Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli

We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli), Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cDNA of Ds-26-16 using the iTRAQ (isobaric tags for relative and absolute quantification) approach. A total of 1,610 proteins were identified, which comprised 39.4% of the whole proteome. Of the 559 differential proteins, 259 were up-regulated and 300 were down-regulated. GO (gene ontology) and KEGG (Kyoto encyclopedia of genes and genomes) enrichment analyses identified 202 major proteins, including those involved in amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS (reactive oxygen species) scavenging, membrane proteins and ABC (ATP binding cassette) transporters, and peptidoglycan synthesis, as well as 5 up-regulated transcription factors. Our iTRAQ data suggest that Ds-26-16 up-regulates the transcription factors in E. coli to enhance salt resistance through osmotic balance, energy metabolism, and oxidative stress protection. Changes in the proteome were also observed in E. coli overexpressing the ORF (open reading frame) of Ds-26-16. Furthermore, pH, nitric oxide and glycerol content analyses indicated that Ds-26-16 overexpression increases nitric oxide content but has no effect on glycerol content, thus confirming that enhanced nitric oxide synthesis via lower intercellular pH was one of the mechanisms by which Ds-26-16 confers salt tolerance to E. coli.


Introduction
Soil salinization is one of the major factors that limit the growth and distribution of organisms. Currently, the worldwide saline soil area is greater than 6% of the total land area [1] and is projected to increase to 50% by the year 2050 [2]. High salinity can lead to ion imbalance and gene Ds-26-16 from a 4 M NaCl-induced D. salina cDNA library and discovered that this gene could confer salt tolerance to broad-spectrum organisms, including E. coli, Haematococcus pluvialis and tobacco [19]. Bioinformatics revealed that Ds-26-16 has homology with a Xenopus zinc finger protein and an Oreochromis niloticus nucleic acid binding factor. Subcellular localization showed that Ds-26-16 localizes to either the nucleus of onion or the nucleoid area of E. coli [19]. Therefore, Ds-26-16 is possibly a salt-stress associated trans-regulatory gene. However, it is unclear how the proteome changes upon overexpression of Ds-26-16 in E. coli. We sought to determine the relationship between these differentially expressed proteins and salt tolerance.
iTRAQ is an isotope labeling quantitative proteomic approach developed by Applied Biosystems Incorporation (Waltham, Massachusetts, USA) in 2004. This method permits multiplex quantitation of up to eight complex protein samples in a single multiplexed analysis [20] with high sensitivity [21]. So far, this technique has been extensively implemented in the field of quantitative proteomics. In this study, we employed iTRAQ to study the proteome of E. coli overexpressing Ds-26-16 and to investigate the molecular mechanisms of Ds-26-16 that conferred salt tolerance to the bacterium. The study provides a basis for elucidating the broadspectrum salt tolerance of this gene.

Protein extraction and iTRAQ analysis
The E. coli cell cultures were harvested by centrifugation, and the pellets were resuspended in 4 mL PBS buffer including 50 μg mL -1 of lysozyme. After incubation at room temperature for 30 min, the bacteria were then subjected to ultrasonic lysis (80 W, pulse duration of 1 min ontime and 1 min off-time). When the solution became clear, the samples were centrifuged at 14,800 g for 15 min. The resulting supernatants were used as the crude/total protein samples and stored at -80°C.
iTRAQ analysis was carried out by PTM Biolabs Inc. (Hangzhou, China). For each sample, 100 μg of protein solution was digested with trypsin (1:20, w/w) at 37°C overnight. The resulting peptides were then dried by vacuum centrifugation, resuspended in 0.5 M EDTA, and labeled using the 6-plex TMT kit (Thermo Scientific, San Jose, CA). Peptides from pET-21b (+), p21-cDNA, p21-ORF were labeled with TMT-129, TMT-130, and TMT-131, respectively. Afterwards, the labeled peptides were fractionated by SCX (strong cation exchange) chromatography, desalinated using a C18 spin column (Thermo Fisher Scientific, San Jose, USA) and freeze-dried. Then, the peptides were redissolved in buffer A [25 mM NaH 2 PO4 in 25% CAN (cerium ammonium nitrate), pH 2.7], loaded onto a 4.6×250 mm Ultremex SCX column (Thermo Fisher Scientific, San Jose, USA), and eluted at 1 mL min -1 with a gradient of buffer A for 10 min, 5-35% of buffer B (25 mM NaH 2 PO 4 , 1 M KCl in 25% CAN, pH 2.7) for 11 min, and 35-50% of buffer B for 1 min. Elutions were collected every minute and monitored by measuring the absorbance at 214 nm, desalted with a StrataX C18 column (Phenomenex, California, USA) and vacuum-dried. The collected peptides were eluted again with buffer A (2% acetonitrile, 0.1% formic acid) for 4 min at 8 μL min -1 followed by buffer B (95% CAN, 0.1% folic acid) for 40 min at 0.3 μL min -1 using the Shimadzu LC-20AD nano HPLC (high performance liquid chromatography) system (Shimadzu, Tokyo, Japan). After coupling Q Exactive Tandem Mass Spectrometry (MS/MS) (Thermo Fisher Scientific, San Jose, USA) with HPLC, the complete peptides were detected in an orbit trap with a resolution of 70,000. After highenergy collision dissociation with a collision energy of 27.0 and normalized collision energy strengthening to 12.0%, the fragment peptides were analyzed with MS/MS in an orbit trap with a resolution of 17,500. The electro spray voltage was 1.6 kV, and the automatic gain control was used to optimize the track spectrum. The range of the MS m/z scan was between 350 Da and 2,000 Da. Peptide and protein identification was performed using the Mascot search engine (ver. 2.3.0, Matrix Science).

Bioinformatics analysis
The abundance of proteins identified from iTRAQ was compared between p21-cDNA and the control. A 1.5-fold cutoff [12] was set to determine up-regulated and down-regulated proteins with a P-value < 0.01. GO (gene ontology) annotation was conducted using the UniProt-GOA database (http://www.ebi.ac.uk/GOA/), and KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment was performed using the KEGG database (www.genome.jp/ kegg/tool/map_pathway2.html). If the P-value < 0.05, either the GO term or KEGG pathway were considered to be significantly enriched. Proteins involved in either the enriched GO term or the KEGG pathway were considered to be major proteins involved in the salt stress signal network in E. coli. The same methods were used to analyze the proteome of p21-ORF.
Cell density, pH, nitric oxide and glycerol content analysis E. coli strains were grown in LB liquid medium containing 100 μg mL -1 ampicillin on a shaker at 37°C. When A 600 reached 0.3, IPTG (final concentration 1 mM) was added. After 1.5 h, the cell cultures were centrifuged at 14,800 g for 3 min. Approximately 4×10 8 cells were transferred to 2×TY medium (1 mM IPTG) with varying NaCl concentrations (0 M, 0.26 M, 0.51 M). After shaking at 37°C for 0 h, 0.5 h, 1 h, 2 h, 4 h or 8 h, the cell density was measured by bloodcell-counting plate. The pH of the ultrasonically lysed cells and the cultured medium was measured with a pH meter (model SP-701, Suntex Instruments Co., Ltd., Taipei, China). Nitric oxide and glycerol content were determined at 0.1 h and 8 h using a nitric oxide assay kit (Beyotime Institute of Biotechnology, Shanghai, China) and a method previously described by Zhao et al. [22], respectively. were identified. Of these, 79% were 6-15 amino acids (S1B Fig). Ultimately, a total of 1,610 proteins were identified (S1 Table), accounting for 39.4% of the entire proteome (4,086) retrieved from UniProtKB. Proteins with a low molecular weight (less than 10 kDa) were also detected, of which 69.6% were 20-70 kDa (S1C Fig). Compared to the control strains, a total of 559 differential proteins were identified in the p21-cDNA strain, of which 259 were up-regulated and 340 were down-regulated (S2 Table).
GO and KEGG enrichment analysis of the differential proteins in p21-cDNA strain To understand the function and feature of the identified proteins, the differentially expressed proteins were annotated according to cellular component, molecular function and biological process. Of the 559 differential proteins, 490 were annotated (S2 Table). Structurally relevant proteins were localized in the cell, membrane and macromolecular complex, accounting for 50%, 30% and 13% of this subset, respectively. Molecularly functional proteins classified into catalytic activity, binding and transporter activity, accounting for 50%, 37% and 9%, respectively. Biological proteins relating to cellular processes and metabolic processes, accounting for 36% and 37%, respectively (S3 Table).
GO enrichment of biological processes was conducted with Fisher's exact P-value. Eleven biological processes were identified with P < 0.05 as the standard (Table 1). Of these, amino acid metabolism, carbohydrate metabolism, ROS metabolism, single-organism membrane organization, transport, and oxidative phosphorylation of organic compounds have been reported to be involved in salt tolerance [9,11,23].
Of the 559 differential proteins, 472 were positioned into KEGG pathways. The enriched pathways were obtained when P < 0.05 (Table 2). Of these, nine were enriched from the upregulated proteins, including ABC (ATP binding cassette) transporters and proteins involved in glyoxylate and dicarboxylate metabolism, sulfur metabolism, taurine and hypotaurine metabolism, butanoate metabolism, and tryptophan metabolism. Six were enriched from the down-regulated proteins, including oxidative phosphorylation, and ribosomal and peptidoglycan biosynthesis. Of these affected processes, oxidative phosphorylation, peptidoglycan biosynthesis and ABC transporters have been reported to relate to salt tolerance [12,24,25].

Analysis of specific differential proteins to salt tolerance
To elucidate the mechanism of Ds-26-16 conferred salt tolerance in E. coli, we investigated the differential proteins that strongly enriched in the above biological processes and KEGG pathways. A total of 202 main proteins were obtained (S4 Table). Of these, proteins associated with membrane proteins and ABC transporters accounted for the largest percentage (31.7%), followed by those involved in amino acid and organic acid metabolism (29.7%), carbohydrate metabolism (16.3%), energy metabolism (10.9%), oxidative stress protection (6.9%), and peptidoglycan biosynthesis (4.5%). Amino acid and organic acid metabolism. Free amino acids such as proline (Pro) and glutamine (Gln) function as osmotically organic solutes under salt stress [26]. Under osmotic stress, the glutamate (Glu), Gln, aspartate (Asp), asparagine (Asn), threonine (Thr), serine (Ser), leucine (Leu) and histidine (His) concentrations were greater in the freezing-tolerant substitution wheat line compared to the sensitive line [27]. The transcription factor AsnC, which is associated with the global regulation of amino acid biosynthesis [28], was up-regulated (2.74-fold) in p21-cDNA. Proteins involved in amino acid metabolism were also differentially expressed. Of these, eleven proteins associated with Ala (alanine), Asp and Glu metabolism (S2A Fig) were altered. For instance, argininosuccinate lyase (catalyzes Asp flow into the tricarboxylic acid cycle) and aspartate carbamoyltransferase (catalyzes Asp flow into pyrimidine metabolism) were down-regulated 0.29-and 0.34-fold, respectively. The down-regulation of these proteins resulted in the accumulation of Asp. Meanwhile, asparaginase II, which catalyzes the inter-conversion between Asn and Asp, was up-regulated 1.77-fold. Similar situations were also observed in Glu/Gln-related pathways. Glutamine-fructose-6-phosphate aminotransferase (catalyzes Gln flow into amino sugar metabolism) and carbamoyl-phosphate synthase small chain (catalyzes the conversion of Gln to carbamoyl-phosphate) were down-regulated, whereas glutaminase (catalyzes the inter-conversion between Gln and Glu) was up-regulated (3.63-fold). The differential expression of these proteins increased the acidic amino acid (Asp and Glu), Asn and Gln contents to maintain the intracellular osmotic balance in p21-cDNA. All proteins involved in arginine (Arg) biosynthesis (except glutaminase) were down-regulated (S2B Fig). Acetylglutamate kinase, acetylornithine/succinyldiaminopimelate aminotransferase, and argininosuccinate lyase were down-regulated 0.26-, 0.27-, and 0.29-fold, respectively. Meanwhile, aspartokinase, bifunctional polymyxin resistance protein ArnA, and acetylornithine/succinyldiaminopimelate aminotransferase, which are involved in the conversion of Asp into Lys, were down-regulated 0.35-, 0.23-, and 0.27-fold, respectively (S2C Fig).
The down-regulation of these proteins decreased the Arg and Lys content and further increased the Asp content.
The regulation of organic acid metabolism also plays a key role in plant tolerance to saline conditions [29]. The accumulation of organic acids may serve to counter increasing cation levels (e.g., Na + ) [30]. Three proteins involved in organic acid synthesis were up-regulated in the p21-cDNA strain (S2A Fig). Glutamate decarboxylase, which catalyzes Glu into GABA (γ-aminobutyric acid); 4-aminobutyrate aminotransferase, which catalyzes the inter-conversion between GABA and succinate semialdehyde; and succinate semialdehyde dehydrogenase, which catalyzes succinate semialdehyde to succinate, were up-regulated 4.11-, 2.98-, and 3.14-fold, respectively. The upregulation of these proteins resulted in an increase in GABA and succinic acid content. THF (tetrahydrofolate), a one-carbon carrier, is essential for the synthesis of purines and pyrimidines and is derived from DHF (dihydrofolate). This reaction is catalyzed by dihydrofolate reductase, whose expression is regulated in a growth-dependent manner [31]. Five proteins involved in folate biosynthesis were down-regulated (S2D Fig), whereas only dihydrofolate reductase was up-regulated (2.62-fold). The up-regulation of dihydrofolate reductase might stimulate the cell growth of the p21-cDNA strain under salt stress. Furthermore, the subunits of nitrate reductase involved in nitric oxide synthesis, including subunit 1α, subunit 2α and 2β, were up-regulated. Nitric oxide is an important signaling molecule involved in various biotic and abiotic stresses [32]. The up-regulation of nitrate reductase increased nitric oxide content to alleviate the damage caused by salt stress.
Energy metabolism. All 21 differential proteins (except FrdB) involved in oxidative phosphorylation were down-regulated in the p21-cDNA strain (S3A Fig). Subunits I (cyoB) and II (cyoA) of cytochrome bo terminal oxidase were down-regulated 0.35-and 0.27-fold, respectively; both subunits A (nuoA) and B (nuoB) of NADH-quinone oxidoreductase were downregulated 0.43-fold. These changes resulted in a decrease in ATP content and increase in NADH levels. Furthermore, 5 differential proteins involved in TCA (tricarboxylic acid) cycle were all down-regulated (S3B Fig). Dihydrolipoamide acetyltransferase, 2-oxoglutarate dehydrogenase, and succinate dehydrogenase and fumarate reductase iron-sulfur protein were down-regulated 0.56-, 0.52-, and 0.54-fold, respectively. Meanwhile, transcription factor ArcA, which has been reported to repress the expression of TCA cycle genes [33] and proton translocating NADH dehydrogenase (oxidative phosphorylation) [34] in E. coli, was also up-regulated (1.55-fold), thus resulting in a decrease in TCA cycle activity and ATP levels in the p21-cDNA strain.
Oxidative stress protection. As key elements in the defense system against ROS, several ROS-scavenging enzymes such as CAT (catalase), SOD (superoxide dismutase), POD (catalase-peroxidase) and osmotically inducible peroxidase OsmC were significantly up-regulated in the p21-cDNA strain (S4 Table). GSH-dependent enzymes such as GPX (glutathione peroxidase), GST (glutathione S-transferase), glutathione transferase, glutathione S-transferase domain protein, and glutaredoxin 2, all of which are vital for maintaining the redox status, were also up-regulated. In addition, the cold shock protein exoribonuclease 2 was up-regulated, as well as the transcription factor IscR, which acts as an activator in response to oxidative stress [35] (2.33-fold).
Membrane proteins and ABC transporters. Membrane proteins primarily include outer membrane pore proteins and outer membrane protein assembly factors (S4 Table). Of the outer membrane proteins, OmpN and OmpC were up-regulated 19.12-and 4.09-fold, respectively, whereas OmpF was down-regulated 0.38-fold. OmpF and OmpC are two of the main porins in the outer membrane of E. coli. The OmpF pore is larger and presents a higher permeability compared to OmpC [36]. OmpN displays similar functional properties (single-channel conductance) to those of OmpC [37]. An increase in OmpC expression and decrease in OmpF expression created a balance that can be reversed when growth conditions improve [37]. Lipoproteins BamA, BamB, and BamD, which comprise the BAM complex [38] and are essential for the assembly of outer membrane protein (OMP) in the outer membrane, were down-regulated 0.57-, 0.58-, and 0.58-fold, respectively, although their corresponding function in salt stress is unknown. Meanwhile, a membrane-related transcription factor BolA that is involved in cellular protection under adverse growth conditions [39] was up-regulated 2.28-fold.
ABC transporters can be distinguished into ABC exporters and ABC importers based on the polarity of transport. ABC importers, the primary transport systems in prokaryotes, actively transport various substances into cells [40]. Twenty-six ABC transporters were differentially expressed in the p21-cDNA strain (S5 Fig). The up-regulated transporters include mineral and organic ion transporters (e.g., extracellular solute-binding protein family 1 PotD, PotF), monosaccharide transporters (e.g., D-ribose transporter subunit RbsB, D-xylose ABC transporter XylF), and phosphate and acid transporters (e.g., phosphate-binding protein PstS, lysine/arginine/ornithine ABC transporter ArgT, cystine transporter FliY). These proteins primarily transport small compatible osmoprotectants into cells. The down-regulated transporters include oligosaccharide, polyol and lipid transporters (ABC transporter-related MalK, Maltose/Maltodextrin transporter MalF), and cationic amino acid transporters (periplasmic binding protein HisJ and arginine transporter subunit ArtJ). These proteins mainly transport large non-compatible substances and cationic amino acids. Meanwhile, a LYSR-type transcriptional regulator related to the transporter system [41] was also up-regulated.
iTRAQ analyses of p21-ORF and its comparison to that of p21-cDNA To confirm the iTRAQ of the p21-cDNA strain, we performed iTRAQ analyses of the p21-ORF strain. A total of 542 differential proteins were identified with respect to the control strain (S2 Table). Of these, 252 were up-regulated and 290 were down-regulated. A total of 591 differential proteins were identified in either the p21-ORF or p21-cDNA strains. Of these, 509 were present in both p21-ORF and p21-cDNA, and the recurrence rate was 86.1% (509/591). The linear equation for fold change of the overlapped proteins was F p21-ORF = 1.0268 × F p21-cDNA , R 2 = 0.8818 ÃÃ (n = 509), suggesting that the differential proteins identified in the two strains were highly consistent. Of the remaining (82) non-overlapped proteins, 32 appeared in the p21-ORF strain. Of these, 30 showed a small change, whereas 2 were not involved in with salt tolerance. Forty-nine identified proteins appeared in the p21-cDNA strain, with 46 showing a small change and 4 not reported to be involved in salt tolerance. Only one conserved protein was down-regulated in the p21-cDNA strain but up-regulated in the p21-ORF strain (S2 Table).
Effect of Ds-26-16 on cell density, pH, nitric oxide and glycerol content in E. coli iTRAQ analyses of the p21-cDNA and p21-ORF strains showed that proteins associated with the synthesis of acidic amino acids, organic acids, nitric oxide, and glycerol were up-regulated. To investigate whether these changes are related to the mechanism of Ds-26-16 in salt tolerance, the intracellular and extracellular pH, nitric oxide, and glycerol contents in these two strains and a control strain were measured under salt stress. The cell densities were first determined under different NaCl concentrations (Fig 2A). Under salt-free condition, the densities of the p21-cDNA and p21-ORF strains were higher than that of the control strain at 0.5 h, 1 h, 2 h and 4 h, but they were nearly equal by 8 h. At 0.26 M NaCl, the temporal kinetics of the cell densities of these strains were almost the same as those in the salt-free conditions, except that those of the p21-cDNA and p21-ORF strains were 1.22-and 1.1-fold higher, respectively, than the control strain at 8 h. At 0.51 M NaCl, the densities of the p21-cDNA and p21-ORF strains were decreased to 90.2% and 94.7% of those of the salt-free strains at 8 h, whereas the control strain density decreased to 63.6% to that of the salt-free control strain at 8 h. These data confirm that overexpression of Ds-26-16 confers salt tolerance to E. coli.
Under varying NaCl conditions, the intracellular pH ( Fig 2B) and extracellular pH (Fig 2C) of the p21-cDNA, p21-ORF, and control strains showed a similar change pattern and stabilized after 2 h (except for the p21-cDNA strain in salt-free conditions). However, the intracellular and extracellular pH of the p21-cDNA and p21-ORF strains (6.40-7.09) were both lower than that of the control strain (7.00-7.46) at each culture time (except for 0 h), and the difference became greater with increasing salt concentration. The intracellular pH values of each strain at each culture time was slightly lower than the corresponding extracellular pH, indicating that the lowered pH derived from the alterations of the cellular metabolism. As the cell growth rate of E. coli under low pH (6.5-7.0) is higher than that under high pH (7.5-7.8) [44][45], the densities of the p21-cDNA and p21-ORF strains were higher than those of the control strain, which was probably due to a relatively lower extracellular pH in the engineered E. coli. Under salt-free conditions, the nitric oxide content of the p21-cDNA and p21-ORF strains were increased 1.23-and 1.32-fold, respectively, of the value in the control strain at 0.1 h, whereas the nitric oxide content was 1.53-and 1.77-fold greater, respectively, compared to the control strain at 8 h (Fig 3A). At 0.26 M NaCl, the nitric oxide content of the p21-cDNA and p21-ORF strains was 1.73-and 1.66-fold, respectively, of the control strain at 0.1 h but was 1.30-and 1.31-fold, respectively, of the control strain at 8 h. At 0.51 M NaCl, the nitric oxide content of the engineered strains was still higher than that in the control strain, suggesting that Bars represent the averages and standard deviations from at least three independent measurements. The difference between p21-cNDA or p21-ORF with pET-21b(+) at the same salt concentration and same culture time were compared respectively. * or ** over the bar indicates significant difference (P < 0.05) or highly significant difference (P < 0.01). overexpression of Ds-26-16 increased nitric oxide synthesis and conferred salt tolerance to E. coli. The nitric oxide contents at 0.26 M and 0.51 M NaCl at 8 h were lower than those in saltfree conditions, indicating that salt stress damaged the nitric oxide synthesis pathway of engineered strains to some extent.
The glycerol content of the p21-cDNA, p21-ORF and control strains at different NaCl stresses at 0.1 h and 8 h showed no significant changes trends (Fig 3B). It appears that at 0.1 h, the glycerol content of the p21-cDNA and p21-ORF strains were clearly higher than that of the control. However, as time progressed, the content of these engineered strains at 8 h was lower than that at 0.1 h and also lower than that of the control strain at 8 h. This trend was not observed in the control strain, and no significant differences were apparent between the 8 h and 0.1 h timepoints. This result suggests that overexpression of Ds-26-16 did not affect glycerol synthesis in E. coli.

Discussion
As a proteomics technology, iTRAQ has higher accuracy than other non-isotope quantitative labeling techniques. It can process several samples concurrently, thus improving the throughput of comparative proteome and reducing inter-assay error to a great extent. Therefore, iTRAQ was chosen to study the proteome of an E. coli strain that overexpressed the full length of Ds-26-16 cDNA. To confirm the reliability of the results, the proteome of an E. coli strain that overexpressed its ORF were also conducted. Possible mechanisms derived from iTRAQ analysis, including nitric oxide and glycerol synthesis, were also characterized in E. coli that overexpressed Ds-26-16.
Ds-26-16 from D. salina is a novel broad-spectrum transregulatory gene that confers salt tolerance to various organisms [19]. In this study, we showed that five transcription factors (a LYSR-type transcription factor that regulates ABC transporters; IscR, which regulates antioxidant enzymes; AsnC, which regulates amino acid biosynthesis; ArcA, which regulates energy metabolism; and BolA, which regulates membrane proteins and peptidoglycan synthesis) were up-regulated in the p21-cDNA strain that overexpressed the cDNA of Ds-26-16, confirming the speculation that Ds-26-16 is a transregulatory gene. Our data suggested that Ds-26-16 may function as a global regulator by up-regulating 5 transcription factors of E. coli to regulate a number of metabolic pathways. The differential proteins identified are primarily involved in amino acid and organic acid metabolism, carbohydrate metabolism, energy metabolism, antioxidant activities, and peptidoglycan synthesis; membrane proteins and ABC transporters were also identified. Based on their function in salt tolerance, these proteins can be further classified into the following three groups: osmotic balance, energy metabolism and oxidative stress protection. Therefore, the possible mechanisms of Ds-26-16 in enhancing salt tolerance of E. coli could be described as follows (Fig 4):
D. salina lacks a rigid cell wall, and the cytoplasmic membrane alone makes the cell susceptible to osmotic pressure. Water flows through the cytoplasmic membrane in response to hypertonic conditions such that the original cell volume is restored [46]. However, peptidoglycan is the primary component of the cell wall in E. coli. Overexpression of Ds-26-16 up-regulated the transcription factor BolA, which resulted in down-regulation of proteins involved in peptidoglycan synthesis. The decrease in the peptidoglycan content enhances the flexibility of the cell wall. Simultaneously, as a morphology gene, BolA renders the cells shorter and rounder, thus causing a decrease in the surface-to-volume ratio and a reduction in the surface area exposed to the stress environment [43]. Thus, overexpression of Ds-26-16 enables E. coli cells to change their morphology to adapt to the environment similar to D. salina.
Moreover, among the enriched differential proteins, the proportion of proteins related to amino acid and organic acid metabolism, as well as membrane proteins and ABC transporters, was the greatest, indicating that regulation of osmotic balance is one of the most important mechanisms for Ds-26-16 to confer the salt tolerance to E. coli.

Energy metabolism
Overexpression of Ds-26-16 in E. coli up-regulated ArcA and then down-regulated TCA cycle activity and oxidative phosphorylation. It was previously reported that TCA cycle activity was decreased in the roots of salt-tolerant rice [47], although the demand for energy is commonly increased under a stress environment [23]. Therefore, a decrease in energy production may also be a mechanism of Ds-26-16 to confer salt tolerance. Meanwhile, succinic acid and GABA synthesis were also upregulated. Therefore, we speculated that overexpression of Ds-26-16 reduced the energy demand and increased the succinic acid content and other organic acids to confer salt tolerance to E. coli.
Glycerol synthesis is an important mechanism of salt tolerance in D. salina. It was reported that a decrease in ATP content stimulated the glycerol synthesis in D. salina [48]. After expressing Ds-26-16 in E. coli, the TCA cycle and oxidative phosphorylation were observed to be down-regulated in conjunction with up-regulation of glycerol-3-phosphate dehydrogenase and other enzymes involved in glycerol synthesis. However, glycerol-3-phosphatase was not up-regulated at the same time, which was reflected in the measurement of the glycerol content (Fig 3B). In D. salina, GPDH (glycerol-3-phosphate dehydrogenase) has two independent functional domains: a dehydrogenase and phosphorylase [6,49]. However, only the phosphorylase domain is present in GPDH from E. coli [50]. Therefore, we proposed that Ds-26-16 in E. coli could not substantially improve the glycerol content because of the deficiency in the phosphorylase, although most enzymes related to glycerol synthesis were up-regulated (Fig 1). As propanoate metabolism was up-regulated, the increase in glycerol-3-phosphate increased the succinate content, which is beneficial for the intercellular osmotic balance under salt stress.

Oxidative stress protection
Overexpression of Ds-26-16 in E. coli also up-regulated IscR followed by upregulation of active oxygen scavenging and other antioxidant enzyme activities, thus protecting the bacterium from the oxidative damage and osmotic injury caused by salt stress. Production of nitritedependent nitric oxide by E. coli via membrane-associated nitrate reductase also contributes to the oxidative stress protection mechanism [51]. Overexpression of Ds-26-16 in E. coli up-regulated nitrate reductase activity and increased the nitric oxide content, which was also demonstrated by nitric oxide analysis showing that the content of the engineered strain was higher than that of the control strain regardless of applied salt stress ( Fig 3A). As nitrate reductase exhibited higher activity at a lower pH [52][53], the decreased intracellular pH possibly improved the nitrate reductase activity and enhanced the nitric oxide content in the engineered strains. Thus, enhancing nitric oxide content by decreasing the intracellular pH is another mechanism by which Ds-26-16 confers salt tolerance to E. coli.
Several major differential proteins with a large change in expression (S4 Table) could not be placed into a specific pathway although they were derived from the enrichment analysis. Some of these proteins had different expression patterns from those of a previous study. For example, ketol-acid reductoisomerase (ilvC), a cold stress-induced protein in Bacillus subtilis [54], was down-regulated to 0.26-fold in this study. C 4 -dicarboxylate transport gene (dctA) and cusB, which are induced by external stimuli and indole, respectively, were both down-regulated in this study in nodule bacteria [55][56]. Others had the same expression pattern as those of a previous study, although their specific role was unclear. For example, 5-methyltetrahydropteroyltriglutamatehomocysteine methyltransferase (metE), a methionine synthase that was readily inactivated in E. coli under oxidative stress [57], was down-regulated in this study (0.22-fold). 2-dehydro-3-deoxyphosphooctonate aldolase (kdsA), whose gene was down-regulated in mulberry leaves under high salt or drought stress [58], was also down-regulated in this study. The lipopolysaccharide transport periplasmic protein LptA, which is implicated in the transport of lipopolysaccharides from the inner membrane to the outer membrane of E. coli [59], was up-regulated in this study.
In summary, the proteome of E. coli that overexpress Ds-26-16 was quantitated using iTRAQ. Our data suggest that Ds-26-16 enhances the salt tolerance of E. coli by 5 transcription factors and regulating a number of metabolic pathways, including amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS scavenging, and peptidoglycan synthesis, as well as the membrane proteins and ABC transporters. The study provides a basis for understanding the mechanism of Ds-26-16 in enhancing salt tolerance in E. coli. Further study regarding these mechanisms in eukaryotes such as yeast and plants is needed to elucidate the broad-spectrum salt tolerance of the gene.  Table. The original protein list containing both identification and quantification information identified in p21-cDNA and p21-ORF strain using iTRAQ. (XLS) S2 Table. Differential proteins identified in p21-cDNA and p21-ORF strain under salt stress. (PDF) S3 Table. GO classification of differential proteins identified in p21-cDNA strain. (PDF) S4 Table. Main differential proteins identified in p21-cDNA strain under salt stress. (PDF)