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Regulation of the expression level of transcription factor XylS reveals new functional insight into its induction mechanism at the Pm promoter.

Zwick F, Lale R, Valla S - BMC Microbiol. (2013)

Bottom Line: Their maximum intracellular concentration and the corresponding output from Pm are limited by the concentration-dependent conversion into inactive aggregates.Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression.The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands Vei 6/8, Trondheim N-7491, Norway. rahmi.lale@ntnu.no.

ABSTRACT

Background: XylS is the positive regulator of the inducible Pm promoter, originating from Pseudomonas putida, where the system controls a biochemical pathway involved in degradation of aromatic hydrocarbons, which also act as inducers. The XylS/Pm positive regulator/promoter system is used for recombinant gene expression and the output from Pm is known to be sensitive to the intracellular XylS concentration.

Results: By constructing a synthetic operon consisting of xylS and luc, the gene encoding luciferase, relative XylS expression levels could be monitored indirectly at physiological concentrations. Expression of XylS from inducible promoters allowed control over a more than 800-fold range, however, the corresponding output from Pm covered only an about five-fold range. The maximum output from Pm could not be increased by introducing more copies of the promoter in the cells. Interestingly, a previously reported XylS variant (StEP-13), known to strongly stimulate expression from Pm, caused the same maximum activity from Pm as wild-type XylS at high XylS expression levels. Under uninduced conditions expression from Pm also increased as a function of XylS expression levels, and at very high concentrations the maximum activity from Pm was the same as in the presence of inducer.

Conclusion: According to our proposed model, which is in agreement with current knowledge, the regulator, XylS, can exist in three states: monomers, dimers, and aggregates. Only the dimers are active and able to induce expression from Pm. Their maximum intracellular concentration and the corresponding output from Pm are limited by the concentration-dependent conversion into inactive aggregates. Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression. The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

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Expression levels from pFS7 for different variants of xylS with silent mutations. Relative expression levels from Pm (measured as maximum ampicillin tolerance at 1 mM m-toluate) are given in grey (error bars = lowest ampicillin concentrations in test on which no growth was observed) and relative luciferase activity as a measure for XylS amounts in black (values from at least two biological replicas). All values (relative ampicillin tolerance and luciferase expression) refer to those of wild type XylS (tolerating 350 μg mL-1), which are both arbitrarily set to 1. Mutations in the variants (1 to 9), the number stands for the base position that has been changed, relative to the translational start site, the character tells the base in the variant. 1: 6- > C; 2: 13- > C; 3: 15- > G; 4: 16- > C; 5: 27- > G; 6: 30- > C; 7: 36- > T; 8: 42- > T; 9: all of the eight mutations.
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Figure 2: Expression levels from pFS7 for different variants of xylS with silent mutations. Relative expression levels from Pm (measured as maximum ampicillin tolerance at 1 mM m-toluate) are given in grey (error bars = lowest ampicillin concentrations in test on which no growth was observed) and relative luciferase activity as a measure for XylS amounts in black (values from at least two biological replicas). All values (relative ampicillin tolerance and luciferase expression) refer to those of wild type XylS (tolerating 350 μg mL-1), which are both arbitrarily set to 1. Mutations in the variants (1 to 9), the number stands for the base position that has been changed, relative to the translational start site, the character tells the base in the variant. 1: 6- > C; 2: 13- > C; 3: 15- > G; 4: 16- > C; 5: 27- > G; 6: 30- > C; 7: 36- > T; 8: 42- > T; 9: all of the eight mutations.

Mentions: With the goal to enable detection of XylS at low concentrations we developed a synthetic operon in which luciferase functions as an indirect indicator of expression of XylS from its native Ps2 promoter. In this design the luc gene is transcriptionally fused to xylS via overlapping stop and start codons and should be translated only when xylS is translated first. The new plasmid was designated as pFS7 (Figure 1). To test the functionality of this construct we used a series of xylS variant sequences which had been synthesized. These variants contain synonymous codon changes relative to the wild type sequence and had been found to activate Pm to varying extents (in the presence of induction). We hypothesized that the effects of the codon changes were caused by variations in xylS mRNA translation, since transcript amounts were found to be similar to the levels of the wild type gene (qRT-PCR, data not shown). Nine such variant sequences were tested in pFS7, and luciferase activities were measured (Figure 2). The values varied in the range from about 20 to 100% of that of the construct containing the wild type xylS.


Regulation of the expression level of transcription factor XylS reveals new functional insight into its induction mechanism at the Pm promoter.

Zwick F, Lale R, Valla S - BMC Microbiol. (2013)

Expression levels from pFS7 for different variants of xylS with silent mutations. Relative expression levels from Pm (measured as maximum ampicillin tolerance at 1 mM m-toluate) are given in grey (error bars = lowest ampicillin concentrations in test on which no growth was observed) and relative luciferase activity as a measure for XylS amounts in black (values from at least two biological replicas). All values (relative ampicillin tolerance and luciferase expression) refer to those of wild type XylS (tolerating 350 μg mL-1), which are both arbitrarily set to 1. Mutations in the variants (1 to 9), the number stands for the base position that has been changed, relative to the translational start site, the character tells the base in the variant. 1: 6- > C; 2: 13- > C; 3: 15- > G; 4: 16- > C; 5: 27- > G; 6: 30- > C; 7: 36- > T; 8: 42- > T; 9: all of the eight mutations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225500&req=5

Figure 2: Expression levels from pFS7 for different variants of xylS with silent mutations. Relative expression levels from Pm (measured as maximum ampicillin tolerance at 1 mM m-toluate) are given in grey (error bars = lowest ampicillin concentrations in test on which no growth was observed) and relative luciferase activity as a measure for XylS amounts in black (values from at least two biological replicas). All values (relative ampicillin tolerance and luciferase expression) refer to those of wild type XylS (tolerating 350 μg mL-1), which are both arbitrarily set to 1. Mutations in the variants (1 to 9), the number stands for the base position that has been changed, relative to the translational start site, the character tells the base in the variant. 1: 6- > C; 2: 13- > C; 3: 15- > G; 4: 16- > C; 5: 27- > G; 6: 30- > C; 7: 36- > T; 8: 42- > T; 9: all of the eight mutations.
Mentions: With the goal to enable detection of XylS at low concentrations we developed a synthetic operon in which luciferase functions as an indirect indicator of expression of XylS from its native Ps2 promoter. In this design the luc gene is transcriptionally fused to xylS via overlapping stop and start codons and should be translated only when xylS is translated first. The new plasmid was designated as pFS7 (Figure 1). To test the functionality of this construct we used a series of xylS variant sequences which had been synthesized. These variants contain synonymous codon changes relative to the wild type sequence and had been found to activate Pm to varying extents (in the presence of induction). We hypothesized that the effects of the codon changes were caused by variations in xylS mRNA translation, since transcript amounts were found to be similar to the levels of the wild type gene (qRT-PCR, data not shown). Nine such variant sequences were tested in pFS7, and luciferase activities were measured (Figure 2). The values varied in the range from about 20 to 100% of that of the construct containing the wild type xylS.

Bottom Line: Their maximum intracellular concentration and the corresponding output from Pm are limited by the concentration-dependent conversion into inactive aggregates.Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression.The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands Vei 6/8, Trondheim N-7491, Norway. rahmi.lale@ntnu.no.

ABSTRACT

Background: XylS is the positive regulator of the inducible Pm promoter, originating from Pseudomonas putida, where the system controls a biochemical pathway involved in degradation of aromatic hydrocarbons, which also act as inducers. The XylS/Pm positive regulator/promoter system is used for recombinant gene expression and the output from Pm is known to be sensitive to the intracellular XylS concentration.

Results: By constructing a synthetic operon consisting of xylS and luc, the gene encoding luciferase, relative XylS expression levels could be monitored indirectly at physiological concentrations. Expression of XylS from inducible promoters allowed control over a more than 800-fold range, however, the corresponding output from Pm covered only an about five-fold range. The maximum output from Pm could not be increased by introducing more copies of the promoter in the cells. Interestingly, a previously reported XylS variant (StEP-13), known to strongly stimulate expression from Pm, caused the same maximum activity from Pm as wild-type XylS at high XylS expression levels. Under uninduced conditions expression from Pm also increased as a function of XylS expression levels, and at very high concentrations the maximum activity from Pm was the same as in the presence of inducer.

Conclusion: According to our proposed model, which is in agreement with current knowledge, the regulator, XylS, can exist in three states: monomers, dimers, and aggregates. Only the dimers are active and able to induce expression from Pm. Their maximum intracellular concentration and the corresponding output from Pm are limited by the concentration-dependent conversion into inactive aggregates. Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression. The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

Show MeSH