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First published online January 19, 2007; 10.1104/pp.106.091736 Plant Physiology 143:1385-1397 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Carbon Cycling in Anabaena sp. PCC 7120. Sucrose Synthesis in the Heterocysts and Possible Role in Nitrogen Fixation1,[OA]Centro de Investigaciones Biológicas, Fundación para Investigaciones Biológicas Aplicadas, 7600 Mar del Plata, Argentina
Nitrogen (N) available to plants mostly originates from N2 fixation carried out by prokaryotes. Certain cyanobacterial species contribute to this energetically expensive process related to carbon (C) metabolism. Several filamentous strains differentiate heterocysts, specialized N2-fixing cells. To understand how C and N metabolism are regulated in photodiazotrophically grown organisms, we investigated the role of sucrose (Suc) biosynthesis in N2 fixation in Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120). The presence of two Suc-phosphate synthases (SPS), SPS-A and SPS-B, directly involved in Suc synthesis with different glucosyl donor specificity, seems to be important in the N2-fixing filament. Measurement of enzyme activity and polypeptide levels plus reverse transcription-polymerase chain reaction experiments showed that total SPS expression is greater in cells grown in N2 versus combined N conditions. Only SPS-B, however, was seen to be active in the heterocyst, as confirmed by analysis of green fluorescent protein reporters. SPS-B gene expression is likely controlled at the transcriptional initiation level, probably in relation to a global N regulator. Metabolic control analysis indicated that the metabolism of glycogen and Suc is likely interconnected in N2-fixing filaments. These findings suggest that N2 fixation may be spatially compatible with Suc synthesis and support the role of the disaccharide as an intermediate in the reduced C flux in heterocyst-forming cyanobacteria.
Nitrogen (N) is the fourth most abundant element in the biosphere. An estimated 80% to 90% of N available to plants in the terrestrial ecosystems originates from the biological conversion of nitrogen gas (N2) to ammonia, an energetically expensive process linked to carbohydrate metabolism (Ludden and Barris, 1986  ). There are only a few prokaryotic microorganisms, including certain free-living or symbiotically associated bacteria and cyanobacteria that carry out N2 assimilation. They do so through the action of the essentially anaerobic enzyme nitrogenase, an activity that requires reductants and ATP (Peters and Meeks, 1989; Crawford et al., 2000).
Diazotrophic cyanobacteria are the only organisms able to simultaneously and independently fix N2 and produce photosynthetic molecular oxygen. Because nitrogenase is inhibited upon exposure to oxygen, different strains have adaptations that include either temporal or spatial separation of these processes (Berman-Frank et al., 2003
Because heterocysts lack ribulose-1,5-diphosphate carboxylase, a key enzyme of the Calvin cycle, they are limited to heterotrophic metabolism and depend on vegetative cells for the generation of carbon (C) skeletons and reducing power (Wolk, 1968
Suc metabolism in Anabaena sp. PCC 7119 and PCC 7120 (also known as Nostoc sp. PCC 7120) has recently been elucidated. It has been demonstrated that Suc is synthesized through two different Suc-P synthases (SPS; EC 2.4.1.14) coupled to Suc-P phosphatase (SPP; EC 3.1.3.24). Suc can either be cleaved by SuS or irreversibly hydrolyzed by two alkaline/neutral invertases (A/N-Inv) when there is high demand for hexoses (Porchia and Salerno, 1996
Studies on the relationship between C and N metabolism in heterocyst-forming cyanobacteria have focused on the role of glycogen in N2 fixation (Ernst and Böger, 1985
The critical role of Suc in C flux modulation in the N2-fixing filaments of Anabaena sp. was recently demonstrated by Curatti et al. (2002)
The presence of two SPSs (SPS-A and SPS-B) with different glucosyl donor specificity (Porchia and Salerno, 1996
SPS Expression Is Higher in Anabaena Cells Grown in N2 Than in Combined N Conditions
To clarify the physiological role of Suc metabolism in heterocyst-forming cyanobacteria, we built on previous work analyzing the expression of enzymes involved in Suc biosynthesis. We showed that total SPS and SPP are only present in soluble protein fractions (Fig. 1
) as reported in plants (Winter and Huber, 2000
This study aimed to relate Suc synthesis to N2 fixation. The experiments were performed in illuminated cells because most N2 fixation takes place under light conditions (Stal, 2003 ). Analysis of the effect of N availability on SPS expression showed that total enzyme activity was maximal in N2-fixing cells (Fig. 2, A and C). Immunoanalysis of the SPS polypeptide level showed a similar pattern of SPS expression (Fig. 2, B and D). Enzyme activity was also higher in the presence of ADP-Glc (SPS-B activity) in cells grown in a diazotrophic versus a N combined environment (Fig. 2C). Transcriptional analysis of spsA and spsB, determined by reverse transcription (RT)-PCR, likewise showed maximal transcript level in N-deficient cells for both genes; the total amount in N-deficient cells was approximately 4 times higher than in the combined N-grown cells (Fig. 3 ). It was also found that spsB mRNA is likely to be predominant during N2 fixation. Independent of the N source, the level of sppA transcripts was in accordance with the increase in sps gene expression (Fig. 3). SPP polypeptide levels, however, were similar in the different N sources provided in the medium (Fig. 2D).
In contrast to the high Suc-synthesizing capacity seen in N2-fixing filaments, the Suc content of the N-2deficient-grown cells was approximately one-half that of the combined N-grown cells (6–10 nmol mg fresh weight–1). Because both UDP-Glc and ADP-Glc can be substrates for Anabaena SPS reactions, expression of the enzymes responsible for their synthesis was also investigated. It was seen that UDP-Glc pyrophosphorylase (UGPase; EC 2.7.7.9) activity was similar in cells grown in N2 and in combined N (approximately 55 nmol UDP-Glc min–1 mg–1), whereas a higher AGPase activity was seen in N2-fixing cells (Fig. 2E). A similar expression pattern was noted in polypeptide level and mRNA content (Fig. 2F; data not shown).
To contribute to an understanding of the interconnection between Suc synthesis and the presence of nucleoside diphosphates, we performed product inhibition assays for the two Anabaena SPSs and modeled C flux under diazotrophic conditions. The formal model was built using the Gepasi framework (Mendes, 1997
Involvement of the two SPSs in Suc production and the relationship between Suc and glycogen metabolism in Anabaena cells diazotrophically grown in the light were investigated through metabolic control analysis (Kacser and Burns, 1973
In all cases, the fluxes and steady-state concentrations of the variable metabolites calculated by the model were in agreement with experimental determinations. Negative fluxes were observed for SuS, hexose-P mutase, and AGPase. This indicated that the reactions catalyzed by these enzymes may operate in vivo in a direction opposite to that outlined in Table I. The flux through SuS should have been toward the cleavage of Suc and the flux through hexose-P mutase toward the formation of Glc-1P, a substrate for the nucleoside-triphosphate-hexose-1P nucleotidyltransferases (AGPase and UGPase). The UGPase reaction operated close to equilibrium, as consistent with previous findings (Roscher et al., 1998 ; Kleczkowski et al., 2004). The simulation showed the SPS-B flux (0.490 nmol min–1 mg fresh weight–1) to be approximately 5 times higher than the SPS-A flux. The sum of the two fluxes agrees with SPP flux, accounting for the entire Suc biosynthesis capacity. SPS-A was inhibited by UDP (approximately Ki UDP of 2.0 ± 0.5 mM). This supports the hypothesis that the preferred substrate for this enzyme is UDP-Glc. On the other hand, SPS-B was inhibited only by ADP, whether UDP-Glc or ADP-Glc (approximately Ki ADP of 1.8 ± 0.3 and 3.9 ± 0.5 mM, respectively) served as the glucosyl donor. When the SPS-A reaction was removed from the model, the net flux through SuS was interrupted (–5 x 10–11 nmol min–1 mg fresh weight–1), whereas the fluxes through invertases and UGPase remained unaltered (0.350 and 6 x 10–11 nmol min–1 mg fresh weight–1, respectively).
Immunoblot analysis and enzyme activity measurements showed that SPS is present not only in the vegetative cells, but also in the heterocysts of two Anabaena strains (Fig. 5 ). This was confirmed by incorporation of labeled C into Suc from UDP-[U-14C]Glc (approximately 3,000 cpm min–1 mg fresh weight–1) or ADP-[U-14C]Glc (approximately 2,500 cpm min–1 mg fresh weight–1) into permeabilized heterocysts in Anabaena sp. PCC 7120 in the presence of Fru-6P. Furthermore, only spsB transcripts, which are accompanied by sppA expression, were detected in the heterocysts (Fig. 6A ).
The localization of SPS-A and SPS-B in N2-fixing filament cells was also evidenced with transcriptional fusions of an optimized version of the green fluorescent protein (GFP) gene (gfp-mut2) to putative promoters of spsA, spsB, and sppA (PspsA, PspsB, and PsppA, respectively). DNA fragments of 950, 477, and 559 bp upstream from the translation start site of spsA, spsB, and sppA, respectively, directed the expression of gfp- mut2 in Anabaena sp. PCC 7120 (Fig. 6B). In diazotrophically grown Anabaena cells, spsA expression was detected only in vegetative cells (Fig. 6C, a–c), whereas spsB and sppA expression was localized to both vegetative cells and heterocysts (Fig. 6C, d–i). Anabaena sp. PCC 7120 cells harboring a plasmid (pAM1000) in which a heterocyst cell-specific promoter (PnifHDK) was fused to gfp is shown as control (Fig. 6C, j–l). No fluorescence signal was detected in Anabaena cells containing a control plasmid (pAMpmt–) carrying a promoterless gfp construct (data not shown).
The finding that only spsB is expressed in the heterocysts of N2-fixing filaments led to investigation of possible mechanisms for its transcriptional regulation. This was undertaken through determination of the RNA 5' ends that correspond to the transcription start points (tsps), mapped by primer extension of RNA obtained from Anabaena sp. PCC 7120 grown under different N conditions. Two RNA sizes (RNAI and RNAII) were observed, starting at the –54 (tspI) and –93 (tspII) nucleotides upstream of the translation initiation site (Fig. 7
). Each tsp was confirmed through similar experiments conducted with two different oligonucleotides. RNAI and RNAII were detected under the three N culture conditions assayed, but spsB expression was greater in cells grown in N2 than in combined N (Fig. 7A). The extension product of RNAII was, however, 4 times more abundant than that of RNAI in N2-fixing cells. Analysis of the upstream sequences of the two putative promoters showed a –10 box similar to those of Escherichia coli
A close relationship between Suc metabolism and the N2 fixation process has been described in legume-Rhizobium symbiosis. Degradation of Suc imported from the plant by SuS is a key initial step in the development and normal function of the nodule and maintenance of nitrogenase activity (Gordon et al., 1999 ). Suc biosynthesis, however, is unlikely to take place in the heterotrophic nodule. A different scenario of N2 fixation is the filament of diazotrophic cyanobacteria, where Suc metabolism has been described (Curatti et al., 2002) and Suc cleavage takes place only in photosynthetic vegetative cells (Curatti et al., 2006). This study focused on the fact that Suc biosynthesis occurs in N2-fixing filaments of Anabaena sp. PCC 7120, modeling the C flux between Suc and glycogen. The presence of an active SPS in the heterocysts (SPS-B) was uncovered in addition to a specific isoform in the vegetative cells (SPS-A), both of which relate to the dynamic mechanism underlying N2 fixation.
Although Suc biosynthesis is higher in diazotrophically grown filaments than in cells grown under combined N conditions (Figs. 2 and 3), a very small amount of Suc accumulation is detectable due to a high rate of disaccharide degradation (Curatti et al., 2002
Whereas the presence of one SPS gene is likely to be characteristic of unicellular cyanobacterium strains, filamentous N2-fixing cyanobacteria were reported to have two genes (spsA and spsB) present (Curatti et al., 1998
Because control of spsB expression may occur at transcriptional initiation (Fig. 7), primer extension experiments were conducted. These indicated that the putative spsB promoter inferred from tspII shares a sequence similar to that of promoters activated by NtcA. The latter is a cAMP receptor protein family transcription factor, which, in the absence of ammonium, promotes the expression of alternative N source assimilation genes (Herrero et al., 2001
Experimental data from this study point to the interconnection between glycogen and Suc metabolism in Anabaena N2-fixing filaments. Glycogen is produced and stored only during the day, serving as the predominant metabolic fuel at night (Stal and Moezelaar, 1997
Suc synthesis in the heterocyst could be paralleled to Suc synthesis in heterotrophic plant tissues. The heterocyst, however, is very distinct from cotton (Gossypium hirsutum) fibers, etiolated hypocotyls, or germinating seeds, for example, in which Suc pool regulation is modulated by simultaneous degradation and resynthesis and Suc cleavage by SuS plays a central role (Geigenberger and Stitt, 1991
Substrate cycling involving carbohydrate turnover in plant tissues has been widely reported, but its mechanisms and functions remain poorly understood (Alonso et al., 2005
Our findings are depicted in the model in Figure 8
. Suc translocated from the vegetative cells into the heterocysts may be the respiratory substrate (Wolk et al., 1994
Ongoing work is focusing on creating spsA and spsB mutant strains to further pinpoint the role of Suc biosynthesis in the N2-fixing filaments. Future studies on the role of Suc during the early and later stages of heterocyst differentiation should help to bring some light on the physiological role of Suc in the N source-dependent development of these specialized cells.
Biological Material and Culture Conditions
Axenic cultures of Anabaena sp. strain PCC 7120 and Anabaena variabilis ATCC 29413 were grown photoautotrophically under a 12-h light/12-h dark cycle with white fluorescent light at 30°C with orbital shaking. Media used for growth were BG11 (prepared with KNO3 instead of NaNO3), BG110 (BG11 medium lacking nitrate), and BG110NH4+/TES [BG11 medium lacking nitrate and supplemented with 2.5 mM NH4Cl and 5 mM N-tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid (TES-KOH buffer, pH 7.5); Curatti et al., 2002
Escherichia coli strains were grown at 37°C in Luria-Bertani liquid or agar medium supplemented with appropriate antibiotics according to standard protocols (Sambrook and Russell, 2001
Membrane and periplasm proteins were purified from 2 L of Anabaena sp. PCC 7120 cultures, according to Norling et al. (1998)
Total cell extracts from Anabaena vegetative cells and heterocysts were prepared as previously reported (Cumino et al., 2001
Because SPS-A and SPS-B show different substrate specificity (both SPSs accept UDP-Glc as the glucosyl donor, but only SPS-B uses ADP-Glc), total SPS activity was assayed in a reaction mixture containing Fru-6P and UDP-Glc, and SPS-B activity was carried out in the presence of ADP-Glc (Porchia and Salerno,1996
The Ki values for UDP and ADP (the enzyme-inhibitor dissociation constant for product-competitive inhibition) for SPS-A and SPS-B were determined with homogeneous preparations of each isoenzyme (Porchia and Salerno, 1996
Filaments of Anabaena sp. PCC 7120 grown autotrophically in BG110 or BG11 medium were permeabilized as previously described (Salerno et al., 2004
Suc and glycogen were determined in ethanol extracts as described by Geigenberger et al. (1996)
Control of the steady-state behavior of the Suc-glycogen metabolism relationship in Anabaena N2-fixing filaments was determined through metabolic control analysis, as developed by Kacser and Burns (1973)
To restrict the model to reactions directly involved in Suc synthesis and degradation, we omitted the Calvin cycle and pentose-P pathway, the major pathway of cyanobacteria Glc catabolism (Summers et al., 1995
The kinetic parameters and thermodynamic data for all enzymes used in the model are summarized in Table I. Kinetic constant values were either taken from the literature, where available, or estimated, as indicated in Table I. The reactions in this model were assumed reversible unless information on irreversibility in vivo had been reported (Rohwer and Botha, 2001
Plasmids were isolated and modified according to standard protocols (Sambrook and Russell, 2001
For RT-PCR analysis, total RNA treated with DNase (RQ1 RNase-free DNase; Promega) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Promega) and specific primers for spsA, spsB, and sppA (Cumino et al., 2001
To construct the spsA promoter::gfp (PspsA::gfp), spsB promoter::gfp (PspsB::gfp), sppA promoter::gfp (PsppA::gfp), and nifHDK promoter::gfp (PnifHDK::gfp) transcriptional reporters, three DNA fragments containing upstream sequences of the translational start codons of spsA, spsB, sppA, and nifHDK were amplified from Anabaena sp. PCC 7120 genomic DNA by PCR using the primers spsA-f (5'-GTCTTTTGATATATACTTTCTGCATAGCTT-3') and spsA-r (5'-ACCACCGGCTTCTTCTTGACCTATTTC-3'); spsB-f (5'-GCTATAGTTGTGATTTCTTACGTATAT-3') and spsB-r (5'-TGCCCTCCAGCTTCTTCTTTGCCAATTT-3'); sppA-f (5'-CCGTCGGGAATCTGAAATAAAGCGTATA-3') and sppA-r (5'-TACTCTGGTAGGTAATGATGCAGCCCTGGC-3'); and nif-f (5'-CTGAGACTGCACATCAAGGTAGAAG-3') and nif-r (5'-TGTTCTCTTTTCCTGCAATTGGTTG-3').
Amplification products were of 950, 477, 559, and 1,000 bp, respectively. Each DNA fragment was ligated to pGEM-T Easy (Promega). After digestion with EcoRI, each resulting DNA fragment was ligated into a shuttle vector derived from pAM505 and pKEN2-GFPmut2 (source of the gfp-mut2 sequence) in frame with the GFP coding sequence (Yoon and Golden, 2001
Cells grown in BG110 were used for photography. Fluorescence and phase contrast micrographs were taken on a Nikon microscope (model E600) with an X100 objective using specific emission filter sets. GFP fluorescence images were taken by illumination with light (450- to 490-nm wavelength) and photographing emission through a filter of 510-nm wavelength narrow band pass with a 3-s exposure. Red emission from photosynthetic pigments was photographed without the 510-nm filter. Images were captured with a Nikon digital camera (model E995) attached to the microscope and processed with Adobe Photoshop version 4.0 software. The presence of a thick cell envelope, changes in cytoplasm granularity, cyanophycin granulate formation at the cell poles, and absence of chlorophyll fluorescence distinguished heterocysts from vegetative cells (Elhai and Wolk, 1990
Primer extension experiments were performed according to Sambrook and Russell (2001) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AJ302071 and AJ302073.
We are grateful to Dr. James W. Golden at Texas A&M University, College Station, Texas, for facilitating our use of the vectors pAM505 and pKEN-GFPmut2; Dr. Horacio G. Pontis and our colleagues at the Centro de Investigaciones Biológicas, Fundación para Investigaciones Biológicas Aplicadas, Mar del Plata, Argentina, for many helpful discussions; and C. Fernández for technical assistance. Received October 21, 2006; accepted January 8, 2006; published January 19, 2007.
1 This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Fundación para Investigaciones Biológicas Aplicadas, and Universidad Nacional de Mar del Plata, Argentina. 
2 Present address: Pioneer Hi-Bred International (a DuPont company), 7300 N.W. 62nd Ave., Johnston, IA 50131. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Graciela L. Salerno (gsalerno{at}fiba.org.ar).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.091736 * Corresponding author; e-mail gsalerno{at}fiba.org.ar; fax 54–223–475–7120.
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ABSTRACT
RESULTS
-1,4-glucosidic chain. It is mainly regulated at the level of ADP-Glc synthesis catalyzed by ADP-Glc pyrophosphorylase (AGPase; EC 2.7.7.27), the enzyme encoded by the agp gene. AGPase activity was shown to be allosterically regulated by 3-phosphoglycerate (activator) and inorganic phosphate (Pi; inhibitor) in Anabaena sp. PCC 7120 (Ballicora et al., 2003
70 promoters (
DE3):pLysS strain (Novagen) was used for the overproduction of SPS-A and SPP proteins of Anabaena sp. PCC 7120, as previously reported by Cumino et al. (2001
-32P]ATP using T4 polynucleotide kinase (Invitrogen). cDNA products were purified and resolved on a sequencing gel along sequencing ladders generated using the same primers as those used in the primer extension experiments. Nucleotide sequencing was carried out using the dideoxy-chain termination method with a Sequenase quick-denature plasmid sequencing kit (USB Corporation).