- Open Access
Subcellular proteomic characterization of the high-temperature stress response of the cyanobacterium Spirulina platensis
Proteome Science volume 7, Article number: 33 (2009)
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.
High temperature stresses are well known to cause protein aggregation and denaturation, and in order to cope with these stress, a cellular response occurs. Proteomics research regarding cellular responses to high temperature stresses was carried out in bacteria. The majority of the differentially expressed proteins belong to the group of proteins including heat shock responsive chaperones and proteases, of which many are also induced in response to gamma irradiation and/or desiccation [1, 2]. In addition to these proteins, some central metabolic proteins were also found by Fourier transform ion cyclotron resonance (FTIR) mass spectrometric proteomics analysis . Thus, it was hypothesized that elevated temperature may induce a general stress response, and could lead to cross-protection against related stresses .
In cyanobacteria, gene regulation mediated by high temperature stresses has been studied less extensively than regulatory responses to low temperature stresses. In some cyanobacteria, such as Synechocystis, Synechococcus and Nostoc, heat shock responses have been investigated [3–5]. An alternative sigma factor (SigH) and heat-shock protein (HSP) were both significantly induced immediately following exposure to heat stress [6, 7].
Since Spirulina cells are grown in outdoor ponds for mass cultivation, they are exposed to various stress conditions, including high temperature stress. During daylight hours in tropical countries, the cells are exposed to high temperatures of around 40°C. The temperature fluctuation in outdoor mass cultivation has a serious effect on biomass yield and the biochemical content of the cells. Some components of Spirulina cells have pharmaceutical benefits, such as unsaturated fatty acids. The level of unsaturated fatty acids in membrane lipids has been shown to play a critical role in response to temperature change in various organisms. Substantial evidence points to an association between fatty acid desaturation and temperature stress . Due to this relationship, the molecular responses to high temperature stress of genes involved in the desaturation process have been well studied in Spirulina. Upon temperature increase from 35°C to 40°C, the level of the polyunsaturated fatty acid γ-linolenic acid (GLA) in Spirulina plantensis decreases approximately 30%, compared to the level found in cells grown at an optimal temperature (35°C) . This highlights the regulation of Spirulina-Δ6 desaturase, which carries out the last step of the Spirulina desaturation process. Thus, the transcriptional levels of the three Spirulina-desaturase genes, desC, desA and desD, were examined .
Despite the heat shock response studies in other cyanobacteria, transcriptomic and proteomic analyses of responses to high temperature stress have not been performed in Spirulina. The lack of a complete Spirulina genome sequence hinders this relevant research. Therefore, the present study focused on the S. platensis response to a temperature upshift at the subcellular level. This analysis was performed by proteomic and transcriptomic analyses, protein clustering (based on protein expression patterns), and protein-protein interaction analysis.
Materials and methods
Organisms and culture conditions
S. platensis strain C1 cultures were grown at 35°C under illumination by a 100 μEm-2s-1 fluorescent light with continuous stirring in 2 L of Zarrouk's medium . The culture was grown until the optical density at 560 nm reached 0.4 (mid-log phase), and subsequently a cell sample was harvested by filtration before shifting the growth temperature (t = 0 min). The growth temperature was then immediately shifted from 35°C to 40°C and the culture was incubated for 45, 90, or 180 min before cell harvesting.
The harvested cells were washed and lysed as described previously . The three subcellular fractions of Spirulina were separated according to the methods described by Murata and Omata, and Hongsthong et al. [11, 12]. It should be noted that the soluble fraction contained cytoplasmic and periplasmic proteins. The purity of thylakoid (TM) and plasma membrane (PM) fractions were tested by scanning absorption spectra and western blot analysis as described previously . The membrane pellet was resuspended in 500 μl of dissolving buffer, containing 2 M thiourea, 8 M urea, 20 mM Tris, 30 mM DTT, 1% (v/v) IPG buffer, 0.05% (w/v) β-dodecyl maltoside, and 4% (w/v) CHAPS, prior to protein precipitation using a 2D-clean up kit (GE Healthcare Biosciences, USA). The protein pellets were then dissolved in dissolving buffer without DTT before determining protein concentrations using a 2D-Quant kit protein assay (GE Healthcare Biosciences).
Protein separation by two-dimensional differential gel electrophoresis (2D-DIGE) and protein profile analyses
The pH of the protein samples was adjusted to 8.5 and 10 μg of each sample, prepared as described above, was labeled with fluorescent dyes, according to the manufacturer's instructions (GE Healthcare Biosciences). The proteins were separated by 2D-DIGE and statistically analyzed for differential expression as described previously . Protein spot picking and in-gel digestion of the proteins of interest were carried out as described .
To study phosphorylated proteins, 2D-PAGE using 7 cm non-linear IPG strips, pH 3-10 and 4-7 (GE Healthcare Biosciences), in the first dimension were performed. Subsequently, the second dimension was conducted as described above, followed by western blot analysis. Three independent experiments were performed.
Protein identification using MALDI-TOF mass spectrometry
After protein digestion with trypsin, peptide samples were analyzed by MALDI-TOF mass spectrometry. For protein identification, the resulting peptide mass fingerprints (PMFs) were analyzed with our in-house software tool, using the unpublished S. platensis C1 database, which was generated from in silico digestion of the S. platensis C1 completed genome sequence. The search parameters and data filtering were carried out as described in detail previously . PMF identification results of a protein spot were required to be reproducible in order to consider the spot as an identified protein.
Western blot analysis
Following 2D-PAGE, detection of phosphorylated proteins was performed by western blot analysis . Phosphorylations on serine, threonine, and tyrosine residues were detected separately using monoclonal antibodies raised against the designated phosphorylated amino acid residues (Santa Cruz, USA, and Assay Designs, USA). Phosvidin and trypsin inhibitor were used as positive and negative controls, respectively. An equal amount of each protein sample was separated by 2D-PAGE, as described previously, and transferred onto a nitrocellulose membrane using a semi-dry electroblotter at a constant voltage of 20 V for 30 min at room temperature. A chemiluminescent western blot detection kit, with an HRP-based system and chemiluminescent molecular weight markers, was used (Pierce, USA) according to the manufacturer's instructions.
The resulting phosphoproteome spot-maps were then matched with the 2D-DIGE spot-maps over the same pH range. Finally, the analysis of differentially expressed proteins containing phosphorylated amino acid residues was performed.
Transcriptional analysis by RT-PCR
The details of Spirulina RNA isolation were described previously . The transcriptional expression levels of the genes of interest were analyzed by RT-PCR using an AccessQuick™ RT-PCR System (Promega, USA). The RT-PCR analysis was carried out according to the manufacturer's instructions. Details on primers are shown [see Additional file 1]. The densities of the RT-PCR product bands were quantified using the Image Quant TL program (GE Healthcare Biosciences). Normalization of the RT-PCR product levels was performed by comparing the density of the designated band to the density of the 16S rRNA band.
Protein clustering based on expression patterns
The protein expression dataset was validated for the input well-form of protein ratio values. The null values and those ratios that were extremely high or low, relative to the threshold value of 1e+-10, were filtered out. K-mean clustering was applied to obtain 23 profiles of the protein expression patterns. A good k-profile number was chosen by simulation, as described by Martin et al. .
Potential protein-protein interaction network
A protein-protein interaction (PPI) network in Spirulina was constructed on the prototype PPI database of Synechocystis from CyanoBase . Prototype construction is based on a graph in which nodes and edges represent proteins and interactions, respectively. Each interaction was experimentally identified by the yeast two-hybrid system. Source nodes represent the bait proteins, and prey proteins are represented by the target nodes. Next, homologous proteins, identified by BLAST similarity searches with significant values less than 1e-10, were mapped to their best-hit Synechocystis protein nodes. Finally, differentially expressed proteins in Spirulina were mapped to their corresponding nodes. The size of each node represents the level of differential expression.
Results and Discussion
In the present study, 2D-DIGE [see fig S1, S2 and S3; Additional file 2] and mass spectrometry were employed to identify differentially expressed proteins at the subcellular level of Spirulina, in response to a temperature increase from 35°C to 40°C.
Differentially expressed proteins
Up- regulated proteins
Expression profile analysis identified 38, 50 and 26 up-regulated proteins in the PM, soluble, and TM fractions, respectively. Of these, 2, 6 and 2 proteins were phosphorylated in the three respective fractions (Tables 1, 2 and 3). Further analysis showed that several of the up-regulated proteins are involved in two-component signal transduction: histidine kinase, Ser/Thr protein kinase and response regulator, including GGDEF (Gly-Gly-Asp-Glu-Phe) domains. It should be noted that a large number of these proteins were detected in the soluble fraction, suggesting the dissociation of these domains from the membrane-bound domains of the two-component systems during sample preparation. Moreover, our study showed phosphorylation of the GGDEF domain in the soluble fraction, consistent with the report by Ryjenkov et al. . Phosphorylation of this domain is required for its activity. Thus, the GGDEF domains represent the output of complex bacterial signal transduction networks, which convert different signals into the production of a secondary messenger, cyclic diguanylic acid [17, 18]. In addition, it has been reported that the GGDEF domain plays a critical role in heterocyst formation .
Three molecular chaperones were found to be up-regulated in two subcellular fractions, GroEL (Hsp60) and ClpB in the soluble fraction and DnaK (Hsp70) in the thylakoid membrane fraction. The major molecular chaperones, such as DnaK/DnaJ, GroES/GroEL and ClpB, are involved in de novo protein folding of newly synthesized polypeptides and solubilising aggregated proteins under high temperature stress conditions .
Although the chaperone proteins have been generally reported to be soluble proteins, membrane-bound chaperones have been identified in many eukaryotes and prokaryotes, including cyanobacteria. This type of chaperone has been proposed to play a role in protein translocation, translational machinery associated with the surface of the thylakoid membrane, and enhancement of membrane fluidity through association with membrane lipids in response to heat stress .
In the case of stress related proteins, glycosyl transferase and ABC transporter were detected in all subcellular fractions, while membrane helicase, LysR and ferredoxin-glutamate synthase were found in the membrane fractions (TM and PM). Glycosyl transferase has been reported to be involved in osmo- and thermoadaptation .
One up-regulated membrane protein, DEAD/DEAH box helicase, is involved in RNA maturation, proof-reading and enhancement of DNA-unwinding . Interestingly, the helicase present in the PM of Spirulina was phosphorylated. Phosphorylation of RNA helicase is rare, and mostly found in plants. This modification is believed to be a direct link between helicase and environmental sensing-signal transduction phosphorylation cascades . To the best of our knowledge, this is the first evidence of helicase phosphorylation in cyanobacteria.
A few of the proteins involved in DNA damage, repair and modification (endonucleases and methylases) were dramatically induced in the membrane fractions upon temperature upshift. Under stress conditions where DNA damage may occur, induction of SbcC is expected. This exonuclease removes unusual DNA structures, such as hairpins, that are generated upon DNA damage .
In contrast to cold stress conditions in Spirulina, the significant induction of DNA gyrase upon induction of heat stress could lead to elevated function of the DNA repair system . Another up-regulated protein that plays a vital role in DNA replication, repair and chromosome stability is chromosome segregation ATPase [26, 27]. These results demonstrate the requirement for DNA replication, modification and repair for cell survival under heat stress conditions in this cyanobacterium.
Two down-regulated proteins were identified in the PM and soluble fractions, while thirteen down-regulated proteins were identified in the TM fraction (Table 4).
UvrD/REP helicase plays a critical role in DNA repair by restarting stalled replication forks. It facilitates this process by displacing the RecA protein from DNA . The UvrD/REP helicase is known to be part of the SOS response induced by ultraviolet light (UV), which induces DNA lesions . The down-regulation of this protein suggests that the DNA damage caused by exposure to heat stress can be rescued by a different DNA repair system than the SOS response.
Finally, the level of Δ9-desaturase was decreased upon high temperature stress in the photosynthetic membrane of Spirulina. This enzyme catalyses the first step of the fatty acid desaturation process in the TM and PM of this cyanobacterium. We observed that the mRNA stability of this gene decreased dramatically in response to high temperature stress [see fig S4; Additional file 2]. Therefore, the reduction in the level of enzyme is likely caused by the decrease in mRNA stability.
Transcriptional analysis of some differentially expressed proteins
RT-PCR was used to analyze the transcriptional expression levels of some differentially expressed proteins (Fig. 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10) [see fig S5; Additional file 2]. The transcriptional expression patterns of DNA gyrase and chromosome segregation ATPase from the soluble fraction, and ABC transporter, S-adenosyl-L-homocysteine hydrolase and Δ9 desaturase from the thylakoid membrane were well correlated with the protein expression patterns. This correlation suggests that these proteins are likely regulated at the transcriptional level. Interestingly, the Δ9 desaturase gene, the first gene in the fatty acid desaturation process of Spirulina, was previously reported to be temperature-independent . However, an earlier study by our group using Northern blot analysis [see fig S4; Additional file 2] demonstrates that this gene is indeed temperature-dependent, in agreement with the results obtained in the present study.
The transcriptional patterns of sensory box/GGDEF and RNA-directed DNA polymerase, both from the soluble fraction, were different than their protein expression patterns. The transcripts of these genes increased throughout the experimental time period (3 hours), while their protein levels initially increased, followed by reduction to steady state protein levels. Importantly, phosphorylation was detected on both of these proteins. This suggests that the post-translational modification might play a role in the function of these proteins in response to the high temperature stress.
In the thylakoid membrane fraction, the transcription patterns of DnaK and N-6 DNA methylase (adenine specific) were not well correlated with their protein expression patterns, and phosphorylation was not detected for these proteins. These results suggest that these proteins might be regulated at either the post-transcriptional level or the post-translational level (except phosphorylation), although further investigation will be required to confirm this hypothesis. There are some interesting facts related to these three proteins in the photosynthetic membrane of Spirulina. It has been reported that the N-6 DNA methylase uses S-adenosyl-methionine as a methyl-donor for its DNA-methylation reaction in plants . It should be noted that the S-adenosyl-L-homocysteine hydrolase, the enzyme responsible for regeneration of S-adenosyl-methionine [31, 32], was also up-regulated in the same fraction.
In Synechococcus sp. PCC7942, photosystem II (PSII) is drastically deactivated at 40°C . The chaperone DnaK (Hsp70) is present in plant-chloroplast as a component of multi-chaperone complex  and plays a critical role in photoprotection and repair of PSII during and after photoinhibition [35, 36]. Thus, it is expected that this chaperone was up-regulated in response to the high temperature stress in Spirulina, although its regulation should be further investigated.
Adenylate cyclase, which is known to localize in the thylakoid membrane of cyanobacteria, plays a key role in cAMP biosynthesis . The level of cAMP is regulated by red/far red light and thus adenylate cyclase works in association with phytochrome , an up-regulated protein found in Spirulina-TM. These proteins are part of the cAMP-dependent light signaling cascade. Adenylate cyclase has also been reported to be regulated at the post-translational level by ligand binding, protein binding and phosphorylation . Together, our results demonstrate the association between high temperature response and the light signaling cascade.
Clustering of protein expression patterns
The proteins with significantly differential expression in each subcellular fraction were clustered, based on their expression patterns [see fig S6; Additional file 2]. According to Lacerda et al., the expression patterns in response to stress can be classified into three major groups: resistance, adaptation and sustained tolerance . The results shown in Fig. 11, 12 and 13 demonstrate that the majority of proteins in every subcellular fraction belong to the sustained tolerance expression pattern. If all differentially expressed proteins are set as 100%, the percentages of the resistance, adaptation and sustained tolerance groups are: (i) 7%, 3% and 46% in the PM fraction, (ii) 9%, 10%, 46% in the soluble fraction and (iii) 18%, 12% and 58% in the TM fraction, respectively. It should be noted that some patterns do not fit into any categories, and these patterns were mostly found in the PM fraction. Moreover, the plasma membrane, where the environmental changes are first encountered, is the only site where the resistance proteins are present at a significantly higher level than the adaptation proteins.
Site-specific DNA methyltransferase (cytosine-specific) is the only resistance protein that was identified in this study. The level of this protein initially increased and subsequently decreased in the TM fraction (Table 4). DNA methylase is involved in the DNA repair system, and it shows the same expression pattern in response to cadmium stress, which is known to induce DNA-damage . Most of the two component signal transduction systems, stress-related proteins and proteins involved in DNA-damage and DNA-repair are classified in the sustained tolerance group (Fig. 11). This suggests the critical role of these proteins in the tolerance to high temperature stress in Spirulina. It is noteworthy that the resistance proteins (short-term only response) were present at a significantly higher level (2-fold) in the thylakoid membrane than in the other two fractions (Fig. 13). Additionally, adaptation proteins (long-term only response) were found at a higher level in the soluble and the thylakoid membrane fractions (Fig. 12 and 13) than the plasma membrane fraction (Fig. 11).
Potential protein-protein interactions
Several differentially expressed proteins identified in this study can be mapped onto the PPI network available on Cyanobase (Fig. 14, 15 and 16). The potential PPIs shown in the three subcellular fractions represent interesting linkages or cross-talks among the three cellular compartments. For example, in the PM fraction, two component system sensory histidine kinase (spot#1564), ABC transporter (spot#2179), ferredoxin-glutamate synthase (spot#1388) and carboxypeptidase (spot#1284) show interactions with the photosynthetic system. In the soluble fraction, the phosphorylated form of multi-sensor signal transduction histidine kinase (spot#1883) interacts with several periplasmic proteins. However, in the TM fraction, the same protein was found in the non-phosphorylated form. The interactions found in the thylakoid membrane also show communication with the other two fractions.
Additionally, PPI networks clearly demonstrate the linkage between high temperature stress and nitrogen and ammonia assimilation in Spirulina. It is well established that photosynthesis and nitrate reduction are closely related in cyanobacteria and plants, via the nitrate reductase requirement of photoreduced ferredoxin [39, 40]. In response to heat stress, inhibition of photosynthesis and nitrate reductase was observed. Moreover, it was reported by Rajaram and Apte  that a Hsp60 family protein, Cpn60, which is induced by heat stress and stabilized by nitrogen supplementation, either from nitrate or ammonia, is essential for the thermal stability of these vital metabolic processes.
The differentially expressed proteins identified in the subcellular fractions of Spirulina in response to high temperature stress can be functionally classified into 5 major groups: two component systems, stress-related proteins, DNA damage/DNA repair system, translational machinery and proteins with conserved motifs. The transcriptional expression levels of several proteins were studied by RT-PCR. Several of the differentially expressed proteins, such as DNA gyrase and ABC transporter, were regulated at the transcriptional level. Some proteins, such as sensory box/GGDEF domain and RNA-directed DNA polymerase, were found to be regulated at the post-translational level. Finally, other proteins, such as DnaK and adenylate cyclase, were found to be regulated at the post-transcriptional level.
All the differentially expressed proteins were subjected to protein clustering, based on their expression pattern in the three cellular compartments. The clustering data assists in grouping the up- or down-regulated proteins into three major trends: resistance proteins, adaptation proteins and sustained tolerance proteins. The majority of the differentially expressed proteins from all subcellular fractions were found to be sustained tolerance proteins, suggesting the critical role of these proteins in the tolerance of Spirulina to high temperature stress. A group of resistance proteins (short-term only expression) in the photosynthetic membrane was present at 2-fold higher levels than in either of the other two fractions. This is well correlated with the report  that photosynthetic systems are rapidly affected by high temperature (40°C) in the present of light.
According to the data obtained from the PPI network construction, the cross-talk and linkages between the three cellular compartments, via protein-protein interactions, were substantial. The data give clear evidence that the nitrogen and ammonia assimilation processes are affected by exposure to heat stress.
In terms of applications, the present proteomic analysis and PPI network construction are part of an attempt to control and manipulate conditions to maximize polyunsaturated fatty acid (PUFA) biosynthesis in this cyanobacterium. Taken together with the data obtained in our cold-shock response study of S. platensis [11, 13], several proteins involved in fatty acid biosynthesis, such as histidine kinases, (3R)-hydroxymyristoyl- [acyl-carrier-protein]-dehydratase or FabZ, acyl carrier protein (ACP) and Δ9-desaturase, were revealed to be differentially expressed. The knowledge obtained can be applied at the industrial level, for manipulation of PUFA production, as well as in future studies of various aspects of Spirulina. In addition to valuable product biosynthesis in Spirulina, the results from the PPI network are beneficial to the functional annotation of some Spirulina platensis-ORFs. For example, AP07850026 (spot#2410), annotated as a two component system, could possibly be functionally annotated to a two component system in the Nar family, due to its interaction with a protein involved with nitrogen assimilation. Finally, future proteomic analysis of Spirulina will involve analyzing the complete proteome of Spirulina by combining the techniques of 2D-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
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This research was funded by a grant from the National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok, Thailand.
The authors declare that they have no competing interests.
AH carried out the proteome analysis and the protein-protein interaction analysis, conceived of the study, and participated in its design and coordination. MS participated in the proteome analysis. RY participated in the proteome analysis. JS carried out the potential protein-protein interaction construction and others statistical analysis. PK carried out the molecular genetic studies. SC participated in the design of the study. MT participated in the design of the study. All authors read and approved the final manuscript.
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Additional file 2: Additional figures 1-6. The data provided represent spot map of 2D-DIGE of all protein fractions, quantitative analysis of protein, of which mRNAs were analyzed by RT-PCR, and protein clustering based on their expression level. (DOC 3 MB)
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Hongsthong, A., Sirijuntarut, M., Yutthanasirikul, R. et al. Subcellular proteomic characterization of the high-temperature stress response of the cyanobacterium Spirulina platensis . Proteome Sci 7, 33 (2009). https://doi.org/10.1186/1477-5956-7-33
- Heat Stress
- Subcellular Fraction
- Histidine Kinase
- High Temperature Stress