Serine/Threonine Protein Kinase SpkG Is a Candidate for High Salt Resistance in the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

Background Seven serine/threonine kinase genes have been predicted in unicellular cyanobacterium Synechocystis sp. PCC6803. SpkA and SpkB were shown to be required for cell motility and SpkE has no kinase activity. There is no report whether the other four STKs are involved in stress-mediated signaling in Synechocystis PCC6803. Methodology/Principal Findings In this paper, we examined differential expression of the other four serine/threonine kinases, SpkC, SpkD, SpkF and SpkG, at seven different stress conditions. The transcriptional level was up-regulated of spkG and down-regulated of spkC under high salt stress condition. Two spk deletion mutants, ΔspkC and ΔspkG, were constructed and their growth characteristic were examined compared to the wild strain. The wild strain and ΔspkC mutant were not affected under high salt stress conditions. In contrast, growth of spkG mutant was completely impaired. To further confirm the function of spkG, we also examined the effect of mutation of spkG on the expression of salt stress-inducible genes. We compared genome-wide patterns of transcription between wild-type Synechocystis sp. PCC6803 and cells with a mutation in the SpkG with DNA microarray analysis. Conclusion In this study, we first study the spkG gene as sensor of high salt signal. We consider that SpkG play essential roles in Synechocystis sp. for sensing the high salt signal directly, rather than mediating signals among other kinases. Our microarray experiment may help select relatively significant genes for further research on mechanisms of signal transduction of Synechocystis sp. PCC6803 under high salt stress.


Introduction
Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, so their signal transduction systems, which perceive and transduce environmental signals, are important in the acclimation to the environmental changes. In prokaryotes, one-component and two-component signal transduction systems are the pre-eminent mechanisms for signal transduction [1,2,3]. In contrast, the Ser/Thr-specific protein kinases (STK) serve as the backbone of the eukaryotes transduction network [4]. However, with the first identification of an STK in Myxococcus xanthus in 1991 [5], regulatory STKs have been repeatedly identified in prokaryotes, and some of them have been shown to regulate various cellular functions, such as development [6,7,8], stress responses [9,10] and pathogenicity [11]. Protein phosphorylation on serine/threonine residues in cyanobacteria was first revealed by radioactive labeling of proteins in 1994 [12]. Now, a large number of genes are emerging with the availability of whole genome sequences. A total of 286 putative STK genes have been identified from 21 species of cyanobacteria, and functions of most of them are still unknown [13]. The entire nucleotide sequence of the Synechocystis sp. PCC6803, a strain bearing the ability to be transformed naturally [14], was the first cyanobacterial genome to be sequenced. Genome sequence data of Synechocystis has predicted the presence of 7 serine/threonine kinase (PKN2 type) genes, named spkA (sll1574-1575), spkB (slr1697), spkC (slr0599), spkD (sll0776), spkE (slr1443), spkF (slr1225) and spkG [15]. Six of them have been biochemically characterized with the exception of SpkG which was not expressed in Escherichia coli. [15,16,17]. SpkA, SpkB, SpkC, SpkD and SpkF were demonstrated as autophosphorylation and phosphorylation of the general substrate proteins. SpkE did not show any protein kinase activities, which was consistent with its lack of several key amino acid residues. But only SpkA and SpkB were confirmed to be essential for the motility of Synechocystis cells [15,16]. In high plant, environmental stresses trigger a wide variety of plant responses, ranging from altered gene expression and cellular metabolism to changes in growth rate and plant productivity [18]. Here, we examined probable physiological roles of SpkC, SpkD, SpkF and SpkG in Synechocystis . To investigate whether the four STKs are involved in stress-mediated signaling in Synechocystis PCC6803, the expression of spkC, spkD, spkF and spkG were examined under a variety of stress conditions by semi-quantitative RT-PCR. We found the expression of spkC was down-regulated but expression level of spkG was increased to a significantly extent at high salt conditions. In order to better understand the function of spkG and spkC and their importance in salt stress, we constructed a knockout mutant and compared the feature of this strain to that of the wild-type in the presence and absence of high salt 855 mM NaCl (0.5 g/l NaCl). The growth of the spkG mutant strain was completely impaired at 855 mM NaCl conditions in contrast to the wild strain and DspkC mutant, which were unaffected. Based on the above results, the microarrays were used for a highprecision look at differential gene expression when comparing wild-type to a mutant or when comparing differently environmental conditions. We obtained the regulatory genes under high salt stress were induced or repressed by spkG.

Semi-quantitative reverse transcription PCR
To investigate whether the four STKs are involved in stressmediated signaling in Synechocystis PCC6803, the expression of spkC, spkD, spkF and spkG were examined under a variety of stress conditions by semi-quantitative RT-PCR. Total RNA isolated from Synechocystis PCC6803 untreated and stress-treated cells was used to amplify spkC, spkD, spkF and spkG cDNA. The amplified RT-PCR fragment was confirmed by cloning and sequencing. Semi-quantitative RT-PCR expression analysis showed that under standard conditions, spkC and spkD showed relatively high levels compared to spkG (Fig. 1), and there was no detective expression of spkF. A severe decrease was observed in mRNA accumulation for spkC after the treatment of high salt stress and a moderate decrease was detected after the treatment of nitrogen deprivation and high light stress. The levels of spkC transcripts under other stress conditions did not vary significantly. This suggested that the expression of spkC were down-regulated under high salt conditions. The expression levels of spkD did not vary significantly under normal and different stress conditions but was relatively higher compared to the others, consistent with the fact that it is essential for survival,which could not be knocked out completely [17]. No expression of spkF could be detected under any condition (data not shown). After exposure to high salt stress, the expression level of spkG was increased to a significantly extent. A densitometry  showed that the induced level of spkG transcripts s about 1.360.02-fold higher than the constitutive level( Fig. 1), while the other stress conditions had no effect on the transcription level. The up-regulated expression of spkG suggested a mechanism of positive regulation under high salt stress. In two-component signal transduction systems, most histidine kinases are known to respond to stress by post-translational alterations and regulatory proteins could be affected at both the transcriptional and posttranscriptional levels [19][20][21][22]. Also, in Rice, calmodulin-dependent serine/threonine phosphatase was reported to be involved in the process of salt stress tolerance [23]. We examined the transcriptional levels of serine/threonine kinases using RT-PCR and our results suggested that, in Synechocystis at least, STKs could respond to stress conditions by controlling their expression levels.

The detection of mutants and growth curve
To further affirm the probable functions of SpkC and SpkG in high salt stress, two mutants, DspkC and DspkG, were generated by replacing part sequences of these two genes with kanamycin resistance cassettes ( Fig. 2A) and the complete segregation of mutants were verified by PCR analysis (Fig. 2B). We examined the growth of DspkC and DspkG mutants under standard and high salt-stress conditions compared to that of the wild strain. The growth properties of the two mutants were not significant changed compared to the wild strain under normal conditions. The presence of 684 mM NaCl, the same condition with RT-PCR, in the growth medium has no effect on the growth of DspkC, DspkG and wild strain (data not shown). Whereas, the growth of the DspkG mutant strain was almost completely impaired at 855 mM NaCl conditions in contrast to the wild strain and DspkC mutant, which were unaffected (Fig. 3). During their long evolution, cyanobacteria have adapted to aquatic habitats with various salt concentrations [24]. Synechocystis sp. PCC6803 has the ability to regulate essential metabolic processes to enable survival in high salt environments [25]. This result indicates that SpkG may be involved in adaptation to high salt stress condition. In our previous work, we predicted that SpkG has four strong transmembrane helices by the TMbase (www.ch.embnet.org/software/TMPRED_ form.html) [13]. So we predicted that SpkG could sense the high salt signal directly, rather than mediate signals among other kinases.

Examination of the genome-wide expression of high saltstress genes
To elucidate the effect of spkG on the adaptation to high salt condition, we used DNA microarrays to investigate gene expression of salt-stress Synechocystis, wild types of cells and DspkG mutants were treated with high salt-stress at 855 mM NaCl conditions for 30 min. The results of the first screening were confirmed by a second screening. The raw data of microarray in this study was released in a MIAME compliant database with accession number A-MEXP-1953. The results are summarized in Fig. 4. The average signal intensities of treated samples are plotted versus average signal intensities of control samples and the two reference lines are corresponding to ratios of expression of 2.0 and 0.5 in Figure 4. The putative differentially expressed genes were finally selected based on the expression profiles and the following criteria: the data pointing outside this range were regarded as genes that are salt stress-inducible or repressible genes in our DNA microarray experiments. Thus, approximately 25% of the genes in Synechocystis responded positively to high salt stress. 401 genes were induced more than twofold and 358 genes were repressed more than twofold after high salt-stress for wild type Synechocystis, while 401 genes were induced more than twofold and 240 genes were repressed more than twofold for DspkG mutants.
To identify genes whose expression is affected by spkG upon exposure of cells to high salt stress, we compared genome-wide patterns of transcription between wild-type and DspkG cells. The  putative differentially expressed genes were finally selected based on the expression profiles and the following criteria: if the average fold change between wild type and mutants cells was more than or equal to twofold. Sixty genes have been found expressed differently in wild strain and DspkG mutant, the genes that could be divided into four groups: (i) Group 1, genes that were normally saltregulated genes was no longer regulated by salt stress genes in DspkG mutants. (ii) Group 2, genes whose salt inducibility was moderately diminished in DspkG mutants. (iii) Group 3, genes whose salt regulation was heightened in DspkG mutants. (iv) Group4, genes were regulated by salt stress only in DspkG mutants (Table S1).
Gene whose expression was induced or repressed by salt-stress was regulated by spkG Using a DNA microarray, we compared the genome-wide patterns of transcription in wild-type and DspkG cells upon high salt stress to demonstrate the effect of the mutation in spkG on the high salt regulation genes. We found expression of 15 of the normally salt-regulated genes was no longer regulated by salt stress in DspkG mutants (Table S1, group 1). Many of the genes whose salt inducibility were diminished by mutation of spkG. It appeared that the regulation of expression of particular sets of genes by high salt in wild type was controlled by spkG. spkG was essential for the regulation of the expression of 15 genes (group 1) and was partially involved in the regulation of 12 genes (group 2). This group includes genes for proteins whose functions are related to transporters and mechanisms. nrtC and nrtD were reported as a member of the ABC (ATP-binding cassette) superfamily of active transporters. It is essential for the growth of the cyanobacterium at physiological concentrations of nitrate and has been shown to be involved in the active transport of nitrite as well [26] OprB is considered a central component of carbohydrate transport and described as a carbohydrate-selective porin [27]. The ferric uptake regulator (Fur) protein, as originally described in Escherichia coli, is a global regulator that acts as a transcriptional repressor when it binds ferrous ion. Fur also activates the expression of many genes by either indirect or direct mechanisms [28].
Genes whose salt stress-regulated expression were not regulated by spkG The salt inducibility of a large group of genes was unaffected by mutations in spkG, shows the functionally characterized and most strongly salt-induced members of this group. In particular, the fourth group of genes was regulated by salt stress only in DspkG mutants. This group included the following genes: plsC for 1-acylsn-glycerol-3-phosphate acyltransferase; amiA for N-acetylmuramoyl-L-alanine amidase Enzyme catalyzing the preferential transfer of unsaturated fatty acids to the 2-position of the glycerol backbone of phospholipids and triacylglycerols ; ndhA for NADH dehydrogenase subunit I; a Trans-Isoprenyl Diphosphate Synthases Squalene/phytoene synthase(sll0513) synthesis; a C-methyltransferase(slr1610) and a number of other genes for protein with known and unknown function. It is possible that there are several sensory mechanisms in the perception of high salt stress. When the presence of spkG, spkG protein could sense the high salt signal directly and regulated some genes to response to high salt. However, when the absence of spkG, an additional mechanism was activated. Hik16, Hik33, Hik34 and Hik41, have been reported to respond to salt stress and regulate the expression of only one-fifth of the salt-inducible genes [29]. The induction of the remaining 80% salt-inducible genes was unaffected by any histidine kinases mutants, suggesting an additional mechanism absent in Synechocystis 6803. However, the mechanisms involved in sensing specific salt stress signals are not well resolved [20]. Thus, it is probable that complex mechanisms involved in the regulation of the response to salt stress remain to be identified and characterized. In the future, more analysis will be necessary for defining the A. Gene expression in wild-type cells that had been exposed to 855 mM NaCl for 30 min was compared with that in unstressed cells. B. Gene expression in DspkG cells that had been exposed to 855 mM NaCl for 30 min was compared with that in unstressed cells. Dots correspond to genes whose fold change .2 are beyond the reference lines. doi:10.1371/journal.pone.0018718.g004 functions of the many unknown proteins regulated in salt stress cells, which will help us to identify the complex salt-sensing mechanisms in Synechocystis sp. PCC6803.

Strains and growth conditions
The Synechocystis sp. PCC6803 strain was kindly provided by Pro. Xudong Xu (Institute of Hydrobiology, Chinese Academy of Science). Wild-type and derived mutant cells of Synechocystis sp. PCC6803 were grown photoautotrophically in BG11 medium [30] at 30uC and 30 mmol m 22 s 21 white light. Solid BG11medium was supplemented with 1% agar, 8 mM TES (PH8.2) and 5 mM sodium thiosulfate. For mutants, kanamycin was added to a final concentration of 30 mg/ml in the solid medium and 50 mg/ ml in the liquid medium. Cells grew to mid-logarithmic growth phase (optical density at 730 nm of 0.6,0.8) before different stress treatment. For high light treatment, cells were diluted with fresh medium to an OD730 of ,0.3, and then were exposed to high light (500 mmol m 22 s 21 ) for 30 min. For temperature shock experiments, cells were shifted to 15uC and 42uC for 30 min separately. For high salt stress, cells were exposure to 684 mM NaCl for 30 min. In the nutrient deprivation experiment, cells were cultivated in BG-11 medium lacking carbon (2C), nitrogen (2N), or phosphorus (2P) for 2 h. Growth characteristics under high salt stress were examined in BG11 medium with 684 mM and 855 mM NaCl.

Isolation of total RNA
RNA was isolated from 50 ml cultures of wild-type cells. The cells were collected by centrifugation (5000 g for 5 min) and the cell pellets were cooled in liquid nitrogen. Then cooled cells were grinded into powder under the liquid nitrogen and were resuspended in 1 ml Biozol reagent and Chloroform (ratio of 1:5 to Biozol). An equal volume of isopropyl alcohol were added to the aqueous phase after centrifuged at 12000 g for 15 min at 4uC, and the solution was incubated for 2 h at 220uC. Precipitated RNA was collected by centrifugation at 12000 g for 15 min at 4uC and washed with 70% ethanol, resuspended in DEPC-H 2 O. The total RNA was treated with RNase-free DNase I (Promega) at 37uC for 30 min to remove DNA.

RT-PCR analysis
RT-PCR primers were designed to amplify 350-400 bp of internal coding region of each gene (Table 1). Reverse transcription reactions were performed with the random hexamer primers using M-MLV Reverse Transcriptase (Promega). The resulting cDNA was used as the template for RT-PCR. Amplified products were electrophoretically examined on 1% agarose gels. The transcript abundance of rnpA (slr1469) encoding RNase P were used as external standards.

DNA manipulation and mutant construction and culvation
To inactivate spkC gene, two fragments (upstream and downstream) were separately amplified by PCR. Each primer was added a different restriction site at the 59 end (Table 1). These two fragments were then cloned into the pMD-18T vector (TAKARA) one by one after double-digested, resulting in plasmid pMD-CUD. The plasmid pEGFP-1 (BD) was used as template to amplify the selectable cassette encoding the kna genes for resistance to kanamycin. This 1.0 kb kna fragment was then cloned into plasmid pMD-CUD via BamHI and SalI sites added at the ends of the primers, yielding plasmid pCUKD. The plasmid pGUKD to inactive spkG gene was constructed with the same method. Cells of Synechocytis sp. PCC6803 were transformed with plasmids pCUKD and pGUKD according to Williams [31]. Transformants were selected by screening for resistance to 30 mg of kanamycin/ml in BG-11 solid medium supplemented with 10 mM glucose. Segregation of the inactivated spkC and spkG genes were monitored by PCR using genomic DNA of transformants used as the template and primers that recognize sequences upstream or downstream of the inserted kna gene.

MicroArray analysis
A Synechocystis DNA microarray (CyanoCHIP) was purchased from TaKaRa Bio Co. Ltd (Kyoto). This microarray covered 3,079 of the 3,168 ORFs of Synechocystis, excluding genes for transposases. As hybridization probes, Cy3 dyelabeled or Cy5 dyelabeled cDNAs were used, which were synthesized by reverse transcription of total RNA using an RNA Fluorescence Labeling kit (MMLV) provided by TaKaRa. The method of hybridization was performed as described previously [32]. Hybridization of the dye-labeled cDNAs was evaluated with an array scanner. For quantification with the IIlumina BeadArry Reader, the local background of each spot was subtracted and the signal was normalized by transforming it to the ratio of the spot-specific intensity relative to the total intensity of signals from all genes with the exception of rDNA genes. Therefore, changes in the level of transcript of each gene relative to the total level of mRNAs were calculated. Each gene is spotted twice on the microarray, allowing signal evaluation and error exclusion. The gene expression in cells exposed for 30 min to 855 mM NaCl was analyzed by two DNAmicroarray experiments. All data is MIAME compliant and the raw data has been deposited in a MIAME compliant database.