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• W2023357287 abstract "Two gene clusters each encoding the cyanophycin-metabolism enzymes cyanophycin synthetase and cyanophycinase are found in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. In cluster cph1, the genes cphB1 and cphA1 were expressed in media containing ammonium, nitrate, or N2 as nitrogen sources, but expression was higher in the absence of combined nitrogen taking place both in vegetative cells and heterocysts. Both genes were cotranscribed from three putative promoters located upstream of cphB1, and, additionally, the cphA1 gene was expressed monocistronically from at least two promoters located in the intergenic cphB1-cphA1 region. Both constitutive promoters and promoters dependent on the global nitrogen control transcriptional regulator NtcA were identified. In cluster cph2, the cphB2 and cphA2 genes, which are found in opposite orientations, were expressed as monocistronic messages in media containing ammonium, nitrate, or N2, but expression was higher in the absence of ammonium. Expression of the cph2 genes was lower than that of cph1 genes. Analysis of cph gene insertional mutants indicated that cluster cph1 genes contributed more than cluster cph2 genes to cyanophycin accumulation in the whole filament as well as in heterocysts. Diazotrophic growth was more severely impaired in cyanophycinase than in cyanophycin synthetase mutants, indicating that cyanophycin, although normally synthesized in the heterocysts, is not required for heterocyst function and that the inability to degrade this polymer is detrimental for the diazotrophic growth of the cyanobacterium. Two gene clusters each encoding the cyanophycin-metabolism enzymes cyanophycin synthetase and cyanophycinase are found in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. In cluster cph1, the genes cphB1 and cphA1 were expressed in media containing ammonium, nitrate, or N2 as nitrogen sources, but expression was higher in the absence of combined nitrogen taking place both in vegetative cells and heterocysts. Both genes were cotranscribed from three putative promoters located upstream of cphB1, and, additionally, the cphA1 gene was expressed monocistronically from at least two promoters located in the intergenic cphB1-cphA1 region. Both constitutive promoters and promoters dependent on the global nitrogen control transcriptional regulator NtcA were identified. In cluster cph2, the cphB2 and cphA2 genes, which are found in opposite orientations, were expressed as monocistronic messages in media containing ammonium, nitrate, or N2, but expression was higher in the absence of ammonium. Expression of the cph2 genes was lower than that of cph1 genes. Analysis of cph gene insertional mutants indicated that cluster cph1 genes contributed more than cluster cph2 genes to cyanophycin accumulation in the whole filament as well as in heterocysts. Diazotrophic growth was more severely impaired in cyanophycinase than in cyanophycin synthetase mutants, indicating that cyanophycin, although normally synthesized in the heterocysts, is not required for heterocyst function and that the inability to degrade this polymer is detrimental for the diazotrophic growth of the cyanobacterium. Cyanobacteria are oxygenic photoautotrophs that make an important contribution to primary productivity in our planet. They fix CO2 through the reductive penthose phosphate pathway and preferentially assimilate inorganic sources of nitrogen. Many cyanobacteria are able to carry out the fixation of atmospheric nitrogen, and, as a way to protect the nitrogen fixation machinery from oxygen, some filamentous strains differentiate cells called heterocysts where nitrogenase is confined (1Wolk C.P. Ernst A. Elhai J. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers, The Netherlands1994: 769-823Crossref Google Scholar). Nitrogen fixed in the heterocysts is donated, probably in the form of amino acids, to the rest of cells in the filament by a mechanism not yet understood that may involve the operation of amino acid uptake permeases (2Montesinos M.L. Herrero A. Flores E. J. Bacteriol. 1995; 177: 3150-3157Crossref PubMed Google Scholar) and that might be related to the accumulation of cyanophycin (see below) in the heterocyst. The differentiation of heterocysts and the assimilation of nitrogen in cyanobacteria are subjected to nutritional repression by ammonium (3Flores E. Herrero A. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 487-517Crossref Google Scholar). In the absence of ammonium, the transcriptional regulator NtcA activates expression of many genes encoding elements of the pathways for the assimilation of nitrogen or involved in the differentiation and function of the heterocyst (4Herrero A. Muro-Pastor A.M. Flores E. J. Bacteriol. 2001; 183: 411-425Crossref PubMed Scopus (542) Google Scholar). NtcA is homologous to transcriptional regulators of the CAP family and directly binds to the promoter regions of nitrogen-regulated genes at specific DNA sites with the consensus sequence GTAN8TAC frequently centered at -41.5 and located upstream from a σ70-consensus -10 box in the form TAN3T (4Herrero A. Muro-Pastor A.M. Flores E. J. Bacteriol. 2001; 183: 411-425Crossref PubMed Scopus (542) Google Scholar). Cyanophycin is a nonribosomically synthesized peptide composed of multi-l-arginyl-poly-l-aspartate (α-amino groups of arginine residues linked to β-carboxyl groups of a polyaspartate backbone) that until very recently was considered exclusive of cyanobacteria. In these organisms, cyanophycin accumulates in the cytoplasm in the form of granules mainly under unbalanced growth conditions (e.g. under stationary phase or under starvation conditions that do not involve nitrogen starvation) (5Allen M.M. Annu. Rev. Microbiol. 1984; 38: 1-25Crossref PubMed Scopus (218) Google Scholar, 6Simon R.D. Fay P. van Baalen C. The Cyanobacteria. Elsevier, Amsterdam1987: 199-225Google Scholar). It is considered that cyanophycin can represent a dynamic reserve of nitrogen; e.g. in Cyanothece sp. ATCC 51142, which temporarily separates N2 fixation and photosynthesis, cyanophycin is degraded during the light period and is formed during the period of darkness, when N2 fixation is operative (7Sherman L.A. Meunier P. Colón-López M.S. Photosyn. Res. 1998; 58: 25-42Crossref Scopus (84) Google Scholar). In the N2-fixing heterocyst, cyanophycin accumulates, forming conspicuous deposits in the cellular poles adjacent to the vegetative cells (e.g. see Ref. 8Sherman D.M. Tucker D. Sherman L.A. J. Phycol. 2000; 36: 932-941Crossref Scopus (48) Google Scholar). A cyanophycin-synthesizing enzyme, called cyanophycin synthetase, has been identified that could add both l-aspartic acid and l-arginine to a cyanophycin primer (9Simon R.D. Biochim. Biophys. Acta. 1976; 422: 407-418Crossref PubMed Scopus (98) Google Scholar). Recently, cyanophycin synthetase has been purified from the heterocystforming Anabaena variabilis ATCC 29413 and characterized as a homodimer of a 100-kDa subunit (10Ziegler K. Diener A. Herpin C. Richter R. Deutzmann R. Lockau W. Eur. J. Biochem. 1998; 254: 154-159Crossref PubMed Scopus (155) Google Scholar). Based on the amino acid sequence from this protein, the corresponding gene, cphA, has been identified in the genomic sequence of the unicellular cyanobacterium Synechocystis sp. PCC 6803 and then in Anabaena variabilis (10Ziegler K. Diener A. Herpin C. Richter R. Deutzmann R. Lockau W. Eur. J. Biochem. 1998; 254: 154-159Crossref PubMed Scopus (155) Google Scholar). On the other hand, based on analysis of the products of cyanophycin degradation by crude or fractionated cell extracts of different cyanobacteria, two different enzymatic activities have been implicated in cyanophycin degradation: an exopeptidase, called cyanophycinase, that would produce β-Asp-Arg dipeptides (11Gupta M. Carr N.G. J. Gen. Microbiol. 1981; 125: 17-23Google Scholar) and a peptidase that would hydrolyze this peptide. Recently, the gene cphB encoding a cyanophycinase has been identified in the genomic sequence of Synechocystis sp. PCC 6803 (12Richter R. Hejazi M. Kraft R. Ziegler K. Lockau W. Eur. J. Biochem. 1999; 263: 163-169Crossref PubMed Scopus (96) Google Scholar), and ORFs 1The abbreviations used are: ORF, open reading frame; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; tsp, transcription start point. of both Synechocystis sp. and Anabaena sp. have been described to encode plant-type asparaginases able to hydrolyze β-Asp-Arg bonds and that, thus, may be responsible for the last step of cyanophycin degradation (13Hejazi M. Piotukh K. Mattow J. Deutzmann R. Volkmer-Engert R. Lockau W. Biochem. J. 2002; 364: 129-136Crossref PubMed Scopus (62) Google Scholar). The cphB and cphA genes have also been identified in whole genome sequence projects of several cyanobacteria. Putative cyanophycin synthetase genes have recently been identified in the genomic sequences of a number of eubacteria different from cyanobacteria (14Krehenbrink M. Oppermann-Sanio F.B. Steinbüchel A. Arch. Microbiol. 2002; 177: 371-380Crossref PubMed Scopus (96) Google Scholar), and those from Acinetobacter sp. strain DSM 587 (14Krehenbrink M. Oppermann-Sanio F.B. Steinbüchel A. Arch. Microbiol. 2002; 177: 371-380Crossref PubMed Scopus (96) Google Scholar) and Desulfitobacterium hafniense (15Ziegler K. Deutzmann R. Lockau W. Z. Naturforsch. C. 2002; 57: 522-529Crossref PubMed Scopus (81) Google Scholar) have proven able to direct the synthesis of cyanophycin-like polymers when expressed in Escherichia coli. Moreover, 11 eubacteria different from cyanobacteria have been found to be able to utilize cyanophycin as a carbon source for growth based on the action of extracellular cyanophycinases. The product of the so-called cphE gene from Pseudomonas anguilliseptica strain BI, although exhibiting only 27–28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria, catalyzes a very specific degradation of cyanophycin to β-Asp-Arg dipeptides (16Obst M. Oppermann-Sanio F.B. Luftmann H. Steinbüchel A. J. Biol. Chem. 2002; 277: 25096-25105Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Thus, cyanophycin appears to occur in a wide range of bacteria that make use of diverse metabolic options including phototrophy, aerobic and anaerobic respiration, fermentation, and chemolithoautotrophy, where it can serve, perhaps among other functions, as a nitrogen reserve or an external carbon source. Therefore, cyanophycin seems to have a role in the biology of bacteria much wider than previously recognized. The present work deals with the genetic systems for cyanophycin synthesis and degradation in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, which is amenable to genetic manipulation and whose entire genomic sequence is available (17Kaneko T. Nakamura Y. Wolk C.P. Kuritz T. Sasamoto S. Watanabe A. Iriguchi M. Ishikawa A. Kawashima K. Kimura T. Kishida Y. Kohara M. Matsumoto M. Matsuno A. Muraki A. Nakazaki N. Shimpo S. Sugimoto M. Takazawa M. Yamada M. Yasuda M. Tabata S. DNA Res. 2001; 8: 227-253Crossref Google Scholar). The regulation of the expression of cyanophycin metabolism genes with regard to the nitrogen regime and cell type in the diazotrophic filament has been studied, and the involvement of cyanophycin in diazotrophic metabolism has been investigated through the generation of cyanophycin metabolism gene mutants. Strains and Growth Conditions—This study was carried out with the heterocyst forming cyanobacterium Anabaena sp. PCC 7120 and an insertional mutant of the ntcA gene, strain CSE2 (18Frías J.E. Flores E. Herrero A. Mol. Microbiol. 1994; 14: 823-832Crossref PubMed Scopus (177) Google Scholar). Anabaena sp. PCC 7120 was grown axenically in BG11 medium, which contains 17.6 mm NaNO3 (19Rippka R. Deruelles J. Waterbury J.B. Herdman M. Stanier R.Y. J. Gen. Microbiol. 1979; 111: 1-61Crossref Google Scholar), in BG110 (nitrogen-free) medium or in BG110 medium supplemented with 8 mm NH4Cl and 16 mm TES-NaOH buffer (pH 7.5). Strain CSE2 was grown in ammonium-containing medium in the presence of 2 μg ml-1 of streptomycin and 2 μg ml-1 of spectinomycin. For plates, the medium was solidified with 1% separately autoclaved agar (Difco). Liquid cultures were incubated at 30 °C in the light (75 microeinsteins m-2 s-1), with shaking (80–90 rpm). Anabaena mutants carrying gene cassette C.K3 (20Mazodier P. Cossart P. Giraud E. Gasser F. Nucleic Acids Res. 1985; 13: 195-205Crossref PubMed Scopus (140) Google Scholar) were routinely grown in medium supplemented with 5–25 μg ml-1 neomycin, mutants carrying cassette C.S3 (21Prentki P. Krisch H.M. Gene (Amst.). 1984; 29: 303-313Crossref PubMed Scopus (1343) Google Scholar) were grown in medium supplemented with 2–5 μgml-1 Sm and Sp, and mutants carrying cat and erm genes, encoding for CmR and EmR, respectively, (SalI-XhoI fragment from plasmid pRL271) (22Black T.A. Cai Y. Wolk C.P. Mol. Microbiol. 1993; 9: 77-84Crossref PubMed Scopus (319) Google Scholar) were grown in medium supplemented with 5 μgml-1 Em. Growth rates were estimated from the increase in protein concentration of the cultures, determined by a modified Lowry procedure (23Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5333) Google Scholar) in 0.2-ml aliquots periodically withdrawn from the cultures. The growth rate constant (μ) corresponded to ln 2/td, where td represents the doubling time. Mutant Construction—To inactivate gene cphB1, a 1.63-kb DNA fragment from the cph1 region of Anabaena sp. PCC 7120 was amplified by PCR using oligonucleotides CB1–1 (corresponding to nucleotides +8 to +29 with respect to the translation start of cphB1) and CA1–3 (complementary to nucleotides +536 to +519 relative to the translation start of cphA1) and genomic DNA from strain PCC 7120 as template. PCR products were cloned in vector pGEM-T (Promega) to generate plasmid pCSS37. The C.K3 gene cassette excised with SmaI (24Elhai J. Wolk C.P. Gene (Amst.). 1988; 68: 119-138Crossref PubMed Scopus (374) Google Scholar) was inserted into the BclI site that is present in the Anabaena DNA insert of pCSS37 to generate plasmid pCSS38. The SpeI-ScaI fragment from pCSS38, filled in with Klenow enzyme, was ligated to the sacB-containing vector pRL277 (22Black T.A. Cai Y. Wolk C.P. Mol. Microbiol. 1993; 9: 77-84Crossref PubMed Scopus (319) Google Scholar) linearized with EcoRV, rendering plasmid pCSS39. The sacB gene determines sensitivity to sucrose and can be counterselected for in Anabaena sp., allowing positive selection for double recombinants (25Cai Y.P. Wolk C.P. J. Bacteriol. 1990; 172: 3138-3145Crossref PubMed Scopus (406) Google Scholar). To inactivate gene cphA1, a 2-kb DNA fragment from the cph1 region of Anabaena sp. PCC 7120 was amplified by PCR using oligonucleotides CA1–1 (corresponding to nucleotides +43 to +63 relative to the translation start of cphA1) and CA1–2 (complementary to nucleotides +2029 to +2009 relative to the translation start of cphA1) and genomic DNA from strain PCC 7120 as template. PCR products were cloned in vector pGEM-T to generate plasmid pCSS33. A 15-bp DNA fragment from pCSS33 was excised with EcoRV and substituted by the C.S3 gene-cassette excised with HindIII (24Elhai J. Wolk C.P. Gene (Amst.). 1988; 68: 119-138Crossref PubMed Scopus (374) Google Scholar) and filled in with Klenow enzyme, rendering plasmid pCSS34. The SalI-NcoI fragment from pCSS34, filled in with Klenow enzyme, was ligated to the sacB-containing vector pRL278 (22Black T.A. Cai Y. Wolk C.P. Mol. Microbiol. 1993; 9: 77-84Crossref PubMed Scopus (319) Google Scholar) digested with NruI, rendering plasmid pCSS35. To inactivate gene cphB2, a 2-kb DNA fragment from the cph2 region of Anabaena sp. PCC 7120 was amplified by PCR using oligonucleotides CB2-1 (complementary to nucleotides +1654 to +1636 with respect to the translation start of cphB2) and CB2-2 (corresponding to nucleotides -386 to -366 with respect to the translation start of cphB2) and genomic DNA from strain PCC 7120 as template. PCR products were cloned in vector pGEM-T to generate plasmid pCSS50. The C.S3 gene cassette excised with HindIII was inserted into the HindIII site that is present in the Anabaena DNA insert of pCSS50, rendering plasmid pCSS51. The PvuII fragment from pCSS51 was ligated to the sacB-containing vector pRL271 (22Black T.A. Cai Y. Wolk C.P. Mol. Microbiol. 1993; 9: 77-84Crossref PubMed Scopus (319) Google Scholar), digested with BglII, and filled in with Klenow enzyme, rendering plasmid pCSS52. To inactivate gene cphA2, a 1.7-kb DNA fragment from the cph2 region of Anabaena sp. PCC 7120 was amplified by PCR using oligonucleotides CA2–2-XhoI (complementary to nucleotides +1783 to +1762 relative to the translation start of cphA2 and ended with a XhoI restriction site) and CA2-3-XhoI (corresponding to nucleotides +60 to +77 relative to the translation start of cphA2 and ended with a XhoI restriction site). PCR products were cloned in vector pGEM-T to generate plasmid pCSS53. The EmR/CmR gene cassette (containing erm and cat genes) excised with XbaI from plasmid pCSE52, which contains the SalI-XhoI fragment from pRL271 cloned in vector pIC20R, and filled with Klenow enzyme was inserted into the Eco47III site that is present in the Anabaena DNA insert of pCSS53, rendering plasmid pCSS54. The XhoI fragment from pCSS54 was ligated to the sacB-containing vector pRL278 digested with XhoI, rendering plasmid pCSS55. Constructs generated in vitro bearing a gene cassette inserted into cphB1, cphA1, cphB2, or cphA2 and cloned in sacB-containing vectors were transferred by conjugation (26Elhai J. Wolk C.P. Methods Enzymol. 1988; 167: 747-754Crossref PubMed Scopus (445) Google Scholar) to Anabaena sp. to generate strains bearing mutations in the cph1 or/and cph2 genomic regions. For generation of strains CSS7, CSS13, CSS21, and CSS25, E. coli strain HB101 containing plasmid pCSS35, pCSS39, pCSS52, or pCSS55 and helper plasmids pRL528 (26Elhai J. Wolk C.P. Methods Enzymol. 1988; 167: 747-754Crossref PubMed Scopus (445) Google Scholar) and pRL591-W45 (27Elhai J. Cai Y. Wolk C.P. J. Bacteriol. 1994; 176: 5059-5067Crossref PubMed Google Scholar) or pRL623 (28Elhai J. Vepritskiy A. Muro-Pastor A.M. Flores E. Wolk C.P. J. Bacteriol. 1997; 179: 1998-2005Crossref PubMed Scopus (263) Google Scholar) was mixed with E. coli ED8654 carrying the conjugative plasmid pRL443 and thereafter with Anabaena sp. For the generation of double mutants, plasmids pCSS39 and pCSS55 were transferred to strain CSS7 to generate strains CSS35 and CSS27, respectively, plasmid pCSS52 was transferred to strain CSS13 to generate strain CSS23, and plasmid pCSS55 was transferred to strain CSS21 to generate strain CSS36. Exconjugants were isolated (26Elhai J. Wolk C.P. Methods Enzymol. 1988; 167: 747-754Crossref PubMed Scopus (445) Google Scholar), and double recombinants were identified as clones resistant to the antibiotic for which resistance was encoded in the inserted gene cassette, resistant to sucrose, and sensitive to the antibiotic for which the resistance determinant was present in the vector portion of the transferred plasmid and were confirmed by PCR analysis. Homozygous mutant clones were selected for this study. DNA Isolation and Analysis—Total DNA from Anabaena sp. PCC 7120 and its derivatives was isolated as previously described (25Cai Y.P. Wolk C.P. J. Bacteriol. 1990; 172: 3138-3145Crossref PubMed Scopus (406) Google Scholar). For sequencing ladders used in primer extension analysis, sequencing was carried out by the dideoxy chain termination method, using a T7-Sequencing™ kit (Amersham Biosciences) and [α-35S]thio-dATP. DNA fragments were purified from agarose gels with the Geneclean II kit (BIO 101). Southern blot analysis, plasmid isolation from E. coli, transformation of E. coli, digestion of DNA with restriction endonucleases, ligation with T4 ligase, and PCR were performed by standard procedures (29Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Current Protocols in Molecular Biology. Green/Wiley-Interscience, New York2003Google Scholar). Band Shift Assays—DNA fragments to be used in electrophoretic mobility shift assays were obtained by PCR amplification. Oligonucleotides CB1–4 (corresponding to positions -697 to -679 relative to the translation start of cphB1), CB1–5 (complementary to positions -250 to -272 relative to the translation start of cphB1), CB1–7 (complementary to positions -458 to -477 relative to the translation start of cphB1), and CB1–9 (corresponding to positions -485 to -465 relative to the translation start of cphB1) and plasmid pCSS68, containing the cphB1 promoter sequence (cloned by PCR with oligonucleotides CB1–4 and CB1–5 in vector pGEM-T), were used to obtain DNA fragments of the cphB1 upstream region. Oligonucleotides CA1–6 (corresponding to positions -214 to -196 relative to the translation start of cphA1) and CA1–5 (complementary to positions +16 to -3 relative to the translation start of cphA1) and plasmid pCSS37 as a template were used for PCR amplification of the cphA1 upstream region. For competition tests, a DNA fragment from the region upstream from glnA containing an NtcA-binding site (18Frías J.E. Flores E. Herrero A. Mol. Microbiol. 1994; 14: 823-832Crossref PubMed Scopus (177) Google Scholar) was used as a specific binding fragment, and a DNA fragment of the pBluescript vector was used as a nonspecific fragment. In the case of the glnA upstream region, oligonucleotides GA3 (corresponding to positions -238 to -215 relative to the translation start of glnA) and GA6 (complementary to positions -70 to -87 relative to the translation start of glnA) and plasmid pAN503 (30Tumer N.E. Robinson S.J. Haselkorn R. Nature. 1983; 306: 337-342Crossref Scopus (140) Google Scholar) were used for PCR. In the case of the pBluescript DNA fragment, oligonucleotides Forward and Reverse were used, rendering a DNA fragment of 220 nucleotides. DNA fragments were end-labeled with T4 polynucleotide kinase (Roche Applied Science) and [γ-32P]dATP as described previously (29Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Current Protocols in Molecular Biology. Green/Wiley-Interscience, New York2003Google Scholar). Assays were carried out as described previously (31Luque I. Flores E. Herrero A. EMBO J. 1994; 13: 2862-2869Crossref PubMed Scopus (161) Google Scholar) with 1 fmol of labeled fragment and 0.1–3 μm of NtcA purified from E. coli strain DH5α (pCSAM61) bearing the strain PCC 7120 ntcA gene cloned in pTrc99A vector (Amersham Biosciences) and thus expressed from the trc promoter. Images of radioactive gels were obtained using a Cyclone storage phosphor system (Packard). RNA Isolation and Analysis—Cells used for RNA isolation were exponentially growing in the light (75 microeinsteins m-2 s-1) in liquid BG11 or BG110 media (19Rippka R. Deruelles J. Waterbury J.B. Herdman M. Stanier R.Y. J. Gen. Microbiol. 1979; 111: 1-61Crossref Google Scholar) or in medium BG110 containing 8 mm NH4Cl and 16 mm TES-NaOH buffer (pH 7.5), supplemented with 10 mm of NaHCO3 and bubbled with air and CO2 (1%, v/v). Alternatively, filaments growing in ammonium-containing medium were harvested at room temperature and either used directly or washed with and resuspended in BG110 medium and further incubated under culture conditions for the number of hours indicated in each experiment. For the isolation of RNA from heterocysts, cells were grown in ammonium-containing medium until they reached a chlorophyll a concentration of 3–5 μg ml-1. Filaments were then washed with and resuspended in nitrogen-free medium (BG110) and further incubated until mature heterocysts were observed (19 h). Heterocysts were then isolated as described (32Golden J.W. Whorff L.L. Wiest D.R. J. Bacteriol. 1991; 173: 7098-7105Crossref PubMed Google Scholar). Total RNA from whole filaments or from isolated heterocysts was isolated in the presence of ribonucleoside-vanadyl complex as previously described (33Muro-Pastor A.M. Valladares A. Flores E. Herrero A. Mol. Microbiol. 2002; 44: 1377-1385Crossref PubMed Scopus (129) Google Scholar). For Northern analysis, 40–70 μg of RNA were loaded per lane and subjected to electrophoresis in 1% agarose denaturating formaldehyde gels. Transfer and fixation to Hybond-N+ membranes (Amersham Biosciences) were carried out using 0.1 m NaOH. Hybridization was performed at 65 °C according to the recommendations of the manufacturer of the membranes. The cph probes were internal fragments of these genes amplified by PCR, using plasmid pCSS33 as a template and oligonucleotides CA1–1 and CA1–2 (see above) in the case of the cphA1 probe; plasmid pCSS37 as a template and oligonucleotides CB1–1 (see above) and CB1–2 (complementary to nucleotides +885 to +864 with respect to the translation start of cphB1) for the cphB1 probe; plasmid pCSS53 as a template and oligonucleotides CA2–2 and CA2–3 (see above) for the cphA2 probe; and plasmid pCSS50 as a template and oligonucleotides CB2–3 (complementary to nucleotides +858 to +840 with respect to the translation start of cphB2) and CB2–4 (corresponding to nucleotides +76 to +95 with respect to the translation start of cphB2) in the case of the cphB2 probe. All probes were 32P-labeled with a Ready to Go™ DNA labeling kit (Amersham Biosciences) using [α-32P]dCTP. Images of radioactive filters and gels were obtained with a Cyclone storage phosphor system and OptiQuant image analysis software (Packard). The rnpB gene, which encodes a stable RNA (34Vioque A. Nucleic Acids Res. 1997; 25: 3471-3477Crossref PubMed Scopus (73) Google Scholar), was used as an RNA loading and transfer control. Primer extension analysis of the cph transcripts was carried out as previously described (35Muro-Pastor A.M. Valladares A. Flores E. Herrero A. J. Bacteriol. 1999; 181: 6664-6669Crossref PubMed Google Scholar) with 32P-labeled oligonucleotides CB1–6 (complementary to nucleotides -198 to -220 with respect to the translation start of cphB1) and CB1–5 (see above) for the case of cphB1 and CA1–4 (complementary to nucleotides +74 to +55 with respect to the translation start of cphA1) and CA1–5 (see above) for the case of cphA1. Cyanophycin Measurements—Cells subjected to a treatment to force cyanophycin accumulation were used. Cells of 150-ml liquid cultures in BG11 medium supplemented with 10 mm NaHCO3 and bubbled with air and CO2 (1%) were harvested in the exponential growth phase (2–5 μg of Chl ml-1), washed twice with BG110 medium, and used to inoculate 150-ml cultures in BG110 medium supplemented with bicarbonate and bubbled with air and CO2 (1%). These cultures were then incubated for about 8 h under culture conditions. After this incubation, NH4NO3 was added at 4 mm final concentration, and the cultures were incubated under dim light (5 microeinsteins m-2 s-1) for 12–14 h. Cells from 100 ml of these cultures were collected at room temperature, washed twice with, and resuspended in, milliQ-purified and autoclaved H2O, and disrupted with a French Press (twice at 20,000 p.s.i.). After measuring the obtained volume of cell extract, chlorophyll a was determined in a 100-μl sample. The remnant of each extract was centrifuged for 15 min at 15,000 rpm in a SS34 rotor, the resulting supernatants were discarded, and the pellets were washed twice with 11 ml of milliQ-purified, autoclaved H2O and resuspended in 1 ml of 0.1 m HCl. After 2–4 h of incubation at room temperature and centrifugation under the same conditions, the resulting supernatants were stored. The pellets were resuspended in 1 ml of 0.1 m HCl, incubated overnight at room temperature, and again subjected to centrifugation. The obtained supernatants were combined with those obtained after the first centrifugation, stored at 4 °C, for no longer than 1 week, and used for arginine determination, which was carried out by the Sakaguchi method as modified by Messineo (36Messineo L. Arch. Biochem. Biophys. 1966; 117: 534-540Crossref Scopus (46) Google Scholar). Microscopy—Cells grown during 7–10 days in shaken BG110 liquid medium were observed and photographed with a Zeiss Axioscop microscope equipped with an MC 80 camera. Identification of Two Clusters of Cyanophycin Metabolism Genes—In the genomic sequence of Anabaena sp. PCC 7120 (17Kaneko T. Nakamura Y. Wolk C.P. Kuritz T. Sasamoto S. Watanabe A. Iriguchi M. Ishikawa A. Kawashima K. Kimura T. Kishida Y. Kohara M. Matsumoto M. Matsuno A. Muraki A. Nakazaki N. Shimpo S. Sugimoto M. Takazawa M. Yamada M. Yasuda M. Tabata S. DNA Res. 2001; 8: 227-253Crossref Google Scholar), two clusters of ORFs showing homology to cyanophycin synthetase or cyanophycinase-encoding genes could be identified (Fig. 1). We have named cluster 1 to that containing genes more similar to those identified in other cyanobacteria (e.g. see Refs. 10Ziegler K. Diener A. Herpin C. Richter R. Deutzmann R. Lockau W. Eur. J. Biochem. 1998; 254: 154-159Crossref PubMed Scopus (155) Google Scholar, 12Richter R. Hejazi M. Kraft R. Ziegler K. Lockau W. Eur. J. Biochem. 1999; 263: 163-169Crossref PubMed Scopus (96) Google Scholar, and 37Kaneko 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 (2122) Google Scholar). Gene cphB1 would encode a cyanophycinase of 298 amino acids showing 99% identity to CphB from A. variabilis (12Richter R. Hejazi M. Kraft R. Ziegler K. Lockau W. Eu" @default.
• W2023357287 created "2016-06-24" @default.
• W2023357287 creator A5055766909 @default.
• W2023357287 creator A5059683644 @default.
• W2023357287 creator A5060098521 @default.
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• W2023357287 date "2004-03-01" @default.
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