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• W2000283762 abstract "It has been reported that an RNA polymerase σ factor, SigC, mainly contributes to specific transcription from the promoter PglnB-54,-53 under nitrogen-deprived conditions during the stationary phase of cell growth in the cyanobacterium Synechocystis sp. strain PCC 6803 (Asayama, M., Imamura, S., Yoshihara, S., Miyazaki, A., Yoshida, N., Sazuka, T., Kaneko, T., Ohara, O., Tabata, S., Osanai, T., Tanaka, K., Takahashi, H., and Shirai, M. (2004) Biosci. Biotechnol. Biochem. 68, 477-487). In this study, we further examined the functions of group 2 σ factors of RNA polymerase in NtcA-dependent nitrogen-related gene expression in PCC 6803. Results indicated that SigB and SigC contribute to the transcription from PglnB-54,-53 with a σ factor replaced in a growth phase-dependent manner. We also confirmed the contribution of SigB and SigC to the transcription of other NtcA-dependent genes, glnA, sigE, and amt1, as in the case of glnB. On the other hand, the transcription of glnN was dependent on SigB and SigE. In the SigB and SigC-based regulation, the level of SigB increased, but that of SigC was constant under conditions of nitrogen deprivation. Furthermore, it was found that SigC negatively and positively regulates the level of SigB in the log and stationary phase, respectively. SigC also had a positive effect on the level of sigB transcript during the stationary phase. In contrast, SigB acts positively on SigC levels in both growth phases. These results and previous findings indicated that multiple group 2 σ factors take part in the control of NtcA-dependent nitrogen-related gene expression in cooperation with a group 1 σ factor, SigA. It has been reported that an RNA polymerase σ factor, SigC, mainly contributes to specific transcription from the promoter PglnB-54,-53 under nitrogen-deprived conditions during the stationary phase of cell growth in the cyanobacterium Synechocystis sp. strain PCC 6803 (Asayama, M., Imamura, S., Yoshihara, S., Miyazaki, A., Yoshida, N., Sazuka, T., Kaneko, T., Ohara, O., Tabata, S., Osanai, T., Tanaka, K., Takahashi, H., and Shirai, M. (2004) Biosci. Biotechnol. Biochem. 68, 477-487). In this study, we further examined the functions of group 2 σ factors of RNA polymerase in NtcA-dependent nitrogen-related gene expression in PCC 6803. Results indicated that SigB and SigC contribute to the transcription from PglnB-54,-53 with a σ factor replaced in a growth phase-dependent manner. We also confirmed the contribution of SigB and SigC to the transcription of other NtcA-dependent genes, glnA, sigE, and amt1, as in the case of glnB. On the other hand, the transcription of glnN was dependent on SigB and SigE. In the SigB and SigC-based regulation, the level of SigB increased, but that of SigC was constant under conditions of nitrogen deprivation. Furthermore, it was found that SigC negatively and positively regulates the level of SigB in the log and stationary phase, respectively. SigC also had a positive effect on the level of sigB transcript during the stationary phase. In contrast, SigB acts positively on SigC levels in both growth phases. These results and previous findings indicated that multiple group 2 σ factors take part in the control of NtcA-dependent nitrogen-related gene expression in cooperation with a group 1 σ factor, SigA. The RNA polymerase holoenzyme of eubacteria consists of a core enzyme and σ factor (1Ishihama A. J. Bacteriol. 1993; 175: 2483-2489Crossref PubMed Google Scholar). The core enzyme is capable of undergoing transcriptional elongation, and the σ factor is required for the initiation of transcription from a specific promoter sequence. Multiple σ factors are usually encoded by a eubacterial genome, and they have been generally classified into three groups (2Lonetto M. Gribskov M. Gross C.A. J. Bacteriol. 1992; 174: 3843-3849Crossref PubMed Scopus (740) Google Scholar). Group 1 comprises principal σ factors that are responsible for transcription from a number of housekeeping promoters and are eventually crucial for cell viability. Group 2 and group 3 σ factors are alternative types. Group 2 σ factors are similar to the group 1 types in molecular structure but are nonessential for cell viability. Group 3 σ factors are structurally different from proteins of group 1 and group 2 and are sometimes involved in the transcription of regulons for survival under stress. The cyanobacterium Synechocystis sp. strain PCC 6803 used in this study possesses nine species of σ factors assigned to group 1 (SigA), group 2 (SigB, SigC, SigD, and SigE), and group 3 (SigF, SigG, SigH, and SigI) (3Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Crossref PubMed Scopus (2140) Google Scholar, 4Imamura S. Yoshihara S. Nakano S. Shiozaki N. Yamada A. Tanaka K. Takahashi H. Asayama M. Shirai M. J. Mol. Biol. 2003; 325: 857-872Crossref PubMed Scopus (106) Google Scholar). The functions of some of these σ factors have been recently revealed. For example, SigD and SigB are light- and dark-responsive σ factors (4Imamura S. Yoshihara S. Nakano S. Shiozaki N. Yamada A. Tanaka K. Takahashi H. Asayama M. Shirai M. J. Mol. Biol. 2003; 325: 857-872Crossref PubMed Scopus (106) Google Scholar, 5Imamura S. Asayama M. Takahashi H. Tanaka K. Takahashi H. Shirai M. FEBS Lett. 2003; 554: 357-362Crossref PubMed Scopus (59) Google Scholar, 6Imamura S. Asayama M. Shirai M. Genes Cells. 2004; 9: 1175-1187Crossref PubMed Scopus (24) Google Scholar). SigB is also identified as a heat-shock σ factor (4Imamura S. Yoshihara S. Nakano S. Shiozaki N. Yamada A. Tanaka K. Takahashi H. Asayama M. Shirai M. J. Mol. Biol. 2003; 325: 857-872Crossref PubMed Scopus (106) Google Scholar, 6Imamura S. Asayama M. Shirai M. Genes Cells. 2004; 9: 1175-1187Crossref PubMed Scopus (24) Google Scholar). SigE is a σ factor required for positive regulation of sugar catabolic pathways (7Osanai T. Kanesaki Y. Nakano T. Takahashi H. Asayama M. Shirai M. Kanehisa M. Suzuki I. Murata N. Tanaka K. J. Biol. Chem. 2005; 280: 30653-30659Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Cyanobacteria, blue-green algae, are prokaryotes that perform oxygenic evolving photosynthesis like plants and mainly use inorganic nitrogen sources, ammonium and nitrate. The nitrate is reduced by nitrate reductase and nitrite reductase, and the resulting ammonium is usually incorporated with glutamine synthetase (GS) and glutamate synthase (GOGAT), a pathway commonly known as the GS-GOGAT cycle (8Merrick M.J. Edwards R.A. Microbiol. Rev. 1995; 59: 604-622Crossref PubMed Google Scholar). In this cycle, 2-oxoglutarate (2-OG), 2The abbreviations used are: 2-OG, 2-oxoglutarate; RNAP, RNA polymerase; QRT-PCR, quantitative real time PCR; IS, integration site; ppGpp, guanosine 3,5-(bis)pyrophosphate; ΔB, ΔC, ΔD, and ΔE, sigB, sigC, sigD, and sigE knock-out strain, respectively; Log, midexponential phase; Sta, stationary phase; -N and +N, without and with nitrogen, respectively; GS, glutamine synthase; GOGAT, glutamate synthase. which is synthesized by isocitrate dehydrogenase from isocitrate, is used as a carbon skeleton for nitrogen assimilation. A remarkable feature of the intermediary metabolism of cyanobacteria is a lack of 2-OG dehydrogenase (9Stanier R.Y. Cohen-Bazire G. Annu. Rev. Microbiol. 1977; 31: 225-274Crossref PubMed Scopus (711) Google Scholar). Consequently, 2-OG is a main substrate for nitrogen assimilation in cyanobacteria. The system regulating nitrogen levels is well characterized in enteric bacteria (8Merrick M.J. Edwards R.A. Microbiol. Rev. 1995; 59: 604-622Crossref PubMed Google Scholar, 10Arcondeguy T. Jack R. Merrick M. Microbiol. Mol. Biol. Rev. 2001; 65: 80-105Crossref PubMed Scopus (352) Google Scholar). Expression of glnA (encoding glutamine synthetase) and other nitrogen-related genes is required for RNAP-containing RpoN (σ54), an alternative σ factor whose molecular structure and transcriptional mechanism are quite different from those of group 1-3 σ factors (11Reitzer L.J. Magasanik B. Cell. 1986; 45: 785-792Abstract Full Text PDF PubMed Scopus (303) Google Scholar). The expression is regulated by a two-component regulatory system, NtrB/NtrC, the activity of which is controlled by the uridylylation status of PII (8Merrick M.J. Edwards R.A. Microbiol. Rev. 1995; 59: 604-622Crossref PubMed Google Scholar, 10Arcondeguy T. Jack R. Merrick M. Microbiol. Mol. Biol. Rev. 2001; 65: 80-105Crossref PubMed Scopus (352) Google Scholar). The uridylylation or nonuridylylation of PII is coordinated by the ratio of intracellular concentrations of 2-OG and glutamine. PII itself binds 2-OG; therefore, it senses the status of the cells and plays a central role in the assimilation of nitrogen. The regulation of nitrogen assimilation could differ between enteric bacteria and cyanobacteria, because no homologues of RpoN-type σ factor, NtrB/NtrC, and glutamine synthetase adenylyltransferase have been identified in cyanobacteria. In fact, a cAMP receptor protein family transcription factor, NtcA, plays a central role in nitrogen assimilation in cyanobacteria. A consensus sequence needed for the binding of NtcA to DNA (TGAN8TAC) has been reported, and the motif is generally located about 40 bp upstream from the transcription start point (12Herrero A. Muro-Pastor A.M. Flores E. J. Bacteriol. 2001; 183: 411-425Crossref PubMed Scopus (552) Google Scholar). The promoters activated by NtcA exhibit a conserved sequence, TAN3T, as a -10 promoter hexamer but do not possess a -35 hexamer. Under nitrogen-deprived conditions, 2-OG directly binds to NtcA and increases the DNA binding affinity of NtcA (13Vazquez-Bermudez M.F. Herrero A. Flores E. FEBS Lett. 2002; 512: 71-74Crossref PubMed Scopus (153) Google Scholar, 14Tanigawa R. Shirokane M. Maeda S. Omata T. Tanaka K. Takahashi H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4251-4255Crossref PubMed Scopus (164) Google Scholar). NtcA with 2-OG activates the expression of nitrogen assimilation-related genes. Although 2-OG and NtcA play key roles in the assimilation of nitrogen, a molecular study of NtcA-dependent transcription has been performed only in a few cases in cyanobacteria. For example, Muro-Pastor et al. (15Muro-Pastor A.M. Herrero A. Flores E. J. Bacteriol. 2001; 183: 1090-1095Crossref PubMed Scopus (78) Google Scholar) have reported that the group 2 σ factor gene, sigE (rpoD2-V), possesses the NtcA-binding motif upstream of its promoter, and its transcription is induced under nitrogen-deprived conditions. Expression of glnN, a type-3 glutamine synthase gene, was impaired in strains bearing an inactivated copy of the sigE gene of Synechocystis sp. PCC 6803 (15Muro-Pastor A.M. Herrero A. Flores E. J. Bacteriol. 2001; 183: 1090-1095Crossref PubMed Scopus (78) Google Scholar). Our recent study revealed that transcription from the glnB (encoding PII) (16Forchhammer K. FEMS Microbiol. Rev. 2004; 28: 319-333Crossref PubMed Scopus (206) Google Scholar) promoter (PglnB-54,-53) is due to specific recognition by a PCC 6803 group 2 σ factor, SigC, in the stationary (postexponential) growth phase under nitrogen-deprived conditions (17Asayama M. Imamura S. Yoshihara S. Miyazaki A. Yoshida N. Sazuka T. Kaneko T. Ohara O. Tabata S. Osanai T. Tanaka K. Takahashi H. Shirai M. Biosci. Biotechnol. Biochem. 2004; 68: 477-487Crossref PubMed Scopus (44) Google Scholar). This raised the possibility that another σ factor recognizes the glnB promoter in the logarithmic (exponential) growth phase and that a “σ-switch” for the nitrogen-promoter recognition occurs during the log to stationary phase. However, which σ factor recognizes the glnB promoter under the log phase has remained to be elucidated. Here, we presented data for resolving this issue. We also characterized the specificity with which PCC 6803 group 2 σ factors recognize other NtcA-dependent promoters, glnA, sigE, amt, and glnN. We summarize these results and present a possible regulatory network of group 1 and group 2 σ factors for the transcription of NtcA-dependent nitrogen-related genes. Strains and Growth Conditions—Synechocystis sp. strain PCC 6803 (Kazusa strain) was grown at 30 °C with shaking (120 rpm, NR-30, TAITEC, Tokyo, Japan) under white light (35 μmol m-2 s-1 photon flexture) in BG11 medium (18Rippka R. Methods Enzymol. 1988; 167: 3-27Crossref PubMed Scopus (683) Google Scholar), supplemented with 15 μg/ml kanamycin sulfate and/or 40 μg/ml spectinomycin if required. For nitrogen-starved conditions, PCC 6803 was cultivated in BG11 medium without NaNO3 for 6 h, as described previously (17Asayama M. Imamura S. Yoshihara S. Miyazaki A. Yoshida N. Sazuka T. Kaneko T. Ohara O. Tabata S. Osanai T. Tanaka K. Takahashi H. Shirai M. Biosci. Biotechnol. Biochem. 2004; 68: 477-487Crossref PubMed Scopus (44) Google Scholar). Isolation of RNA and Primer Extension Analysis—Procedures were performed as described previously (19Asayama M. Tanaka K. Takahashi H. Sato A. Aida T. Shirai M. Gene (Amst.). 1996; 181: 213-217Crossref PubMed Scopus (23) Google Scholar). The oligonucleotides used in the primer extension for glnB, glnA, amt1, glnN, sigB, and sigC were glnB-R2 (17Asayama M. Imamura S. Yoshihara S. Miyazaki A. Yoshida N. Sazuka T. Kaneko T. Ohara O. Tabata S. Osanai T. Tanaka K. Takahashi H. Shirai M. Biosci. Biotechnol. Biochem. 2004; 68: 477-487Crossref PubMed Scopus (44) Google Scholar), glnA-R (5′-CGTCTTGGATCCACTTGAGGACTTC-3′), amt1-R4 (5′-CTACATTGTTCTACGAAAG-3′), glnN-R2 (5′-GCCAGAAGATAGAGGTCGA-3′), sigB-R2 (4Imamura S. Yoshihara S. Nakano S. Shiozaki N. Yamada A. Tanaka K. Takahashi H. Asayama M. Shirai M. J. Mol. Biol. 2003; 325: 857-872Crossref PubMed Scopus (106) Google Scholar), and 0184V-R3 (5′-TCGTTGCTTGGTTTAGTC-3′), respectively. The products of reverse transcription were dissolved in 7 μl of a dye solution and denatured at 95 °C for 3 min. Then an aliquot of 3 μl was resolved on a 7% polyacrylamide gel containing 8 m urea followed by autoradiography. Quantitative Real Time PCR Analysis—Quantitative real time PCR (QRT-PCR) was performed as described previously (17Asayama M. Imamura S. Yoshihara S. Miyazaki A. Yoshida N. Sazuka T. Kaneko T. Ohara O. Tabata S. Osanai T. Tanaka K. Takahashi H. Shirai M. Biosci. Biotechnol. Biochem. 2004; 68: 477-487Crossref PubMed Scopus (44) Google Scholar). The set of oligonucleotides used for glnB, glnA, glnN, sigE, amt1, and rrn16Sa was as follows: glnB-RT-F/glnB-RT-R (17Asayama M. Imamura S. Yoshihara S. Miyazaki A. Yoshida N. Sazuka T. Kaneko T. Ohara O. Tabata S. Osanai T. Tanaka K. Takahashi H. Shirai M. Biosci. Biotechnol. Biochem. 2004; 68: 477-487Crossref PubMed Scopus (44) Google Scholar), glnA-RT-F (5′-CCAAACACCGCCACCATC-3′/glnA-RT-R (5′-GCTGGAGTTTTCCGTTTGG-3′), glnN-RT-F2 (5′-CCTGGAAGATATGTGGGCTG-3′)/glnN-RT-R (5′-GTAGGCAGGACTGGTTAC-3′), sigE-RT-F (5′-AAGAAATGGCCCGCTATCCC-3′)/sigE-RT-R (5′-TTCGTTCCAGTTGTTGGGTG-3′), amt1-RT-F (5′-GGCAGCAGTGGCAATCCC-3′)/amt1-RT-R (5′-GCTACAGCACCGGAAACA-3′), and 16srRNA-RTF (5′-CTGAAGATGGGCTCGCGT-3′)/16srRNA-RTR (5′-CGTATTACCGCGGCTGCT-3′), respectively. Standard curves for each gene were also constructed with serial dilutions (1 to 1 × 5-3) of cDNA, synthesized with total RNA extracted from wild-type cells under nitrogen-deprived conditions at the log phase. Respective relative levels of transcripts were calculated with the relevant standard curve. Assays without cDNA were conducted for each experiment as a negative control. All assays were done in triplicate. Plasmids and a Strain for Complementation Tests—The plasmid DNA, pOXL6803-COMP-B, used in this study was constructed as follows. A plasmid, pOXL6803-2 (20Aoki S. Kondo T. Ishiura M. J. Microbiol. Methods. 2002; 49: 265-274Crossref PubMed Scopus (29) Google Scholar), was digested with SmaI and KpnI followed by MungBean treatment, and then a resultant 10.9-kb fragment was self-ligated to create pOXL-6803-3. Fragments annealed with oligonucleotides, GATCCCCGGGGGTACCA and GGGCCCCCATGGTCTAG (double underlines, underlines, and italic type indicate a sequence that can unite with a BglII site, an SmaI site, and a KpnI site, respectively) were restricted with BglII and cloned into the same restriction enzyme cutting site of pOXL6803-3 to yield pOXL6803-4-6. A PCR-amplified BglII-SacII 1538-bp segment containing the PCC 6803 sigB gene and its promoter region (-500 to +1038, +1 as the initiation codon) was cloned into the same restriction enzyme site of pOXL6803-4-6 to make pOXL6803-COMP-B. For the construction of pOXL6803-COMP-C, a PCR-amplified KpnI-SacII 1715-bp segment containing the PCC 6803 sigC gene and its promoter region (-500 to +1215) was cloned into the same restriction enzyme site of pOXL6803-4-6. Natural transformation (21Golden S.S. Brusslan J. Haselkorn R. Methods Enzymol. 1987; 153: 215-231Crossref PubMed Scopus (286) Google Scholar, 22Ito Y. Asayama M. Shirai M. Biosci. Biotechnol. Biochem. 2003; 67: 1382-1390Crossref PubMed Scopus (11) Google Scholar, 23Shibato J. Agrawal G.K. Kato H. Asayama M. Shirai M. Mol. Genet. Genomics. 2002; 267: 684-694Crossref PubMed Scopus (22) Google Scholar) was carried out with pOXL6803-COMP plasmids, and transformants were selected on BG 11 plates containing spectinomycin (40 μg/ml) and kanamycin (15 μg/ml). Polyclonal Antibody for NtcA and Western Blot Analysis—Overexpression and purification of PCC 6803 NtcA were achieved as described previously (17Asayama M. Imamura S. Yoshihara S. Miyazaki A. Yoshida N. Sazuka T. Kaneko T. Ohara O. Tabata S. Osanai T. Tanaka K. Takahashi H. Shirai M. Biosci. Biotechnol. Biochem. 2004; 68: 477-487Crossref PubMed Scopus (44) Google Scholar). The purified NtcA was subjected to SDS-PAGE and recovered from the gel. The gel was splintered off and mixed with adjuvant, and this mixture was injected into a rabbit whose serum (1:500 dilution) did not cross-react to PCC 6803 total protein (50 μg) during Western blotting. The Western blotting was performed as described previously (4Imamura S. Yoshihara S. Nakano S. Shiozaki N. Yamada A. Tanaka K. Takahashi H. Asayama M. Shirai M. J. Mol. Biol. 2003; 325: 857-872Crossref PubMed Scopus (106) Google Scholar). The dilution for rabbit serums of the antibody was 1:1,000, 1:500, 1:1,000, 1:1,000, 1:1,000, 1:500, and 1:1,000 for PCC 6803 SigA, SigB, SigC, SigD, SigE, RpoB, and NtcA, respectively. RNA Polymerase Core Enzyme and σ Factors—Purification of the reconstituted PCC 6803 core enzyme with each recombinant subunit was performed as reported previously (6Imamura S. Asayama M. Shirai M. Genes Cells. 2004; 9: 1175-1187Crossref PubMed Scopus (24) Google Scholar) but with some improvements to the renaturation and reconstitution steps. Previously, the renaturation and reconstitution of RNAP core enzyme were done at the same time in one tube. During these steps, γ (RpoC1) subunits in particular tended to aggregate and consequently the RNAP sometimes lacked the γ subunit. Therefore, for the renaturation of RNAP subunits, purified RpoA-His (His tag attached at the C-terminal domain) or crude RpoB, RpoC1, and RpoC2 dissolved in Buffer G were separately dialyzed against the reconstitution buffer at 4 °C for 16 h (6Imamura S. Asayama M. Shirai M. Genes Cells. 2004; 9: 1175-1187Crossref PubMed Scopus (24) Google Scholar). After clearance of the debris by centrifugation, each supernatant was mixed in the following molar ratio, α:β:β′:γ = 1:1:2:4, and incubated at 30 °C for 14 h to reconstitute the PCC 6803 core enzyme. After incubation, the reconstituted core enzyme was purified as described previously (6Imamura S. Asayama M. Shirai M. Genes Cells. 2004; 9: 1175-1187Crossref PubMed Scopus (24) Google Scholar), and the purified fractions were concentrated by centrifugation using an Amicon Ultra-4 filter unit (100-kDa molecular mass cut-off; Millipore Corp.). PCC 6803 σ factors were also prepared by methods reported previously (4Imamura S. Yoshihara S. Nakano S. Shiozaki N. Yamada A. Tanaka K. Takahashi H. Asayama M. Shirai M. J. Mol. Biol. 2003; 325: 857-872Crossref PubMed Scopus (106) Google Scholar, 6Imamura S. Asayama M. Shirai M. Genes Cells. 2004; 9: 1175-1187Crossref PubMed Scopus (24) Google Scholar). In Vitro Transcription Analysis—Multiple-round run-off assays were performed as described previously (6Imamura S. Asayama M. Shirai M. Genes Cells. 2004; 9: 1175-1187Crossref PubMed Scopus (24) Google Scholar, 24Shibato J. Asayama M. Shirai M. Biochim. Biophys. Acta. 1998; 1442: 296-303Crossref PubMed Scopus (23) Google Scholar). The assay mixture (40 μl) comprised 50 mm Tris-HCl (pH 7.9), 5 mm MgCl2, 0.05 mm EDTA·2Na, 0.5 mm dithiothreitol, 0.2 mm each ATP/CTP/GTP/UTP, 25 nm RNAP core enzyme, 100 nm σ factor, 2.5 nm template DNA, and/or 300 nm NtcA and 3 mm 2-OG. The constructs of template DNA used in this study are as follows: pGLN9B (17Asayama M. Imamura S. Yoshihara S. Miyazaki A. Yoshida N. Sazuka T. Kaneko T. Ohara O. Tabata S. Osanai T. Tanaka K. Takahashi H. Shirai M. Biosci. Biotechnol. Biochem. 2004; 68: 477-487Crossref PubMed Scopus (44) Google Scholar); pYS1756, a 419-bp segment of the PCR-amplified SmaI-BglII PCC 6803 glnA promoter region (-375 to +44) cloned into the same restriction enzyme site of pUC119B (25Asayama M. Hayasaka Y. Kabasawa M. Shirai M. Ohyama A. J. Biochem. (Tokyo). 1999; 125: 460-468Crossref PubMed Scopus (22) Google Scholar); pAMT1, pKK-T1, a 550-bp segment of the PCR-amplified BglII-BglII PCC 6803 amt1 promoter region (-550 to +50) cloned into pKK223-3 the same as pKK-A2 (6Imamura S. Asayama M. Shirai M. Genes Cells. 2004; 9: 1175-1187Crossref PubMed Scopus (24) Google Scholar), digested with HindIII followed by Klenow treatment, and then digested with BglII and a 466-bp fragment containing the amt1 promoter region (-416 to +50) cloned the same as the glnA promoter region; and pGLNN, a 341-bp segment of the PCR-amplified SmaI-BglII PCC 6803 glnN promoter region (-241 to +100), cloned the same as the glnA promoter region. The mixture was incubated at 30 °C for 15 min, and then the reaction was stopped with the addition of 60 μl of stop solution (40 mm EDTA·2Na, 300 μg/ml Escherichia coli tRNA, and 300 mm LiCl). The RNA products were precipitated with 2-propanol and dissolved in 10 μl of RNase-free water. The samples were subjected to primer extension as mentioned above. Growth Phase-dependent Regulation of the glnB Transcript by SigB and SigC—Our previous study has indicated that SigC controls synthesis of the glnB transcript from PglnB-54,-53 induced by nitrogen deprivation in the stationary phase. However, which group 2 σ factors recognize and regulate the glnB promoter during the log phase? To answer this question, we examined the glnB transcript using primer extension in all other knock-out strains missing the group 2 σ factors SigB, SigD, and SigE (Fig. 1). PCC 6803 glnB possesses two transcription start points: PglnB-33, an E. coli RpoD-type promoter, transcription from which is constitutive, and PglnB-54,-53, an NtcA-dependent promoter, transcription from which is induced under nitrogen-deprived conditions (Table 1). Primer extension analyses showed that the amount of transcript synthesized from PglnB-54,-53 in the sigB knock-out strain (ΔsigB) was significantly reduced during the log phase (Log) regardless of nitrogen levels. A slight reduction was observed in the stationary phase (Sta) under nitrogen-deprived conditions (-N). These results indicate that SigB mainly contributes to the expression of glnB in the log phase. In contrast, similar amounts of the glnB transcript were observed in the ΔsigD and ΔsigE strains, compared with wild-type cells during both phases, indicating that SigD and SigE do not contribute to glnB expression. In this situation, SigA drives the transcription from PglnB-33, because the transcripts were almost constitutively expressed even in the knock-out strains. This supports previous results (17Asayama M. Imamura S. Yoshihara S. Miyazaki A. Yoshida N. Sazuka T. Kaneko T. Ohara O. Tabata S. Osanai T. Tanaka K. Takahashi H. Shirai M. Biosci. Biotechnol. Biochem. 2004; 68: 477-487Crossref PubMed Scopus (44) Google Scholar).TABLE 1A nucleotide sequence alignment of the NtcA-activated promoter regions in PCC 6803 Boxes, -10 hexamers; double underlines, NtcA-binding motifs; bold face letters, consensus nucleotide sequences; lower case letters, transcription start sites. Open table in a new tab Complementation of SigB Function in the Knock-out Strain—To confirm that SigB and SigC actually function in cells, a complementation test was conducted for glnB expression. Plasmids pOXL6803-COMP-B and pOXL6803-COMP-C (see “Experimental Procedures”), carrying sigB and sigC, respectively, were constructed and introduced into the knock-out strains by a method of natural transformation (21Golden S.S. Brusslan J. Haselkorn R. Methods Enzymol. 1987; 153: 215-231Crossref PubMed Scopus (286) Google Scholar, 22Ito Y. Asayama M. Shirai M. Biosci. Biotechnol. Biochem. 2003; 67: 1382-1390Crossref PubMed Scopus (11) Google Scholar, 23Shibato J. Agrawal G.K. Kato H. Asayama M. Shirai M. Mol. Genet. Genomics. 2002; 267: 684-694Crossref PubMed Scopus (22) Google Scholar). After homologous recombination between the plasmids and PCC 6803 genomes (Fig. 2A), we obtained a transformant called “Comp-ΔsigB,” which is resistant to kanamycin and spectinomycin. Unfortunately, we could not obtain a transformant of “Comp-ΔsigC,” suggesting that some factor would not allow the transformation. To verify the recombination in Comp-ΔsigB, we conducted PCR and Western blot analyses. The results are shown in Fig. 2, B and C. When a set of primers, 0306II-F and 0306II-R (4Imamura S. Yoshihara S. Nakano S. Shiozaki N. Yamada A. Tanaka K. Takahashi H. Asayama M. Shirai M. J. Mol. Biol. 2003; 325: 857-872Crossref PubMed Scopus (106) Google Scholar), which can amplify a region of sigB (1.0 kb) were used, bands of 1.0 and 2.5 kb were detected with Comp-ΔsigB genomic DNA (Fig. 2B, left). However, when a set of primers, IS-F (5′-TTGGAGGTCATCGAGTTTGG-3′) and IS-R (5′-GAAGATCTCAGGCAAAAGCCAATGAGTG-3′), which amplify a region around the integration site (IS) of ssl0410, were used, a band of 3.6 kb was detected with Comp-ΔsigB genomic DNA (Fig. 2B, right), confirming that this recombination involved a double-crossover reaction. We further confirmed the complementation of sigB expression at the protein level in Comp-ΔsigB (Fig. 2C). The RpoB antibody was used as a loading control for all proteins in Western blotting. Finally, we verified that SigB compensated for the transcription from PglnB-54,-53 under Log/-N, in which SigB significantly influences the expression of glnB as shown in Fig. 1 (Fig. 2D). Although the amount of transcript from PglnB-33 was the same in the three strains, the amount from PglnB-54,-53 was restored in Comp-ΔsigB to the level observed in the wild type. Thus, we concluded that SigB contributes to the expression of glnB under nitrogen-deprived conditions. SigB and SigC Contribute to Nitrogen-related Gene Expression—The glnB transcript was further analyzed by QRT-PCR. Amounts of the transcript were reduced ∼30% relative to levels in wild-type cells in the ΔsigB strain during the log and stationary phases (Fig. 3A). On the other hand, there was also a 45% reduction in the ΔsigC strain in the stationary phase (Fig. 3A). These results well support the data shown in Fig. 1 and again indicate that SigB and SigC mainly contribute to transcription from PglnB-53,-54 under conditions of nitrogen deprivation in a growth phase-dependent manner. In this case, the QRT-PCR analysis was useful for measuring the amount of glnB transcript although the gene possesses multiple promoters (Fig. 3A, top), PglnB-54,-53 (an NtcA-dependent nitrogen deprivation-responsive promoter recognized by SigB and SigC) and PglnB-33 (a constitutive promoter recognized by SigA), and could practically resolve the decrease in transcription from the NtcA-dependent promoter in the knock-out strains. To clarify whether SigB and SigC contribute to the expression of other nitrogen-related genes, we characterized another four NtcA-dependent promoters (Table 1), transcription from which is induced under nitrogen-deprived conditions in PCC 6803: glnA, a type-1 glutamine synthase gene; sigE, a group 2 σ factor gene; amt1, an ammonium permease gene; and glnN, a type-3 glutamine synthase gene, in respective knock-out strains. QRT-PCR analyses also revealed that the transcription of glnA, sigE, and amt1 decreased ∼30-40% in the sigB knock-out strain in the log phase and ∼34-50% in the sigB or sigC knock-out strain in the stationary phase under conditions of nitrogen deprivation (Fig. 3, B-D). We used rrn16Sa as a control and observed an almost constant level of expression in the knock-out strains (Fig. 3F). These results were similar to those found in the case of glnB, indicating that the contribution of SigB and SigC to the NtcA-dependent promoters might be conserved in PCC 6803. The profile of transcription of glnN in the sigB or sigC knock-out strain was different from that of glnB, glnA, sigE, or amt1 (Fig. 3E). The level of the glnN transcript was increased about 1.8- and 1.5-fold in the sigB and sigC knock-out strains, respectively, in the log phase. In contrast, the level was reduced about 25% in the sigB and sigE knock-out strains during the stationary phase and both phases, respectively. Muro-Pastor et al. (15Muro-Pastor A.M. Herrero A. Flores E. J. Bacteriol. 2001; 183: 1090-1095Crossref PubMed Scopus (78) Google Scholar) reported that SigE contributes to glnN expression under nitrogen-deprived conditions. Our results support theirs. The distinct roles of S" @default.
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