Improved Accumulation and Tolerant to Heavy Metals Triggered by Over-Expressing AtGCS from Arabidopsis Thaliana in E. coli

GSH is one of the most ubiquitous proteins known to provide protection against toxic heavy metals. A gene-AtGCS encoding Arabidopsis γ-glutamylcysteine synthetase was introduced into Escherichia coli (BL21) by over-expression of TrxA-AtGCS fusion protein and analyzed the tolerant capacity to heavy metal by transformed E. coli. As comparison, the strain over-expressing TrxA was selected as the control. The results showed that the growth of E. coli cells over-expressing TrxA-AtGCS was much better than the control cells expressing TrxA under 1 mM Cd, Zn and Cu heavy metals respectively. Meanwhile, the higher bioaccumulation of Cd, Zn, Cu and GSH content were observed in the strain over-expressing TrxA-AtGCS. It could be concluded that overexpression of AtGCS offered a promising heavy metals resistance of E. coli with superior heavy metals accumulation and GSH content.


INTRODUCTION
Heavy metals pollution of soils and waters, mainly caused by mining and the burning of fossil fuels, is a major environmental problem, which impacted many organisms by stress-induced formation of Reactive Oxygen Species (ROS) (Nies, 1999;Nriagu and Pacyna, 1998).The best-known detoxification molecule, which serves as storage place as well as transportation molecules in cells, is glutathione (L-γglutamyl-L-cysteinylglycine, GSH) (Chan and Cherian, 1992;Foyer and Noctor, 2005;Meister, 1995).GSH seems to be the first defense against metal toxicity.GSH functions as an antioxidant itself and as a component of the detoxifying enzyme system containing oxidase and reductase.Glutathione is also required for synthesis of proteins and nucleic acids, maintenance of enzymes in their active forms and maintenance of cell membranes.Production of GSH has been suggested to play a protective role against metal toxicity (Szalai et al., 2009).Thus, increase of glutathione content may increase the cells' tolerance to heavy metal stress.In eukaryotes, GSH is synthesized intracellular in two ATP-dependent steps catalyzed by γ-glutamylcysteine synthetase (GSH1, γ-GCS; EC 6.3.2.2) and glutathione synthetase (GSH2, GS; EC 6.3.2.3) and γ-ECS is feedback inhibited by GSH.The two enzymes have been isolated and characterized from several plants, yeast and etc. (Kim et al., 2003;Rawlins et al., 1995).
In the past decades, in order to sequester, precipitate or alter such oxidation state of various heavy metals, the use of microorganisms has been extensively studied.Attempts to enhance the metal content of bacterial cells have been made by expression of Metallothioneins (MTs) to increase the affinity and biosorptive capabilities for heavy metals (Chaturvedi et al., 2012;Sauge-Merle et al., 2012), over-expressing metal-binding peptides or proteins such as polyhistidines (Sousa et al., 1996) and metal binding motif (Pazirandeh et al., 1998).Nevertheless, few researches focus on manipulating the expression of enzymes involved in glutathione, which maybe a good approach to enhance heavy-metal tolerance in E. coli.
To gain more insight into the protective role of AtGCS gene in heavy metal tolerance of E. coli and whether and how it could enhance the metal-retaining ability of bacterial cells more evidently, AtGCS was expressed as a fusion protein with TrxA to obtain a stable over-expressed protein.The experiment was carried out with transformed E. coli in 1 mM Cd 2+ , Zn 2+  and Cu 2+ respectively.The heavy metal tolerance and accumulation were examined with the glutathione content assay comprehensively.

MATERIALS AND METHODS
Materials: E. coli TransDH5α and BL21, used as expression host strains, were cultured aerobically in Luria-Bertani medium (LB).The expression vector used in this study was pETM-20, which contains a lac promoter for isopropyl β-D-Thiogalactopyranoside (IPTG) inducible, high-level TrxA-fusion protein expression and a cleavage site for Tobacco Etch Virus (TEV) protease.

Expression and purification of recombinant fusion
protein: E. coli BL21 harboring plasmid pETM-20-AtGCS was used for expression and purification of TrxA-AtGCS.Cells transfected with pETM-20 of deletion RAGE were used for expression of TrxA as a control.Cells were grown to OD 600 ~0.5 at 37°C and induced by adding IPTG to a final concentration of 1 mM overnight at 30°C with gentle shaking.Cell pellets were resuspended in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0) supplemented with 2 mg/mL lysozyme.The translucent suspension was centrifuged at 15000 g for 20 min.Proteins were purified by Ni-NTA resin affinity column chromatography.AtGCS recombinant proteins were obtained from the TrxA-fusion proteins by cleavage with TEV protease.

Heavy metal tolerance analysis of transformed E. coli:
For assessment of heavy metal tolerance on solidified medium, E. coli cultures were incubated at 37°C until cells reached the midlog phase (OD 600 = 0.5).Each strain was separately inoculated into 100 ml of LB medium containing 1 mM CdCl 2 , 1 mM ZnCl 2 or 1 mM CuCl 2 with or without 1 mM IPTG induction for the duration of the experiment.Growth rate was analyzed by monitoring the increase in absorbance at 600 nm at 4 h intervals for 24 h.

Heavy metal accumulation assay and intracellular glutathione determination:
Overnight cultures of E. coli BL21 were diluted to OD 600 = 0.5 and incubated For determination of intracellular glutathione, the pellets were resuspended and intracellular GSH measurements were made by using a GSH assay kit (Sigma, USA).The results are expressed as nmol of intracellular glutathione per mg of total protein.

Statistical analysis:
All data were subjected to statistical analysis.All experiments were carried out in triplicate and three samples in each individual determination were examined each time.The values shown in the figures and table are mean values ±SD.

Heavy metal tolerance of E. coli cells overexpressing TrxA-StGCS-GS fusion protein:
To determine whether AtGCS could enhance the heavy metal tolerance of E. coli, the effects of heavy metal stress on growth of E. coli cells transformed with TrxA-AtGCS were examined.Under normal conditions, there were no differences in growth rate among E. coli cells transformed with TrxA and TrxA-AtGCS, whether or not there was IPTG induction (data not shown).When there was a heavy metal stress, the growth rate of all four transformed strains was seriously damaged to almost the same low level without IPTG induction.On the contrary, although there was a little enhancement of cells expression of TrxA, expression of AtGCS reached a higher grown stage within 24 h (Fig. 2).The results suggested that the cells over-expressing AtGCS could overcome the deleterious effects of cadium, zinc and copper toxicity to some content.In E. coli, a similar function has also been shown for MTs, poly-histidines and metal binding motif proteins (Chaturvedi et al., 2012;Sauge-Merle et al., 2012;Sousa et al., 1996;Pazirandeh et al., 1998;An et al., 2006).

Heavy metal bioaccumulation and cellular GSH level by transformed strains over-expressing TrxA-AtGCS:
To determine why and how the expression of AtGCS recombinant protein enhanced the growth of E. coli under heavy metal stress, the ability of cells expressing TrxA-AtGCS fusion to remove heavy metals from solutions was tested and compared to that of the control bacteria.Induced E. coli cells were incubated for 24 h under 1 mM Cd 2+ , Zn 2+ and Cu 2+ pressure.As shown in Table 1, higher levels of bioaccumulation were measured with the strains over-expressing TrxA-AtGCS under such three kinds of heavy metal ions.No obvious accumulation of heavy metal ions was detected in the control cells over-expressing TrxA.The differences were more distinctive when cells suffered from Cd 2+ stress.It could be concluded that the overexpression of TrxA-AtGCS could more efficiently remove Cd 2+ , Zn 2+ and Cu 2+ .
Furthermore, the effects of heavy metal on cellular glutathione levels of the two E. coli strains were evaluated (Table 1).The GSH concentrations of cells over-expressing TrxA-AtGCS were relatively stable and GSH was about five-fold that produced by the strains over-expressing TrxA under the same heavy metal stressful conditions in 24 h.The results indicated that the high content of cellular GSH could prevent E. coli cells from harmful heavy metal stress damage to some degree.It can be speculated that such high glutathione levels by over-expression of AtGCS overcoming the inhibition feedback by GSH, resulted in the higher accumulation of heavy metal ions in E. coli cells.These results coincided with the previous study that GSH was important for residual metal resistance in E. coli (Helbig et al., 2008).

CONCLUSION
The applications of genetic engineering in the modification of the microorganisms have become a promising way for increasing the efficiency of the bioremediation processes for heavy metals removal (Kim et al., 2005).GSH, an important antioxidant involved in the heavy metal stress response, is synthesized by the consecutive actions of γ-glutamylcysteine synthetase and glutathione synthetase (Anderson, 1998).Because the activity of the former is feedback regulated by GSH, it partially restores impaired growth of E. coli.Nevertheless, by over-production of γ-GCS, there might be no feedback inhibition of GSH.Thus, we try to analyze the relative importance of over-expression of AtGCS in enhancement of E. coli under heavy metal stressful conditions.
In TrxA-AtGCS-transformed cells, production of recombinant protein allowed E. coli to survive under Cd 2+ , Zn 2+ and Cu 2+ more consistently, which was lethal to cells expressing TrxA.This improved resistance of E. coli cells to heavy metal stresses was relative to more efficient bioaccumulation of Cd 2+ , Zn 2+ and Cu 2+ and higher levels of glutathione content.It could be concluded that AtGCS played an essential role in protecting E. coli cells against the toxic effects of Cd 2+ , Zn 2+ and Cu 2+ .

Fig. 1 :
Fig. 1: Expression and purification of recombinant proteins as followed by SDS-PAGE.(A) TrxA, (B) TrxA-AtGCS.Lane 1 and 2, total crude extract of uninduced or induced cells; lane 3, cell lysate supernatant of induced cells; lane 4 and 5, purified TrxA-AtGCS and AtGCS cleavage by TEV protease indicated by arrows.M, molecular mass markers at 37°C for 24 h containing 1 mM IPTG and metal ions at the desired concentration.The biomass was separated by centrifugation at 5 000g for 10 min and the supernatant were analyzed for residual metal concentration on an atomic absorption spectroscopy (Thermo Electron Corp. USA).Heavy metal content was expressed as the weight of metal per weight of the wet cells (milligram per milligram of wet weight).For determination of intracellular glutathione, the pellets were resuspended and intracellular GSH measurements were made by using a GSH assay kit (Sigma, USA).The results are expressed as nmol of intracellular glutathione per mg of total protein.

Table 1 :
Accumulation of heavy metal ions and glutathione content by E. coli BL21 strains over-expressing TrxA-AtGCS Strains Amount of heavy metal accumulated by bacteria [mg of metal/mg of cells (wet weight)] a -