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• W2045496696 abstract "The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (Ci), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the QB site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by Ci limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in Ci conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon. The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (Ci), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the QB site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by Ci limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in Ci conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon. Regulatory RNAs are key transcriptional and post-transcriptional regulators of gene expression in all domains of life. In plants, small RNA-based mechanisms control almost all aspects of plant biology, including chromatin structure, genome stability, gene expression, and defense. The functions and phylogenetic distribution of plant microRNAs, one particular class of RNA regulators, have been studied comparatively well (for a review, see Ref. 1Cuperus J.T. Fahlgren N. Carrington J.C. Evolution and functional diversification of MIRNA genes.Plant Cell. 2011; 23: 431-442Crossref PubMed Scopus (555) Google Scholar), whereas the functions of longer noncoding RNAs are only beginning to emerge (2De Lucia F. Dean C. Long non-coding RNAs and chromatin regulation.Curr. Opin. Plant Biol. 2011; 14: 168-173Crossref PubMed Scopus (71) Google Scholar). Transcripts, which originate from the reverse complementary strand of an annotated gene and hence fully or partially overlap with their respective mRNAs, are known as cis-natural antisense transcripts or antisense RNAs (asRNAs). 3The abbreviations used are: asRNAantisense RNAncRNAnoncoding RNAPSIIphotosystem IILClow carbon (0.038% CO2 in air)HChigh carbon (3% CO2 in air)Ciinorganic carbonntnucleotide(s)CCMCO2-concentrating mechanismFlvflavodiironRubiscoribulose-bisphosphate carboxylase/oxygenase. Natural asRNAs are abundant in the plant nuclear genome (3Zhou X. Sunkar R. Jin H. Zhu J.K. Zhang W. Genome-wide identification and analysis of small RNAs originated from natural antisense transcripts in Oryza sativa.Genome Res. 2009; 19: 70-78Crossref PubMed Scopus (101) Google Scholar, 4Zhang X. Xia J. Lii Y.E. Barrera-Figueroa B.E. Zhou X. Gao S. Lu L. Niu D. Chen Z. Leung C. Wong T. Zhang H. Guo J. Li Y. Liu R. Liang W. Zhu J.K. Zhang W. Jin H. Genome-wide analysis of plant nat-siRNAs reveals insights into their distribution, biogenesis and function.Genome Biol. 2012; 13: R20Crossref PubMed Scopus (99) Google Scholar). Plant asRNAs have been more frequently observed to be associated with mRNAs of nucleus-encoded plastid and mitochondrial proteins than with other eukaryotic mRNAs (5Jin H. Vacic V. Girke T. Lonardi S. Zhu J.K. Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis.BMC Mol. Biol. 2008; 9: 6Crossref PubMed Scopus (114) Google Scholar), a fact that may be taken as a hint for the possible role of bacterial asRNAs during evolution and even after endosymbiosis. Indeed, recent observations for plant chloroplasts indicated asRNAs to be associated to 35% of all genes (6Zhelyazkova P. Sharma C.M. Förstner K.U. Liere K. Vogel J. Börner T. The primary transcriptome of barley chloroplasts: numerous non-coding RNAs and the dominating role of the plastid-encoded RNA polymerase.Plant Cell. 2012; 24: 123-136Crossref PubMed Scopus (155) Google Scholar). antisense RNA noncoding RNA photosystem II low carbon (0.038% CO2 in air) high carbon (3% CO2 in air) inorganic carbon nucleotide(s) CO2-concentrating mechanism flavodiiron ribulose-bisphosphate carboxylase/oxygenase. In cyanobacteria, the evolutionary ancestors of plant chloroplasts, asRNAs summing up to 26% of all genes for the unicellular Synechocystis sp. PCC 6803 (hereafter Synechocystis) (7Georg J. Voss B. Scholz I. Mitschke J. Wilde A. Hess W.R. Evidence for a major role of antisense RNAs in cyanobacterial gene regulation.Mol. Syst. Biol. 2009; 5: 305Crossref PubMed Scopus (146) Google Scholar, 8Mitschke J. Georg J. Scholz I. Sharma C.M. Dienst D. Bantscheff J. Voss B. Steglich C. Wilde A. Vogel J. Hess W.R. An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC 6803.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 2124-2129Crossref PubMed Scopus (291) Google Scholar) and to 39% of all genes in the nitrogen-fixing Anabaena sp. PCC 7120 (hereafter Anabaena) (9Mitschke J. Vioque A. Haas F. Hess W.R. Muro-Pastor A.M. Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 20130-20135Crossref PubMed Scopus (192) Google Scholar) were reported. However, the functional relevance of specific antisense transcripts in plants, chloroplasts, and bacteria has barely been addressed. In cyanobacteria, the functions of only two asRNAs have been studied in molecular detail thus far. In Anabaena, furA, the gene for the ferric uptake regulator, is covered by a long asRNA (10Hernández J.A. Muro-Pastor A.M. Flores E. Bes M.T. Peleato M.L. Fillat M.F. Identification of a furA cis antisense RNA in the cyanobacterium Anabaena sp. PCC 7120.J. Mol. Biol. 2006; 355: 325-334Crossref PubMed Scopus (87) Google Scholar) whose knock-out mutation results in an iron deficiency phenotype (11Hernández J.A. Alonso I. Pellicer S. Luisa Peleato M. Cases R. Strasser R.J. Barja F. Fillat M.F. Mutants of Anabaena sp. PCC 7120 lacking alr1690 and α-furA antisense RNA show a pleiotropic phenotype and altered photosynthetic machinery.J. Plant Physiol. 2010; 167: 430-437Crossref PubMed Scopus (30) Google Scholar). In Synechocystis, the 177-nt asRNA IsrR controls the expression of isiA, which encodes the iron stress-induced protein A, in a co-degradation mechanism (12Dühring U. Axmann I.M. Hess W.R. Wilde A. An internal antisense RNA regulates expression of the photosynthesis gene isiA.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 7054-7058Crossref PubMed Scopus (185) Google Scholar). Cyanobacteria are often challenged by changes in biotic and abiotic factors in their natural environments. In particular, changes in cellular functions triggered by fluctuations in the availability of inorganic carbon (Ci) have been a subject of studies for years (13Benschop J.J. Badger M.R. Dean Price G. Characterisation of CO2 and HCO3− uptake in the cyanobacterium Synechocystis sp. PCC6803.Photosynth. Res. 2003; 77: 117-126Crossref PubMed Scopus (28) Google Scholar, 14McGinn P.J. Price G.D. Maleszka R. Badger M.R. Inorganic carbon limitation and light control the expression of transcripts related to the CO2-concentrating mechanism in the cyanobacterium Synechocystis sp. strain PCC6803.Plant Physiol. 2003; 132: 218-229Crossref PubMed Scopus (105) Google Scholar, 15Woodger F.J. Badger M.R. Price G.D. Inorganic carbon limitation induces transcripts encoding components of the CO2-concentrating mechanism is Synechococcus sp. PCC7942 through a redox-independent pathway.Plant Physiol. 2003; 133: 2069-2080Crossref PubMed Scopus (79) Google Scholar, 16Wang H.L. Postier B.L. Burnap R.L. Alterations in global patterns of gene expression in Synechocystis sp. PCC 6803 in response to inorganic carbon limitation and the inactivation of ndhR, a LysR family regulator.J. Biol. Chem. 2004; 279: 5739-5751Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 17Zhang P. Battchikova N. Jansen T. Appel J. Ogawa T. Aro E.M. Expression and functional roles of the two distinct NDH-1 complexes and the carbon acquisition complex NdhD3/NdhF3/CupA/Sll1735 in Synechocystis sp. PCC 6803.Plant Cell. 2004; 16: 3326-3340Crossref PubMed Scopus (186) Google Scholar, 18Woodger F.J. Badger M.R. Price G.D. Sensing of inorganic carbon limitation in Synechococcus PCC7942 is correlated with the size of the internal inorganic carbon pool and involves oxygen.Plant Physiol. 2005; 139: 1959-1969Crossref PubMed Scopus (67) Google Scholar, 19Eisenhut M. Kahlon S. Hasse D. Ewald R. Lieman-Hurwitz J. Ogawa T. Ruth W. Bauwe H. Kaplan A. Hagemann M. The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria.Plant Physiol. 2006; 142: 333-342Crossref PubMed Scopus (107) Google Scholar, 20Eisenhut M. von Wobeser E.A. Jonas L. Schubert H. Ibelings B.W. Bauwe H. Matthijs H.C. Hagemann M. Long-term response toward inorganic carbon limitation in wild type and glycolate turnover mutants of the cyanobacterium Synechocystis sp. strain PCC 6803.Plant Physiol. 2007; 144: 1946-1959Crossref PubMed Scopus (103) Google Scholar, 21Eisenhut M. Huege J. Schwarz D. Bauwe H. Kopka J. Hagemann M. Metabolome phenotyping of inorganic carbon limitation in cells of the wild type and photorespiratory mutants of the cyanobacterium Synechocystis sp. strain PCC 6803.Plant Physiol. 2008; 148: 2109-2120Crossref PubMed Scopus (67) Google Scholar, 22Battchikova N. Vainonen J.P. Vorontsova N. Keranen M. Carmel D. Aro E.M. Dynamic changes in the proteome of Synechocystis 6803 in response to CO2 limitation revealed by quantitative proteomics.J. Proteome. Res. 2010; 9: 5896-5912Crossref PubMed Scopus (61) Google Scholar, 23Schwarz D. Nodop A. Hüge J. Purfürst S. Forchhammer K. Michel K.P. Bauwe H. Kopka J. Hagemann M. Metabolic and transcriptomic phenotyping of inorganic carbon acclimation in the cyanobacterium Synechococcus elongatus PCC 7942.Plant Physiol. 2011; 155: 1640-1655Crossref PubMed Scopus (65) Google Scholar). Most striking is the induction of the CO2-concentrating mechanism (CCM) in cyanobacterial cells after a shift from high (>1% CO2 in air) to low (atmospheric 0.038% CO2 in air) levels of Ci. By coordinated action of different CCM components, comprising specialized Ci uptake mechanisms and the Rubisco-containing carboxysomes, cyanobacteria manage to lower the CO2 compensation point and thus overcome the otherwise limiting Ci availability (for reviews, see Refs. 24Kaplan A. Reinhold L. CO2 concentrating mechanisms in photosynthetic microorganisms.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (577) Google Scholar, 25Badger M.R. Price G.D. CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution.J. Exp. Bot. 2003; 54: 609-622Crossref PubMed Scopus (598) Google Scholar, 26Giordano M. Beardall J. Raven J.A. CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution.Annu. Rev. Plant Biol. 2005; 56: 99-131Crossref PubMed Scopus (1060) Google Scholar, 27Price G.D. Badger M.R. Woodger F.J. Long B.M. Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants.J. Exp. Bot. 2008; 59: 1441-1461Crossref PubMed Scopus (5) Google Scholar). Furthermore, flavodiiron (Flv) proteins have recently been shown to be involved in the low carbon (0.038% CO2 in air; LC) acclimation process (28Zhang P. Allahverdiyeva Y. Eisenhut M. Aro E.M. Flavodiiron proteins in oxygenic photosynthetic organisms: photoprotection of photosystem II by Flv2 and Flv4 in Synechocystis sp. PCC 6803.PLoS One. 2009; 4: e5331Crossref PubMed Scopus (126) Google Scholar, 29Allahverdiyeva Y. Ermakova M. Eisenhut M. Zhang P. Richaud P. Hagemann M. Cournac L. Aro E.M. Interplay between flavodiiron proteins and photorespiration in Synechocystis sp. PCC 6803.J. Biol. Chem. 2011; 286: 24007-24014Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 30Zhang P. Eisenhut M. Brandt A.M. Carmel D. Silén H.M. Vass I. Allahverdiyeva Y. Salminen T.A. Aro E.M. Operon flv4-flv2 provides cyanobacteria with a novel photoprotection mechanism.Plant Cell. 2012; 24: 1952-1971Crossref PubMed Scopus (109) Google Scholar). The fully sequenced (31Kaneko 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. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions.DNA Res. 1996; 3: 109-136Crossref PubMed Scopus (2121) Google Scholar) cyanobacterial model organism Synechocystis contains four genes encoding the proteins Flv1, Flv2, Flv3, and Flv4. The expression of the flv2, flv3, and flv4 genes becomes up-regulated under LC conditions with flv2 and flv4 showing the strongest induction (16Wang H.L. Postier B.L. Burnap R.L. Alterations in global patterns of gene expression in Synechocystis sp. PCC 6803 in response to inorganic carbon limitation and the inactivation of ndhR, a LysR family regulator.J. Biol. Chem. 2004; 279: 5739-5751Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 20Eisenhut M. von Wobeser E.A. Jonas L. Schubert H. Ibelings B.W. Bauwe H. Matthijs H.C. Hagemann M. Long-term response toward inorganic carbon limitation in wild type and glycolate turnover mutants of the cyanobacterium Synechocystis sp. strain PCC 6803.Plant Physiol. 2007; 144: 1946-1959Crossref PubMed Scopus (103) Google Scholar, 28Zhang P. Allahverdiyeva Y. Eisenhut M. Aro E.M. Flavodiiron proteins in oxygenic photosynthetic organisms: photoprotection of photosystem II by Flv2 and Flv4 in Synechocystis sp. PCC 6803.PLoS One. 2009; 4: e5331Crossref PubMed Scopus (126) Google Scholar). Although the Flv1 and Flv3 proteins participate in the Mehler-like reaction (29Allahverdiyeva Y. Ermakova M. Eisenhut M. Zhang P. Richaud P. Hagemann M. Cournac L. Aro E.M. Interplay between flavodiiron proteins and photorespiration in Synechocystis sp. PCC 6803.J. Biol. Chem. 2011; 286: 24007-24014Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 32Vicente J.B. Gomes C.M. Wasserfallen A. Teixeira M. Module fusion in an A-type flavoprotein from the cyanobacterium Synechocystis condenses a multiple-component pathway in a single polypeptide chain.Biochem. Biophys. Res. Commun. 2002; 294: 82-87Crossref PubMed Scopus (79) Google Scholar, 33Helman Y. Tchernov D. Reinhold L. Shibata M. Ogawa T. Schwarz R. Ohad I. Kaplan A. Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria.Curr. Biol. 2003; 13: 230-235Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), the Flv2 and Flv4 proteins were demonstrated to have a crucial role in photoprotection of photosystem II (PSII) under LC conditions (28Zhang P. Allahverdiyeva Y. Eisenhut M. Aro E.M. Flavodiiron proteins in oxygenic photosynthetic organisms: photoprotection of photosystem II by Flv2 and Flv4 in Synechocystis sp. PCC 6803.PLoS One. 2009; 4: e5331Crossref PubMed Scopus (126) Google Scholar). We have shown recently (30Zhang P. Eisenhut M. Brandt A.M. Carmel D. Silén H.M. Vass I. Allahverdiyeva Y. Salminen T.A. Aro E.M. Operon flv4-flv2 provides cyanobacteria with a novel photoprotection mechanism.Plant Cell. 2012; 24: 1952-1971Crossref PubMed Scopus (109) Google Scholar) that under these conditions the small membrane protein Sll0218, which is also encoded by the flv4-2 operon, stabilizes the PSII dimer and enables the Flv2/Flv4 heterodimer to accept electrons from PSII. Thus, the products of the flv4-2 operon provide β-cyanobacteria with a unique and novel photoprotection mechanism. Despite numerous studies and continuous progress (15Woodger F.J. Badger M.R. Price G.D. Inorganic carbon limitation induces transcripts encoding components of the CO2-concentrating mechanism is Synechococcus sp. PCC7942 through a redox-independent pathway.Plant Physiol. 2003; 133: 2069-2080Crossref PubMed Scopus (79) Google Scholar, 16Wang H.L. Postier B.L. Burnap R.L. Alterations in global patterns of gene expression in Synechocystis sp. PCC 6803 in response to inorganic carbon limitation and the inactivation of ndhR, a LysR family regulator.J. Biol. Chem. 2004; 279: 5739-5751Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 18Woodger F.J. Badger M.R. Price G.D. Sensing of inorganic carbon limitation in Synechococcus PCC7942 is correlated with the size of the internal inorganic carbon pool and involves oxygen.Plant Physiol. 2005; 139: 1959-1969Crossref PubMed Scopus (67) Google Scholar, 20Eisenhut M. von Wobeser E.A. Jonas L. Schubert H. Ibelings B.W. Bauwe H. Matthijs H.C. Hagemann M. Long-term response toward inorganic carbon limitation in wild type and glycolate turnover mutants of the cyanobacterium Synechocystis sp. strain PCC 6803.Plant Physiol. 2007; 144: 1946-1959Crossref PubMed Scopus (103) Google Scholar, 23Schwarz D. Nodop A. Hüge J. Purfürst S. Forchhammer K. Michel K.P. Bauwe H. Kopka J. Hagemann M. Metabolic and transcriptomic phenotyping of inorganic carbon acclimation in the cyanobacterium Synechococcus elongatus PCC 7942.Plant Physiol. 2011; 155: 1640-1655Crossref PubMed Scopus (65) Google Scholar, 34Woodger F.J. Bryant D.A. Price G.D. Transcriptional regulation of the CO2-concentrating mechanism in a euryhaline, coastal marine cyanobacterium, Synechococcus sp. strain PCC 7002: role of NdhR/CcmR.J. Bacteriol. 2007; 189: 3335-3347Crossref PubMed Scopus (64) Google Scholar, 35Nishimura T. Takahashi Y. Yamaguchi O. Suzuki H. Maeda S. Omata T. Mechanism of low CO2-induced activation of the cmp bicarbonate transporter operon by a LysR family protein in the cyanobacterium Synechococcus elongatus strain PCC 7942.Mol. Microbiol. 2008; 68: 98-109Crossref PubMed Scopus (64) Google Scholar, 36Lieman-Hurwitz J. Haimovich M. Shalev-Malul G. Ishii A. Hihara Y. Gaathon A. Lebendiker M. Kaplan A. A cyanobacterial AbrB-like protein affects the apparent photosynthetic affinity for CO2 by modulating low-CO2-induced gene expression.Environ. Microbiol. 2009; 11: 927-936Crossref PubMed Scopus (63) Google Scholar), the understanding of the Ci-controlled gene expression dynamics is still incomplete. Here we report the identification of three asRNAs and one noncoding RNA (ncRNA) associated with the flv4-2 operon. These transcripts were primarily detected by microarray analysis (7Georg J. Voss B. Scholz I. Mitschke J. Wilde A. Hess W.R. Evidence for a major role of antisense RNAs in cyanobacterial gene regulation.Mol. Syst. Biol. 2009; 5: 305Crossref PubMed Scopus (146) Google Scholar) and 454 sequencing (8Mitschke J. Georg J. Scholz I. Sharma C.M. Dienst D. Bantscheff J. Voss B. Steglich C. Wilde A. Vogel J. Hess W.R. An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC 6803.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 2124-2129Crossref PubMed Scopus (291) Google Scholar). We verified the existence of these ncRNAs by Northern blotting and characterized the asRNA As1_flv4 in more detail. The inversely correlated accumulation of As1_flv4 transcript with the transcripts and proteins from the flv4-2 operon and the results obtained from artificial modulation of As1_flv4 levels suggest a stoichiometric function of As1_flv4 to control the expression of the flv4-2 operon according to the environmental Ci availability. Furthermore, the direct or indirect repression by the AbrB-like transcriptional regulator Sll0822 and the control of the promoter activity by the Ci level support the assumption that ncRNAs play a significant role in the Ci-regulatory network in Synechocystis. The glucose-tolerant strain of cyanobacterium, Synechocystis sp. PCC 6803, served as the WT. Cultivation of mutants was performed at 50 μg ml−1 kanamycin and 20 μg ml−1 spectinomycin, respectively. For the experiments, axenic cultures of the cyanobacteria were grown photoautotrophically under 50 μmol photons m−2 s−1 (white light) at 30 °C. Cells were cultivated in BG-11 medium (pH 7.5) and aerated by shaking in the presence of CO2-enriched air (3% CO2 in air; high carbon (HC)) or ambient air CO2 (LC). In the case of the LC shift experiment, the cells were collected by centrifugation (2 min at 1730 × g at room temperature) and resuspended in fresh BG-11, and the OD750 measured with a Spectronic Genesys 2 spectrophotometer (Thermo Fisher Scientific, Madison, WI) was adjusted to 0.8. After precultivation at HC conditions for 1 h, cultures were transferred to LC conditions. In analogous experiments, cells were aerated directly by continuous bubbling with LC or HC. For the asRNA overexpression experiments, the two overexpression mutants As1_flv4(+)/2 and As1_flv4(+)/3 and a control strain (mutant in spkA) were precultivated in Cu2+-containing BG-11 medium and bubbled with HC. For induction of petJ promoter activity by Cu2+ depletion (43Tous C. Vega-Palas M.A. Vioque A. Conditional expression of RNase P in the cyanobacterium Synechocystis sp. PCC6803 allows detection of precursor RNAs. Insight in the in vivo maturation pathway of transfer and other stable RNAs.J. Biol. Chem. 2001; 276: 29059-29066Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), the cells were spun down and washed with and resuspended in Cu2+-free BG-11 medium. Subsequently, cultures were treated as described above. 300- and 700-nt promoter regions of the genes encoding the asRNA As1_flv4 and the flv4-2 operon, respectively, were amplified by PCR using chromosomal DNA and specific primers (supplemental Table S1). After digestion with KpnI, the respective promoter fragment was ligated into the unique KpnI site of the promoter test vector pILA (37Kunert A. Hagemann M. Erdmann N. Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB.J. Microbiol. Methods. 2000; 41: 185-194Crossref PubMed Scopus (83) Google Scholar). The vector pILA allows transcriptional fusion of the promoter sequence with the luxAB genes and its stable integration into the chromosome at a neutral site (37Kunert A. Hagemann M. Erdmann N. Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB.J. Microbiol. Methods. 2000; 41: 185-194Crossref PubMed Scopus (83) Google Scholar). Plasmids with correct promoter insertion direction relative to the reporter genes were selected for subsequent transformation of Synechocystis. Completely segregated clones were checked by PCR analysis as described (19Eisenhut M. Kahlon S. Hasse D. Ewald R. Lieman-Hurwitz J. Ogawa T. Ruth W. Bauwe H. Kaplan A. Hagemann M. The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria.Plant Physiol. 2006; 142: 333-342Crossref PubMed Scopus (107) Google Scholar) and subsequently used for promoter activity measurements. 1-ml cell aliquots were taken at selected time points from shaking cultures with an OD750 of 0.5. Subsamples of 100 μl were transferred in triplicate to white 96-well microtiter plates (Thermo Fisher Scientific). To start the measurement, 100 μl of 2 mm decanal ready-to-use solution was added, and the plate was immediately placed into the plate reader (Wallac Victor 2 1420 multilabel counter, PerkinElmer Life Sciences). 100 mm decanal stock solution was prepared in methanol and freshly diluted with BG-11 for 2 mm decanal ready-to-use solution. Bioluminescence was measured for 30 min at 25 °C. The maximum light emission (around 10 min after the start) was used as the bioluminescence value and related to the OD750. Results are presented in relative bioluminescence units. Experiments were repeated three times. All primers used for plasmid preparation in this work are listed in supplemental Table S1. To generate the overexpression construct, a 563-nt DNA fragment beginning from the mapped as1_flv4 transcriptional start site (nucleotide 166849 according to Ref. 7Georg J. Voss B. Scholz I. Mitschke J. Wilde A. Hess W.R. Evidence for a major role of antisense RNAs in cyanobacterial gene regulation.Mol. Syst. Biol. 2009; 5: 305Crossref PubMed Scopus (146) Google Scholar) was fused with the petJ promoter and integrated with a kanamycin resistance cassette in the spkA gene. The spkA gene can be used as an uncommitted integration site because this gene is disrupted by a frameshift mutation in the Synechocystis strain used (44Kamei A. Yuasa T. Orikawa K. Geng X.X. Ikeuchi M. A eukaryotic-type protein kinase, SpkA, is required for normal motility of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803.J. Bacteriol. 2001; 183: 1505-1510Crossref PubMed Scopus (68) Google Scholar). The DNA fragment is longer than the asRNA transcript to allow for transcription termination at its own terminator. To prevent eventual read-through, the λ phage oop terminator was fused to the 3′-end of the fragment. First a platform for the integration of a ncRNA between the petJ promoter and the oop terminator was constructed. The primers “5′ApaI_petJ” and “3′petJ_AsuII_oop_SalI” were used to amplify the petJ promoter fragment. The construct contained ApaI and SalI restriction sites for integration in the pJet-spk plasmid and an AsuII restriction site for the integration of an ncRNA between petJ promoter and oop terminator. The new pJet-spk-petJp plasmid was AsuII-digested for the integration of the AsuII-digested as1_flv4 fragment generated with the as_flv4_asuII_for and as_flv4_asuII_rev primers. The sense orientation of the fragment was tested with the as_flv4_asuII_rev primer and the spK_seg_for primer. The segregation of the construct in the genome was tested with the spK_seg_for and spK_seg_rev primers. A schematic presentation of the cloning strategy is shown in supplemental Fig. S1A. Total RNA was isolated with TRIzol reagent (Invitrogen) and treated with a TURBO DNase kit (Invitrogen) to remove genomic DNA. To characterize small RNAs, RNA samples (5 μg) were mixed with RNA loading dye (Fermentas, St. Leon-Rot, Germany), denatured for 10 min at 70 °C, separated in 10% polyacrylamide-urea gels, and transferred to Hybond-N nylon membranes (GE Healthcare) by electroblotting for 1 h. For the mRNA studies, RNA samples were mixed with denaturation solution, incubated for 10 min at 70 °C, separated in 1.3% agarose gels containing 7% formaldehyde in MOPS, and transferred to Hybond-N nylon membranes by capillary blotting with 10× SSC overnight (38Hagemann M. Schoor A. Jeanjean R. Zuther E. Joset F. The stpA gene from Synechocystis sp. strain PCC 6803 encodes the glucosylglycerol-phosphate phosphatase involved in cyanobacterial osmotic response to salt shock.J. Bacteriol. 1997; 179: 1727-1733Crossref PubMed Google Scholar). After UV cross-linking, even loading and blotting were checked by methylene blue staining (0.5 m sodium acetate, pH 5.2, 0.04% methylene blue). The membranes were hybridized with [α-32P]UTP-incorporated transcripts or [α-32P]CTP-labeled DNA probes. In vitro transcription was performed with the MAXIscript kit (Invitrogen) as described (39Steglich C. Futschik M.E. Lindell D. Voss B. Chisholm S.W. Hess W.R. The challenge of regulation in a minimal photoautotroph: non-coding RNAs in Prochlorococcus.PloS Genet. 2008; 4: e1000173Crossref PubMed Scopus (118) Google Scholar) and labeled DNA probes obtai" @default.
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• W2045496696 date "2012-09-01" @default.
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• W2045496696 title "The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply" @default.
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