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Expression, purification, and functional analysis of novel RelE operon from X. nematophila.

Rathore JS, Gautam LK - ScientificWorldJournal (2014)

Bottom Line: Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified.This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 303 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA ; School of Biotechnology, Gautam Buddha University, Greater Noida, Yamuna Expressway, Uttar Pradesh 201312, India.

ABSTRACT
Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

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Related in: MedlinePlus

Endogenous toxicity assay. All the experiments were performed in triplicate, and mean values were used to show the results in percentage (%) growth at different time intervals. Bacterial growth was monitored by determining optical density at 600 nm in the presence of arabinose. (◆) JSL strain [WT (wild type) strain + empty vector (control)]; (▲) JSL3 strain [WT (wild type) strain + Xn-relE toxin]; and (■) JSL4 strain [WT (wild type) + operon (Xn-relE + Xn-relEAT)].
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Related In: Results  -  Collection


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fig4: Endogenous toxicity assay. All the experiments were performed in triplicate, and mean values were used to show the results in percentage (%) growth at different time intervals. Bacterial growth was monitored by determining optical density at 600 nm in the presence of arabinose. (◆) JSL strain [WT (wild type) strain + empty vector (control)]; (▲) JSL3 strain [WT (wild type) strain + Xn-relE toxin]; and (■) JSL4 strain [WT (wild type) + operon (Xn-relE + Xn-relEAT)].

Mentions: Type II toxin acts in a number of ways, but toxin activity is exerted most if it acts as an endonuclease/interferase [27, 28] while antitoxin usually inhibits the toxin by downregulating toxin expression. Under stress condition transcription of TA operon leads to formation of TA complex [10, 14]. Simultaneous expression of Lon protease and its action over labile antitoxin that is susceptible to be degraded ultimately unleash toxin from the toxin-antitoxin complex. Toxins are less susceptible to proteases and their toxic effect is exerted on host cells either by inhibiting cell wall formation or by halting protein synthesis [29]. Therefore, to study the endogenous toxic effect of putative protein from novel identified Xn-relE gene as well as neutralization effect by putative Xn-relEAT gene, both were cloned under tightly regulated ara promoter. pJSL3 recombinant plasmid harbouring Xn-relE gene and pJSL4 recombinant plasmid containing complete TA operon (Xn-relE + Xn-relEAT) were transformed in E. coli Top 10 cells. In the endogenous toxic assay, control strain JSL containing pBAD vector alone was considered as 100% based on the growth profile after induction with arabinose when compared with arabinose-induced JSL3 strain containing Xn-relE toxin only as well as with JSL4 strain with full operon (Xn-relE + Xn-relEAT). Results show that there was no change in growth profile of JSL3 strain after the first hour of induction; however, there is gradual decrease in the growth following time intervals. It was inhibited by 25% by the third hour of arabinose induction graph showing steep fall at initial time points and was further declined to 8% after 8 hours in JSL3 strain as compared to control as shown in Figure 4. However, ara induced full length operon was also growth inhibition, but it was less inhibited than that of Xn-relE toxin alone as only ~55% inhibition was observed after eight hours of induction. Reduction in growth inhibition is due to the expression of antitoxin Xn-relEAT from the operon under ara promoter although the expression of antitoxin Xn-relEAT is not able to completely neutralize the toxic effect of Xn-relE gene. Less expression of antitoxin Xn-relEAT which might be incapable of neutralizing the toxicity of Xn-relE makes a clue behind the difference in the growth inhibition profile of arabinose-induced culture. It also indicates that action of toxin is much rapid that it exerts its effects before antitoxin molecule makes a TA complex and neutralizes it. Recent studies showed that free toxins in the cell are very less due to the negative transcriptional control loop as well as tight complex formation between toxin and antitoxin. Moreover, in E. coli TA module RelBE they form heterotrimer complex, consisting of two antitoxins (RelB) and one toxin molecule (RelE) bounded together [24, 30]. Therefore, it can be assumed that toxic effect of Xn-relE toxin neutralization may require more than one Xn-relEAT protein molecule. Hence, in endogenous toxicity assay, difference in growth to significant extent was observed between JSL strain [(wild type) strain + empty vector (control)] and JSL4 strain [WT (wild type) + operon (Xn-relE + Xn-relEAT)].


Expression, purification, and functional analysis of novel RelE operon from X. nematophila.

Rathore JS, Gautam LK - ScientificWorldJournal (2014)

Endogenous toxicity assay. All the experiments were performed in triplicate, and mean values were used to show the results in percentage (%) growth at different time intervals. Bacterial growth was monitored by determining optical density at 600 nm in the presence of arabinose. (◆) JSL strain [WT (wild type) strain + empty vector (control)]; (▲) JSL3 strain [WT (wild type) strain + Xn-relE toxin]; and (■) JSL4 strain [WT (wild type) + operon (Xn-relE + Xn-relEAT)].
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4265723&req=5

fig4: Endogenous toxicity assay. All the experiments were performed in triplicate, and mean values were used to show the results in percentage (%) growth at different time intervals. Bacterial growth was monitored by determining optical density at 600 nm in the presence of arabinose. (◆) JSL strain [WT (wild type) strain + empty vector (control)]; (▲) JSL3 strain [WT (wild type) strain + Xn-relE toxin]; and (■) JSL4 strain [WT (wild type) + operon (Xn-relE + Xn-relEAT)].
Mentions: Type II toxin acts in a number of ways, but toxin activity is exerted most if it acts as an endonuclease/interferase [27, 28] while antitoxin usually inhibits the toxin by downregulating toxin expression. Under stress condition transcription of TA operon leads to formation of TA complex [10, 14]. Simultaneous expression of Lon protease and its action over labile antitoxin that is susceptible to be degraded ultimately unleash toxin from the toxin-antitoxin complex. Toxins are less susceptible to proteases and their toxic effect is exerted on host cells either by inhibiting cell wall formation or by halting protein synthesis [29]. Therefore, to study the endogenous toxic effect of putative protein from novel identified Xn-relE gene as well as neutralization effect by putative Xn-relEAT gene, both were cloned under tightly regulated ara promoter. pJSL3 recombinant plasmid harbouring Xn-relE gene and pJSL4 recombinant plasmid containing complete TA operon (Xn-relE + Xn-relEAT) were transformed in E. coli Top 10 cells. In the endogenous toxic assay, control strain JSL containing pBAD vector alone was considered as 100% based on the growth profile after induction with arabinose when compared with arabinose-induced JSL3 strain containing Xn-relE toxin only as well as with JSL4 strain with full operon (Xn-relE + Xn-relEAT). Results show that there was no change in growth profile of JSL3 strain after the first hour of induction; however, there is gradual decrease in the growth following time intervals. It was inhibited by 25% by the third hour of arabinose induction graph showing steep fall at initial time points and was further declined to 8% after 8 hours in JSL3 strain as compared to control as shown in Figure 4. However, ara induced full length operon was also growth inhibition, but it was less inhibited than that of Xn-relE toxin alone as only ~55% inhibition was observed after eight hours of induction. Reduction in growth inhibition is due to the expression of antitoxin Xn-relEAT from the operon under ara promoter although the expression of antitoxin Xn-relEAT is not able to completely neutralize the toxic effect of Xn-relE gene. Less expression of antitoxin Xn-relEAT which might be incapable of neutralizing the toxicity of Xn-relE makes a clue behind the difference in the growth inhibition profile of arabinose-induced culture. It also indicates that action of toxin is much rapid that it exerts its effects before antitoxin molecule makes a TA complex and neutralizes it. Recent studies showed that free toxins in the cell are very less due to the negative transcriptional control loop as well as tight complex formation between toxin and antitoxin. Moreover, in E. coli TA module RelBE they form heterotrimer complex, consisting of two antitoxins (RelB) and one toxin molecule (RelE) bounded together [24, 30]. Therefore, it can be assumed that toxic effect of Xn-relE toxin neutralization may require more than one Xn-relEAT protein molecule. Hence, in endogenous toxicity assay, difference in growth to significant extent was observed between JSL strain [(wild type) strain + empty vector (control)] and JSL4 strain [WT (wild type) + operon (Xn-relE + Xn-relEAT)].

Bottom Line: Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified.This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 303 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA ; School of Biotechnology, Gautam Buddha University, Greater Noida, Yamuna Expressway, Uttar Pradesh 201312, India.

ABSTRACT
Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

Show MeSH
Related in: MedlinePlus