Hexane effect on Synechocystis sp. PCC 6803

The growth of Synechocystis supplemented with 0, 0.7%, 0.8% and 0.9% hexane was assessed to determine an appropriate hexane concentration for proteomic studies (Figure 1). The results showed that the hexane concentration that caused an approximately 50% growth inhibition was found to be 0.8% (v/v) at both 24 h and 48 h (corresponding to middle-exponential and exponential-stationary transition phases, respectively), and was selected for the proteomics analysis in this study. The tolerance level of Synechocystis to hexane was similar to what has been reported for Aeromonas hydrophila and Pseudomonas aeruginosa[38]. Cell morphology examination showed no visible change after hexane treatment (data not shown). For proteomic analysis, two independent cultivations for both control and 0.8% hexane treatment were conducted, and cells were collected by centrifugation (8,000 x g for 10 min at 4°C) at 24 h and 48 h, resulting in two biological replicates for each time point of control and hexane-treated samples (Figure 1).

Overview of quantitative proteomics

A total of 167,191 spectra were obtained from the iTRAQ - LC-MS/MS proteomic analysis. After data filtering to eliminate low-scoring spectra, a total of 24,162 unique spectra that met the strict confidence criteria for identification were matched to 1,491 unique proteins, representing approximately 41.8% of the 3569 predicted proteins in the Synechocystis genome ( Additional file 1: Table S1). In terms of protein molecular weight (MW) distribution, good coverage (averages of 30–45% of the total proteins in each MW group) was obtained for a wide MW range for proteins larger than 10 kDa (Figure 2A). In addition, most of the proteins were identified with good peptide coverage, of which ~65% of the proteins were with more than 10% of the sequence coverage, and ~44% were with 20% of the sequence coverage (Figure 2B). Among all the proteins detected, 1,181 and 1,172 were identified from the samples of 24 h and 48 h, respectively. The proteins that were identified only in control or treated samples so that ratio calculation is not available were excluded from the analysis. Based on the number of unique proteins identified in each functional category, the most frequently detected functional categories were “general function prediction only” and “signal transduction mechanisms”, representing 11.1% and 10.85% of all the protein identified, respectively (Figure 2C). Proteins involved in signal transduction network are generally with low abundance, quick protein turnover time and membrane-bound [39], a high coverage of the group of signal proteins also suggested the methodology used in the study is with high sensitivity and very reliable. The high percentage of functionally unknown proteins identified is not unreasonable, considering more than 40% of proteins in the Synechocystis genome are still annotated as hypothetical proteins without any functional prediction. Other most frequently detected functional categories included “amino acid transportation and metabolism” (9.17%), “energy production and conversion” (7.55%), and “translation, ribosomal structure and biogenesis” (7.55%) and “cell wall/membrane/envelope biogenesis” (7.24%), suggested that the proteins in these functional categories were highly expressed and may be active during the growth and treatment conditions.

Reproducibility of the proteomic analysis was assessed in two types of comparisons (Figure 3). First we labeled and mixed two biological replicates of a given condition directly for proteomic analysis (i.e. biological replicate 1 and 2 of control at 24 h, replicate 1 and 2 of control at 48 h, biological replicate 1 and 2 of hexane treatment at 24 h, biological replicate 1 and 2 of hexane treatment at 48 h), the difference was plotted verse the percentage of the proteins identified, the results showed that approximately 60% of the proteins with difference less than delta error of 0.1, and more than 95% of the proteins with difference less than delta error of 0.5 (Figure 3-I). Second we labeled and mixed each pair of hexane-treated sample and its control for proteomic analysis, the difference between different biological pairs was plotted in Figure 3-II. The dispersion of the iTRAQ ratios of the quantified proteins (i.e. 1,181 and 1,172 for 24 h and 48 h, respectively) was found with very similar trends between four biological replicates at either 24 h (Figure 3-II-A) or 48 h (Figure 3-II-B), suggesting that the biological noise was reasonably low.

Pathway enrichment analysis of the hexane-responsive proteins

Using a cutoff of 1.5-fold change and a p-value less than 0.05, we determined that 140 and 148 unique proteins were differentially regulated between control and hexane treatments conditions at 24 h and 48 h, respectively ( Additional file 1: Table S1). Among them, a total of 164 and 77 proteins were up-regulated and down-regulated by the hexane treatments, respectively. Forty-two up-regulated and 4 down-regulated proteins were shared between 24 h and 48 h, while more of the responsive proteins were unique for each of the time points, consistent with the expected physiological differences between middle-exponential and transition phases of the cell growth (Figure 1).

Metabolic pathway enrichment analysis was carried out for the differentially regulated proteins to determine the affected cellular metabolism ( Additional file 2: Table S2). The analysis was performed by matching the responsive proteins to the proteins annotated with KEGG Pathway database, and then comparing the frequencies of the responsive proteins in each KEGG pathway to determine statistically authenticity of the involvement of that KEGG pathway in hexane response. A series of KEGG pathways affected by the hexane treatment, with p-value less than 0.05 as cut off were identified. The results showed that at 24 h after hexane treatment, four KEGG pathways were differentially regulated by the hexane treatment: they are “Ribosome” (KO03010), “Sulfur relay system” (KO04122), “Photosynthesis” (KO00195) and “Arachidonic acid metabolism” (KO00590). All four pathways were enriched by at least two out of four biological replicates, with “Ribosome” (KO03010) enriched with good confidence in all four replicates and “Photosynthesis” (KO00195) enriched in two replicates, consistent with the relatively high expression of ribosomal proteins and photosynthetic proteins, and the sensitivity of protein biosynthesis and photosynthetic processes to stress. Most of the ribosomal proteins identified were down-regulated, suggesting an overall slowdown of the protein biosynthesis and possible slow metabolism. At 48 h, five KGG pathways, “Sulfur relay system” (KO04122), “ABC transporters” (KO02010), “Photosynthesis” (KO00195), “Steroid biosynthesis” (KO00100), and “Biosynthesis of ansamycins” (KO01051) were enriched by the differentially expressed proteins. Although these KEGG pathways were enriched in only two out of four biological replicates, the p-values were reasonably low, suggesting in general the reliability of the data analysis. The enrichment of “Ribosome” pathway (KO03010) at only 24 h was interesting, which may implicate that modification of the primary metabolism, such as protein biosynthesis, may be one of the major strategies that cells used to deal with stress during middle-exponential phase. In this study, we found that several ribosomal proteins, Sll1816 (rpsM), Sll1803 (rplV) and Ssr0482 (rpsP) were down-regulated under hexane treatment. Interestingly, these three genes, rpsM, rplV, and rpsP, were named as targets as they were also down-regulated in E. coli against organic solvent also indicated [35].

“Sulfur relay system” (KO04122) was differentially regulated at both 24 and 48 h, in three out of four biological replicates at 24 h, and two out of four biological replicates at 48 h, respectively ( Additional file 2: Table S2). The sulfur-relay system classified in tRNA modification has been demonstrated to modify a few nucleotides of tRNA molecules, their increased expression has been reported contributing to stabilization of their structure, and was required for survival at an extremely high temperature in E. coli[40] and Thermous thermophilus[41]. In addition, ubiquitin (Ub) like proteins are signaling messengers that control many cellular functions in bacteria. It has been proposed that the Ub-protein modification evolved from prokaryotic sulfurtransfer systems [42]. Molybdenum cofactor (Moco) and thiamin are sulfur-containing cofactors whose biosynthesis includes a key sulfur transfer step that uses unique sulfur carrier proteins, MoaD and ThiS. Detailed analysis showed that upon hexane stress, two proteins in “Sulfur relay system” pathway, Slr0821 and Ssl1707 with homologies to sulfur carrier proteins MoaD and sulfur-accepting protein TusA were up-regulated, consistent with their possible roles against stress.

“Arachidonic acid metabolism” pathway (KO00590) was differentially regulated by hexane exposure at 24 h, in two out of four biological replicates ( Additional file 2: Table S2). Detailed analysis showed two proteins of glutathione peroxidase (Slr1992 and Slr1171) were up-regulated and thus contributed to the enrichment of this pathway (Table 1). Peroxidases are heme-cofactored enzymes responsible for hydrogen peroxide removal, cytoplasmic glutathione peroxidase (Gpx) has been found involved in oxidative defense in many bacteria [46].

Membrane-bound proteins significantly induced by hexane

Pathway enrichment analysis showed that “ABC transporters” (KO02010) was differentially regulated by hexane exposure, among which four ABC transporters were matched to the KEGG pathway KO02010 at 48 h ( Additional file 2: Table S2). Further analysis, however, showed that more putative transporters were up-regulated by the hexane treatment (Table 1). Induction of transporters by stress or toxic solvent has been reported in many microbes, such as acrAB-tolC in E. coli and srpABC in Pseudomonas putida which have been shown to export hexane, heptane, octane, octanol and nonane, and the enhanced expression of these genes were related to high tolerance [48, 49]. Up-regulation of transporters has been reported for various stresses in many other cyanobacterial species, such as arsenate resistance in Anabaena variabilis, salinity in Synechocystis[50–53]. In Synechocystis sp. PCC 6803, we recently found that five putative transporters involved in transporting of different substrates (i.e. polar amino acid, bicarbonate, iron, Na⁺/K⁺) were up-regulated by ethanol exposure [45]. Upon hexane exposure, a spectrum of putative transporters, including five transporters involved in bicarbonate transporting (Slr0041, Slr0043, Sll0834, Slr1512, Sll1734), two involved in cation transporting (Slr2131, Sll0672), two involved in Na⁺ and K⁺ transporting (Sll0689, Sll0493), one involved in mercuric transporting (Ssr2857), were up-regulated (Table 1). In Synechocystis sp. PCC 6803, the slr004 0, slr0041, slr0043, and slr0044 genes, forming an operon with a putative porin gene (slr0042), were induced under low CO₂ stress conditions to increase the bicarbonate transporting [54]. Slr1512 was previously found essential to Na⁺-dependent HCO₃^- transport and may play a crucial role in carbon acquisition when CO₂ supply is limited [55]. sll0493 encodes an NAD⁺-binding peripheral membrane protein (KtrA) that by working with a K⁺ transporters (KtrB; Slr1509) and KtrE (Slr1508), played a crucial role in the early phase of cell turgor regulation after hyperosmotic shock in Synechocystis[56]. Although the preliminary evidence suggested that these transporters may directly involve in hexane transporting, their up-regulation implicated the important roles for the hexane tolerance.

In addition, our proteomic analysis showed that many proteins located on cell membrane were up-regulated (Table 1), including a plasma membrane protein essential for thylakoid formation Sll0617 (vipp1) with a similar gene, pspA, in E. coli up-regulated by organic solvents [35], a periplasmic iron-binding protein (Slr0513) [57], four periplasmic proteins with unknown function (Slr1160, Slr2144, Slr1513, Sll0319), a salt-enhanced periplasmic protein (Sll1549). Meanwhile, several membrane-bound proteins were also found down-regulated by hexane exposure (Table 2). Although the exact function of these proteins was mostly unknown, the significant changes occurred at the cell membrane level may represent an important resistance strategy against hexane toxicity.

Bacteria use a variety of secretion systems to transport proteins beyond their cell membrane in order to interact with their environment. In E. coli and other gram-negative bacteria, one of the major translocation systems is the twin arginine translocation pathway consisted of TatA, TatB and TatC to export folded proteins across the cytoplasmic membrane [58]; and another is the Sec-dependent protein translocation system whose complex molecular machine comprises a flexible transmembrane conduit coupled to a motor-like component [59]. Our proteomics analysis showed that hexane treatment induced the expression of both systems in Synechocystis sp. PCC 6803. The putative TatA protein (Slr1046) of twin arginine translocation pathway and SecF (Slr0775) of the Sec-dependent protein translocation system were both up-regulated (Table 1).

Regulatory systems regulated by hexane

Two-component system (TCS) is an important signal transduction mechanism employed by prokaryotes to survive the complex and volatile environments [60], and has been involved in various stress responses in cyanobacteria [61, 62]. In our previous work, two response regulators (Slr1783, Slr1909) were found up-regulated by ethanol exposure; however, these two response regulators were not differentially regulated by hexane under our experimental conditions [45]; meanwhile, four different regulatory proteins were up-regulated, and two regulatory proteins were down-regulated by hexane exposure (Table 1, 2). Interestingly, up-regulated Slr1697 of a serine/threonine kinase and up-regulated Sll0043 of a positive phototaxis hybrid histidine kinase (homologous to chemotaxis protein CheA) were both required for cell motility and chemotaxis [63, 64]. Chemotaxis and flagellar motility are essential mechanisms by which bacteria use to adapt to and survive in environments suffered with various natural stresses [65], our results showed that the Synechocystis cells may adapt the similar mechanism in dealing with the stress caused by hexane. In addition, the fact that a different set of regulatory proteins were involved in hexane stress from those in ethanol stress implicated that variable resistance mechanisms were used by the cells for the biofuel products of different chemical properties [45].

Bacterial growth condition and hexane treatment

Synechocystis sp. PCC 6803 was obtained from American Type Culture Collection (ATCC) and grown in BG11 medium (pH 7.5) under a light intensity of approximately 50 μmol photons m^-2 s^-1 in an illuminating incubator operated at 130 rpm and at 30°C (HNY-211B Illuminating Shaker, Honour, China) [73–75]. Cell density was measured on a UV-1750 spectrophotometer (Shimadzu, Japan). For growth and hexane treatment, the 10 mL fresh cells of OD₇₃₀ 0.5 were collected by centrifugation at 8000 x g for 10 min at 4°C, and then were inoculated into 50 mL BG11 liquid medium in a 250-mL flask. Hexane of varying concentration was added at the beginning of cultivation. 1 mL of culture samples were collected and measured with a spectrophotometer at OD₇₃₀ every 12 h. Morphology of Synechocystis under control and treatment conditions was observed using a BX43 fluorescence microscope (Olympus, Japan). Growth experiments were repeated at least three times to confirm the growth patterns. Cells for proteomics analysis were collected by centrifugation at 8,000 x g for 10 min at 4°C.

Protein preparation and digestion

For each sample, 10 mg of cells were frozen by liquid nitrogen immediately after centrifugation and washed with phosphate buffer (pH 7.2). The cells were broken with sonication cracking at low temperature. Cell pellets were then resuspended in a lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris–HCl), with 1 mM PMSF and 2 mM Ethylenediaminetetraacetic acid (EDTA) (final concentration). After 5 min of vigorously vortex, dithiothreitol (DTT) was also added to a final concentration of 10 mM. After mix, the sample were centrifuged for 20 min at 20,000 x g, and the supernatant was mixed well with ice-cold acetone (1:4, v/v) with 30 mM DTT. After repeating this step twice, supernatants were combined and precipitated at −20°C overnight, and stored at −80°C prior to sample if not for immediate use. For digestion, protein pellet from previous step was resuspended in digestion buffer (100 mM triethylammonium bicarbonate TEAB, 0.05% w / v sodium dodecyl sulfate, SDS) to a final concentration of 1 mg/mL (total protein measured by bicinchonic acid assay (Sigma, St. Louis, MO)). Equal aliquots (500 μg) from each lysate were then digested with trypsin overnight at 37°C (Sigma; 1:40 w/w added at 0 and 2 h) and lyophilized.

iTRAQ labeling

The iTRAQ labeling of peptide samples derived from control and hexane treatment conditions were performed using iTRAQ reagent Multiplex kit (Applied Biosystems, Foster City, CA) according to manufacturer’s protocol. For each time point (i.e. 24 h or 48 h), four samples (two biological replicates for control and two biological replicates for hexane-treated samples) were iTRAQ labeled. The peptides labeled with respective isobaric tags, incubated for 2 h and vacuum centrifuged to dryness. The labeled control and hexane treatment replicate samples were 1:1 pooled, and generating four combinations of samples for each time point, which were reconstituted in Buffer A (10 mM KH₂PO₄, 25% acetonitrile, pH 2.85). The iTRAQ labeled peptides were fractionated using PolySULFOETHYL ATM SCX column (200 x 4.6 mm, 5 μm particle size, 200 A° pore size) by HPLC system (Shimadzu, Japan) at flow rate 1.0 ml min^-1. The 50 min HPLC gradient consisted of 100% buffer A (10 mM KH₂PO₄, 25% acetonitrile, pH 2.85) for 5 min; 0-20% buffer B (10 mM KH₂PO₄, 25% ACN, 500 mM KCL, pH 3.0) for 15 min; 20-40% buffer B for 10 min; 40-100% buffer B for 5 min followed by 100% buffer A for 10 min. The chromatograms were recorded at 218 nm. The collected fractions were desalted with Sep-Pak® Vac C18 cartridges (Waters, Milford, Massachusetts), concentrated to dryness using vacuum centrifuge and reconstituted in 0.1% formic acid for LC-MS/MS analysis.

LC-MS/MS proteomic analysis

The mass spectroscopy analysis was performed using a AB SCIEX TripleTOF™ 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA), coupled with online micro flow HPLC system (Shimadzu, JAPAN) as described before [76, 77]. The peptides were separated using nanobored C18 column with a picofrit nanospray tip (75 μm ID x 15 cm, 5 μm particles) (New Objectives, Wubrun, MA). The separation was performed at a constant flow rate of 20 μl min^-1, with a splitter to get an effective flow rate of 0.2 μl min^-1. The mass spectrometer data acquired in the positive ion mode, with a selected mass range of 300–2000 m/z. Peptides with +2 to +4 charge states were selected for MS/MS. The three most abundant peptides above a 5 count threshold were selected for MS/MS and dynamically excluded for 30 s with ±30 mDa mass tolerance. Smart information-dependent acquisition (IDA) was activated with automatic collision energy and automatic MS/MS accumulation. The fragment intensity multiplier was set to 20 and maximum accumulation time was 2 s. The peak areas of the iTRAQ reporter ions reflect the relative abundance of the proteins in the samples.

Proteomic data analysis

where N is the number of all proteins with KEGG pathway annotation information, n is the number of the differentially regulated proteins with KEGG pathway annotation information, M is the number of proteins with a given KEGG pathway annotation, m is the number of the differentially regulated proteins with a given KEGG pathway annotation. The KEGG pathways with p-value less than 0.05 were considered as enriched KEGG pathways by the hexane responsive proteins.