Pathway-Level Acceleration of Glycogen Catabolism by aResponse Regulator in the Cyanobacterium SynechocystisSpecies PCC 68031[W]

Takashi Osanai, Akira Oikawa, Keiji Numata, Ayuko Kuwahara, Hiroko Iijima, Yoshiharu Doi,Kazuki Saito, and Masami Yokota Hirai*

RIKEN Center for Sustainable Resource Science, Tsurumi-ku, Yokohama, Kanagawa 230–0045, Japan (T.O.,A.O., K.N., A.K., H.I., Y.D., K.S., M.Y.H.); PRESTO, Japan Science and Technology Agency, Kawaguchi,Saitama 332–0012, Japan (T.O.); Yamagata University, Faculty of Agriculture, Wakaba-machi, Tsuruoka-shi,Yamagata 997–8555, Japan (A.O.); and Graduate School of Pharmaceutical Sciences, Chiba University,Chuo-ku, Chiba 260–8522, Japan (K.S.)

Response regulators of two-component systems play pivotal roles in the transcriptional regulation of responses to environmentalsignals in bacteria. Rre37, an OmpR-type response regulator, is induced by nitrogen depletion in the unicellular cyanobacteriumSynechocystis species PCC 6803. Microarray and quantitative real-time polymerase chain reaction analyses revealed that genesrelated to sugar catabolism and nitrogen metabolism were up-regulated by rre37 overexpression. Protein levels of GlgP(slr1367),one of the two glycogen phosphorylases, in the rre37-overexpressing strain were higher than those of the parental wild-typestrain under both nitrogen-replete and nitrogen-depleted conditions. Glycogen amounts decreased to less than one-tenth byrre37 overexpression under nitrogen-replete conditions. Metabolome analysis revealed that metabolites of the sugar catabolicpathway and amino acids were altered in the rre37-overexpressing strain after nitrogen depletion. These results demonstrate thatRre37 is a pathway-level regulator that activates the metabolic flow from glycogen to polyhydroxybutyrate and the hybridtricarboxylic acid and ornithine cycle, unraveling the mechanism of the transcriptional regulation of primary metabolism in thisunicellular cyanobacterium.

The study of carbon metabolism of photosynthetic or-ganisms including cyanobacteria is indispensable forbiology and biotechnology. Synechocystis sp. PCC 6803(hereafter Synechocystis 6803) is a non-nitrogen-fixing,unicellular cyanobacterium that performs oxygenic pho-tosynthesis. The Synechocystis 6803 genome was identi-fied in 1996, and the cells synthesize glycogen for carbonstorage through the Calvin cycle and gluconeogenesisusing CO2 and light energy (Kaneko et al., 1996). Glyco-gen synthesis is also activated during nutrient-limited con-ditions such as nitrogen starvation (Osanai et al., 2006).Glycogen is degraded to supply carbon sources and re-ductants for producing ATP by respiration under darkconditions (Osanai et al., 2007). Therefore, glycogen me-tabolism is important for the acclimation of cyanobacteriaduring environmental changes in light or nutrientconditions.

The transcriptional regulators that control genes re-lated to sugar catabolism in Synechocystis 6803 havebeen recently revealed (Osanai et al., 2007). The RNApolymerase sigma factor SigE widely regulates sugarcatabolism (Osanai et al., 2005, 2011). In particular, SigEregulates the gene expression related to glycogen ca-tabolism, including glycogen phosphorylase (encodedby glgP), isoamylase (encoded by glgX), and the oxida-tive pentose phosphate (OPP) pathway includingglucose-6-phosphate dehydrogenase (G6PD; encodedbyzwf), a positive regulator of G6PD (encoded by opcA),6-phosphogluconate dehydrogenase (6PGD; encoded bygnd), and transaldolase (encoded by tal; Osanai et al.,2005, 2011). SigE also activates polyhydroxyalkanoate(PHA) biosynthetic genes encoding b-ketothiolase(encoded by phaA), acetoacetyl-CoA reductase (encodedby phaB), and PHAsynthase (encoded by phaC and phaE)and enhances polyhydroxybutyrate (PHB) levels duringprolonged nitrogen starvation (Osanai et al., 2013).

A gene encoding the response regulator Rre37(sll1330)is induced by the depletion of nitrogen or the addition ofGlc (Osanai et al., 2006; Tabei et al., 2012). The disruptionof rre37 leads to defective growth under light-activatedheterotrophic conditions, while the mutant cells nor-mally grow under photoautotrophic conditions (Tabeiet al., 2012). Glycolytic genes including glk (encodingglucokinase), pfkA(sll1196) (encoding phosphofructoki-nase), and fbaA(sll0018) (encoding Fru-bisP aldolase) aredown-regulated by rre37 knockout, particularly under

1 This work was supported by the Ministry of Education, Culture,Sports, Science, and Technology, Japan, by the Japan Science andTechnology Agency (Grant-in-Aid to T.O.), and by Core Researchfor Evolutional Science and Technology (Grant-in-Aid to M.Y.H.).

* Address correspondence to masami.hirai@riken.jp.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Masami Yokota Hirai (masami.hirai@riken.jp).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.113.232025

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light andGlc-supplemented conditions (Tabei et al., 2007).Transcriptome analysis revealed that glgP(slr1367), glgX(slr1857), and gap1 (encoding glyceraldehyde-3-phosphatedehydrogenase) are repressed in the rre37-disruptedmutant, particularly under nitrogen-starved conditions(Azuma et al., 2011). Rre37 mediates light and nutrientsignals to control glycolytic gene expression (Tabei et al.,2009, 2012; Azuma et al., 2011); however, the role of Rre37in primary metabolism has not been examined to datebecause no metabolome analysis has been performedusing rre37 mutants.

In this study, we constructed a strain overexpressingrre37, and compared with the wild type, we observedincreased expression of genes related to sugar catabo-lism and nitrogen metabolism and decreased glycogenlevels under nitrogen-replete conditions. Metabolomeanalysis demonstrated changes in carbon metabolitesrelated to sugar metabolism and amino acids in re-sponse to the nitrogen status. With biochemical andphysiological analyses, we demonstrated that rre37overexpression accelerated sugar catabolism, PHB pro-duction, and the hybrid tricarboxylic acid and Orn cyclein Synechocystis 6803.

RESULTS

Construction of the rre37-Overexpressing Strain andMicroarray Analysis

The rre37 open reading frame (ORF) was integratedinto pTKP2031 fusedwith the psbAII promoter (encodingthe PSII D1 protein) and integrated into a neutral site ofthe Synechocystis 6803 genome; the resultant strain wasnamed ROX370 (Fig. 1A). Protein levels of Rre37 inROX370 were higher than those of the parental Glc-tolerant (GT) strain under both nitrogen-replete andnitrogen-depleted conditions (Fig. 1B).

Microarray analysis was performed to compare thetranscript profiles of GT and rre37-overexpressingstrains grown under nitrogen-replete conditions (Fig. 1C).Among the top 20 genes, mainly the expression of genesrelated to primary carbon, nitrogen, and nucleotidemetabolism was up-regulated by rre37 overexpression(Supplemental Table S1). Quantitative real-time PCRwas first performed to measure the transcript levelsof 14 genes up-regulated by rre37 overexpression(Supplemental Fig. S1). The transcript levels of fivegenes down-regulated by rre37were also measured byquantitative real-time PCR (Supplemental Fig. S2).

Quantitative real-time PCR was then performed with12 genes related to sugar catabolism (Fig. 2). It wasdemonstrated that five of the 12 genes, glgP(slr1367),pfkA(sll1196), gap1, phaA, and phaB, were up-regulatedby rre37 overexpression (Fig. 2). In contrast, four OPPpathway genes, zwf, opcA, gnd, and tal, were down-regulated in the rre37-overexpressing strain (Fig. 2).

Changes in the Expression of Sugar Catabolic Genes andPrimary Carbon Metabolism by rre37 Overexpression

Following transcriptome analysis, protein levels ofthe four glycogen catabolic enzymes [GlgP(sll1356),GlgP(slr1367), GlgX(slr0237), and GlgX(slr1857)] underboth nitrogen-replete and nitrogen-depleted conditionswere measured by immunoblotting. GlgP(slr1367) andGlgX(slr1857) levels increased as a result of rre37 over-expression under nitrogen-replete conditions (Fig. 3).GlgP(slr1367) protein levels in the rre37-overexpressingstrain were maintained at a higher level than those inthe GT strain under nitrogen starvation, whereas GlgX(slr1857) protein levels were less than those in theGT strain after 3 d of nitrogen depletion (Fig. 3). GlgP(sll1356) and GlgX(slr0237) levels were similar betweenthe two strains under nitrogen-replete and nitrogen-

Figure 1. A, Plasmid for rre37 over-expression in Synechocystis 6803. Therre37 coding region was ligated withthe psbAII promoter. A kanamycin re-sistance cassette is located upstream ofthe psbAII promoter and flanked byslr2030 and slr2031. B, Levels of Rre37proteins in GT and ROX370 cells.Immunoblotting was performed with10 mg of total protein from cells grownunder nitrogen-replete and nitrogen-depleted conditions. C, Microarrayanalysis of gene expression comparingtranscript profiles of the GT andROX370 cells. Each point representsthe ROX370-GT expression ratio, andthe signal intensity in the GT strainfor an ORF fragment on the array isshown. Experiments were performedfive times with biologically indepen-dent RNA. Data from the means of fiveexperiments are shown.

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depleted conditions (Fig. 3). Glycogen levels in therre37-overexpressing strain decreased to 7.3% of thelevels in the GT strain under nitrogen-replete conditions(Table I). Glycogen similarly increased during nitrogenstarvation in both the strains, although a 10% decreaseby rre37 overexpression was observed after 3 d of nitrogendepletion (Table I).The levels of G6PD and 6PGD proteins, the two key

enzymes of the OPP pathway, were similarly measuredby immunoblotting (Supplemental Fig. S3). G6PD pro-teins were reduced by rre37 overexpression undernitrogen-replete and nitrogen-depleted conditions (1 d),while 6PGD proteins increased under nitrogen-repleteconditions (Supplemental Fig. S3). The protein levels ofSigE decreased in the rre37-overexpressing strain undernitrogen-replete and nitrogen-depleted conditions (1 d;Supplemental Fig. S3). The protein levels of Gln synthe-tase (GS; encoded by glnA) were similar between the GTand rre37-overexpressing strains (Supplemental Fig. S3).Toquantify themetabolite levels, capillaryelectrophoresis-

mass spectrometry (CE-MS) analysiswasperformedusingcells grown under nitrogen-replete or nitrogen-depletedconditions for 4 h. A total of 104 peaks were annotatedas knownmetabolites (Supplemental Table S2). Statisticalanalysis demonstrated that 16 and 34 metabolite levels

were altered by rre37 overexpression under nitrogen-replete and nitrogen-depleted conditions, respectively.(Supplemental Table S2). After nitrogen depletion, thelevels of metabolites of sugar metabolism (Glc-6-P/Fru-6-P/Man-6-P, glucosamine-6-phosphate, N-acetyl-glucosamine-6-phosphate, and GDP-Man), the OPPpathway (sedoheptulose-7-phosphate), glycolysis (di-hydroxyacetone phosphate and 2-phosphoglycerate),and the tricarboxylic acid cycle (malate and fumarate) inthe rre37-overexpressing strain were lower than those intheGT strain (Table II). Fru-1,6-bisP/Glc-1,6-bisP increasedas a result of rre37 overexpression during nitrogen starva-tion (Table II). CE-MS analysis also revealed that the sevenpurine and pyrimidine nucleotide levels (AMP, dAMP,CMP, CTP, GMP, dTDP, and dTTP) in the rre37-over-expressing strain were higher than those in the GT strainafter 4 h of nitrogen depletion (Supplemental Table S2).

Additional CE-MS analysis was then performedusing cells grown under nitrogen-replete or nitrogen-depleted conditions for 1 d (Supplemental Table S3).A total of 70 peaks were annotated as knownmetabolites(Supplemental Table S3). The levels of several sugarmetabolites, such as 6-phosphogluconate, ribulose-5-phosphate/xylulose-5-phosphate, and raffinose, werelower in the rre37-overexpressing strain than in the

Figure 2. Quantitative real-time PCR analysis with the rre37-overexpressing strain. The relative transcript levels of genes involved inglycogen catabolism [glgP(sll1356), glgP(slr1367), glgX(slr0237), and glgX(slr1857)], glycolysis [pfkA(sll0745), pfkA(sll1196), gap1,and gap2], the OPP pathway (zwf, opcA, gnd, and tal), and PHA biosynthesis (phaA, phaB, phaC, and phaE) are shown. Datarepresent means6 SD from four to five independent experiments. Levels were calibrated relative to that of the GT strain (set at 100%).Statistically significant differences between GT and ROX370 are marked by asterisks (Student’s t test; *P , 0.05, **P , 0.005).

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GT strain after 1 d of nitrogen depletion (SupplementalTable S3). In contrast, sugar metabolites, including Glc-1,6-bisP, lactate, citrate, and GDP-Man, increased byrre37 overexpression after 1 d of nitrogen depletion(Supplemental Table S3). CE-MSalsodemonstrated thatlevels of two pyrimidine nucleotides (dTTP andUTP) inthe rre37-overexpressing strain were higher than thosein the GT strain, while levels of two purine and pyrim-idine nucleotides (AMP and CMP) were lower after1 d of nitrogen depletion (Supplemental Table S3).

Levels of 18 amino acids (Cys and Arg could not bemeasured by our analysis), Orn, and glutathione werethen quantified by gas chromatography-mass spec-trometry (GC-MS; Fig. 4; Supplemental Table S4). Asplevels increased more than three times by rre37 over-expression under nitrogen-replete conditions (Fig. 4).Compared with the GT strain, levels of eight aminoacids (Ala, Gly, Val, Leu, Ile, Met, His, and Tyr) werelower in the rre37-overexpressing strain after 4 or 24 hof nitrogen depletion (Fig. 4).

Physiological and Biochemical Experiment toDemonstrate the Involvement of Rre37 in SugarCatabolic Regulation

Sugar catabolism mutants fail to grow under mixo-trophic and heterotrophic conditions (Osanai et al.,2005, 2011; Singh and Sherman, 2005). The growth ofthe rre37-overexpressing strain was similar to that of

the GT strain under photoautotrophic and mixotrophicconditions (addition of 5 mM Glc; Fig. 5). However, therre37-overexpressing cells were not viable under con-tinuous dark conditions (for 4 d), whereas GT cellssurvived in the presence of Glc (Fig. 5).

Direct involvement of Rre37 in gene expression wasexamined in vitro. Glutathione S-transferase (GST)-tagged Rre37 proteins (GST-Rre37) were expressed inEscherichia coli and were purified by affinity chroma-tography (Supplemental Fig. S4A). Purified GST-Rre37bound with the promoter region, containing the 2200-to 21-bp region from the translation initiation site ofglgP(slr1367), unlike GST proteins (Supplemental Fig.S4B). A weak interaction of GST-Rre37 to the promoterregion of pfkA(sll1196) and phaA was observed; how-ever, no interaction was observed in the case of gap1

Figure 3. Levels of glycogen catabolicproteins. Levels of GlgP (sll1356 andslr1367) and GlgX (slr0237 andslr1857) in the GT and ROX370 strainsunder nitrogen-limited conditions wereassayed by immunoblotting (total pro-tein at 14 mg). Bands show the relativeexpression of the four proteins forthe two strains under nitrogen-repleteconditions (+N) and after 1 and 3 d ofnitrogen depletion (2N). Data repre-sent means 6 SD from three to four in-dependent experiments. Protein levelswere calibrated relative to that of theGT strain under nitrogen-replete condi-tions (set at 100%). Statistically significantdifferences between GT and ROX370are marked by asterisks (Student’s t test;*P , 0.05).

Table I. Glycogen levels of rre37 overexpression

Data represent means 6 SD from four independent experiments.Glycogen levels were calibrated relative to that of the GT strain undernitrogen-replete conditions (set at 100%). Statistically significant dif-ferences between GT and ROX370 are marked by asterisks (Student’st-test; *P , 0.05, **P , 0.005).

StrainRelative Glycogen Amount

+Nitrogen 2Nitrogen, 1 d 2Nitrogen, 3 d

GT 100 6 7.4 2,173 6 142 2,850 6 137ROX370 7.3 6 5.3** 2,054 6 163 2,556 6 67*

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(Supplemental Fig. S4B). The glgP(slr1367) transcrip-tion start site was located at 2101 bp from the trans-lation initiation site (Mitschke et al., 2011). It wasdemonstrated that GST-Rre37 bound to the region2180 to 2141 bp from the translation initiation sites,although GST-Rre37 did not bind to any 30-bp frag-ment in the 2190- to 2131-bp region (SupplementalFig. S4C). Therefore, the2180- to2141-bp region fromthe translation initiation site was required for GST-Rre37 binding (Supplemental Fig. S4D).An additional electrophoretic mobility shift assay

(EMSA) was performed using the promoter regions ofgenes whose expression was up-regulated by rre37overexpression. The results of EMSA with the pro-moter regions of seven genes showed smear bands inthe presence of GST-Rre37 (Supplemental Fig. S5),similar to the results of pfkA(sll1196) and phaA; thus, itis not clear whether GST-Rre37 binds to the promoterregions, with the exception of the glgP(slr1367) region.

Increased PHB Biosynthesis during Nitrogen Starvation byrre37 Overexpression

Following transcriptome analysis (Figs. 1C and 2),protein levels of PHA biosynthetic enzymes (PhaA,

PhaB, PhaC, and PhaE) in the rre37 overexpressionstrain were measured by immunoblotting. PhaC andPhaE levels were quantified with insoluble fractionsbecause they were integrated into the PHB super-complex (Osanai et al., 2013). PhaB protein levelsincreased by rre37 overexpression under nitrogen-replete conditions, whereas PhaA, PhaC, and PhaElevels were enhanced after nitrogen depletion byrre37 overexpression (Supplemental Fig. S6). PHBlevels after nitrogen starvation increased by rre37overexpression (Supplemental Fig. S7A). The Mr andmonomer units of PHB were not affected by rre37overexpression (Supplemental Fig. S7, B and C).

A strain overexpressing both rre37 and sigE wasgenerated to investigate the relationship betweenRre37 and SigE (Fig. 6A). Immunoblotting confirmedincreased levels of SigE and Rre37 compared with theGT strain (Fig. 6B). The transcripts of all four PHAbiosynthetic enzymes (phaA, phaB, phaC, and phaE)increased by double overexpression of rre37 and sigE(Fig. 6C). Compared with the rre37-overexpressingstrain, PHB levels were further increased by doubleoverexpression of rre37 and sigE (Supplemental Fig. S7A).The Mr and monomer units of PHB were not alteredby the double overexpression (Supplemental Fig S7,B and C).

Table II. Selected metabolite levels of primary metabolism in GT and ROX370

Data represent means 6 SD from five independent experiments. The data of the GT strain were obtainedfrom our previous study (Osanai et al., 2014). Metabolite levels were calibrated relative to that of the GTstrain under nitrogen-replete conditions (set at 100%). Statistically significant differences between GT andROX370 are marked by asterisks (Student’s t test; *P , 0.05, **P , 0.005).

Metabolites

Relative Metabolite Levels

GT ROX370

+Nitrogen 2Nitrogen, 4 h +Nitrogen 2Nitrogen, 4 h

Sugar metabolismGlc-1-P 100 6 29.1 119.1 6 27.1 87.9 6 17.8 96.6 6 20.1Glc-6-P/Fru-6-P/Man-6-P 100 6 27.5 148.2 6 12.6 60.8 6 10.8* 118.0 6 16.0*Glucosamine-6-phosphate 100 6 39.7 64.7 6 25.6 99.4 6 48.7 8.7 6 5.1**Gluconate 100 6 28.9 172.7 6 10.1 78.5 6 14.1 118.3 6 7.4**GlcNAc 100 6 35.0 57.1 6 23.8 159.0 6 15.3* 54.3 6 6.6GlcNAc-6-P 100 6 45.9 87.2 6 19.9 87.0 6 21.6 59.6 6 21.4*GDP-Man 100 6 34.7 168.0 6 12.7 88.6 6 26.5 98.1 6 12.1**

GlycolysisFru-1,6-bisP/Glc-1,6-bisP 100 6 32.4 82.2 6 6.9 111.3 6 35.7 150.9 6 18.1**Dihydroxyacetone

phosphate100 6 29.7 149.8 6 14.3 76.5 6 9.3 118.6 6 18.3*

3-Phosphoglycerate 100 6 28.6 138.6 6 21.6 112.0 6 39.2 151.4 6 31.82-Phosphoglycerate 100 6 53.8 155.0 6 27.6 107.1 6 31.9 76.3 6 13.0**Phosphoenolpyruvate 100 6 51.7 104.4 6 19.5 124.2 6 41.5 78.4 6 30.8Acetyl-CoA 100 6 38.3 83.1 6 23.6 90.8 6 30.9 108.5 6 35.7

OPP pathwaySedoheptulose-7-phosphate 100 6 35.9 121.0 6 5.8 70.3 6 12.9 107.1 6 9.6**

Tricarboxylic acid cycleCitrate 100 6 32.4 59.1 6 38.8 91.0 6 25.9 92.4 6 29.1Cis- and trans-aconitate 100 6 53.7 114.8 6 55.4 53.9 6 28.7 46.4 6 25.8Isocitrate 100 6 82.7 197.0 6 157.7 31.2 6 29.0 28.0 6 29.0Succinate 100 6 23.9 398.3 6 94.0 97.0 6 19.5 355.6 6 39.4Fumarate 100 6 57.0 1,014.8 6 93.1 146.0 6 89.3 386.7 6 58.0**Malate 100 6 40.9 875.6 6 83.4 172.3 6 83.1 372.9 6 52.6**

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DISCUSSION

In this study, rre37 overexpression altered transcriptprofiles and, particularly, activated glycogen catabolicgenes (Fig. 2; Supplemental Table S1). Glycogen levelsdecreased severely under nitrogen-replete conditionsowing to rre37 overexpression (Table I), and metabo-lites in the sugar catabolic pathway decreased after 4 hof nitrogen depletion (Fig. 7; Table II; SupplementalTable S2). Compared with the GT strain, metabolitesincluding citrate, lactate, N-acetyl-Glu, and PHB in-creased in the rre37-overexpressing strain after pro-longed nitrogen depletion (Fig. 7; Supplemental Table S3;Supplemental Fig. S7). Our results revealed that sugarcatabolism is accelerated by genetic engineering ofthe Rre37 response regulator at the pathway level inSynechocystis 6803 (Fig. 7). Rre37 is important for reg-ulating the proper distribution of carbon storage dur-ing nitrogen starvation in this cyanobacterium.

The results of this study indicate a correlation amongtranscripts, proteins, metabolites, and phenotypes. Boththe transcript andprotein levels ofglgP(sll1356) increasedby rre37 overexpression (Figs. 2 and 3), and the glyco-gen levels decreased in the rre37-overexpressing strain(Table I). Thus, glycogenphosphorylase encoded by glgP(sll1356) is a key enzyme in determining glycogen levels

in Synechocystis 6803. On the other hand, GlgX(slr1857)proteins increased as a result of rre37 overexpression(Fig. 3), whereas the transcripts decreased (Fig. 2), sug-gesting that GlgX(slr1857) is regulated at posttranscrip-tional levels. Glycogen levels decreased by less than one-tenth by rre37 overexpression (Table I); however, therewas a relatively lower induction of glycogen catabolicenzymes (Fig. 3). This pathway-level acceleration ofsugar catabolism, including the activation of glycolysis,may be a reason for decreased glycogen in the rre37-overexpressing strain. The protein levels of three PHAbiosynthetic enzymes (PhaA, PhaC, and PhaE) were en-hanced by rre37 overexpression after nitrogen depletion(Supplemental Fig. S6), which is positively correlatedwith the increase in PHB levels (Supplemental Fig. S7).Compared with the wild type, Fru-1,6-bisP levels werehigher in the rre37-overexpressing strain after nitrogendepletion (Table II; Fig. 7). The transcription of pfkA(sll1196) is activated by Rre37 (Fig. 2), possibly leading tothe accumulation of Fru-1,6-bisPs in the rre37-over-expressing strain. Phosphofructokinase is known to cat-alyze one of the rate-limiting steps in glycolysis (Boscaand Corredor, 1984), and our metabolome analysisalso suggests that phosphofructokinase is a key enzymein glycolysis in this cyanobacterium. The dark-sensitive

Figure 4. Levels of 18 amino acids, Orn, and glutathione. Data represent means 6 SD from four independent experiments.Levels were calibrated relative to that of the GT strain (set at 100%). Statistically significant differences between GT andROX370 are marked by asterisks (Student’s t test; *P , 0.05, **P , 0.005).

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phenotype of the rre37-overexpressing strain (Fig. 5)seems to be due to aberrant sugar catabolism, whichhas been demonstrated by several mutants in Syne-chocystis 6803 (Osanai et al., 2005, 2011; Singh andSherman, 2005; Kahlon et al., 2006; Nagarajan et al.,2012).

Redundant glycogen catabolic and glycolytic genesare present in the Synechocystis 6803 genome. The twoglycogen phosphorylase genes glgP(sll1356) and glgP(slr1367) are present in Synechocystis 6803, and Rre37 se-lectively up-regulates glgP(slr1367) (Fig. 2; SupplementalTable S1). In contrast, SigE preferentially activated glgP(sll1356) expression (Osanai et al., 2011); therefore, thetwo glgPs are differentially regulated by two transcrip-tional regulators. Biochemical analysis revealed thatGST-Rre37was directly associatedwith the glgP(slr1367)promoter (Supplemental Fig. S4B), although the bindingsequencewasnot clearly identified. In thenitrogen-fixingcyanobacterium Anabaena sp. PCC 7120, an ortholog ofRre37, named NrrA, bound to the promoter regions ofnitrogen-fixing and sugar catabolic genes [includingCTT(A/G)AT(G/T)T] (Ehira and Ohmori, 2006, 2011). A sim-ilar AT-rich tandem repeat of octamers T(C/T)AAC(T/A)(G/A)T occurswithin the promoter region of glgP(sll1356) (Supplemental Fig. S4D), and further analysis isrequired todetermine theRre37-binding sequences. rre37overexpression up-regulated the glycolytic genes pfkA(sll1196) and gap1, whereas theOPPpathwaygeneswererepressed (Fig. 2). SigE particularly activated gene ex-pression in the OPP pathway at the mRNA, protein, andenzymatic activity levels (Osanai et al., 2011); therefore,the balance of Rre37 and SigE protein levels may deter-mine which Glc degradation pathway (either glycolysisor the OPP pathway) is activated. Rre37 preferentiallyactivates the expression of phaA and phaB, whereas SigEspecifically up-regulates the expression of phaC and phaE(Osanai et al., 2013; Fig. 2), indicating that multipletranscriptional regulatorsare involved inPHBbiosynthesis.

Figure 5. Growth on a modified BG-11 plate. Each spot contains 2 mLof culture diluted to A730 = 1, 0.5, or 0.2. The cells were grown with orwithout 5 mM Glc under continuous light conditions for 3 d (top) orunder continuous dark for 4 d and light for 5 d (bottom).

Figure 6. Construction of the strain overexpressing both rre37 and sigE. A, Plasmid for sigE overexpression in Synechocystis 6803. The sigEcoding region was ligated with the psbAII promoter. A gentamycin resistance cassette is located upstream of the psbAII promoter andflanked by the sll0945ORF. B, SigE and Rre37 protein levels in theGT strain and the rre37/sigE double-overexpressing strain. C, Quantitativereal-time PCR analysis with the rre37/sigE double-overexpressing strain. The relative transcript levels of genes involved in PHA biosynthesis(phaA, phaB, phaC, and phaE) are shown. Data represent means 6 SD from five to six independent experiments. Levels were calibratedrelative to that of the GT strain (set at 100%). Statistically significant differences are marked by asterisks (Student’s t test; **P , 0.005).

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The network of sigma factors in Synechocystis, which isstudied by many researchers (Foster et al., 2007; Pollariet al., 2008, 2011), may also be important for regulatingprimary metabolism.

In addition to sugar catabolism, Rre37 plays pivotalroles in nitrogen metabolism. The levels of Asp increasedby rre37 overexpression under nitrogen-replete condi-tions (Fig. 4), which may be due to the up-regulation ofthe expression of aspC (encoding Asp aminotransferase),indicating the positive correlation between transcriptsand metabolites related to Asp biosynthesis (Fig. 7;Supplemental Fig. S1). The data combining tran-scriptome and metabolome analyses suggest the activa-tion of nitrogen assimilation by the GS-Glu synthase cycleand carbamoyl phosphate synthase, constituting “thehybrid tricarboxylic acid and ornithine cycle” duringnitrogen depletion (Fig. 7). This hybrid tricarboxylic acidand Orn cycle is likely to increase ammonium assimilationduring early nitrogen starvation because a single round of

this cycle assimilates two molecules of ammonium ionsand generates an Arg, which could be used for storingnitrogen (Fig. 7). Future metabolomic analysis is requiredto support the rationale of metabolic flow.

CE-MS analysis revealed that several purine andpyrimidine nucleotides increased as a result of rre37overexpression under nitrogen-replete or early nitrogen-starved conditions (Supplemental Table S2). Transcrip-tome analysis revealed that the expression of carA andcarB, encoding a subunit of a carbamoyl phosphatesynthase, which catalyzed the first reaction of pyrimidinebiosynthesis from Gln, increased by rre37 overexpression(Supplemental Table S1; Supplemental Fig. S1). Car-bamoyl phosphate is used to produce orotate via threeenzymatic reactions, and orotate and 5-phospho-a-D-ribosyl 1-pyrophosphate produce orotidine-5-phosphate,which is a precursor of UMP. The levels of 5-phospho-a-D-ribosyl 1-pyrophosphate were not increased in therre37-overexpressing strain (Supplemental Tables S2 and

Figure 7. Schematic model of the metabolic changes by rre37 overexpression. Metabolites that increased and decreased byrre37 overexpression under nitrogen-depleted conditions (4 h) are marked with red and blue, respectively. Glycogen partic-ularly decreased under nitrogen-replete conditions, and PHB increased after prolonged nitrogen starvation by rre37 over-expression. DHAP, Dihydroxyacetone phosphate; 2-OG, 2-oxoglutarate; 2-PG, 2-phosphoglycerate; TCA, tricarboxylic acid.

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S3); therefore, the reason for increased pyrimidinenucleotides may be the increased supply of carbamoylphosphate as a result of rre37 overexpression.

CONCLUSION

We demonstrated that the metabolic flow fromglycogen to PHB or the hybrid tricarboxylic acid andOrn cycle was regulated at the pathway level by Rre37.The study of global regulators of primary metabolismis useful for basic science to help better understand theregulation of primary metabolism and possibly forapplied science to increase interest in metabolites suchas bioplastics and biofuels in the future.

MATERIALS AND METHODS

Bacterial Strains and Culture Conditions

Among the GT strains of Synechocystis sp. PCC 6803 (isolated by Williams[1988]), the GT-I strain was used in this study (Kanesaki et al., 2012). The cells weregrown in modified BG-11 medium (Rippka, 1988), which is BG-110 liquid mediumcontaining 5 mM NH4Cl (buffered with 20 mM HEPES-KOH, pH 7.8). Liquidcultures were bubbled with 1% (v/v) CO2 in air at 30°C under continuous whitelight (approximately 50–70 mmol photons m–2 s–1). Growth and cell densities weremeasured at A730 using the Hitachi U-3310 spectrophotometer.

Plasmid Construction for rre37 Overexpression

A region of the Synechocystis 6803 genome encoding the rre37(sll1330) ORFwas amplified by PCR using KOD polymerase (Toyobo) and the specific primers59-ATTATTCATATGAATCCAGTCTACATA-39 and 59-AAAGGGGTTAACC-TAGGTAAGTACAGAGAC-39. The amplified PCR fragments were digestedwith NdeI and HpaI (Takara Bio) and inserted into the NdeI-HpaI sites of thepTKP2031V vector (Osanai et al., 2011) using the DNA Ligation Kit (Takara Bio).The resultant plasmid was confirmed by sequencing. The plasmids were inte-grated into the GT-I strain by natural transformation as described previously(Osanai et al., 2011). The rre37-overexpressing strain was named ROX370.

RNA Isolation

Cells were suspended in 400 mL of TES buffer (5 mM Tris-HCl, pH 8,0.5 mM EDTA, and 0.5% (w/v) SDS) followed by the addition of 400 mL ofacid-phenol. After incubation for 10 min at 65°C, the mixtures werecentrifuged at 20,500g for 5 min and the supernatants were transferred to a1.5-mL tube. A 400-mL aliquot of phenol:chloroform:isoamyl alcohol(25:24:1) was then added, and the tubes were centrifuged for an additional 3min. RNAs were concentrated by isopropanol precipitation, followed byadding 100 mL of TE buffer (10 mM Tris-HCl, pH 8, and 1 mM EDTA). Fol-lowing this, 1 mL of Isogen (Wako) was added, and the tubes were incu-bated for 5 min at room temperature, followed by centrifugation for 15 min.The supernatants were transferred to a 1.5-mL tube, topped up with 400 mLof phenol:chloroform:isoamyl alcohol (25:24:1), and centrifuged at 20,500gfor 3 min. Eight extractions with phenol:chloroform:isoamyl alcohol wereperformed in total. RNAs were resuspended in 20 mL of sterilized waterafter isopropanol precipitation.

Microarray Analysis

Residual DNA was excluded by reaction with TURBO DNase (Life Tech-nologies Japan) for 2 h at 37°C. Microarray analysis was performed asdescribed previously (Osanai et al., 2011). RNA (24 mg) was used for com-plementary DNA synthesis using the RNA Fluorescence Labeling Core Kit(Takara Bio). Signals were quantified using ImaGene software version 4.0(BioDiscovery). Data were deposited to the Gene Expression Omnibus publicdatabase (accession no. GSE47091).

Quantitative Real-Time PCR

Residual DNA was excluded by reaction with TURBO DNase for 5 h at37°C. Complementary DNA was synthesized from 2 mg of total RNA usingthe SuperScript III First-Strand Synthesis System (Life Technologies Japan).Quantitative real-time PCR was performed using StepOne Plus (Life Tech-nologies Japan), according to the manufacturer’s instruction, using the primerslisted in Supplemental Table S5. The expression level of rnpB (encodingRNaseP subunit B) was used as an internal standard as described previously(Schlebusch and Forchhammer, 2010).

Production of Antisera and Immunoblotting

We previously produced antisera against Rre37, GlgX(slr0237), GlgX(slr1857), GlgP(sll1356), GlgP(slr1367), PhaA, PhaB, PhaC, PhaE, and SigE(Azuma et al., 2011; Osanai et al., 2011, 2013). Cells were dissolved in 0.5 mLof PBS-T (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, and0.05% (w/v) Tween 20, pH 7.4) supplemented with the protease inhibitorComplete Mini (Roche Diagnostics; one tablet per 10 mL in PBS-T). Cells weredisrupted by sonication using the Bioruptor UCD-250 (CosmoBio) instrument.Following centrifugation at 20,500g for 5 min, 300 mL of the supernatant wasmixed with 100 mL of the SDS-PAGE sample buffer (250 mM Tris-HCl, pH 6.8,20% (w/v) 2-mercaptoethanol, 8% (w/v) SDS, 20% (w/v) Suc, and 1% (w/v)bromphenol blue) and boiled for 4 min at 98°C. The centrifuged pellet wasresuspended in 300 mL of PBS-T with 0.5% (w/v) Tween 20 (Wako) for in-soluble fraction immunoblotting. In total, 200 mL of the suspension was mixedwith 200 mL of water and 400 mL of SDS-PAGE sample buffer, followed by4 min of boiling at 98°C. Protein concentrations were measured using the BCAProtein Assay Reagent (Thermo Scientific) with bovine serum albumin as astandard. Immunoblotting was performed as described previously (Osanaiet al., 2014). Proteins were visualized using one-step nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Thermo Scientific) by alkaline phos-phatase reaction. Bands were quantified with NIH ImageJ software.

Glycogen Measurement

Glycogen levels were measured by the Biotechnology Center of AkitaPrefectural University. Cells grown under nitrogen-replete or nitrogen-depleted conditions were concentrated in 1 mL of methanol with A730 = 6,followed by mixing for 10 min using a vortex mixer. After centrifugation, thesupernatant was transferred to a 1.5-mL tube and dried at 65°C. The driedglycogen was enzymatically degraded with glucoamylase, and the resultantGlcs were quantified by measuring changes in A340 during reactions withhexokinase and G6PD.

CE-MS Analysis

Cells were collected by centrifugation at 9,800g for 2 min, followed byfreezing in liquid nitrogen. Cells (50–100 mg fresh weight) were suspendedin 600 mL of a mixture containing 60% (v/v) methanol and 200 mM each10-camphorsulfonic acid and trimesic acid as internal standards, and it wasmixed using an MT-200 microtube mixer (Tomy) for 20 min at room tem-perature, followed by centrifugation at 20,500g for 5 min at 4°C. A 300-mLaliquot of the supernatant was centrifuged through a Millipore 5-kD cutofffilter at 10,000g for 90 min. A 250-mL aliquot of the filtrate was dried in acentrifugal concentrator (drying time, 120 min). The residue was dissolved in20 mL of water and subjected to CE-MS analysis. The CE-MS system andconditions were as described previously (Oikawa et al., 2011).

Amino Acid Analysis with GC-MS

Equal amounts of cells (50 mL of cell culture with A730 = 1) were harvestedby rapid filtration using a previously described method (Osanai et al., 2014). Intotal, 300 mL of the upper phase was transferred to a new tube and vacuumdried. Samples were suspended in 500 mL of methanol with 1 mM nor-Val as aninternal standard and derivatized using the EZ:faast kit (Phenomenex;Badawy et al., 2008). GC-MS was performed using a GCMS-QP2010Plus(Shimadzu) equipped with a 10-m 3 0.25-mm ZB-AAA capillary gas chro-matography column. The injection volume was 1 mL at a carrier gas flow of1.17 mL min21 helium with a split ratio of 1:15. The initial column oventemperature of 110°C was maintained for 1 min and raised to 300°C at

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20°C min21. The interface and ion source temperatures were 280°C and 240°C,respectively.

Purification of GST-Tagged Rre37 and EMSA

The region of the Synechocystis 6803 genome encoding rre37 was amplifiedby PCR using KOD polymerase and the specific primers 59-GAAGGT-CGTGGGATCATGAATCCAGTCTACATA-39 and 59-GATGCGGCCGCTC-GAGCTAGGTAAGTACAGAGAC-39. The amplified DNA fragments weredigested with BamHI and XhoI and inserted into the BamHI-XhoI sites ofpGEX5X-1 (GE Healthcare Japan) using the In-Fusion HD Cloning Kit (TakaraBio). Purification was performed as described previously (Osanai et al., 2009).

Double-stranded DNAs were synthesized by PCR (for 200-bp fragments) orannealing of synthesized primers (for 30- or 40-bp fragments) with the primerslisted in Supplemental Table S5. PCR was performed with KOD plus neopolymerase (Toyobo), and purification from agarose gels was performed usingthe Wizard SV Gel and PCR Clean-up System (Promega). The primers (final10 pmol mL21 for each primer) were suspended in TE buffer (10 mM Tris-HCland 1 mM EDTA) with 100 mM NaCl for annealing and incubated for 10 min at96°C, followed by switching off the incubator to gradually bring the tem-perature down to room temperature.

The double-stranded DNAs and GST-Rre37 proteins were mixed in 15 mLof EMSA buffer (20 mM Tris-HCl, pH 8, 50 mM NaCl, 10 mM MgCl2, 1 mM

dithiothreitol, and 4% (w/v) glycerol) and incubated for 20 min at roomtemperature. After adding 1.5 mL of loading buffer (40% (w/v) Suc, 0.02%(w/v) xylene cyanol, and 0.02% (w/v) bromphenol blue), the mixtures wereresolved by acrylamide gel electrophoresis in 0.53 TBE buffer (44.5 mM Tris,44.5 mM borate, and 1 mM EDTA) using precast gels (SuperSepAce 5%–20%(w/v); Wako). DNA bands were visualized by ethidium bromide and UVillumination.

Extraction, Purification, and Analysis of PHB

Precultured cells were diluted to A730 = 0.2 in 70 mL of BG-110 mediumcontaining 3 mM NH4Cl. Cells were cultured for 9 d and collected by cen-trifugation at 18,000g for 2 min. Cells were freeze dried with FDU-2200(EYELA) at 280°C for 3 d. Extraction, quantification, and determination ofthe Mr and monomer units of PHB have been described (Osanai et al., 2013).

Construction of a Strain Overexpressing Both rre37and sigE

First, the kanamycin resistance cassette of pTKP2031Vwas deleted by digestionwith XhoI and AatII (Takara Bio). The gentamycin resistance cassette frompVZ322 (Zinchenko et al., 1999) was amplified by PCR using KOD polymeraseand the specific primers 59-AAATTTCTCGAGTGTAAGCAGACAGTTTTA-39and 59-AAACCCGACGTCTGTTAGGTGGCGGTACTT-39, digested with XhoIand AatII, and inserted into the XhoI-AatII sites of pTKP2031V. The resultantplasmid was named pTGP2031. A region of the Synechocystis 6803 genome en-coding one of two glycogen synthases, glgA(sll0945), including +1 to +1,180 bp fromthe translation initiation codon was amplified by PCR using KOD polymerase andthe specific primers 59-TTCCGCATGCATGAAGATTTTATTTGTGGC-39 and59-TTAAGAATTCCATTGATAGGATCGTAGAA-39. The amplified PCR frag-ments were digested with SphI and EcoRI (Takara Bio) and inserted into the SphI-EcoRI sites of the pUC119 vector (Takara Bio). The resultant plasmid was digestedwith ApaI, and the region including the gentamycin resistance cassette, psbAIIpromoter, and NdeI-HpaI cloning sites of pTGP2031 was amplified using KODpolymerase and the specific primers 59-TTTGCTTCATCGCTCGAG-39 and59-ATCCAATGTGAGGTTAAC-39 and integrated into theApaI site of the plasmid.The resultant plasmid was named pTGP0945. The sigE coding region was obtainedby PCR as described previously (Osanai et al., 2011), except for the reverse primer(59-AAAGGGGTTAACCTATAACCAACCTTTGAG-39), which was newly synthe-sized and contained the HpaI site. The amplified sigE coding region digested withNdeI and HpaI was integrated into the NdeI-HpaI sites of pTGP0945 (Fig. 6A). Trans-formation was performed as well with pTKP2031V-rre37, except that 3 mg mL21

gentamycin was used instead of 50 mg mL21 kanamycin.

Statistical Analyses

Statistical analyses were performed using StatPlus:macLE software forMacOSX (Analyst Soft) or Microsoft Excel version 14.3.2 (Microsoft). P values

were determined using paired two-tailed Student’s t tests. A 95% confidenceinterval was used to determine significance.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Results of quantitative real-time PCR 1.

Supplemental Figure S2. Results of quantitative real-time PCR 2.

Supplemental Figure S3. Levels of G6PD, 6PGD, SigE, and GS.

Supplemental Figure S4. EMSA analysis 1.

Supplemental Figure S5. EMSA analysis 2.

Supplemental Figure S6. Levels of PHA biosynthetic proteins.

Supplemental Figure S7. Levels of PHB.

Supplemental Table S1. Results of microarray analysis.

Supplemental Table S2. Results of CE-MS analysis (4 h after nitrogendepletion).

Supplemental Table S3. Results of CE-MS analysis (1 d after nitrogendepletion).

Supplemental Table S4. Levels of amino acids.

Supplemental Table S5. Primers used for quantitative real-time PCR.

Received November 4, 2013; accepted February 10, 2014; published February12, 2014.

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