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Histidine kinases are common sensory proteins used to detect environmental changes in bacteria. They respond to specific stimuli via a signal-input domain and alter gene expression through a cognate response regulator. The modulation/control of transcriptional regulation in cyanobacteria is important to reinforce the production of useful target compounds via photosynthesis without altering the growth profiles. For instance, heavy metal ions (Ni2+ and Cu2+), chemical inducers (IPTG), and a volatile compound (toluene) have been previously applied to regulate gene expression in cyanobacteria. However, most systems/regulators are only able to regulate gene expression once because it is impossible to eliminate them from the medium. To construct a reversible regulation system, a chimeric sensor, VanN_SphS, was developed by fusing the signal input domain of a quorum-sensing (QS) sensor, VanN, from Vibrio anguillarum, responding N-3-hydroxyhexanoyl-L-homoserine lactone (OHC6-HSL), with the kinase domain of SphS, a phosphate-deficiency sensor from the cyanobacterium Synechocystis sp. PCC 6803. After expression of the chimeric sensor in Synechocystis cells, responses to the various N-acyl-homoserine-lactones (AHLs) were evaluated by measuring the alkaline phosphatase (AP) activity, which is regulated by SphS. VanN_SphS responded only to OHC6-HSL and repressed AP activity. Then, the coexpression of the AHLs-degradation enzyme, Aii20J, a lactonase from Tenacibaculum sp. 20J, resumed the activity. This is the first report on the use of AHL-mediated transcriptional regulation in Synechocystis, which could be used in the future for the controlled production of useful compounds in the cyanobacterium.

The availability of inorganic carbon (C

Recently, microalgae have attracted attention as sources of biomass energy. However, fatty acids from the microalgae are mainly unsaturated and show low stability in oxygenated environments, due to oxidation of the double bonds. The branched-chain fatty acid, 10-methyl stearic acid, is synthesized from oleic acid in certain bacteria; the fatty acid is saturated, but melting point is low. Thus, it is stable in the presence of oxygen and is highly fluid. We previously demonstrated that BfaA and BfaB in Mycobacterium chlorophenolicum are involved in the synthesis of 10-methyl stearic acid from oleic acid. In this study, as a consequence of the introduction of bfaA and bfaB into the cyanobacterium, Synechocystis sp. PCC 6803, we succeeded in producing 10-methyl stearic acid, with yields up to 4.1% of the total fatty acid content. The synthesis of 10-methyl stearic acid in Synechocystis cells did not show a significant effect on photosynthetic activity, but the growth of the cells was retarded at 34 °C. We observed that the synthesis of 10-methylene stearic acid, a precursor of 10-methyl stearic acid, had an inhibitory effect on the growth of the transformants, which was mitigated under microoxic conditions. Eventually, the amount of 10-methyl stearic acid present in the sulfoquinovosyl diacylglycerol and phosphatidylglycerol of the transformants was remarkably higher than that in the monogalactosyldiacylglycerol and digalactosyldiacylglycerol. Overall, we successfully synthesized 10-methyl stearic acid in the phototroph, Synechocystis, demonstrating that it is possible to synthesize unique modified fatty acids via photosynthesis that are not naturally produced in photosynthetic organisms.

Transport systems, which perform a pivotal function in regulating the import and export of various substances via cellular membranes, are important machinery in all living organisms. The ATP-binding cassette (ABC) transporter, one of such transporters, has a crucial role in the high-affinity uptake of various nutrients in bacteria. In the cyanobacterium Synechocystis sp. PCC 6803, a model cyanobacterium, its response to phosphate (Pi)-depleted conditions and the consequential regulation of the Pi transport system have been well-studied. However, its responses and the Pi uptake under Pi-repleted conditions are not comprehended yet. This study observed the peculiar stimulation of the Pi uptake following severe chlorosis in the Synechocystis cells grown under Pi-repleted conditions. The Synechocystis cells grown under high Pi conditions (approximately 4 mM) showed the rapid Pi uptake after 3 d of cultivation, and significantly severe degradation of chlorophyll was observed. Remarkably, the Synechocystis cells showed more than 16-fold higher Pi uptake at that time. Furthermore, we revealed that sulfate (S) deficiency-induced Pi uptake and that magnesium deficiency caused chlorosis and released the intracellularly accumulated Pi. Moreover, reverse genetics revealed that the rapid Pi uptake occurred via an ABC transporter induced by S deficiency, namely, a putative sulfate transporter, SbpA-CysTWA. Our findings provide a new perspective on the Pi uptake under high Pi conditions in cyanobacteria and bacteria.

Microalgae have been used to produce useful compounds. In many cases in which target compounds are produced from CO2 via photosynthesis, there is a trade-off between the proliferation of cells and the production of the target compounds. This is due to carbon partitioning between the synthesis of cellular components and the target compounds, and/or the toxicity of the target products. Therefore, there is a requirement to develop a gene switch that can modulate the metabolism from the growth phase of cells to the production phase of the target molecules. In this study, we attempted to develop an artificial sensory protein that can respond to insoluble gaseous compounds and regulate gene expression in the model cyanobacterium Synechocystis sp. PCC 6803. We constructed a chimeric histidine kinase by fusing the signal input domain of TodS, a toluene sensor from Pseudomonas putida, and the kinase domain of SphS, a phosphate-deficient sensor from Synechocystis. Because TodS contains two kinase domains, we substituted either of the kinase domains of TodS with the kinase domain of SphS to obtain TodSS_SphS and TodSL_SphS. We introduced a gene for the chimeric sensor to Synechocystis cells and evaluated their response to toluene by measuring alkaline phosphatase (AP) activity and phoA mRNA expression. Although the cells harboring TodSL_SphS did not exhibit AP activity, both in the presence or absence of toluene, the cells harboring TodSS_SphS accumulated the transcript of the phoA gene after 5-h exposure to toluene, and only exhibited AP activity after exposure to toluene.

Eukaryotic Δ6-desaturases are microsomal enzymes which balance the synthesis of ω-3 and ω-6 C18-polyunsaturated-fatty-acids (PUFA) accordingly to their specificity. In several microalgae, includingO. tauri, plastidic C18-PUFA are specifically regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFA. Sequence retrieval fromO. tauridesaturases, highlighted two putative Δ6/Δ8-desaturases sequences clustering, with other microalgal homologs, apart from other characterized Δ-6 desaturases. Their overexpression in heterologous hosts, includingN. benthamianaandSynechocystis, unveiled their Δ6-desaturation activity and plastid localization.O. taurilines overexpressing these Δ6-desaturases no longer adjusted their plastidic C18-PUFA amount under phosphate starvation but didn’t show any obvious physiological alterations. Detailed lipid analyses from the various overexpressing hosts, unravelled that the substrate features involved in the Δ6-desaturase specificity importantly involved the lipid head-group and likely the non-substrate acyl-chain, in addition to the overall preference for the ω-class of the substrate acyl-chain. The most active desaturase displayed a broad range substrate specificity for plastidic lipids and a preference for ω-3 substrates, while the other was selective for ω-6 substrates, phosphatidylglycerol and 16:4-galactolipid species specific to the native host. The distribution of plastidial Δ6-desaturase products in eukaryotic hosts suggested the occurrence of C18-PUFA export from the plastid.One sentence summaryOsteococcus tauri plastidic lipid C18-PUFA remodelling involves two plastid-located cytochrome-b5 fused Δ6-desaturases with distinct preferences for both head-group and acyl-chain.

Microalgae have received much attention as a next-generation source of biomass energy. However, most of the fatty acids (FAs) from microalgae are multiply unsaturated; thus, the biofuels derived from them are fluid, but vulnerable to oxidation. In this study, we attempted to synthesize cyclopropane FAs in the cyanobacterium Synechocystis sp. PCC 6803 by expressing the cfa gene for cyclopropane FA synthase from Escherichia coli with the aim of producing FAs that are fluid and stable in response to oxidization. We successfully synthesized cyclopropane FAs in Synechocystis with a yield of ~30% of total FAs. Growth of the transformants was altered, particularly at low temperatures, but photosynthesis and respiration were not significantly affected. C16:1(∆9) synthesis in the desA(-)/desD(-) strain by expression of the desC2 gene for sn-2 specific ∆9 desaturase positively affected growth at low temperatures via promotion of various cellular processes, with the exceptions of photosynthesis and respiration. Estimation of the apparent activities of desaturases suggested that some acyl-lipid desaturases might recognize the lipid side chain.

Photoautotrophic bacteria have developed mechanisms to maintain K + homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K + uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K + (Kdp). The contributions of each of these K + transport systems to cellular K + homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli , where Kdp conferred K + uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K + uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K + depletion. Correspondingly, Kdp-mediated K + uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19 , encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K + required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K + concentration under conditions of limited K + in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K + availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K + levels under fluctuating environmental conditions.

The coccolithophorid Emiliania huxleyi is a bloom-forming marine phytoplankton thought to play a key role as a biological pump that transfers carbon from the surface to the bottom of the ocean, thus contributing to the global carbon cycle. This alga is also known to accumulate a variety of polyunsaturated fatty acids. At 25 °C, E. huxleyi produces mainly 14:0, 18:4n − 3, 18:5n − 3 and 22:6n − 3. When the cells were transferred from 25 °C to 15 °C, the amount of unsaturated fatty acids, i.e. 18:1n − 9, 18:3n − 3 and 18:5n − 3, gradually increased. Among the predicted desaturase genes whose expression levels were up-regulated at low temperature, we identified a gene encoding novel ∆15 fatty acid desaturase, EhDES15, involved in the production of n − 3 polyunsaturated fatty acids in E. huxleyi. This desaturase contains a putative transit sequence for localization in chloroplasts and a ∆6 desaturase-like domain, but it does not contain a cytochrome b5 domain nor typical His-boxes found in ∆15 desaturases. Heterologous expression of EhDES15 cDNA in cyanobacterium Synechocystis sp. PCC 6803 cells increased the level of n − 3 fatty acid species, which are produced at low levels in wild-type cells grown at 30 °C. The orthologous genes are only conserved in the genomes of prasinophytes and cryptophytes. The His-boxes conserved in orthologues varied from that of the canonical ∆15 desaturases. These results suggested the gene encodes a novel ∆15 desaturase responsible for the synthesis of 18:3n − 3 from 18:2n − 6 in E. huxleyi.

A conserved hypothetical protein, Sll1130, is a novel transcription factor that regulates the expression of major heat-responsive genes in Synechocystis sp. PCC6803. Synechocystis exhibited an increased thermotolerance due to disruption of sll1130. Δsll1130 cells recovered much faster than wild-type cells after they were subjected to heat shock (50°C) for 30 min followed by recovery at 34°C for 48 h. In Δsll1130 cultures, 70% of the cells were viable compared with the wild-type culture in which only 30% of the cells were viable. DNA microarray analysis revealed that in Δsll1130, expression of the heat-responsive genes such as htpG, hspA, isiA, isiB and several hypothetical genes were up-regulated. Sll1130 binds to a conserved inverted-repeat (GGCGATCGCC) located in the upstream region of the above genes. In addition, both the transcript and protein levels of sll1130 were immediately down-regulated upon shift of wild-type cells from 34 to 42°C. Collectively the results of the present study suggest that Sll1130 is a heat-responsive transcriptional regulator that represses the expression of certain heat-inducible genes at optimum growth temperatures. Upon heat shock, a quick drop in the Sll1130 levels leads to de-repression of the heat-shock genes and subsequent thermal acclimation. On the basis of the findings of the present study, we present a model which describes the heat-shock response involving Sll1130.

Histidine kinase Hik33 responds to a variety of stress conditions and regulates the expression of stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. However, the mechanisms of response and regulation remain unknown. Generally, a histidine kinase perceives a specific signal via its N-terminal region. Hik33 has two transmembrane helices, a periplasmic loop, and HAMP and PAS domains in its N-terminal region, all of which might be involved in signal perception. To investigate the functions of these subdomains in vivo, we expressed a chimeric histidine kinase (Hik33n-SphSc) by fusing the N-terminal region of Hik33 with the C-terminal region of a sensory histidine kinase that is activated under phosphate-deficient conditions, SphS. Hik33n-SphSc responded to several stimuli that are perceived by intact Hik33 and regulated expression of the phoA gene for alkaline phosphatase, which is normally regulated under phosphate-deficient conditions by SphS. We introduced genes for modified versions of Hik33n-SphSc into Synechocystis and monitored expression of phoA under standard and stress conditions. Hik33n-SphSc lacking either the transmembrane helices or both the HAMP and PAS domains had no kinase activity, whereas Hik33n-SphSc lacking the HAMP or the PAS domain enhanced expression of phoA. Moreover, variants of Hik33n-SphSc, in which the membrane-localizing region was replaced by those of other histidine kinases, also responded to stress conditions. Thus, transmembrane helices, regardless of sequence, appear to be essential for the function of Hik33, while the HAMP and PAS domains play important roles in regulating kinase activity in vivo.

Cyanobacteria are the only prokaryotes possessing plasma, thylakoid, and outer membranes. The plasma membrane of a cyanobacterial cell is essential for the biogenesis of cyanobacterial photosystems and serves as a barrier against environmental stress. We previously identified dozens of salt-responsive proteins in the plasma membrane of Synechocystis sp. PCC 6803. Five histidine kinases (Hiks) including Hik33 were also proposed to be involved in the perception of salt stress in Synechocystis. In this study, we analyzed proteomic profiles of the plasma membrane from a hik33-knockout mutant (ΔHik33) under normal and salt-stress conditions. Using 2D-DIGE followed by mass spectrometry analysis, we identified 26 differentially expressed proteins in ΔHik33 mutant cells. Major changes, due to the Hik33 mutation, included the substrate-binding proteins of ABC transporters, such as GgtB and FutA1, regulatory proteins including MorR and Rre13, as well as several hypothetical proteins. Under salt-stress conditions, the Hik33 mutation reduced levels of 7 additional proteins, such as NrtA, nitrate/sulfonate/bicarbonate-binding protein and LexA, and enhanced levels of 9 additional proteins including SphX. These observations suggest a substantial rearrangement in the plasma membrane proteome of Synechocystis due to the loss of hik33. Furthermore, a comprehensive molecular network was revealed in ΔHik33 mutant coping with salt stress.

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.

InSynechocystissp. PCC 6803 the histidine kinase SphS (sll0337) is involved in transcriptional activation of the phosphate (Pi)-acquisition system which includes alkaline phosphatase (AP). The N-terminal region of SphS contains both a hydrophobic region and a Per-Arnt-Sim (PAS) domain. The C-terminal region has a highly conserved transmitter domain. Immunological localization studies on heterologously expressed SphS inEscherichia coliindicate that the hydrophobic region is important for membrane localization. In order to evaluate the function of the N-terminal region of SphS, deletion mutants under the control of the native promoter were analysed forin vivoAP activity. Deletion of the N-terminal hydrophobic region resulted in loss of AP activity under both Pi-deficient and Pi-sufficient conditions. Substitution of the hydrophobic region of SphS with that from the Ni2+-sensing histidine kinase, NrsS, resulted in the same induction characteristics as SphS. Deletion of the PAS domain resulted in the constitutive induction of AP activity regardless of Piavailability. To characterize the PAS domain in more in detail, four amino acid residues conserved in the PAS domain were substituted with Ala. Among the mutants R121A constitutively expressed AP activity, suggesting that R121 is important for the function of the PAS domain. Our observations indicated that the presence of a transmembrane helix in the N-terminal region of SphS is critical for activity and that the PAS domain is involved in perception of Piavailability.

In the cyanobacterium Synechocystis sp. PCC 6803, the histidine kinase Hik33 regulates the expression of several stress-inducible genes. Recently, a yeast two-hybrid screen revealed a specific interaction between Hik33 and a small protein, Ssl3451. To investigate the function of Ssl3451, we developed an assay to monitor the autophosphorylation of Hik33 in vitro. Addition of Ssl3451 to the reaction mixture dramatically enhanced the autophosphorylation activity of Hik33. Pulse-chase experiments revealed that Ssl3451 stimulated the autophosphorylation of Hik33 but did not affect its dephosphorylation. These findings indicated that Ssl3451 might be an activator of Hik33. When the amount of Hik33 was kept constant and the amount of Ssl3451 was increased in the reaction mixture, the extent of autophosphorylation of Hik33 reached a plateau when equimolar concentrations were present, suggesting that Ssl3451 enhances the activity of Hik33 by associating with it with a 1 : 1 stoichiometry. Disruption of the gene for Ssl3451 resulted in increased expression of the hliB gene, which is induced by Hik33 under standard growth conditions, but it did not affect the levels of the hliB mRNA at low temperature. Together, these results suggest that Ssl3451 might enhance the activity of Hik33 both in vitro and in vivo.

Proteomic analysis of the heat shock response of wild type and a mutant of the histidine kinase 34 gene (Deltahik34), which shows increased thermal tolerance, has been performed in the cyanobacterium Synechocystis sp. PCC6803. In vivo radioactive labelling demonstrates that major proteomic changes occur within 1 h of heat shock. 2-D DIGE and MS have been used to quantify changes in specific proteins following heat shock in the wild type and the mutant. Over 100 spots, corresponding to 65 different proteins alter following heat shock. Changes occur not only in the classical heat shock proteins but also in the protein biosynthetic machinery, amino acid biosynthetic enzymes, components of the light and dark acts of photosynthesis and energy metabolism. The Deltahik34 cells have elevated levels of heat shock proteins under both non-heat shock and heat shock conditions, in comparison to the wild type, consistent with Hik34, or a down stream component, being a negative regulator of heat shock-responsive genes.

Histidine kinases (Hiks) in Synechocystis sp. PCC 6803 are involved in the transduction of signals associated with various kinds of environmental stress. To examine the potential role in thermotolerance of Hiks, we used genome microarray analysis to screen a Hik knockout library for mutations that affected the expression of genes for heat shock proteins. Mutation of the hik34 gene enhanced the levels of transcripts of a number of heat shock genes, including htpG and groESL1. Overexpression of the hik34 gene repressed the expression of these heat shock genes. In addition, the cells with a mutant gene for Hik34 (ΔHik34 cells) survived incubation at 48°C for 3 h, while wild-type cells and cells with mutations in other Hiks were killed. However, mutation of the hik34 gene had only an insignificant effect on the global expression of genes upon incubation of the mutant cells at 44°C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that levels of GroES and HspA were elevated in ΔHik34 cells after incubation of cells at 42°C for 60 min. We overexpressed recombinant Hik34 protein in Escherichia coli and purified it. We found that the protein was autophosphorylated in vitro at physiological temperatures, but not at elevated temperatures, such as 44°C. These results suggest that Hik34 might negatively regulate the expression of certain heat shock genes that might be related to thermotolerance in Synechocystis.

In previous studies, we characterized five histidine kinases (Hiks) and the cognate response regulators (Rres) that control the expression of approximately 70% of the hyperosmotic stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we screened a gene knock-out library of Rres by RNA slot-blot hybridization and with a genome-wide DNA microarray and identified three Hik-Rre systems, namely, Hik33-Rre31, Hik10-Rre3, and Hik16-Hik41-Rre17, as well as another system that included Rre1, that were involved in perception of salt stress and transduction of the signal. We found that these Hik-Rre systems were identical to those that were involved in perception and transduction of the hyperosmotic stress signal. We compared the induction factors of the salt stress- and hyperosmotic stress-inducible genes that are located downstream of each system and found that these genes responded to the two kinds of stress to different respective extents. In addition, the Hik33-Rre31 system regulated the expression of genes that were specifically induced by hyperosmotic stress, whereas the system that included Rre1 regulated the expression of one or two genes that were specifically induced either by salt stress or by hyperosmotic stress. Our observations suggest that the perception of salt and hyperosmotic stress by the Hik-Rre systems is complex and that salt stress and hyperosmotic stress are perceived as distinct signals by the Hik-Rre systems.

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-phosphogluconate 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.

The photosynthetic machinery and, in particular, the photosystem II (PSII) complex are susceptible to strong light, and the effects of strong light are referred to as photodamage or photoinhibition. In living organisms, photodamaged PSII is rapidly repaired and, as a result, the extent of photoinhibition represents a balance between rates of photodamage and the repair of PSII. In this study, we examined the roles of electron transport and ATP synthesis in these two processes by monitoring them separately and systematically in the cyanobacterium Synechocystis sp. PCC 6803. We found that the rate of photodamage, which was proportional to light intensity, was unaffected by inhibition of the electron transport in PSII, by acceleration of electron transport in PSI, and by inhibition of ATP synthesis. By contrast, the rate of repair was reduced upon inhibition of the synthesis of ATP either via PSI or PSII. Northern blotting and radiolabeling analysis with [35S]Met revealed that synthesis of the D1 protein was enhanced by the synthesis of ATP. Our observations suggest that ATP synthesis might regulate the repair of PSII, in particular, at the level of translation of the psbA genes for the precursor to the D1 protein, whereas neither electron transport nor the synthesis of ATP affects the extent of photodamage.

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.

The cyanobacterium Synechocystis sp. PCC 6803 accumulates the compatible solute glucosylglycerol (GG) and sucrose under salt stress. Although the molecular mechanisms for GG synthesis including regulation of the GG-phosphate synthase (ggpS) gene, which encodes GgpS, has been intensively investigated, the role of GG in protection against salt stress remains poorly understood. In our study of the role of GG in the tolerance to salt stress, we found that salt stress due to 450 mm NaCl inhibited cell division and significantly increased cell size in ΔggpS mutant cells, whereas the inhibition of cell division and increase in cell size were observed in wild-type cells at high concentrations of NaCl, such as 800 mm. Electron microscopy revealed that, in ΔggpS cells, separation of daughter cells was incomplete, and aborted division could be recognized by the presence of a structure that resembled a division ring. The addition of GG to the culture medium protected ΔggpS cells against salt stress and reversed the adverse effects of NaCl on cell division and cell size. These observations suggest that GG is important for salt tolerance and thus for the proper division of cells under salt stress conditions.

The unicellular cyanobacteria Synechocystis sp. (PCC6803) has become a model organism for a range of biochemical and molecular biology studies aimed at investigating environmental stress responses. In this study the soluble proteins of Synechocystis were analysed using narrow pH range (pH 4.5-5.5) zoom gels, automated matrix-assisted laser desorption/ionization mass spectrometry acquisition, spectral processing and database searching. The work sets the foundation for investigations of proteomic changes following stress treatment. One hundred and ninety-two protein spots were analysed and 105 proteins identified, of these 37 were novel proteins not previously seen on two-dimensional gels. Proteins involved in amino acid biosynthesis, energy metabolism and protein modification were identified using this fully automated procedure demonstrating that automated acquisition and processing will be a useful tool for proteomic analyses on this organism.

Mn is an essential component of the oxygen-evolving machinery of photosynthesis and is an essential cofactor of several important enzymes, such as Mn-superoxide dismutase and Mn-catalase. The availability of Mn in the environment varies, and little is known about the mechanisms for maintaining cytoplasmic Mn(2+) ion homeostasis. Using a DNA microarray, we screened knockout libraries of His kinases and response regulators of Synechocystis sp PCC 6803 to identify possible participants in this process. We identified a His kinase, ManS, which might sense the extracellular concentration of Mn(2+) ions, and a response regulator, ManR, which might regulate the expression of the mntCAB operon for the ABC-type transporter of Mn(2+) ions. Furthermore, analysis with the DNA microarray and by reverse transcription PCR suggested that ManS produces a signal that activates ManR, which represses the expression of the mntCAB operon. At low concentrations of Mn(2+) ions, ManS does not generate a signal, with resulting inactivation of ManR and subsequent expression of the mntCAB operon.

A histidine kinase, Hik33, appears to sense decreases in temperature and to regulate the expression of certain cold-inducible genes in the cyanobacterium Synechocystis sp. PCC6803. To examine the role of Hik33 in the regulation of gene expression, we analysed a ΔHik33 mutant using the DNA microarray technique. In wild-type cells, genes that were strongly induced at low temperature encoded proteins that were predominantly subunits of the transcriptional and translational machinery. Most cold-repressible genes encoded components of the photosynthetic machinery. Mutation of the hik33 gene suppressed the expression of some of these cold-regulated genes, which could be divided into three groups according to the effect of the mutation of hik33. In the first group, regulation of gene expression by low temperature was totally abolished; in the second group, the extent of such regulation was reduced by half; and, in the third group, such regulation was totally unaffected. These results suggest that expression of the genes in the first group is regulated solely by Hik33, expression of genes in the third group is regulated by an as yet unidentified cold sensor, and expression of genes in the second group is regulated by both these cold sensors.

The gene products of sll0337 and slr0081 in Synechocystis sp. PCC 6803 have been identified as the homologues of the Escherichia coli phosphate-sensing histidine kinase PhoR and response regulator PhoB, respectively. Interruption of sll0337, the gene encoding the histidine protein kinase, by a spectinomycin-resistance cassette blocked the induction of alkaline phosphatase activity under phosphate-limiting conditions. A similar result was obtained when slr0081, the gene encoding the response regulator, was interrupted with a cassette conferring resistance to kanamycin. In addition, the phosphate-specific transport system was not up-regulated in our mutants when phosphate was limiting. Unlike other genes for bacterial phosphate-sensing two-component systems, sll0337 and slr0081 are not present in the same operon. Although there are three assignments for putative alkaline phosphatase genes in the Synechocystissp. PCC 6803 genome, only sll0654 expression was detected by northern analysis under phosphate limitation. This gene codes for a 149 kDa protein that is homologous to the cyanobacterial alkaline phosphatase reported in Synechococcussp. PCC 7942 [Ray, J.M., Bhaya, D., Block, M.A. and Grossman, A.R. (1991) J. Bact. 173: 4297‐4309]. An alignment identified a conserved 177 amino acid domain that was found at the N-terminus of the protein encoded by sll0654 but at the C-terminus of the protein in Synechococcussp. PCC 7942.

Low temperature is an important environmental factor that has effects on all living organisms. Various low-temperature-inducible genes encode products that are essential for acclimation to low temperature, but low-temperature sensors and signal transducers have not been identified. However, systematic disruption of putative genes for histidine kinases and random mutagenesis of almost all the genes in the genome of the cyanobacterium Synechocystis sp. PCC 6803 have allowed us to identify two histidine kinases and a response regulator as components of the pathway for perception and transduction of low-temperature signals. Inactivation, by targeted mutagenesis, of the gene for each of the two histidine kinases and inactivation of the gene for the response regulator depressed the transcription of several lowtemperature-inducible genes.

The role of unsaturated fatty acids in membrane lipids in the tolerance of the photosynthetic machinery to salt stress was studied by comparing thedesA−/desD−mutant ofSynechocystissp. PCC 6803, which contained monounsaturated fatty acids, with the wild-type strain, which contained a full complement of polyunsaturated fatty acids. In darkness, the loss of oxygen-evolving photosystem II activity in the presence of 0.5 M NaCl or 0.5 M LiCl was much more rapid indesA−/desD−cells than in wild-type cells. Oxygen-evolving activity that had been lost during incubation with 0.5 M NaCl in darkness returned when cells were transferred to conditions that allowed photosynthesis or respiration. Recovery was much greater in wild-type than indesA−/desD−cells, and it was prevented by lincomycin. Thus, the unsaturation of fatty acids is important in the tolerance of the photosynthetic machinery to salt stress. It appears also that the activity and synthesis of the Na+/H+antiporter system might be suppressed under high-salt conditions and that this effect can be reversed, in part, by the unsaturation of fatty acids in membrane lipids.

Histidine kinases are sensory proteins involved in the perception of environmental changes. Here, we characterized one of three essential histidine kinases, Hik2, in the cyanobacterium Synechocystis sp. PCC 6803 by constructing a fused sensor, Hik2n–Hik7c, which has the signal input domain of Hik2 and the kinase domain of the phosphate-deficiency sensor Hik7. The coding region of the hik7 gene was replaced with the fused sensor to evaluate the signalling activity in vivo as the activity of alkaline phosphatase (AP), which is regulated by Hik7. Cells expressing Hik2n–Hik7c had weak AP activities under standard growth conditions. Saline stress by NaCl induced AP activity in a dose-dependent manner. Analysis of the effects of several salt compounds on induction of AP activity indicated that Hik2n–Hik7c responded to Cl− concentration. Amino acid substitution in the signal input domain of Hik2 resulted in loss of this responsiveness. These results suggest that the signal input domain of Hik2 responds to environmental Cl− concentration in Synechocystis .

CyanoPhyChe is a user friendly database that one can browse through for physico-chemical properties, structure and biochemical pathway information of cyanobacterial proteins. We downloaded all the protein sequences from the cyanobacterial genome database for calculating the physico-chemical properties, such as molecular weight, net charge of protein, isoelectric point, molar extinction coefficient, canonical variable for solubility, grand average hydropathy, aliphatic index, and number of charged residues. Based on the physico-chemical properties, we provide the polarity, structural stability and probability of a protein entering in to an inclusion body (PEPIB). We used the data generated on physico-chemical properties, structure and biochemical pathway information of all cyanobacterial proteins to construct CyanoPhyChe. The data can be used for optimizing methods of expression and characterization of cyanobacterial proteins. Moreover, the 'Search' and data export options provided will be useful for proteome analysis. Secondary structure was predicted for all the cyanobacterial proteins using PSIPRED tool and the data generated is made accessible to researchers working on cyanobacteria. In addition, external links are provided to biological databases such as PDB and KEGG for molecular structure and biochemical pathway information, respectively. External links are also provided to different cyanobacterial databases. CyanoPhyChe can be accessed from the following URL: http://bif.uohyd.ac.in/cpc.

A glucose-tolerant strain of the cyanobacterium Synechocystis sp. PCC 6803, generally referred to as wild type, produces a hemolysin-like protein (HLP) located on the cell surface. To analyze the function of HLP, we constructed a mutant in which the hlp gene was disrupted. The growth rate of the mutant was reduced when the cells were stressed by treatment with CuSO(4), CdCl(2), ZnCl(2), ampicillin, kanamycin, or sorbitol in liquid medium, suggesting that HLP may increase cellular resistance to the inhibitory effects of these compounds. Uptake assays with (109)Cd(2+) using the silicone-oil layer centrifugation technique revealed that both wild type and mutant cells were labeled with (109)Cd(2+) within 1 min. Although the total radioactivity was much higher in the wild-type cells, (109)Cd(2+) incorporation was clearly much higher in the mutant cells after adsorbed (109)Cd(2+) was removed from the cell surface by washing with EDTA. These findings suggest that HLP functions as a barrier against the adsorption of toxic compounds.

Background: Photosystem II (PSII) is the most thermally sensitive component of photosynthesis. Thermal acclimation of this complex activity is likely to be critically important to the ability of photosynthetic organisms to tolerate temperature changes in the environment. Methodology/Findings: We have analysed gene expression using whole-genome microarrays and monitored alterations in physiology during acclimation of PSII to elevated growth temperature in Synechocystis sp. PCC 6803. PSII acclimation is complete within 480 minutes of exposure to elevated temperature and is associated with a highly dynamic transcriptional response. 176 genes were identified and classified into seven distinct response profile groups. Response profiles suggest the existence of an early transient phase and a sustained phase to the acclimation response. The early phase was characterised by induction of general stress response genes, including heat shock proteins, which are likely to influence PSII thermal stability. The sustained phase consisted of acclimation-specific alterations that are involved in other cellular processes. Sustained responses included genes involved in phycobillisome structure and modification, photosynthesis, respiration, lipid metabolism and motility. Approximately 60% of genes with sustained altered expression levels have no known function. The potential role of differentially expressed genes in thermotolerance and acclimation is discussed. We have characterised the acclimation physiology of selected gene ‘knockouts’ to elucidate possible gene function in the response. Conclusions/Significance: All mutants show lower PSII rates under normal growth conditions. Basal PSII thermotolerance was affected by mutations in clpB1, cpcC2, hspA, htpG and slr1674. Final PSII thermotolerance was affected by mutations in cpcC2, hik34, hspA and hypA1, suggesting that these gene products play roles in long-term thermal acclimation of PSII.

ThecrhRgene for RNA helicase, CrhR, was one of the most highly induced genes when the cyanobacteriumSynechocystissp. PCC 6803 was exposed to a downward shift in ambient temperature. Although CrhR may be involved in the acclimatization of cyanobacterial cells to low-temperature environments, its functional role during the acclimatization is not known. In the present study, we mutated thecrhRgene by replacement with a spectinomycin-resistance gene cassette. The resultant ΔcrhRmutant exhibited a phenotype of slow growth at low temperatures. DNA microarray analysis of the genome-wide expression of genes, and Northern and Western blotting analyses indicated that mutation of thecrhRgene repressed the low-temperature-inducible expression of heat-shock genesgroEL1andgroEL2, at the transcript and protein levels. The kinetics of thegroESLco-transcript and thegroEL2transcript after addition of rifampicin suggested that CrhR stabilized these transcripts at an early phase, namely 5–60 min, during acclimatization to low temperatures, and enhanced the transcription of these genes at a later time, namely 3–5 h. Our results suggest that CrhR regulates the low-temperature-inducible expression of these heat-shock proteins, which, in turn, may be essential for acclimatization ofSynechocystiscells to low temperatures.

Changes in the supercoiling of genomic DNA play an important role in the regulation of gene expression. We compared the genome-wide expression of genes in cells of the cyanobacterium Synechocystis sp. PCC 6803 when they were subjected to salt, cold, and heat stress, in the presence of novobiocin, an inhibitor of DNA gyrase, and in its absence. The analysis revealed that the expression of a large number of stress-inducible genes depends on the extent of genomic DNA supercoiling. The function of the two-component regulatory systems, which are known as sensors and transducers of salt, cold, and heat stress, depends on, and might be controlled by, the degree of supercoiling of the genomic DNA. These results suggest that stress-induced changes in superhelicity of genomic DNA provide an important permissive background for successful acclimatization of cyanobacterial cells to stress conditions.

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

Previous studies showed that a Ser/Thr protein kinase, SpkA, in Synechocystis sp. strain PCC 6803 is involved in cell motility. The present study, in which DNA microarray analysis and electron microscopy were used, demonstrated that SpkA regulates the expression of putative pilA9-pilA10-pilA11 -slr2018, pilA5-pilA6 , and pilA1-pilA2 operons and is essential for the formation of thick pili.

When cells of the cyanobacterium Synechocystis sp. PCC 6803 are exposed to high temperature they perceive changes in the growth conditions and regulate the expression of genes and synthesize heat-inducible proteins as a response to the heat stress. DNA microarray analysis revealed that genes for chaperonins and proteases, such as groESL1, groEL2, htpG, hspA, and clpB1 were transiently induced after incubation of the cells at 44 degrees C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that the levels of these chaperonins and proteases were elevated after incubation of cells at 44 degrees C for 60 min. These findings indicated that levels of the mRNAs and proteins of chaperonins were well correlated in the cells of Synechocystis. However, the level of elongation factors are mainly regulated at the protein level. These results indicated that acclimation to the heat-shock conditions might be governed by transcriptional and translational regulation in Synechocystis.

The reason(s) for glucose sensitivity in certain cyanobacterial strains is poorly understood. Inactivation of genes encoding the putative sensor kinase Hik31 inSynechocystissp. strain PCC 6803 resulted in a mutant unable to grow in the presence ofd-glucose. Sensitivities tod-glucose, its analogue 2-deoxy-d-glucose, and fructose, were alleviated in mutants in whichglcP, encoding the glucose transporter, was inactivated. These data indicate that permeation of these substrates is required to inflict cell death. The mutant Δhik31, and the glucose-sensitive strain ofSynechocystis, do not possess glucokinase activity, although a transcript originating fromglk, encoding glucokinase, is present. Inactivation ofglkled to severe sensitivity to glucose, indicating that the presence of glucose itself, within the cells, inflicted this sensitivity. On the other hand, sensitivity to 2-deoxy-d-glucose was lower in Δglk, thus distinguishing between the effect of glucose itself and that of its analogue, which, in the absence of glucokinase activity, may not be phosphorylated. Addition of glucose led to a small rise in glucose-6-phosphate dehydrogenase activity in the wild type, but constitutive activity was observed in the Δhik31mutant regardless of the presence of glucose. Microarray analyses showed only small changes in the abundance of global transcripts inSynechocystisfollowing glucose addition, but the transcription levels of several genes, includingicfG, but notglk, were strongly affected by inactivation ofhik31. The mechanism(s) whereby Hik31 is involved in glucose sensing and response is discussed.

Osmotic stress causes water molecules to efflux from cells through the cytoplasmic membrane. This study reveals that targeted mutation of the aqpZ gene, encoding an aquaporin water channel protein, in the cyanobacterium Synechocystis sp. PCC 6803 prevents the osmotic shrinkage of cells, suggesting that it is the water channel rather than the lipid bilayer that is primarily responsible for water transition through the membrane of this organism. The observations suggest that the aquaporin-mediated shrinkage of the Synechocystis cells plays an important role in changes of gene expression in response to hyperosmotic stress.

Living organisms respond to phosphate limitation by expressing various genes whose products maintain an appropriate range of phosphate concentrations within each cell. We identified previously a two component system, which consists of histidine kinase SphS and its cognate response regulator SphR, which regulates the expression of the phoA gene for alkaline phosphatase under phosphate-limiting conditions in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we used DNA microarrays to investigate the role of SphS and SphR in the regulation of the genome-wide expression of genes in response to phosphate limitation. In wild-type cells, phosphate limitation strongly induced the expression of 12 genes with induction factors greater than 7. These genes were included in three clusters of genes, namely, the pst1 and pst2 clusters that encode phosphate transporters; the phoA gene and the nucH gene for the extracellular nuclease. Phosphate limitation strongly repressed the expression of only the urtA gene with induction factors below 0.2. Inactivation of either of SphS or SphR completely eliminated the phosphate limitation-inducible expression of the 12 genes and the phosphate limitation-repressible expression of the urtA gene. These results suggest that the SphS-SphR two component system in Synechocystis sp. PCC 6803 is the dominant sensory system that controls gene expression in response to phosphate limitation.

A sudden decrease in ambient temperature induces the expression of a number of genes in poikilothermic organisms. We report here that the cold inducibility of gene expression in Synechocystis sp. PCC 6803 was enhanced by the rigidification of membrane lipids that was engineered by disruption of genes for fatty acid desaturases. DNA microarray analysis revealed that cold-inducible genes could be divided into three groups according to the effects of the rigidification of membrane lipids. The first group included genes whose expression was not induced by cold in wild-type cells but became strongly cold-inducible upon rigidification of membrane lipids. This group included certain heat-shock genes, genes for subunits of the sulfate transport system, and the hik34 gene for a histidine kinase. The second group consisted of genes whose cold inducibility was moderately enhanced by the rigidification of membrane lipids. Most genes in this group encoded proteins of as yet unknown function. The third group consisted of genes whose cold inducibility was unaffected by the rigidification of membrane lipids. This group included genes for an RNA helicase and an RNA-binding protein. DNA microarray analysis also indicated that the rigidification of membrane lipids had no effect on the heat inducibility of gene expression. Hik33, a cold-sensing histidine kinase, regulated the expression of most genes in the second and third groups but of only a small number of genes in the first group, an observation that suggests that the cold-inducible expression of genes in the first group might be regulated by a cold sensor that remains to be identified.

Acclimation of microorganisms to environmental stress is closely related to the expression of various genes. We report here that salt stress and hyperosmotic stress have different effects on the cytoplasmic volume and gene expression in Synechocystis sp. PCC 6803. DNA microarray analysis indicated that salt stress strongly induced the genes for some ribosomal proteins. Hyperosmotic stress strongly induced the genes for 3-ketoacyl-acyl carrier protein reductase and rare lipoprotein A. Genes whose expression was induced both by salt stress and by hyperosmotic stress included those for heat-shock proteins and the enzymes for the synthesis of glucosylglycerol. We also found that each kind of stress induced a number of genes for proteins of unknown function. Our findings suggest that Synechocystis recognizes salt stress and hyperosmotic stress as different stimuli, although mechanisms common to the responses to each form of stress might also contribute to gene expression.

Microorganisms respond to hyperosmotic stress via changes in the levels of expression of large numbers of genes. Such responses are essential for acclimation to a new osmotic environment. To identify factors involved in the perception and transduction of signals caused by hyperosmotic stress, we examined the response of Synechocystis sp. PCC 6803, which has proven to be a particularly useful microorganism in similar analyses. We screened knockout libraries of histidine kinases (Hiks) and response regulators (Rres) in Synechocystis by DNA microarray and slot-blot hybridization analyses, and we identified several two-component systems, which we designated Hik-Rre systems, namely, Hik33-Rre31, Hik34-Rre1, and Hik10-Rre3, as well as Hik16-Hik41-Rre17, as the transducers of hyperosmotic stress. We also identified Hik2-Rre1 as a putative additional two-component system. Each individual two-component system regulated the transcription of a specific group of genes that were responsive to hyperosmotic stress.

The hybrid sensory histidine kinase Slr1759 of the cyanobacterium Synechocystis sp. strain PCC 6803 contains multiple sensory domains and a multi-step phosphorelay system. Immuno blot analysis provided evidence that the histidine kinase Slr1759 is associated with the cytoplasmic membrane. The gene slr1759 is part of an operon together with slr1760, encoding a response regulator. A comparative investigation was performed on Synechocystis sp. strain PCC 6803 wild type (WT) and an insertionally inactivated slr1759-mutant (Hik14) which also lacks the transcript for the response regulator Slr1760. The mutant Hik14 grew significantly slower than WT in the early growth phase, when both were inoculated with a low cell density into BG11 medium without additional buffer and when aerated with air enriched with 2% CO2. Since the aeration with CO2-enriched air results in a decrease of the pH value in the medium, the growth experiments indicated that Hik14 is not able to adjust its metabolic activities as rapidly as WT to compensate for a larger decrease of the pH value in the medium. No significant differences in growth between Hik14 and WT were observed when cells were inoculated with a higher cell density in BG11 medium or when the BG11 medium contained 50 mm Epps-NaOH, pH 7.5, to prevent the pH drop. This Hik14 phenotype has so far only been seen under the above defined growth condition. Results of photosynthetic activity measurements as well as Northern blot-, immuno blot-, and metabolite analyses suggest that the two-component system Slr1759/Slr1760 has a function in the coordination of several metabolic activities which is in good agreement with the complex domain structure of Slr1759. The direct targets of this two-component system have so far not been identified.