Hik28-dependent and Hik28-independent ABC transporters were revealed by proteome-wide analysis of ΔHik28 under combined stress.

Synechocystis histidine kinase, Sll0474: Hik28, a signal protein in a two-component signal transduction system, plays a critical role in responding to a decrease in growth temperature and is also involved in nitrogen metabolism. In the present study, under combined stress from non-optimal growth temperature and nitrogen depletion, a comparative proteomic analysis of the wild type (WT) and a deletion mutant (MT) of Synechocystis histidine kinase, Sll0474: Hik28, in a two-component signal transduction system identified the specific groups of ABC transporters that were Hik28-dependent, e.g., the iron transporter, and Hik28-independent, e.g., the phosphate transporter. The iron transporter, AfuA, was found to be upregulated only in the WT strain grown under the combined stress of high temperature and nitrogen depletion. Whereas, the expression level of the phosphate transporter, PstS, was increased in both the WT and MT strains. Moreover, the location in the genome of the genes encoding Hik28 and ABC transporters in Synechocystis sp. PCC6803 were analyzed in parallel with the comparative proteomic data. The results suggested the regulation of the ABC transporters by the gene in a two-component system located in an adjacent location in the genome.

Revealing the key point of the temperature stress response of Arthrospira platensis C1 at the interconnection of C- and N- metabolism by proteome analyses and PPI networking.

BACKGROUND: Growth-temperature stress causes biochemical changes in the cells and reduction of biomass yield. Quantitative proteome of Arthrospira platensis C1 in response to low- and high temperature stresses was previously analysed to elucidate the stress response mechanism. The data highlighted the linkage of signaling proteins and proteins involved in nitrogen and ammonia assimilation, photosynthesis and oxidative stress. RESULTS: After phosphoproteome analysis was carried out in this study, the tentative temperature response cascade of A. platensis C1 was drawn based on data integration of quantitative proteome and phosphoproteome analysis and protein-protein interaction (PPI) networks. The integration revealed 31 proteins regulated at the protein-expression and post-translational levels; thus, this group of proteins was designated bi-level regulated proteins. PPI networks were then constructed based on A. platensis C1 gene inference from publicly available interaction data. The key two-component system (TCS) proteins, SPLC1_S082010 and SPLC1_S230960, were identified as bi-level regulated proteins and were linked to SPLC1_S270380 or glutamate synthase, an important enzyme in nitrogen assimilation that synthesizes glutamate from 2-oxoglutarate, which is known as the signal compound that regulates the carbon/nitrogen (C/N) balance of cells. Moreover, the role of the p-site in the PPIs of some phosphoproteins of interest was determined using site-directed mutagenesis and a yeast two-hybrid system. Evidence showing the critical role of the p-site in the PPI was observed for the multi-sensor histidine kinase SPLC1_S041070 (Hik28) and glutamate synthase. PPI subnetwork also showed that the Hik28 involved with the enzymes in fatty acid desaturation and nitrogen metabolism. The effect of Hik28-deletion was validated by fatty acid analysis and measurement of photosynthetic activity under nitrogen depletion. CONCLUSIONS: Taken together, the data clearly represents (i) the multi-level regulation of proteins involved in the stress response mechanism and (ii) the key point of the temperature stress response at the interconnection of C- and N- metabolism.

Identification of regulatory regions and regulatory protein complexes of the Spirulina desD gene under temperature stress conditions: role of thioredoxin as an inactivator of a transcriptional repressor GntR under low-temperature stress.

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.

Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components.

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.

Identification of a heat shock-responsive cis-acting DNA sequence and its transcriptional regulator: Their roles in the expression of the Spirulina-desD gene in response to heat stress.

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.

Subcellular proteomic characterization of the high-temperature stress response of the cyanobacterium Spirulina platensis.

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.

Truncation mutants highlight a critical role for the N- and C-termini of the Spirulina Delta(6) desaturase in determining regioselectivity.

The results of our previous study on heterologous expression in Escherichia coli of the gene desD, which encodes Spirulina Delta(6) desaturase, showed that co-expression with an immediate electron donor-either cytochrome b ( 5 ) or ferredoxin-was required for the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6) desaturase. Since a system for stable transformation of Spirulina is not available, studies concerning Spirulina-enzyme characterization have been carried out in heterologous hosts. In this present study, the focus is on the role of the enzyme's N- and C-termini, which are possibly located in the cytoplasmic phase. Truncated enzymes were expressed in E. coli by employing the pTrcHisA expression system. The truncation of the N- and C-terminus by 10 (N10 and C10) and 30 (N30 and C30) amino acids, respectively, altered the enzyme's regioselective mode from one that measures from a preexisting double bond to that measuring from the methyl end of the substrate.

Isolation and functional characterization of Spirulina D6D gene promoter: role of a putative GntR transcription factor in transcriptional regulation of D6D gene expression.

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.

Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.

When the gene desD encoding Spirulina Delta(6)-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 (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-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 Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-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 FADH(2) (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.

Revealing the complementation of ferredoxin by cytochrome b (5) in the Spirulina- (6)-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b (5) domain and Spirulina- (6)-acyl-lipid desaturase.

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.

Functional expression of Spirulina-Delta6 desaturase gene in yeast, Saccharomyces cerevisiae.

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.