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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: Only the dimers are active and able to induce expression from Pm.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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Effects of variations in wild type or variant XylS expression on Pm activity. Upper host ampicillin tolerance levels as a function of the expression level of wild type XylS (pFZ2B1) or variant StEP-13 (pFZ2B1.StEP-13), using two different copy number variants (pFS15 and pFS15.271) of the target plasmid. Pm activity was measured as upper relative ampicillin tolerance on agar medium. The tolerance for cells containing pFZ2B1 + pFS15, no cyclohexanone, was arbitrarily set to 1 and corresponds to about 650 μg mL-1 ampicillin resistance. The relative XylS expression was measured as luciferase activity and was also set to 1 for the same data point. The data points indicate the highest ampicillin concentration on which growth occurred, while the lowest concentration on which no growth was observed is indicated by error bars. Shapes that are half grey and half black indicate identical data points for both wild type and StEP-13. 1 mM m-toluate was added to all samples, cyclohexanone concentrations leading to the measured XylS expression levels (from left to right): 0, 0.25, 0.5, 1 and 2 mM, respectively.
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Figure 3: Effects of variations in wild type or variant XylS expression on Pm activity. Upper host ampicillin tolerance levels as a function of the expression level of wild type XylS (pFZ2B1) or variant StEP-13 (pFZ2B1.StEP-13), using two different copy number variants (pFS15 and pFS15.271) of the target plasmid. Pm activity was measured as upper relative ampicillin tolerance on agar medium. The tolerance for cells containing pFZ2B1 + pFS15, no cyclohexanone, was arbitrarily set to 1 and corresponds to about 650 μg mL-1 ampicillin resistance. The relative XylS expression was measured as luciferase activity and was also set to 1 for the same data point. The data points indicate the highest ampicillin concentration on which growth occurred, while the lowest concentration on which no growth was observed is indicated by error bars. Shapes that are half grey and half black indicate identical data points for both wild type and StEP-13. 1 mM m-toluate was added to all samples, cyclohexanone concentrations leading to the measured XylS expression levels (from left to right): 0, 0.25, 0.5, 1 and 2 mM, respectively.

Mentions: The experiments described above as well as previously published studies [21,31] demonstrate that expression from Pm can be increased by producing more XylS, and to determine what the maximum level is we decided to use the inducible Pb promoter from Acinetobacter sp. to express XylS. Pb, like Pm, can be used to regulate expression of genes in a continuously graded manner [33]. It is positively regulated by the ChnR protein, which also belongs to the AraC/XylS transcription factor family, in the presence of its inducer cyclohexanone. The xylS-luc operon expressed from Pb and the gene of the activator protein, chnR, were cloned into pBBR1MCS-5 [34], generating pFZ2B1, and pFS15 was used as target plasmid for XylS harboring the Pm promoter, as described above. Cells containing both of these plasmids were plated on agar medium, supplemented with varying amounts of ampicillin, cyclohexanone and m-toluate. As expected, cells with only one of the two plasmids (either pFZ2B1 or pFS15) reacted only marginally to the addition of the inducers. However, in the presence of both plasmids the ampicillin tolerance of the host cells varied as a function of both the cyclohexanone and m-toluate concentrations. At a fixed 1 mM m-toluate concentration the host ampicillin tolerance correlated well with both the concentration of cyclohexanone and the luciferase activity, which reflects XylS expression (Figure 3, grey squares). However, at the two highest concentrations of cyclohexanone tested (1 and 2 mM) the upper ampicillin tolerances were similar (3500 μg mL-1) and about 5.4 times higher than in the absence of the Pb inducer.


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)

Effects of variations in wild type or variant XylS expression on Pm activity. Upper host ampicillin tolerance levels as a function of the expression level of wild type XylS (pFZ2B1) or variant StEP-13 (pFZ2B1.StEP-13), using two different copy number variants (pFS15 and pFS15.271) of the target plasmid. Pm activity was measured as upper relative ampicillin tolerance on agar medium. The tolerance for cells containing pFZ2B1 + pFS15, no cyclohexanone, was arbitrarily set to 1 and corresponds to about 650 μg mL-1 ampicillin resistance. The relative XylS expression was measured as luciferase activity and was also set to 1 for the same data point. The data points indicate the highest ampicillin concentration on which growth occurred, while the lowest concentration on which no growth was observed is indicated by error bars. Shapes that are half grey and half black indicate identical data points for both wild type and StEP-13. 1 mM m-toluate was added to all samples, cyclohexanone concentrations leading to the measured XylS expression levels (from left to right): 0, 0.25, 0.5, 1 and 2 mM, respectively.
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Related In: Results  -  Collection

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Figure 3: Effects of variations in wild type or variant XylS expression on Pm activity. Upper host ampicillin tolerance levels as a function of the expression level of wild type XylS (pFZ2B1) or variant StEP-13 (pFZ2B1.StEP-13), using two different copy number variants (pFS15 and pFS15.271) of the target plasmid. Pm activity was measured as upper relative ampicillin tolerance on agar medium. The tolerance for cells containing pFZ2B1 + pFS15, no cyclohexanone, was arbitrarily set to 1 and corresponds to about 650 μg mL-1 ampicillin resistance. The relative XylS expression was measured as luciferase activity and was also set to 1 for the same data point. The data points indicate the highest ampicillin concentration on which growth occurred, while the lowest concentration on which no growth was observed is indicated by error bars. Shapes that are half grey and half black indicate identical data points for both wild type and StEP-13. 1 mM m-toluate was added to all samples, cyclohexanone concentrations leading to the measured XylS expression levels (from left to right): 0, 0.25, 0.5, 1 and 2 mM, respectively.
Mentions: The experiments described above as well as previously published studies [21,31] demonstrate that expression from Pm can be increased by producing more XylS, and to determine what the maximum level is we decided to use the inducible Pb promoter from Acinetobacter sp. to express XylS. Pb, like Pm, can be used to regulate expression of genes in a continuously graded manner [33]. It is positively regulated by the ChnR protein, which also belongs to the AraC/XylS transcription factor family, in the presence of its inducer cyclohexanone. The xylS-luc operon expressed from Pb and the gene of the activator protein, chnR, were cloned into pBBR1MCS-5 [34], generating pFZ2B1, and pFS15 was used as target plasmid for XylS harboring the Pm promoter, as described above. Cells containing both of these plasmids were plated on agar medium, supplemented with varying amounts of ampicillin, cyclohexanone and m-toluate. As expected, cells with only one of the two plasmids (either pFZ2B1 or pFS15) reacted only marginally to the addition of the inducers. However, in the presence of both plasmids the ampicillin tolerance of the host cells varied as a function of both the cyclohexanone and m-toluate concentrations. At a fixed 1 mM m-toluate concentration the host ampicillin tolerance correlated well with both the concentration of cyclohexanone and the luciferase activity, which reflects XylS expression (Figure 3, grey squares). However, at the two highest concentrations of cyclohexanone tested (1 and 2 mM) the upper ampicillin tolerances were similar (3500 μg mL-1) and about 5.4 times higher than in the absence of the Pb inducer.

Bottom Line: Only the dimers are active and able to induce expression from Pm.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
Related in: MedlinePlus