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Originally published In Press as doi:10.1074/jbc.M505043200 on June 8, 2005

J. Biol. Chem., Vol. 280, Issue 35, 30653-30659, September 2, 2005
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Positive Regulation of Sugar Catabolic Pathways in the Cyanobacterium Synechocystis sp. PCC 6803 by the Group 2 {sigma} Factor SigE*{boxs}

Takashi Osanai{ddagger}, Yu Kanesaki{ddagger}, Takayuki Nakano{ddagger}, Hiroyuki Takahashi{ddagger}, Munehiko Asayama**, Makoto Shirai**, Minoru Kanehisa§, Iwane Suzuki¶, Norio Murata¶, and Kan Tanaka{ddagger}||

From the {ddagger}Institute of Molecular and Cellular Biosciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, **College of Agriculture, Ibaraki University, Ami, Inashiki, Ibaraki 300-0393, §Institute for Chemical Research, Kyoto University, Uji 611-0011, and the Department of Regulation Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan

Received for publication, May 9, 2005 , and in revised form, June 1, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sigE gene of Synechocystis sp. PCC 6803 encodes a group 2 {sigma} factor for RNA polymerase and has been proposed to function in transcriptional regulation of nitrogen metabolism. By using microarray and Northern analyses, we demonstrated that the abundance of transcripts derived from genes important for glycolysis, the oxidative pentose phosphate pathway, and glycogen catabolism is reduced in a sigE mutant of Synechocystis maintained under the normal growth condition. Furthermore, the activities of the two key enzymes of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phophogluconate dehydrogenase, encoded by the zwf and gnd genes were also reduced in the sigE mutant. The dark enhancements in both enzyme activity and transcript abundance apparent in the wild type were eliminated by the mutation. In addition, the sigE mutant showed a reduced rate of glucose uptake and an increased intracellular level of glycogen. Moreover, it was unable to proliferate under the light-activated heterotrophic growth conditions. These results indicate that SigE functions in the transcriptional activation of sugar catabolic pathways in Synechocystis sp. PCC 6803.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyanobacteria, which constitute one of the largest taxonomic groups of eubacteria, perform oxygenic photosynthesis similar to higher plants and algae. Despite the diversity in their morphology, physiology, and cellular development, all cyanobacteria are able to assimilate inorganic carbon via the reductive pentose phosphate cycle by using light energy. Moreover, in the absence of light, cyanobacteria utilize assimilated organic carbon, and some species of cyanobacterium are even capable of utilizing exogenous organic carbon. The cyanobacterium Synechocystis sp. PCC 6803 grows photoautotrophically under the light conditions and survives in the dark by catabolizing storage carbohydrates such as glycogen. Although the original isolate of this strain was not able to grow in the presence of glucose, a glucose-tolerant (GT)1 mutant was subsequently isolated (1). In the presence of glucose, this GT strain can grow mixotrophically under the light conditions as well as heterotrophically in the dark if provided with a daily pulse of white light. The latter type of growth is referred to as light-activated heterotrophic growth (LAHG) (2).

Similar to other eubacteria, the genome of cyanobacteria encodes multiple {sigma} factors for RNA polymerase. These factors recognize gene promoters and determine the specificity of transcription by RNA polymerase. In general, eubacterial {sigma} factors are classified into three categories. Group 1 {sigma} factors are unique to each strain and correspond to the essential principal {sigma} factor. Group 2 {sigma} factors show high sequence similarity to group 1 {sigma} factors. They also exhibit overlapping promoter recognition specificity with group 1 {sigma} factors but are nonessential for growth. Group 3 includes all other types of {sigma} factors. The existence of multiple {sigma} factors is thought to allow switching of gene expression patterns in response to changes in the environment or developmental stage (3).

In comparison to other eubacteria, cyanobacteria have unusually large number of group 2 {sigma} factors (4). Group 2 {sigma} factors of cyanobacteria were first discovered in Anabaena sp. PCC 7120, Synechococcus sp. PCC 7942, Synechococcus sp. PCC 7002, and Microcystis sp. (5–8). Genome analyses of Synechocystis sp. PCC 6803 and subsequent cyanobacterial genome projects revealed at least four group 2 {sigma} factors in each strain (9). A similar number of group 2 {sigma} factors had only been found previously in Actinomycetes (10). In Anabaena sp. PCC 7120, the genes for the group 2 {sigma} factors SigB and SigC are induced by nitrogen or sulfur deprivation, suggesting that these factors play a role in adaptation to nutrient limitation (5). In Synechococcus sp. PCC 7002, the abundance of the sigB transcript is increased by either nitrogen or carbon depletion, whereas that of the sigC transcript initially increased and then declined under the same conditions (7). Moreover, SigE of Synechococcus sp. PCC 7002 was shown to be responsible for transcription of the dpsA gene, which encodes a nucleoid protein, during the stationary phase of growth (11). In the case of nitrogen-fixing cyanobacteria, SigH of Nostoc punctiforme is implicated in symbiosis with plant hosts (12). SigD, SigE, and SigF of Anabaena sp. PCC 7120 contribute to cellular differentiation under diazotrophic growth conditions (13). Four group 2 {sigma} factors, RpoD2, RpoD3, RpoD4, and SigC, of Synechococcus sp. PCC 7942 are important for circadian rhythmicity of transcription (14, 15).

Four group 2 {sigma} factors, SigB, SigC, SigD, and SigE, have been identified in Synechocystis sp. PCC 6803. The expression patterns of sigB and sigD differ during the dark to light transition. Both sigB and sigD transcripts are also induced under various stress conditions (16, 17). SigC activates transcription of glnB, which encodes the carbon-nitrogen sensor PII, in the stationary phase (18, 19). Transcription of sigE increases by nitrogen depletion in a manner dependent on the global nitrogen regulator NtcA (20). Disruption of sigE results in a loss of viability under conditions of nitrogen deprivation as well as in a reduction in the expression of glnN (20), which encodes a type III glutamine synthetase. However, expression of most NtcA-dependent genes is not affected by the sigE mutation, and the relation between SigE and nitrogen regulation is still unclear. Moreover, the abundance of SigE is affected by the light to dark transition (16), suggesting that this {sigma} factor is important in these aspects of cell physiology that are unrelated to nitrogen availability.

In the present study, we investigated the targets of the regulatory function of SigE in Synechocystis sp. PCC 6803 and found that this {sigma} factor plays an important role in the regulation of sugar catabolic pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Culture Conditions—The GT strain of Synechocystis sp. PCC 6803 (1) and its derivatives were used in this study. Cells were grown in BG-11 medium containing 10 or 5 mM NH4Cl as a nitrogen source for plates and liquid culture, respectively. Medium was also supplemented with 20 mM HEPES-KOH (pH 8.0). The cells were cultured under continuous white light (~70 µmol photons m–2 s–1) at 30 °C in an atmosphere of 2% (v/v) CO2. Liquid cultures were aerated with the same gas mixture (21). The dark condition was achieved by wrapping culture vessels with aluminum foil. For LAHG, BG-11 plates further containing 5 mM glucose were incubated in the dark with a daily pulse of white light for 15 min. Cell growth and density were determined by measurement of OD750 with a spectrophotometer (Beckman model DU640). For the construction of a sigE mutant, the sigE (sll1689) coding region of Synechocystis sp. PCC 6803 was isolated by digestion of M13 phage clone ps00320736 (CyanoBase positions 1,301,322–1,303,755) (22) with PvuII and was cloned into the HincII site of pUC118 (Takara). The gene was interrupted at the unique BglII site by insertion of a BamHI kanamycin resistance cassette derived from pUC4K (Amersham Biosciences). The resulting construct was used to transform the GT strain. Colonies resistant to kanamycin (5 µg/ml) were selected, and isolation of a single colony was repeated three times. The disruption of sigE was confirmed by PCR with specific primers (Table SI) and also by immunoblot analysis with specific antiserum. The hik8 mutant was as described previously (23).

Isolation of RNA and Microarray Analysis—Cells of mid-exponential phase cultures of Synechocystis sp. PCC 6803 (OD750, 0.5–0.7) grown in BG-11-based medium were collected by centrifugation at 6,600 x g for 5 min. RNA was isolated from the cells by the previously described acid phenol-chloroform method (24). DNA microarray analysis was performed with CyanoChip version 1.6, which contained PCR fragments of full length or each 1 kbp of the COOH-terminal part of the ORFs (Takara), as described previously (23). Labeling of cDNA with Cy3-dUTP or Cy5-dUTP (Amersham Biosciences) was performed with an RNA fluorescence labeling core kit (Moloney murine leukemia virus version) version 2.0 (Takara). After hybridization, the microarray was rinsed with 0.2x SSC and then scanned with an array scanner (GMS418, Affymetrix) (18). Signals were quantified and analyzed with ImaGene version 4.0 software (BioDiscovery) as described previously (25). Three biologically independent microarray experiments were performed, and similar results were obtained.

Northern Blot Analysis—Total RNA was extracted using the same method as for microarray analysis, and Northern blot analysis was performed as described previously (26). Gene-specific probes were constructed with specific primers (Table SI) and Synechocystis genomic DNA as template (27).

Assay of Glucose Uptake—Cells were collected at mid-exponential phase by centrifugation at 17,400 x g for 1 min and were resuspended in BG-11 supplemented with 5 mM NH4Cl and 2 mM glucose to obtain an OD750 of 1.0. Glucose uptake was assayed by measurement of the concentration of glucose in the medium with a Glucose CII kit (Wako Pure Chemicals). Portions (100 µl) of the culture were harvested every 2 h and centrifuged at 17,400 x g for 1 min, followed by the determination of glucose concentration of the resulting supernatant.

Analysis of G6PD and 6PGD Activities—Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities were measured by monitoring the glucose 6-phosphate- or 6-phosphogluconate-dependent increase in NADPH concentration at A340 with a Beckman DU640 spectrophotometer. For assay of G6PD activity, cells were suspended in 1 ml of buffer A (55 mM Tris-HCl (pH 8.0), 3.4 mM MgCl2) and disrupted by sonication. The lysate was centrifuged at 17,400 x g for 5 min, and the resulting supernatant was used for the enzyme assay. The reaction was initiated at 25 °C in a mixture containing 45 mM Tris-HCl (pH 8.0), 2.8 mM MgCl2, 1 mM NADP+, and 10 mM glucose 6-phosphate, and the change in A340 was monitored for 1 min. As a control, the change in A340 in the absence of glucose 6-phosphate was measured and subtracted from the experimental values. The activity of 6PGD was measured according to the protocol provided by Oriental Yeast Co. Ltd. with some modifications.

Determination of Glycogen Abundance—Assay of intracellular glycogen was performed as described previously (28) with some modifications. Cells (~6 x 108) were suspended in 100 µl of 3.5% (v/v) sulfuric acid and boiled for 40 min. Glucose produced by acid hydrolysis was quantified with the use of o-toluidine according to the protocol recommended by Sigma.

Production of Rabbit Antiserum to SigE and Immunoblot Analysis— The ORF of sigE was amplified by PCR with specific primers (Table SI) and cloned into pET21b (Novagen) with the use of the 5' NdeI and 3' XhoI sites included in the primers. After confirming the structure by sequencing, the resulting plasmid was used to transform Escherichia coli BL21 (DE3), and expression of the hexahistidine-tagged recombinant SigE protein was induced in cells cultivated at 37 °C in LB medium by exposure to 1 mM isopropyl-{beta}-D-thiogalactopyranoside (Wako Pure Chemicals) for 2 h. Cells were harvested from 500-ml cultures by centrifugation (2,400 x g for 10 min), resuspended in 25 ml of sonication buffer (50 mM sodium phosphate (pH 8.0), 300 mM NaCl), and disrupted by sonication. The SigE protein was present in inclusion bodies and was collected by centrifugation of the crude extract at 10,000 x g for 15 min. The resulting pellet was washed once with 25 ml of sonication buffer containing 4% (v/v) Triton X-100, dissolved in 5 ml of denaturation buffer (6 M guanidine hydrochloride, 0.1 M sodium phosphate, 0.01 M Tris-HCl (pH 8.0)), incubated at 37 °C for 1 h, and centrifuged again at 10,000 x g for 15 min to remove debris. The recombinant protein was purified from the resulting supernatant by affinity chromatography with nickel-nitrilotriacetic acid-agarose (Qiagen). After application of the sample and washing of the column with denaturation buffer, proteins were eluted with a solution containing 8 M urea, 0.1 M sodium phosphate, and 0.01 M Tris-HCl and a pH gradient of 8.0 to 4.5. SigE was eluted at pH 4.5, and its abundance was quantified with a Bio-Rad protein assay. The purified protein (1 mg) was mixed with the same amount of Freund's complete adjuvant (Sigma) and injected into a rabbit for the generation of polyclonal antibodies. Immunoblot analysis with the resulting antiserum was performed as described previously (29).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Microarray Analysis for Identification of Potential SigE Targets—To provide insight into SigE function, we constructed a targeting vector by inserting a kanamycin resistance cassette into the BglII site of the gene, and we used the resulting construct to transform the wild-type (GT) strain of Synechocystis sp. PCC 6803 (Fig. 1A). The disruption of endogenous sigE in the transformed strain, designated G50, was confirmed by PCR (Fig. 1B) and immunoblot analysis with antiserum specific for SigE (Fig. 1C). The growth of G50 under photoautotrophic or photomixotrophic conditions did not appear to differ from that of the wild type (data not shown).

To compare the transcriptomes of GT and G50, we extracted total RNA from both strains cultured under the normal growth condition and processed it for microarray analysis with a gene chip including fragments of cyanobacterial ORF (Fig. 1D). Of the 3,076 genes represented on the array, the expression level of 67 genes was reduced over 2-fold by the sigE mutation (Table SII). The down-regulated genes included several genes whose products contribute to sugar catabolism, including enzymes that participate in glycolysis, the oxidative pentose phosphate (OPP) pathway, or glycogen breakdown (Table I). These results thus suggest that the expression of these genes is dependent on SigE.


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  		TABLE I
Genes whose products contribute to sugar catabolism and whose expression was affected by the sigE mutation

Data represent the ratio of the transcript level in G50 to that in wild-type cells. The array included at least two spots for each ORF.

 



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  		FIG. 1.
Disruption of sigE in Synechocystis sp. PCC 6803 and its effect on the transcriptome. A, The sigE gene was interrupted by insertion of a kanamycin resistance (Kmr) cassette (~1.2 kbp) at a unique BglII site. The orientation of the kanamycin resistance gene was opposite to that of sigE. B and C, disruption of sigE in the insertion mutant (G50) was confirmed by PCR with specific primers indicated in A and by immunoblot analysis with antiserum to SigE, respectively. The wild-type arrows in B indicate the specific PCR products. GT strain was analyzed for comparison. D, comparison of gene expression patterns of the sigE mutant and the wild-type strain by microarray analysis. Each point represents the G50/GT expression ratio and the signal intensity in G50 for an ORF fragment on the array. Experiments were performed three times with biologically independent RNAs.

 
Positive Regulation of Glycolysis by SigE—The genes whose expression was reduced by the sigE mutation and whose products contribute to glycolysis included those for phosphofructokinase (pfkA (sll1196)), glyceraldehyde-3-phosphate dehydrogenase (gap1), and pyruvate kinase (pyk1) (Table I). Northern hybridization confirmed that the abundance of transcripts of these three genes was reduced in G50 cells under the normal growth condition (Fig. 2A). The genome of Synechocystis sp. PCC 6803 contains two structural genes for each of these enzymes. Phosphofructokinase and pyruvate kinase catalyze irreversible reactions in glycolysis. Induction of the expression of either of the two structural genes for each of these enzymes would be expected to increase glycolytic flux. Of the two genes for glyceraldehyde-3-phosphate dehydrogenase, gap1 encodes an NAD+-dependent enzyme that has been shown to be responsible for the glycolytic reaction (30). Induction of transcription of pfkA (sll1196), gap1, and pyk1 by SigE thus likely increases the rate of glycolysis in Synechocystis sp. PCC 6803.

Given that sugar catabolism is especially important in the absence of light, we also examined the effects of a shift from light to dark on the amounts of the transcripts of these three genes. Northern blot analysis revealed that, for GT cells, the abundance of pyk1 transcripts was increased slightly, whereas that of pfkA (sll1196) and gap1 transcripts remained unchanged 1 h after the dark shift. However, the amount of pyk1 transcripts was decreased slightly and that of pfkA (sll1196) and gap1 transcripts was reduced markedly after 4 h (Fig. 2A). Consistent with a role for SigE in regulation of the expression of these genes, in G50, although a slight induction of pyk1 expression was observed after the dark shift, the transcript levels of all three genes remained reduced compared with those in the wild type (Fig. 2A).

Positive Regulation of the OPP Pathway by SigE—Microarray analysis suggested that transcription of OPP pathway genes was reduced by the sigE mutation (Table I). Northern blot analysis confirmed that under the normal growth condition and after the dark shift the abundance of transcripts derived from gnd (which encodes 6PGD, a key enzyme of the OPP pathway) and derived from tal (which encodes transaldolase, another enzyme of the OPP pathway) was greatly reduced in G50 cells compared with GT cells (Fig. 2B). The amounts of transcripts derived from zwf (which encodes G6PD, another key enzyme of the OPP pathway) and from opcA (which encodes a protein that is conserved only among cyanobacteria and is required for oligomerization of G6PD) were also reduced by the sigE mutation under both light and dark conditions (Fig. 2B). Consistent with these differences in transcript levels, the activities of both G6PD and 6PGD were decreased by the sigE mutation under the normal growth condition and after the dark shift (Fig. 2, C and D). In GT cells, the enhancement in the activities of these enzymes 4 h after dark shift was also reduced (G6PD) or actually reversed by the sigE mutation (Fig. 2, C and D). These results thus indicate that metabolic flux through the OPP pathway is down-regulated in G50 cells.



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  		FIG. 2.
Down-regulation of the expression of glycolytic and OPP pathway enzymes in G50 cells. A and B, Northern blot analysis of transcripts derived from genes that contribute to glycolysis (pfkA (sll1196), gap1, and pyk1) or to the OPP pathway (zwf, opcA, gnd, and tal), respectively. GT or G50 cells were grown under the normal growth condition, and RNA was isolated either before or 1 or 4 h after a shift from light (L) to dark. Total RNA (10 µg) was then subjected to Northern analysis with probes specific for the indicated genes. The positions of molecular size markers (in kilobases) are indicated. 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 the activities of G6PD and 6PGD. Data are expressed relative to the value for GT cells under the normal growth condition and are means ± S.D. of values from three independent experiments.

 
Reduced Rate of Glucose Uptake in the sigE Mutant—Current observations indicate that glycolysis and the OPP pathway are positively regulated by SigE, suggesting that the rate of glucose catabolism might be reduced by the sigE mutation. To examine this possibility, we cultured GT and G50 in medium supplemented with glucose, and we measured the rate of glucose uptake by the cells. As predicted, in comparison to wild type, the rate of glucose uptake in G50 cells was reduced (~20–30%) (Fig. 3), suggesting that the rate of glucose catabolism is indeed reduced by the sigE mutation.

Positive Regulation of Glycogen Catabolism by SigE—Microarray analysis suggested that the abundance of transcripts derived from the genes for glycogen isoamylase (glgX (slr0237)) and glycogen phosphorylase (glgP (sll1356)) was reduced by the sigE mutation (Table I). In eubacteria, most glycogen is degraded through the action of glycogen phosphorylase. However glycogen isoamylase is also required for the complete digestion of glycogen (31). The genome of Synechocystis sp. PCC 6803 contains two glgX and two glgP genes, and we examined the abundance of the transcripts of all four genes by Northern analysis. Consistent with the microarray data, the amounts of glgX (slr0237) and glgP (sll1356) transcripts were reduced in G50 cells under the normal growth condition (Fig. 4A). The sigE deficiency did not markedly affect the levels of glgX (slr1857) and glgP (slr1367) transcripts under the normal growth condition. However, the amount of glgP (slr1367) transcripts in the dark condition was reduced in G50 compared with that in GT cells (Fig. 4A). We further examined whether these effects of the sigE mutation resulted in a difference in the extent of glycogen accumulation between GT and G50 cells. Under the normal growth condition, the amount of glycogen in G50 was greater than that in GT cells (Fig. 4B). Transfer of both strains to the dark resulted in reduction in the amount of glycogen. However, the rate of glycogen utilization was reduced by ~10–20% in the sigE mutant (Fig. 4B). These results indicated that glycogen catabolism was reduced by the sigE mutation.



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  		FIG. 3.
Reduced rate of glucose uptake in G50 cells. GT or G50 cells in mid-logarithmic phase were suspended in BG-11 supplemented with 10 mM NH4Cl and 2 mM glucose, and the decrease in the glucose concentration of the medium was determined at the indicated times thereafter. Data are means ± S.D. of values from three independent experiments.

 
Impaired Heterotrophic Growth and Dark Survival of the sigE Mutant—Finally, we examined G50 cells for phenotypes related to sugar catabolism. GT and G50 cells were grown to mid-logarithmic phase, and serial dilutions of the cultures were then spotted onto plates supplemented with 5 mM glucose and were incubated under the LAHG condition. In contrast to GT cells, G50 cells were incapable of proliferation under this condition (Fig. 5A). We also compared the abilities of the two strains to survive in the dark by spotting them onto plates supplemented with glucose as for the LAHG condition and then incubating them in the dark for 4 days. The cells were then exposed to light, and their growth was monitored. The appearance of visible colonies was markedly delayed by the sigE mutation (Fig. 5B). Although it is unclear whether this effect of the mutation was because of a loss of cell viability or to lengthening of the latent growth period, both phenotypes are consistent with the defect in sugar catabolism in G50 cells.

Regulatory Relationship between SigE and Hik8 —Very recently, it has been reported that a histidine kinase Hik8 (Sll0750) positively regulates the expression of sugar catabolic and anabolic genes in Synechocystis sp. PCC 6803 (32). They also showed that transcript levels of cph1 (encoding a cyanobacterial phytochrome (slr0473)) and rcp1 (encoding a response regulator cotranscribed with cph1 (slr0474)) were decreased by the hik8 disruption (32). In the current investigation, microarray analysis also suggested the SigE dependence of these gene expressions (Table SII), which was further confirmed by Northern hybridization analysis (Fig. 6A). Because target genes of Hik8 were partially identical to those of SigE, we examined the regulatory relationship between SigE and Hik8. The result of microarray analysis indicated that the hik8 transcript levels were almost identical in GT and G50 cells (expression ratio (G50/GT) was 0.976; Table SII), suggesting that Hik8 expression is not under the control of SigE. We also found that the amount of SigE was not affected by the hik8 mutation (Fig. 6B), indicating that Hik8 is not required for the SigE expression. Thus, Hik8 and SigE presumably activate these gene transcriptions independently or cooperatively.



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  		FIG. 4.
Down-regulation of the expression of glycogen catabolic enzymes in G50 cells. A, Northern blot analysis of transcripts derived from glycogen catabolic genes. GT or G50 cells were grown under the normal growth condition, and RNA was isolated either before or 1 or 4 h after a dark shift. Total RNA (10 µg) was subjected to Northern analysis with probes specific for the indicated genes. The positions of molecular size standards (in kilobases) are indicated. L, light. B, analysis of intracellular glycogen abundance. The amount of glycogen in cells under the normal growth condition or 1, 4, or 6 h after a dark shift was determined. Data are expressed relative to the value for GT cells under the normal growth condition and are means ± S.D. of values from three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the current investigation, microarray and Northern analyses have revealed that the abundance of transcripts derived from genes important for glycolysis, the OPP pathway, and glycogen catabolism is decreased in a sigE mutant (G50) of Synechocystis sp. PCC 6803 (Fig. 7). Furthermore, the rate of glucose utilization was reduced, and glycogen accumulation was increased in the mutant cells, indicating that SigE positively regulates sugar catabolism in Synechocystis.

The expression of various genes other than those important for sugar catabolism was also reduced in G50 cells compared with the parental GT strain. It is therefore possible that the changes in expression of some of these other genes contribute to the inability of the mutant cells to grow under the LAHG condition and/or to the increased sensitivity of the cells to the dark. However, in Nostoc sp., mutants deficient in the OPP pathway have been shown previously to be unable to grow under the heterotrophic condition (33) and to manifest a decreased viability after incubation in the dark in Synechococcus sp. PCC 7942 (34). It is thus likely that the defect in the OPP pathway in G50 cells may be partly responsible for these growth phenotypes of the sigE mutant. Indeed, the OPP pathway was shown to be a major route of glucose catabolism under the heterotrophic condition in Synechocystis sp. PCC 6803 (35), and the major role of the OPP pathway is to provide a reductant (NADPH) under this growth condition.



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  		FIG. 5.
Phenotypic effects of the sigE mutation. A, cultures of GT or G50 cells grown to mid-exponential phase were spotted onto BG-11 plates containing both 10 mM NH4Cl as a nitrogen source and 5 mM glucose. Each spot consisted of 1 µl of culture diluted to an OD750 of 0.1, 0.05, or 0.01, as indicated. The plates were incubated in the dark with the exception of exposure to light for 15 min each day. Plates were photographed at the indicated times after inoculation. B, cells were spotted onto plates as in A, incubated in continuous darkness for 4 days (no light stimulation), and then transferred to the light. Plates were photographed at the indicated times after the shift from dark to light.

 



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  		FIG. 6.
A, Northern blot analysis of transcripts derived from cph1-rcp1 genes. GT or G50 cells were grown under the normal growth condition, and RNA was isolated either before or 1 or 4 h after a dark shift. Total RNA (10 µg) was subjected to Northern analysis with probes for cph1. The positions of molecular size standards (in kilobases) are indicated. L, light. B, the amount of SigE proteins in GT and hik8 mutant. GT and hik8 mutant cells were grown at normal growth condition, and proteins were obtained by disrupting the cells by sonication. Total protein (5 µg) was subjected to immunoblotting with antiserum to SigE.

 



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  		FIG. 7.
Glycolysis, gluconeogenesis, the OPP pathway, and glycogen metabolism in Synechocystis. The depicted scheme was predicted from the data in Ref. 35 and the KEGG data base (www.genome.jp/kegg/pathway.html). Genes whose transcripts were reduced in abundance by the sigE mutation are shown in boldface. The asterisk indicates that the effect of the sigE mutation on the expression of slr1367 was apparent only after a dark shift. G1P, glucose-1-phosphate; G6P, glucose-6-P; 6PGL, 6-phosphogluconolactone; 6PG, 6-phosphogluconate; F6P, fructose-6-P; F1,6P2, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3-P; DHAP, dihydroxyacetone-P; G1,3P2, 1,3-bisphosphoglycerate; G3P, 3-phosphoglycerate; G2P, 2-phosphoglycerate; PEP, phosphoenolpyruvate; PYK, pyruvate; RU5P, ribulose-5-P; R5P, ribose-5-P; S7P, sedoheptulose-7-P; X5P, xylulose-5-P; E4P, erythrose-4-P.

 
Although SigE is a transcriptional regulator implicated in the activation of sugar catabolic pathways in Synechocystis, transcription of these sugar catabolic genes was still apparent and, in some cases, was still increased by a dark shift even in the absence of SigE, suggesting that other transcriptional regulators also participate in the control of their expression. The Hik8 that was recently found to control common regulatory targets with SigE (32) is a probable candidate for such regulation (Fig. 8), and this kind of complexity may increase the metabolic versatility of cyanobacteria.

Some genes for glycolytic and glycogen catabolic enzymes are present in two copies in the genome of Synechocystis sp. PCC 6803. However, with the exception of the two glyceraldehyde-3-phosphate dehydrogenases, the functional differences between the encoded isozymes have not been clarified. Although Gap1 catalyzes the glycolytic reaction, Gap2 catalyzes the reverse reaction in gluconeogenesis (30). We discovered that SigE activates the transcription of gap1 but not that of gap2 (Table I), suggesting that SigE contributes only to catabolism, not to anabolism, of glucose. There are also two phosphofructokinase genes (sll0745 and sll1196) and two pyruvate kinase genes (sll0587 and sll1275) in Synechocystis sp. PCC 6803, and the transcription of only one of each of these pairs of genes (sll1196 and sll0587) was decreased by the sigE mutation (Table I and Fig. 2A, and data not shown). These results thus suggest that members of each of these pairs of isozymes are deployed preferentially under specific physiological conditions. With the use of in silico analysis, Mrazek et al. (36) predicted that each of the two phosphofructokinase genes and the two pyruvate kinase genes of Synechocystis sp. PCC 6803 would be expressed at different levels on the basis of their codon usage, which may also imply the functional difference.



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  		FIG. 8.
Schematic model for the SigE function. SigE and Hik8 positively regulate gene expression for sugar catabolism (including glycolysis, the OPP pathway, and glycogen catabolism) and cph1-rcp1 probably under control of the diurnal rhythm. In addition Hik8 but not SigE positively regulates sugar anabolic genes. Activation by these components could be cooperative or independent.

 
Global analysis of the circadian pattern of gene expression in Synechocystis sp. PCC 6803 (37) recently revealed that transcription of sigE manifests robust circadian oscillation, peaking around the end of the day (circadian time 9–10 h). The expression of sugar catabolic genes, including pfkA (sll1196), gap1, zwf, opcA, gnd, tal, and glgX (slr0237), was also found to peak between the end of the day and the start of the night (circadian time 11–14 h). These findings are thus consistent with our results showing that expression of these sugar catabolic genes is dependent on SigE.

In this study, we additionally analyzed the regulatory position of Hik8 that was recently shown to be involved in the activation of common genes with SigE (32). Hik8 is an ortholog of SasA of Synechococcus sp. PCC7942 that is essential to sustain the robust circadian oscillation (32, 38). Considering the circadian oscillation of SigE and the corresponding target genes, we suspected some regulatory interactions between SigE and Hik8. It was speculated that SigE regulates Hik8 or vice versa. However, this was not the case (Fig. 6B), and the relationship is summarized in Fig. 8. Further analysis should be required to clarify the regulatory circuits.

SigE was first recognized as a nitrogen-regulated {sigma} factor, and its expression was suggested to be under the control of the global nitrogen regulator NtcA (20). However, the present results suggest that SigE is unlikely to contribute directly to the regulation of nitrogen metabolism. A requirement for the OPP pathway during nitrogen deficiency has been suggested previously in nitrogen-fixing cyanobacteria (39, 40), and the activities of G6PD and 6PGD are actually increased by nitrogen depletion in Synechocystis sp. PCC 6803.2 SigE might thus play a role in communication between carbon and nitrogen metabolism.


   FOOTNOTES
 
* This work was supported in part by Grants-in-aid for Scientific Research on Priority Areas "Genome Biology" 13206011 and for Creative Scientific Research 16GS0304 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Tables SI and SII. Back

|| To whom correspondence should be addressed: Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Tel.: 81-3-5841-7825; Fax: 81-3-5841-8476; E-mail: kntanaka{at}iam.u-tokyo.ac.jp.

1 The abbreviations used are: GT, glucose-tolerant; LAHG, light-activated heterotrophic growth; G6PD, glucose-6-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; OPP, oxidative pentose phosphate; ORF, open reading frame. Back

2 T. Osanai, Y. Kanesaki, T. Nakano, H. Takahashi, M. Kanehisa, I. Suzuki, N. Murata, and K. Tanaka, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Poonam Bahatia for editing the manuscript.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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