Journal of Bacteriology, February 2004, p . 740-749, Vol . 186, No . 3

Rhodobacter capsulatus nifA1 Promoter: High-GC -10 Regions in High-GC Bacteria and the Basis for Their Transcription

Cynthia L . Richard, Animesh Tandon, and Robert G . Kranz^*

Department of Biology, Washington University, St . Louis, Missouri 63130

Received 27 August 2003/ Accepted 29 October 2003

 
It was previously shown that the Rhodobacter capsulatus NtrC enhancer-binding protein activates the R . capsulatus housekeeping RNA polymerase but not the Escherichia coli RNA polymerase at the nifA1 promoter . We have tested the hypothesis that this activity is due to the high G+C content of the -10 sequence . A comparative analysis of R . capsulatus and other -proteobacterial promoters with known transcription start sites suggests that the G+C content of the -10 region is higher than that for E . coli . Both in vivo and in vitro results obtained with nifA1 promoters with -10 and/or -35 variations are reported here . A major conclusion of this study is that -proteobacteria have evolved a promiscuous sigma factor and core RNA polymerase that can transcribe promoters with high-GC -10 regions in addition to the classic E . coli Pribnow box . To facilitate studies of R . capsulatus transcription, we cloned and overexpressed all of the RNA polymerase subunits in E . coli, and these were reconstituted in vitro to form an active, recombinant R . capsulatus RNA polymerase with properties mimicking those of the natural polymerase . Thus, no additional factors from R . capsulatus are necessary for the recognition of high-GC promoters or for activation by R . capsulatus NtrC . The addition of R . capsulatus ⁷⁰ to the E . coli core RNA polymerase or the use of -10 promoter mutants did not facilitate R . capsulatus NtrC activation of the nifA1 promoter by the E . coli RNA polymerase . Thus, an additional barrier to activation by R . capsulatus NtrC exists, probably a lack of the proper R . capsulatus NtrC-E . coli RNA polymerase (protein-protein) interaction(s) .

 
Rhodobacter capsulatus is an anoxygenic photosynthetic -proteobacterium that can fix nitrogen under anaerobic or microaerobic conditions . The R . capsulatus two-component nitrogen regulatory system (NtrB-NtrC) is similar to the well-characterized enteric system with respect to sensing and phosphorelay . R . capsulatus NtrC also binds to tandem sites located greater than 100 nucleotides upstream of the transcription start site . However, the two systems differ in how phosphorylated R . capsulatus NtrC activates its target promoters . R . capsulatus NtrC activates the ⁷⁰ housekeeping RNA polymerase (RNAP) in a manner that requires ATP binding but not ATP hydrolysis (4, 7) . This activation mechanism may represent a transition between the ⁷⁰- and ⁵⁴-dependent transcriptional activators (4) .

The -proteobacteria characteristically have a genomic G+C content of 65% or greater and are predicted to have diverged from the -proteobacteria (e.g., Escherichia coli) approximately 500 million years ago (5) . Kranz et al . previously noted that R . capsulatus and other -proteobacteria (see reference 15 for a review) may have -10 features different from those of organisms with a lower G+C content (3, 4) . For another -proteobacterium, Sinorhizobium meliloti, most of the ⁷⁰ promoters that have been characterized are not transcribed by the E . coli RNAP in vivo or in vitro, but the S . meliloti RNAP can initiate transcription at typical E . coli ⁷⁰ promoters (23) . The -proteobacterium Caulobacter crescentus ⁷³ RNAP recognizes E . coli ⁷⁰ promoters lacUV5 and neo, whereas E . coli does not recognize typical C . crescentus ⁷³ promoters (28) . For Rhodobacter sphaeroides, a similar situation was shown with the rrnB promoter (14) . The R . capsulatus ⁷⁰ RNAP also was shown to recognize typical E . coli ⁷⁰ promoters, while the E . coli ⁷⁰ RNAP does not recognize some R . capsulatus promoters (4, 8) . The present study is intended to address the basis for these differences by using the well-characterized nifA1 promoter .

Bowman and Kranz previously noted that the E . coli RNAP was not activated by R . capsulatus NtrC at the nifA1 promoter, even when the -35 hexamer was changed toward the consensus sequence (4) . It was reasoned that this effect could be due to (i) nucleotides in the -10 region being incompatible with the E . coli RNAP, (ii) another missing cofactor which copurifies with the R . capsulatus RNAP, and/or (iii) a site(s) of interaction with the R . capsulatus NtrC protein that coevolved with the R . capsulatus RNAP but is absent in the E . coli RNAP . In the present study, we analyzed the nifA1 -10 region to address the first possibility . A difference in recognition of the nifA1 -10 region between the E . coli and the R . capsulatus RNAPs was investigated further . Using recombinant R . capsulatus RNAP subunits (, ß, ß', ⁷⁰, and ) produced in E . coli and R . capsulatus ßß'⁷⁰ subunits assembled in vitro, we ruled out the involvement of another factor(s) from R . capsulatus, suggesting that the site(s) of interaction between R . capsulatus NtrC and the RNAP may be a major determinant in R . capsulatus NtrC activation . Richard et al . recently reported that the specific site of interaction with R . capsulatus NtrC is the R . capsulatus ß' subunit and that the E . coli ß' subunit lacks this site (24) .

 
Growth media and strains. E . coli strains were grown in Luria broth at 37°C, and R . capsulatus strains were grown in RB (1) broth at 34°C . Carbenicillin (Sigma), when needed, was used at a concentration of 150 µg/ml . Tetracycline, when required, was used at concentrations of 1 µg/ml for R . capsulatus strains and at 12.5 µg/ml for E . coli strains . The source of fixed nitrogen for nitrogen-free RB broth was glutamate (10 mM) . R . capsulatus SB1003 is a spontaneous rifampin-resistant wild-type strain (29) . E . coli strains BL21(DE3), Bl21(DE3)/pLysS, and Tuner(DE3)/pLacI were purchased from Novagen, and E . coli CC118 (18) is a lacZ phoA mutant .

In vitro transcription templates. Plasmids pnifA1_mut1, pnifA1_mut2, and pnifA1_mut3 were described previously (8) . pnifA1_mut3A was made by PCR of mutant 3 of the nifA1 promoter (nifA1_mut3) with the upstream oligonucleotide 5'-AGCGGATAACAATTTCACACAGGAAACAGC-3' and the downstream oligonucleotide 5'-CCGGATCCTGCAGTCGGGACTTCTGCACTGACTATAGGGC-3', with the downstream oligonucleotide containing a G-to-A change in the -10 sequence (bold) (mutant 3A of nifA1 [nifA1_mut3A]) . The 0.3-kb product was digested with EcoRI/PstI and ligated to EcoRI/PstI-digested pUC118 . pnifA1_mut3AA was made by PCR of the nifA1_mut3 promoter with the upstream oligonucleotide 5'-AGCGGATAACAATTTCACACAGGAAACAGC-3' and the downstream oligonucleotide 5'-CCGGATCCTGCAGTCGGGACTTCTGCACTGATTATAGGGC-3', with the downstream oligonucleotide containing a GG-to-AA change in the -10 sequence (bold) (mutant 3AA of nifA1 [nifA1_mut3AA]) . The 0.3-kb product was digested with EcoRI/PstI and ligated to EcoRI/PstI-digested pUC118 . RNAI is a promoter present on the transcription template that served as an internal control .

lacZ promoter fusions. nifA1 and mutant nifA1 promoter-lacZ fusion plasmids were generated in the following manner . Plasmid A1Z118 (372-bp nifA1 promoter fused to the lacZYA operon from pSKS104 [6] in pUC118) was digested with PstI, yielding a fragment containing 194 bp of the nifA1 promoter downstream of the -10 sequence fused to the lacZYA operon . The 6.4-kb PstI fragment was ligated to PstI-linearized pnifA1 to yield pnifA1lacZYA . pPUCAnifA1 was generated by excising a KpnI/HindIII fragment containing the 6.6-kb nifA1-lacZYA fusion and the 124-bp cassette from pnifA1lacZYA and ligating it to KpnI/HindIII-digested pUCA12 . pUCA12 is a derivative of pRK2013 (9)-mobilizable Tet^r pUCA10 (10) and contains an expanded multiple cloning site . All of the pUCA-lacZ fusion plasmids (pUCAnifA1, pUCAnifA1_mut1, pUCAnifA1_mut2, pUCAnifA1_mut3, pUCAnifA1_mut3A, and pUCAnifA1_mut3AA) were generated from their respective in vitro transcription templates, described above, in the same manner as pUCAnifA1 .

Protein overexpression plasmids. Plasmids pRGK301, pHTT7f1-NH, pMKSe2, and pT7ß' were described previously (3, 4, 25, 27, 30) . pRGK325, which allows overexpression of the R . capsulatus subunit of RNAP, was made by PCR of the rpoA gene from the chromosome of R . capsulatus SB1003 by use of the upstream oligonucleotide 5'-GAGGCAGAGCATATGATCCACAAGAATTGG-3' and the downstream oligonucleotide 5'-CGGGATCCTCAGAACTGGTCTTCGAAGCGCTTGGCC-3' . The 1-kb product was digested with NdeI and BamHI and ligated in frame to the amino-terminal six-histidine tag encoded by pET15b (Novagen) . pRGK326, which allows overexpression of the R . capsulatus ß subunit of RNAP, was made by PCR of the rpoB gene from the chromosome of SB1003 by use of the upstream oligonucleotide 5'-CGACGACCATGGCTCAAGCTTACGTTGGTCAG-3' and the downstream oligonucleotide 5'-GCGGATCCTCACTCTTCCTCCGAATCCAGGAG-3' . The 4.1-kb product was digested with NcoI/BamHI and ligated to NcoI/BamHI-digested pETBlue-2 (Novagen) . pRGK327, which allows overexpression of the R . capsulatus ß' subunit of RNAP, was made by PCR of the rpoC gene from the chromosome of SB1003 by use of the upstream oligonucleotide 5'-GGAAAGATCCCATGGACCAGGAAATCACCAACAAC-3' and the downstream oligonucleotide 5'-GGGGTACCTCAATCGCGGCTTTCCGGGGTTC-3' . The 4.2-kb product was digested with NcoI/KpnI and ligated to NcoI/KpnI-digested pETBlue-2 . pRGK328, which allows overexpression of the R . capsulatus subunit of RNAP, was made by PCR of the rpoZ gene from the chromosome of SB1003 by use of the upstream oligonucleotide 5'-CTGGAGTTGCCCATGGCCCGCGTGACGGTTGAA-3' and the downstream oligonucleotide 5'-CATGCCTCGAGTCAGTCGCGGCCTTGCGCGTC-3' . The 0.4-kb product was digested with NcoI/XhoI and ligated in frame to the carboxy-terminal six-histidine tag encoded by pETBlue-2 . pRGK329, which allows overexpression of the E . coli ⁷⁰ subunit of RNAP, was made by PCR of the rpoD gene from the chromosome of E . coli K-12 by use of the upstream oligonucleotide 5'-CGTCTCCCATGGAGCAAAACCCGCAGTCACAG-3' and the downstream oligonucleotide 5'-AAGGAGAGGAGCGGCCGCTTAATCGTCCAGGAAGCTACGCAGCAC-3' . The 1.8-kb product was digested with BsmBI/NotI and ligated to NcoI/NotI-digested pET30a (Novagen) with an in-frame fusion to the pET30a-encoded amino-terminal six-histidine tag .

Purification of R . capsulatus ⁷⁰, maltose-binding protein-NtrB, R . capsulatus NtrC, and R . capsulatus RNAP. Purification of the six-histidine-tagged R . capsulatus ⁷⁰ subunit and maltose-binding protein-NtrB was described previously (4, 7) . R . capsulatus NtrC was purified by the method of Cullen et al . (7) . R . capsulatus RNAP was isolated and purified as described by Cullen et al . (8) .

Purification of the R . capsulatus subunit of RNAP. The R . capsulatus subunit was purified by a modification of the procedure of Tang et al . (27) . The six-histidine-tagged subunit of R . capsulatus was overexpressed in E . coli strain BL21(DE3) containing pRGK325 by exposure to 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 3 h at 37°C . Cells were harvested by centrifugation at 3,000 x g for 10 min and sonicated in 20 ml of buffer A (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 5 mM imidazole) . The lysate was cleared by centrifugation at 16,000 x g for 20 min at 4°C in a Sorvall centrifuge . As determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis, the supernatant contained a major polypeptide of approximately 43 kDa that was not present in the uninduced sample . The volume of the supernatant was adjusted to 50 ml with buffer A, and the R . capsulatus subunit was precipitated by the addition of (NH₄)₂SO₄ to 60% . The R . capsulatus subunit was collected by centrifugation at 16,000 x g for 20 min at 4°C and solubilized in 20 ml of buffer B (6 M urea, 20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 5 mM imidazole) . The sample was loaded onto a His-Bind (Novagen) column that had been washed after Ni²⁺ charging with buffer B, and the column then was washed with 10 column volumes of buffer B followed by 6 column volumes of buffer C (6 M urea, 20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 30 mM imidazole) . The six-histidine-tagged R . capsulatus subunit of RNAP was eluted with 6 column volumes of buffer D (6 M urea, 20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 500 mM imidazole), and 1-ml fractions were collected . The protein concentrations were determined with Coomassie Plus-200 protein assay reagent (Pierce) . The protein fractions were stored at -80°C and were found to be stable for reconstitution for up to 3 months .

Preparation of crude recombinant R . capsulatus ß and ß' subunits. The R . capsulatus ß and ß' inclusion bodies were prepared by a modification of the method of Tang et al . (27) . The ß and ß' subunits of R . capsulatus RNAP were overexpressed in E . coli strain BL21(DE3)/pLysS containing pRGK326 and pRGK327, respectively, by exposure of cultures that had been grown to an optical density at 600 nm of 0.6 to 1.0 to 1 mM IPTG for 3 h at 37°C . Cells were harvested at 3,000 x g for 15 min at 4°C and sonicated in 16 ml of buffer E (40 mM Tris-HCl [pH 8.0], 300 mM KCl, 10 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) with the addition of 0.2 mg of lysozyme/ml and 0.2% (wt/vol) sodium deoxycholate . Following incubation on ice with gentle shaking for 20 min, the inclusion bodies were harvested by centrifugation at 38,000 x g for 30 min at 4°C, washed once with buffer E containing 0.2% (wt/vol) n-ocytl-ß-D-glucoside, and washed once with buffer E . Each wash step included sonication, incubation on ice for 30 min, and centrifugation at 38,000 x g for 30 min . The washed inclusion bodies were solubilized in buffer F (6 M urea, 50 mM Tris-HCl [pH 8.0], 10 mM MgCl₂, 10 µM ZnCl₂, 1 mM EDTA, 10 mM dithiothreitol, 10% [vol/vol] glycerol) by standing on ice for 1 h . Following solubilization, the supernatant was cleared by centrifugation at 10,000 x g for 10 min at 4°C . The protein concentrations were determined as described above, and the crude R . capsulatus ß and ß' preparations were stored at -80°C . The washed inclusion bodies were found to be stable for reconstitution for up to 3 months .

Purification of the subunit of R . capsulatus RNAP. The six-histidine-tagged subunit of R . capsulatus was overexpressed in E . coli strain Tuner(DE3)/pLacI containing pRGK328 . The culture was grown to an optical density at 600 nm of 0.6 to 1.0, and protein expression was induced by exposure to 1 mM IPTG for 3 h at 37°C . The cells were harvested at 3,000 x g for 15 min at 4°C and sonicated in 20 ml of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl [pH 7.9]) . After centrifugation at 12,000 x g for 20 min at 4°C in a Sorvall centrifuge, the supernatant contained a major polypeptide of approximately 16 kDa that was not present in the uninduced sample . The cleared supernatant was loaded onto a His-Bind column, and the column then was washed with 10 column volumes of binding buffer followed by 6 column volumes of wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl [pH 7.9]) . The six-histidine-tagged R . capsulatus subunit was eluted in 6 column volumes of elution buffer (250 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl [pH 7.9]), and 1-ml fractions were collected . The protein concentrations of the fractions were determined as described above, and the fractions were stored at -80°C for up to 3 months .

Reconstitution of R . capsulatus ßß'⁷⁰. Recombinant reconstituted R . capsulatus holoenzyme (R . capsulatus ßß'⁷⁰) was prepared by a modification of the method of Tang et al . (27) . The amounts of RNAP subunits added per 2-ml reconstitution mixtures were as follows: 60 µg of six-histidine-tagged R . capsulatus , 300 µg of crude R . capsulatus ß, 600 µg of crude R . capsulatus ß', 60 µg of six-histidine-tagged R . capsulatus , and 120 µg of six-histidine-tagged R . capsulatus ⁷⁰ . The R . capsulatus RNAP subunits were combined in Snakeskin dialysis tubing (Pierce), the final reconstitution volume was brought to 2 ml with buffer F, and the samples were dialyzed overnight against two changes of buffer G (50 mM Tris-HCl [pH 8.0], 200 mM KCl, 10 mM MgCl₂, 10 µM ZnCl₂, 1 mM EDTA, 5 mM 2-mercaptoethanol, 20% [vol/vol] glycerol) . After the dialyzed samples were cleared by centrifugation at 16,000 x g for 10 min at 4°C, the RNAPs were mixed with 0.2 ml of Ni²⁺-charged His-Bind that had been equilibrated in buffer H (50 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 5% [vol/vol] glycerol) . Following adsorption for 2 h at 4°C with gentle rotation, the resin was washed three times with 1.5 ml of buffer H containing 5 mM imidazole . Each wash step included 1 min of mixing and 1 min of centrifugation at 1,000 x g . The reconstituted recombinant RNAPs were eluted with 0.25 ml of buffer H containing 150 mM imidazole by gentle rotation at 4°C for 1 h . The RNAPs were concentrated to 100 µl by centrifugal ultrafiltration (Centricon-100) . Following concentration, the final glycerol concentration was brought to 50% . RNAPs prepared in this way were stored at -20°C and were found to be stable for at least 1 month .

Other methods. In vitro transcription assays were performed as described by Bowman and Kranz (4) . Transcripts were quantitated by digitizing the autoradiograms with a Fuji luminescent image analyzer (LAS-1000 Plus) and analyzing the bands with Fuji Image Gauge software (version 3.4) . Both the digitizing and the software analysis could readily distinguish differences in transcript levels of twofold or more . Conjugation with R . capsulatus SB1003 and J61 was carried out as described previously (1) . ß-Galactosidase activities were determined from sonicated cell extracts as previously described (10) . Protein measurements for the ß-galactosidase assays were determined with Pierce Coomassie Plus-200 protein reagent and a microplate protocol at A₅₇₅ .

 
Effects of mutations of the nifA1_mut3 -10 sequence on in vitro and in vivo transcription. Unlike the R . capsulatus RNAP, the E . coli RNAP could not be activated by R . capsulatus NtrC in vitro at any nifA1 promoter, including nifA1_mut1, mutant 2 of nifA1 (nifA1_mut2), and mutant 3 of nifA1 (nifA1_mut3), when -35 nucleotides were changed (4); Fig . 1 shows the promoter sequences used . The E . coli RNAP exhibits only a low level of basal transcription from the nifA1_mut3 promoter (Fig . 1B, lane 6), which is close to the consensus -35 hexamer and has 4 out of the 6 nucleotides in the -10 consensus sequence (TATGGT versus TATAAT; Fig . 1A) . To address whether the R . capsulatus RNAP holoenzyme may be more efficient than the E . coli RNAP at transcribing the nifA1 -10 sequence because of the neighboring GG nucleotides, we mutated the nifA1_mut3 -10 sequence to either TATAGT or TATAAT . To determine whether the nucleotides in the nifA1 -35 and -10 sequences play a role in determining the differential transcription of these promoters in vivo, lacZ was expressed from the nifA1 mutant promoters . Table 1 shows the lacZ-encoded ß-galactosidase activities from all of the nifA1 promoters in E . coli CC118, R . capsulatus SB1003 (NtrC⁺), and R . capsulatus J61 (NtrC^-) . As shown in vitro previously (4), the nifA1, nifA1_mut1, and nifA1_mut2 promoters are still responsive to R . capsulatus NtrC activation, but the nifA1_mut3 promoter is constitutive and fully expressed in R . capsulatus NtrC^- and NtrC⁺ strains . In a comparison of nifA1_mut3 in E . coli and R . capsulatus, R . capsulatus expresses nifA1_mut3 at a level approximately 25-fold higher than E . coli (7,714 versus 308 units of ß-galactosidase activity) . Changing GG to AG (nifA1_mut3A) or AA (nifA1_mut3AA) increases transcription four- to fivefold in E . coli but only twofold in R . capsulatus . These results are consistent with the hypothesis that R . capsulatus can naturally transcribe -10 regions with a higher G+C content .

 
To confirm the results in vitro and examine the biochemical basis for the results, we analyzed the above promoters using E . coli and R . capsulatus RNAPs . We quantitated the ability of the E . coli RNAP and the R . capsulatus RNAP to transcribe the nifA1_mut3, nifA1_mut3A, and nifA1_mut3AA supercoiled templates in vitro (Fig . 1B to D) . Table 2 shows in vitro transcription of these promoters (relative to control RNAI transcription) for different RNAPs at limiting concentrations . The results obtained with the native holoenzymes show that the R . capsulatus RNAP is 36-fold more effective than the E . coli RNAP at transcribing the nifA1_mut3 promoter template (TATGGT) and that changing GG to AG or AA facilitated increased transcription by the E . coli RNAP (Fig . 1B to D, lanes 6, and Table 2) .

 
Analysis of 40 known promoters from a Rhodobacter sp . for which a transcriptional start site has been identified revealed a -35 region similar to that of E . coli . A consensus -35 sequence (percentage of each base is shown as a subscript) of T₇₇T₈₈G₈₀C₄₅C/G₃₅N was identified and is consistent with that previously analyzed by site-directed mutagenesis for R . capsulatus by Cullen et al . (8) . A consensus -10 sequence was not as apparent . A region with the sequence T₃₅A₆₅T₄₅A₄₅A₃₈T₅₃ (compared to the E . coli sequence T₈₀A₉₅T₄₅A₆₀A₅₀T₉₆) was found 5 to 9 nucleotides upstream of the transcription start site . Interestingly, 33 of the 40 ⁷⁰ promoters analyzed have at least 50% G+C at position 4 or 5, and 8 have a G or a C at both positions . The two most conserved positions of the -10 sequence, 2 (A; 65%) and 6 (T; 53%), are the same as in the E . coli -10 consensus sequence . These two positions may be the most important nucleotides for sequence-specific binding of the RNAP subunit, while positions 4 and 5 have been found to be important in single-stranded DNA as part of the transcription bubble (21) .

Comparison of R . capsulatus RNAP and E . coli RNAP with heterologous ⁷⁰ and -10 effects. To analyze the role of the R . capsulatus ⁷⁰ subunit in promoter recognition and melting, we used the E . coli RNAP core enzyme and R . capsulatus ⁷⁰ for in vitro transcription with the nifA1_mut3 template . The addition of R . capsulatus ⁷⁰ to the E . coli RNAP core enzyme facilitated an eightfold increase in the transcription of the nifA1_mut3 -10 sequence (Fig . 1B, compare lane 5 with lane 6, and Table 2) . Conversely, we added E . coli ⁷⁰ to the R . capsulatus RNAP core enzyme and analyzed this RNAP in the same manner . The R . capsulatus RNAP core enzyme with E . coli ⁷⁰ is approximately 2-fold less efficient at transcribing the nifA1_mut3 -10 sequence than the homologous R . capsulatus holoenzyme but still 21-fold more efficient than the homologous E . coli holoenzyme (Fig . 1B, compare lane 2 with lane 3 and lane 3 with lane 6) . These results suggest that optimal transcription of the -10 sequence with TATGGT is significantly increased because of R . capsulatus ⁷⁰ but is not due solely to the ⁷⁰ subunit . These findings also suggest a more promiscuous role for R . capsulatus ⁷⁰ in recognition and melting of the -10 sequence, a role that has been proposed for E . coli ^S as well (11, 16) . R . capsulatus ⁷⁰ has evolved the ability to transcribe promoters with a higher G+C content while retaining the ability to transcribe promoters that are more A+T rich . This conclusion is consistent with the analysis of the 40 promoters discussed earlier . It has been noted that the regions of R . capsulatus ⁷⁰ corresponding to regions 2.3, 2.4, and 2.5 of E . coli ⁷⁰ (22) are identical . These regions are implicated in DNA strand melting and are responsible for sequence-specific recognition of both double- and single-stranded DNA within the -10 and "extended -10" promoter elements (2, 19, 26, 31) . Therefore, the ability to facilitate transcription of the high-GC -10 regions may be due to other properties of the ⁷⁰ subunit . This notion is similar to an observation made in a study of another GC-rich organism, Chlamydia, when the promoters were tested in vivo in E . coli (20) .

If temperature affects the ability of the E . coli RNAP to transcribe the -10 sequence of the nifA1_mut3 promoter, then the melting of TATGGT may be an important element . We performed in vitro transcription assays with the nifA1_mut3 template at a variety of temperatures (Fig . 2) . The E . coli holoenzyme is approximately fourfold more efficient at transcribing the nifA1_mut3 promoter at 37°C than at 20 or 25°C (Fig . 2) . At 20 through 37°C, the level of transcription is significantly increased when R . capsulatus ⁷⁰ is added to the E . coli RNAP core enzyme (Fig . 2), again suggesting an important role for the R . capsulatus ⁷⁰ subunit . At 20°C, the R . capsulatus RNAP core enzyme with added E . coli ⁷⁰ transcribes the nifA1_mut3 promoter less efficiently than does this enzyme with added R . capsulatus ⁷⁰ (Fig . 2) . However, at higher temperatures, the difference disappears, again suggesting that the R . capsulatus RNAP core also plays a role in optimal transcription of this promoter . It is possible that increased temperatures (e.g., at least 30°C) aid strand separation, thus making transcription less dependent on E . coli ⁷⁰ in melting the neighboring GG nucleotides (13) . This notion is also suggested by results obtained with nifA1_mut3 -10 promoter sequences in which GG is changed to AG or AA . As the -10 sequence is changed toward the consensus sequence, E . coli ⁷⁰ can more efficiently transcribe the nifA1_mut3 promoters . While the R . capsulatus RNAP activity approximately doubles when GG is changed to AG or AA, the E . coli RNAP activity increases over 30-fold (Table 1) . We conclude that both the R . capsulatus RNAP core and the R . capsulatus ⁷⁰ subunit facilitate transcription of the TATGGT -10 promoter . It is likely that in addition to ⁷⁰ promiscuity, the core enzyme has coevolved this property with the high-GC nature of this -proteobacterium .

 
R . capsulatus ⁷⁰ can be replaced with E . coli ⁷⁰ and activated by R . capsulatus NtrC. The second possible barrier for the E . coli RNAP in activation by R . capsulatus NtrC at the nifA1promoter is that another factor (such as R . capsulatus ⁷⁰ or an additional factor) is required . To test this possibility and to begin to analyze the protein-protein interactions of R . capsulatus NtrC and the RNAP, we performed in vitro transcription and R . capsulatus NtrC activation assays with supercoiled nifA1_mut1, nifA1_mut2, and nifA1_mut3 templates and various RNAP preparations (Fig . 3) . All three nifA1 mutant promoter templates demonstrate that R . capsulatus NtrC is required for activation, with significant basal transcription of the nifA1_mut3 template by the R . capsulatus RNAP (Fig . 3A, lane 2), as shown earlier (4) . There is <8% basal transcription of the nifA1_mut1 and nifA1_mut2 templates (Fig . 3B, lanes 2 and 3, and Fig . 3C, lanes 2 and 3), but upon the addition of activators, the level of the nifA1_mut1 transcript is approximately equivalent to that of the RNAI transcript (Fig . 3B, lanes 4 and 5) and the level of the nifA1_mut2 transcript is approximately 50% of that of the RNAI transcript (Fig . 3C, lanes 4 and 5) . Activated transcription occurs with either R . capsulatus ⁷⁰ or E . coli ⁷⁰ used with the R . capsulatus RNAP core enzyme (Fig . 3, lanes 4 and 5) . The E . coli RNAP holoenzyme is not activated by R . capsulatus NtrC when the nifA1_mut3, nifA1_mut1, or nifA1_mut2 promoter template is used (Fig . 3, lanes 9) . The barrier to R . capsulatus NtrC activation is not overcome by the R . capsulatus ⁷⁰ subunit (Fig . 3, lanes 10), suggesting that the R . capsulatus ⁷⁰ subunit is not sufficient to allow activation by R . capsulatus NtrC .

 
Other factors are not required for R . capsulatus NtrC activation, as determined by in vitro reconstitution of the recombinant R . capsulatus ⁷⁰ holoenzyme. Specific R . capsulatus RNAP subunits and/or some other factor that copurifies with the R . capsulatus RNAP enzyme most likely are involved in activation by R . capsulatus NtrC at the nifA1 promoter . To address whether other, minor factors from R . capsulatus are involved, we assembled in vitro active R . capsulatus RNAP (R . capsulatus ßß'⁷⁰) from purified recombinant subunits . We used a slightly modified version of the method of Tang et al . (27), which was previously used to efficiently reconstitute the E . coli RNAP core enzyme and RNAP holoenzyme . This method uses the six-histidine tag on the R . capsulatus subunit, facilitating RNAP purification when crudely prepared R . capsulatus ß and ß' subunits overexpressed in E . coli are used . While our study was in progress, this method was shown to work efficiently with the subunits from other -proteobacterial organisms for in vitro transcription with the E . coli ß and ß' subunits (17, 23) . The R . capsulatus subunit (45% identical to the E . coli subunit) is overexpressed as an approximately 43-kDa subunit and purified by using an Ni²⁺ affinity column with urea . The R . capsulatus ß and ß' subunits (58 and 59% identical to the E . coli ß and ß' subunits, respectively) are overexpressed with no affinity tag and are crudely purified from inclusion bodies as approximately 150-kDa proteins . The R . capsulatus subunit (39% identical to the E . coli subunit) also is overexpressed as a C-terminal six-histidine-tagged protein of approximately 16 kDa and purified by using an Ni²⁺ affinity column . Analysis of the purified R . capsulatus RNAP subunits by SDS-PAGE showed that the ⁷⁰, , and subunits (Fig . 4A, lanes 2, 3, and 6) are >90% pure, while the ß and ß' subunits (Fig . 4A, lanes 4 and 5) are >80% pure . The subunits are mixed, and the urea is removed by dialysis . The recombinant holoenzyme is assembled and then purified by using an Ni²⁺ affinity column . Following the assembly and purification, the characteristic pattern of the holoenzyme polypeptides is observed (Fig . 4B) . The reconstituted recombinant R . capsulatus ßß'⁷⁰ RNAP obtained with this procedure typically is greater than 95% pure .

 
Because the R . capsulatus RNAP subunits are overexpressed in E . coli for in vitro reconstitution, the possibility exists for contaminating E . coli RNAP subunits to be reconstituted into active RNAPs . The strain of R . capsulatus (SB1003) used to engineer all of the RNAP subunits is rifampin resistant (Rif^r), whereas the E . coli strain used for overexpression is rifampin sensitive (Rif^s) . Rifampin inhibits bacterial transcription by binding to the ß subunit of RNAP (12) . The Rif^r property of SB1003 was previously characterized (8) and found to contain the same amino acid mutation as that which confers resistance to E . coli, a QL substitution at amino acid 532 which is homologous to E . coli QL at amino acid 513 . To confirm that the principal ß subunit present in our reconstituted recombinant R . capsulatus RNAP preparations is derived from the recombinant R . capsulatus ß subunit and not from a contaminating E . coli ß subunit, we used the Rif^r property of the R . capsulatus ß subunit . In vitro transcription reactions were performed in the presence of 62.5 ng of rifampin/µl with reconstituted R . capsulatus ßß' RNAP, reconstituted E . coli core RNAP, and the nifA1_mut1 template . The E . coli core RNAP has high levels of the RNAI transcript but, upon addition of rifampin, no RNAI transcript can be detected (Fig . 4C, compare lanes 11 and 12 with lanes 13 and 14) . Upon addition of rifampin to the R . capsulatus ßß' RNAP, RNAI and nifA1_mut1 transcripts are detected (Fig. 4C, compare lanes 4 and 5 with lanes 6 and 7) . These results indicate that a negligible amount of E . coli ß subunit contaminates our R . capsulatus ß-subunit preparations . The R . capsulatus ßß' RNAP core enzyme and the R . capsulatus natural RNAP core enzyme show similar levels of R . capsulatus NtrC activation of nifA1 when either R . capsulatus ⁷⁰ or E . coli ⁷⁰ is added (data not shown) .

The recombinant R . capsulatus ßß'⁷⁰ RNAP enzyme is as active at transcribing the RNAI control as the R . capsulatus natural RNAP holoenzyme when approximately equal amounts of the RNAPs are used (compare Fig . 5A, lanes 1 and 2, RNAI, with Fig . 3A, lane 2, RNAI) . R . capsulatus ßß'⁷⁰ RNAP also demonstrates an ability to transcribe the nifA1_mut3 promoter comparable to that of the R . capsulatus natural RNAP (compare Fig . 5A, lane 1, with Fig . 3A, lane 2; 58 versus 71%) as well as equivalent NtrC activation patterns for the nifA1_mut3 promoter (compare Fig. 5A, lane 2, with Fig . 3A, lane 4; approximately 2-fold), the nifA1_mut1 promoter (compare Fig . 5B, lane 2, with Fig . 3B, lane 4; >6-fold), and the nifA1_mut2 promoter (compare Fig . 5C, lane 2, with Fig . 3C, lane 4; >20-fold) . Richard et al . recently reported that reconstitution of recombinant R . capsulatus RNAP requires the subunit, unlike the E . coli enzyme (24) . Additionally, when a hybrid recombinant enzyme approach is used, it can be concluded that the R . capsulatus ß' subunit but not the R . capsulatus or ß subunit is necessary for R . capsulatus NtrC activation (24) .

 
We thank Bill Bowman for some in vitro transcription templates and Nathaniel Sloan for technical assistance .

This research was supported by USDA NRI grant 99-35305-8647 to R.G.K .

 
* Corresponding author . Mailing address: Washington University, Department of Biology, Campus Box 1137, 1 Brookings Dr., St . Louis, MO 63130 . Phone: (314) 935-4278 . Fax: (314) 935-4432 . E-mail: kranz@biology.wustl.edu.

1. Avtges, P., R . G . Kranz, and R . Haselkorn. 1985 . Isolation and organization of genes for nitrogen fixation in Rhodopseudomonas capsulata . Mol . Gen . Genet . 201:363-369.
2. Barne, K . A., J . A . Bown, S . J . Busby, and S . D . Minchin. 1997 . Region 2.5 of the Escherichia coli RNA polymerase sigma70 subunit is responsible for the recognition of the ‘extended-10’ motif at promoters . EMBO J . 16:4034-4040 .
3. Bowman, W . C., S . Du, C . E . Bauer, and R . G . Kranz. 1999. In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus . Mol . Microbiol . 33:429-437.
4. Bowman, W . C., and R . G . Kranz. 1998 . A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase . Genes Dev . 12:1884-1893 .
5. Brock, T . D., and M . T . Madigan. 1991 . Biology of microorganisms, 6th ed . Prentice Hall, Inc., Englewood Cliffs, N.J.
6. Casadaban, M . J., A . Martinez-Arias, S . K . Shapira, and J . Chou. 1983 . Beta-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast . Methods Enzymol . 100:293-308.
7. Cullen, P . J., W . C . Bowman, and R . G . Kranz. 1996 . In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system . J . Biol . Chem . 271:6530-6536 .
8. Cullen, P . J., C . K . Kaufman, W . C . Bowman, and R . G . Kranz. 1997 . Characterization of the Rhodobacter capsulatus housekeeping RNA polymerase . In vitro transcription of photosynthesis and other genes . J . Biol . Chem . 272:27266-27273 .
9. Ditta, G., S . Stanfield, D . Corbin, and D . R . Helinski. 1980 . Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti . Proc . Natl . Acad . Sci . USA 77:7347-7351.
10. Foster-Hartnett, D., and R . G . Kranz. 1992 . Analysis of the promoters and upstream sequences of nifA1 and nifA2 in Rhodobacter capsulatus: activation requires ntrC but not rpoN . Mol . Microbiol . 6:1049-1060.
11. Gaal, T., W . Ross, S . T . Estrem, L . H . Nguyen, R . R . Burgess, and R . L . Gourse. 2001 . Promoter recognition and discrimination by EsigmaS RNA polymerase . Mol . Microbiol . 42:939-954.
12. Jin, D . J., and C . A . Gross. 1988 . Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance . J . Mol . Biol . 202:45-58.
13. Juang, Y . L., and J . D . Helmann. 1994 . A promoter melting region in the primary sigma factor of Bacillus subtilis . Identification of functionally important aromatic amino acids . J . Mol . Biol. 235:1470-1488.
14. Karls, R . K., D . J . Jin, and T . J . Donohue. 1993 . Transcription properties of RNA polymerase holoenzymes isolated from the purple nonsulfur bacterium Rhodobacter sphaeroides . J . Bacteriol . 175:7629-7638.
15. Kranz, R . G., W . C . Bowman, and N . R . Sloan. 1999 . Nitrogen fixation: from molecules to crop productivity, p . 79-82 . Proceedings of the 12th International Congress on Nitrogen Fixation . Kluwer Academic Press, Dordrecht, The Netherlands.
16. Lee, S . J., and J . D . Gralla. 2001 . Sigma38 (rpoS) RNA polymerase promoter engagement via -10 region nucleotides . J . Biol . Chem . 276:30064-30071 .
17. Lohrke, S . M., S . Nechaev, H . Yang, K . Severinov, and S . J . Jin. 1999 . Transcriptional activation of Agrobacterium tumefaciens virulence gene promoters in Escherichia coli requires the A . tumefaciens rpoA gene, encoding the alpha subunit of RNA polymerase . J . Bacteriol . 181:4533-4539 .
18. Manoil, C., and J . Beckwith. 1985 . TnphoA: a transposon probe for protein export signals . Proc . Natl . Acad . Sci . USA 82:8129-8133.
19. Marr, M . T., and J . W . Roberts. 1997 . Promoter recognition as measured by binding of polymerase to nontemplate strand oligonucleotide . Science 276:1258-1260 .
20. Mathews, S . A., and R . S . Stephens. 1999 . DNA structure and novel amino and carboxyl termini of the Chlamydia sigma 70 analogue modulate promoter recognition . Microbiology 145:1671-1681.
21. Matlock, D . L., and T . Heyduk. 2000 . Sequence determinants for the recognition of the fork junction DNA containing the -10 region of promoter DNA by E . coli RNA polymerase . Biochemistry 39:12274-12283.
22. Pasternak, C., W . Chen, C . Heck, and G . Klug. 1996 . Cloning, nucleotide sequence and characterization of the rpoD gene encoding the primary sigma factor of Rhodobacter capsulatus . Gene 176:177-184.
23. Peck, M . C., T . Gaal, R . F . Fisher, R . L . Gourse, and S . R . Long. 2002 . The RNA polymerase alpha subunit from Sinorhizobium meliloti can assemble with RNA polymerase subunits from Escherichia coli and function in basal and activated transcription both in vivo and in vitro . J . Bacteriol . 184:3808-3814 .
24. Richard, C . L., A . Tandon, N . R . Sloan, and R . G . Kranz. 2003 . RNA polymerase subunit requirements for activation by the enhancer-binding protein Rhodobacter capsulatus NtrC . J . Biol . Chem . 278:31701-31708 .
25. Severinov, K., M . Soushko, A . Goldfarb, and V . Nikiforov. 1993 . Rifampicin region revisited . New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Escherichia coli RNA polymerase . J . Biol . Chem . 268:14820-14825 .
26. Siegele, D . A., J . C . Hu, W . A . Walter, and C . A . Gross. 1989 . Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase . J . Mol . Biol . 206:591-603.
27. Tang, H., K . Severinov, A . Goldfarb, and R . H . Ebright. 1995 . Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase . Proc . Natl . Acad . Sci . USA 92:4902-4906.
28. Wu, J., N . Ohta, A . K . Benson, A . J . Ninfa, and A . Newton. 1997 . Purification, characterization, and reconstitution of DNA-dependent RNA polymerases from Caulobacter crescentus . J . Biol . Chem . 272:21558-21564 .
29. Yen, H . C., and B . Marrs. 1977 . Growth of Rhodopseudomonas capsulata under anaerobic dark conditions with dimethyl sulfoxide . Arch . Biochem . Biophys . 181:411-418.
30. Zalenskaya, K., J . Lee, C . N . Gujuluva, Y . K . Shin, M . Slutsky, and A . Goldfarb. 1990 . Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products . Gene 89:7-12.
31. Zuber, P., J . Healy, H . L . Carter III, S . Cutting, C . P . Moran, Jr., and R . Losick. 1989 . Mutation changing the specificity of an RNA polymerase sigma factor . J . Mol . Biol . 206:605-614.

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