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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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Visualization of the hypothesis explaining XylS behaviour at various intracellular concentrations. The numbers of DNA or XylS molecules are not meant to represent the actual numbers in the cells. Only aggregates formed from active dimers of the protein are considered. At low XylS concentrations a certain percentage of the dimerized XylS molecules will activate transcription (a); the amount of activated Pm promoters will increase proportionally to XylS amounts up to a certain treshold value (b); when the threshold value is exceeded, XylS dimers will aggregate and become inactive, while the amount of active dimers remains constant (c). For StEP-13 a higher percentage of XylS molecules will dimerize at low XylS concentrations, resulting in more transcribed DNA (d); when the saturating concentration for wild type XylS is reached, there will already be some aggregation of dimers in case of StEP-13 (e), and as for wild type this will increase further as more XylS is expressed (f). In the absence of m-toluate, only a very small fraction of the XylS molecules will form dimers and these will activate transcription from Pm, aggregation does not start at the XylS expression levels depicted here (g, h, i).
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Figure 6: Visualization of the hypothesis explaining XylS behaviour at various intracellular concentrations. The numbers of DNA or XylS molecules are not meant to represent the actual numbers in the cells. Only aggregates formed from active dimers of the protein are considered. At low XylS concentrations a certain percentage of the dimerized XylS molecules will activate transcription (a); the amount of activated Pm promoters will increase proportionally to XylS amounts up to a certain treshold value (b); when the threshold value is exceeded, XylS dimers will aggregate and become inactive, while the amount of active dimers remains constant (c). For StEP-13 a higher percentage of XylS molecules will dimerize at low XylS concentrations, resulting in more transcribed DNA (d); when the saturating concentration for wild type XylS is reached, there will already be some aggregation of dimers in case of StEP-13 (e), and as for wild type this will increase further as more XylS is expressed (f). In the absence of m-toluate, only a very small fraction of the XylS molecules will form dimers and these will activate transcription from Pm, aggregation does not start at the XylS expression levels depicted here (g, h, i).

Mentions: In the absence of m-toluate XylS is mainly present in a monomeric state, which probably is not able to activate Pm, while in the presence of m-toluate an unknown fraction of these monomers are converted to dimers, which activate transcription from Pm[5,6]. At low XylS concentrations formation of active dimers probably depends on m-toluate concentrations (Figure 6a), and this assumption can explain the well known fact that expression from Pm correlates with the concentration of inducer at fixed levels of XylS expression (usually from Ps2). In contrast, above a certain threshold value for XylS expression (illustrated in Figure 6b) the activity from Pm does not increase any further, and this can be explained by formation of XylS in a third state, as aggregated and not active molecules (Figure 6c). Alternatively, the threshold value might also be caused by saturation of the Pm targets available in the cell. However, this explanation does not fit with the observation that introduction of more Pm copies does not lead to a corresponding stimulation of expression even if total XylS levels are increased beyond the threshold value (Figure 3). Therefore, the upper maximum level of active dimers in the cells seems to be the result of inherent properties of the XylS molecule itself.


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)

Visualization of the hypothesis explaining XylS behaviour at various intracellular concentrations. The numbers of DNA or XylS molecules are not meant to represent the actual numbers in the cells. Only aggregates formed from active dimers of the protein are considered. At low XylS concentrations a certain percentage of the dimerized XylS molecules will activate transcription (a); the amount of activated Pm promoters will increase proportionally to XylS amounts up to a certain treshold value (b); when the threshold value is exceeded, XylS dimers will aggregate and become inactive, while the amount of active dimers remains constant (c). For StEP-13 a higher percentage of XylS molecules will dimerize at low XylS concentrations, resulting in more transcribed DNA (d); when the saturating concentration for wild type XylS is reached, there will already be some aggregation of dimers in case of StEP-13 (e), and as for wild type this will increase further as more XylS is expressed (f). In the absence of m-toluate, only a very small fraction of the XylS molecules will form dimers and these will activate transcription from Pm, aggregation does not start at the XylS expression levels depicted here (g, h, i).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4225500&req=5

Figure 6: Visualization of the hypothesis explaining XylS behaviour at various intracellular concentrations. The numbers of DNA or XylS molecules are not meant to represent the actual numbers in the cells. Only aggregates formed from active dimers of the protein are considered. At low XylS concentrations a certain percentage of the dimerized XylS molecules will activate transcription (a); the amount of activated Pm promoters will increase proportionally to XylS amounts up to a certain treshold value (b); when the threshold value is exceeded, XylS dimers will aggregate and become inactive, while the amount of active dimers remains constant (c). For StEP-13 a higher percentage of XylS molecules will dimerize at low XylS concentrations, resulting in more transcribed DNA (d); when the saturating concentration for wild type XylS is reached, there will already be some aggregation of dimers in case of StEP-13 (e), and as for wild type this will increase further as more XylS is expressed (f). In the absence of m-toluate, only a very small fraction of the XylS molecules will form dimers and these will activate transcription from Pm, aggregation does not start at the XylS expression levels depicted here (g, h, i).
Mentions: In the absence of m-toluate XylS is mainly present in a monomeric state, which probably is not able to activate Pm, while in the presence of m-toluate an unknown fraction of these monomers are converted to dimers, which activate transcription from Pm[5,6]. At low XylS concentrations formation of active dimers probably depends on m-toluate concentrations (Figure 6a), and this assumption can explain the well known fact that expression from Pm correlates with the concentration of inducer at fixed levels of XylS expression (usually from Ps2). In contrast, above a certain threshold value for XylS expression (illustrated in Figure 6b) the activity from Pm does not increase any further, and this can be explained by formation of XylS in a third state, as aggregated and not active molecules (Figure 6c). Alternatively, the threshold value might also be caused by saturation of the Pm targets available in the cell. However, this explanation does not fit with the observation that introduction of more Pm copies does not lead to a corresponding stimulation of expression even if total XylS levels are increased beyond the threshold value (Figure 3). Therefore, the upper maximum level of active dimers in the cells seems to be the result of inherent properties of the XylS molecule itself.

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
Related in: MedlinePlus