Light-induced gene expression of fructose 1,6-bisphosphate aldolase during heterotrophic growth in a cyanobacterium, Synechocystis sp PCC 6803 Yosuke Tabei, Katsuhiko Okada, Nobuaki Makita and Mikio Tsuzuki School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Japan Keywords cyanobacteria; glucose degradation; light; signal transduction; Synechocystis Correspondence K Okada, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Tokyo 192-0392, Japan Fax: +81 42 676 6721 Tel: +81 42 676 6716 E-mail: kokada@ls.toyaku.ac.jp (Received August 2008, revised 18 October 2008, accepted 30 October 2008) doi:10.1111/j.1742-4658.2008.06772.x Synechocystis sp PCC 6803 exhibits light-activated heterotrophic growth (LAHG) under dark conditions with glucose as a carbon source The light activation is remarkable at a late period of photoautotrophic preculture, such as the late-linear and stationary growth phases To understand the physiological effects of light irradiation and glucose under LAHG conditions, their effects on the expression of soluble proteins were analyzed by means of 2D-PAGE Various soluble proteins, which were minimal under photoautotrophic preculture conditions, were observed clearly under LAHG conditions, suggesting that proteins were synthesized actively under these conditions Fructose 1,6-bisphosphate aldolase, one of the glycolytic enzymes, was found to be induced under LAHG conditions on 2D-PAGE The activity of fructose 1,6-bisphosphate aldolase, which had decreased during photoautotrophic preculture, also increased under LAHG conditions, similar to the mRNA level of the encoding gene, fbaA In addition, we found that a deletion mutant of sll1330, a putative gene containing a helix-turn-helix DNA-binding motif, could not grow under LAHG conditions, whereas it could grow photoautotrophically The increases in the protein level of FbaA and fbaA gene expression observed in wild-type cells under LAHG conditions were greatly inhibited in the deletion mutant These results suggest that the regulation of fbaA gene expression by way of sll1330 is one of the important processes in Synechocystis sp PCC 6803 under light pulse LAHG conditions In photosynthetic organisms, light activates various signal transduction pathways regulating the growth rate, the expression of genes involved in various metabolic processes and the activation of enzymes related to energy processes In higher plants, various phenomena concerning photogermination, phototropism and photoperiodism, including flower initiation, are regulated by light as a signal via phytochromes and ⁄ or other photoreceptors [1,2] In cyanobacteria, resetting of the circadian rhythm [3] and phototaxis [4] have been analyzed at the gene expression level In the light, cyanobacteria assimilate CO2 via the reductive pentose phosphate pathway using ATP and NAD(P)H generated through photosynthesis to produce glycogen and other organic compounds In the dark, glucose residues derived from glycogen are catabolized via glycolysis, the oxidative pentose phosphate pathway (OPPP) and the incomplete tricarboxylic acid cycle, leading to the production of NAD(P)H and biosynthetic intermediates for maintenance and growth [5,6] Both Abbreviations EMSA, electrophoretic mobility shift assay; FBA, fructose 1,6-bisphosphate aldolase; G6PD, glucose 6-phosphate dehydrogenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GK, glucokinase; HTH, helix-turn-helix; LAHG, light-activated heterotrophic growth; OPPP, oxidative pentose phosphate pathway; PGI, glucose 6-phosphate isomerase; PK, pyruvate kinase FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS 187 Activation of expression of fbaA in Synechocystis Y Tabei et al metabolic processes occur in the same compartment in prokaryotic cells [7] Synechocystis sp PCC 6803 (hereafter referred to as Synechocystis), a unicellular cyanobacterium, is a useful model species for research on biological responses to the environment because its whole genome sequence has been determined [8] and molecular techniques are available, including transformation and homologous recombination Exhaustive analyses of gene expression (such as of the transcriptome and proteome) are now possible for this organism For example, new insights into the role of thioredoxin in the regulation of cellular processes and the function of the cell membrane in cyanobacteria have been obtained through proteomic analysis [9] Moreover, Synechocystis can grow heterotrophically on glucose Light irradiation, however, is required for heterotrophic growth, which occurs even with a low light intensity at which photosynthesis can scarcely proceed The cells can grow even with occasional light pulses (e.g for every 24 h) [10] Therefore, so far, this phenomenon has been designated as light-activated heterotrophic growth (LAHG) Although hik8 and sigE have been reported to be necessary for LAHG [11,12], the exact mechanism underlying LAHG has not yet been determined Genome sequencing for several cyanobacteria has revealed the presence of all the genes required for glucose metabolism, and biochemical evidence of the functional roles of the gene products has been obtained [8,13] The genes and ⁄ or enzymes involved in glycolysis and the OPPP, as studied to date in some detail in cyanobacteria, include glucose 6-phosphate dehydrogenase (G6PD) [14], 6-phosphogluconate dehydrogenase [15], glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) [16–18], pyruvate kinase (PK; EC 2.7.1.40) [19], phosphoenolpyruvate carboxylase [20], fructose 1,6-bisphosphate aldolase (FBA; EC 4.1.2.13) [21] and phosphofructokinase [6,22] However, the regulation of glucose metabolism under dark conditions and the coordination of its control in cyanobacteria remain poorly understood It is important to note that the reductive pentose phosphate pathway as well as the glycolysis and gluconeogenesis pathways function in the same compartment of the cyanobacterial cells In addition, cyanobacterial thylakoid membranes harbor both photosynthetic and respiratory electron transport chains, and the same components are shared by the two processes [23] Therefore, it is important to strictly regulate the direction of reactions in the anabolic and catabolic pathways in response to environmental conditions For characterization of LAHG, the analysis of gene expression involved in carbon metabolism that is 188 induced by light, especially of glycolytic enzymes, is important In the present study, we provide clear evidence of LAHG with cells at the late-linear and stationary growth phases of photoautotrophic preculture In addition, we show that, under LAHG conditions FBA, a glycolytic enzyme, is regulated by light and glucose via sll1330, a putative gene containing a helix-turn-helix (HTH) DNA-binding motif Results Growth of Synechocystis and its glucose utilization under heterotrophic growth conditions Photoautotrophically grown cells at the late-linear growth phase were cultured heterotrophically with or without short periods (5 min) of light every h, which is considered as LAHG in the present study The cells grew with approximately 30 and 66 h of doubling time on culture with or without light pulses, respectively They grew to a two-fold higher level with 10 mm glucose (6.0 · 108 cellsỈmL)1) than with mm glucose in approximately week (2.8 · 108 cellsỈmL)1) (Fig 1) However, in continuous darkness (dark heterotrophic conditions), the cells did not grow so much, even in the presence of either or 10 mm glucose Without glucose, the cells could not grow either with or without the light pulses Whereas glucose in the medium was almost completely consumed under LAHG conditions in days, glucose remained in the medium even after cell growth had ceased under dark heterotrophic conditions (Fig 1B) These results suggest that the supply of glucose in the medium was the sole carbon and energy source for the cells to grow and that the cells could not fully utilize glucose until they were exposed to light pulses During culture, the amount of chlorophyll a in the cells grown photoautotrophically decreased under both LAHG and dark heterotrophic conditions from 3.0 to 0.9 · 10)14 gỈcell)1 in days The amount of soluble protein was maintained at approximately 2.0 · 10)14 gỈcell)1 under LAHG conditions, but decreased to 0.8 · 10)14 gỈcell)1 in days under dark heterotrophic conditions The results obtained suggest that soluble protein synthesis continued at a normal level under LAHG conditions, but not in the dark Dependence of LAHG on the cell conditions for photoautotrophic preculture The cell conditions for photoautotrophic preculture greatly affected the subsequent LAHG and heterotrophic growth profiles (Fig 2) The cells harvested at the FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Tabei et al Activation of expression of fbaA in Synechocystis at a late period of culture, such as the late-linear growth phase, where reactivation of some processes by light pulses may occur under LAHG conditions Cells harvested on day of photoautotrophic preculture (Fig 2E) were used for subsequent experiments Cell concentration (A730) A 1.5 Proteome analysis of cells grown under photoautotrophic preculture, LAHG and dark heterotrophic conditions 0.5 0 8 Time (days) Glucose concentration (mM) B 10 0 Time (days) Fig Growth of Synechocystis under heterotrophic conditions with (open circles and open squares) or without (filled circles and filled squares) light pulses for every h (A) and the glucose concentration in the medium during culture (B) The cultures grown photoautotrophically at the late-linear growth phase were incubated in BG-11 in the presence of mM (open circles and filled circles) or 10 mM (open squares and filled squares) glucose, and in the absence of glucose (open triangles and filled triangles) at 30 °C The decrease in the glucose concentration was determined at the indicated times thereafter Error bars are for three separately grown cultures logarithmic growth phase could grow fast heterotrophically either with or without light pulses (Fig 2B) By contrast, those harvested at the stationary growth phase of the preculture scarcely grew, even in the presence of glucose in the dark (Fig 2F) Cells harvested at any phase of the preculture could grow under LAHG conditions, although the induction period was longer with a longer preculture period (Fig 2C–E) Therefore, the difference in cell growth between LAHG and dark heterotrophic conditions was obvious The compositions of soluble proteins on culture under LAHG, dark heterotrophic and the respective preculture conditions were analyzed by 2D-PAGE Under photoautotrophic preculture and LAHG conditions (Fig 3A,B), over 90 protein spots were observed, whereas less than half this number was observed under heterotrophic conditions These results imply that some of the proteins appearing during photoautotrophic preculture disappeared under dark heterotrophic conditions We also found that 32 of the spots observed were thicker for LAHG than the corresponding spots for photoautotrophic preculture, suggesting a greater synthesis of the respective proteins with the addition of glucose Moreover, 20 of these 32 spots were thicker than the corresponding spots under dark heterotrophic conditions, suggesting a greater synthesis of the respective proteins with light in the presence of glucose The putative genes and their products for individual spots that were increased by the glucose supply with and without the short periods of light were determined from the molecular weights of fragments by means of MALDI-TOF MS (Table 1) Protein syntheses with various categories of genes in relation to metabolism, transport and the machinery of gene expression were enhanced under LAHG conditions The proteins involved in cellular processes, GroEL1 (spot 6), Tig (8) and GreP (32), were induced under LAHG conditions HtpG (spot 3), DnaK2 (4) and GroEL2 (5, 7) appeared under both LAHG and dark heterotrophic conditions Rps1a (spot 20) and Tsf (26) involved in the translation machinery were induced under LAHG conditions, whereas Fus (spot 2), Tuf A (14) and Rpl12 (31) were induced under dark heterotrophic conditions These results suggest that cellular processes and translation machinery function differently with or without light in the presence of glucose AtpB (spot 9), NatB (10), UrtA (11), RbcL (17) and CpcB (29), formerly regarded as representing peripheral membrane proteins [24], were abundant in the soluble fraction of the cells grown under LAHG conditions They might have been extracted during our sample preparation due to good solubilization, or the association between the proteins and the membrane might change with growth conditions FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS 189 Activation of expression of fbaA in Synechocystis Cell concentration (A730) A B 1.0 C 1.0 4d 5d 0.8 0.6 0.4 0.4 0.2 8d 0.8 0.6 12d 0.2 7d 5d 4d 0 0 12 Time (days) E 1.0 D 1.0 Cell concentration (A730) Y Tabei et al F1.0 8d 7d 12d 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 Time (days) 8 10 12 14 Fig Effects of pre-culture conditions on the heterotrophic growth of Synechocystis Growth of Synechocystis on photoautotrophic preculture (A) Growth of Synechocystis under the heterotrophic conditions (B–F) with (open circles and open squares) or without (filled circles and filled squares) light pulses for every h The cultures were carried out in BG-11 in the presence of mM glucose (open circles and filled circles) and in the absence of glucose (open squares and filled squares) (B–F) Growth when the cells started to be cultured in the presence of glucose at 4, 5, 7, and 12 days, respectively, in (A) The data are representative of three separate cultures 2D-PAGE also revealed that some proteins, including RbcL (spot 17), Gap2 (24) and Prk (19) in photosynthetic carbon metabolism, and FbaA (18) in glucose metabolism, were synthesized to a greater extent under LAHG conditions than under any of the other conditions The reason why photosynthetic genes were induced by glucose was probably a result of the higher protein-synthetic activity Enzymes involved in glycolysis and the OPPP other than FbaA were not included in Table 1, probably due to their low contents or to them being less changeable in their amounts The increase in the protein level of FbaA under LAHG conditions compared to under dark heterotrophic conditions suggested that glucose degradation was induced by light and glucose in Synechocystis The activity of FBA, which decreased from 88 to 46 unitsỈmg)1 protein during autotrophic preculture, was determined for both LAHG and dark heterotrophic cultures (Fig 4) The FBA activity increased under LAHG conditions, whereas, under dark heterotrophic conditions, it remained constant for days and then decreased gradually This finding is consistent with the protein level of FBA, which was increased by light pulses in the presence of glucose 190 Possible enhancement of glucose metabolism during LAHG When mm d-[14C(U)]glucose was supplied to cells that had been incubated in continuous darkness with mm glucose for days, most of the radioactivity incorporated was found in phosphate esters and a slight amount in glutamate at h after the supply of d-[14C(U)]glucose (data not shown) Thus, we presumed that even the cells cultured in continuous darkness metabolized it to phosphorylated sugars, but to a lesser extent to other organic compounds 2D TLC of a cell extract with detection with ninhydrin revealed that glutamate was a major amino acid in cells incubated under both LAHG and dark heterotrophic conditions, but that the amount under the former was much higher than that under the latter (see Fig S1) The l-glutamate contents in the cells at and days under LAHG, and at days in continuous darkness, were 0.13, 0.20 and 0.05 · 10)11 gỈcell)1, respectively Considering that glutamate is synthesized from 2-oxoglutarate via the tricarboxylic acid cycle by way of an aminotransferase reaction, we could deduce that the glucose metabolism is more active under FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Tabei et al Activation of expression of fbaA in Synechocystis Autotrophic conditions A kDa B pI LAHG conditions 250 75 50 25 C Dark heterotrophic conditions E 14 15 16 13 19 18 22 21 20 32 25 26 27 28 10 11 12 23 30 D kDa Δsll1330 LAHG conditions 17 24 29 31 Fig Proteome analyses of the soluble protein fraction of Synechocystis under various conditions The soluble proteins were extracted from Synechocystis cultured under photoautotrophic preculture (A), LAHG (B) and dark heterotrophic (C) conditions for days Equivalent amounts of proteins were separated by 2D-PAGE and stained with Coomassie brilliant blue The profile shown in (E) is identical to (B) with circles and numbers for annotation purposes The soluble proteins of Dsll1330 cultured under LAHG conditions for days (D) were also analyzed The positions of molecular size markers are indicated in kDa on the left FBA is indicated by an arrow LAHG conditions than under dark heterotrophic conditions Changes in some enzymatic activities on glucose metabolism The maximum enzymatic activites of glycolytic enzymes under dark heterotrophic and LAHG conditions for days were determined in the presence of dithiothreitol (Table 2) The activities of glucokinase (GK; EC 2.7.1.2) and PK were constantly maintained under the three sets of conditions The glucose 6-phosphate isomerase (PGI; EC 5.3.1.9) and FBA activities were relatively higher than those of the other enzymes The total activity of FBA under LAHG conditions was higher than the initial level, whereas that under dark heterotrophic conditions was lower The activity of GAPDH was increased by the addition of glucose Therefore, we concluded that FBA was enhanced at both the protein level and with respect to its activity FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS 191 Activation of expression of fbaA in Synechocystis Y Tabei et al Table Proteins were observed under LAHG conditions to a 1.5-fold greater extent than under autotrophic conditions in Synechocystis Category ORF Putative gene product Amino acid biosynthesis sll1931 sll1363 sll0430 Serine hydroxymethyltrasferase (GlyA) Ketol-acid reductoisomerase (IlvC) Heat shock protein 90 molecular chaperone (HtpG) 70 kDa heat shock protein (DnaK2) 60 kDa chaperonin (GroEL2) 60 kDa chaperonin (GroEL1) Trigger factor (Tig) Heat shock protein GrpE (GreP) Adenosylhomocysteinase ATP synthase b subunit (AtpB) Phycocyanine b subunit (CpcB) Superoxide dismutase (SodB) Water-soluble carotenoid protein Ribulose bisphosphate carboxylase large subunit (RbcL) Phosphoribulokinase (Prk) Glyceraldehyde 3-phosphate dehydrogenase (Gap2) Fructose 1,6-bisphosphate aldolase (FbaA) Periplasmic binding protein of ABC transporter for natural amino acids (NatB) Periplasmic protein, ABC-type urea transporter system substrate binding protein (UrtA) Nitrate ⁄ nitrite transporter system substrate binding protein (NrtA) Type I restriction-modification system S subunit DNA primase (DnaG) 30s ribosomal protein S1 (Rps1a) Elongation factor Ts (Tsf) 50s ribosomal protein L12 (Rpl12) Elongation factor (Fus) Elongation factor Tu (TufA) Antioxidant protein AhpC ⁄ TSA family protein, Cellular processes Energy metabolism Photosynthesis and respiration, energy metabolism sll0170 sll0416 slr2076 sll0533 sll0057 sll1234 slr1329 sll1577 slr1561 slr1963 slr0009 sll1525 sll1342 sll0018 Transport and binding proteins slr0559 sll1447 sll1450 DNA replication, restriction, modification, recombination, and repair Translation Others a sll8006 sll1868 slr1356 sll1261 sll1749 slr1463 sll1099 slr1198 sll1621 Unidentified Unidentified Spot no on 2D-PAGE Light induciblea 15 21 s 5, 32 16 29 29 22 17 s s s s s s s s s 19 24 s s 18 s 10 s 11 13 12 20 26 31 14 27 28 25 23 s s s s s s Genes enhanced under LAHG conditions to a 1.5-fold greater extent than under dark conditions are indicated (s) under LAHG conditions compared to in continuous darkness This result is consistent with the enhancement of fbaA gene expression by light pulses in the presence of glucose [25] Changes in the transcript level of fbaA during heterotrophic culture Figure shows the changes in the mRNA level of fbaA under LAHG and dark heterotrophic conditions Considering that the cells at days in Fig had been 192 grown under photoautotrophic conditions, and that the mRNA level of fbaA increased under LAHG conditions, we deduced that the increase in the mRNA level of fbaA was induced by both light pulses and glucose On the other hand, the mRNA level of fbaA under dark heterotrophic conditions increased slightly until days, and then decreased gradually The level under the dark heterotrophic conditions was approximately one-tenth of that under LAHG conditions after days These results suggested that light irradiation was required for the expression of fbaA FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Tabei et al Activation of expression of fbaA in Synechocystis 100 LAHG Dark heterotrophic FBA activity (mU·mg–1 protein) 8 (days) fbaA 80 rRNA 60 Fig Northern analysis of fbaA gene expression in Synechocystis under LAHG and dark heterotrophic conditions in BG-11 containing mM glucose RNA was isolated from cells grown photoautotrophically on day (0 days) and cells incubated under LAHG and dark heterotrophic conditions for 2, 4, and days Total RNA (10 lg) was then subjected to northern hybridization with a probe specific for the fbaA gene The lower panel shows rRNA stained with ethidium bromide as a control The data are representative of three independent experiments 40 20 0 Time (days) mutagenesis with a spectinomycin resistance cassette A resultant sll1330 deletion mutant, Dsll1330, could grow under photoautotrophic and mixotrophic (i.e under continuous light illumination with glucose) conditions as fast as the wild-type (Fig 6A,B) However, Dsll1330 did not grow well either under LAHG or dark heterotrophic conditions compared to the wildtype (Fig 6C,D) This can be explained by a deficiency in the ability to use glucose as a carbon source for growth Proteome analysis of Dsll1330 showed relatively lower contents of various proteins [e.g Gap2 (spot 24) and Tsf (spot 26)] The FbaA protein (spot 18; Fig 3, arrow) was also less in Dsll1330 (Fig 3B,D) and was as high as that in wild-type cells incubated under dark conditions The FbaA protein appeared to be more stable than its mRNA In any case, these results, when taken together with those obtained previously [25], suggest that one of the reasons for the inability of LAHG by Dsll1330 is a lack of enhancement of the protein level of FBA with light pulses in the presence of glucose Fig The FBA activities of Synechocystis under LAHG and heterotrophic conditions in BG-11 containing mM glucose Cells were incubated under LAHG (white bars) and dark heterotrophic (black bars) conditions for 2, 4, and days (0 days indicates the activity of FBA on photoautotrophic preculture on day 8) The error bars represent three separately grown cultures LAHG and the protein composition of Dsll1330, encoding a two-component system response regulator Because many transcriptional factors contain the HTH DNA-binding motif, there is a possibility that some HTH DNA-binding proteins may be involved in the fbaA gene expression induced by light and glucose We have searched the Synechocystis genome sequence databank and Cyanobase, and identified 57 ORFs encoding putative HTH motif-containing transcriptional factors Among these candidates, we successfully mutated the sll1330 gene encoding a putative twocomponent response regulator by means of targeted Table Glycolytic enzyme activities of cells grown under dark heterotrophic and LAHG conditions for days Activity (mmg)1 protein ± SE) Enzyme ORF Initial (0 days) Dark heterotroph (8 days) LAHG (8 days) GK PGI FBA (total) FBA (class-I)a GAPDH Enolase PK sll0593 slr1349 sll0018, slr0943 slr0943 slr0884 sll0752 sll0587, sll1275 12.0 370 45.9 7.4 2.8 2.9 13.3 8.8 363 31.7 3.2 12.1 1.2 12.2 9.9 204 61.6 5.7 12.2 3.1 10.2 a ± ± ± ± ± ± ± 2.9 26 4.5 0.8 0.8 1.3 2.4 ± ± ± ± ± ± ± 2.6 17 5.6 1.2 3.4 0.9 1.4 ± ± ± ± ± ± ± 1.8 11 7.2 0.8 5.0 1.2 1.2 Class-II was inhibited by the addition of mM EDTA FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS 193 Activation of expression of fbaA in Synechocystis 0.6 Y Tabei et al the fbaA gene was found to bind with Sll1330, which was overexpressed in Escherichia coli (see Fig S2) These results suggest that Sll1330 binds to this putative promoter region of the fbaA gene in PCC 6803 cells A 0.4 0.2 Discussion 3.0 8 8 B Cell concentration (A730) 2.0 1.0 0 C 1.5 1.0 0.5 0 0.5 D 0.4 0.3 0.2 0.1 0 Time (days) Fig Growth of the wild-type (circles) and Dsll1330 (triangles) under photoautotrophic (continuous light; A), mixotrophic (continuous light + 10 mM glucose; B), LAHG (light pulses + 10 mM glucose; C) and dark heterotrophic (dark + 10 mM glucose; D) conditions Error bars indicate the SD for three independent experiments Interaction of the Sll1330 protein with the fbaA promoter region was additionally examined by means of the electrophoretic mobility shift assay (EMSA) using DNA fragments of the upstream region of fbaA (see Fig S2) The DNA fragment comprising )331 to )186 bp from the putative transcription start point of 194 In the present study, the effects of light on heterotrophic growth of Synechocystis were precisely investigated First, we confirmed that Synechocystis could not grow in complete darkness, as reported previously [10] However, the cells at the logarithmic growth phase could grow heterotrophically even without illumination This would be because glucose-degrading machineries were active in the photoautotrophic preculture at relatively early stages The total FBA activity in the late-linear growth phase was lower than that in the exponential growth phase (Table 2) This is consistent with the fact that the mRNA levels of some glycolytic genes were suppressed at the stationary growth phase compared to at the exponential growth phase [26] Physiological changes occur at different growth phases with respect to the accumulation of inhibitory metabolites [27,28] and gene expression [29] The expression of 10% of the genes was different in the linear growth phase compared to in the logarithmic growth phase [30] Considering the close correlation between nitrogen and carbon metabolism [13], we assume the decrease in gene expression during growth is due to the lower level of nitrogen in the medium Alternatively, because of the higher concentration of cells during photoautotrophic preculture, the amount of light absorbed by each cell decreased in these conditions In any case, when using cells precultured at a late period of the preculture (i.e after days of culture), an effect of light on the heterotrophic growth was clearly observed Therefore, LAHG might be due to reactivation of some biochemical activities We have attempted proteome analysis using the combination of 2D-PAGE and MALDI-TOF MS of tryptic digests Considerable amounts of soluble proteins were found in the cells under LAHG conditions compared to those after days of photoautotrophic preculture Considering that various kinds of proteins were present at higher levels under LAHG conditions than under dark heterotrophic conditions, we deduced that the light pulses triggered the transcription and translation of a whole set of proteins essential for growth with glucose In this respect, the transcriptional levels of molecular chaperones groESL and cpn60 are known to be significantly increased by light irradiation in Synechocystis [30] Moreover, the DnaK2 protein is one of the abundant soluble proteins, with dnaK gene expression having FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Tabei et al been reported to obey up-regulation with the lights on and down-regulation with the lights off (i.e in the manner of a circadian rhythm) [31], which is consistent with the results shown in Fig DnaK2 and rehydrin may protect the cells from continuous light irradiation and facilitate the functional assembly of photosynthetic machinery proteins [32] The SodB protein (Spot 29) catalyzes the dismutation of O2) to H2O2 and O2, in which sodB gene and SodB protein expression are induced by continuous light irradiation [33] Light induction of putative 30S ribosomal protein S1 (sll1356) and elongation factor Ts (sll1261), which are involved in translation processes, suggests that protein synthesis is enhanced during cell growth under LAHG conditions This is supported by the appearance of more spots on 2D-PAGE of cells under LAHG conditions than under heterotrophic conditions In the present study, the expression of GroEL1, Tig and GreP was also up-regulated under LAHG conditions compared to under both photoautotrophic and dark heterotrophic conditions These results suggest the higher activities of cellular processes (e.g protein synthesis and metabolic processes) under LAHG conditions FbaA, one of the components of the glucosemetabolism machinery, was also up-regulated by light under glucose-utilizing conditions The protein level of FbaA was high compared to that of other glycolytic enzymes The increase in the maximum Fba activity in days (Fig 4) under LAHG conditions, but not under dark heterotrophic conditions, suggested the light activation of Fba synthesis This is also supported by the higher content of glutamate in the cells Therefore, cells under LAHG conditions may be more active than without light We found also that, in the sll1330 deletion mutant (Dsll1330), which could not grow heterotrophically (Fig 6), the protein level of FbaA was repressed (Fig 3D), even under LAHG conditions, and the increases in the mRNA levels of five glycolytic genes, including fbaA, were repressed by light and glucose [25] We also found binding of Sll1330 with the promoter region of the fbaA gene Because the Dsll1330 mutant could grow photoautotrophically with the expression of fbaA, the sll1330 gene is not required for the expression of fbaA under photoautotrophic conditions This means that fbaA gene expression is controlled through two signal pathways: presumably photosynthetic processes and light pulses in the presence of glucose Gene expression of fbaA may be important for survival in various environments, including both under photoautotrophic and heterotrophic conditions In the latter case, glucose induces the expression of several genes, such as those for GK, phosphofructokinase, phosphoglycerate mutase Activation of expression of fbaA in Synechocystis and PK, including sll1330 [34] In this respect, glucose degradation as a whole may be regulated by the expression of glycolytic genes via sll1330 Experimental procedures Algal cells and culture conditions A glucose tolerant strain of Synechocystis [35] was cultivated photoautotrophically in BG-11 containing 20 mm Tes-KOH (pH 8.2) with continuous illumination by a bank of fluorescent lamps at 10 WỈm)2 and bubbled with 2% (v ⁄ v) CO2 in glass tubes or flasks [36] The LAHG cultures were incubated in the dark with of light irradiation (5 WỈm)2), eight times a day in the presence of and 10 mm glucose, whereas the heterotrophic cultures were incubated without light irradiation in the presence of glucose Growth and cell density were followed by measuring the absorption at 730 nm with a spectrophotometer (DU640; Beckman Coulter, Fullerton, CA, USA) Cell concentrations were determined with a hemacytometer (Burker-Turk; Erma Optiă cal Works, Tokyo, Japan) Glucose uptake was assayed by measurement of the concentration of glucose in the medium with a glucose CII kit (Wako Pure Chemical Industries, Osaka, Japan) In the mutant, the wild-type gene for sll1330 was disrupted by the insertion of a spectinomycin resistance cassette [25] Determination of intracellular chlorophyll a, soluble protein and L-glutamate Chlorophyll a was measured by the procedure described by Mackinney [37], with some modifications Soluble protein was assayed by measurement of the concentration of a particular protein with a BCA protein assay reagent kit (Pierce Biotechnology, Rockford, IL, USA) Cells were harvested at a certain time, centrifuged, and then disrupted with a French press at 28 000 p.s.i The lysate was centrifuged and the resulting supernatant was used for the measurement of soluble protein Intracellular l-glutamate was assayed by measurement of the concentration of l-glutamate with a l-glutamate determination kit (Yamasa Corp., Chiba, Japan) 2D-PAGE and in-gel digestion The materials precipitated from 300 lg of soluble protein were resuspended in m urea, 1% (v ⁄ v) Triton X-100, ampholine (pH 3.5–10.0) and 2-mercaptoethanol The mixture was centrifuged at 10 000 g for 10 at °C The supernatant was applied to a linear immobilized pH gradient strip (pH 4.0–8.0) Isoelectric focusing and subsequent SDS ⁄ PAGE were then performed Proteins were stained with Coomassie brilliant blue and then scanned using an FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS 195 Activation of expression of fbaA in Synechocystis Y Tabei et al image scanner In-gel digestion was performed as described for an in-gel digest kit (Millipore, Billerica, MA, USA) The gel pieces were re-swollen by adding a trypsin solution Samples were incubated overnight at 30 °C and the protein fragments were extracted MALDI-TOF analysis Peptide extracts were loaded onto the target plate by the dried droplet method using a-cyano-4-hydroxycinnamic acid as a matrix MALDI-TOF analysis was performed in the reflector mode with a Voyager-DE PRO mass spectrometer (Applied Biosystems, Foster City, CA, USA) Database search Proteins were identified by searching the Synechocystis database using ms-fit (Protein Prospector; UCSF, San Francisco, CA, USA) The following criteria were used for a positive identification in the ms-fit database search: (a) at least four matching peptide masses; (b) at least 50% of the measured masses must match the theoretical masses; and (c) 0.01% or better mass accuracy RNA isolation and northern hybridization Total RNA was extracted and purified by phenol–chloroform extraction [39] For northern hybridization, the DNA fragments of fbaA were amplified by PCR, using primers 5¢-ATTTCGATCATGCAGGCCG-3¢ and 5¢-GGAAGAAC CGTGCATTACC-3¢, and labeled with [a-32P]dCTP using a Megaprime labeling kit (Amersham Pharmacia, Piscataway, NJ, USA) Hybridization signals were detected with a BAS2000 bio-imaging analyzer (Fuji Film, Tokyo, Japan) Expression of the Sll1330 protein in E coli Overexpression of Sll1330 was carried out as described previously [40] with some modification The sll1330 gene was cloned from PCC 6803 genomic DNA after PCR amplification using specific oligonucleotides The sll1330 gene was amplified using primers 1330OX-F (5¢-GTCTAGACAT ATGAATCCAG-3¢), comprising nucleotides )10 to +10 of the sll1330 coding region, and 1330OX-R (5¢-CGGGA TCCAGGGTAACAGGTTTCTCCG-3¢), comprising nucleotides +704 to +730 of the sll1330 coding region NdeI and BamHI sites were introduced into the 1330OX-F and 1330OX-R primers, respectively (underlined) Assaying enzyme activity The cells cultivated under LAHG and heterotrophic conditions were harvested by centrifugation The pellets were resuspended and then lysed by three passages through a French press at 28 000 p.s.i., followed by clarification by centrifugation at 35 000 g for 10 The enzyme activities were measured using previously described procedures [7,21,38] with some modifications The assay mixtures comprised: GK [50 mm Hepes-KOH (pH 8.0), mm dithiothreitol, 10 mm MgCl2, 2.5 mm glucose, mm ATP, 0.5 mm NAD+ and unitsỈmL)1 G6PD]; PGI [50 mm Hepes-KOH (pH 8.0), mm dithiothreitol, 10 mm MgCl2, 1.7 mm NAD+, mm fructose 6-phosphate and unitsỈmL)1 G6PD]; PFK [50 mm Hepes-KOH (pH 8.0), mm dithiothreitol, 10 mm MgCl2, 0.15 mm NADH, mm fructose 6-phosphate, 2.5 mm ATP, unitỈmL)1 aldolase, 10 unitsỈmL)1 triosephosphate isomerase and unitỈmL)1 glycerophosphate dehydrogenase]; FBA [50 mm Tris–HCl (pH 7.5), 0.2 mm CoCl2, 0.2 mm NADH, unitỈmL)1 glycerol 3-phosphate dehydrogenase, unitỈmL)1 triosephosphate isomerase and mm fructose 1,6-bisphosphate]; GAPDH [50 mm Tris–HCl (pH 7.5), mm MgCl2, mm 3-phosphoglycerate, mm EDTA, unitsỈmL)1 3-phosphoglycerate kinase, mm ATP and 0.2 mm NADH]; enolase [50 mm Hepes-KOH (pH 8.0), mm dithiothreitol, 10 mm MgCl2, 0.15 mm NADH, mm ADP, mm 2-phosphoglycerate, unitsỈmL)1 PK and unitsỈmL)1 lactate dehydrogenase]; and PK [50 mm Hepes-NaOH (pH 7.0), 30 mm MgCl2, 0.15 mm NADH, 2.5 mm phosphoenolpyruvate, mm ADP and unitsỈmL)1 lactate dehydrogenase] 196 EMSA A 135-bp DNA fragment, prom1, corresponding to positions )86 to +49 upstream of the translation initiation site of the fbaA gene was amplified, using forward primer 5¢-GCAGAAACTAGCCTAAGATG-3¢ and reverse primer 5¢-CCATTTTCCGCCGCATGGTC-3¢ A 121-bp DNA fragment, prom2, corresponding to positions )190 to )70 upstream of the translation initiation site of the fbaA gene was amplified with forward primer 5¢-CTCAGCCATAGA CTAGGGTG-3¢ and reverse primer 5¢-ATCTTAGGCTA GTTTCTGC-3¢ A 126-bp fragment, prom3, corresponding to positions )311 to )186 upstream of the translation initiation site of the fbaA gene was amplified with forward primer 5¢-GCTAAAGGAAGTATTTGCTAC-3¢ and reverse primer 5¢-CTGAGTTAACCAAGTCCAGG-3¢ A 134 bp DNA fragment, prom4, corresponding to positions )438 to )305 upstream of the translation initiation site of the fbaA gene was amplified with forward primer 5¢-CA ATAAATTTGCCCGTTTCC-3¢ and reverse primer 5¢-CCTTAGCGACGGCAATGGTC-3¢ EMSA was carried out as described previously [41] Acknowledgements The authors are indebted to Dr S Fujiwara for helpful discussion and Mr N J Halewood for his kind help in the correction of the English They are also indebted to Mr Masato Sasahara, Ms Yuka Katsuki and FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Tabei et al Ms Miwako Itoh for their experimental support This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan; the Promotion and Mutual Aid Corporation for Private Schools to M T.; and a Sasakawa Scientific Research Grant from the Japan Science Society to Y T Activation of expression of fbaA in Synechocystis 13 14 References Briggs WR & Christie JM (2002) Phototropins and 2: versatile plant blue-light receptors Trends Plant Sci 7, 204–210 Gyula P, Schafer E & Nagy F (2003) Light perception ă and signaling in higher plants Curr Opin Plant Biol 6, 446–452 Mackey SR & Golden SS (2007) Winding up the cyanobacterial circadian clock Trends Microbiol 15, 381–388 Yoshihara S & Ikeuchi M (2004) Phototactic motility in the unicellular cyanobacterium Synechocystis sp PCC 6803 Photochem Photobiol Sci 3, 512–518 Stal LJ & Moezelaar R (1997) Fermentation in cyanobacteria FEMS Microbiol Rev 21, 179–211 Knowles VL & Plaxton WC (2003) From genome to enzyme: analysis of key glycolytic and oxidative pentose-phosphate pathway enzymes in the cyanobacterium Synechocystis sp PCC 6803 Plant Cell Physiol 44, 758– 763 Ball SG & Morell MK (2003) From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule Annu Rev Plant Biol 54, 207–233 Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S et al (1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp strain PCC 6803 II Sequence determination of the entire genome and assignment of potential proteincoding regions DNA Res 3, 109–136 ´ ´ ´ Florencio FJ, Perez-Perez ME, Lopez-Maury L, Mata-Cabana A & Lindahl M (2006) The diversity and complexity of the cyanobacterial thioredoxin systems Photosynth Res 89, 157–171 10 Anderson SL & McIntosh L (1991) Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp strain PCC 6803: a blue-light-requiring process J Bacteriol 173, 2761–2767 11 Singh AK & Sherman LA (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 12 Osanai T, Kanesaki Y, Nakano T, Takahashi H, Asayama M, Shirai M, Kanehisa M, Suzuki I, Murata N & Tanaka K (2005) Positive regulation of sugar catabolic pathways in the cyanobacterium Synechocystis sp PCC 15 16 17 18 19 20 21 22 23 24 6803 by the group sigma factor SigE J Biol Chem 280, 30653–30659 Yang C, Hua 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 Scanlan DJ, Newman J, Sebaihia M, Mann NH & Carr NG (1992) Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942 Plant Mol Biol 19, 877–880 Broedel SE Jr & Wolf RE Jr (1990) Genetic tagging, cloning, and DNA sequence of the Synechococcus sp strain PCC 7942 gene (gnd) encoding 6-phosphogluconate dehydrogenase J Bacteriol 172, 4023–4031 Valverde F, Losada M & Serrano A (1997) Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase of Synechocystis sp PCC 6803 J Bacteriol 179, 4513–4522 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 Figge RM, Cassier-Chauvat C, Chauvat F & Cerff R (2000) The carbon metabolism-controlled Synechocystis gap2 gene harbours a conserved enhancer element and a Gram-positive-like –16 promoter box retained in some chloroplast genes Mol Microbiol 36, 44–54 Knowles VL, Smith CS, Smith CR & Plaxton WC (2001) Structural and regulatory properties of pyruvate kinase from the cyanobacterium Synechococcus PCC 6301 J Biol Chem 276, 20966–20972 Luinenburg I & Coleman JR (1990) A requirement for phosphoenolpyruvate carboxylase in the cyanobacterium Synechococcus PCC 7942 Arch Microbiol 154, 471–474 Nakahara K, Yamamoto H, Miyake C & Yokota A (2003) Purification and characterization of class-I and class-II fructose-1,6-bisphosphate aldolases from the cyanobacterium Synechocystis sp PCC 6803 Plant Cell Physiol 44, 326–333 Pelory RA, Levine GA & Bassham JA (1976) Kinetics of light-dark CO2 fixation and glucose assimilation by Aphanocapsa 6714 J Bacteriol 128, 633–643 Scherer S (1990) Do photosynthetic and respiratory electron transport chains share redox proteins? Trends Biochem Sci 15, 458–462 Wang Y, Sun J & Chitnis PR (2000) Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp PCC 6803 Electrophoresis 21, 1746–1754 FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS 197 Activation of expression of fbaA in Synechocystis Y Tabei et al 25 Tabei Y, Okada K & Tsuzuki M (2007) Sll1330 controls the expression of glycolytic genes in Synechocystis sp PCC 6803 Biochem Biophys Res Commun 355, 1045–1050 26 Foster JS, Singh AK, Rothschild LJ & Sherman LA (2007) Growth-phase dependent differential gene expression in Synechocystis sp strain PCC 6803 and regulation by a group sigma factor Arch Microbiol 187, 265–279 27 Siegele DA & Kolter R (1992) Life after log J Bacteriol 174, 345–348 28 Nystrom T (2004) Stationary-phase physiology Annu ă Rev Microbiol 58, 161181 29 Tani TH, Khodursky A, Blumenthal RM, Brown PO & Matthews RG (2002) Adaptation to famine: a family of stationary-phase genes revealed by microarray analysis Proc Natl Acad Sci USA 99, 13471–13476 ´ ´ 30 Glatz A, Horvath I, Varvasovszki V, Kovacs E, Torok ă ă Z & Vigh L (1997) Chaperonin genes of the Synechocystis PCC 6803 are differentially regulated under lightdark transition during heat stress Biochem Biophys Res Commun 239, 291–297 31 Aoki S, Kondo T & Ishiura M (1995) Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp strain PCC 6803 J Bacteriol 177, 5606–5611 32 Choi JS, Kim DS, Lee J, Kim SJ, Kim SI, Kim YH, Hong J, Yoo JS, Suh KH & Park YM (2000) Proteome analysis of light-induced proteins in Synechocystis sp PCC 6803: identification of proteins separated by 2D-PAGE using N-terminal sequencing and MALDITOF MS Mol Cells 10, 705–711 33 Kim JH & Suh KH (2005) Light-dependent expression of superoxide dismutase from cyanobacterium Synechocystis sp strain PCC 6803 Arch Microbiol 183, 218–223 34 Lee S, Ryu JY, Kim SY, Jeon JH, Song JY, Cho HT, Choi SB, Choi D, de Marsac NT & Park YI (2007) Transcriptional regulation of the respiratory genes in the cyanobacterium Synechocystis sp PCC 6803 during the early response to glucose feeding Plant Physiol 145, 1018–1030 35 Williams JGK (1988) Construction of specific mutants in photosystem II photosynthetic reaction center by 198 36 37 38 39 40 41 genetic engineering methods in Synechocystis 6803 Methods Enzymol 167, 766–778 Rippka R, Deruelles J, Waterbury JB, Herdman M & Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria J Gen Microbiol 111, 1–61 Mackinney G (1941) Absorption of light by chlorophyll solutions J Biol Chem 140, 315–322 Sparla F, Pupillo P & Trost P (2002) The C-terminal extension of glyceraldehyde-3-phosphate dehydrogenase subunit B acts as an autoinhibitory domain regulated by thioredoxins and nicotinamide adenine dinucleotide J Biol Chem 277, 44946–44952 Los DA, Ray MK & 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 Gutekunst K, Phunpruch S, Schwarz C, Schuchardt S, Schulz-Friedrich R & Appel J (2005) LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp PCC 6803 as a transcription activator Mol Microbiol 58, 810–823 Oliveira P & Lindblad P (2005) LexA, a transcription regulator binding in the promoter region of the bidirectional hydrogenase in the cyanobacterium Synechocystis sp PCC 6803 FEMS Microbiol Lett 251, 59–66 Supporting information The following supplementary material is available: Fig S1 Amino acid compositions of soluble fractions of Synechocystis Fig S2 EMSA analysis of Sll1330 and the promoter region of the fbaA gene This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 276 (2009) 187–198 ª 2008 The Authors Journal compilation ª 2008 FEBS ... 5¢-GGAAGAAC CGTGCATTACC-3¢, and labeled with [a- 32P]dCTP using a Megaprime labeling kit (Amersham Pharmacia, Piscataway, NJ, USA) Hybridization signals were detected with a BAS2000 bio-imaging analyzer... protein Intracellular l-glutamate was assayed by measurement of the concentration of l-glutamate with a l-glutamate determination kit (Yamasa Corp., Chiba, Japan) 2D-PAGE and in- gel digestion The materials... translation initiation site of the fbaA gene was amplified with forward primer 5¢-GCTAAAGGAAGTATTTGCTAC-3¢ and reverse primer 5¢-CTGAGTTAACCAAGTCCAGG-3¢ A 134 bp DNA fragment, prom4, corresponding