 499-518Crossref PubMed Scopus (453) Google Scholar, 7Gross C.A. Lonetto M. Losick R. McKnight S.L. Yamamoto K.R. Transcription Regulation. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 129-176Google Scholar, 8Merrick M.J. Mol. Microbiol. 1993; 10: 903-909Crossref PubMed Scopus (341) Google Scholar). The RNA polymerase core (E) associated with a sigma factor of the σ70 family is capable of binding to a promoter and initiating transcription without any ancillary factor. In contrast, Eσ54 holoenzyme is unable to form open complex in the absence of an activator protein (9Popham D.L. Szeto D. Keener J. Kustu S. Science. 1989; 243: 629-635Crossref PubMed Scopus (315) Google Scholar, 10Sasse-Dwight S. Gralla J.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8934-8938Crossref PubMed Scopus (190) Google Scholar, 11Morett E. Buck M. J. Mol. Biol. 1989; 210: 65-77Crossref PubMed Scopus (181) Google Scholar). Eσ54 binds to highly conserved promoters with the consensus sequence TGGCACN5TTGCW, of which the most conserved positions are the GG and GC located at -24 and -12 nucleotides upstream from the transcription initiation site (12Beynon J. Cannon M. Buchanan-Wollaston V. Cannon F. Cell. 1983; 34: 665-671Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 13Barrios H. Valderrama B. Morett E. Nucleic Acids Res. 1999; 27: 4305-4313Crossref PubMed Scopus (303) Google Scholar). Mutation of the GG or GC dinucleotides in any σ54 promoter strongly reduces transcription, confirming its relevance in the transcriptional initiation process (14Merrick M. Chambers S. J. Bacteriol. 1992; 174: 7221-7226Crossref PubMed Google Scholar). The high degree of conservation of the σ54-dependent promoters in many bacteria allows the σ54 factor from Aquifex aeolicus to successfully recognize the σ54-dependent promoter glnHp2 from E. coli (15Studholme D.J. Wigneshwereraraj S.R. Gallegos M.T. Buck M. J. Bacteriol. 2000; 182: 1616-1623Crossref PubMed Scopus (13) Google Scholar). As mentioned above, Eσ54 strictly requires the presence of an activator protein to carry out the transition from closed to open complex. Experimental evidence obtained from in vitro studies using heteroduplex DNA fragments containing a σ54 promoter sequence mimicking a fork junction suggests that the inability of Eσ54 holoenzyme to spontaneously melt DNA is based on the interaction between the N-terminal region of σ54 and a repressive DNA fork formed transitorily by melted DNA at the -11- and -10-positions, 2The promoter numbering given in this work considers the dinucleotide GC as the -13- and -12-positions, respectively. which are adjacent to the GC dinucleotide. This interaction inhibits the ability of the holoenzyme to spread melting and keeps it inactive to initiate transcription. Therefore, it has been proposed that the bases in the -12 box have a regulatory role in the isomerization process (16Guo Y. Wang L. Gralla J.D. EMBO J. 1999; 18: 3736-3745Crossref PubMed Scopus (50) Google Scholar, 17Guo Y. Lew C.M. Gralla J.D. Genes Dev. 2000; 14: 2242-2255Crossref PubMed Scopus (64) Google Scholar, 18Casaz P. Gallegos M.T. Buck M. J. Mol. Biol. 1999; 292: 229-239Crossref PubMed Scopus (31) Google Scholar, 19Cannon W.V. Gallegos M.T. Buck M. Nat. Struct. Biol. 2000; 7: 594-601Crossref PubMed Scopus (93) Google Scholar, 20Wigneshweraraj S.R. Chaney M.K. Ishihama A. Buck M. J. Mol. Biol. 2001; 306: 681-701Crossref PubMed Scopus (30) Google Scholar). Transcriptional activators of Eσ54 usually bind to sequence motifs located 100 bp upstream of the promoter sequence and for this reason are commonly known as enhancer-binding proteins (EBPs). 3The abbreviations used are: EBP, enhancer-binding protein; WT, wild type; UAS, upstream activation site. These proteins belong to the family of AAA+ ATPases and couple the energy derived from ATP hydrolysis to remodel the nucleoprotein complex formed by the N terminus of σ54 bound to the DNA fork structure, relieving the inhibitory interactions and allowing open complex formation (21Buck M. Gallegos M.T. Studholme D.J. Guo Y. Gralla J.D. J. Bacteriol. 2000; 182: 4129-4136Crossref PubMed Scopus (353) Google Scholar, 22Xu H. Hoover T.R. Curr. Opin. Microbiol. 2001; 4: 138-144Crossref PubMed Scopus (99) Google Scholar, 23Zhang X. Chaney M. Wigneshweraraj S.R. Schumacher J. Bordes P. Cannon W. Buck M. Mol. Microbiol. 2002; 45: 895-903Crossref PubMed Scopus (136) Google Scholar). By sequence alignment, σ54 has been divided in three regions. Region I is located at the N terminus of the protein and contains determinants for nucleating DNA melting, for inhibiting open complex formation, and to mediate the response to the EBPs (24Cannon W. Gallegos M.T. Casaz P. Buck M. Genes Dev. 1999; 13: 357-370Crossref PubMed Scopus (66) Google Scholar, 25Syed A. Gralla J.D. J. Bacteriol. 1998; 180: 5619-5625Crossref PubMed Google Scholar). Recently, it has been established that Region I makes a major contribution to bind the fork junction structure located at the -11-position in heteroduplex probes (16Guo Y. Wang L. Gralla J.D. EMBO J. 1999; 18: 3736-3745Crossref PubMed Scopus (50) Google Scholar, 17Guo Y. Lew C.M. Gralla J.D. Genes Dev. 2000; 14: 2242-2255Crossref PubMed Scopus (64) Google Scholar, 18Casaz P. Gallegos M.T. Buck M. J. Mol. Biol. 1999; 292: 229-239Crossref PubMed Scopus (31) Google Scholar, 19Cannon W.V. Gallegos M.T. Buck M. Nat. Struct. Biol. 2000; 7: 594-601Crossref PubMed Scopus (93) Google Scholar, 20Wigneshweraraj S.R. Chaney M.K. Ishihama A. Buck M. J. Mol. Biol. 2001; 306: 681-701Crossref PubMed Scopus (30) Google Scholar). Region II is variable, and although its role in transcription is still unclear, it has been implicated in assisting σ54 binding to DNA and melting (26Cannon W. Chaney M. Buck M. Nucleic Acids Res. 1999; 27: 2478-2486Crossref PubMed Scopus (19) Google Scholar, 27Southern E. Merrick M. Nucleic Acids Res. 2000; 28: 2563-2570Crossref PubMed Scopus (10) Google Scholar). At the C terminus, Region III shows a helixturn-helix motif and a very conserved amino acid sequence named the RpoN box; both have been proposed to interact with DNA (21Buck M. Gallegos M.T. Studholme D.J. Guo Y. Gralla J.D. J. Bacteriol. 2000; 182: 4129-4136Crossref PubMed Scopus (353) Google Scholar). Recently, it was suggested that some residues in the RpoN box interact with the -24 promoter sequence (28Burrows 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, 29Doucleff M. Malak L.T. Pelton J.G. Wemmer D.E. J. Biol. Chem. 2005; 280: 41530-41536Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Results obtained using (p-bromoacetamidobenzyl)-EDTA Fe-σ54 conjugates indicate that in addition to σ54 Region I, the residue Arg336, which lies in Region III, is also close to the -12 promoter box (20Wigneshweraraj S.R. Chaney M.K. Ishihama A. Buck M. J. Mol. Biol. 2001; 306: 681-701Crossref PubMed Scopus (30) Google Scholar). This result agrees with previous observations, where mutants in Arg336, as well as mutants in Region I, were defective in controlling open complex formation. Therefore, two different structural elements of σ54 have been proposed to be part of the regulatory center that controls melting (30Wang L. Gralla J.D. J. Biol. Chem. 2001; 276: 8979-8986Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 31Chaney M. Buck M. Mol. Microbiol. 1999; 33: 1200-1209Crossref PubMed Scopus (49) Google Scholar). The complete genome sequence of several microorganisms has shown that the rpoN gene (encoding the σ54 factor) is widely distributed among eubacteria. A few organisms have two copies of rpoN, such as Bradyrhizobium japonicum, Rhizobium etli (32Kullik I. Fritsche S. Knobel H. Sanjuan J. Hennecke H. Fischer H.M. J. Bacteriol. 1991; 173: 1125-1138Crossref PubMed Google Scholar, 33Michiels J. Moris M. Dombrecht B. Verreth C. Vanderleyden J. J. Bacteriol. 1998; 180: 3620-3628Crossref PubMed Google Scholar), Ralstonia solanacearum, and Burkholderia fungorum. In B. japonicum, these copies are highly similar, and they are functionally interchangeable (32Kullik I. Fritsche S. Knobel H. Sanjuan J. Hennecke H. Fischer H.M. J. Bacteriol. 1991; 173: 1125-1138Crossref PubMed Google Scholar). Rhodobacter sphaeroides is the only known bacterium with four copies of rpoN in its genome. This unusual high number of rpoN gene copies does not seem to be the result of recent events of duplication, since the products of these genes show a degree of similarity ranging from 48 to 51%. We have previously shown that these copies are not functionally interchangeable (34Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2002; 46: 75-85Crossref PubMed Scopus (31) Google Scholar, 35Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2005; 58: 969-983Crossref PubMed Scopus (43) Google Scholar). RpoN1 is involved in the expression of the genes required for nitrogen fixation, whereas RpoN2 is required for the transcription of the class II and class III flagellar genes. The molecular determinants that confer this specificity have not yet been investigated. In this work, we show evidence that the promoter sequences recognized by RpoN1 and RpoN2 have subtle differences that are used by these proteins to bind differentially; furthermore, our results indicate that each sigma factor is activated only by its cognate activator protein (EBP). Bacterial Strains, Plasmids, and Growth Conditions—Plasmids and strains of R. sphaeroides and E. coli used in this work are listed in Table 1. R. sphaeroides was grown in Sistrom's minimal medium (36Sistrom W.R. J. Gen. Microbiol. 1962; 28: 607-616Crossref PubMed Scopus (151) Google Scholar) or in minimal medium with malate (0.4%) and glutamate as sole carbon and nitrogen sources, respectively (MG medium) (34Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2002; 46: 75-85Crossref PubMed Scopus (31) Google Scholar). For the N+-aerobic growth conditions, a saturated culture was diluted 1:10 with fresh Sistrom's medium in a 250-ml Erlenmayer flask. The cultures were incubated in the dark with strong shaking (200 rpm). To achieve nitrogen fixing conditions, R. sphaeroides was grown as described before (34Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2002; 46: 75-85Crossref PubMed Scopus (31) Google Scholar). Briefly, a culture grown under heterotrophic conditions was collected when it reached an A600 of 0.4. After two washes with minimal medium, the cells were suspended in the original volume of MG medium. This culture was used to completely fill screw cap tubes that were incubated under constant illumination for a period of 12 h. Antibiotics were used at the following concentrations: for R. sphaeroides, spectinomycin (50 μg/ml), kanamycin (25 μg/ml), tetracycline (1 μg/ml), and gentamycin (5 μg/ml); for E. coli, spectinomycin (100 μg/ml), kanamycin (50 μg/ml), tetracycline (15 μg/ml), gentamycin (30 μg/ml), and ampicillin (100 μg/ml). E. coli strains were grown in LB medium at 37 °C with shaking at 200 rpm.TABLE 1Bacterial strains and plasmids used in this studyStrain or plasmidGenotype or descriptionReference/SourceR. sphaeroidesWS8Wild-type strain, NalrRef. 61Sockett R.E. Foster J.C.A. Armitage J.P. FEMS Symp. 1990; 53: 473-479Google ScholarSP7WS8 derivative, rpoN2Δ::KanRef. 34Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2002; 46: 75-85Crossref PubMed Scopus (31) Google ScholarSP13WS8 derivative, fleQΔ::KanRef. 35Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2005; 58: 969-983Crossref PubMed Scopus (43) Google ScholarSP16WS8 derivative, nifAΔ::KanThis workSP17WS8 derivative nifAΔ::Kan rpoN1Δ::aadAThis workE. coliJM103hsdR4 Δ(lac-pro) F′ traD36 proAB lacIqZΔM15Ref. 62Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1987Google ScholarS17-1recA endA thi hsdR RP4-2 Tc::Mu::Tn7, Tpr SmrRef. 63Simon R. Priefer U. Pühler A. Bio/Technology. 1983; 1: 784-791Crossref Scopus (5643) Google ScholarLMG174ΔlacX74 galE thi rpsL ΔphoA (PvuII) Δara714 leu::Tn10InvitrogenMX848Δ(pro-lac) galE ilv-680 rpoN73::Tn5 thi-1Ref. 64Castaño I. Bastarrachea F. Mol. Gen. Genet. 1984; 195: 228-233Crossref PubMed Scopus (24) Google ScholarLMG174-1Same as LMG174 but rpoN73::Tn5This workPlasmidspTZ19RCloning vector, Apr; pUC derivativeFermentaspJQ200mp18Vector used for gene replacementRef. 65Quandt J. Hynes M.F. Gene (Amst.). 1993; 127: 15-21Crossref PubMed Scopus (838) Google ScholarpUC4KSource of the Kanr cassetteAmersham BiosciencespRK415pRK404 used for expression in R. sphaeroidesRef. 66Keen N.T. Tamaki S. Kobayashi D. Trollinger D. Gene (Amst.). 1988; 70: 191-197Crossref PubMed Scopus (1272) Google ScholarpBBMCS53Transcriptional uidA fusion vector, GmrRef. 67Girard L. Brom S. Davalos A. López O. Soberón M. Romero D. Mol. Plant. Microbe Interact. 2000; 13: 1283-1292Crossref PubMed Scopus (60) Google ScholarpBAD/HisCExpression vector; Apr N-terminal His6 tagInvitrogenpBOppBBMCS53 derivative, fliOp-uidA gene fusionRef. 34Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2002; 46: 75-85Crossref PubMed Scopus (31) Google ScholarpRS210pRK415 carrying fleT+Ref. 35Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2005; 58: 969-983Crossref PubMed Scopus (43) Google Scholar Open table in a new tab Recombinant DNA Techniques—Plasmid DNA preparations were carried out with a minicolumn plasmid purification kit (Qiagen Inc., Valencia, CA). Restriction enzymes were used according to the recommendations from the manufacturer. Standard methods were used for transformation, ligation, and other related techniques. To isolate SP16 and SP17 strains, the wild-type nifA gene was obtained by PCR using total DNA from WS8 cells and the oligonucleotides nifA1 (5′-GCTCTAGACATCGCTCGCCCCTTCGTGCG-3′) and nifAr1 (5′-ACCGAATTCACCTTCACCAGC-3′). These oligonucleotides were designed in accordance with the R. sphaeroides 2.4.1 genomic sequence. The PCR product was cloned in pTZ19R, generating pRS310. From this plasmid, most of the coding region of nifA was deleted by inverse PCR, using the oligonucleotides nifArev1 (5′-GGTACCCCGGACGTGTCCATCACCAGAC-3′) and nifArev3 (5′-GGTACCCCGAGAAGTTCTAGCCCGCCAC-3′), which included a KpnI restriction site. The PCR product was gel-purified and digested with KpnI. The resultant product was ligated with the Kanr cassette obtained from pUC4K. The fragment carrying the nifAΔ::Kan allele was then subcloned in pJQ200mp18 and introduced to R. sphaeroides WS8 wild type or into SP8 strain (34Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2002; 46: 75-85Crossref PubMed Scopus (31) Google Scholar). Allelic exchange was confirmed by Southern blot or PCR. The upstream region of fliOp wild type or that of the mutant versions of fliOp was substituted with the upstream region of nifUp following the next steps. First, the region upstream nifU was amplified by PCR using chromosomal DNA from WS8 cells and the oligonucleotides nif3Eco 5′-GGAATTCGCTCCCGGCAGGCGTCGTCC-3′ and nifBgl 5′-GAGATCTCACTGCAGGGAGTTGCGGAGG-3′. The amplification product was gel-purified and digested with EcoRI and BglII. The plasmids carrying fliOp wild type and the mutant versions of fliOp were amplified by PCR deleting the region upstream fliOp, using the oligonucleotides fliOBgl (5′-TGAGATCTCGCGGGCGGGCGGCACGGATG-3) and pTZ19Eco (5′-GAATTCACTGGCCGTCGTTTTACAACG-3′). The products were gel-purified and digested with EcoRI and BglII, and each of these products was ligated with the PCR product carrying the upstream region of nifUp. The presence of the insert as well as the correct mutation in fliOp was confirmed by DNA sequencing. Finally, the complete fragment was transferred into pBBRMCS53, and the appropriate orientation was confirmed. Site-directed Mutagenesis—Site-directed mutagenesis was performed according to the method of Kunkel (37Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar) with a uracil-containing single-stranded DNA as template. The plasmid carrying the fliOp promoter consists of a 391-bp DNA fragment, cloned in pTZ19R. This DNA fragment carries 254 bp upstream of the transcriptional start site (38Poggio S. Aguilar C. Osorio A. González-Pedrajo B. Dreyfus G. Camarena L. J. Bacteriol. 2000; 182: 5787-5792Crossref PubMed Scopus (15) Google Scholar). The oligonucleotides used for mutagenesis were as follows: 5′-GTCCCCCTCCGCNGCAACATCCGTGCCG-3′, 5′-CAACATCCGTGCCAAGCGCCCGCGTGAGGATC-3′, and 5′-GTCCCCCTCCGCAGCACGATCCGTGCCAAGCGCC-3′. The presence of the desired mutation in the resultant plasmid was confirmed by sequencing. Finally, the fragments carrying the wild-type promoter or its derivatives were subcloned into pBBRMCS53 in the appropriate orientation. Conjugation—Plasmid DNA was mobilized into R. sphaeroides by conjugation according to previously reported procedures (39Davis J. Donohue T.J. Kaplan S. J. Bacteriol. 1988; 170: 320-329Crossref PubMed Google Scholar). β-Glucuronidase Assay—β-Glucuronidase was determined from sonicated cell-free extracts using 4-methyl-umbelliferyl-β-d-glucuronide as substrate and following a previously reported protocol (40Jefferson R.A. Burgess S.M. Hirsh D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8447-8451Crossref PubMed Scopus (824) Google Scholar). Briefly, cell-free extracts were incubated at 37 °C in reaction buffer. Samples of 100 μl were taken at three different time points and mixed with 0.9 ml of stop buffer (0.2 m Na2CO3). Fluorometric determinations were made in a PerkinElmer Life Sciences LA-5 apparatus (excitation wavelength, 360 nm; emission wavelength, 446 nm). The fluorometer was calibrated using 4-methylumbelliferone standards. Specific activities are expressed as nmol of 4-methylum-belliferone formed/min/mg of protein. DNA and Proteins—Synthetic PAGE-purified oligonucleotides, corresponding to the -59 to +22 sequence of the fliOp promoter (38Poggio S. Aguilar C. Osorio A. González-Pedrajo B. Dreyfus G. Camarena L. J. Bacteriol. 2000; 182: 5787-5792Crossref PubMed Scopus (15) Google Scholar) or the -58 to +23 sequence of the predicted nifUp promoter were used to construct the heteroduplex molecules used in this work (see Fig. 4). DNA probes were prepared as follows. The top strand was labeled with [γ-32P]ATP and mixed with the complementary strand. The mixture containing 4 pmol of 32P-end-labeled DNA and 6 pmol of complementary strand in 20 mm Tris-HCl, pH 7.5, and 80 mm NaCl was boiled briefly and cooled slowly to room temperature. The resulting annealed probes were then diluted in TE (10 mm Tris-HCl, pH 8.0, 1 mm EDTA) containing 80 mm NaCl. To obtain RpoN1 and RpoN2 proteins, the coding regions of rpoN1 and rpoN2 were amplified by PCR and cloned into pBADHis-C vector. The resultant plasmids were introduced to the LMG174-1 strain, and different growth temperatures and arabinose concentrations were tested in order to determine the optimal condition to overproduce these polypeptides. Both proteins remained in the insoluble fraction in all tested conditions, and for this reason, the proteins were purified from the inclusion bodies. The procedure for protein purification is based on previously reported methods (41Cannon W. Missailidis S. Smith C. Cottier A. Austin S. Moore M. Buck M. J. Mol. Biol. 1995; 248: 781-803Crossref PubMed Scopus (62) Google Scholar) with minor modifications. Briefly, after 3 h of induction at 30 °C with 0.02% arabinose, the cells were lysed by sonication at 4 °C. The insoluble fraction was extracted with TGED buffer containing 1 m NaCl and 1 mm phenylmethylsulfonyl fluoride or a mixture of protease inhibitors manufactured by Roche Applied Science, and a second extraction with 1% Triton X-100 was carried out. Detergent was removed by washing three times with TGED. Finally, the pellet was carefully resuspended in 8 m urea buffered with TGED. The protein was dialyzed overnight at 4 °C against TGED buffer containing 250 mm NaCl, followed by a further 6-h dialysis with fresh TGED buffer containing 100 mm NaCl. Insoluble material was removed by centrifugation. The oligonucleotides 5′-AGATCTGGTGCGCGTGCCCGAGGGGGC-3′ and 5′-AGATCTGCTGCGCCTGCCGCCGGCGCC-3′ were used as forward primers to obtain the PCR product coding for RpoN1ΔI and RpoN2ΔI. These products were cloned into pBAD/His-C and introduced into LMG174-1 strain. To obtain the polypeptides corresponding to Region I of RpoN1 and RpoN2, the DNA region encoding the first 57 residues of these proteins was amplified by PCR using as reverse primers the 57Nif or 57Fli oligonucleotide (5′-GGAATTCTCACTCGAGGCAGGGATTTTC-3′ or 5′-GAATTCTCATTCGATGAAAGGGTTTTC-3′, respectively), which include TCA bases as the stop codon. These proteins were purified following the procedure described above. Circular Dichroism—Previous to CD analysis, the proteins were dialyzed against 10 mm sodium phosphate buffer, pH 8.0, 10 mm NaCl, and 20% glycerol, with three changes of the dialysis buffer, each of 100 times the sample volume. Dialysis was carried out over a period of 24 h at 4 °C. Molar ellipticity values were obtained using an Aviv Biomedical CD spectrophotometer model 202-01. The resulting spectra were analyzed using CDPro (available on the World Wide Web at lamar.colostate.edu/~sreeram/CDPro/main.html). Gel Mobility Shift Assay—Binding reactions were carried out at 30 °C in STA buffer (24Cannon W. Gallegos M.T. Casaz P. Buck M. Genes Dev. 1999; 13: 357-370Crossref PubMed Scopus (66) Google Scholar) in a total volume of 20 μl. The reactions included 1 μg of poly(dI-dC) as nonspecific competitor, DNA probe labeled with 32P, and a 50, 100, or 200 μm concentration of the indicated protein. After 20 min, the samples were loaded on a 4.5% native polyacrylamide gel and run at 12 mA in Tris-glycine buffer. After electrophoresis, radioactivity was visualized and quantified using a PhosphorImager apparatus (Bio-Rad) or by autoradiography using ImageJ software. Analysis of the RpoN1- and RpoN2-dependent Promoters from the Complete Genome of R. sphaeroides—To investigate if there were any significant differences in the promoter sequences recognized by RpoN1 and RpoN2, we compared the promoter sequences that are recognized by these sigma factors. For this, the logo sequence of each set of flagellar (fli) and nitrogen fixation (nif) promoters predicted from the genome sequence was obtained using the information content software developed by Schneider and Stephens (42Schneider T.D. Stephens R.M. Nucleic Acids Res. 1990; 18: 6097-6100Crossref PubMed Scopus (2439) Google Scholar). Comparison of the logo sequences revealed some differences (Fig. 1), in particular a higher conservation at the -26-, -27-, and -28-positions for the nif promoters as well as a strong preference for T or A at the -11-position in the nif or fli promoters, respectively, suggesting that RpoN1 recognizes a larger promoter sequence than RpoN2 and that the -11-position could be important for specificity. Evaluation of the Contribution of the Promoter Sequence Bias to Sigma Factor Specificity—To test if the differences in the promoter sequences mentioned above might be involved in the differential recognition and expression mediated by RpoN1 and RpoN2, we mutagenized the nucleotides of fliOp for those present in the sequence of nifUp. Transcriptional fusions of these promoter versions with the reporter gene uidA (encoding β-glucuronidase) were constructed using pBBMCS53. The amount of β-glucuronidase expressed from these plasmids was determined for WS8 cells grown under N+-aerobic conditions, which would maintain a low level of RpoN1 (43Meijer W.G. Tabita F.R. J. Bacteriol. 1992; 174: 3855-3866Crossref PubMed Google Scholar). Compared with the level of β-glucuronidase produced by the wild-type fliOp promoter, a 5-fold reduction was observed when changes were introduced at positions -28, -27, and -26, and a 9-fold reduction was observed when changes were introduced at positions -15 and -16. When these changes where combined in a single construction, the promoter activity was reduced by only 5-fold (Fig. 2). It is noteworthy that when the wild-type A at the -11-position was substituted by any other nucleotide, a reduction of 50-100-fold in the promoter activity was observed, suggesting a major contribution of this particular nucleotide in the activity of the fliOp promoter. For simplicity, the changes at -28, -27, and -26 will be referred to hereafter as up-24, and the changes at -16 and -15 will be referred to as up-12. All previous constructions were introduced into the SP7/pRS210 strain, which carries a mutation in rpoN2 but properly expresses the fleT and fleQ genes encoding the flagellar EBPs (35Poggio S. Osorio A. Dreyfus G. Camarena L. Mol. Microbiol. 2005; 58: 969-983Crossref PubMed Scopus (43) Google Scholar). As shown in Fig. 2, in this strain, the activities of all of these promoters were strongly reduced, indicating that RpoN2 was responsible for the activity detected in WS8 cells. As mentioned above, the expression of RpoN1 would be at a low level under the growth conditions used in the previous experiments (43Meijer W.G. Tabita F.R. J. Bacteriol. 1992; 174: 3855-3866Crossref PubMed Google Scholar). Therefore, to correctly evaluate if RpoN1 could promote transcription from these promoters, we cultured these strains under nitrogen-fixing conditions, as described under “Experimental Procedures.” As an induction control, the plasmid carrying the fusion nifUp-uidA was included in the assay. All of the fliOp promoter versions tested in the WS8 strain showed a similar amount of β-glucuronidase activity to that observed under N+-aerobic conditions (Fig. 3), indicating that these promoters are not significantly expressed by RpoN1. A slight increase in the activity level could be detected in SP7/pRS210 cells grown under nitrogen-fixing conditions, when the fliOp promoter more closely resembled nifUp (Fig. 3D), although the β-glucuronidase activity is only 5% of tha" @default.
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• W2000873407 date "2006-09-01" @default.
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• W2000873407 title "Transcriptional Specificity of RpoN1 and RpoN2 Involves Differential Recognition of the Promoter Sequences and Specific Interaction with the Cognate Activator Proteins" @default.
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• W2000873407 doi "https://doi.org/10.1074/jbc.m601735200" @default.
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