Molecular Cloning, Expression, and Function of Synechocystis PCC6803 type ii Peroxiredoxin (sll1621) Gene in Escherichia coli Cells under salinity stress Conditions

Microorganism’s cycle exposure to reactive oxidants from internal metabolism and abiotic stress conditions, e.g. oxidative stress, salinity, drought, low temperature, high temperature, and high light. Synechocystis PCC6803 genomes typically encode different types of antioxidant scavenging enzymes including Peroxiredoxins (Prxs). There are five genes similar to Prxs were found inthe Synechocystis PCC 6803 genome. Based on sequence homology analysis of Synechocystis PCC 6803sll1621 gene, it is categorized into type II Prx (PrxII). The presumed amino acid sequence of Synechocystis PCC6803 Prxii protein exhibited identity about 44%-99% to other PrxII proteins from human, plants, algae, and other different cyanobacterial cells. In the last decade, the genetically controllable model organism E. coli has been introduced as a viable biotechnological model for genetically modified microorganisms. The Synechocystis PCC6803 sll1621 gene was overexpressed in pTYB21 expression vector and the resulting was named as pTYB21/sll1621. The pTYB21/sll1621 was overexpressed in Escherichia coli BL21 (DE3) host cell. The overexpressed protein of PrxII gave the recombinant E. coli cells the ability to survive under high concentrations of salinity stress, whereas the viability of wild type cells was completely inhibited at the same high concentrations of salinity stress. In conclusion, the present research documented the expressing of the sll1621 gene into E. coli cells. This result approved the absence of species barrier in relations to the function of Synechocystis PCC 6803 PrxII protein in different microorganism.


INTRODUCTION
Peroxiredoxins (Prxs) are a universal group of antioxidant proteins that promote the scavenging of several hydroperoxides 1 . In the past decades, Prxs have gotten extensive consideration as a group of thiol-specific antioxidant proteins. Additionally, they named as thioredoxin peroxidases and/or alkylhydroperoxide reductase proteins 2 . It started back to 1989 when an alkyl hydroperoxide reductase activity was isolated from Salmonellatyphimurium and E. coli. The enzyme was able to decrease cumene hydroperoxide as an electron acceptor with NADH or NADPH as an electron donor 3 . Yet, in a way autonomous of selenium, glutathione and heme can replace NADH and NADPH4. Thus, in such situation, two enzymes named AhpF (52 kDa) and AhpC (22 kDa) correspondingly made the activity 4 . According to their genetic background and catalytic activities, there are four different groups of enzymes that belong to Prxs which are 1-Cys Prx, 2-Cys Prx, PrxII, and PrxQ 5 .
Synechocystis PCC 6803 contains five Prxs genes 6 . These genes are a part of each reported groups as sll1621 (PrxII), sll0755 (2-Cys Prx), slr1198 (1-Cys Prx), and slr0242, sll0221 (Prx Q) 6 . Investigations of cyanobacterial Prxs mutant strains propose that the physiological roles of these proteins are focused on the adjustment of the growth of the cyanobacterial cells at high light intensities, however the catalytic activity may not generally include peroxide detoxification 1,7 . Interestingly, Synechocystis PCC 6803 PrxII disrupting mutant strain showed a seriously diminished development and growth rate comparative to the growth rate of wild type cells under even normal light intensities 1,7 . Interestingly, a mutant strain of Anabaena PCC 7120 was failed to express one of its four PrxQ, displayed a slow growth rate at moderate light intensities and was hypersensitive to methyl viologen 8 . Perez-Perez et al. 9 stated that all five enzymes of Synechocystis PCC 6803 Prxs could utilize thioredoxins as an electron donor. The most noteworthy catalytic ability was acquired for the two enzymes of PrxII and TrxQ with thioredoxin as an electron donor 9 . Kobayashi et al. 7 indicated that the expression level of Synechocystis PCC6803 sll1621 gene was significantly induced under oxidative stress through the treatment of Synechocystis PCC6803 wild-type cells to methyl viologen for 15 min under high light intensities. Additionally, numerous defensive mechanism genes are liable for adaptation under a biotic stress conditions in Synechocystis PCC6803 cells. For example, 1-Cys Prx (slr1198) and PrxII (sll1621) genes are overexpressed under highlight treatment 10 . Also,another antioxidant enzyme in Synechocystis PCC6803, NADPH-dependent glutathione peroxidase-like protein (slr1992), was found to be overexpressed and enhance the transgenic Arabidopsis plants under several abiotic stress including salinity, drought, chilling and highlight 11,12 . Additionally, it was reported that the mRNA levels of sll1621 under conditions of paraqout-induced oxidative stress increased seven times more than the untreated cells 13 . All these data clearly show the importance of the sll1621 gene to scavenge the free radicals of reactive oxygen species (ROS) particularly under different abiotic stress conditions such as the exposure to high light and methyl viologen.
The aim of the present researchis to further analyze the effect of the overexpression of the Synechocystis PCC 6803 PrxII protein on the growth of E. coli BL21 cells under salt stress condition that induced oxidative stress. The present data will help to more emphasizing of the physiological role of Synechocystis PCC 6803 PrxII under salt stress and its use for the scavenging of ROS that generated under salt stress.

Chemicals and bacterial strains
Ampicillin, X-gal and IPTG purchased from UFC Biotechnology (USA). The restriction enzyme EcoRI and E. coli BL21 strain were acquired from NEB (USA). Other different synthetic compounds were obtained with the highest caliber financially accessible. Cloning vector (pGEM-T easy) was bought from Promega (USA); whereas, the expression vector pTVB21 was gotten from NEB (USA).

Growth conditions
Synechocystis sp. PCC 6803 cells was grown in Allen's medium 14 under dim light condition with shaking for five to seven days. Cloning E. coli host cell (DH5a) and the expression E. coli host cells (BL21 DE3) were cultured in LB broth media at 36°C. Cells harboring recombinant plasmid was cultured and kept on LB media accompanied with ampicillin (100 mg/mL).

Gene expression of Synechocystis PCC6803 sll1621 in E. coli
Total DNA of Synechocystis PCC6803 cells was isolated as described previously 15 . The specific sll1621 gene was isolated using PCR with the following primer: 5'-CATATGACCCCC-GAACGAGTTCC-3' (forward primer) and 5'-CTCGAGTTAGCCG-ACAAAAGCTTTAACG-3' (reverse primer). Both primers were designed to introduce restriction enzymes sites of NdeI in forward primer and XhoI in reverse primer. The purified PCR product was successfully cloned into E. coli DH5a host cells (Promega, USA) using pGEM-T easy vector according to the instruction of the supplier. The positive transformed colony was confirmed by PCR and DNA sequencer. Next, the confirmed sequenced gene was over expressed in E. coli BL21 (DE3) (NEB, USA) host cell using pTYB21 vector (NEB, USA). The resulting construct was designated as pTYB21/sll1621.

Production of the recombinant protein
The recombinant pTYB21/sll1621vector was cultured in five mL LB broth accompanied with ampicillin (100 mg/mL)at two different temperatures (16°C or 36°C), then left overnight. Next, the overnight cultures were inoculated to a new 100 mL of LB broth. When the OD 600 = 0.4, IPTG (400 mmol/L) was added and the culture cells were grown for additional three hours to induce the recombinant protein. In parallel, wild type strain harboring empty pTYB21 vector was cultured. Both cells were harvested, and SDS-PAGE was achieved in 12% (w/v) as descripted before 14 .

Evaluate of salt stress tolerance of recombinant E. coli cells
The recombinant (pTYB21/sll1621) and wild type (pTYB21/empty) cells were cultured in 50 mL LB broth accompanied with ampicillin (100 mg/ mL) at 36°C with shaking. After the induction of the recombinant protein, both cells were collected by centrifugation at 5000 rpm/10 min. Next, the collected cells were re-inoculated in five ml of LB broth and distributed by equal concentration on LB agar medium including several concentrations of NaCl (0, 400, 800, and 1000mM) and were grown overnight at 36°C.

Protein sequence evaluation
T h e a m i n o a c i d s s e q u e n c e o f Synechocystis PCC 6803 PrxIIwas alignment with other PrxIIproteins from different organisms using ClustalW program 16 . The molecular information of PrxII protein (amino acids composition, molecular weight and pI value)wereestimatedusing the ProtParam resourcesfrom ExPASy website as described previously 15 .

RESULTS AND DISCUSSION Characterization of the sll1621 gene
To defend the cells and organ against oxidative stress and ROS, aerobic organisms have developed a profoundly classy and complex antioxidant prevention system 17 . It includes a diversity of components, both endogenous and exogenous in source. Both components work collaboratively to remove free radicals 17 . Type II Prxs are one of the most important peroxiredoxins proteins that can catalyze the reduction of various hydroperoxides 1,2 . Investigations of cyanobacterial Prxs mutant cells propose that these proteins work in adjustment to growth and development of cyanobacteria at high-light intensity, however the catalytic activity may not generally contain peroxide detoxification 1 . It was found that the interruption of the sll1621 gene had a reduction consequence on the survival growth rate of Synechocystis PCC 6803 cells under normal or dim light circumstances, suggesting that PrxII enzyme is important for the viability of this cells7. The purpose of the present research is to further explore the effect of the overexpression of Synechocystis PCC6803 sll1621 genein E. coli  (Fig. 1). All PrxIIs proteins share high degree of the critical catalytic cysteine residue inside the active site consensus sequence of PGAFTP(T/G)CS 18 . Moreover, all PrxIIs proteins have two cysteine residues. The second cysteine Amino acid sequences were aligned by CLUSTALW method using MAFFT v7.429. The active site is pointed out by black frame, and the two conserved cysteine residues are marked by red asterisk. residue separated from the first one by the same number of amino acid residues (Fig. 1) 18 .
According to the phylogenetic tree analysis of PrxII proteins (Fig. 2), the Synechocystis PCC 6803 PrxII was clustered in same group with the similar PrxII proteins from other prokaryotic algae, including Synechocystis PCC 6714 and Synechococcus PCC 7003. The other PrxII proteins from eukaryotic algae, plants and human were clustered together in second group. Among all of these proteins, Synechocystis PCC 6803 PrxII was most nearly to PrxII from Synechocystis PCC 6714 (Fig. 2). The PrxII proteins of plants, Oryza sativa and Jatropha curcas, have an N-terminal extension about 38 amino acids than Synechocystis PCC 6803 PrxII (Fig. 1). Also, the human PrxII protein have an N-terminal about 25 amino acids than Synechocystis PCC 6803 PrxII. These extensions in plants or human PrxII proteins might be coded for a transit peptide anticipated to address the protein to chloroplast or mitochondria, respectively.

Protein expression of PrxII in E. coli cells
We amplify the sll1621 gene using PCR and the resulted specific product was used for cloning in E. coli DH5a cells (Fig. 3). Thesequence of the PCR product (570 base pair, Fig. 3A lane 1) was checkedusing an automated DNA sequencer apparatus. The results of DNA alignments of the gene productwith other genes in NCBI website indicated that the gene was 100% homologous to Synechocystis PCC 6803 sll1621 gene (data not shown). To further ensure about the full sequence of sll1621 gene, we performed a restriction gene map using EcoRI restriction enzyme. As expected, the restriction map of the sll1621 after cutting with EcoRI was given three fragments with lengths of 259 bp, 229 bp and 123 bp (Fig. 3A, lane 2).
The restriction sites of NdeI and XhoI that involved in the gene product were used to overexpress sll1621 gene into pTYB21 vector. The resulting was namedaspTYB21/sll1621, which was transformed into E. coli BL21 (DE3) cells to obtain there combinant protein. Subsequently, we examined the best conditions for the growth of BL21 (DE3) cells to produce PrxII protein with high expression (Fig. 3B). In this case two types of temperatures were used (15°C and 36°C). The reason for choosing 15°C, as it is recommended for the overexpression of the Intein protein, of the Saccharomyces cerevisiae, that fused at the N-terminus of pTYB21 vector (56 kDa). The recombinant protein was successfully expressed with a sufficient level in BL21 (DE3) cells at 36°C (Fig. 3B). The predicted molecular weight of the recombinant PrxII protein (21.167 kDa) was detected (Fig. 3B, lane 3). Whereas, we could not detect any protein band related to PrxII molecular weight in E. coli control cells (Fig.3B, lane 4). Interestingly, at 15°C, we detect a protein with a molecular weight of 77 kDa in recombinant BL21 (DE3) cells that harboring pTYB21/sll1621 vector (Fig. 3B, lane 2). This molecular weight is considered for the total molecular weight of the Intein protein (56 KDa) that expressed by pTYB21 at 15°C plus PrxII protein (21.16 KDa).

Synechocystis PCC 6803 Prxii protein is necessary for improve growth of E. coli under salt stress
Peroxiredoxins are proteins that have molecular weight around 20 to 30 kDa and widely spread in living organisms 19 . Initially, Prxs were identified according to its ability to protect cells especially proteins from abiotic stress particularly oxidative damage that leads to an increase of ROS 20 . Previously, Prxs was termed as the "protector protein" or "thiol-specific antioxidant" before being name again as Prxs [21][22][23][24] . Five different Prxs genes including sll1621 were found in the genome of Synechocystis PCC 68036. Also, Kobayashi et al. 7 stated that the mRNA level of the sll1621 gene is remarkably up-regulated when Synechocystis PCC 6803 are exposed to methylviologen with light conditions. Also, Hosoya-Matsuda et al. 1 concluded that PrxII is significantly important as scavenging ROS enzyme. They found that the growth of the knockout mutant cells lacking sll1621 gene was remarkably weak against oxidative stress 1 .
Oxidative injury to microorganism cells frequently happens under different abiotic stress conditions during bioprocess 11,15 . Thus, it has been addressed either the peroxiredoxin activity of PrxII protein assumes a significant useful role in recombinant E. coli, and additionally whether such activity might be useful under salinity stress. In the present research, we used the model microorganism, E. coli, to examine the ability of Synechocystis PCC 6803 sll1621 gene to increase the viability of growth of E. coli recombinant cells under salt stress. Salt stress was generated in-vitro by culturing the recombinant and wild type E. coli cells on LB agar provided with various concentrations of NaCl (Fig. 4). The pTYB21/empty cells were sensitive to 400mM NaCl, while, the growth was completely deleted in the presence of 800mM and 1000mM of NaCl (Fig. 4). On the other hand, the recombinant pTYB21/sll1621 cells increased the growth efficiency under the high concentration of NaCl (1000mM), demonstrating that the growth was, for some reason, improved compare to those of the pTYB21/emptycells (Fig.  4). Accordingly, this survival test under different concentration of NaCl showed that the sll1621 gene from Synechocystis PCC 6803 could function as an antioxidant protector enzyme to defend cells from oxidative damage under salt stress (Fig. 4). Similar results documented that the expression of PrxIIgene are typically affected by oxidative stress and abiotic stress that caused by hyperoxia, peroxides, UV, and ionizing radiation [25][26][27] .

CONCLUSIONS
In the present work, the Synechocystis PCC 6803 sll1621 gene was successfully over expressed in the cytoplasm of E. coli BL21 (DE3) cells. The over expressed protein was verified by SDS-PAGE and observ ableviability test. The present data specify that Synechocystis PCC 6803 sll1621 gene confers tolerance of recombinant E. coli cells to high concentration of salt stress. Consequently, these results are strong evidence for the success of gene expression and the absence of species barrier among different microorganisms.