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Journal of Bacteriology, February 2004, p. 740-749, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.740-749.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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


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ABSTRACT
 
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 {alpha}-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 {alpha}-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 {sigma}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).


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INTRODUCTION
 
Rhodobacter capsulatus is an anoxygenic photosynthetic {alpha}-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 {sigma}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 {sigma}70- and {sigma}54-dependent transcriptional activators (4).

The {alpha}-proteobacteria characteristically have a genomic G+C content of 65% or greater and are predicted to have diverged from the {gamma}-proteobacteria (e.g., Escherichia coli) approximately 500 million years ago (5). Kranz et al. previously noted that R. capsulatus and other {alpha}-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 {alpha}-proteobacterium, Sinorhizobium meliloti, most of the {sigma}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 {sigma}70 promoters (23). The {alpha}-proteobacterium Caulobacter crescentus {sigma}73 RNAP recognizes E. coli {sigma}70 promoters lacUV5 and neo, whereas E. coli does not recognize typical C. crescentus {sigma}73 promoters (28). For Rhodobacter sphaeroides, a similar situation was shown with the rrnB promoter (14). The R. capsulatus {sigma}70 RNAP also was shown to recognize typical E. coli {sigma}70 promoters, while the E. coli {sigma}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 ({alpha}, ß, ß', {sigma}70, and {omega}) produced in E. coli and R. capsulatus {alpha}ßß'{sigma}70{omega} 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).


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MATERIALS AND METHODS
 
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 pnifA1mut1, pnifA1mut2, and pnifA1mut3 were described previously (8). pnifA1mut3A was made by PCR of mutant 3 of the nifA1 promoter (nifA1mut3) 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 [nifA1mut3A]). The 0.3-kb product was digested with EcoRI/PstI and ligated to EcoRI/PstI-digested pUC118{Omega}. pnifA1mut3AA was made by PCR of the nifA1mut3 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 [nifA1mut3AA]). The 0.3-kb product was digested with EcoRI/PstI and ligated to EcoRI/PstI-digested pUC118{Omega}. 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{Omega}. pPUCAnifA1 was generated by excising a KpnI/HindIII fragment containing the 6.6-kb nifA1-lacZYA fusion and the 124-bp {Omega} cassette from pnifA1lacZYA{Omega} and ligating it to KpnI/HindIII-digested pUCA12. pUCA12 is a derivative of pRK2013 (9)-mobilizable Tetr pUCA10 (10) and contains an expanded multiple cloning site. All of the pUCA-lacZ fusion plasmids (pUCAnifA1, pUCAnifA1mut1, pUCAnifA1mut2, pUCAnifA1mut3, pUCAnifA1mut3A, and pUCAnifA1mut3AA) were generated from their respective in vitro transcription templates, described above, in the same manner as pUCAnifA1.

Protein overexpression plasmids. Plasmids pRGK301, pHTT7f1-NH{alpha}, pMKSe2, and pT7ß' were described previously (3, 4, 25, 27, 30). pRGK325, which allows overexpression of the R. capsulatus {alpha} 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 {omega} 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 {sigma}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 {sigma}70, maltose-binding protein-NtrB, R. capsulatus NtrC, and R. capsulatus RNAP. Purification of the six-histidine-tagged R. capsulatus {sigma}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 {alpha} subunit of RNAP. The R. capsulatus {alpha} subunit was purified by a modification of the procedure of Tang et al. (27). The six-histidine-tagged {alpha} 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 {alpha} subunit was precipitated by the addition of (NH4)2SO4 to 60%. The R. capsulatus {alpha} 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 {alpha} 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 {omega} subunit of R. capsulatus RNAP. The six-histidine-tagged {omega} 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 {omega} 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 {alpha}ßß'{sigma}70{omega}. Recombinant reconstituted R. capsulatus holoenzyme (R. capsulatus {alpha}ßß'{sigma}70{omega}) 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 {alpha}, 300 µg of crude R. capsulatus ß, 600 µg of crude R. capsulatus ß', 60 µg of six-histidine-tagged R. capsulatus {omega}, and 120 µg of six-histidine-tagged R. capsulatus {sigma}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.


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RESULTS AND DISCUSSION
 
Effects of mutations of the nifA1mut3 -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 nifA1mut1, mutant 2 of nifA1 (nifA1mut2), and mutant 3 of nifA1 (nifA1mut3), 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 nifA1mut3 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 nifA1mut3 -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, nifA1mut1, and nifA1mut2 promoters are still responsive to R. capsulatus NtrC activation, but the nifA1mut3 promoter is constitutive and fully expressed in R. capsulatus NtrC- and NtrC+ strains. In a comparison of nifA1mut3 in E. coli and R. capsulatus, R. capsulatus expresses nifA1mut3 at a level approximately 25-fold higher than E. coli (7,714 versus 308 units of ß-galactosidase activity). Changing GG to AG (nifA1mut3A) or AA (nifA1mut3AA) 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.



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  		FIG. 1. DNA sequences of nifA1 and engineered nifA1promoters and in vitro transcription reactions at mutant nifA1mut3 promoters. (A) DNA sequences of nifA1 promoters. The transcriptional start site is designated +1, and the -10 and -35 sequences are underlined. Shaded bars indicate the R. capsulatus NtrC tandem binding sites. The DNA sequences of consensus E. coli -10 and -35 sequences are aligned for comparison with the nifA1 promoter sequences. (B to D) In vitro transcription of nifA1mut3 and its -10 derivatives. The natural RNAP core enzyme and whether {sigma}70 has been added (+ or -) are noted above each set of transcription reactions. The templates are noted to the right of each panel. The internal control transcript, RNAI, is also noted to the right of each set of in vitro transcription reactions. Rc, R. capsulatus; Ec, E. coli.


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  		TABLE 1. In vivo influence of variations in the -35 and -10 sequences on nifA1 promoter activity in E. coli and R. capsulatus

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 nifA1mut3, nifA1mut3A, and nifA1mut3AA 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 nifA1mut3 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).


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  		TABLE 2. Quantitation of nifA1mut3, nifA1mut3A and nifA1mut3AA in vitro transcripts

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 {sigma}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 {sigma} 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 {sigma}70 and -10 effects. To analyze the role of the R. capsulatus {sigma}70 subunit in promoter recognition and melting, we used the E. coli RNAP core enzyme and R. capsulatus {sigma}70 for in vitro transcription with the nifA1mut3 template. The addition of R. capsulatus {sigma}70 to the E. coli RNAP core enzyme facilitated an eightfold increase in the transcription of the nifA1mut3 -10 sequence (Fig. 1B, compare lane 5 with lane 6, and Table 2). Conversely, we added E. coli {sigma}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 {sigma}70 is approximately 2-fold less efficient at transcribing the nifA1mut3 -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 {sigma}70 but is not due solely to the {sigma}70 subunit. These findings also suggest a more promiscuous role for R. capsulatus {sigma}70 in recognition and melting of the -10 sequence, a role that has been proposed for E. coli {sigma}S as well (11, 16). R. capsulatus {sigma}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 {sigma}70 corresponding to regions 2.3, 2.4, and 2.5 of E. coli {sigma}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 {sigma}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 nifA1mut3 promoter, then the melting of TATGGT may be an important element. We performed in vitro transcription assays with the nifA1mut3 template at a variety of temperatures (Fig. 2). The E. coli holoenzyme is approximately fourfold more efficient at transcribing the nifA1mut3 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 {sigma}70 is added to the E. coli RNAP core enzyme (Fig. 2), again suggesting an important role for the R. capsulatus {sigma}70 subunit. At 20°C, the R. capsulatus RNAP core enzyme with added E. coli {sigma}70 transcribes the nifA1mut3 promoter less efficiently than does this enzyme with added R. capsulatus {sigma}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 {sigma}70 in melting the neighboring GG nucleotides (13). This notion is also suggested by results obtained with nifA1mut3 -10 promoter sequences in which GG is changed to AG or AA. As the -10 sequence is changed toward the consensus sequence, E. coli {sigma}70 can more efficiently transcribe the nifA1mut3 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 {sigma}70 subunit facilitate transcription of the TATGGT -10 promoter. It is likely that in addition to {sigma}70 promiscuity, the core enzyme has coevolved this property with the high-GC nature of this {alpha}-proteobacterium.



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  		FIG. 2. Effects of temperature and {sigma}70 on nifA1mut3 promoter transcription. Preparations of native core RNAPs and added sigma factors are noted. The y axis represents the percentage of in vitro transcription of the nifA1mut3 promoter relative to the internal standard RNAI. The x axis represents the temperatures used for in vitro transcription assays. Other in vitro transcription reactions in this study were performed at 23°C. Error bars represent the standard deviation for 10 independent in vitro transcription assays. Rc, R. capsulatus; Ec, E. coli.

R. capsulatus {sigma}70 can be replaced with E. coli {sigma}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 {sigma}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 nifA1mut1, nifA1mut2, and nifA1mut3 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 nifA1mut3 template by the R. capsulatus RNAP (Fig. 3A, lane 2), as shown earlier (4). There is <8% basal transcription of the nifA1mut1 and nifA1mut2 templates (Fig. 3B, lanes 2 and 3, and Fig. 3C, lanes 2 and 3), but upon the addition of activators, the level of the nifA1mut1 transcript is approximately equivalent to that of the RNAI transcript (Fig. 3B, lanes 4 and 5) and the level of the nifA1mut2 transcript is approximately 50% of that of the RNAI transcript (Fig. 3C, lanes 4 and 5). Activated transcription occurs with either R. capsulatus {sigma}70 or E. coli {sigma}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 nifA1mut3, nifA1mut1, or nifA1mut2 promoter template is used (Fig. 3, lanes 9). The barrier to R. capsulatus NtrC activation is not overcome by the R. capsulatus {sigma}70 subunit (Fig. 3, lanes 10), suggesting that the R. capsulatus {sigma}70 subunit is not sufficient to allow activation by R. capsulatus NtrC.



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  		FIG. 3. Effect of {sigma}70 on nifA1 mutant promoter transcription and R. capsulatus NtrC activation. Each panel represents in vitro transcription assays with the native core RNAP, {sigma}70, and R. capsulatus NtrB or NtrC as noted. The positions of the RNAI transcript and the mutant nifA1 promoter are shown to the right of each panel. (A) In vitro transcription with the nifA1mut3 template. (B) In vitro transcription with the nifA1mut1 template. (C) In vitro transcription with the nifA1mut2 template. Figure 1A shows the sequences of the mutant nifA1 promoters. The percentage of the designated nifA1 mutant transcript relative to the RNAI transcript is given in parentheses below the lane number and represents the mean of at least three independent experiments (e.g., 100% would indicate that the RNAI and nifA1 mutant transcripts are equal). Rc, R. capsulatus; Ec, E. coli.

Other factors are not required for R. capsulatus NtrC activation, as determined by in vitro reconstitution of the recombinant R. capsulatus {sigma}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 {alpha}ßß'{sigma}70{omega}) 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 {alpha} 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 {alpha} subunits from other {alpha}-proteobacterial organisms for in vitro transcription with the E. coli ß and ß' subunits (17, 23). The R. capsulatus {alpha} subunit (45% identical to the E. coli {alpha} 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 {omega} subunit (39% identical to the E. coli {omega} 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 {sigma}70, {alpha}, and {omega} 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 {alpha}ßß'{sigma}70{omega} RNAP obtained with this procedure typically is greater than 95% pure.



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  		FIG. 4. Recombinant R. capsulatus RNAP subunits, reconstitution, and effects of rifampin on transcription. (A) SDS-12.5% PAGE with 25 µg of protein sample/lane. Size standards (in thousands) are shown on the left (Bio-Rad), and purified R. capsulatus RNAP subunits are shown at the top. Lane 1, low-molecular-weight size standards; lane 2, six-histidine-tagged {sigma}70; lane 3, six-histidine-tagged {alpha} subunit; lane 4, washed and solubilized inclusion bodies of ß subunit; lane 5, washed and solubilized inclusion bodies of ß' subunit; lane 6, six-histidine-tagged {omega} subunit. (B) SDS-12.5% PAGE of purified recombinant reconstituted R. capsulatus {alpha}ßß'{sigma}70{omega} RNAP. Size standards are shown on the left, and the positions of the ß, ß', {sigma}70, {alpha}, and {omega} subunits are shown on the right. (C) In vitro transcription assays of R. capsulatus {alpha}ßß'{sigma}70{omega} and reconstituted E. coli core RNAP with the nifA1mut1 template. The positions of the RNAI and nifA1mut1 transcripts are shown on the right. Rifampin (Rif) was added at 62.5 µg/ml. Rc, R. capsulatus; Ec, E. coli.

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 (Rifr), whereas the E. coli strain used for overexpression is rifampin sensitive (Rifs). Rifampin inhibits bacterial transcription by binding to the ß subunit of RNAP (12). The Rifr 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 Q->L substitution at amino acid 532 which is homologous to E. coli Q->L 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 Rifr 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 {alpha}ßß'{omega} RNAP, reconstituted E. coli core RNAP, and the nifA1mut1 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 {alpha}ßß'{omega} RNAP, RNAI and nifA1mut1 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 {alpha}ßß'{omega} 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 {sigma}70 or E. coli {sigma}70 is added (data not shown).

The recombinant R. capsulatus {alpha}ßß'{sigma}70{omega} 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 {alpha}ßß'{sigma}70{omega} RNAP also demonstrates an ability to transcribe the nifA1mut3 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 nifA1mut3 promoter (compare Fig. 5A, lane 2, with Fig. 3A, lane 4; approximately 2-fold), the nifA1mut1 promoter (compare Fig. 5B, lane 2, with Fig. 3B, lane 4; >6-fold), and the nifA1mut2 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 {omega} 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 {alpha} or ß subunit is necessary for R. capsulatus NtrC activation (24).



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  		FIG. 5. In vitro transcriptional activation by R. capsulatus NtrC of reconstituted R. capsulatus holoenzyme. In vitro transcription assays of R. capsulatus {alpha}ßß'{sigma}70{omega} were carried out with nifA1mut3 (A), nifA1mut1 (B), and nifA1mut2 (C) templates. The percentages of RNAI are given in parentheses below the lane numbers. Rc, R. capsulatus.


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ACKNOWLEDGMENTS
 
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.


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FOOTNOTES
 
* 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{at}biology.wustl.edu. Back


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Journal of Bacteriology, February 2004, p. 740-749, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.740-749.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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