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DNA Research Advance Access originally published online on October 17, 2006
DNA Research 2006 13(5):185-195; doi:10.1093/dnares/dsl010
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© The Author 2006. Kazusa DNA Research Institute.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Nitrogen Induction of Sugar Catabolic Gene Expression in Synechocystis sp. PCC 6803

Takashi Osanai1, Sousuke Imamura1,2, Munehiko Asayama2, Makoto Shirai2, Iwane Suzuki3, Norio Murata3 and Kan Tanaka1,*

1 Institute of Molecular and Cellular Biosciences, The University of Tokyo 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2 Laboratory of Molecular Genetics, College of Agriculture, Ibaraki University, Ami Inashiki, Ibaraki 300-0393, Japan
3 Department for Regulation Biology, National Institute for Basic Biology Myodaiji, Okazaki 444-8585, Japan

Received 7 June 2006; revised 18 September 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and Methods
 3. Results
 4. Discussion
 Acknowledgements
 REFERENCES
 
Nitrogen starvation requires cells to change their transcriptome in order to cope with this essential nutrient limitation. Here, using microarray analysis, we investigated changes in transcript profiles following nitrogen depletion in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Results revealed that genes for sugar catabolic pathways including glycolysis, oxidative pentose phosphate (OPP) pathway, and glycogen catabolism were induced by nitrogen depletion, and activities of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), two key enzymes of the OPP pathway, were demonstrated to increase under this condition. We recently showed that a group 2 sigma factor SigE, which is under the control of the global nitrogen regulator NtcA, positively regulated these sugar catabolic pathways. However, increases of transcript levels of these sugar catabolic genes under nitrogen starvation were still observed even in a sigE-deficient mutant, indicating the involvement of other regulatory element(s) in addition to SigE. Since these nitrogen activations were abolished in an ntcA mutant, and since these genes were not directly included in the NtcA regulon, we suggested that sugar catabolic genes were induced by nitrogen depletion under complex and redundant regulations including SigE and other unknown factor(s) under the control of NtcA.

Key words: cyanobacteria; sugar catabolism; NtcA; SigE; nitrogen starvation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and Methods
 3. Results
 4. Discussion
 Acknowledgements
 REFERENCES
 
Cyanobacteria performing oxygenic photosynthesis constitute one of the largest taxonomic groups of eubacteria. Although cyanobacterial species are morphologically and physiologically divergent, their fundamental metabolism is generally conserved. To maintain cellular integrity, they must coordinate their metabolisms in order to acclimate to their environmental conditions such as nutrient limitation. In the absence of combined nitrogen, diazotrophic cyanobacteria fix molecular nitrogen to avoid nitrogen deprivation.1Go Non-diazotrophic cyanobacteria including Synechocystis sp. PCC 6803, degrade their light harvesting apparatus called phycobilisome to provide nitrogen resources under nitrogen-limited conditions.2Go This phycobilisome degradation is called bleaching (or chlorosis), resulting in a change in color of cultures from blue-green to yellow. In addition to physiological changes, it has been clarified that genes for mechanisms such as nitrogen transport and nitrogen assimilation are induced under nitrogen starvation in unicellular cyanobacteria.3Go,4Go However, genome-wide transcript profiles following nitrogen depletion have not been examined in detail.

In cyanobacteria, NtcA is known as a global nitrogen regulator belonging to the CRP (cAMP receptor protein) family.5Go NtcA was discovered during genetic screening of mutants that failed to activate nitrogen assimilatory enzymes in Synechococcus sp. PCC 7942.6Go In vivo and in vitro analyses revealed that 2-oxoglutarate, which is a signaling molecule for nitrogen starvation in cyanobacteria, promotes NtcA binding to the DNA promoter of glnA (encoding type I glutamine synthetase), and in transcription initiation by RNA polymerase.7Go–9Go A DNase I footprinting study uncovered that NtcA interacted with a consensus DNA sequence containing the palindromic motif GTAN8TAC.10Go In Synechocystis sp. PCC 6803, purified NtcA binds to promoter regions of amt1 (encoding an ammonium transporter), glnA, glnB (encoding PII), icd (encoding isocitrate dehydrogenase), and sigE (encoding a group 2 sigma factor).3Go,11Go–14Go Although a completely segregated ntcA mutant of Synechocystis sp. PCC 6803 has not been obtained, a partially segregated mutant showed abolished activations of glnA, glnB and glnN (encoding Type III glutamine synthetase) promoters under nitrogen starvation.15Go

Nine genes for RNA polymerase sigma factors sigA–sigI, exist in the Synechocystis sp. PCC 6803 genome.16Go Of these sigma factor genes, sigE (sll1689) was shown to be induced by nitrogen depletion under the control of NtcA.14Go SigE is also regulated by light/dark signals,17Go and the circadian rhythm.18Go Recently, we found that SigE positively regulated expression of sugar catabolic genes such as those involved in glycolysis, oxidative pentose phosphate (OPP) pathway and glycogen catabolism.19Go For photosynthetic organisms such as cyanobacteria, sugar catabolism is essential for survival under dark conditions,20Go,21Go and consistently, a sigE mutant cannot grow under light-activated heterotrophic conditions.19Go In this study, we found that sugar catabolic pathways were activated under the control of NtcA during nitrogen depletion, and SigE and other regulatory elements simultaneously mediated their activations. The physiological significance of nitrogen activation of sugar catabolic genes is also discussed.


   2. Materials and Methods
Top
 Abstract
 1. Introduction
 2. Materials and Methods
3. Results
 4. Discussion
 Acknowledgements
 REFERENCES
 
2.1. Bacterial strains and culture conditions
The glucose-tolerant (GT) strain of Synechocystis sp. PCC 6803, isolated by Williams,22Go and the cognate sigE and ntcA mutants were grown in BG-110 liquid medium (BG-11 without nitrogen source) with 5 mM NH4Cl (buffered with 20 mM Hepes-KOH, pH 8.0). We refer to this medium as ‘modified BG-11 medium’ in this study. Liquid cultures were bubbled with 2% (v/v) CO2 in air, at 30°C under continuous white light (ca. 70 µmol photons m–2 s–1).23Go For plate cultures, BG-11 medium with 17.5 mM NaNO3 and 20 mM Hepes-KOH, pH 8.0 was solidified using 1.5% (w/v) agar (Nissui), and incubated in air containing 2% (v/v) CO2 at 30°C under continuous white light (ca. 70 µmol photons m–2 s–1). For sigE and ntcA mutants, 20 µg/ml kanamycin (SIGMA) and 80 µg/ml chloramphenicol (SIGMA) were supplemented in modified BG-11 liquid medium. Growth and cell densities were measured at A750 with a Beckman DU640 spectrophotometer.

2.2. Construction of an ntcA mutant
The ntcA (sll1423) coding region was amplified by PCR using Pfu DNA polymerase (Promega), phosphorylated by T4 polynucleotide kinase (TaKaRa), and cloned into pUC119 vectors digested with SmaI (Toyobo) using blunt end ligation (Primers: forward, 5'-CCTGAGCTAATTCTAGGC-3'; reverse, 5'-GTTGTTCAATTGAGCCC-3'). The resulting plasmids were named pNtcA. The ntcA gene was interrupted at the BsmI site (bases 392 and 506) with a chloramphenicol resistance cassette derived from pKRP10,24Go and was digested by SmaI. pUC119 vectors containing the interrupted ntcA gene were used for transformation of GT Synechocystis cells, and cells resistant to 5 µg/ml chloramphenicol were selected. After replating three times, mutant cells were maintained on plates containing 80 µg/ml chloramphenicol.

2.3. Isolation of RNA and microarray analysis
Cells of mid-exponential phase cultures of Synechocystis sp. PCC 6803 (A750, 0.5–0.7) grown in modified BG-11 medium were collected by centrifugation at 6600 g for 5 min. RNA was isolated by the acid phenol–chloroform method, as previously described.25Go Microarray analysis was performed, as described by Suzuki et al.26Go Signals were quantified using the ImaGene version 4.0 software (Bio Discovery).27Go

2.4. Northern blot analysis
Cells of mid-exponential phase cultures of Synechocystis sp. PCC 6803 (A750, 0.5–0.7) grown in modified BG-11 medium were collected by filtration with 0.45 µm MF-membrane filters (Millipore), and were resuspended in BG-110 medium. After cultivation for 0 or 4 h, cells were collected, and RNA was extracted by the acid phenol–chloroform method.25Go Methods for northern blot analysis have been described previously.28Go Gene-specific probes were constructed as described previously.19Go,29Go

2.5. Enzymatic activities of G6PD and 6PGD
Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities were measured by monitoring glucose-6-phosphate- or 6-phosphogluconate-dependent increases in NADPH A340 using a Beckman DU640, as previously described.19Go

2.6. Estimation of glycogen levels
Determination of cellular glycogen was performed as previously described.19Go In brief, about 109 cells monitored using a spectrometer were suspended in 100 µl 3.5% (v/v) sulfuric acid solution, and were boiled for 40 min. Glucose produced by acid hydrolysis was quantified using o-toluidine, following protocols by Sigma Diagnostics (procedure No. 635; Sigma).

2.7. Immunoblot analysis
Immunoblot analysis was performed as described previously.30Go Antisera against SigE and NtcA were produced by Osanai et al.19Go and Imamura et al.,31Go respectively.

2.8. Complementation of sigE and ntcA mutations
A DNA fragment containing the sigE gene was amplified by PCR using the forward primer 5'-CTAGGGTTATTGACTGATTTG-3' and the reverse primer 5'-AGCTACGCTAACTTGAAG-3'. The 5' positions of the forward and reverse primers were set to correspond to 500 bp upstream of the initiation codon and to 501 bp downstream of the termination codon, respectively. The resulting 2.1 kb amplified fragment was phosphorylated by T4 polynucleotide kinase (TaKaRa), and was inserted into the cyanobacterial autonomous replication plasmid pVZ32232Go digested with SmaI (Toyobo). Resultant complementation vectors were called pVZ322:sigE, and were introduced into sigE mutant cells using a triparental gene transfer method.32Go

Constructs for ntcA complementation were prepared as follows: pNtcA plasmids were partially digested with DraI (TaKaRa), ligated with a kanamycin cassette derived from pUC4K (Pharmacia), digested by PstI (TaKaRa), and blunt-ended. Plasmids in which the kanamycin cassette was inserted in the preferred DraI site (located 120 bp downstream of the termination codon of ntcA) of pNtcA were chosen, and used to transform an ntcA mutant. Transformants were selected on BG-11 plates containing 5 µg/ml kanamycin, and were checked for chloramphenicol sensitivity.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results
4. Discussion
 Acknowledgements
 REFERENCES
 
3.1. Sugar catabolic genes are induced by nitrogen depletion
To provide insights into transcript profiles during nitrogen starvation, we compared transcriptomes of GT cells cultivated for 0 or 4 h in nitrogen-depleted media. Total RNA extracted from both cells was utilized for microarray analysis with a gene chip containing fragments of cyanobacterial ORF [CyanoChip version 1.6 (TaKaRa)]. Of the 3076 genes represented on the array, expression levels of 306 genes were increased over 2-fold by nitrogen depletion for 4 h (Fig. 1, Supplementary Table S1). Microarray analysis demonstrated that genes for nitrogen transport (such as amt1, amt2, amt3 and nrtABCD) and nitrogen assimilation (glnA and glnN) were induced, consistent with previous studies.3Go,11Go,33Go Moreover, we found that genes encoding enzymes involved in sugar catabolism such as those acting in glycolysis, OPP pathway and glycogen metabolism, which were included in the SigE regulon,19Go were up-regulated (Table 1). Northern blot analysis confirmed that transcript levels of two glycolytic genes, glyceraldehyde-3-phosphate dehydrogenase (gap1) and pyruvate kinase (pyk1), were increased by nitrogen depletion in GT (Fig. 2A). On the other hand, expression of phosphofructokinase (pfkA; sll1196) that was also included in the SigE regulon showed little changes during nitrogen starvation. In addition to glycolytic genes, four genes for the OPP pathway, i.e. G6PD (zwf), a positive regulator of G6PD (opcA), 6PGD (gnd), and transaldolase (tal) were induced by nitrogen deprivation (Fig. 2B). Subsequently, we examined expressions of these genes in a previously constructed a sigE mutant (G50).19Go In G50, it was shown that transcript levels of sugar catabolic genes were decreased in both light and dark conditions compared with GT.19Go Northern blot analysis demonstrated that gap1, pyk1, zwf, opcA, gnd and tal were still induced regardless of sigE disruption, while pfkA (sll1196) was markedly repressed after nitrogen depletion in G50 (Fig. 2A and B). Consistent with transcriptional analyses, enzymatic activities of G6PD and 6PGD, two key enzymes of the OPP pathway, were also enhanced by nitrogen depletion in both GT and G50 (Fig. 2C and D).


Figure 1
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  		Figure 1. Genome-wide analysis of gene expression following induction or repression by nitrogen depletion. GT strain cultivated in ammonium medium was transferred into nitrogen-deprived medium by filtration. After 0 or 4 h, cells were collected, and their transcript profiles were compared. Each point represents expression ratio of 4 h/0 h after nitrogen depletion, and signal intensity in the GT strain at 0 h for an ORF fragment on the array is shown. Experiments were performed three times with biologically independent RNAs. Data from one of three independent experiments are shown.

 

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  		Table 1. Sugar catabolic genes induced by nitrogen depletion (4 h)

 

Figure 2
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  		Figure 2. Up-regulated expressions of glycolytic and OPP pathway enzymes. (A and B) Northern blot analyses of transcripts derived from three glycolytic genes (pfkA, gap1, and pyk1) or four OPP pathway genes (zwf, opcA, gnd and tal), respectively. GT or G50 cells were grown in modified BG-11, and their RNA was isolated at 0 or 4 h after a shift to nitrogen-deprived medium. Total RNA (10 µg) was then subjected to northern blot analysis with probes specific to the indicated genes. Arrows indicate the positions of the molecular size markers (in kilobases). The lower panels show rRNA stained with methylene blue as a loading control. (C and D) Enzyme activities of G6PD and 6PGD, respectively. Cells treated as described above were assayed for activities of G6PD and 6PGD. Data (means ± SD of values from four or three independent experiments, respectively) are expressed relative to the value for GT cells at 0 h after nitrogen depletion.

 
The genome of Synechocystis sp. PCC 6803 includes two genes for glycogen isoamylase (glgX), and two genes for glycogen phosphorylase (glgP). Microarray and northern blot analyses unraveled that expressions of the two glgX genes (slr1857 and slr0237) and one glgP gene (slr1367) were induced by nitrogen depletion (Table 1, Fig. 3A). In G50, glgX (slr0237) was still induced by nitrogen deprivation; however, expression of one glgX (slr1857) or the other glgP (slr1367) was not or less increased in G50 than in GT, respectively (Fig. 3A). In Synechococcus sp. PCC 7942, increased glycogen level was previously reported during nitrogen starvation.34Go Therefore, in this study, we examined glycogen accumulation in Synechocystis, and demonstrated that glycogen levels were expectedly increased by nitrogen deprivation in both GT and G50 (Fig. 3B).


Figure 3
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  		Figure 3. (A) Northern blot analysis of transcripts derived from four glycogen catabolic genes. GT or G50 cells were grown in modified BG-11, and their RNA was isolated at 0 or 4 h after a shift to nitrogen-deprived medium. Total RNA (10 µg) was then subjected to northern blot analysis with probes specific to the indicated genes. Arrows indicate positions of molecular size markers (in kilobases). The lower panels show rRNA stained with methylene blue as a loading control. (B) Analysis of intracellular glycogen amount. Amounts of glycogen in cells at 0, 4, 8 or 12 h after a shift to nitrogen-deprived medium were determined. Data (means ± SD of values from three independent experiments) are expressed relative to the value for GT cells at 0 h after nitrogen depletion.

 
In order to genetically confirm that decreased expression of sugar catabolic genes in G50 actually resulted from sigE deficiency, and not from unknown secondary mutation(s), we performed a complementation experiment with a plasmid including the wild-type sigE gene, pVZ322:sigE (Fig. 4A). After introduction of the plasmid, expression of SigE protein was confirmed by immunoblot analysis with an antiserum against SigE (Fig. 4A). Northern blot analysis showed that wild-type sigE gene restored transcript levels of sigE, zwf, gnd (Fig. 4B) and other genes (data not shown).


Figure 4
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  		Figure 4. Complementation of a sigE mutant. (A) Schematic representation of complementation vectors. A DNA fragment containing sigE was inserted into pVZ322 vector, and transformed into G50 cells. (B) Amounts of SigE proteins in GT, G50 and G50 containing pVZ322-SigE vectors. Cells were grown in modified BG-11, and proteins were obtained by disrupting cells with glass beads. Total protein (5 µg) was subjected to immunoblotting with an antiserum against SigE. (C) Northern blot analysis (10 µg RNA per lane) with probes specific to sigE, zwf and gnd. Positions of molecular size markers are indicated in kilobases.

 
3.2. Induction of sugar catabolic genes by nitrogen depletion is abolished by ntcA mutation
Data obtained with a sigE mutant indicated that other transcriptional regulators were potentially involved in induction of sugar catabolic genes during nitrogen starvation. In Synechococcus sp. PCC 7942, two-dimensional PAGE analysis suggested that most protein syntheses in response to nitrogen deprivation were dependent on NtcA either directly or indirectly.35Go Hence, we constructed an ntcA mutant of Synechocystis sp. PCC 6803 to examine the role of this central nitrogen regulator (Fig. 5A). Consistent with a previous study,15Go ntcA could not be deleted, and thus mutant cells contained both the wild-type and disrupted copies of the ntcA gene, resulting in about 50% decrease in NtcA protein compared with the parental strain (Fig. 5A). The resultant ntcA mutant was named GN20. GN20 cells grew slowly in modified BG-11 medium compared with the parental GT strain (growth rate of GN20 was about one-third to one-fourth of GT cells) (data not shown). Northern blot analyses of sugar catabolic genes gap1, pyk1, zwf, gnd, opcA, tal, glgX (slr0237) and glgP (slr1367), in GN20 showed that transcript levels were decreased even under normal growth conditions (similar to G50), but not or less increased by nitrogen depletion (different from G50), compared with GT (Fig. 5B). Since NtcA is thought to be a positive regulator of SigE,14Go we considered that decreased transcripts for sugar catabolic genes under normal growth conditions were due to reduction in SigE protein levels in GN20. Northern blot analysis and immunoblotting confirmed that both sigE transcript and protein were decreased by ntcA mutation (Fig. 6A and B). In addition, induction of SigE protein during nitrogen starvation was not observed in GN20 (Fig. 6C). On the other hand, contrary to G50, GN20 showed abolished inductions of sugar catabolic genes by nitrogen depletion (Fig. 5B). Sequence motif analyses by us (data not shown) and by others36Go did not demonstrate any NtcA-binding site within promoter regions of the corresponding sugar catabolic genes; therefore, NtcA appeared to be indirectly involved in their expression (See Discussion).


Figure 5
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  		Figure 5. Decreased inductions of sugar catabolic genes by nitrogen depletion in an ntcA mutant (GN20). (A) The ntcA gene was interrupted by a deletion with a chloramphenicol resistance (Cmr) cassette (1.0 kb) at unique BsmI sites. Orientation of the chloramphenicol resistance gene was the same as that of ntcA (data not shown). The right panel indicates amounts of NtcA protein in GT and GN20. GT and GN20 cells were grown in modified BG-11, and proteins were obtained by disrupting cells by vortexing with glass beads. Total protein (5 µg) was subjected to immunoblotting with an antiserum against NtcA. (B) Northern blot analysis of transcripts derived from glnB and sugar catabolic genes. GT or GN20 cells were grown in modified BG-11, and their RNA was isolated at 0 or 4 h after a shift to nitrogen-deprived medium. Total RNA (10 µg) was then subjected to northern blot analysis with probes specific to the appropriate genes. Arrows indicate positions of molecular size markers (in kilobases). The lower panels show rRNA stained with methylene blue as a loading control.

 

Figure 6
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  		Figure 6. Transcript and protein levels of SigE in GN20. (A) Northern blot analysis with a sigE-specific probe. GT or GN20 cells were grown in modified BG-11, and their RNA was isolated at 0 or 4 h after a shift to nitrogen-deprived medium. Total RNA (10 µg) was then subjected to northern blot analysis with a probe specific to sigE. Arrows indicate positions of molecular size markers (in kilobases). The lower panels show rRNA stained with methylene blue as a loading control. (B and C) Amount of SigE proteins in GT and GN20. GT and GN20 cells were grown in modified BG-11 (B) or nitrogen-depleted medium for indicated times (C). Proteins were obtained by disrupting the cells by vortexing with glass beads. Total protein (5 µg) was subjected to immunoblotting with an antiserum against SigE.

 
To genetically confirm that abolished inductions of sugar catabolic genes in GN20 actually resulted from ntcA deficiency, a complementation test was performed. The wild-type ntcA gene was integrated into GN20 by homologous recombination to restore the chromosomal ntcA gene. The resultant ntcA-complemented strain was named GN20Cp. Immunoblot analysis using an NtcA-antiserum demonstrated that protein level of NtcA was restored by complementation (Fig. 7A). Immunoblotting and northern blot analysis also confirmed that the wild-type ntcA gene restored SigE protein level, and induction of sugar catabolic genes under nitrogen starvation (Fig. 7B and C, and data not shown).


Figure 7
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  		Figure 7. Complementation of an ntcA mutant. (A) The ntcA gene was re-integrated within the ntcA mutant with a kanamycin resistance (Kmr) cassette (1.2 kb) at unique DraI sites. The lower panel indicates amounts of NtcA proteins in GT, GN20 and GN20Cp. The three strains were grown in modified BG-11, and proteins were obtained by disrupting cells by vortexing with glass beads. Total protein (5 µg) was subjected to immunoblotting with an antiserum against NtcA. (B) Immunoblotting with SigE antiserum. Total protein (5 µg) was subjected to immunoblotting with an antiserum against SigE. (C) Northern blot analysis of transcripts derived from sugar catabolic genes. GT, GN20 or GN20Cp cells were grown in modified BG-11, and their RNA was isolated at 0 or 4 h after a shift to nitrogen-deprived medium. Total RNA (5 µg) was then subjected to northern blot analysis with probes specific to appropriate genes. Arrows indicate positions of molecular size markers (in kilobases). The lower panels show rRNA stained with methylene blue as a loading control.

 
Since Hik8 is a histidine kinase whose mutation results in decreased transcript levels of sugar catabolic genes,37Go we also examined involvement of Hik8 in nitrogen induction. We found that although transcript levels of the examined genes were rather decreased, induced expression by nitrogen depletion was not affected by hik8 mutation as well as sigE and hik8 double mutation (Fig. 8 and data not shown). Thus, Hik8 is not responsible for the nitrogen-induced expression.


Figure 8
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  		Figure 8. Northern blot analysis of a hik8 mutant. Northern blot analysis of transcripts derived from four sugar catabolic genes. GT or hik8 mutant cells were grown in modified BG-11, and their RNA was isolated at 0 or 4 h after a shift to nitrogen-deprived medium. Total RNA (10 µg) was then subjected to northern blot analysis with probes specific to appropriate genes. Arrows indicate positions of molecular size markers (in kilobases). The lower panels show rRNA stained with methylene blue as a loading control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results
 4. Discussion
Acknowledgements
 REFERENCES
 
In this study, we analyzed transcript profiles of nitrogen-deprived cells, and revealed that amounts of mRNAs derived from genes for glycolysis, OPP pathway and glycogen catabolism were increased by nitrogen depletion in Synechocystis sp. PCC 6803 (Table 1, Figs 2A and B and 3A). Although two pfkA genes were less induced, OPP pathway genes such as zwf, opcA, gnd and tal were strongly induced by nitrogen depletion (Table 1, and Fig. 2A and B), implying that glucose was degraded mainly through the OPP pathway when nitrogen was limited, similar to heterotrophic conditions.38Go However, in the case of glycogen metabolism, not only glycogen catabolic genes such as glgX and glgP but also glycogen anabolic genes glgB (1,4-alpha-glucan branching enzyme) were induced by nitrogen depletion (Table 1, Supplementary Table S1, and Fig. 3A). Glycogen level was increased during nitrogen starvation, and hence, net flow of glycogen metabolism was that of anabolism (Fig. 3B). Thus, current data suggest that glucose is utilized for both glucose catabolism and glycogen synthesis.

Transcriptome analysis also revealed that genes for gluconeogenesis and the reductive pentose phosphate (Calvin–Benson–Basham) cycle were repressed by nitrogen deprivation (Supplementary Table S1). For example, gap2 (sll1342) (encoding glyceraldehyde-3-phosphate dehydrogenase, catalyzing only anabolic reactions39Go) and rbcL, S, X (slr0009, slr0011 and slr0012 encoding the large and small subunits and chaperone of ribulose-1,5-bis phosphate carboxylase, respectively) were repressed by nitrogen depletion. Expression ratios (GT 4 h/0 h after nitrogen depletion) were 0.45, 0.07, 0.16 and 0.19, respectively for gap2, rbcL, S, and X. (Table 1 and Supplementary Table S1). Genes for carbon concentration mechanism proteins encoded by the operon sll1028-sll1032, were also repressed by nitrogen depletion (Supplementary Table S1), and thus, inorganic carbon fixation was likely to be repressed. As a whole, net increase of cellular carbon content appeared to be arrested to compensate for the carbon/nitrogen (C/N) imbalance in response to nitrogen depletion.

Prior to this study, transcriptional interaction between glucose catabolism and nitrogen metabolism has been suggested in some cyanobacteria. In the nitrogen-fixing cyanobacterium Nostoc punctiforme strain ATCC 29133, the zwf-opcA operon involved in the OPP pathway is induced by nitrogen depletion.40Go Specific enzymatic activities for the OPP pathway were shown to increase up to 70-folds in heterocysts, that are specific cells for nitrogen fixation in filamentous cyanobacteria,41Go and it was suggested that G6PD and 6PGD produced the reducing powers for nitrogen fixation instead of the photosynthetic linear electron flow. In the unicellular cyanobacterium Synechococcus sp. PCC 7942, PSII activity rapidly declined after nitrogen starvation,34Go indicating the loss of reducing power production by photosynthesis. Thus, the OPP pathway may provide the reducing power, instead of photosynthesis during nitrogen starvation.

Synechocystis sp. PCC 6803 genomes encode each two isozymes for several sugar metabolic steps, and some of these sets are differentially regulated. In a previous study, we showed that one of two pyruvate kinase genes, pyk1 (sll0587) was positively regulated by SigE, while the other pyk2 (sll1275) was not.19Go In this study, we also found that pyk1 was induced by nitrogen depletion while pyk2 was rather repressed (Table 1, Fig. 2A). Of the two phosphofructokinase genes, only pfkA (sll1196) showed SigE-dependent expression, and required SigE to keep a constant transcript level after nitrogen depletion (Fig. 2A). In the case of two glycogen phosphorylases, one glgP (slr1367) was more induced than the other glgP (sll1356) by nitrogen depletion (Table 1, Fig. 3A), suggesting that GlgP encoded by slr1367 was the major glycogen phosphorylase under nitrogen-limited conditions. With regard to glycogen isoamylases, transcript levels of both two glgX genes were increased by nitrogen depletion (Table 1, Fig. 3A). However, induction of one glgX (slr1857) was diminished by sigE mutation, whereas the other glgX (slr0237) was still induced in a sigE mutant (Fig. 3A). Thus, these results suggested that differential regulatory pathways were necessary for maintaining metabolic integrity under various environmental conditions.

Induction of sugar catabolic genes in response to nitrogen depletion was still observed in a sigE mutant, whereas it was abolished in an ntcA mutant (Table 2). Bioinformatic analysis suggested that sugar catabolic genes are not included in the NtcA regulon.36Go Indeed, no sequence motifs recognized by NtcA were found in promoter regions of sugar catabolic genes (data not shown). Therefore, other regulator(s), which is (are) under the control of NtcA, is (are) probably involved in inductions of sugar catabolic genes during nitrogen starvation (Fig. 9). Northern blot analysis demonstrated that Hik8 was not relevant to these nitrogen-responsive inductions (Fig. 8 and data not shown). Su et al.36Go predicted that the transcriptional regulators including histidine kinase (slr1147) and three response regulators (sll1330, sll1291 and sll1624) were included in the NtcA regulon. Among them, two response regulators, i.e. sll1330 and sll1291 were actually induced by nitrogen depletion (Supplementary Table S1, and data not shown). Thus, future studies will unravel regulatory networks responsible for expressions of sugar catabolic genes under nitrogen starvation.


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  		Table 2. List of changes in transcript amounts for each gene in GT, G50 (sigE mutant) or GN20 (ntcA mutant) cells by nitrogen depletion for 4 h

 

Figure 9
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  		Figure 9. Schematic model for the regulation of sugar catabolic gene expression. SigE and Hik8 positively regulate gene expression in sugar catabolism under normal growth conditions, as previously demonstrated.19Go,37Go In this study, it was demonstrated that sugar catabolic gene expression was enhanced by nitrogen depletion. Besides SigE, other factor(s), which are under the control of NtcA, is (are) involved in catabolic gene expression during nitrogen starvation.

 
In the upstream region of gnd, we noticed a palindrome sequence AGAAATTTCT at –205 to –196 with respect to the first position of the start codon, and similar sequences, AGAAATTTTT and AAAAATTTCT, were also found in the upstream regions of zwf and opcA, respectively (data not shown). This palindrome sequence motif was not found in upstream of other sugar catabolic genes, implying this motif is for genes for the OPP pathway. Upstream region of gnd in Anabaena sp. PCC 7120 also contains AGAAATTTT (data not shown). Thus, future studies will unravel cis- and trans-factors responsible for expressions of sugar catabolic genes under nitrogen starvation in Synechocystis and other cyanobacteria.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results
 4. Discussion
 Acknowledgements
REFERENCES
 
This work was supported by a Grant-in-Aid for Creative Scientific Research (16GS0304 to K.T.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a Grant-in-Aid for Takashi Osanai for Scientific Research for Plant Graduate Student from Nara Institute of Science and Technology, supported by The Ministry of Education, Culture, Sports, Science, and Technology, Japan.

Supplementary Data:: Supplementary data are available online at http://dnaresearch.oxfordjournals.org.


   Footnotes
 
*To whom correspondence should be addressed. Tel. +81-3-5841-7825, Fax. +81-3-5841-8476, E-mail: kntanaka{at}iam.u-tokyo.ac.jp

Communicated by Satoshi Tabata


   REFERENCES
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results
 4. Discussion
 Acknowledgements
 REFERENCES
 

  1. Golden, J. W. and Yoon, H. S. 2003, Heterocyst development in Anabaena, Curr. Opin. Microbiol., 1, 623–629.
  2. Grossman, A. R., Schaefer, M. R., Chiang, G. G., Collier, J. L. 1994, The responses of cyanobacteria to environmental conditions: light and nutrients, In Bryant, D. A. (Ed.). The Molecular Biology of CyanobacteriaDordrecht Kluwer Publishers pp. 641–675.
  3. Reyes, J. C., Muro-Pastor, M. I., Florencio, F. J. 1997, Transcription of glutamine synthetase genes (glnA and glnN) from the cyanobacterium Synechocystis sp. strain PCC 6803 under nonexponential growth conditions, J. Bacteriol., 179, 2678–2689.[Abstract/Free Full Text]
  4. Suzuki, I., Horie, N., Sugiyama, T., Omata, T. 1995, Identification and characterization of two nitrogen-regulated genes of the cyanobacterium Synechococcus sp. strain PCC 7942 required for maximum efficiency of nitrogen assimilation, J. Bacteriol., 177, 290–296.[Abstract/Free Full Text]
  5. Herrero, A., Muro-Pastor, A. M., Flores, E. 2001, Nitrogen control in cyanobacteria, J. Bacteriol., 183, 411–425.[Free Full Text]
  6. Vega-Palas, M. A., Flores, E., Herrero, A. 1992, NtcA, a global nitrogen regulator from the cyanobacterium Synechococcus that belongs to the Crp family of bacterial regulators, Mol. Microbiol., 6, 1853–1859.[CrossRef][Web of Science][Medline]
  7. Muro-Pastor, M. I., Reyes, J. C., Florencio, F. J. 2001, Cyanobacterium perceive nitrogen status by sensing intracellular 2-oxoglutaraet levels, J. Biol. Chem., 276, 38320–38328.[Abstract/Free Full Text]
  8. Vazquez-Bermudez, M. F., Herrero, A., Flores, E. 2002, 2-oxoglutarate increases the binding affinity of the NtcA (nitrogen control) transcription factor for the Synechococcus glnA promoter, FEBS Lett., 512, 71–74.[CrossRef][Web of Science][Medline]
  9. Tanigawa, R., Shirokane, M., Maeda, S., Omata, T., Tanaka, K., Takahashi, H. 2002, Transcriptional activation of NtcA-dependent promoters of Synechococcus sp. PCC 7942 by 2-oxoglutarate in vitro, Proc. Natl Acad. Sci. USA, 99, 4251–4255.[Abstract/Free Full Text]
  10. Luque, I., Flores, E., Herrero, A. 1994, Molecular mechanism for the operation of nitrogen control in cyanobacteria, EMBO J., 13, 2862–2869.[Web of Science][Medline]
  11. Montesinos, M. L., Muro-Pastor, A. M., Herrero, A., Flores, E. 1998, Ammonium/methylammonium permeases of a cyanobacterium. Identification and analysis of three nitrogen-regulated amt genes in Synechocystis sp. PCC 6803, J. Biol. Chem., 273, 31463–31470.[Abstract/Free Full Text]
  12. Garcia-Dominguez, M. J. and Florencio, F. J. 1997, Nitrogen availability and electron transport control the expression of glnB gene (encoding PII protein) in the cyanobacterium Synechocystis sp. PCC 6803, Plant Mol. Biol., 35, 723–734.[CrossRef][Web of Science][Medline]
  13. Muro-Pastor, M. I., Reyes, J. C., Florencio, F. J. 1996, The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria, J. Bacteriol., 178, 4070–4076.[Abstract/Free Full Text]
  14. Muro-Pastor, A. M., Herrero, A., Flores, E. 2001, Nitrogen-regulated group 2 sigma factor from Synechocystis sp. strain PCC 6803 involved in survival under nitrogen stress, J. Bacteriol., 183, 1090–1095.[Abstract/Free Full Text]
  15. Garcia-Dominguez, M. J., Reyes, J. C., Florencio, F. J. 2000, NtcA represses transcription of gifA and gifB, genes that encode inhibitors of glutamine synthetase type I from Synechocystis sp. PCC 6803, Mol. Microbiol., 35, 1192–1201.[CrossRef][Web of Science][Medline]
  16. Kaneko, T., Sato, S., Kotani, H., et al. 1996, 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., 3, 109–136.[Abstract]
  17. Imamura, S., Asayama, M., Takahashi, H., Tanaka, K., Takahashi, H., Shirai, M. 2003, Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria, FEBS Lett., 554, 357–362.[CrossRef][Web of Science][Medline]
  18. Kucho, K., Okamoto, K., Tsuchiya, Y., et al. 2005, Global analysis of circadian expression in the cyanobacterium Synechocystis sp. strain PCC 6803, J. Bacteriol., 187, 2190–2199.[Abstract/Free Full Text]
  19. Osanai, T., Kanesaki, Y., Nakano, T., et al. 2005, Positive regulation of sugar catabolic pathways in the cyanobacterium Synechocystis sp. PCC 6803 by the group 2 {sigma} factor SigE, J. Biol. Chem., 280, 30653–30659.[Abstract/Free Full Text]
  20. Summers, M. L., Wallis, J. G., Campbell, E. L., Meeks, J. C. 1995, Genetic evidence of a major role for glucose-6-phosphate dehydrogenase in nitrogen fixation and dark growth of the cyanobacterium Nostoc sp. strain ATCC 29133, J. Bacteriol., 177, 6184–6194.[Abstract/Free Full Text]
  21. Scanlan, D. J., Sundaram, S., Newman, J., Mann, N. H., Carr, N. G. 1995, Characterization of a zwf mutant of Synechococcus sp. strain PCC 7942, J. Bacteriol., 177, 2550–2553.[Abstract/Free Full Text]
  22. Williams, J.G. K. 1988, Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803, Methods Enzymol., 167, 766–778.[CrossRef][Web of Science]
  23. Rippka, R. 1988, Isolation and purification of cyanobacteria, Methods Enzymol., 167, 3–27.[Web of Science][Medline]
  24. Reece, K. S. and Phillips, G. J. 1995, New plasmids carrying antibiotic-resistance cassettes, Gene, 165, 141–142.[CrossRef][Web of Science][Medline]
  25. Los, D. A., Ray, M. K., Murata, N. 1997, Differences in the control of the temperature-dependent expression of four genes for desaturases in Synechocystis sp. PCC 6803, Mol. Microbiol., 25, 1167–1175.[CrossRef][Web of Science][Medline]
  26. Suzuki, S., Ferjani, A., Suzuki, I., Murata, N. 2004, The SphS-SphR two component system is the exclusive sensor for the induction of gene expression in response to phosphate limitation in Synechocystis, J. Biol. Chem., 279, 13234–13240.[Abstract/Free Full Text]
  27. Kanesaki, Y., Suzuki, I., Allakhverdiev, S. I., Mikami, K., Murata, N. 2002, Salt stress and hyperosmotic stress regulate the expression of different sets of genes in Synechocystis sp. PCC 6803, Biochem. Biophys. Res. Commun., 20, 339–348.
  28. Kanamaru, K., Nagashima, A., Fujiwara, M., et al. 2001, An Arabidopsis sigma factor (SIG2)-dependent expression of plastid-encoded tRNAs in chloroplasts, Plant Cell Physiol., 42, 1034–1043.[Abstract/Free Full Text]
  29. Osanai, T., Sato, S., Tabata, S., Tanaka, K. 2005, Identification of PamA as a PII-binding membrane protein important in nitrogen-related and sugar-catabolic gene expression in Synechocystis sp. PCC 6803, J. Biol. Chem., 280, 34684–34690.[Abstract/Free Full Text]
  30. Tanaka, K., Takayanagi, Y., Fujita, N., Ishihama, A., Takahashi, H. 1993, Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli, Proc. Natl. Acad. Sci. USA, 90, 3511–3515.[Abstract/Free Full Text]
  31. Imamura, S., Tanaka, K., Shirai, M., Asayama, M. 2006, Growth phase-dependent activation of nitrogen-related genes by a control network of group 1 and group 2 sigma factors in a cyanobacterium, J. Biol. Chem., 281, 2668–2675.[Abstract/Free Full Text]
  32. Zinchenko, V. V., Piven, I. V., Melnik, V. A., Shestakov, S. V. Rus. J. Genet., 1999, 35, 228–232.
  33. Aichi, M., Takatani, N., Omata, T. 2001, Role of NtcB in activation of nitrate assimilation genes in the cyanobacterium Synechocystis sp. strain PCC 6803, J. Bacteriol., 183, 5840–5847.[Abstract/Free Full Text]
  34. Görl, M., Sauer, J., Baier, T., Forchhammer, K. 1998, Nitrogen-starvation-induced chlorosis in Synechococcus PCC 7942: adaptation to long-term survival, Microbiology, 144, 2449–2458.[Abstract/Free Full Text]
  35. Aldehni, M. F., Sauer, J., Spielhaupter, C., Schmid, R., Forchhammer, K. 2003, Signal transduction protein PII is required for NtcA-regulated gene expression during nitrogen deprivation in the cyanobacterium Synechococcus elongatus strain PCC 7942, J. Bacteriol., 185, 2582–2591.[Abstract/Free Full Text]
  36. Su, Z., Olman, V., Mao, F., Xu, Y. 2005, Comparative genomics analysis of NtcA regulons in cyanobacteria: regulation of nitrogen assimilation and its coupling to photosynthesis, Nucleic Acids Res., 33, 5156–5171.[Abstract/Free Full Text]
  37. Singh, A. K. and Sherman, L. A. 2005, Pleiotropic effect of a histidine kinase on carbohydrate metabolism in Synechocystis sp. strain PCC 6803 and its requirement for heterotrophic growth, J. Bacteriol., 187, 2368–2376.[Abstract/Free Full Text]
  38. Yang, C., Hau, Q., Shimizu, K. 2002, Integration of the information from gene expression and metabolic fluxes for the analysis of the regulatory mechanisms in Synechocystis, Appl. Microbiol. Biotechnol., 58, 813–822.[CrossRef][Web of Science][Medline]
  39. Koksharova, O., Schubert, M., Shestakov, S., Cerff, R. 1998, Genetic and biochemical evidence for distinct key functions of two highly divergent GAPDH genes in catabolic and anabolic carbon flow of the cyanobacterium Synechocystis sp. PCC 6803, Plant Mol. Biol., 36, 183–194.[CrossRef][Web of Science][Medline]
  40. Summers, M. L. and Meeks, J. C. 1996, Transcriptional regulation of zwf, encoding glucose-6-phosphate dehydrogenase, from the cyanobacterium Nostoc punctiforme strain ATCC 29133, Mol. Microbiol., 22, 473–480.[CrossRef][Web of Science][Medline]
  41. Winkenbach, F. and Wolk, C. P. 1973, Activities of enzymes of the oxidative and the reductive pentose phosphate pathways in heterocysts of a blue-green alga, Plant Physiol., 52, 480–483.[Abstract/Free Full Text]

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