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Additional file 11. Fatty acid analysis profile obtained by using gas chromatography of Synechocystis sp. PCC6803 WT and Δsll0474 mutant.

Additional file 6. PPI subnetwork of Hik28. The subnetwork was constructed by using STRING. The A. platensis C1 proteins were inferred to that of the A. platensis NIES39 via orthologous group.

Additional file 2. PPI subnetworks of four groups of the differentially expressed proteins; (A) proteins upregulated at 22 °C and downregulated at 40 °C, (B) proteins downregulated at 22 °C and upregulated at 40 °C, (C) proteins upregulated at both temperatures (D) proteins downregulated at both temperatures. The subnetworks were constructed by using STRING. The A. platensis C1 proteins were inferred to that of the A. platensis NIES39 via orthologous group.

Additional file 8. PPI subnetwork of the bi-level regulated proteins. The subnetwork was constructed by using STRING. The A. platensis C1 proteins were inferred to that of the A. platensis NIES39 via orthologous group.

Additional file 7. PPI subnetwork of Hik28 and enzymes involved in fatty acid desaturation. The subnetwork was constructed by using STRING. The A. platensis C1 proteins were inferred to that of the Synechocystis sp. PCC6803 via orthologous group.

Additional file 5. Protein sequence alignment of the eukaryotic type Ser/Thr kinase, PkA, and the Ser/Thr kinases of A. platensis C1; (A) SPLC1_S240280, SPLC1_S032990 and SPLC1_S280030, and (B) SPLC1_S200190, SPLC1_S208550, SPLC1_S532860, SPLC1_S580170 and SPLC1_S541370. The conserved protein domains are labeled.

In the present study, electrophoretic mobility shift assays were used to identify temperature responsive elements in the 5′ upstream region (5′ UTR) of the Spirulina desD gene. Overlapping, synthetic oligonucleotides of both sense and anti-sense strands that spanned the entire 5′ UTR of the gene were analyzed. The responsive DNA-binding protein complexes were identified using liquid chromatography–tandem mass spectrometry. The results indicated that the cold-responsive elements were located at –453 to –247, –197 to –151, –105 to –76, and –50 to –1, whereas the low-temperature specific regulatory regions were located at –372 to –352. Moreover, the heat-responsive elements were located at –347 to –243, –197 to –151, and –124 to –1, whereas the high-temperature specific elements were located between –130 to –101 and –30 to –1. In terms of regulatory protein complexes under the two stress conditions, Trx was only detected in the low-temperature responsive protein complex, and divalent cations were essential for the binding of the protein complex to the regulatory elements. Furthermore, Trx was shown to play a critical role as a reducing agent that inactivates the Spirulina desD repressor, GntR. Consequently, the desD gene expression is induced under the low-temperature condition.

Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield gamma-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b (5). The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-(6) desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-(6) desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b (5) domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-(6) desaturase fused or co-expressed with cytochrome b (5) in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b (5). Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b (5) domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b (5) domain in the desaturation process of Spirulina-(6) desaturase. Furthermore, the free form of cytochrome b (5) domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.

The present study focused on comparative proteome analyses of low- and high-temperature stresses and potential protein-protein interaction networks, constructed by using a bioinformatics approach, in response to both stress conditions. The data revealed two important points: first, the results indicate that low-temperature stress is tightly linked with oxidative stress as well as photosynthesis; however, no specific mechanism is revealed in the case of the high-temperature stress response. Second, temperature stress was revealed to be linked with nitrogen and ammonia assimilation. Moreover, the data also highlighted the cross-talk of signaling pathways. Some of the detected signaling proteins, e.g., Hik14, Hik26 and Hik28, have potential interactions with differentially expressed proteins identified in both temperature stress conditions. Some differentially expressed proteins found in the Spirulina protein-protein interaction network were also examined for their physical interactions by a yeast two hybrid system (Y2H). The Y2H results obtained in this study suggests that the potential PPI network gives quite reliable potential interactions for Spirulina. Therefore, the bioinformatics approach employed in this study helps in the analysis of phenomena where proteome analyses of knockout mutants have not been carried out to directly examine for specificity or cross-talk of signaling components.

This study addresses the importance of a heat-shock-responsive cis-acting DNA element and its transcriptional regulator, which play key roles in the regulation of the Spirulina-desD gene on exposure to high temperatures. Temperature response analysis studies showed that the AT-rich region that is located between nt − 98 to − 80 of the Spirulina-desD gene promoter serves as a binding site for its transcriptional regulator. LC-MS/MS analysis of the DNA-binding protein complex revealed that the amino acid sequences of the bound proteins were homologous to those of several proteins, including a DNA-binding protein, heat shock protein-90 (Hsp90 or HtpG), GroEL and various protein kinases. In addition, western blot analysis indicated that the chaperones GroEL and Hsp90 and a dephosphorylation reaction played a role in the response to elevated temperatures. We conclude that the regulatory DNA segments and the corresponding regulatory binding proteins are distinct for each particular stress condition. This is true, irrespective of whether the regulatory mechanisms that govern the expression of the cold- and heat-regulated desD gene depend on similar phosphorylation- and dephosphorylation-dependent conformational changes that modulate the association of the co-chaperone.

Delta6-Desaturase (D6D) is a key enzyme that catalyzes the synthesis of gamma-linolenic acid (GLA), an essential polyunsaturated fatty acid. We report here the isolation and first functional characterization of the D6D gene promoter from Spirulina platensis C1. Functional analysis of this isolated promoter showed that the Spirulina promoter was functional in Escherichia coli. Site-specific mutation studies demonstrated that the -10 sequence (TATAAT), located at -33bp relative to the translation start site, was essential for D6D promoter function. Temperature responsive deletion analysis studies identified the minimal core promoter within the region -51 to +1, which was sufficient for basal D6D promoter activity, and several cold-shock responsive cis-acting elements with activating and repressing activity. Electrophoretic mobility shift assay and LC-MS/MS studies demonstrated that an 'AT-rich Inverted Repeat' (-192 to -164) served as a target-binding site for a transcriptional regulator (probably a member of the GntR family) from Spirulina. Western blot analysis studies revealed that the DNA-binding transcriptional regulator underwent phosphorylation after a temperature downshift and possibly associated with transcriptional regulation of D6D gene expression. Taken together, our results suggest complex regulation of D6D gene expression in Spirulina.

When the gene desD encoding Spirulina Δ6-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b 5 domain from Mucor rouxii, the results showed the production of GLA (γ-linolenic acid), the product of the reaction catalyzed by Δ6-desaturase. The results revealed that in E. coli cells, where cytochrome b 5 is absent and ferredoxin, a natural electron donor of Δ6-desaturase, is present at a very low level, the cytochrome b 5 domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Δ6-desaturase in E. coli. In comparison to the co-expression of cytochrome b 5 with the Δ6-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Δ6-desaturase co-expressed with cytochrome b 5 and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH2 (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.

Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty acid desaturation process of Spirulina to yield gamma-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Delta(6) desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Delta(6)-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole as their functional group) in the conserved clusters play a critical role in Delta(6)-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker's yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor for the Delta(6) desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Delta(6) desaturation in yeast is more likely to be cytochrome b(5), which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo.

The present study examined the changes in protein expression in Spirulina platensis upon exposure to high temperature, with the changes in expression analyzed at the subcellular level. In addition, the transcriptional expression level of some differentially expressed proteins, the expression pattern clustering, and the protein-protein interaction network were analyzed. The results obtained from differential expression analysis revealed up-regulation of proteins involved in two-component response systems, DNA damage and repair systems, molecular chaperones, known stress-related proteins, and proteins involved in other biological processes, such as capsule formation and unsaturated fatty acid biosynthesis. The clustering of all differentially expressed proteins in the three cellular compartments showed: (i) the majority of the proteins in all fractions were sustained tolerance proteins, suggesting the roles of these proteins in the tolerance to high temperature stress, (ii) the level of resistance proteins in the photosynthetic membrane was 2-fold higher than the level in two other fractions, correlating with the rapid inactivation of the photosynthetic system in response to high temperature. Subcellular communication among the three cellular compartments via protein-protein interactions was clearly shown by the PPI network analysis. Furthermore, this analysis also showed a connection between temperature stress and nitrogen and ammonia assimilation.