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Identification of glutathione S-transferase (GST) genes from a dark septate endophytic fungus (Exophiala pisciphila) and their expression patterns under varied metals stress.

Shen M, Zhao DK, Qiao Q, Liu L, Wang JL, Cao GH, Li T, Zhao ZW - PLoS ONE (2015)

Bottom Line: Moreover, the variable expression patterns of these EpGSTs were observed under different heavy metal stresses at their effective concentrations for inhibiting growth by 50% (EC50).These results indicate that E.Pisciphila harbored a diverse of GST genes and the up-regulated EpGSTs are closely related to the heavy metal tolerance of E. pisciphila.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Conservation and Utilization for Bioresources and Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan University, Kunming, Yunnan, China.

ABSTRACT
Glutathione S-transferases (GSTs) compose a family of multifunctional enzymes that play important roles in the detoxification of xenobiotics and the oxidative stress response. In the present study, twenty four GST genes from the transcriptome of a metal-tolerant dark septate endophyte (DSE), Exophiala pisciphila, were identified based on sequence homology, and their responses to various heavy metal exposures were also analyzed. Phylogenetic analysis showed that the 24 GST genes from E. pisciphila (EpGSTs) were divided into eight distinct classes, including seven cytosolic classes and one mitochondrial metaxin 1-like class. Moreover, the variable expression patterns of these EpGSTs were observed under different heavy metal stresses at their effective concentrations for inhibiting growth by 50% (EC50). Lead (Pb) exposure caused the up-regulation of all EpGSTs, while cadmium (Cd), copper (Cu) and zinc (Zn) treatments led to the significant up-regulation of most of the EpGSTs (p < 0.05 to p < 0.001). Furthermore, although heavy metal-specific differences in performance were observed under various heavy metals in Escherichia coli BL21 (DE3) transformed with EpGSTN-31, the over-expression of this gene was able to enhance the heavy metal tolerance of the host cells. These results indicate that E. Pisciphila harbored a diverse of GST genes and the up-regulated EpGSTs are closely related to the heavy metal tolerance of E. pisciphila. The study represents the first investigation of the GST family in E. pisciphila and provides a primary interpretation of heavy metal detoxification for E. pisciphila.

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Growth curves of the transformed BL21 strains with empty pGEX-4T-1(negative control) and recombinant pGEX-4T-1-EpGSTN-31 plasmids.A total of 200 mg L-1 IPTG was added to each culture to induce the expression of the recombinant protein. (a) 50 mg L-1 Cd2+ (b) 100 mg L-1 Pb2+ (c) 70 mg L-1 Cu2+ and (d) 100mg L-1 Zn 2+. Each treatment was conducted independently four times. Asterisks indicate significant differences (p < 0.05*, p < 0.01**, p < 0.001***) compared to the control according to Independent-Samples T test.
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pone.0123418.g003: Growth curves of the transformed BL21 strains with empty pGEX-4T-1(negative control) and recombinant pGEX-4T-1-EpGSTN-31 plasmids.A total of 200 mg L-1 IPTG was added to each culture to induce the expression of the recombinant protein. (a) 50 mg L-1 Cd2+ (b) 100 mg L-1 Pb2+ (c) 70 mg L-1 Cu2+ and (d) 100mg L-1 Zn 2+. Each treatment was conducted independently four times. Asterisks indicate significant differences (p < 0.05*, p < 0.01**, p < 0.001***) compared to the control according to Independent-Samples T test.

Mentions: There are currently no reports in the literature describing heavy metal tolerance in association with the GST N-3 class. According to the results of transcriptome analysis, the top differentially expressed gene was EpGSTN-31 among 575 detected genes [9]. In addition, the qRT-PCR analysis revealed that EpGSTN-31 was the most highly expressed among the 24 EpGSTs under Cd stress. Taken together, we carried out the functional verification of the EpGSTN-31. The results (Fig 3) indicated that compared with the control strain containing the empty pGEX-4T-1 vetors, the cell growth of the transformed BL21 strain expressing EpGSTN-31 was significantly enhanced at most of the exposure times under various heavy metal stresses. However, the transformed strain responded differently to Pb, Zn, Cu and Cd treatments; EpGSTN-31 appeared to confer better Pb and Cu tolerances compared to those of Cd and Zn, particularly following long-term exposure (Fig 3). In addition, we observed maximal levels of heavy metal tolerance in the transformed cells after 8 h of exposure to Cu stress compared to the control cells, but the transformed cells under Cd and Pb stresses did not show maximal tolerance levels until the 20th and 24th hours, respectively. Taken together, these results demonstrated that the over-expression of EpGSTN-31 was able to enhance the heavy metal tolerance of the host cells, but heavy metal-specific differences in performance were observed among the various heavy metals.


Identification of glutathione S-transferase (GST) genes from a dark septate endophytic fungus (Exophiala pisciphila) and their expression patterns under varied metals stress.

Shen M, Zhao DK, Qiao Q, Liu L, Wang JL, Cao GH, Li T, Zhao ZW - PLoS ONE (2015)

Growth curves of the transformed BL21 strains with empty pGEX-4T-1(negative control) and recombinant pGEX-4T-1-EpGSTN-31 plasmids.A total of 200 mg L-1 IPTG was added to each culture to induce the expression of the recombinant protein. (a) 50 mg L-1 Cd2+ (b) 100 mg L-1 Pb2+ (c) 70 mg L-1 Cu2+ and (d) 100mg L-1 Zn 2+. Each treatment was conducted independently four times. Asterisks indicate significant differences (p < 0.05*, p < 0.01**, p < 0.001***) compared to the control according to Independent-Samples T test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401685&req=5

pone.0123418.g003: Growth curves of the transformed BL21 strains with empty pGEX-4T-1(negative control) and recombinant pGEX-4T-1-EpGSTN-31 plasmids.A total of 200 mg L-1 IPTG was added to each culture to induce the expression of the recombinant protein. (a) 50 mg L-1 Cd2+ (b) 100 mg L-1 Pb2+ (c) 70 mg L-1 Cu2+ and (d) 100mg L-1 Zn 2+. Each treatment was conducted independently four times. Asterisks indicate significant differences (p < 0.05*, p < 0.01**, p < 0.001***) compared to the control according to Independent-Samples T test.
Mentions: There are currently no reports in the literature describing heavy metal tolerance in association with the GST N-3 class. According to the results of transcriptome analysis, the top differentially expressed gene was EpGSTN-31 among 575 detected genes [9]. In addition, the qRT-PCR analysis revealed that EpGSTN-31 was the most highly expressed among the 24 EpGSTs under Cd stress. Taken together, we carried out the functional verification of the EpGSTN-31. The results (Fig 3) indicated that compared with the control strain containing the empty pGEX-4T-1 vetors, the cell growth of the transformed BL21 strain expressing EpGSTN-31 was significantly enhanced at most of the exposure times under various heavy metal stresses. However, the transformed strain responded differently to Pb, Zn, Cu and Cd treatments; EpGSTN-31 appeared to confer better Pb and Cu tolerances compared to those of Cd and Zn, particularly following long-term exposure (Fig 3). In addition, we observed maximal levels of heavy metal tolerance in the transformed cells after 8 h of exposure to Cu stress compared to the control cells, but the transformed cells under Cd and Pb stresses did not show maximal tolerance levels until the 20th and 24th hours, respectively. Taken together, these results demonstrated that the over-expression of EpGSTN-31 was able to enhance the heavy metal tolerance of the host cells, but heavy metal-specific differences in performance were observed among the various heavy metals.

Bottom Line: Moreover, the variable expression patterns of these EpGSTs were observed under different heavy metal stresses at their effective concentrations for inhibiting growth by 50% (EC50).These results indicate that E.Pisciphila harbored a diverse of GST genes and the up-regulated EpGSTs are closely related to the heavy metal tolerance of E. pisciphila.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Conservation and Utilization for Bioresources and Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan University, Kunming, Yunnan, China.

ABSTRACT
Glutathione S-transferases (GSTs) compose a family of multifunctional enzymes that play important roles in the detoxification of xenobiotics and the oxidative stress response. In the present study, twenty four GST genes from the transcriptome of a metal-tolerant dark septate endophyte (DSE), Exophiala pisciphila, were identified based on sequence homology, and their responses to various heavy metal exposures were also analyzed. Phylogenetic analysis showed that the 24 GST genes from E. pisciphila (EpGSTs) were divided into eight distinct classes, including seven cytosolic classes and one mitochondrial metaxin 1-like class. Moreover, the variable expression patterns of these EpGSTs were observed under different heavy metal stresses at their effective concentrations for inhibiting growth by 50% (EC50). Lead (Pb) exposure caused the up-regulation of all EpGSTs, while cadmium (Cd), copper (Cu) and zinc (Zn) treatments led to the significant up-regulation of most of the EpGSTs (p < 0.05 to p < 0.001). Furthermore, although heavy metal-specific differences in performance were observed under various heavy metals in Escherichia coli BL21 (DE3) transformed with EpGSTN-31, the over-expression of this gene was able to enhance the heavy metal tolerance of the host cells. These results indicate that E. Pisciphila harbored a diverse of GST genes and the up-regulated EpGSTs are closely related to the heavy metal tolerance of E. pisciphila. The study represents the first investigation of the GST family in E. pisciphila and provides a primary interpretation of heavy metal detoxification for E. pisciphila.

Show MeSH
Related in: MedlinePlus